# Patent

## Title
Compositions for use in identification of bacteria

## Abstract

The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 11/409,535, filed April 21, 2006, which is a continuation-in-part of U.S. application Ser. No. 11/060,135, filed Feb. 17, 2005 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/545,425 filed Feb. 18, 2004; U.S. Provisional Application Ser. No. 60/559,754, filed Apr. 5, 2004; U.S. Provisional Application Ser. No. 60/632,862, filed Dec. 3, 2004; U.S. Provisional Application Ser. No. 60/639,068, filed Dec. 22, 2004; and U.S. Provisional Application Ser. No. 60/648,188, filed Jan. 28, 2005. U.S. application Ser. No. 11/409,535 is a also continuation-in-part of U.S. application Ser. No. 10/728,486, filed Dec. 5, 2003 now U.S. Pat. No. 7,718,354 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/501,926, filed Sep. 11, 2003. U.S. application Ser. No. 11/409,535 also claims the benefit of priority to: U.S. Provisional Application Ser. No. 60/674,118, filed Apr. 21, 2005; U.S. Provisional Application Ser. No. 60/705,631, filed Aug. 3, 2005; U.S. Provisional Application Ser. No. 60/732,539, filed Nov. 1, 2005; and U.S. Provisional Application Ser. No. 60/773,124, filed Feb. 13, 2006. Each of the above-referenced U.S. Applications is incorporated herein by reference in its entirety. Methods disclosed in U.S. application Ser. Nos. 09/891,793, 10/156,608, 10/405,756, 10/418,514, 10/660,122, 10/660,996, 10/660,997, 10/660,998, 10/728,486, 11/060,135, and 11/073,362, are commonly owned and incorporated herein by reference in their entirety for any purpose.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made with United States Government support under CDC contracts RO1 CI000099-01. The United States Government has certain rights in the invention.


SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled DIBIS0083USC3SEQ.txt, created on Mar. 6, 2007 which is 252 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.
BACKGROUND OF THE INVENTION
A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al. Philos. Trans. R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals.
Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific primers and probes designed to selectively detect certain pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of infectious disease agents and there are equally important biosafety and security concerns for agriculture.
A major conundrum in public health protection, biodefense, and agricultural safety and security is that these disciplines need to be able to rapidly identify and characterize infectious agents, while there is no existing technology with the breadth of function to meet this need. Currently used methods for identification of bacteria rely upon culturing the bacterium to effect isolation from other organisms and to obtain sufficient quantities of nucleic acid followed by sequencing of the nucleic acid, both processes which are time and labor intensive.
Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism.
The present invention provides oligonucleotide primers and compositions and kits containing the oligonucleotide primers, which define bacterial bioagent identifying amplicons and, upon amplification, produce corresponding amplification products whose molecular masses provide the means to identify bacteria, for example, at and below the species taxonomic level.
SUMMARY OF THE INVENTION
The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 288.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1269.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 288 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1269.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 698.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1420.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 698 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1420.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 217.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1167
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 217 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1167.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 399.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1041.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 399 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1041.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 430.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1321.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 430 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1321.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 174.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 853.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 174 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 853.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 172.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1360.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 172 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1360.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 288:1269, 698:1420, 217:1167, 399:1041, 430:1321, 174:853, and 172:1360.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 315.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1379.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 315 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1379.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 346.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 955.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 346 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 955.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 504.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1409.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 504 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1409.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 323.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1068.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 323 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1068.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 479.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 938.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 479 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 938.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 315:1379, 346:955, 504:1409, 323:1068, 479:938.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 583.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 923.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 583 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 923.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 454.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1418.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 454 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1418.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 250.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 902.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 250 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 902.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 384.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 878.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 384 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 878.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 694.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1215.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 694 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1215.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 194.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1173.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 194 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1173.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 375.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 890.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 375 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 890.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 656.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1224.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 656 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1224.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 618.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1157.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 618 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1157.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 302.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 852.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 302 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 852.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 199.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 889.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 199 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 889.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 596.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1169.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 596 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1169.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 150.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1242.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 150 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1242.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1069.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1069.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1168.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1168.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 583 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 923 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 454:1418, 250:902, 384:878, 694:1215, 194:1173, 375:890, 656:1224, 618:1157, 302:852, 199:889, 596:1169, 150:1242, 166:1069 and 166:1168.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 437.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1137.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 437 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1137.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 530.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 891.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 530 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 891.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 474.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 869.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 474 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 869.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 268.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1284.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 268 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1284.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 418.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1301.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 418 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1301.
An embodiment is an oligonucleotide primer pair comprising a forward and a reverse primer, each comprising between 13 and 35 linked nucleotides in length, designed to generate an amplicon that is between about 45 and about 200 linked nucleotides in length, wherein said forward primer comprises at least 80% complementarity to a first region within nucleotides 1-286 of a reference sequence, said reference sequence being a sequence extraction of coordinates 830671-831072 of Genbank gi number 21281729 (ttccacgaaacagatgaa gaaattaacaaaaaagcgcacgctattttcaaacatggaatgactccaattatttgtgttggtgaaacagacgaagagc gtgaaagtggtaaagctaacgatgttgtaggtgagcaagttaagaaagctgttgcaggtttatctgaagatcaacttaa atcagttgtaattgcttatgagccaatctgggcaatcggaactggtaaatcatcaacatctgaagatgcaaatgaaatgt gtgcatttgtacgtcaaactattgctgacttatcaagcaaagaagtatcagaagcaactcgtattcaatatggtggtagtg ttaaacctaacaacattaaagaatacatggcacaaactgatattgatggggcattagtaggtggc (SEQ ID NO.: 1465)), and wherein said reverse primer comprises at least 80% complementarity to a second region within nucleotides 1-286 of said reference sequence.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 318.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1300.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 318 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1300.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 440.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1076.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 440 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1076.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 219.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1013.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 219 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1013.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 437 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1137 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 530:891, 474:869, 268:1284, 418:1301, 318:1300, 440:1076 and 219:1013.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 325.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1163.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 325 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1163.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 278.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1039.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 278 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1039.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 465.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1037.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 465 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1037.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 148.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1172.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 148 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1172.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 190.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1254.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 190 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1254.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 266.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1094.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 266 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1094.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 508.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1297.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 508 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1297.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 259.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1060.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 259 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1060.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 325 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1163 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 278:1039: 465:1037, 148:1172, 190:1254, 266:1094, 508:1297 and 259:1060.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 376.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1265.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 376 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1265.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 267.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1341.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 267 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1341.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 705.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1056.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 705 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1056.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 710.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1259.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 710 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1259.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 374.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1111.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 374 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1111.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 545.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 978.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 545 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 978.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 249.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1095.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 249 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1095.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 195.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1376.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 195 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1376.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 311.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1014.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 311 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1014.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 365.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1052.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 365 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1052.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 527.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1071.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 527 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1071.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 490.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1182.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 490 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1182.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 376 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1265 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 267:1341, 705:1056, 710:1259, 374:1111, 545:978, 249:1095, 195:1376, 311:1014, 365:1052, 527:1071 and 490:1182.
In some embodiments, either or both of the primers of a primer pair composition contain at least one modified nucleobase such as 5-propynyluracil or 5-propynylcytosine for example.
In some embodiments, either or both of the primers of the primer pair comprises at least one universal nucleobase such as inosine for example.
In some embodiments, either or both of the primers of the primer pair comprises at least one non-templated T residue on the 5′-end.
In some embodiments, either or both of the primers of the primer pair comprises at least one non-template tag.
In some embodiments, either or both of the primers of the primer pair comprises at least one molecular mass modifying tag.
In some embodiments, the present invention provides primers and compositions comprising pairs of primers, and kits containing the same, and methods for use in identification of bacteria. The primers are designed to produce amplification products of DNA encoding genes that have conserved and variable regions across different subgroups and genotypes of bacteria.
Some embodiments are kits that contain one or more of the primer pair compositions. In some embodiments, each member of the one or more primer pairs of the kit is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from any of the primer pairs listed in Table 2.
Some embodiments of the kits contain at least one calibration polynucleotide for use in quantitiation of bacteria in a given sample, and also for use as a positive control for amplification.
Some embodiments of the kits contain at least one anion exchange functional group linked to a magnetic bead.
In some embodiments, the present invention also provides methods for identification of bacteria. Nucleic acid from the bacterium is amplified using the primers described above to obtain an amplification product. The molecular mass of the amplification product is measured. Optionally, the base composition of the amplification product is determined from the molecular mass. The molecular mass or base composition is compared with a plurality of molecular masses or base compositions of known analogous bacterial identifying amplicons, wherein a match between the molecular mass or base composition and a member of the plurality of molecular masses or base compositions identifies the bacterium. In some embodiments, the molecular mass is measured by mass spectrometry in a modality such as electrospray ionization (ESI) time of flight (TOF) mass spectrometry or ESI Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, for example. Other mass spectrometry techniques can also be used to measure the molecular mass of bacterial bioagent identifying amplicons.
In some embodiments, the present invention is also directed to a method for determining the presence or absence of a bacterium in a sample. Nucleic acid from the sample is amplified using the composition described above to obtain an amplification product. The molecular mass of the amplification product is determined. Optionally, the base composition of the amplification product is determined from the molecular mass. The molecular mass or base composition of the amplification product is compared with the known molecular masses or base compositions of one or more known analogous bacterial bioagent identifying amplicons, wherein a match between the molecular mass or base composition of the amplification product and the molecular mass or base composition of one or more known bacterial bioagent identifying amplicons indicates the presence of the bacterium in the sample. In some embodiments, the molecular mass is measured by mass spectrometry.
In some embodiments, the present invention also provides methods for determination of the quantity of an unknown bacterium in a sample. The sample is contacted with the composition described above and a known quantity of a calibration polynucleotide comprising a calibration sequence. Nucleic acid from the unknown bacterium in the sample is concurrently amplified with the composition described above and nucleic acid from the calibration polynucleotide in the sample is concurrently amplified with the composition described above to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon. The molecular masses and abundances for the bacterial bioagent identifying amplicon and the calibration amplicon are determined. The bacterial bioagent identifying amplicon is distinguished from the calibration amplicon based on molecular mass and comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in the sample. In some embodiments, the base composition of the bacterial bioagent identifying amplicon is determined.
In some embodiments, the present invention provides methods for detecting or quantifying bacteria by combining a nucleic acid amplification process with a mass determination process. In some embodiments, such methods identify or otherwise analyze the bacterium by comparing mass information from an amplification product with a calibration or control product. Such methods can be carried out in a highly multiplexed and/or parallel manner allowing for the analysis of as many as 300 samples per 24 hours on a single mass measurement platform. The accuracy of the mass determination methods in some embodiments of the present invention permits allows for the ability to discriminate between different bacteria such as, for example, various genotypes and drug resistant strains of Staphylococcus aureus. 



BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing summary of the invention, as well as the following detailed description of the invention, is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation.
FIG. 1: process diagram illustrating a representative primer pair selection process.
FIG. 2: process diagram illustrating an embodiment of the calibration method.
FIG. 3: common pathogenic bacteria and primer pair coverage. The primer pair number in the upper right hand corner of each polygon indicates that the primer pair can produce a bioagent identifying amplicon for all species within that polygon.
FIG. 4: a representative 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples (labeled NHRC samples) closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.
FIG. 5: a representative mass spectrum of amplification products indicating the presence of bioagent identifying amplicons of Streptococcus pyogenes, Neisseria meningitidis, and Haemophilus influenzae obtained from amplification of nucleic acid from a clinical sample with primer pair number 349 which targets 23S rRNA. Experimentally determined molecular masses and base compositions for the sense strand of each amplification product are shown.
FIG. 6: a representative mass spectrum of amplification products representing a bioagent identifying amplicon of Streptococcus pyogenes, and a calibration amplicon obtained from amplification of nucleic acid from a clinical sample with primer pair number 356 which targets rplB. The experimentally determined molecular mass and base composition for the sense strand of the Streptococcus pyogenes amplification product is shown.
FIG. 7: a representative mass spectrum of an amplified nucleic acid mixture which contained the Ames strain of Bacillus anthracis, a known quantity of combination calibration polynucleotide (SEQ ID NO: 1464), and primer pair number 350 which targets the capC gene on the virulence plasmid pX02 of Bacillus anthracis. Calibration amplicons produced in the amplification reaction are visible in the mass spectrum as indicated and abundance data (peak height) are used to calculate the quantity of the Ames strain of Bacillus anthracis. 



DEFINITIONS
As used herein, the term “abundance” refers to an amount. The amount may be described in terms of concentration which are common in molecular biology such as “copy number,” “pfu or plate-forming unit” which are well known to those with ordinary skill. Concentration may be relative to a known standard or may be absolute.
As used herein, the term “amplifiable nucleic acid” is used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” also comprises “sample template.”
As used herein the term “amplification” refers to a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out. Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Qβ replicase, MDV-1 RNA is the specific template for the replicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acid will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (D. Y. Wu and R. B. Wallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989]).
As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification, excluding primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
As used herein, the term “analogous” when used in context of comparison of bioagent identifying amplicons indicates that the bioagent identifying amplicons being compared are produced with the same pair of primers. For example, bioagent identifying amplicon “A” and bioagent identifying amplicon “B”, produced with the same pair of primers are analogous with respect to each other. Bioagent identifying amplicon “C”, produced with a different pair of primers is not analogous to either bioagent identifying amplicon “A” or bioagent identifying amplicon “B”.
As used herein, the term “anion exchange functional group” refers to a positively charged functional group capable of binding an anion through an electrostatic interaction. The most well known anion exchange functional groups are the amines, including primary, secondary, tertiary and quaternary amines.
The term “bacteria” or “bacterium” refers to any member of the groups of eubacteria and archaebacteria.
As used herein, a “base composition” is the exact number of each nucleobase (for example, A, T, C and G) in a segment of nucleic acid. For example, amplification of nucleic acid of Staphylococcus aureus strain carrying the lukS-PV gene with primer pair number 2095 (SEQ ID NOs: 456:1261) produces an amplification product 117 nucleobases in length from nucleic acid of the lukS-PV gene that has a base composition of A35 G17 C19 T46 (by convention—with reference to the sense strand of the amplification product). Because the molecular masses of each of the four natural nucleotides and chemical modifications thereof are known (if applicable), a measured molecular mass can be deconvoluted to a list of possible base compositions. Identification of a base composition of a sense strand which is complementary to the corresponding antisense strand in terms of base composition provides a confirmation of the true base composition of an unknown amplification product. For example, the base composition of the antisense strand of the 139 nucleobase amplification product described above is A46 G19 C17 T35.
As used herein, a “base composition probability cloud” is a representation of the diversity in base composition resulting from a variation in sequence that occurs among different isolates of a given species. The “base composition probability cloud” represents the base composition constraints for each species and is typically visualized using a pseudo four-dimensional plot.
In the context of this invention, a “bioagent” is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus. Examples of bioagents include, but are not limited, to cells, (including but not limited to human clinical samples, bacterial cells and other pathogens), viruses, fungi, protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered. In the context of this invention, a “pathogen” is a bioagent which causes a disease or disorder.
As used herein, a “bioagent division” is defined as group of bioagents above the species level and includes but is not limited to, orders, families, classes, clades, genera or other such groupings of bioagents above the species level.
As used herein, the term “bioagent identifying amplicon” refers to a polynucleotide that is amplified from a bioagent in an amplification reaction and which 1) provides sufficient variability to distinguish among bioagents from whose nucleic acid the bioagent identifying amplicon is produced and 2) whose molecular mass is amenable to a rapid and convenient molecular mass determination modality such as mass spectrometry, for example.
As used herein, the term “biological product” refers to any product originating from an organism. Biological products are often products of processes of biotechnology. Examples of biological products include, but are not limited to: cultured cell lines, cellular components, antibodies, proteins and other cell-derived biomolecules, growth media, growth harvest fluids, natural products and bio-pharmaceutical products.
The terms “biowarfare agent” and “bioweapon” are synonymous and refer to a bacterium, virus, fungus or protozoan that could be deployed as a weapon to cause bodily harm to individuals. Military or terrorist groups may be implicated in deployment of biowarfare agents.
In context of this invention, the term “broad range survey primer pair” refers to a primer pair designed to produce bioagent identifying amplicons across different broad groupings of bioagents. For example, the ribosomal RNA-targeted primer pairs are broad range survey primer pairs which have the capability of producing bacterial bioagent identifying amplicons for essentially all known bacteria. With respect to broad range primer pairs employed for identification of bacteria, a broad range survey primer pair for bacteria such as 16S rRNA primer pair number 346 (SEQ ID NOs: 202:1110) for example, will produce an bacterial bioagent identifying amplicon for essentially all known bacteria.
The term “calibration amplicon” refers to a nucleic acid segment representing an amplification product obtained by amplification of a calibration sequence with a pair of primers designed to produce a bioagent identifying amplicon.
The term “calibration sequence” refers to a polynucleotide sequence to which a given pair of primers hybridizes for the purpose of producing an internal (i.e.: included in the reaction) calibration standard amplification product for use in determining the quantity of a bioagent in a sample. The calibration sequence may be expressly added to an amplification reaction, or may already be present in the sample prior to analysis.
The term “clade primer pair” refers to a primer pair designed to produce bioagent identifying amplicons for species belonging to a clade group. A clade primer pair may also be considered as a “speciating” primer pair which is useful for distinguishing among closely related species.
The term “codon” refers to a set of three adjoined nucleotides (triplet) that codes for an amino acid or a termination signal.
In context of this invention, the term “codon base composition analysis,” refers to determination of the base composition of an individual codon by obtaining a bioagent identifying amplicon that includes the codon. The bioagent identifying amplicon will at least include regions of the target nucleic acid sequence to which the primers hybridize for generation of the bioagent identifying amplicon as well as the codon being analyzed, located between the two primer hybridization regions.
As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) related by the base-pairing rules. For example, for the sequence “5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid strand, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand.
The term “complement of a nucleic acid sequence” as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5′ end of one sequence is paired with the 3′ end of the other, is in “antiparallel association.” Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. Where a first oligonucleotide is complementary to a region of a target nucleic acid and a second oligonucleotide has complementary to the same region (or a portion of this region) a “region of overlap” exists along the target nucleic acid. The degree of overlap will vary depending upon the extent of the complementarity.
In context of this invention, the term “division-wide primer pair” refers to a primer pair designed to produce bioagent identifying amplicons within sections of a broader spectrum of bioagents For example, primer pair number 352 (SEQ ID NOs: 687:1411), a division-wide primer pair, is designed to produce bacterial bioagent identifying amplicons for members of the Bacillus group of bacteria which comprises, for example, members of the genera Streptococci, Enterococci, and Staphylococci. Other division-wide primer pairs may be used to produce bacterial bioagent identifying amplicons for other groups of bacterial bioagents.
As used herein, the term “concurrently amplifying” used with respect to more than one amplification reaction refers to the act of simultaneously amplifying more than one nucleic acid in a single reaction mixture.
As used herein, the term “drill-down primer pair” refers to a primer pair designed to produce bioagent identifying amplicons for identification of sub-species characteristics or confirmation of a species assignment. For example, primer pair number 2146 (SEQ ID NOs: 437:1137), a drill-down Staphylococcus aureus genotyping primer pair, is designed to produce Staphylococcus aureus genotyping amplicons. Other drill-down primer pairs may be used to produce bioagent identifying amplicons for Staphylococcus aureus and other bacterial species.
The term “duplex” refers to the state of nucleic acids in which the base portions of the nucleotides on one strand are bound through hydrogen bonding the their complementary bases arrayed on a second strand. The condition of being in a duplex form reflects on the state of the bases of a nucleic acid. By virtue of base pairing, the strands of nucleic acid also generally assume the tertiary structure of a double helix, having a major and a minor groove. The assumption of the helical form is implicit in the act of becoming duplexed.
As used herein, the term “etiology” refers to the causes or origins, of diseases or abnormal physiological conditions.
The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide or a precursor. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.
The terms “homology,” “homologous” and “sequence identity” refer to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is otherwise identical to another 20 nucleobase primer but having two non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of a primer 20 nucleobases in length would have 15/20=0.75 or 75% sequence identity with the 20 nucleobase primer. In context of the present invention, sequence identity is meant to be properly determined when the query sequence and the subject sequence are both described and aligned in the 5′ to 3′ direction. Sequence alignment algorithms such as BLAST, will return results in two different alignment orientations. In the Plus/Plus orientation, both the query sequence and the subject sequence are aligned in the 5′ to 3′ direction. On the other hand, in the Plus/Minus orientation, the query sequence is in the 5′ to 3′ direction while the subject sequence is in the 3′ to 5′ direction. It should be understood that with respect to the primers of the present invention, sequence identity is properly determined when the alignment is designated as Plus/Plus. Sequence identity may also encompass alternate or modified nucleobases that perform in a functionally similar manner to the regular nucleobases adenine, thymine, guanine and cytosine with respect to hybridization and primer extension in amplification reactions. In a non-limiting example, if the 5-propynyl pyrimidines propyne C and/or propyne T replace one or more C or T residues in one primer which is otherwise identical to another primer in sequence and length, the two primers will have 100% sequence identity with each other. In another non-limiting example, Inosine (I) may be used as a replacement for G or T and effectively hybridize to C, A or U (uracil). Thus, if inosine replaces one or more C, A or U residues in one primer which is otherwise identical to another primer in sequence and length, the two primers will have 100% sequence identity with each other. Other such modified or universal bases may exist which would perform in a functionally similar manner for hybridization and amplification reactions and will be understood to fall within this definition of sequence identity.
As used herein, “housekeeping gene” refers to a gene encoding a protein or RNA involved in basic functions required for survival and reproduction of a bioagent. Housekeeping genes include, but are not limited to genes encoding RNA or proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like.
As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the Tm of the formed hybrid. “Hybridization” methods involve the annealing of one nucleic acid to another, complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the “hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology.
The term “in silico” refers to processes taking place via computer calculations. For example, electronic PCR (ePCR) is a process analogous to ordinary PCR except that it is carried out using nucleic acid sequences and primer pair sequences stored on a computer formatted medium.
As used herein, “intelligent primers” are primers that are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and, upon amplification, yield amplification products which ideally provide enough variability to distinguish individual bioagents, and which are amenable to molecular mass analysis. By the term “highly conserved,” it is meant that the sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity among all, or at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of species or strains.
The “ligase chain reaction” (LCR; sometimes referred to as “Ligase Amplification Reaction” (LAR) described by Barany, Proc. Natl. Acad. Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wu and Wallace, Genomics 4:560 (1989) has developed into a well-recognized alternative method for amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, that hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, hybridization and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes. However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
The term “locked nucleic acid” or “LNA” refers to a nucleic acid analogue containing one or more 2′-O, 4′-C-methylene-β-D-ribofuranosyl nucleotide monomers in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The primers of the present invention may contain LNA modifications.
As used herein, the term “mass-modifying tag” refers to any modification to a given nucleotide which results in an increase in mass relative to the analogous non-mass modified nucleotide. Mass-modifying tags can include heavy isotopes of one or more elements included in the nucleotide such as carbon-13 for example. Other possible modifications include addition of substituents such as iodine or bromine at the 5 position of the nucleobase for example.
The term “mass spectrometry” refers to measurement of the mass of atoms or molecules. The molecules are first converted to ions, which are separated using electric or magnetic fields according to the ratio of their mass to electric charge. The measured masses are used to identity the molecules.
The term “microorganism” as used herein means an organism too small to be observed with the unaided eye and includes, but is not limited to bacteria, virus, protozoans, fungi; and ciliates.
The term “multi-drug resistant” or multiple-drug resistant”refers to a microorganism which is resistant to more than one of the antibiotics or antimicrobial agents used in the treatment of said microorganism.
The term “multiplex PCR” refers to a PCR reaction where more than one primer set is included in the reaction pool allowing 2 or more different DNA targets to be amplified by PCR in a single reaction tube.
The term “non-template tag” refers to a stretch of at least three guanine or cytosine nucleobases of a primer used to produce a bioagent identifying amplicon which are not complementary to the template. A non-template tag is incorporated into a primer for the purpose of increasing the primer-duplex stability of later cycles of amplification by incorporation of extra G-C pairs which each have one additional hydrogen bond relative to an A-T pair.
The term “nucleic acid sequence” as used herein refers to the linear composition of the nucleic acid residues A, T, C or G or any modifications thereof, within an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double stranded, and represent the sense or antisense strand
As used herein, the term “nucleobase” is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).
The term “nucleotide analog” as used herein refers to modified or non-naturally occurring nucleotides such as 5-propynyl pyrimidines (i.e., 5-propynyl-dTTP and 5-propynyl-dTCP), 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogs and comprise modified forms of deoxyribonucleotides as well as ribonucleotides.
The term “oligonucleotide” as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 13 to 35 nucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5′-end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′-end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends. A first region along a nucleic acid strand is said to be upstream of another region if the 3′ end of the first region is before the 5′ end of the second region when moving along a strand of nucleic acid in a 5′ to 3′ direction. All oligonucleotide primers disclosed herein are understood to be presented in the 5′ to 3′ direction when reading left to right. When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3′ end of one oligonucleotide points towards the 5′ end of the other, the former may be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide. Similarly, when two overlapping oligonucleotides are hybridized to the same linear complementary nucleic acid sequence, with the first oligonucleotide positioned such that its 5′ end is upstream of the 5′ end of the second oligonucleotide, and the 3′ end of the first oligonucleotide is upstream of the 3′ end of the second oligonucleotide, the first oligonucleotide may be called the “upstream” oligonucleotide and the second oligonucleotide may be called the “downstream” oligonucleotide.
In the context of this invention, a “pathogen” is a bioagent which causes a disease or disorder.
As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
The term “peptide nucleic acid” (“PNA”) as used herein refers to a molecule comprising bases or base analogs such as would be found in natural nucleic acid, but attached to a peptide backbone rather than the sugar-phosphate backbone typical of nucleic acids. The attachment of the bases to the peptide is such as to allow the bases to base pair with complementary bases of nucleic acid in a manner similar to that of an oligonucleotide. These small molecules, also designated anti gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63). The primers of the present invention may comprise PNAs.
The term “polymerase” refers to an enzyme having the ability to synthesize a complementary strand of nucleic acid from a starting template nucleic acid strand and free dNTPs.
As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.” With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
The term “polymerization means” or “polymerization agent” refers to any agent capable of facilitating the addition of nucleoside triphosphates to an oligonucleotide. Preferred polymerization means comprise DNA and RNA polymerases.
As used herein, the terms “pair of primers,” or “primer pair” are synonymous. A primer pair is used for amplification of a nucleic acid sequence. A pair of primers comprises a forward primer and a reverse primer. The forward primer hybridizes to a sense strand of a target gene sequence to be amplified and primes synthesis of an antisense strand (complementary to the sense strand) using the target sequence as a template. A reverse primer hybridizes to the antisense strand of a target gene sequence to be amplified and primes synthesis of a sense strand (complementary to the antisense strand) using the target sequence as a template.
The primers are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which ideally provide enough variability to distinguish each individual bioagent, and which are amenable to molecular mass analysis. In some embodiments, the highly conserved sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity, or between about 99-100% identity. The molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region. Thus design of the primers requires selection of a variable region with appropriate variability to resolve the identity of a given bioagent. Bioagent identifying amplicons are ideally specific to the identity of the bioagent.
Properties of the primers may include any number of properties related to structure including, but not limited to: nucleobase length which may be contiguous (linked together) or non-contiguous (for example, two or more contiguous segments which are joined by a linker or loop moiety), modified or universal nucleobases (used for specific purposes such as for example, increasing hybridization affinity, preventing non-templated adenylation and modifying molecular mass) percent complementarity to a given target sequences.
Properties of the primers also include functional features including, but not limited to, orientation of hybridization (forward or reverse) relative to a nucleic acid template. The coding or sense strand is the strand to which the forward priming primer hybridizes (forward priming orientation) while the reverse priming primer hybridizes to the non-coding or antisense strand (reverse priming orientation). The functional properties of a given primer pair also include the generic template nucleic acid to which the primer pair hybridizes. For example, identification of bioagents can be accomplished at different levels using primers suited to resolution of each individual level of identification. Broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division (e.g., an order, family, genus or other such grouping of bioagents above the species level of bioagents). In some embodiments, broad range survey intelligent primers are capable of identification of bioagents at the species or sub-species level. Other primers may have the functionality of producing bioagent identifying amplicons for members of a given taxonomic genus, clade, species, sub-species or genotype (including genetic variants which may include presence of virulence genes or antibiotic resistance genes or mutations). Additional functional properties of primer pairs include the functionality of performing amplification either singly (single primer pair per amplification reaction vessel) or in a multiplex fashion (multiple primer pairs and multiple amplification reactions within a single reaction vessel).
As used herein, the terms “purified” or “substantially purified” refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An “isolated polynucleotide” or “isolated oligonucleotide” is therefore a substantially purified polynucleotide.
The term “reverse transcriptase” refers to an enzyme having the ability to transcribe DNA from an RNA template. This enzymatic activity is known as reverse transcriptase activity. Reverse transcriptase activity is desirable in order to obtain DNA from RNA viruses which can then be amplified and analyzed by the methods of the present invention.
The term “ribosomal RNA” or “rRNA” refers to the primary ribonucleic acid constituent of ribosomes. Ribosomes are the protein-manufacturing organelles of cells and exist in the cytoplasm. Ribosomal RNAs are transcribed from the DNA genes encoding them.
The term “sample” in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc. Environmental samples include environmental material such as surface matter, soil, water, air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention. The term “source of target nucleic acid” refers to any sample that contains nucleic acids (RNA or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen.
As used herein, the term “sample template” refers to nucleic acid originating from a sample that is analyzed for the presence of “target” (defined below). In contrast, “background template” is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is often a contaminant. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
A “segment” is defined herein as a region of nucleic acid within a target sequence.
The “self-sustained sequence replication reaction” (3SR) (Guatelli et al., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum at Proc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based in vitro amplification system (Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177 [1989]) that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5′ end of the sequence of interest. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).
As used herein, the term “sequence alignment” refers to a listing of multiple DNA or amino acid sequences and aligns them to highlight their similarities. The listings can be made using bioinformatics computer programs.
In context of this invention, the term “speciating primer pair” refers to a primer pair designed to produce a bioagent identifying amplicon with the diagnostic capability of identifying species members of a group of genera or a particular genus of bioagents. Primer pair number 2249 (SEQ ID NOs: 430:1321), for example, is a speciating primer pair used to distinguish Staphylococcus aureus from other species of the genus Staphylococcus. 
As used herein, a “sub-species characteristic” is a genetic characteristic that provides the means to distinguish two members of the same bioagent species. For example, one viral strain could be distinguished from another viral strain of the same species by possessing a genetic change (e.g., for example, a nucleotide deletion, addition or substitution) in one of the viral genes, such as the RNA-dependent RNA polymerase. Sub-species characteristics such as virulence genes and drug-are responsible for the phenotypic differences among the different strains of bacteria.
As used herein, the term “target” is used in a broad sense to indicate the gene or genomic region being amplified by the primers. Because the present invention provides a plurality of amplification products from any given primer pair (depending on the bioagent being analyzed), multiple amplification products from different specific nucleic acid sequences may be obtained. Thus, the term “target” is not used to refer to a single specific nucleic acid sequence. The “target” is sought to be sorted out from other nucleic acid sequences and contains a sequence that has at least partial complementarity with an oligonucleotide primer. The target nucleic acid may comprise single- or double-stranded DNA or RNA. A “segment” is defined as a region of nucleic acid within the target sequence.
The term “template” refers to a strand of nucleic acid on which a complementary copy is built from nucleoside triphosphates through the activity of a template-dependent nucleic acid polymerase. Within a duplex the template strand is, by convention, depicted and described as the “bottom” strand. Similarly, the non-template strand is often depicted and described as the “top” strand.
As used herein, the term “Tm” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. Several equations for calculating the Tm of nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr. Thermodynamics and NMR of internal G. T mismatches in DNA. Biochemistry 36, 10581-94 (1997) include more sophisticated computations which take structural and environmental, as well as sequence characteristics into account for the calculation of Tm.
The term “triangulation genotyping analysis” refers to a method of genotyping a bioagent by measurement of molecular masses or base compositions of amplification products, corresponding to bioagent identifying amplicons, obtained by amplification of regions of more than one gene. In this sense, the term “triangulation” refers to a method of establishing the accuracy of information by comparing three or more types of independent points of view bearing on the same findings. Triangulation genotyping analysis carried out with a plurality of triangulation genotyping analysis primers yields a plurality of base compositions that then provide a pattern or “barcode” from which a species type can be assigned. The species type may represent a previously known sub-species or strain, or may be a previously unknown strain having a specific and previously unobserved base composition barcode indicating the existence of a previously unknown genotype.
As used herein, the term “triangulation genotyping analysis primer pair” is a primer pair designed to produce bioagent identifying amplicons for determining species types in a triangulation genotyping analysis.
The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as “triangulation identification.” Triangulation identification is pursued by analyzing a plurality of bioagent identifying amplicons produced with different primer pairs. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
In the context of this invention, the term “unknown bioagent” may mean either: (i) a bioagent whose existence is known (such as the well known bacterial species Staphylococcus aureus for example) but which is not known to be in a sample to be analyzed, or (ii) a bioagent whose existence is not known (for example, the SARS coronavirus was unknown prior to April 2003). For example, if the method for identification of coronaviruses disclosed in commonly owned U.S. patent Ser. No. 10/829,826 (incorporated herein by reference in its entirety) was to be employed prior to April 2003 to identify the SARS coronavirus in a clinical sample, both meanings of “unknown” bioagent are applicable since the SARS coronavirus was unknown to science prior to April, 2003 and since it was not known what bioagent (in this case a coronavirus) was present in the sample. On the other hand, if the method of U.S. patent Ser. No. 10/829,826 was to be employed subsequent to April 2003 to identify the SARS coronavirus in a clinical sample, only the first meaning (i) of “unknown” bioagent would apply since the SARS coronavirus became known to science subsequent to April 2003 and since it was not known what bioagent was present in the sample.
The term “variable sequence” as used herein refers to differences in nucleic acid sequence between two nucleic acids. For example, the genes of two different bacterial species may vary in sequence by the presence of single base substitutions and/or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another. In the context of the present invention, “viral nucleic acid” includes, but is not limited to, DNA, RNA, or DNA that has been obtained from viral RNA, such as, for example, by performing a reverse transcription reaction. Viral RNA can either be single-stranded (of positive or negative polarity) or double-stranded.
The term “virus” refers to obligate, ultramicroscopic, parasites that are incapable of autonomous replication (i.e., replication requires the use of the host cell's machinery). Viruses can survive outside of a host cell but cannot replicate.
The term “wild-type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified”, “mutant” or “polymorphic” refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
As used herein, a “wobble base” is a variation in a codon found at the third nucleotide position of a DNA triplet. Variations in conserved regions of sequence are often found at the third nucleotide position due to redundancy in the amino acid code.
DETAILED DESCRIPTION OF EMBODIMENTS
A. Bioagent Identifying Amplicons
The present invention provides methods for detection and identification of unknown bioagents using bioagent identifying amplicons. Primers are selected to hybridize to conserved sequence regions of nucleic acids derived from a bioagent, and which bracket variable sequence regions to yield a bioagent identifying amplicon, which can be amplified and which is amenable to molecular mass determination. The molecular mass then provides a means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent. The molecular mass or corresponding base composition signature of the amplification product is then matched against a database of molecular masses or base composition signatures. A match is obtained when an experimentally-determined molecular mass or base composition of an analyzed amplification product is compared with known molecular masses or base compositions of known bioagent identifying amplicons and the experimentally determined molecular mass or base composition is the same as the molecular mass or base composition of one of the known bioagent identifying amplicons. Alternatively, the experimentally-determined molecular mass or base composition may be within experimental error of the molecular mass or base composition of a known bioagent identifying amplicon and still be classified as a match. In some cases, the match may also be classified using a probability of match model such as the models described in U.S. Ser. No. 11/073,362, which is commonly owned and incorporated herein by reference in entirety. Furthermore, the method can be applied to rapid parallel multiplex analyses, the results of which can be employed in a triangulation identification strategy. The present method provides rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent detection and identification.
Despite enormous biological diversity, all forms of life on earth share sets of essential, common features in their genomes. Since genetic data provide the underlying basis for identification of bioagents by the methods of the present invention, it is necessary to select segments of nucleic acids which ideally provide enough variability to distinguish each individual bioagent and whose molecular mass is amenable to molecular mass determination.
Unlike bacterial genomes, which exhibit conservation of numerous genes (i.e. housekeeping genes) across all organisms, viruses do not share a gene that is essential and conserved among all virus families. Therefore, viral identification is achieved within smaller groups of related viruses, such as members of a particular virus family or genus. For example, RNA-dependent RNA polymerase is present in all single-stranded RNA viruses and can be used for broad priming as well as resolution within the virus family.
In some embodiments of the present invention, at least one bacterial nucleic acid segment is amplified in the process of identifying the bacterial bioagent. Thus, the nucleic acid segments that can be amplified by the primers disclosed herein and that provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass determination are herein described as bioagent identifying amplicons.
In some embodiments of the present invention, bioagent identifying amplicons comprise from about 45 to about 150 nucleobases (i.e. from about 45 to about 200 linked nucleosides), although both longer and short regions may be used. One of ordinary skill in the art will appreciate that the invention embodies compounds of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, and 150 nucleobases in length, or any range therewithin.
It is the combination of the portions of the bioagent nucleic acid segment to which the primers hybridize (hybridization sites) and the variable region between the primer hybridization sites that comprises the bioagent identifying amplicon. Thus, it can be said that a given bioagent identifying amplicon is “defined by” a given pair of primers.
In some embodiments, bioagent identifying amplicons amenable to molecular mass determination which are produced by the primers described herein are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible with a means of providing a predictable fragmentation pattern in order to obtain predictable fragments of a length compatible with the particular mode of molecular mass determination. Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with chemical reagents, restriction enzymes or cleavage primers, for example. Thus, in some embodiments, bioagent identifying amplicons are larger than 150 nucleobases and are amenable to molecular mass determination following restriction digestion. Methods of using restriction enzymes and cleavage primers are well known to those with ordinary skill in the art.
In some embodiments, amplification products corresponding to bioagent identifying amplicons are obtained using the polymerase chain reaction (PCR) that is a routine method to those with ordinary skill in the molecular biology arts. Other amplification methods may be used such as ligase chain reaction (LCR), low-stringency single primer PCR, and multiple strand displacement amplification (MDA). These methods are also known to those with ordinary skill.
B. Primers and Primer Pairs
In some embodiments, the primers are designed to bind to conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which provide variability sufficient to distinguish each individual bioagent, and which are amenable to molecular mass analysis. In some embodiments, the highly conserved sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity, or between about 99-100% identity. The molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region. Thus, design of the primers involves selection of a variable region with sufficient variability to resolve the identity of a given bioagent. In some embodiments, bioagent identifying amplicons are specific to the identity of the bioagent.
In some embodiments, identification of bioagents is accomplished at different levels using primers suited to resolution of each individual level of identification. Broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division (e.g., an order, family, genus or other such grouping of bioagents above the species level of bioagents). In some embodiments, broad range survey intelligent primers are capable of identification of bioagents at the species or sub-species level. Examples of broad range survey primers include, but are not limited to: primer pair numbers: 346 (SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 SEQ ID NOs: 706:895), and 361 (SEQ ID NOs: 697:1398) which target DNA encoding 16S rRNA, and primer pair numbers 349 (SEQ ID NOs: 401:1156) and 360 (SEQ ID NOs: 409:1434) which target DNA encoding 23S rRNA.
In some embodiments, drill-down primers are designed with the objective of identifying a bioagent at the sub-species level (including strains, subtypes, variants and isolates) based on sub-species characteristics which may, for example, include single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs), deletions, drug resistance mutations or any other modification of a nucleic acid sequence of a bioagent relative to other members of a species having different sub-species characteristics. Drill-down intelligent primers are not always required for identification at the sub-species level because broad range survey intelligent primers may, in some cases provide sufficient identification resolution to accomplishing this identification objective. Examples of drill-down primers include, but are not limited to: confirmation primer pairs such as primer pair numbers 351 (SEQ ID NOs: 355:1423) and 353 (SEQ ID NOs: 220:1394), which target the pX01 virulence plasmid of Bacillus anthracis. Other examples of drill-down primer pairs are found in sets of triangulation genotyping primer pairs such as, for example, the primer pair number 2146 (SEQ ID NOs: 437:1137) which targets the arcC gene (encoding carmabate kinase) and is included in an 8 primer pair panel or kit for use in genotyping Staphylococcus aureus, or in other panels or kits of primer pairs used for determining drug-resistant bacterial strains, such as, for example, primer pair number 2095 (SEQ ID NOs: 456:1261) which targets the pv-luk gene (encoding Panton-Valentine leukocidin) and is included in an 8 primer pair panel or kit for use in identification of drug resistant strains of Staphylococcus aureus. 
A representative process flow diagram used for primer selection and validation process is outlined in FIG. 1. For each group of organisms, candidate target sequences are identified (200) from which nucleotide alignments are created (210) and analyzed (220). Primers are then designed by selecting appropriate priming regions (230) to facilitate the selection of candidate primer pairs (240). The primer pairs are then subjected to in silico analysis by electronic PCR (ePCR) (300) wherein bioagent identifying amplicons are obtained from sequence databases such as GenBank or other sequence collections (310) and checked for specificity in silico (320). Bioagent identifying amplicons obtained from GenBank sequences (310) can also be analyzed by a probability model which predicts the capability of a given amplicon to identify unknown bioagents such that the base compositions of amplicons with favorable probability scores are then stored in a base composition database (325). Alternatively, base compositions of the bioagent identifying amplicons obtained from the primers and GenBank sequences can be directly entered into the base composition database (330). Candidate primer pairs (240) are validated by testing their ability to hybridize to target nucleic acid by an in vitro amplification by a method such as PCR analysis (400) of nucleic acid from a collection of organisms (410). Amplification products thus obtained are analyzed by gel electrophoresis or by mass spectrometry to confirm the sensitivity, specificity and reproducibility of the primers used to obtain the amplification products (420).
Many of the important pathogens, including the organisms of greatest concern as biowarfare agents, have been completely sequenced. This effort has greatly facilitated the design of primers for the detection of unknown bioagents. The combination of broad-range priming with division-wide and drill-down priming has been used very successfully in several applications of the technology, including environmental surveillance for biowarfare threat agents and clinical sample analysis for medically important pathogens.
Synthesis of primers is well known and routine in the art. The primers may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed.
In some embodiments primers are employed as compositions for use in methods for identification of bacterial bioagents as follows: a primer pair composition is contacted with nucleic acid (such as, for example, bacterial DNA or DNA reverse transcribed from the rRNA) of an unknown bacterial bioagent. The nucleic acid is then amplified by a nucleic acid amplification technique, such as PCR for example, to obtain an amplification product that represents a bioagent identifying amplicon. The molecular mass of each strand of the double-stranded amplification product is determined by a molecular mass measurement technique such as mass spectrometry for example, wherein the two strands of the double-stranded amplification product are separated during the ionization process. In some embodiments, the mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) or electrospray time of flight mass spectrometry (ESI-TOF-MS). A list of possible base compositions can be generated for the molecular mass value obtained for each strand and the choice of the correct base composition from the list is facilitated by matching the base composition of one strand with a complementary base composition of the other strand. The molecular mass or base composition thus determined is then compared with a database of molecular masses or base compositions of analogous bioagent identifying amplicons for known viral bioagents. A match between the molecular mass or base composition of the amplification product and the molecular mass or base composition of an analogous bioagent identifying amplicon for a known viral bioagent indicates the identity of the unknown bioagent. In some embodiments, the primer pair used is one of the primer pairs of Table 2. In some embodiments, the method is repeated using one or more different primer pairs to resolve possible ambiguities in the identification process or to improve the confidence level for the identification assignment.
In some embodiments, a bioagent identifying amplicon may be produced using only a single primer (either the forward or reverse primer of any given primer pair), provided an appropriate amplification method is chosen, such as, for example, low stringency single primer PCR (LSSP-PCR). Adaptation of this amplification method in order to produce bioagent identifying amplicons can be accomplished by one with ordinary skill in the art without undue experimentation.
In some embodiments, the oligonucleotide primers are broad range survey primers which hybridize to conserved regions of nucleic acid encoding the hexon gene of all (or between 80% and 100%, between 85% and 100%, between 90% and 100% or between 95% and 100%) known bacteria and produce bacterial bioagent identifying amplicons.
In some cases, the molecular mass or base composition of a bacterial bioagent identifying amplicon defined by a broad range survey primer pair does not provide enough resolution to unambiguously identify a bacterial bioagent at or below the species level. These cases benefit from further analysis of one or more bacterial bioagent identifying amplicons generated from at least one additional broad range survey primer pair or from at least one additional division-wide primer pair. The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as triangulation identification.
In other embodiments, the oligonucleotide primers are division-wide primers which hybridize to nucleic acid encoding genes of species within a genus of bacteria. In other embodiments, the oligonucleotide primers are drill-down primers which enable the identification of sub-species characteristics. Drill down primers provide the functionality of producing bioagent identifying amplicons for drill-down analyses such as strain typing when contacted with nucleic acid under amplification conditions. Identification of such sub-species characteristics is often critical for determining proper clinical treatment of viral infections. In some embodiments, sub-species characteristics are identified using only broad range survey primers and division-wide and drill-down primers are not used.
In some embodiments, the primers used for amplification hybridize to and amplify genomic DNA, and DNA of bacterial plasmids.
In some embodiments, various computer software programs may be used to aid in design of primers for amplification reactions such as Primer Premier 5 (Premier Biosoft, Palo Alto, Calif.) or OLIGO Primer Analysis Software (Molecular Biology Insights, Cascade, Colo.). These programs allow the user to input desired hybridization conditions such as melting temperature of a primer-template duplex for example. In some embodiments, an in silico PCR search algorithm, such as (ePCR) is used to analyze primer specificity across a plurality of template sequences which can be readily obtained from public sequence databases such as GenBank for example. An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs. In some embodiments, the hybridization conditions applied to the algorithm can limit the results of primer specificity obtained from the algorithm. In some embodiments, the melting temperature threshold for the primer template duplex is specified to be 35° C. or a higher temperature. In some embodiments the number of acceptable mismatches is specified to be seven mismatches or less. In some embodiments, the buffer components and concentrations and primer concentrations may be specified and incorporated into the algorithm, for example, an appropriate primer concentration is about 250 nM and appropriate buffer components are 50 mM sodium or potassium and 1.5 mM Mg2+.
One with ordinary skill in the art of design of amplification primers will recognize that a given primer need not hybridize with 100% complementarity in order to effectively prime the synthesis of a complementary nucleic acid strand in an amplification reaction. Moreover, a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event. (e.g., for example, a loop structure or a hairpin structure). The primers of the present invention may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any of the primers listed in Table 2. Thus, in some embodiments of the present invention, an extent of variation of 70% to 100%, or any range therewithin, of the sequence identity is possible relative to the specific primer sequences disclosed herein. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is identical to another 20 nucleobase primer having two non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of primer 20 nucleobases in length would have 15/20=0.75 or 75% sequence identity with the 20 nucleobase primer.
Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some embodiments, complementarity of primers with respect to the conserved priming regions of viral nucleic acid is between about 70% and about 75% 80%. In other embodiments, homology, sequence identity or complementarity, is between about 75% and about 80%. In yet other embodiments, homology, sequence identity or complementarity, is at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100%.
In some embodiments, the primers described herein comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range therewithin) sequence identity with the primer sequences specifically disclosed herein.
One with ordinary skill is able to calculate percent sequence identity or percent sequence homology and able to determine, without undue experimentation, the effects of variation of primer sequence identity on the function of the primer in its role in priming synthesis of a complementary strand of nucleic acid for production of an amplification product of a corresponding bioagent identifying amplicon.
In one embodiment, the primers are at least 13 nucleobases in length. In another embodiment, the primers are less than 36 nucleobases in length.
In some embodiments of the present invention, the oligonucleotide primers are 13 to 35 nucleobases in length (13 to 35 linked nucleotide residues). These embodiments comprise oligonucleotide primers 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleobases in length, or any range therewithin. The present invention contemplates using both longer and shorter primers. Furthermore, the primers may also be linked to one or more other desired moieties, including, but not limited to, affinity groups, ligands, regions of nucleic acid that are not complementary to the nucleic acid to be amplified, labels, etc. Primers may also form hairpin structures. For example, hairpin primers may be used to amplify short target nucleic acid molecules. The presence of the hairpin may stabilize the amplification complex (see e.g., TAQMAN MicroRNA Assays, Applied Biosystems, Foster City, Calif.).
In some embodiments, any oligonucleotide primer pair may have one or both primers with less then 70% sequence homology with a corresponding member of any of the primer pairs of Table 2 if the primer pair has the capability of producing an amplification product corresponding to a bioagent identifying amplicon. In other embodiments, any oligonucleotide primer pair may have one or both primers with a length greater than 35 nucleobases if the primer pair has the capability of producing an amplification product corresponding to a bioagent identifying amplicon.
In some embodiments, the function of a given primer may be substituted by a combination of two or more primers segments that hybridize adjacent to each other or that are linked by a nucleic acid loop structure or linker which allows a polymerase to extend the two or more primers in an amplification reaction.
In some embodiments, the primer pairs used for obtaining bioagent identifying amplicons are the primer pairs of Table 2. In other embodiments, other combinations of primer pairs are possible by combining certain members of the forward primers with certain members of the reverse primers. An example can be seen in Table 2 for two primer pair combinations of forward primer 16S_EC—789—810_F (SEQ ID NO: 206), with the reverse primers 16S_EC—880—894_R (SEQ ID NO: 796), or 16 S_EC—882—899_R or (SEQ ID NO: 818). Arriving at a favorable alternate combination of primers in a primer depends upon the properties of the primer pair, most notably the size of the bioagent identifying amplicon that would be produced by the primer pair, which preferably is between about 45 to about 150 nucleobases in length. Alternatively, a bioagent identifying amplicon longer than 150 nucleobases in length could be cleaved into smaller segments by cleavage reagents such as chemical reagents, or restriction enzymes, for example.
In some embodiments, the primers are configured to amplify nucleic acid of a bioagent to produce amplification products that can be measured by mass spectrometry and from whose molecular masses candidate base compositions can be readily calculated.
In some embodiments, any given primer comprises a modification comprising the addition of a non-templated T residue to the 5′ end of the primer (i.e., the added T residue does not necessarily hybridize to the nucleic acid being amplified). The addition of a non-templated T residue has an effect of minimizing the addition of non-templated adenosine residues as a result of the non-specific enzyme activity of Taq polymerase (Magnuson et al., Biotechniques, 1996, 21, 700-709), an occurrence which may lead to ambiguous results arising from molecular mass analysis.
In some embodiments of the present invention, primers may contain one or more universal bases. Because any variation (due to codon wobble in the 3rd position) in the conserved regions among species is likely to occur in the third position of a DNA (or RNA) triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a “universal nucleobase.” For example, under this “wobble” pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal nucleobases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides, 1995, 14, 1053-1056) or the purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).
In some embodiments, to compensate for the somewhat weaker binding by the wobble base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs that bind with greater affinity than the unmodified nucleotide. Examples of these analogs include, but are not limited to, 2,6-diaminopurine which binds to thymine, 5-propynyluracil (also known as propynylated thymine) which binds to adenine and 5-propynylcytosine and phenoxazines, including G-clamp, which binds to G. Propynylated pyrimidines are described in U.S. Pat. Nos. 5,645,985, 5,830,653 and 5,484,908, each of which is commonly owned and incorporated herein by reference in its entirety. Propynylated primers are described in U.S. Pre-Grant Publication No. 2003-0170682, which is also commonly owned and incorporated herein by reference in its entirety. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each of which is incorporated herein by reference in its entirety. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which is incorporated herein by reference in its entirety.
In some embodiments, primer hybridization is enhanced using primers containing 5-propynyl deoxy-cytidine and deoxy-thymidine nucleotides. These modified primers offer increased affinity and base pairing selectivity.
In some embodiments, non-template primer tags are used to increase the melting temperature (Tm) of a primer-template duplex in order to improve amplification efficiency. A non-template tag is at least three consecutive A or T nucleotide residues on a primer which are not complementary to the template. In any given non-template tag, A can be replaced by C or G and T can also be replaced by C or G. Although Watson-Crick hybridization is not expected to occur for a non-template tag relative to the template, the extra hydrogen bond in a G-C pair relative to an A-T pair confers increased stability of the primer-template duplex and improves amplification efficiency for subsequent cycles of amplification when the primers hybridize to strands synthesized in previous cycles.
In other embodiments, propynylated tags may be used in a manner similar to that of the non-template tag, wherein two or more 5-propynylcytidine or 5-propynyluridine residues replace template matching residues on a primer. In other embodiments, a primer contains a modified internucleoside linkage such as a phosphorothioate linkage, for example.
In some embodiments, the primers contain mass-modifying tags. Reducing the total number of possible base compositions of a nucleic acid of specific molecular weight provides a means of avoiding a persistent source of ambiguity in determination of base composition of amplification products. Addition of mass-modifying tags to certain nucleobases of a given primer will result in simplification of de novo determination of base composition of a given bioagent identifying amplicon from its molecular mass.
In some embodiments of the present invention, the mass modified nucleobase comprises one or more of the following: for example, 7-deaza-2′-deoxyadenosine-5-triphosphate, 5-iodo-2′-deoxyuridine-5′-triphosphate, 5-bromo-2′-deoxyuridine-5′-triphosphate, 5-bromo-2′-deoxycytidine-5′-triphosphate, 5-iodo-2′-deoxycytidine-5′-triphosphate, 5-hydroxy-2′-deoxyuridine-5′-triphosphate, 4-thiothymidine-5′-triphosphate, 5-aza-2′-deoxyuridine-5′-triphosphate, 5-fluoro-2′-deoxyuridine-5′-triphosphate, O6-methyl-2′-deoxyguanosine-5′-triphosphate, N2-methyl-2′-deoxyguanosine-5′-triphosphate, 8-oxo-2′-deoxyguanosine-5′-triphosphate or thiothymidine-5′-triphosphate. In some embodiments, the mass-modified nucleobase comprises 15N or 13C or both 15N and 13C.
In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with a plurality of primer pairs. The advantages of multiplexing are that fewer reaction containers (for example, wells of a 96- or 384-well plate) are needed for each molecular mass measurement, providing time, resource and cost savings because additional bioagent identification data can be obtained within a single analysis. Multiplex amplification methods are well known to those with ordinary skill and can be developed without undue experimentation. However, in some embodiments, one useful and non-obvious step in selecting a plurality candidate bioagent identifying amplicons for multiplex amplification is to ensure that each strand of each amplification product will be sufficiently different in molecular mass that mass spectral signals will not overlap and lead to ambiguous analysis results. In some embodiments, a 10 Da difference in mass of two strands of one or more amplification products is sufficient to avoid overlap of mass spectral peaks.
In some embodiments, as an alternative to multiplex amplification, single amplification reactions can be pooled before analysis by mass spectrometry. In these embodiments, as for multiplex amplification embodiments, it is useful to select a plurality of candidate bioagent identifying amplicons to ensure that each strand of each amplification product will be sufficiently different in molecular mass that mass spectral signals will not overlap and lead to ambiguous analysis results.
C Determination of Molecular Mass of Bioagent Identifying Amplicons
In some embodiments, the molecular mass of a given bioagent identifying amplicon is determined by mass spectrometry. Mass spectrometry has several advantages, not the least of which is high bandwidth characterized by the ability to separate (and isolate) many molecular peaks across a broad range of mass to charge ratio (m/z). Thus mass spectrometry is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons.
In some embodiments, intact molecular ions are generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent identifying amplicon. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.
The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole, magnetic sector, Q-TOF, and triple quadrupole.
D. Base Compositions of Bioagent Identifying Amplicons
Although the molecular mass of amplification products obtained using intelligent primers provides a means for identification of bioagents, conversion of molecular mass data to a base composition signature is useful for certain analyses. As used herein, “base composition” is the exact number of each nucleobase (A, T, C and G) determined from the molecular mass of a bioagent identifying amplicon. In some embodiments, a base composition provides an index of a specific organism. Base compositions can be calculated from known sequences of known bioagent identifying amplicons and can be experimentally determined by measuring the molecular mass of a given bioagent identifying amplicon, followed by determination of all possible base compositions which are consistent with the measured molecular mass within acceptable experimental error. The following example illustrates determination of base composition from an experimentally obtained molecular mass of a 46-mer amplification product originating at position 1337 of the 16S rRNA of Bacillus anthracis. The forward and reverse strands of the amplification product have measured molecular masses of 14208 and 14079 Da, respectively. The possible base compositions derived from the molecular masses of the forward and reverse strands for the B. anthracis products are listed in Table 1.







TABLE 1







Possible Base Compositions for B. anthracis 46mer Amplification Product












Calc. Mass
Mass Error
Base
Calc. Mass
Mass Error
Base


Forward
Forward
Composition of
Reverse
Reverse
Composition of


Strand
Strand
Forward Strand
Strand
Strand
Reverse Strand





14208.2935
0.079520
A1 G17 C10 T18
14079.2624
0.080600
A0 G14 C13 T19


14208.3160
0.056980
A1 G20 C15 T10
14079.2849
0.058060
A0 G17 C18 T11


14208.3386
0.034440
A1 G23 C20 T2
14079.3075
0.035520
A0 G20 C23 T3


14208.3074
0.065560
A6 G11 C3 T26
14079.2538
0.089180
A5 G5 C1 T35


14208.3300
0.043020
A6 G14 C8 T18
14079.2764
0.066640
A5 G8 C6 T27


14208.3525
0.020480
A6 G17 C13 T10
14079.2989
0.044100
A5 G11 C11 T19


14208.3751
0.002060
A6 G20 C18 T2
14079.3214
0.021560
A5 G14 C16 T11


14208.3439
0.029060
A11 G8 C1 T26
14079.3440
0.000980
A5 G17 C21 T3


14208.3665
0.006520
A11 G11 C6 T18
14079.3129
0.030140
A10 G5 C4 T27


14208.3890
0.016020
A11 G14 C11 T10
14079.3354
0.007600
A10 G8 C9 T19


14208.4116
0.038560
A11 G17 C16 T2
14079.3579
0.014940
A10 G11 C14 T11


14208.4030
0.029980
A16 G8 C4 T18
14079.3805
0.037480
A10 G14 C19 T3


14208.4255
0.052520
A16 G11 C9 T10
14079.3494
0.006360
A15 G2 C2 T27


14208.4481
0.075060
A16 G14 C14 T2
14079.3719
0.028900
A15 G5 C7 T19


14208.4395
0.066480
A21 G5 C2 T18
14079.3944
0.051440
A15 G8 C12 T11


14208.4620
0.089020
A21 G8 C7 T10
14079.4170
0.073980
A15 G11 C17 T3


—
—
—
14079.4084
0.065400
A20 G2 C5 T19


—
—
—
14079.4309
0.087940
A20 G5 C10 T13









Among the 16 possible base compositions for the forward strand and the 18 possible base compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary base compositions, which indicates the true base composition of the amplification product. It should be recognized that this logic is applicable for determination of base compositions of any bioagent identifying amplicon, regardless of the class of bioagent from which the corresponding amplification product was obtained.
In some embodiments, assignment of previously unobserved base compositions (also known as “true unknown base compositions”) to a given phylogeny can be accomplished via the use of pattern classifier model algorithms. Base compositions, like sequences, vary slightly from strain to strain within species, for example. In some embodiments, the pattern classifier model is the mutational probability model. On other embodiments, the pattern classifier is the polytope model. The mutational probability model and polytope model are both commonly owned and described in U.S. patent application Ser. No. 11/073,362 which is incorporated herein by reference in entirety.
In one embodiment, it is possible to manage this diversity by building “base composition probability clouds” around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A “pseudo four-dimensional plot” can be used to visualize the concept of base composition probability clouds. Optimal primer design requires optimal choice of bioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by a triangulation identification process using bioagent identifying amplicons not affected by overlap of base composition probability clouds.
In some embodiments, base composition probability clouds provide the means for screening potential primer pairs in order to avoid potential misclassifications of base compositions. In other embodiments, base composition probability clouds provide the means for predicting the identity of a bioagent whose assigned base composition was not previously observed and/or indexed in a bioagent identifying amplicon base composition database due to evolutionary transitions in its nucleic acid sequence. Thus, in contrast to probe-based techniques, mass spectrometry determination of base composition does not require prior knowledge of the composition or sequence in order to make the measurement.
The present invention provides bioagent classifying information similar to DNA sequencing and phylogenetic analysis at a level sufficient to identify a given bioagent. Furthermore, the process of determination of a previously unknown base composition for a given bioagent (for example, in a case where sequence information is unavailable) has downstream utility by providing additional bioagent indexing information with which to populate base composition databases. The process of future bioagent identification is thus greatly improved as more BCS indexes become available in base composition databases.
E. Triangulation Identification
In some cases, a molecular mass of a single bioagent identifying amplicon alone does not provide enough resolution to unambiguously identify a given bioagent. The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as “triangulation identification.” Triangulation identification is pursued by determining the molecular masses of a plurality of bioagent identifying amplicons selected within a plurality of housekeeping genes. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
In some embodiments, the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using the polymerase chain reaction (PCR), such as multiplex PCR where multiple primers are employed in the same amplification reaction mixture, or PCR in multi-well plate format wherein a different and unique pair of primers is used in multiple wells containing otherwise identical reaction mixtures. Such multiplex and multi-well PCR methods are well known to those with ordinary skill in the arts of rapid throughput amplification of nucleic acids. In other related embodiments, one PCR reaction per well or container may be carried out, followed by an amplicon pooling step wherein the amplification products of different wells are combined in a single well or container which is then subjected to molecular mass analysis. The combination of pooled amplicons can be chosen such that the expected ranges of molecular masses of individual amplicons are not overlapping and thus will not complicate identification of signals.
F. Codon Base Composition Analysis
In some embodiments of the present invention, one or more nucleotide substitutions within a codon of a gene of an infectious organism confer drug resistance upon an organism which can be determined by codon base composition analysis. The organism can be a bacterium, virus, fungus or protozoan.
In some embodiments, the amplification product containing the codon being analyzed is of a length of about 35 to about 200 nucleobases. The primers employed in obtaining the amplification product can hybridize to upstream and downstream sequences directly adjacent to the codon, or can hybridize to upstream and downstream sequences one or more sequence positions away from the codon. The primers may have between about 70% to 100% sequence complementarity with the sequence of the gene containing the codon being analyzed.
In some embodiments, the codon base composition analysis is undertaken
In some embodiments, the codon analysis is undertaken for the purpose of investigating genetic disease in an individual. In other embodiments, the codon analysis is undertaken for the purpose of investigating a drug resistance mutation or any other deleterious mutation in an infectious organism such as a bacterium, virus, fungus or protozoan. In some embodiments, the bioagent is a bacterium identified in a biological product.
In some embodiments, the molecular mass of an amplification product containing the codon being analyzed is measured by mass spectrometry. The mass spectrometry can be either electrospray (ESI) mass spectrometry or matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Time-of-flight (TOF) is an example of one mode of mass spectrometry compatible with the analyses of the present invention.
The methods of the present invention can also be employed to determine the relative abundance of drug resistant strains of the organism being analyzed. Relative abundances can be calculated from amplitudes of mass spectral signals with relation to internal calibrants. In some embodiments, known quantities of internal amplification calibrants can be included in the amplification reactions and abundances of analyte amplification product estimated in relation to the known quantities of the calibrants.
In some embodiments, upon identification of one or more drug-resistant strains of an infectious organism infecting an individual, one or more alternative treatments can be devised to treat the individual.
G. Determination of the Quantity of a Bioagent
In some embodiments, the identity and quantity of an unknown bioagent can be determined using the process illustrated in FIG. 2. Primers (500) and a known quantity of a calibration polynucleotide (505) are added to a sample containing nucleic acid of an unknown bioagent. The total nucleic acid in the sample is then subjected to an amplification reaction (510) to obtain amplification products. The molecular masses of amplification products are determined (515) from which are obtained molecular mass and abundance data. The molecular mass of the bioagent identifying amplicon (520) provides the means for its identification (525) and the molecular mass of the calibration amplicon obtained from the calibration polynucleotide (530) provides the means for its identification (535). The abundance data of the bioagent identifying amplicon is recorded (540) and the abundance data for the calibration data is recorded (545), both of which are used in a calculation (550) which determines the quantity of unknown bioagent in the sample.
A sample comprising an unknown bioagent is contacted with a pair of primers that provide the means for amplification of nucleic acid from the bioagent, and a known quantity of a polynucleotide that comprises a calibration sequence. The nucleic acids of the bioagent and of the calibration sequence are amplified and the rate of amplification is reasonably assumed to be similar for the nucleic acid of the bioagent and of the calibration sequence. The amplification reaction then produces two amplification products: a bioagent identifying amplicon and a calibration amplicon. The bioagent identifying amplicon and the calibration amplicon should be distinguishable by molecular mass while being amplified at essentially the same rate. Effecting differential molecular masses can be accomplished by choosing as a calibration sequence, a representative bioagent identifying amplicon (from a specific species of bioagent) and performing, for example, a 2-8 nucleobase deletion or insertion within the variable region between the two priming sites. The amplified sample containing the bioagent identifying amplicon and the calibration amplicon is then subjected to molecular mass analysis by mass spectrometry, for example. The resulting molecular mass analysis of the nucleic acid of the bioagent and of the calibration sequence provides molecular mass data and abundance data for the nucleic acid of the bioagent and of the calibration sequence. The molecular mass data obtained for the nucleic acid of the bioagent enables identification of the unknown bioagent and the abundance data enables calculation of the quantity of the bioagent, based on the knowledge of the quantity of calibration polynucleotide contacted with the sample.
In some embodiments, construction of a standard curve where the amount of calibration polynucleotide spiked into the sample is varied provides additional resolution and improved confidence for the determination of the quantity of bioagent in the sample. The use of standard curves for analytical determination of molecular quantities is well known to one with ordinary skill and can be performed without undue experimentation.
In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with multiple primer pairs which also amplify the corresponding standard calibration sequences. In this or other embodiments, the standard calibration sequences are optionally included within a single vector which functions as the calibration polynucleotide. Multiplex amplification methods are well known to those with ordinary skill and can be performed without undue experimentation.
In some embodiments, the calibrant polynucleotide is used as an internal positive control to confirm that amplification conditions and subsequent analysis steps are successful in producing a measurable amplicon. Even in the absence of copies of the genome of a bioagent, the calibration polynucleotide should give rise to a calibration amplicon. Failure to produce a measurable calibration amplicon indicates a failure of amplification or subsequent analysis step such as amplicon purification or molecular mass determination. Reaching a conclusion that such failures have occurred is in itself, a useful event.
In some embodiments, the calibration sequence is comprised of DNA. In some embodiments, the calibration sequence is comprised of RNA.
In some embodiments, the calibration sequence is inserted into a vector that itself functions as the calibration polynucleotide. In some embodiments, more than one calibration sequence is inserted into the vector that functions as the calibration polynucleotide. Such a calibration polynucleotide is herein termed a “combination calibration polynucleotide.” The process of inserting polynucleotides into vectors is routine to those skilled in the art and can be accomplished without undue experimentation. Thus, it should be recognized that the calibration method should not be limited to the embodiments described herein. The calibration method can be applied for determination of the quantity of any bioagent identifying amplicon when an appropriate standard calibrant polynucleotide sequence is designed and used. The process of choosing an appropriate vector for insertion of a calibrant is also a routine operation that can be accomplished by one with ordinary skill without undue experimentation.
H. Identification of Bacteria
In other embodiments of the present invention, the primer pairs produce bioagent identifying amplicons within stable and highly conserved regions of bacteria. The advantage to characterization of an amplicon defined by priming regions that fall within a highly conserved region is that there is a low probability that the region will evolve past the point of primer recognition, in which case, the primer hybridization of the amplification step would fail. Such a primer set is thus useful as a broad range survey-type primer. In another embodiment of the present invention, the intelligent primers produce bioagent identifying amplicons including a region which evolves more quickly than the stable region described above. The advantage of characterization bioagent identifying amplicon corresponding to an evolving genomic region is that it is useful for distinguishing emerging strain variants or the presence of virulence genes, drug resistance genes, or codon mutations that induce drug resistance.
The present invention also has significant advantages as a platform for identification of diseases caused by emerging bacterial strains such as, for example, drug-resistant strains of Staphylococcus aureus. The present invention eliminates the need for prior knowledge of bioagent sequence to generate hybridization probes. This is possible because the methods are not confounded by naturally occurring evolutionary variations occurring in the sequence acting as the template for production of the bioagent identifying amplicon. Measurement of molecular mass and determination of base composition is accomplished in an unbiased manner without sequence prejudice.
Another embodiment of the present invention also provides a means of tracking the spread of a bacterium, such as a particular drug-resistant strain when a plurality of samples obtained from different locations are analyzed by the methods described above in an epidemiological setting. In one embodiment, a plurality of samples from a plurality of different locations is analyzed with primer pairs which produce bioagent identifying amplicons, a subset of which contains a specific drug-resistant bacterial strain. The corresponding locations of the members of the drug-resistant strain subset indicate the spread of the specific drug-resistant strain to the corresponding locations.
I. Kits
The present invention also provides kits for carrying out the methods described herein. In some embodiments, the kit may comprise a sufficient quantity of one or more primer pairs to perform an amplification reaction on a target polynucleotide from a bioagent to form a bioagent identifying amplicon. In some embodiments, the kit may comprise from one to fifty primer pairs, from one to twenty primer pairs, from one to ten primer pairs, or from two to five primer pairs. In some embodiments, the kit may comprise one or more primer pairs recited in Table 2.
In some embodiments, the kit comprises one or more broad range survey primer(s), division wide primer(s), or drill-down primer(s), or any combination thereof. If a given problem involves identification of a specific bioagent, the solution to the problem may require the selection of a particular combination of primers to provide the solution to the problem. A kit may be designed so as to comprise particular primer pairs for identification of a particular bioagent. A drill-down kit may be used, for example, to distinguish different genotypes or strains, drug-resistant, or otherwise. In some embodiments, the primer pair components of any of these kits may be additionally combined to comprise additional combinations of broad range survey primers and division-wide primers so as to be able to identify a bacterium.
In some embodiments, the kit contains standardized calibration polynucleotides for use as internal amplification calibrants. Internal calibrants are described in commonly owned U.S. patent application Ser. No. 60/545,425 which is incorporated herein by reference in its entirety.
In some embodiments, the kit comprises a sufficient quantity of reverse transcriptase (if RNA is to be analyzed for example), a DNA polymerase, suitable nucleoside triphosphates (including alternative dNTPs such as inosine or modified dNTPs such as the 5-propynyl pyrimidines or any dNTP containing molecular mass-modifying tags such as those described above), a DNA ligase, and/or reaction buffer, or any combination thereof, for the amplification processes described above. A kit may further include instructions pertinent for the particular embodiment of the kit, such instructions describing the primer pairs and amplification conditions for operation of the method. A kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like. A kit may also comprise reagents or other materials for isolating bioagent nucleic acid or bioagent identifying amplicons from amplification, including, for example, detergents, solvents, or ion exchange resins which may be linked to magnetic beads. A kit may also comprise a table of measured or calculated molecular masses and/or base compositions of bioagents using the primer pairs of the kit.
Some embodiments are kits that contain one or more survey bacterial primer pairs represented by primer pair compositions wherein each member of each pair of primers has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by any of the primer pairs of Table 5. The survey primer pairs may include broad range primer pairs which hybridize to ribosomal RNA, and may also include division-wide primer pairs which hybridize to housekeeping genes such as rplB, tufb, rpoB, rpoC, valS, and infb, for example.
In some embodiments, a kit may contain one or more survey bacterial primer pairs and one or more triangulation genotyping analysis primer pairs such as the primer pairs of Tables 8, 12, 14, 19, 21, 23, or 24. In some embodiments, the kit may represent a less expansive genotyping analysis but include triangulation genotyping analysis primer pairs for more than one genus or species of bacteria. For example, a kit for surveying nosocomial infections at a health care facility may include, for example, one or more broad range survey primer pairs, one or more division wide primer pairs, one or more Acinetobacter baumannii triangulation genotyping analysis primer pairs and one or more Staphylococcus aureus triangulation genotyping analysis primer pairs. One with ordinary skill will be capable of analyzing in silico amplification data to determine which primer pairs will be able to provide optimal identification resolution for the bacterial bioagents of interest.
In some embodiments, a kit may be assembled for identification of strains of bacteria involved in contamination of food. An example of such a kit embodiment is a kit comprising one or more bacterial survey primer pairs of Table 5 with one or more triangulation genotyping analysis primer pairs of Table 12 which provide strain resolving capabilities for identification of specific strains of Campylobacter jejuni. 
Some embodiments of the kits are 96-well or 384-well plates with a plurality of wells containing any or all of the following components: dNTPs, buffer salts, Mg2+, betaine, and primer pairs. In some embodiments, a polymerase is also included in the plurality of wells of the 96-well or 384-well plates.
Some embodiments of the kit contain instructions for PCR and mass spectrometry analysis of amplification products obtained using the primer pairs of the kits.
Some embodiments of the kit include a barcode which uniquely identifies the kit and the components contained therein according to production lots and may also include any other information relative to the components such as concentrations, storage temperatures, etc. The barcode may also include analysis information to be read by optical barcode readers and sent to a computer controlling amplification, purification and mass spectrometric measurements. In some embodiments, the barcode provides access to a subset of base compositions in a base composition database which is in digital communication with base composition analysis software such that a base composition measured with primer pairs from a given kit can be compared with known base compositions of bioagent identifying amplicons defined by the primer pairs of that kit.
In some embodiments, the kit contains a database of base compositions of bioagent identifying amplicons defined by the primer pairs of the kit. The database is stored on a convenient computer readable medium such as a compact disk or USB drive, for example.
In some embodiments, the kit includes a computer program stored on a computer formatted medium (such as a compact disk or portable USB disk drive, for example) comprising instructions which direct a processor to analyze data obtained from the use of the primer pairs of the present invention. The instructions of the software transform data related to amplification products into a molecular mass or base composition which is a useful concrete and tangible result used in identification and/or classification of bioagents. In some embodiments, the kits of the present invention contain all of the reagents sufficient to carry out one or more of the methods described herein.
While the present invention has been described with specificity in accordance with certain of its embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.
EXAMPLES
Example 1
Design and Validation of Primers that Define Bioagent Identifying Amplicons for Identification of Bacteria
For design of primers that define bacterial bioagent identifying amplicons, a series of bacterial genome segment sequences were obtained, aligned and scanned for regions where pairs of PCR primers would amplify products of about 45 to about 150 nucleotides in length and distinguish subgroups and/or individual strains from each other by their molecular masses or base compositions. A typical process shown in FIG. 1 is employed for this type of analysis.
A database of expected base compositions for each primer region was generated using an in silico PCR search algorithm, such as (ePCR). An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs.
Table 2 represents a collection of primers (sorted by primer pair number) designed to identify bacteria using the methods described herein. The primer pair number is an in-house database index number. Primer sites were identified on segments of genes, such as, for example, the 16S rRNA gene. The forward or reverse primer name shown in Table 2 indicates the gene region of the bacterial genome to which the primer hybridizes relative to a reference sequence. In Table 2, for example, the forward primer name 16 S_EC—1077—1106_F indicates that the forward primer (_F) hybridizes to residues 1077-1106 of the reference sequence represented by a sequence extraction of coordinates 4033120.4034661 from GenBank gi number 16127994 (as indicated in Table 3). As an additional example: the forward primer name BONTA_X52066—450—473 indicates that the primer hybridizes to residues 450-437 of the gene encoding Clostridium botulinum neurotoxin type A (BoNT/A) represented by GenBank Accession No. X52066 (primer pair name codes appearing in Table 2 are defined in Table 3. One with ordinary skill knows how to obtain individual gene sequences or portions thereof from genomic sequences present in GenBank. In Table 2, Tp=5-propynyluracil; Cp=5-propynylcytosine; *=phosphorothioate linkage; I=inosine. T. GenBank Accession Numbers for reference sequences of bacteria are shown in Table 3 (below). In some cases, the reference sequences are extractions from bacterial genomic sequences or complements thereof.







TABLE 2







Primer Pairs for Identification of Bacteria













Primer


Forward


Reverse


Pair

Forward
SEQ

Reverse
SEQ


Number
Forward Primer Name
Sequence
ID NO:
Reverse Primer Name
Sequence
ID NO:
















1
16S_EC_1077_1106_F
GTGAGATGTTG
134
16S_EC_1175_1195_R
GACGTCATCCCCA
809




GGTTAAGTCCC


CCTTCCTC




GTAACGAG


 


2
16S_EC_1082_1106_F
ATGTTGGGTTA
38
16S_EC_1175_1197_R
TTGACGTCATCCC
1398




AGTCCCGCAAC


CACCTTCCTC




GAG


 


3
16S_EC_1090_1111_F
TTAAGTCCCGC
651
16S_EC_1175_1196_R
TGACGTCATCCCC
1159




AACGATCGCAA


ACCTTCCTC


 


4
16S_EC_1222_1241_F
GCTACACACGT
114
16_EC_1303_1323_R
CGAGTTGCAGACT
787




GCTACAATG


GCGATCCG


 


5
16S_EC_1332_1353_F
AAGTCGGAATC
10
16S_EC_1389_1407_R
GACGGGCGGTGTG
806




GCTAGTAATCG


TACAAG


 


6
16S_EC_30_54_F
TGAACGCTGGT
429
16S_EC_105_126_R
TACGCATTACTCA
897




GGCATGCTTAA


CCCGTCCGC




CAC


 


7
16S_EC_38_64_F
GTGGCATGCCT
136
16S_EC_101_120_R
TTACTCACCCGTC
1365




AATACATGCAA


CGCCGCT




GTCG


 


8
16S_EC_49_68_F
TAACACATGCA
152
16S_EC_104_120_R
TTACTCACCCGTC
1364




AGTCGAACG


CGCC


 


9
16S_EC_683_700_F
GTGTAGCGGTG
137
16S_EC_774_795_R
GTATCTAATCCTG
839




AAATGCG


TTTGCTCCC


 


10
16S_EC_713_732_F
AGAACACCGAT
21
16S_EC_789_809_R
CGTGGACTACCAG
798




GGCGAAGGC


GGTATCTA


 


11
16S_EC_785_806_F
GGATTAGAGAC
118
16S_EC_880_897_R
GGCCGTACTCCCC
830




CCTGGTAGTCC


AGGCG


 


12
16S_EC_785_810_F
GGATTAGATAC
119
16S_EC_880_897_2_R
GGCGTACTCCCC
830




CCTGGTAGTCA


AGGCG




CACGC


 


13
16S_EC_789_810_F
TAGATACCCTG
206
16S_EC_880_894_R
CGTACTCCCCAGG
796




GTAGTCCACGC


CG


 


14
16S_EC_960_981_F
TTCGATGCAAC
672
16S_EC_1054_1073_R
ACGAGCTGACGAC
735




GCGAAGAACCT


AGCCATG


 


15
16S_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1061_1078_R
ACGACCACGAGCTG
734




CTTACC


ACGAC


 


16
23S_EC_1826_1843_F
CTGACACCTGC
80
23S_EC_1906_1924_R
GACCGTTATAGTT
805




CCGGTGC


ACGGCC


 


17
23S_EC_2645_2669_F
TCTGTCCCTAG
408
23S_EC_2744_2761_R
TGCTTAGATGCTT
1252




TACGAGAGGAC


TCAGC




CGG


 


18
23S_EC_2645_2669_2_F
CTGTCCCTAGT
83
23S_EC_2751_2767_R
GTTTCATGCTTAG
846




ACGAGAGGACC


ATGCTTTCAGC




GG


 


19
23S_EC_493_518_F
GGGGAGTGAAA
125
23S_EC_551_571_R
ACAAAAGGTACGC
717




GAGATCCTGAA


CGTCACCC




ACCG


 


20
23S_EC_493_518_2_F
GGGGAGTGAAA
125
23S_EC_551_571_2_R
ACAAAAGGCACGC
716




GAGATCCTGAA


CATCACCC




ACCG


 


21
23S_EC_971_992_F
CGAGAGGGAAA
66
23S_EC_1059_1077_R
TGGCTGCTTCTAA
1282




CAACCCAGACC


GCCAAC


 


22
CAPC_BA_104_131_F
GTTATTTAGCA
139
CAPC_BA_180_205_R
TGAATCTTGAAAC
1150




CTCGTTTTTAA


ACCATACGTAAC




TCAGCC


G


 


23
CAPC_BA_114_133_F
ACTCGTTTTTA
20
CAPC_BA_185_205_R
TGAATCTTGAAAC
1149




ATCAGCCCG


ACCATACG


 


24
CAPC_BA_274_303_F
GATTATTGTTA
109
CAPC_BA_349_376_R
GTAACCCTTGTCT
837




TCCTGTTATGC


TTGAATTGTATTT




CATTTGAG


GC


 


25
CAPC_BA_276_296_F
TTATTGTTATC
663
CAPC_BA_358_377_R
GGTAACCCTTGTC
834




CTGTTATGCC


TTTGAAT


 


26
CAPC_BA_281_301_F
GTTATCCTGTT
138
CAPC_BA_361_378_R
TGGTAACCCTTGT
1298




ATGCCATTTG


CTTTG


 


27
CAPC_BA_315_334_F
CCGTGGTATTG
59
CAPC_BA_361_378_R
TGGTAACCCTTGT
1298




GAGTTATTG


TCTTTG


 


28
CYA_BA_1055_1072_F
GAAAGAGTTCG
92
CYA_BA_1112_1130_R
TGTTGACCATGCT
1352




GATTGGG


TCTTAG


 


29
CYA_BA_1349_1370_F
ACAACGAAGTA
12
CYA_BA_1447_1426_R
CTTCTACATTTTT
800




CAATACAAGAC


AGCCATCAC


 


30
CYA_BA_1353_1379_F
CGAAGTACAAT
64
CYA_BA_1448_1467_R
TGTTAACGGCTTC
1342




ACAAGACAAAA


AAGACCC




GAAGG


 


31
CYA_BA_1359_1379_F
ACAATACAAGA
13
CYA_BA_1447_1461_R
CGGCTTCAAGACC
794




CAAAAGAAGG


CC


 


32
CYA_BA_914_937_F
CAGGTTTAGTA
53
CYA_BA_999_1026_R
ACCACTTTTAATA
728




CCAGAACATG


AGGTTTGTAGCTA







CAGAC


 


33
CYA_BA_916_935_F
GGTTTAGTACC
131
CYA_BA_1003_1025_R
CCACTTTTAATAA
768




AGAACATGC


GGTTTGTAGC


 


34
INFB_EC_1365_1393_F
TGCTCGTGGTG
524
INFB_EC_1439_1467_R
TGCTGCTTTCGCA
1248




CACAAGTAACG


TGGTTAATTGCTT




GAT




ATTA


CAA


 


35
LEF_BA_1033_1052_F
TCAAGAAGAAA
254
LEF_BA_1119_1135_R
GAATATCAATTTG
803




AAGAGC


TAGC


 


36
LEF_BA_1036_1066_F
CAAGAAGAAAA
44
LEF_BA_1119_1149_R
AGATAAAGAATCA
745




AGAGCTTCTAA


CGAATATCAATTT




AAAGAATAC


GTAGC


 


37
LEF_BA_756_781_F
AGCTTTTGCAT
26
LEF_BA_843_872_R
TCTTCCAAGGATA
1135




ATTATATCGAG


ATTTATTTCTTG




CCAC


TTCG


 


38
LEF_BA_758_778_F
CTTTTGCATATT
90
LEF_BA_843_865_R
AGGATAGATTTAT
748




ATATCGAGC


TTCTTGTTCG


 


39
LEF_BA_795_813_F
TTTACAGCTTT
700
LEF_BA_883_900_R
TCTTGACAGCATC
1140




ATGCACCG


CGTTG


 


40
LEF_BA_883_899_F
CAACGGATGCT
43
LEF_BA_939_958_R
CAGATAAAGAATC
762




GGCAAG


GCTCCAG


 


41
PAG_BA_122_142_F
CAGAATCAAGT
49
PAG_BA_190_209_R
CCTGTAGTAGAAG
781




TCCCAGGGG


AGGTAAC


 


42
PAG_BA_123_145_F
AGAATCAAGTT
22
PAG_BA_187_210_R
CCCTGTAGTAGAA
774




CCCAGGGGTT


GAGGTAACCAC




AC


 


43
PAG_BA_269_287_F
AATCTGCTATT
11
PAG_BA_326_344_R
TGATTATCAGCGG
1186




TGGTCAGG


AAGTAG


 


44
PAG_BA_655_675_F
GAAGGATATAC
93
PAG_BA_755_772_R
CCGTGCTCCATTT
778




GGTTGATGTC


TTCAG


 


45
PAG_BA_753_772_F
TCCTGAAAAAT
341
PAG_BA_849_868_R
TCGGATAAGCTGC
1089




GGAGCACGG


CACAAGG


 


46
PAG_BA_763_781_F
TGGAGCACGG
552
PAG_BA_849_868_R
TCGGATAAGCTGC
1089




CTTCTGATC


CACAAGG


 


47
RPOC_EC_1018_1045_F
CAAACTTATTA
39
RPOC_EC_1095_1124_R
TCAAGCGCCATTT
959




AGGTAAGCGTG


CTTTTGGTAAACC




TTGACT


ACAT


 


48
RPOC_EC_1018_1045_2_F
CAAAACTTATT
39
RPOC_EC_1095_1124_2_R
TCAAGCGCCATCT
958




AGGTAAGCGTG


CTTTCGGTAATCC




TTGACT


ACAT


 


49
RPOC_EC_114_140_F
TAAGAAGCCGG
158
RPOC_EC_213_232_R
GGCGCTTGTACTT
831




AAACCATCAAC


ACCGCAC




TACCG


 


50
RPOC_EC_2178_2196_F
TGATTCTGGTG
478
RPOC_EC_2225_2246_R
TTGGCCATCAGGC
1414




CCCGTGGT


CACGCATAC


 


51
RPOC_EC_2178_2196_2_F
TGATTCCGGTG
477
RPOC_EC_2225_2246_2_R
TTGGCCATCAGAC
1413




CCCGTGGT


CACGCATAC


 


52
RPOC_EC_2218_2241_F
CTGGCAGGTAT
81
RPOC_EC_2313_2337_R
CGACCGTGGGTT
790




GCGTGGTCTGA


GAGATGAAGTAC




TG


 


53
RPOC_EC_2218_2241_2_F
CTTGCTGGTAT
86
RPOC_EC_2313_2337_2_R
CGCACCATGCGTA
789




GCGTGGTCTGA


GAGATGAAGTAC




TG


 


54
RPOC_EC_808_833_F
CGTCGGGTGAT
75
RPOC_EC_865_889_R
GTTTTTCGTTGCG
847




TAACCGTAACA


TACGATGATGTC




ACCG


 


55
RPOC_EC_808_833_2_F
CGTCGTGTAAT
76
RPOC_EC_865_891_R
ACGTTTTTCGTTT
741




AACCGTAACA


TGAACGATAATGC




ACCG


T


 


56
RPOC_EC_993_1019_F
CAAAGGTAAGC
41
RPOC_EC_1036_1059_R
CGAACGGCCTGAG
785




AAGGTCGTTTC


TAGTCAACACG




CGTCA


 


57
RPOC_EC_993_1019_2_F
CAAAGGTAAGC
40
RPOC_EC_1036_1059_2_R
CGAACGGCCAGAG
784




AAGGACGTTTC


TAGTCAACACG




CGTCA


 


58
SSPE_BA_115_137_F
CAAGCAAACGC
45
SSPE_BA_197_222_R
TGCACGTCTGTTT
1201




ACAATCAGAA


CAGTTGCAAATTC




GC


 


59
TUFB_EC_239_259_F
TAGACTGCCCA
204
TUFB_EC_283_303_R
GCCGTCCATCTGA
815




GGACACGCTG


GCAGCACC


 


60
TUFB_EC_239_259_2_F
TTGACTGCCCA
678
TUFB_EC__283 _303_2_R
GCCGTCCATTTGA
816




GGTCACGCTG


GCAGCACC


 


61
TUFB_EC_976_1000_F
AACTACCGTC
4
TUFB_EC_1045_1068_R
GTTGTCGCCAGGC
845




CGCAGTTCTAC


ATAACCATTTC




TTCC


 


62
TUFB_EC_976_1000_2_F
AACTACCGTCC
5
TUFB_EC_1045_1068_2_R
GTTGTCACCAGGC
844




TCAGTTCTACT


ATTACCATTTC




TCC


 


63
TUFB_ECG_985_1012_F
CCACAGTTCTA
56
TUFB_EC_1033_1062_R
TCCAGGCATTACC
1006




CTTCCGTACTA


ATTTCTACTCCTT




CTGACG







CTGG


 


66
RPLB_EC_650_679_F
GACCTACAGTA
98
RPLB_E739_762_R
TCCAAGTGCTGGT
999




AGAGGTTCTGT


TTACCCCATGG




AATTGAACC


 


67
RPLB_EC_688_710_F
CATCCACACGG
54
RFLB_EC_736_757_R
GTGCTGGTTTACC
842




TGGTGGTGAAG


CCATGGAGT




G


 


68
RPOC_EC_1036_1060_F
CGTGTTGACTA
78
RPOC_EC_1097_1126_R
ATTCAAGAGCCAT
754




TTCGGGGCGTTC


TTCTTTTGGTAAA




AG


CCAC


 


69
RPOB_EC_3762_3790_F
TCAACAACCTC
248
RPOB_EC_3836_3865_R
TTTCTTGAAGAGT
1435




TTGGAGGTAAA


ATGAGCTGCTCCG




GCTCAGT


TAAG


 


70
RPLB_EC_688_710_F
CATCCACACGG
54
RPLB_EC_743_771_R
TGTTTTGTATCCA
1356




TGGTGGTGAAG


AGTGCTGGTTTAC




G


CCC


 


71
VALS_EC_1105_1124_F
CGTGGCGGCGT
77
VALS_EC_1195_1218_R
CGGTACGAACTGG
795




GGTTATCGA


ATGTCGCCGTT


 


72
RPOB_EC_1845_1866_F
TATCGCTCAGG
233
RFOB_EC_1909_1929_R
GCTGGATTCGCCT
825




CGAACTCCAAC


TTGCTACG


 


73
RPLB_EC_669_698_F
TGTAATGAACC
623
RPLB_BC_735_761_R
CCAAGTGCTGGTT
767




CTAATGACCAT


TACCCCATGGAGT




CCACACGG


A


 


74
RPLB_EC_671_700_F
TAATGAACCCT
169
RPLB_EC_737_762_R
TCCAAGTGCTGGT
1000




AATGACCATCC


TTACCCCATGGAG


 


75
SP101_SPET11_1_29_F
AACCTTAATTG
2
SP101_SPET11_92_116_R
CCTACCCAACGTT
779




GAAAGAAACCC


CACCAAGGGCAG




AAGAAGT


 


76
SP101_SPET11_118_147_F
GCTGGTGAAAA
115
SP101_SPET11_213_238_R
TGTGGCCGATTTC
1340




TAACCCAGATG


ACCACCTGCTCCT




TCGTCTTC


 


77
SP101_SPET11_216_243_F
AGCAGGTGGTG
24
SP101_SPET11_308_333_R
TGCCACTTTGACA
1209




AAATCGGCCAC


ACTCCTGTTGCTG




ATGATT


 


78
SP101_SPET11_266_295_F
CTTGTACTTGT
89
SP101_SFET11_355_380_R
GCTGCTTTGATGG
824




GGCTCACACGG


CTGAATCCCCTTC




CTGTTTGG


 


79
SP101_SPET11_322_344_F
GTCAAAGTGGC
132
SF101SFET11_423_441R
ATCCCTGCTTCT
753




CACGTTTACTG


GCTGCC




GC


 


80
SP101_SPET11_358_387_F
GGGGATTCAGC
126
SP101_SPET11_448_473_R
CCAACCTTTTCCA
766




CATCAAAGCAG


CAACAGAATCAGC




CTATTGAC


 


81
SP101_SPET11_600_629_F
CCTTACTTCGA
62
SP101_SPET11_686_714_R
CCCATTTTTTCAC
772




AACTATGAATC


GCATGCTGAAAAT




TTTTGGAAG


ATC


 


82
SP101_SPET11_658_684_F
GGGGATTGATA
127
SP101_SPET11_756_784_R
GATTGGCGATAAA
813




TCACCATAAG


GTGATATTTTCTA




AAGAA


AAA


 


83
SP101_SPET11_776_801_F
TCGCCAATCAA
364
SP101_SPET11_871_896_R
GCCCACCAGAAAG
814




AACTAAGGGAA


ACTAGCAGGATAA




TGGC


 


84
SP101_SPET11_893_921_F
GGGCAACAGCA
123
SP101_SPET11_988_1012_R
CATGACAGCCAAG
763




GCGGATTGCGA


ACCTCACCCACC




TTGCGCG


 


85
SP101_SPET11_1154_1179_F
CAATACCGCAA
47
SP101_SPET11_1251_1277_R
GACCCCAACCTGG
804




CAGCGGTGGCT


CCTTTTGTCGTTG




TGGG


A


 


86
SP101_SPET11_1314_1336_F
CGCAAAAAAAT
68
SP101_SPET11_1403_1431_R
AAACTATTTTTTT
711




CCAGCTATTAG


AGCTATACTCGAA




C


CAC


 


87
SP101_SPET11_1408_1437_F
CGAGTATAGCT
67
SP101_SPET11_1486_1515_R
GGATAATTGGTCG
828




AAAAAAATAGT


TAACAAGGGATAG




TTATGACA


TGAG


 


88
SP101_SPET11_1688_1716_F
CCTATATTAAT
60
SP101_SPET11_1783_1808_R
ATATGATTATCAT
752




CGTTTACAGAA


TGAACTGCGGCCG




ACTGGCT


 


89
SP101_SPET11_1711_1733_F
CTGGCTAAAA
82
SP101_SPET11_1808_1835_R
GCGTGACAGACCT
821




CTTTGGCAAC


TCTTGAATTGTAA




GG


TCA


 


90
SP101_SPET11_1807_1835_F
ATGATTACAAT
33
SP101_SPET11_1901_1927_R
TTGGACCTGTAAT
1412




TCAAGAAGGTC


CAGCTGAATACTG




GTCACGC


G


 


91
SP101_SPET11_1967_1991_F
TAACGGTTATC
155
SP101_SPET11_2062_2083_R
ATTGCCCAGAAAT
755




ATGGCCCAGAT


CAAATCATC




GGG


 


92
SP101_SPET11_2260_2283_F
CAGAGACCGTT
50
SP101_SPET11_2375_2397_R
TCTGGGTGACCTG
1131




TTATCCTATCA


GTGTTTTAGA




GC


 


93
S9101_SPET11_2375_2399_F
TCTAAAACACC
390
SP101_SPET11_2470_2497_R
AGCTGCTAGATGA
747




AGGTCACCCAG


GCTTCTGCCATGG




AAG


CC


 


94
SF101_SPET11_2468_2487_F
ATGGCCATGGC
35
SP101_SPET11_2543_2570_R
CCATAAGGTCACC
770




AGAAGCTCA


GTCACCATTCAAA







GC


 


95
SP101_SPET11_2961_2984_F
ACCATGACAGA
15
SP101_SPET11_3023_3045_R
GGAATTTACCAGC
827




AGGCATTTTGA


GATAGACACC




CA


 


96
SP101_SPET11_3075_3103_F
GATGACTTTTT
108
SP101_SPET11_3168_3196_R
AATCGACGACCAT
715




AGCTAATGGTC


CTTGGAAAGATTT




AGGCAGC


CTC


 


97
SP101_SPET11_3386_3403_F
AGCGTAAAGGT
25
SP101_SPET11_3480_3506_R
CCAGCAGTTACTG
769




GAACCTT


TCCCCTCATCTTT







G


 


98
SP101_SPET11_3511_3535_F
GCTTCAGGAAT
116
SP101_SPET11_3605_3629_R
GGGTCTACACCTG
832




CAATGATGGAG


CACTTGCATAAC




CAG


 


111
RPOB_EC_3775_3803_F
CTTGGAGGTAA
87
RFOB_EC_3829_3858_R
CGTATAAGCTGCA
797




GTCTCATTTTG


CCATAAGCTTGTA




GTGGGCA


ATGC


 


112
VALS_EC_1833_1850_F
CGACGCGCTGC
65
VALS_EC_1920_1943_R
GCGTTCCACAGCT
822




GCTTCAC


TGTTGCAGAAG


 


113
RPOB_EC_1336_1353_F
GACCACCTCGG
97
RPOB_EC_1438_1455_R
TTCGCTCTCGGCC
1386




CAACCGTA


TGGCC


 


114
TUFB_EC_225_251_F
GCACTATGCAC
111
TUFB_EC_284_309_R
TATAGCACCATCC
930




ACGTAGATTGT


ATCTGAGCGGCAC




CCTGG


 


115
DNAK_EC_428_449_F
CGGCGTACTTC
72
DNAK_EC_503_522_R
CGCGGTCGGCTCG
792




AACGACAGCCA


TTGATGA


 


116
VALS_EC_1920_1943_F
CTTCTGCAACA
85
VALS_EC_1948_1970_R
TCGCAGTTCATCA
1075




AGCTGTGGAAC


GCACGAAGCG




GC


 


117
TUFB_EC_757_474_F
AAGACGACCTG
6
TUFB_EC_849_867_R
GCGCTCCACGTCT
819




CACGGGC


TCACGC


 


118
23S_EC_2646_2667_F
CTGTTCTTAGT
84
23S_EC_2745_2765_R
TTCGTGCTTAGAT
1389




ACGAGAGGACC


GCTTTCAG


 


119
16S_EC_969_985_1P_F
ACGCGAAGAC
19
16S_EC_1061_1078_2P_R
ACGACACGAGCpT
733




CTTACpC


pGACGAC


 


120
16S_EC_972_985_2P_F
CGAAGAACpCp
63
16S_EC_1064_1075_2P_R
ACACGAGCpTpGA
727




TTACC


C


 


121
16S_EC_972_985_F
CGAAGAACCTT
63
16S_EC_1064_1075_R
ACACGAGCTGAC
727




ACC


 


122
TRAN_ILE-
CCTGATAAGGG
61
23S_SC_40_59_R
ACGTCCTTCATCG
740



RRNH_EC_32_50.2_F



CCTCTGA


 


123
23S_EC_−7_15_F
GTTGTGAGGTT
140
23S_EC_430_450_R
CTATCGGTCAGTC
799




AAGCGACTAAG


AGGAGTAT


 


124
23S_EC_−7_15_F
GTTGTGAGGTT
141
23S_EC_891_910_R
TTGCATCGGGTTG
1403




AAGCGACTAAG


GTAAGTC


 


125
23S_EC_430_450_F
ATACTCCTGAC
30
23S_EC_1424_1442_R
AACATAGCCTTCT
712




TGACCGATAG


CCGTCC


 


126
23S_EC_891_910_F
GACTTACCAAC
100
23S_SC_1908_1931_R
TACCTTAGGACCG
893




CCGATGCAA


TTATAGTTACG


 


127
23S_EC_1424_1442_F
GGACGGAGAAG
117
23S_EC_2475_2494_R
CCAAACACCGCCG
765




GCTATGTT


TCGATAT


 


128
23S_EC_1908_1931_F
CGTAACTATAA
73
23S_EC_2833_9852_R
GCTTACACACCCG
826




CGGTCCTAAGG


GCCTATC




TA


 


129
23S_EC_2475_9494_F
ATATCGACGGC
31
TRNA_ASP-
GCGTGACAGGCAG
820




GGTGTTTGG

RRNH_EC_23_41.2_R
GTATTC


 


131
16S_EC_−60_−39_F
AGTCTCAAGAG
28
16S_EC_508_525_R
GCTGCTGGCACGG
823




TGAACACGTAA


AGTTA


 


132
16S_EC_326_345_F
GACACGGTCCA
95
16S_EC_1041_1058_R
CCATGCAGCACCT
771




GACTCCTAC


GTCTC


 


133
16S_EC905_724_F
GATCTGGAGGA
107
16S_EC_1493_3512_R
ACGGTTACCTTGT
739




ATACCGGTG


TACGACT


 


134
16S_EC_1268_1287_F
GAGAGCAAGCG
101
TRNA_ALA-
CCTCCTGCGTGCA
780




GACCTCATA

RRNH_EC_30_40.2_R
AAGC


 


135
16S_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1061_1078.2_R
ACAACACGAGCTG
719




CTTACC


ACGAC


 


137
165_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1061_1078.2_I14_R
ACAACACGAGCTG
721




CCTTACC


ICGAC


 


138
165_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1061_107.2_I12_R
ACAACACGAGCIG
718




CTTACC


CGAC


 


139
16S_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1061_1078.2_I11_R
ACAACACGAGITG
722




CTTACC


ACGAC


 


140
16S_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1061_1078.2_I16_R
ACAACACGAGCTG
720




CTTACC


ACIAC


 


141
16S_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1061_1078.2_2I_R
ACAACACGAICTI
723




CTTACC


ACGAC


 


142
165_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1061_1078.2_3I_R
ACAACACIAICTI
724




CTTACC


ACGAC


 


143
16S_EC_99_985_F
ACGCGAAGAAC
19
16S_EC_1061_1078.2_4I_R
ACACCACIAICTI
725




CTTACC


ACIAC


 


147
23S_EC_2652_2669_F
CTAGTACGAGA
79
23S_EC_2741_2760_R
ACTTAGATGCTTT
743




GGACCGG


CAGCGGT


 


158
16S_EC_683_700_F
GTGTAGCGGTG
137
16S_EC_880_894_R
CGTACTCCCCAGG
796




AAATGCG


CG


 


159
16S_EC_1100_1116_F
CAACGAGCGCA
42
16S_EC_1174_1188_R
TCCCCACCTTCCT
1019




ACCCTT


CC


 


215
SSPE_BA_121_137_F
AACGCACAATC
3
SSPE_BA_197_216_R
TCTGTTTCAGTTG
1132




AGAAGC


CAAATTC


 


220
GROL_EC_941_959_F
TGGAAGATCTG
544
GROL_EC_1039_1060_R
CAATCTGCTGACG
759




GGTCAGGC


GATCTGAGC


 


221
INFB_EC_1103_1124_F
GTCGTGAAAAC
133
INFB_EC_1174_1191_R
CATGATGGTCACA
764




GAGCTGGAAGA


ACCGG


 


222
HFLB_EC_1082_1102_F
TGGCGAACCTG
569
HFLB_EC_1144_1168_R
CTTTCGCTTTCTC
802




GTGAACGAAGC


GAACTCAACCAT


 


223
INFB_EC_1969_1994_F
CGTCAGGGTAA
74
INFB_EC_2038_2058_R
AACTTCGCCTTCG
713




ATTCCGTGAAG


GTCATGTT




TTAA


 


224
GROL_EC_219_242_F
GGTGAAAGAAG
128
GROL_EC_328_350_R
TTCAGGTCCATCG
1377




TTGCCCTCTAA


GGTTCATGCC




AGC


 


225
VALS_EC_1105_1124_F
CGTGGCGGCGT
77
VALS_EC_1195_1214_R
ACGAACTGGATGT
732




GGTTATCGA


CGCCGTT


 


226
16S_EC_556_575_F
CGGAATTACTG
70
16S_EC_683_700_R
CGCATTTCACCGC
791




GGCGTAAAG


TACAC


 


227
RPOC_EC_1256_1277_F
ACCCAGTGCTG
16
RPOC_EC_1295_1315_R
GTTCAAATGCCTG
843




CTGAACCGTGC


GATACCCA


 


228
16S_EC_774_795_F
GGGAGCAAACA
122
16S_EC_880_894_R
CGTACTCCCCAGG
796




GGATTAGATAC


CG


 


229
RPOC_EC_1584_1604_F
TGGCCCGAAAG
567
RPOC_EC_1623_1643_R
ACGCGGGCATGCA
737




AAGCTGAGCG


GAGATGCC


 


230
16S_EC_1082_1100_F
ATGTTGGGTTA
37
16S_EC_1177_1196_R
TGACGTCATCCCC
1158




AGTCCCGC


ACCTTCC


 


231
16S_EC_1389_1407_F
CTTGTACACAC
88
16S_EC_1525_1541_R
AAGGAGGTGATCC
714




CGCCCGTC


AGCC


 


232
16S_EC_1303_1323_F
CGGATTGGAGT
71
16S_EC_1389_1407_R
GACGGCGGTGTG
808




CTGCAACTCG


TACAAG


 


233
23S_EC_23_37_F
GGTGGATGCCT
129
23S_EC_115_130_R
GGGTTTCCCCATT
833




TGGC


CGG


 


234
23S_EC_187_207_F
GGGAACTGAAA
121
23S_EC_242_256_R
TTCGCTCGCCGCT
1385




CATCTAAGTA


AC


 


235
23S_EC_1602_1620_F
TACCCCAAACC
184
23S_EC_1686_1703_R
CCTTCTCCCGAAG
782




GACACAGG


TTACG


 


236
23S_EC_1685_1703_F
CCGTAACTTC
58
23S_EC_1828_1842_R
CACCGGGCAGGCG
760




GGAGAAGG


TC


 


237
23S_EC_1827_1843_F
GACGCCTGCCC
99
23S_EC_1929_1949_R
CCGACAAGGAATT
775




GGTGC


TCGCTACC


 


238
23S_EC_2434_2456_F
AAGGTACTCCG
9
23S_EC_9490_2511_R
AGCCGACATCGAG
746




GGGATAACAGG


GGTGCCAAAC




C


 


239
23S_EC_2599_2616_F
GACAGTTCGGT
96
23S_SC_2653_2669_R
CCGGTCCTCTCGT
777




CCCTATC


ACTA


 


240
23S_EC_2653_2669_F
TAGTACGAGAG
227
23S_EC_2737_2758_R
TTAGATGCTTTCA
1369




GACCGG


GCACTTATC


 


241
23S_ES_−68_−44_F
AAACTAGATAA
1
23S_B_5_21_R
GTGCGCCCTTTCT
841




CAGTAGACATC


AACTT




AC


 


242
16S_EC_8_27_F
AGAGTTTGATC
23
16S_SC_342_358_R
ACTGCTGCCTCCC
742




ATGGCTCAG


GTAG


 


243
16S_EC_314_332_F
CACTGGAACTG
48
16S_EC_556_575_R
CTTTACGCCCAGT
801




AGACACGG


AATTCCG


 


244
16S_EC_518_536_F
CCAGCAGCCGC
57
16S_EC_774_795_R
GTATCTAATCCTG
839




GGTAATAC


TTTGCTCCC


 


245
16S_EC_683_700_F
GTGTAGCGGTG
137
16S_EC_967_985_R
GGTAAGGTTCTTC
835




AAATGCG


GCGTTG


 


246
16S_EC_937_954_F
AAGCGGTGGAG
7
16S_EC_1220_1240_R
ATTGTAGCACGTG
757




CATGTGG


TGTAGCCC


 


247
16S_EC_1195_1213_F
CAAGTCATCAT
46
16S_EC_1525_1541_R
AAGGAGGTGATCC
714




GGCCCTTA


AGCC


 


248
16S_EC_8_27_F
AGAGTTTGATC
23
16S_EC_1525_1541_R
AAGGAGGTGATCC
714




ATGGCTCAG


AGCC


 


249
23S_EC_1831_1849_F
ACCTGCCCAGT
18
23S_EC_1919_1936_R
TCGCTACCTTAGG
1080




GCTGGAAG


ACCGT


 


250
16S_EC_1387_1407_F
GCCTTGTACAC
112
16S_EC_1494_1513_R
CACGGCTACCTTG
761




ACCTCCCGTC


TTACGAC


 


251
16S_EC_1390_1411_F
TTGTACACACC
693
16S_EC_1486_1505_R
CCTTGTTACGACT
783




GCCCGTCATAC


TCACCCC


 


252
16S_EC_1367_1387_F
TACGGTGAATA
191
16S_EC_1485_1506_R
ACCTTGTTACGAC
731




CGTTCCCGGG


TTCACCCCA


 


253
16S_EC_804_822_F
ACCACGCCGTA
14
16S_EC_909_929_R
CCCCCGTCAATTC
773




AACGATGA


CTTTGAGT


 


254
16S_EC_791_812_F
GATACCCTGGT
106
16S_EC_886_04_R
GCCTTGCGACCGT
817




AGTCCACACCG


ACTCCC


 


255
16S_EC_789_810_F
TAGATACCCTG
206
16S_EC_882_899_R
GCGACCGTACTCC
818




GTAGTCCACGC


CCAGG


 


256
16S_EC_1092_1109_F
TAGTCCCGCAA
228
16S_EC_1174_1195_R
GACGTCATCCCCA
810




CGAGCGC


CCTTCCTCC


 


257
23S_EC_2586_2607_F
TAGAACGTCGC
203
23S_EC_2658_2677_R
AGTCCATCCCGGT
749




GAGACAGTTCG


CCTCTCG


 


258
RNASEP_SA_31_49_F
GAGGAAAGTCC
103
RNASEP_SA_358_379_R
ATAAGCCATGTTC
750




ATGCTCAC


TGTTCCATC


 


258
RNASEP_SA_31_49_F
GAGGAAAGTCC
103
RNASEP_EC_345_362_R
ATAAGCCGGGTTC
751




ATGCTCAC


TGTCG


 


258
RNASEP_SA_31_49_F
GAGGAAAGTCC
103
RNASEP_BS_363_384_R
GTAAGCCATGTTT
838




ATGCTCAC


TGTTCCATC


 


258
RNASEP_BS_43_61_F
GAGGAAAGTCC
104
RNASEP_SA_358_379_R
ATAAGCCATGTTC
750




ATGCTCGC


TGTTCCATC


 


258
RNASEP_BS_43_61_F
GAGGAAAGTCC
104
RNASEP_EC_345_362_R
ATAAGCCGGGTTC
751




ATGCTCGC


TGTCG


 


258
RNASEP_BS_43_31_F
GAGGAAAGTCC
104
RNASEP_ES_363_384_R
GTAAGCCATGTTT
838




ATGCTCGC


TGTTCCATC


 


258
RNASEP_EC_61_77_F
GAGGAAAGTCC
105
RNASEP_SA_358_379_R
ATAAGCCATGTTC
750




GGGCTC


TGTTCCATC


 


258
RNASEP_EC_61_77_F
GAGGAAAGTCC
105
RNASEP_EC_345_362_R
ATAAGCCGGGTTC
751




GGGCTC


TGTCG


 


258
RNASEP_EC_61_77_F
GAGGAAAGTCC
105
RNASEP_BS_363_384_R
GTAAGCCATGTTT
838




GGGCTC


TGTTCCATC


 


259
RNASEP_ES_4331_F
GAGGAAAGTCC
104
RNASEP_ES_363_384_R
GTAAGCCATGTTT
838




ATGCTCGC


TGTTCCATC


 


260
RNASEP_EC_61_77_F
GAGGAAAGTCC
105
RNASEP_EC_345_362_R
ATAAGCCGGGTTC
751




GGGCTC


TGTCG


 


262
RNASEP_SA_31_49_F
GAGGAAAGTCC
103
RNASEP_SA_358_379_R
ATAAGCCATGTTC
750




ATGCTCAC


TGTTCCATC


 


263
16S_EC_1082_1100_F
ATGTTGGGTTA
37
16S_EC_1525_1541_R
AAGGAGGTGATCC
714




AGTCCCGC


AGCC


 


264
16S_EC_556_575_F
CGGAATTACTG
70
16S_EC_774_795_R
GTATCTAATCCTG
839




GGCGTAAAG


TTGCTCCC


 


265
16S_EC_1082_1100_F
ATGTTGGGTTA
37
16S_EC_1177_1196_10G_R
TGACGCATGCCC
1160




AGTCCCGC


ACCTTCC


 


266
16S_EC_1082_1100_F
ATGTTGGGTTA
37
16S_EC_1177_1196_10G_11G_R
TGACGTCATGGCC
1161




AGTCCCGC


ACCTTCC


 


268
YAED_EC_513_532_F_MOD
GGTGTTAAATA
130
TRNA_ALA-
AGACCTCCTGCGT
744




GCCTGGCAG

RRNA_EC_30_49_F_MOD
GCAAAGC


 


269
16S_EC_1082_1100_F_MOD
ATGTTGGGTTA
37
16S_EC_1177_1196_R_MOD
TGACGCATCCCC
1158




AGTCCCGC


ACCTTCC


 


270
23S_EC_2586_2607_F_MOD
TAGAACGTCGC
203
23S_EC_2658_2677_R_MOD
AGTCCATCCCGGT
749




GAGACAGTTCG


CCTCTCG


 


272
16S_EC_969_985_F
ACGCGAAGAAC
19
16S_EC_1389_1407_R
GACGGGCGGTGTG
807




CTTACC


TACAAG


 


273
16S_EC_683_700_F
GTGTAGCGGTG
137
16S_EC_1303_1323_R
CGAGTTGCAGACT
788




AAATGCG


GCGATCCG


 


274
169_EC_49_68_F
TAACACATGCA
152
16S_EC_880_894_R
CGTACTCCCCAGG
796




AGTCGAACG


CG


 


275
16S_EC_49_68_F
TAACACATGCA
152
16S_EC_1061_1078_R
ACGACACGAGCTG
734




AGTCGAACG


ACGAC


 


277
CYA_BA_1349_1370_F
ACAACGAAGTA
12
CYA_BA_1426_1447_R
CTTCTACATTTTT
800




CAATACAAGAC


AGCCATCAC


 


278
16S_EC_1090_1111_2_F
TTAAGTCCCGC
650
16S_EC_1175_1196_R
TGACGTCATCCCC
1159




AACGAGCGCAA


ACCTTCCTC


 


279
16S_EC_405_432_F
TGAGTGATGAA
464
16S_EC_507_527_R
CGGCTGCTGGCAC
793




GGCCTTAGGGT


GAAGTTAG




TGTAAA


 


280
GROL_EC_496_518_F
ATGGACAAGGT
34
GROL_EC_577_596_R
TAGCCGCGGTCGA
914




TGGCAAGGAAG


ATTGCAT




G


 


281
GROL_EC_511_536_F
AAGGAAGGCGT
8
GROL_EC_571_593_R
CCGCGGTCGAATT
776




GATCACCGTTG


GCATGCCTTC




AAGA


 


288
RPOB_EC_3802_3821_F
CAGCGTTTCGG
51
RPOB_EC_3862_3885_R
CGACTTGACGGTT
786




CGAAATGGA


AACATTTCCTG


 


289
RPOB_EC_3799_3821_F
GGGCAGCGTTT
124
RPOB_EC_3862_3888_R
GTCCGACTTGACG
840




CGGCGAAATG


GTCAACATTTCCT




GA


G


 


290
RPOC_EC_2146_2174_F
CAGGAGTCGTT
52
RPOC_EC_2227_2245_R
ACGCCATCAGGCC
736




CAACTCGATCT


ACGCAT




ACATGAT


 


291
ASPS_EC_405_422_F
GCACAACCTGC
110
ASPS_EC_521_538_R
ACGGCACGAGGTA
738




GGCTGCG


GTCGC


 


292
RPOC_EC_1374_1393_F
CGCCGACTTCG
69
RPOC_EC_1437_1455_R
GAGCATCAGCGTG
811




ACGGTGACC


CGTGCT


 


293
TUFB_EC_957_979_F
CCACACGCCGT
55
TUFB_EC_1034_1058_R
GGCATCACCATTT
829




TCTTCAACAAC


CCTTGTCCTTCG




T


 


294
16S_EC_7_33_F
GAGAGTTTGAT
102
16S_EC_101_122_R
TGTTACTCACCCG
1345




CCTGGCTCAGA


TCTGCCACT




ACGAA


 


295
VALS_EC_610_649_F
ACCGAGCAAGG
17
VALS_EC_705_727_R
TATAACGCACATC
929




AGACCAGC


GTCAGGGTGA


 


344
16S_EC_971_990_F
GCGAAGAACCT
113
16S_EC_1043_1062_R
ACAACCATGCACC
726




TACCAGGTC


ACCTGTC


 


346
16S_EC_713_732_TMOD_F
TAGAACACCGA
202
16S_EC_789_809_TMOD_R
TCGTGGACTACCA
1110




TGGCGAAGGC


GGGTATCTA


 


347
16S_EC_785_806_TMOD_F
TGGATTAGAGA
560
16S_EC_880_897_TMOD_R
TGGCCGTACTCCC
1278




CCCTGGTAGTC


CAGGCG




C


 


348
16S_EC_960_981_TMOD_F
TTTCGATGCAA
706
16S_EC_1054_1073_TMOD_R
TACGAGCTGACGA
895




CGCGAAGAACC


CAGCCATG




T


 


349
23S_EC_1826_1843_TMOD_F
TCTGACACCTG
401
23S_EC_1906_1924_TMOD_R
TGACCGTTATAGT
1156




CCCGGTGC


TACGGCC


 


350
CAPC_BA_274_303_TMOD_F
TGATTATTGTT
476
CAPC_BA_349_376_TMOD_R
TGTAACCCTTGTC
1314




ATCCTGTTATG


TTTGAATTGTAT




CCATTTGAG


TGC


 


351
CYA_BA_1353_1379_TMOD_F
TCGAAGTACAA
355
CYA_BA_1448_1467_TMOD_R
TTGTTAACGGCTT
1423




TACAAGACAAA


CAAGACCC




AGAAGG


 


352
INFB_EC_1365_1393_TMOD_F
TTGCTCGTGGT
687
INFB_EC_1439_1467_TMOD_R
TTGCTGCTTTCGC
1411




GCACAAGTAAC


ATGGTTAATTGCT




GGATATTA


TCAA


 


353
LEF_BA_756_781_TMOD_F
TAGCTTTTGCA
220
LEF_BA_843_872_TMOD_R
TTCTTCCAAGGAT
1394




TATTATATCGA


AGATTTATTTCTT




GCCAC


GTTCG


 


354
RPOC_EC_2218_2241_TMOD_F
TCTGGCAGGTA
405
RPOC_EC_2313_2337_TMOD_R
TCGCACCGTGGGT
1072




TGCGTGGTCTG


TGAGATGAAGTAC




ATG


 


355
SSPE_BA_115_137_TMOD_F
TCAAGCAAACG
255
SSPE_BA_197_222_TMOD_R
TTGCACGTCTGTT
1402




CACAATCAGAA


TCAGTTGCAAATT




GC


C


 


356
RPLB_EC_650_679_TMOD_F
TGACCTACAGT
449
RPLB_EC_739_762_TMOD_R
TTCCAAGTGCTGG
1380




AAGAGGTTCTG


TTTACCCCATGG




TAATGAACC


 


357
RPLB_EC_688_710_TMOD_F
TCATCCACACG
296
RPLB_EC_736_757_TMOD_R
TGTGCTGGTTTAC
1337




GTGGTGGTGAA


CCCATGGAGT




GG


 


358
VALS_EC_1105_1124_TMOD_F
TCGTGGCGGCG
385
VALS_EC_1195_1218_TMOD_R
TCGGTACGAACTG
1093




TGGTTATCGA


GATGTCGCCGTT


 


359
RPOB_EC_1845_1866_TMOD_F
TTATCGCTCAG
659
RPOB_EC_1909_1929_TMOD_R
TGCTGGATTCGCC
1250




GCGAACTCCAA


TTTGCTACG




C


 


360
23SEC_2646_2667_TMOD_F
TCTGTTCTTAG
409
23S_EC_2745_2765_TMOD_R
TTTCGTGCTTAGA
1434




TACGAGAGGAC


TGCTTTCAG




C


 


361
16S_EC_1090_1111_2_TMOD_F
TTTAAGTCCCG
697
16S_EC_1175_1196_TMOD_R
TTGACGTCATCCC
1398




CAACGAGCGCA


CACCTTCCTC




A


 


362
RPOB_EC_3799_3821_TMOD_F
TGGGCAGCGTT
581
RPOB_EC_3862_3888_TMOD_R
TGTCCGACTTGAC
1325




TCGGCGAAATG


GGTCAACATTTCC




GA


TG


 


363
RPOC_EC_2146_2174_TMOD_F
TCAGGAGTCGT
284
EPOC_EC_2227_2245_TMOD_R
TACGCCATCAGGC
898




TCAACTCGATC


CACGCAT




TACATGAT


 


364
RPOC_EC_1374_1393_TMOD_F
TCGCCGACTTC
367
RPOC_EC_1437_1455_TMOD_R
TGAGCATCAGCGT
1166




GACGGTGACC


GCGTGCT


 


367
TUFB_ECG_957_979_TMOD_F
TCCACACGCCG
308
TUFB_EC_1034_1058_TMOD_R
TGGCATCACCATT
1276




TTCTTCAACAA


TCCTTGTCCTTCG




CT


 


423
SP101_SPET11_893_921_TMOD_F
TGGGCAACAGC
580
SP101_SPET11_988_1012_TMOD_R
TCATGACAGCCAA
990




AGCGGATTGCG


GACCTCACCCACC




ATTGCGCG


 


424
SP101_SPET11_1154_1179—
TCAATACCGCA
258
SP101_SPET11_1251_1277_TMOD_R
TGACCCCAACCTG
1155



TMOD_F
ACAGCGGTGGC


GCCTTTTGTCGTT




TTGGG


GA


 


425
SP101_SPET11_118_147_TMOD_F
TGCTGGTGAAA
528
SP101_SPET11_213_238_TMOD_R
TTGTGGCCGATTT
1422




ATAACCCAGAT


CACCACCTGCTCC




GTCGTCTTC


T


 


426
SP101_SPET11_1314_1336_TMOD_F
TCGCAAAAAAA
363
SP101_SPET11_1403_1431_TMOD_R
TAAACTATTTTTT
849




TCCAGCTATTA


TAGCTATACTCGA




GC


ACAC


 


427
SP101_SPET11_1408_1437—
TCGAGTATAGC
359
SP101_SPET11_1486_1515_TMOD_R
TGGATAATTGGTC
1268



TMOD_F
TTAAAAAAATA


GTAACAAGGGATA




GTTTATGACA


GTGAG


 


428
SP101_SPET11_1688_1716_TMOD_F
TCCTATATTAA
334
SP101_SPET11_1783_1808_TMOD_R
TATATGATTATCA
932




TCGTTTACAGA


TTGAACTGCGGCC




AACTGGCT


G


 


429
SP101_SPET11_1711_1733—
TCTGGCTAAAA
406
SP101_SPET11_1808_1835_TMOD_R
TGCGTGACGACCT
1239



TMOD_F
CTTTGGCAACG


TCTTGAATTGTAA




GT


TCA


 


430
SP101_SPET11_1807_1835_TMOD_F
TATGATTACAA
235
SP101_SPET11_1901_1927_TMOD_R
TTTGGACCTGTAA
1439




TTCAAGAAGGT


TCAGCTGAATACT




CGTCACGC


GG


 


431
SP101_SPET11_1967_1991—
TTAACGGTTAT
649
SP101_SPET11_2082_2083_TMOD_R
TATTGCCCAGAAA
940



TMOD_F
CATGGCCCAGA


TCAAATCATC




TGGG


 


432
SP101_SPET11_216_243_TMOD_F
TAGCAGGTGGT
210
SP101_SPET11_308_333_TMOD_R
TTGCCACTTTGAC
1404




GAAATCGGCCA


AACTCCTGTTGCT




CATGATT


G


 


433
SP101_SPET11_2260_2283—
TCAGAGACCGT
272
SP101_SPET11_2375_2397_TMOD_R
TTCTGGGTGACCT
1393



TMOD_F
TTTATCCTATC


GGTGTTTTAGA




AGC


 


434
SP101_SPET11_2375_2399—
TTCTAAAACAC
675
SP101_SPET11_2470_2497_TMOD_R
TAGCTGCTAGATG
918



TMOD_F
CAGGTCACCCA


AGCTTCTGCCATG




GAAG


GCC


 


435
SP101_SPET11_2468_2487—
TATGGCCATGG
238
SP101_SPET11_2543_2579_TMOD_R
TCCATAAGGTCAC
1007



TMOD_F
CAGAAGCTCA


CGTCACCATTCAA







AGC


 


436
SP101_SFET11_266_295_TMOD_F
TCTTGTACTTG
417
SP101_SPET11_355_380_TMOD_R
TGCTGCTTTGATG
1249




TGGCTCACACG


GCTGAATCCCCTT




GCTGTTTGG


C


 


437
SP101_SPET11_2961_2984—
TACCATGACAG
183
SP101_SPET11_3023_3045_TMOD_R
TGGAATTTACCAG
1264



TMOD_F
AAGGCATTTTG


CGATAGACACC




ACA


 


438
SP101_SPET11_3075_3103—
TGATGACTTTT
473
SP101_SPET11_3168_3196_TMOD_R
TAATCGACGACCA
875



TMOD_F
TAGCTAATGGT


TCTTGGAAAGATT




CAGGCAGC


TCTC


 


439
SP101_SPET11_322_344_TMOD_F
TGTCAAAGTGG
631
SP101_SPET11_423_441_TMOD_R
TATCCCCTGCTTC
934




CACGTTTACTG


TGCTGCC




GC


 


440
SP101_SPET11_3386_3403—
TAGCGTAAAGG
215
SP101_SPET11_3480_3506_TMOD_R
TCCAGCAGTTACT
1005



TMOD_F
TGAACCTT


GTCCCCTCATCTT







TG


 


441
SP101_SPET11_3511_3535—
TGCTTCAGGAA
531
SP101_SPET11_3605_3629_TMOD_R
TGGGTCTACACCT
1294



TMOD_F
TCAATGATGGA


GCACTTGCATAAC




GCAG


 


442
SP101_SPET11_358_387_TMOD_F
TGGGGATTCAG
588
SP101_SPET11_448_473_TMOD_R
TCCAACCTTTTCC
998




CCATCAAAGCA


ACAACAGAATCAG




GCTATTGAC


C


 


443
SP101_SPET11_600_628_TMOD_F
TCCTTACTTCG
348
SP101_SPET11_886_714_TMOD_R
TCCCATTTTTTCA
1018




AACTATGAATC


CGCATGCTGAAAA




TTTTGGAAG


TATC


 


444
SP101_SPET11_658_684_TMOD_F
TGGGGATTGAT
589
SP101_SPET11_756_784_TMOD_R
TGATTGGCGATAA
1189




ATCACCGATAA


AGTGATATTTTCT




GAAGAA


AAAA


 


445
SP101_SPET11_776_801_TMOD_F
TTCGCCAATCA
673
SP101_SPET11_871_896_TMOD_R
TGCCCACCAGAAA
1217




AAACTAAGGGA


GACTAGCAGGATA




ATGGC


A


 


446
SP101_SPET11_1_29_TMOD_F
TAACCTTAATT
154
SP101_SPET11_92_116_TMOD_R
TCCTACCCAACGT
1044




GGAAAGAAACC


TCACCAAGGGCAG




CAAGAAGT


 


447
SP101_SPET11_364_385_F
TCAGCCATCAA
276
SP101_SPET11_448_471_R
TACCTTTTCCACA
894




AGCAGCTATTG


ACAGAATCAGC


 


448
SP101_SPET11_3085_3104_F
TAGCTAATGGT
216
SP101_SPET11_3170_3194_R
TCGACGACCATCT
1066




CAGGCAGCC


TGGAAAGATTTC


 


449
RPLB_EC_990_710_F
TCCACACGGTG
309
RPLB_EC_737_758_R
TGTGCTGGTTTAC
1336




GTGGTGAAGG


CCCATGGAG


 


481
BONTA_X52066_538_552_F
TATGGCTCTAC
239
BONTA_X52066_647_660_R
TGTTACTGCTGGA
1346




TCAA


T


 


482
BONTA_X5206_538_552P_F
TA*TpGGC*Tp
143
BONTA_X52066_647_660P_R
TG*Tp*TpA*Cp*
1146




*Cp*TpA*Cp*


TpG*Cp*TpGGAT




Tp*CpAA


 


483
BONTA_X5206_701_720_F
GAATAGCAATT
94
BONTA_X5206_759_775_R
TTACTTCTAACCC
1367




AATCCAAAT


ACTC


 


484
BONTA_X52066_701_720P_F
GAA*TpAG*Cp
91
BONTA_X52066_759_775P_R
TTA*Cp*Tp*Tp*
1359




AA*Tp*TpAA*


Cp*TpAA*Cp*Cp




Tp*Cp*CpAAAT


*CpA*Cp*TpC


 


485
BONTA_X5206_450_473_F
TCTAGTAATAA
393
BONTA_X5206_517_539_R
TAACCATTTCGCG
859




TAGGACCCTCA


TAAGATTCAA




GC


 


486
BONTA_X52066_450_473P_F
T*Cp*TpAGTA
142
BONTA_X5206_517_539P_R
TAACCA*Tp*Tp*
857




ATAATAGGA*C


Tp*CpGCGTAAGA




pCp*Cp*Tp*


*Tp*Tp*CpAA




C


 


487
BONTA_X52066_591_620_F
TGAGTCACTTG
463
BONTA_X52066_644_671_R
TCATGTGCTAATG
992




AAGTTGATACA


TTACTGCTGGATC




AATCCTCT


TG


 


608
SSPE_BA_156_168P_F
TGGTpGCpTpA
616
SSPE_BA_243_255P_R
TGCpAGCpTGATp
1241




GCpATT


TpGT


 


609
SSPE_BA_75_89P_F
TACpAGAGTpT
192
SSPE_BA_163_177P_R
TGTGCTpTpTpGA
1338




pTpGCpGAC


ATpGCpT


 


610
SSPE_BA_150_168P_F
TGCTTCTGGTp
533
SSPE_BA_243_264P_R
TGATTGTTTTGCp
1191




GCpTpAGCpAT


AGCpTGATpTpGT




T


 


611
SSPE_BA_72_89P_F
TGGTACpAGAG
602
SSPE_BA_163_182P_R
TCATTTGTGCTpT
995




TpTpTpGCpGA


pTpGAATpGCpT




C


 


612
SSPE_BA_114_137P_F
TCAAGCAAACG
255
SSPE_BA_196_222P_R
TTGCACGTCpCpG
1401




CACAATpCpAG


TTTCAGTTGCAAA




AAGC


TTC


 


699
SSPE_BA_123_153_F
TGCACAATCAG
488
SSPE_BA_202_231_R
TTTCACAGCATGC
1431




AAGCTAAGAAA


ACGTCTGTTTCAG




GCGCAAGCT


TTGC


 


700
SSPE_BA_156_168_F
TGGTGCTAGCA
612
SSPE_BA_243_255_R
TGCAGCTGATTGT
1202




TT


 


701
SSPE_BA_75_89_F
TACAGAGTTTG
179
SSPE_BA_163_177_R
TGTGCTTTGAATG
1338




CGAC


CT


 


702
SSPE_BA_150_168_F
TGCTTTCTGGT
533
SSPE_BA_243_264_R
TGATTGTTTTGCA
1190




GCTAGCATT


GCTGATTGT


 


703
SSPE_BA_72_89_F
TGGTACAGAGT
600
SSPE_BA_163_182_R
TCATTTGTGCTTT
995




TTGCGAC


GAATGCT


 


704
SSPE_BA_146_168_F
TGCAAGCTTCT
484
SSPE_BA_242_267_R
TTGTGATTGTTTT
1421




GGTGCTAGCAT


GCAGCTGATTGTG




T


 


705
SSPE_BA_63_89_F
TGCTAGTTATG
518
SSPE_BA_163_191_R
TCATAACTAGCAT
986




GTACAGAGTTT


TTGTGCTTTGAAT




GCGAC


GCT


 


706
SSPE_BA_114_137_F
TCAAGCAAACG
255
SSPE_BA_196_222_R
TTGCACGTCTGTT
1402




CACAATCAGAA


TCAGTTGCAAAT




GC


C


 


770
PLA_AF053945_7377_7402_F
TGACATCCGGC
442
PLA_AF053945_7434_7462_R
TCTAAATTCCGCA
1313




TCACGTTATTA


AAGACTTTGGCAT




TGGT


TA


 


771
PLA_AF053945_7382_7404_F
TCCGGCTCACG
327
PLA_AF053945_7482_7502_R
TGGTCTGAGTACC
1304




TTATTATGGTA


TCCTTTGC




C


 


772
PLA_AF053945_7481_7503_F
TGCAAAGGAGG
481
PLA_AF053945_7539_7562_R
TATTGGAAATACC
943




TACTCAGACCA


GGCAGCATCTC




T


 


773
PLA_AF053945_7186_7211_F
TTATACCGGAA
657
PLA_AF053945_7257_7280_R
TAATGCGATACTG
879




ACTTCCCGAAA


GCCTGCAAGTC




GGAG


 


774
CAF1_AF053947_33407_33430_F
TCAGTTCCGTT
292
CAF1_AF053947_33494_33514_R
TGCGGGCTGGTTC
1235




ATCGCCATTGC


AACAAGAG




AT


 


775
CAF1_AF053947_33515_33541_F
TCACTCTTACA
270
CAF1_AF053947_33595_33621_R
TCCTGTTTTATAG
1053




TATAAGGAAGG


CCGCCAAGAGTAA




CGCTC


G


 


776
CAF1_AF053947_33435_33457_F
TGGAACTATTG
542
CAF1_AF053947_33499_33517_R
TGATGCGGGCTGG
1183




CAACTGCTAAT


TTCAAC




G


 


777
CAF1_AF053947_33687_33716_F
TCAGGATGGAA
286
CAF1_AF053947_33755_33782_R
TCAAGGTTCTCAC
962




ATAACCACCAA


CGTTTACCTTAGG




TTCACTAC


AG


 


778
INV_U22457_515_539_F
TGGCTCCTTGG
573
INV_U22457_571_598_R
TGTTAAGTGTGTT
1343




TATGACTCTGC


GCGGCTGTCTTTA




TTC


TT


 


779
INV_U22457_699_724_F
TGCTGAGGCCT
525
INV_U22457_753_776_R
TCACGCGACGAGT
976




GGACCGATTAT


GCCATCCATTG




TTAC


 


780
INV_U22457_834_858_F
TTATTTACCTG
664
INV_U22457_942_966_R
TGACCCAAAGCTG
1154




CACTCCCACAA


AAAGCTTTACTG




CTG


 


781
INV_U22457_1558_1581_F
TGGTAACAGAG
597
INV_U22457_1619_1643_R
TTGCGTTGCAGAT
1408




CCTTATAGGCG


TATCTTTACCAA




CA


 


782
LL_NC003143_2366996—
TGTACCCGCTAA
627
LL_NC003143_2367073_2367097_R
TCTCATCCCGATA
1123



2367019_F
AGCACTACCAT


TTACCGCCATGA




CC


 


783
LL_NC003143_2367172—
TGGACGGCATC
550
LL_NC003143_2367249_23672
TGGCAACAGCTCA
1272



2367194_F
ACGATTCTCTA


ACACCTTTGG




C


 


874
RPLB_EC_649_679_F
TGICCIACIGT
620
RPLB_EC_739_762_TMOD_R
TTCCAAGTGCTGG
1380




IIGIGGTTCTG


TTTACCCCATGG




TAATGAACC


 


875
RPLB_EC_642_679P_F
TpCpCpTpTpG
646
RPLB_EC_739_762_TMOD_R
TTCCAAGTGCTGG
1380




ITpGICCIACI


TTTACCCCATGG




GTIIGIGGTTC




TGTAATGAACC


 


876
MECIA_Y14051_3315_3341_F
TTACACATATC
653
MECIA_Y14051_3367_3393_R
TGTGATATGGAGG
1333




GTGAGCAATGA


TGTAGAAGGTGTT




ACTGA


A


 


877
MECA_Y14051_3774_3802_F
TAAAACAAACT
144
MECA_Y14051_3828_3854_R
TCCCAATCTAACT
1015




ACGGTAACATT


TCCACATACCATC




GATCGCA


T


 


878
MECA_Y14051_3645_3670_F
TGAAGTAGAAA
434
MECA_Y14051_3690_3719_R
TGATCCTGAATGT
1181




TGACTGAACGT


TTATATCTTTAAC




CCGA


GCCT


 


879
MECA_Y14051_4507_4530_F
TCAGGTACTGC
288
MECA_Y14051_4555_4581_R
TGGATAGACGTCA
1269




TATCCACCCTC


TATGAAGGTGTGC




AA


T


 


880
MECA_Y14051_4510_4530_F
TGTACTGCTAT
626
MECA_Y14051_4586_4610_R
TATTCTTCGTTAC
939




CCACCCTCAA


TCATGCCATACA


 


881
MECA_Y14051_4669_4698_F
TCACCAGGTTC
262
MECA_Y14051_4765_4793_R
TAACCACCCCAAG
858




AACTCAAAAAA


ATTTATCTTTTG




ATATTAACA


CCA


 


882
MECA_Y14051_4520_4530P_F
TCpCpACpCpC
389
MECA_Y14051_4590_4600P_R
TpACpTpCpATpG
1357




pTpCpAA


CpCpA


 


883
MECA_Y14051_4520_4530P_F
TCpCpACpCpC
389
MECA_Y14051_4600_4610P_R
TpATpTpCpTpTp
1358




pTpCpAA


CpGTpT


 


902
TRPE_AY094355_1467_1491_F
ATGTCGATTGC
36
TRPE_AY094355_1569_1592_R
TGCGCGAGCTTT
1231




AATCCGTACTT


TATTTGGGTTTC




GTG


 


903
TRPE_AY094355_1445_1471_F
TGGATGGCATG
557
TRPE_AY094355_1551_1580_R
TATTTGGGTTTCA
944




GTGAAATGGAT


TTCCACTCAGATT




ATGTC


CT


 


904
TRPE_AY094355_1278_1303_F
TCAAATGTACA
247
TRPE_AY094355_1392_1418_R
TCCTCTTTTCACA
1048




AGGTGAAGTGC


GGCTCTACTTCAT




GTGA


C


 


905
TRPE_AY094355_1064_1086_F
TCGACCTTTGG
357
TRPE_AY094355_1171_1196_R
TACATCGTTTCGC
885




CAGGAACTAGA


CCAAGATCAATCA




C


 


906
TRPE_AY094355_666_688_F
GTGCATGCGGA
135
TRPE_AY094355_769_791_R
TTCAAAATGCGGA
1372




TACAGAGCAGA


GGCGTATGTG


 


907
TRPE_AY094355_757_776_F
TGCAAGCGCGA
483
TRPE_AY094355_864_883_R
TGCCCAGGTACAA
1218




CCACATACG


CCTGCAT


 


908
RECA_AF251469_43_68_F
TGGTACATGTG
601
RECA_AF251469_140_163_R
TTCAAGTGCTTGC
1375




CCTTCATTGAT


TCACCATTGTC




GCTG


 


909
RECA_AF251469_169_190_F
TGACATGCTTG
446
RECA_AF251469_277_300_R
TGGCTCATAAGAC
1280




TCCGTTCAGGC


GCGCTTGTAGA


 


910
PARC_X95819_87_110_F
TGGTGACTCGG
609
PARC_X95819_201_222_R
TTCGGTATAACGC
1387




CATGTTATGAA


ATCGCAGCA




GC


 


911
PARC_X95819_87_110_F
TGGTGACTCGG
609
PARC_X95819_192_219_R
GGTATAACGCATG
836




CATGTTATGAA


GCAGCAAAAGATT




GC


TA


 


912
PARC_X95819_123_147_F
GGCTCAGCCAT
120
PARC_X95819_232_260_R
TCGCTCAGCAATA
1081




TTAGTTACCGC


ATTCACTATAAGC




TAT


CGA


 


913
PARC_X95819_43_63_F
TCAGCGCGTAC
277
PARC_X95819_143_170_R
TTCCCCTGACCTT
1383




AGTGGGTGAT


CGATTAAAGGATA







GC


 


914
OMPA_AY485227_272_301_F
TTACTCCATTA
655
OMPA_AY485227_364_388_R
GAGCTGCGCCAAC
812




TTGCTTGGTTA


GAATAAATCGTC




CACTTTCC


 


915
OMPA_AY485227_379_401_F
TGCGCAGCTCT
509
OMPA_AY485227_492_519_R
TGCCGTAACATAG
1223




TGGTATCGAGT


AAGTTACCGTTGA




T


T


 


916
OMPA_AY485227_311_335_F
TACACAACAAT
178
OMPA_AY485227_424_453_R
TACGTCGCCTTA
901




GGCGGTAAAGA


ACTTGGTTATATT




TGG


CAGC


 


917
OMPA_AY485227_415_441_F
TGCCTCGAAGC
506
OMPA_AY485227_514_546_R
TCGGGCGTAGTTT
1092




TGAATATAACC


TTAGTAATTAAAT




AAGTT


CAGAAGT


 


918
OMPA_AY485227_494_520_F
TCAACGGTAAC
252
OMPA_AY485227_569_596_R
TCGTCGTATTTAT
1108




TTCTATGTTAC


AGTGACCAGCACC




TTCTG


TA


 


919
OMPA_AY485227_551_577_F
TCAAGCCGTAC
257
OMPA_AY485227_658_680_R
TTTAAGCGCCAGA
1425




GTATTATTAGG


AAGCACCAAC




TGCTG


 


920
OMPA_AY485227_555_581_F
TCCGTACGTAT
328
OMPA_AY485227_635_662_R
TCAACACCAGCGT
954




TATTAGGTGCT


TACCTAAAGTACC




GGTCA


TT


 


921
OMPA_AY485227_556_583_F
TCGTACGTATT
379
OMPA_Y485227_659_683_R
TCGTTTAAGCGCC
1114




ATTAGGTGCTG


AGAAAGCACCAA




GTCACT


 


922
OMPA_AY485227_657_679_F
TGTTGGTGCTT
645
OMPA_AY485227_139_765_R
TAAGCCAGCAAGA
871




TCTGGCGCTTA


GCTGTATAGTTCC




A


A


 


923
OMPA_AY485227_660_683_F
TGGTGCTTTCT
613
OMPA_AY485227_786_807_R
TACAGGAGCAGCA
884




GGCGCTTAAAC


GGCTTCAAG




GA


 


924
GYRA_AF100557_4_23_F
TCTGCCCGTGT
402
GYRA_AF100557_119_142_R
TCGAACCGAAGTT
1063




CGTTGGTGA


ACCCTGACCAT


 


925
GYRA_AF100557_70_94_F
TCCATTGTTCG
316
GYRA_AF100557_178_201_R
TGCCAGCTTAGTC
1211




TATGGCTCAAG


ATACGGACTTC


 


926
GYRB_AB008700_19_40_F
TCAGGTGGCT
289
GYRB_AB008700_111_140_R
TATTGCGGATCAC
941




TACACGGCGT


CATGATGATATTC




AG


TTGC


 


927
GYRB_AB008700_265_292_F
TCTTTCTTGAA
420
GYRB_AB008700_369_395_R
TCGTTGAGATGGT
1113




TGCTGGTGTAC


TTTTACCTTCGT




GTATCG


TG


 


928
GYRB_AB008700_368_394_F
TCAACGAAGGT
251
GYRB_A3008700_466_494_R
TTTGTGAAACAGC
1440




AAAAACCATCT


GAACATTTTCTTG




CAACG


GTA


 


929
GYRB_AB008700_477_504_F
TGTTCGCTGTT
641
GYRB_AB008700_611_632_R
TCACGCGCATCAT
977




TCACAAACAAC


CACCAGTCA




ATTCCA


 


930
GYRB_AB008700_760_787_F
TACTTACTTGA
198
GYRB_AB008700_862_888_R
ACCTGCAATATCT
729




GAATCCACAAG


AATGCACTCTTAC




CTGCAA


G


 


931
WAAA_Z96925_2_29_F
TCTTGCTCTTT
416
WAAA_Z96925_115_138_R
CAAGCGGTTTGCC
758




CGTGAGTTCAG


TCAAATAGTCA




TAAATG


 


932
WAAA_Z96925_286_311_F
TCGATCTGGTT
360
WAAA_Z96925_394_412_R
TGGCACGAGCCTG
1274




TCATGCGTTT


ACCTGT


 


939
RPOB_EC_3798_3821_F
TGGGCAGCGTT
581
RPOB_EC_9862_3889_R
TGTCCGACTTGAC
1326




TCGGCGAAATG


GGTCAGCATTTCC




GA


TG


 


940
RPOB_EC_3798_3821_F
TGGGCAGCGTT
581
RPOB_EC_3862_3899_2_R
TGTCCGACTTGAC
1327




TCGGCGAAATG


GGTTAGCATTTCC




GA


TG


 


941
TUFB_EC_275_299_F
TGATCACTGGT
468
TUFB_EC_337_362_R
TGGATGTGCTCAC
1271




GCTGCTCAGAT


GAGTCTGTGGCAT




GGA


 


942
TUFB_EC_251_278_F
TGCACGCCGAC
493
TUFB_EC_337360_R
TATGTGCTCACGA
937




TATGTTAAGAA


GTTTGCGGCAT




CATGAT


 


949
GYRE_AB008700_760_787_F
TACTTACTTGA
198
GYRB_AB008700_862_888_2_R
TCCTGCAATATCT
1050




GAATCCACAAG


AATGCACTCTTAC




CTGCAA


G


 


958
RPOC_EC_2223_2243_F
TGGTATGCGTG
605
RPOC_EC_2329_2352_R
TGCTAGACCTTTA
1243




GTCTGATGGC


CGTGCACCGTG


 


959
RPOC_EC_918_938_F
TCTGGATAACG
404
RPOC_EC_1009_1031_R
TCCAGCAGGTTCT
1004




GTCGTCGCGG


GACGGAAACG


 


960
RPOC_EC_2334_2357_F
TGCTCGTAAGG
523
RPOC_EC_2380_2403_R
TACTAGACGACGG
905




GTCTGGCGGAT


GTCAGGTAACC




AC


 


961
RPOC_EC_917_938_F
TATTGGACAAC
242
RPOC_EC_1009_1034_R
TTACCGAGCAGGT
1362




GGTCGTCGCGG


TCTGACGGAAACG


 


962
RPOB_E2005_2027_F
TCGTTCCTGGA
387
RPOB_EC_2041_2064_R
TTGACGTTGCATG
1399




ACACGATGACG


TTCGAGCCCAT




C


 


963
RPOB_EC_1527_1549_F
TCAGCTGTCGC
282
RPOB_EC_1630_1649_R
TCGTCGCGGACTT
1104




AGTTCATGGAC


CGAAGCC




C


 


964
INFB_EC_1347_1367_F
TGCGTTTACCG
515
INFB_EC_1414_1432_R
TCGGCATCACGCC
1090




CAATGCGTGC


GTCGTC


 


965
VALS_EC_1128_1151_F
TATGCTGACCG
237
VALS_EC_1231_1257_R
TTCGCGCATCCAG
1384




ACCAGTGGTAC


GAGAAGTACATGT




GT


T


 


978
RPOC_EC_2145_2175_F
TCAGGAGTCGT
285
RPOC_EC_2228_2247_R
TTACGCCATCAGG
1363




TCAACTCGATC


CCACGCA




TACATGATG


 


1045
CJST_CJ_1668_1700_F
TGCTCGAGTGA
522
CJST_CJ_1774_1799_R
TGAGCGTGTGGAA
1170




TTGACTTTGCT


AAGGACTTGGATG




AAATTTAGAGA


 


1046
CJST_CJ_2171_2197_F
TCGTTTGGTGG
388
CJST_CJ_2283_2313_R
TCTCTTTCAAAGC
1126




TGGTAGATGAA


ACCATTGCTCATT




AAAGG


ATAGT


 


1047
CJST_CJ_584_616_F
TCCAGGACAAA
315
CJST_CJ_663_692_R
TTCATTTTCTGGT
1379




TGTATGAAAAA


CCAAAGTAAGCAG




TGTCCAAGAAG


TATC


 


1048
CJST_CJ_360_394_F
TCCTGTTATCC
346
CJST_CJ_442_476_R
TCAACTGGTTCAA
955




CTGAAGTAGTT


AAACATTAAGTTG




AATCAAGTTTG


TAATTGTCC




TT


 


1049
CJST_CJ_2636_2668_F
TGCCTAGAAGA
504
CJST_CJ_2753_2777_R
TTGCTGCCATAGC
1409




TCTTAAAAATT


AAAGCCTACAGC




TCCGCCAACTT


 


1050
CJST_CJ_1290_1320_F
TGGCTTATCCA
575
CJST_CJ_1406_1433_R
TTTGCTCATGATC
1437




AATTTAGATCG


TGCATGAAGCATA




TGGTTTTAC


AA


 


1051
CJST_CJ_3267_3293_F
TTTGATTTTAC
707
CJST_CJ_3356_3385_R
TCAAAGAACCCGC
951




GCCGTCCTCCA


ACCTAATTCATCA




GGTCG


TTTA


 


1052
CJST_CJ_5_39_F
TAGGCGAAGAT
222
CJST_CJ_104_137_R
TCCCTTATTTTTC
1029




ATACAAAGAGT


TTTCTACTACCTT




ATTAGAAGCT


CGGATAAT




AGA


 


1053
CJST_CJ_1080_1110_F
TTGAGGGTATG
681
CJST_CJ_1166_1198_R
TCCCTCATGTTT
1022




CACCGTCTTT


AAATGATCAGGAT




TTGATTCTTT


AAAAAGC


 


1054
CJST_CJ_2060_2090_F
TCCCGGACTTA
323
CJST_CJ_2148_2174_R
TCGATCCGCATCA
1068




ATATCAATGAA


CCATCAAAAGCAA




AATTGTGGA


A


 


1055
CJST_CJ_2869_2895_F
TGAAGCTTGTT
432
CJST_CJ_2979_3007_R
TCCTCCTTGTGCC
1045




CTTTAGCAGGA


TCAAAACGCATTT




CTTCA


TTA


 


1056
CJST_CJ_1880_1910_F
TCCCAATTAAT
317
CJST_CJ_1981_2011_R
TGGTTCTTACTTG
1309




TCTGCCATTTT


CTTTGCATAAACT




TCCAGGTAT


TTCCA


 


1057
CJST_CJ_2185_2212_F
TAGATGAAAAG
208
CJST_CJ_2283_2316_R
TGAATTCTTTCAA
1152




GGCGAAGTGGC


AGCACCATTGCTC




TAATGG


ATTATAGT


 


1058
CJST_CJ_1643_1670_F
TTATCGTTTGT
660
CJST_CJ_1724_1752_R
TGCAATGTGTGCT
1198




GGAGCTAGTGC


ATGTCAGCAAA




TTATGC


AAGAT


 


1059
CJST_CJ_2165_2194_F
TGCGGATCGTT
511
CJST_CJ_2247_2278_R
TCCACACTGGATT
1002




TGGTGGTTGTA


GTAATTTACCTTG




GATGAAAA


TTCTTT


 


1060
CJST_CJ_599_632_F
TGAAAAATGTC
424
CJST_CJ_711_743_R
TCCCGAACAATGA
1024




CAAGAAGCATA


GTTGTATCAACTA




GCAAAAAAAGC


TTTTTAC




A


 


1061
CJST_CJ_360_393_F
TCCTGTTATCC
345
CJST_CJ_443_477_R
TACAACTGGTTCA
882




CTGAAGTAGTT


AAAACATTAAGCT




AATCAAGTTTG


GTAATTGTC




T


 


1062
CJST_CJ_2678_2703_F
TCCCCAGGACA
321
CJST_CJ_2760_2787_R
TGTGCTTTTTTTG
1339




CCCTGAAATTT


CTGCCATAGCAAA




CAAC


GC


 


1063
CJST_CJ_1268_1299_F
AGTTATAAACA
29
CJST_CJ_1349_1379_R
TCGGTTTAAGCTC
1096




CGGCTTTCCTA


TACATGATCGTAA




TGGCTTATCC


GGATA


 


1064
CJST_CJ_1680_1713_F
TGATTTTGCTA
479
CJST_CJ_1795_1822_R
TATGTGTAGTTGA
938




AATTTAGAGAA


GCTTACTACATGA




ATTGCGGATGA


GC




A


 


1065
CJST_CJ_2857_2887_F
TGGCATTTCTT
565
CJST_CJ_2965_2998_R
TGCTTCAAAACGC
1253




ATGAAGCTTGT


ATTTTTACATTTT




TCTTTAGCA


CGTTAAAG


 


1070
RNASEP_BKM_580_599_F
TGCGGGTAGGG
512
RNASEP_BKM_665_686_R
TCCGATAAGCCGG
1034




AGCTTGAGC


ATTCTGTGC


 


1071
RNASEP_BKM_616_637_F
TCCTAGAGGAA
333
RNASEP_BKM_665_687_R
TGCCGATAAGCCC
1222




TGGCTGCCACG


GGATTCTGTGC


 


1072
RNASEP_BDP_574_592_F
TGGCACGGCCA
561
RNASEP_BDP_616_635_R
TCGTTTCACCCTG
1115




TCTCCGTG


TCATGCCG


 


1073
23S_BRM_1110_1129_F
TGCGCGGAAGA
510
23S_BRM_1176_1201_R
TCGCAGGCTTACA
1074




TGTAACGGG


GAACGCTCTCCTA


 


1074
23S_BRM_515_536_F
TGCATACAAAC
496
23S_BRM_616_635_R
TCGGACTCGCTTT
1088




AGTCGGAGCCT


CGCTACG


 


1075
RNASEP_CLB_459_487_F
TAAGGATAGTG
162
RNASEP_CLB_498_526_R
TGCTCTTACCTCA
1247




CAACAGAGATA


CCGTTCCACCCTT




TACCGCC


ACC


 


1076
RNASEP_CLB_459_487_F
TAAGGATAGTG
162
RNASEP_CLB_498_522_R
TTTACCTCGCCTT
1426




CAACAGAGATA


TCCACCCTTACC




TACCGCC


 


1077
ICD_CXB_93_120_F
TCCTGACCGAC
343
ICD_CXB_172_194_R
TAGGATTTTTCCA
921




CCATTATTCCC


CGGCGGCATC




TTTATC


 


1078
ICD_CXB_92_120_F
TTCCTGACCGA
671
ICD_CXB_172_194_R
TAGGATTTTTCCA
921




CCCATTATTCC


CGGCGGCATC




CTTTATC


 


1079
ICD_CXB_176_198_F
TCGCCGTGGAA
369
ICD_CXB_224_247_R
TAGCCTTTTCTCC
916




AAATCCTACGC


GGCGTAGATCT




T


 


1080
IS1111A_NC002971_6866—
TCAGTATGTAT
290
IS1111A_NC002971_6928_6954_R
TAAACGTCCGATA
848



6891_F
CCACCGTAGCC


CCAATGGTTCGCT




GTC


C


 


1081
IS1111A_NC002971_7456—
TGGGTGACATT
594
IS1111A_NC002971_7529_7554_R
TCAACAACACCTC
952



7483_F
CATCAATTTCA


CTTATTCCCACTC




TCGTTC


 


1082
RNASEP_RKP_419_448_F
TGGTAAGAGCG
599
RNASEP_RKP_542_565_R
TCAAGCGATCTAC
957




TGGTAACA


CCGCATTACAA


 


1083
RNASEP_RKP_422_443_F
TAAGAGCGCAC
159
RNASEP_RKP_542_565_R
TCAAGCGATCTAC
957




CGGTAAGTTGG


CCGCATTACAA


 


1084
RNASEP_RKP_466_491_F
TCCACCAAGAG
310
RNASEP_RKP_542_565_R
TCAAGCGATCTAC
957




CAAGATCAAAT


CCGCATTACAA




AGGC


 


1085
RNASEP_RKP_264_287_F
TCTAAATGGTC
391
RNASEP_RKP_295_321_R
TCTATAGAGTCCG
1119




GTGCAGTTGCG


GACTTTCCTCGTG




TG


A


 


1086
RNASEP_RKP_426_448_F
TGCATACCGGT
497
RNASEP_RKP_542_565_R
TCAAGCGATCTAC
957




AAGTTGGCAAC


CCGCATTACAA




A


 


1087
OMPB_RKP_860_890_F
TTACAGGAAGT
654
OMPB_RKP_972_996_R
TCCTGCAGCTCTA
1051




TTAGGTGGTAA


CCTGCTCCATTA




TCTAAAAGG


 


1088
OMPB_RKP_1192_1221_F
TCTACTGATTT
392
OMPB_RKP_1288_1315_R
TAGCAgCAAAGT
910




TGGTAATCTTG


TATCACACCTGCA




CAGCACAG


GT


 


1089
OMPB_RKP_3417_3440_F
TGCAAGTGGTA
485
OMPB_RKP_3520_3550_R
TGGTTGTAGTTCC
1310




CTTCAACATGG


TGTAGTTGTTGCA




GG


TTAAC


 


1090
GLTA_RKP_1043_1072_F
TGGGACTTGAA
576
GLTA_RKP_1138_1162_R
TGAACATTTGCGA
1147




GCTATCGCTCT


CGGTATACCCAT




TAAAGATG


 


1091
GLTA_RKP_400_428_F
TCTTCTCATCC
413
GLTA_RKP_499_529_R
TGGTGGGTATCTT
1305




TATGGCTATTA


AGCAATCATTCTA




TGCTTGC


ATAGC


 


1092
GLTA_RKP_1023_1055_F
TCCGTTCTTA
330
GLTA_RKP_1129_1156_R
TTGGCGACGGTAT
1415




AAATAGCAATA


ACCCATAGCTTTA




GAACTTGAAGC


TA


 


1093
GLTA_RKP_1043_1072_2_F
TGGAGCTTGAA
553
GLTA_RKP_1138_1162_R
TGAACATTGCGA
1147




GCTATCGCTCT


CGGTATACCCAT




AAAGATG


 


1094
GLTA_RKP_1043_1072_3_F
TGGAACTTGAA
543
GLTA_RKP_1138_1164_R
TGTGAACATTTGC
1330




GCTCTCGCTCT


GACGGTATACCCA




TAAAGATG


T


 


1095
GLTA_RKP_400_428_F
TCTTCTCATCC
413
GLTA_RKP_505_534_R
TGCGATGGTAGGT
1230




TATGGCTATTA


ATCTTAGCAATCA




TGCTTGC


TTCT


 


1096
CTXA_VBC_117_142_F
TCTTATGCCAA
410
CTXA_VBC_194_218_R
TGCCTAACAAATC
1226




GAGGACAGAGT


CCGTCTGAGTTC




GAGT


 


1097
CTXA_VBC_351_377_F
TGTATTAGGGG
630
CTXA_VBC_441_466_R
TGTCATCAAGCAC
1324




CATACAGTCCT


CCAAAATGAACT




CATCC


 


1098
RNASEP_VBC_331_349_F
TCCGCGGAGTT
325
RNASEP_VBC_388_414_R
TGACTTTCCTCCC
1163




GACTGGGT


CCTTATCAGTCTC







C


 


1099
TOXR_VBC_135_158_F
TCGATTAGGCA
362
TOXR_VBC_221_246_R
TTCAAAACCTTGC
1370




GCAACGAAAGC


TCTCGCCAAACAA




CG


 


1100
ASD_FRT_1_29_F
TTGCTTAAAGT
690
ASD_FRT_86_116_R
TGAGATGTCGAAA
1164




TGGTTTTATTG


AAAACGTTGGCAA




GTTGGCG


AATAC


 


1101
ASD_FRT_43_76_F
TCAGTTTTAAT
295
ASD_FRT_129_156_R
TCCATATTGTTGC
1009




GTCTCGTATGA


ATAAAACCTGTTG




TCGAATCAAAA


GC




G


 


1102
GALE_FRT_168_199_F
TTATCAGCTAG
658
GALE_FRT_241_269_R
TCACCTACAGCTT
973




ACCTTTTAGGT


TAAAGCCAGCAAA




AAAGCTAAGC


ATG


 


1103
GALE_FRT_834_865_F
TCAAAAAGCCC
245
GALE_FRT_901_925_R
TAGCCTTGGCAAC
915




TAGGTAAAGAG


ATCAGCAAAACT




ATTCCATATC


 


1104
GALE_FRT_308_339_F
TCCAAGGTACA
306
GALE_FRT_390_422_R
TCTTCTGTAAAGG
1136




CTAAACTTACT


GTGGTTTATTATT




TGAGCTAATG


CATCCCA


 


1105
IPAH_SGF_258_277_F
TGAGGACCGTG
458
IPAH_SGF_301_327_R
TCCTTCTGATGCC
1055




TCGCGCTCA


TGATGGACCAGGA







G


 


1106
IPAH_SGF_113_134_F
TCCTTGACCGC
350
IPAH_SGF_172_191_R
TTTTCCAGCCATG
1441




CTTTCCGATAC


CAGCGAC


 


1107
IPAH_SGF_462_486_F
TCAGACCATGC
271
IPAH_SGF_522_540_R
TGTCACTCCCGAC
1322




TCGCAGAGAAA


ACGCCA




CTT


 


1111
RNASEP_BRM_461_488_F
TAAACCCCATC
147
RNASEP_BRM_542_561_R
TGCCTCGCGCAAC
1227




GGGAGCAAGAC


CTACCCG




CGAATA


 


1112
RNASEP_BRM_325_347_F
TACCCCAGGGA
185
RNASEP_BRM_402_428_R
TCTCTTACCCCAC
1125




AAGTGCCACAG


CCTTTCACCCTTA




A


C


 


1128
HUPB_CJ_113_134_F
TAGTTGCTCAA
230
HUPB_CJ_157_188_R
TCCCTAATAGTAG
1028




ACAGCTGGGCT


AAATAACTGCATC







AGTAGC


 


1129
HUPB_CJ_76_102_F
TCCCGGAGCTT
324
HUPB_CJ_157_188_R
TCCCTAATAGTAG
1028




TTATGACTAAA


AAATAACTGCATC




GCAGAT


AGTAGC


 


1130
HUPB_CJ_102_F
TCCCGGAGCTT
324
HUPB_CJ_114_135_R
TAGCCCAGCTGTT
913




TTATGACTAAA


TGAGCAACT




GCAGAT


 


1151
AB_MLST-11-
TGAGATTGCTG
454
AB_MLST-11-
TTGTACATTTGAA
1418



OIF007_62_91_F
AACATTTAATG

OIF007_169_203_R
ACAATATGCATGA




CTGATTGA


CATGTGAAT


 


1152
AB_MLST-11-
TATTGTTTCAA
243
AB_MLST-11-
TCACAGGTTCTAC
969



OIF007_185_214_F
ATGTACAAGGT

OIF007_291_324_R
TTCATCAATAATT




GAAGTGCG


TCCATTGC


 


1153
AB_MLST-11-
TGGAACGTTAT
541
AB_MLST-11-
TTGCAATCGACAT
1400



OIF007_260_289_F
CAGGTGCCCCA

OIF007_364_393_R
ATCCATTTCACCA




AAATTCG


TGCC


 


1154
AB_MLST-11-
TGAAGTGCGTG
436
AB_MLST-11-
TCCGCCAAAAACT
1036



OIF007_206_239_F
ATGATATCGAT

OIF007_318_344_R
CCCCTTTTCACAG




GCACTTGATGT


G




A


 


1155
AB_MLST-11-
TCGGTTTAGTA
378
AB_MLST-11-
TTCTGCTTGAGGA
1392



OIF007_522_552_F
AAAGAACGTAT

OIF007_587_610_R
ATAGTGCGTGG




TGCTCAACC


 


1156
AB_MLST-11-
TCAACCTGACT
250
AB_MLST-11-
TACGTTCTACGAT
902



OIF007_547_571_F
GCGTGAATGGT

OIF007_656_686_R
TTCTTCATCAGGT




TGT


ACATC


 


1157
AB_MLST-11-
TCAAGCAGAAG
256
AB_MLST-11-
TACAACGTGATAA
881



OIF007_601_627_F
CTTTGGAAGAA

OIF007_710_736_R
ACACGACCAGAAG




GAAGG


C


 


1158
AB_MLST-11-
TCGTGCCCGCA
384
AB_MLST-11-
TAATGCCGGGTAG
878



OIF007_1202_1225_F
ATTGCATAAA

OIF007_1266_1296_R
TGCAATCCATTCT




GC


TCTAG


 


1159
AB_MLST-11-
TCGTCCCGCA
384
AB_MLST-11-
TGCACCTGCGGTC
1199



OIG007_1202_1225_F
ATTTGCATAAA

OIF007_1299_1316_R
GAGCG




GC


 


1160
AB_MLST-11-
TTGTAGCACAG
694
AB_MLST-11-
TGCCATCCATAAT
1215



OIF007_1234_1264_F
TCCTGAAAC

OIF007_1335_1362_R
CACGCCATACTGA







CG


 


1161
AB_MLST-11-
TAGGTTTACGT
225
AB_MLST-11-
TGCCAGTTTCCAC
1212



OIF007_1327_1356_F
GATTATGG

OIF007_1422_1448_R
ATTTCACGTTCGT







G


 


1162
AB_MLST-11-
TCGTGATTATG
383
AB_MLST-11-
TCGCTTGAGTGTA
1083



OIF007_1345_1369_F
AA

OIF007_1470_1494_R
GTCATGATTGCG


 


1163
AB_MLST-11-
TTATGGATGGC
662
AB_MLST-11-
TCGCTTGAGTGTA
1083



OIF007_1351_1375_F
GT

OIF007_1470_1494_R
GTCATGATTGCG


 


1164
AB_MLST-11-
TCTTTGCCATT
422
AB_MLST-11-
TCGCTTGAGTGTA
1083



OIF007_1387_1412_F
GAAGATGACTT

OIF007_1470_1494_R
GTCATAGATTGCG




AAGC


 


1165
AB_MLST-11-
TACTAGCGGTA
194
AB_MLST-11-
TGAGTCGGGTTCA
1173



OIF007_1542_1569_F
AGCTTAAACAA

OIF007_1656_1680_R
CTTACCTGGCA




GATTGC


 


1166
AB_MLST-11-
TTGCCAATGAT
684
AB_MLST-11-
TGAGTCGGGTTCA
1173



OIF007_1566_1593_F
ATTCGTTGGTT

OIF007_1656_1680_R
CTTTACCTGGCA




AGCAAG


 


1167
AB_MLST-11-
TCGGCGAAATC
375
AB_MLST-11-
TACCGGAAGCACC
890



OIF007_1611_1638_F
CGTATTCCTGA

OIF007_1731_1757_R
AGCGACATTAATA




AAATGA


G


 


1168
AB_MLST-11-
TACCACTATTA
182
AB_MLST-11-
TGCAACTGAATAG
1195



OIF007_1726_1752_F
ATGTCGCTGGT

OIF007_1790_1821_R
ATTGCAGTAAGTT




GCTTC


ATAAGC


 


1169
AB_MLST-11-
TTATAACTTAC
656
AB_MLST-11-
TGAATTATGCAAG
1151



OIF007_1792_1826_F
TGCAATCTATT

OIF007_1876_1909_R
AAGTGATCAATTT




CAGTTGCTG


TCTCACGA




GTG


 


1170
AB_MLST-11-
TTATAACTTAC
656
AB_MLST-11-
TGCCGTAACTAAC
1224



OIF007_1792_1826_F
TGCAATCTATT

OIF007_1895_1927_R
ATAAGAGAATTAT




CAGTTGCTTGG

GCAAGAA




TG


 


1171
AB_MLST-11-
TGGTTATGTAC
618
AB_MLST-11-
TGACGGCATCGA
1157



OIF007_1970_2002_F
CAAATACTTTG

OIF007_2097_2118_R
ACCACCGTC




TCTGAAGAGG


 


1172
RNASEP_BRM_461_488_F
TAAACCCCATC
147
RNASEP_BRM_542_561_2_R
TGCCTCGTGCAAC
1228




GGGAGCAAGAC


CCACCCG




CGAATA


 


2000
CTXB_NC002505_46_70_F
TCAGCGTATGC
278
CTSB_NC002505_132_162_R
TCCGGCTAGAGAT
1039




ACATGGAACTC


TCTGTATACGAC




CTC


AATATC


 


2001
FUR_NC002505_87_113_F
TGAGTGCCAAC
465
FUR_NC002505_205_228_R
TCCGCCTTCAAAA
1037




ATATCAGTGCT


TGGTGGCGAGT




GAAGA


 


2002
FUR_NC002505_87_113_F
TGAGTGCCAAC
465
FUR_NC002505_178_205_R
TCACGATACCTGC
974




ATATCAGTGCT


ATCATCAAATTG




GAAGA


GTT


 


2003
GAPA_NC002505_533_560_F
TCGACAACACC
356
GAPA_NC002505_646_671_R
TCAGAATCGATGC
980




ATTATCTATGG


CAAATGCGTCATC


 


2004
GAPA_NC002505_505_19_721_F
TCAATGAACGA
259
GAPA_NC002505_769_798_R
TCCTCTATGCAAC
1046




CCAACAAGTGA


TTAGTATCAACA




TTGATG


GAAT


 


2005
GAPA_NC002505_753_782_F
TGCTAGTCAAT
517
GAPA_NC002505_856_881_R
TCCATCGCAGTCA
1011




CTATCATTCCG


CGTTTACTGTTGG




GTTGATAC


 


2006
GYRB_NC002505_2_32_F
TGCCGGACAAT
501
GYRB_NC002505_109_134_R
TCCACCACCTCAA
1003




TACGATTCATC


AGACCATGTGGTG




GAGTATTAA


 


2007
GYRB_NC002505_123_152_F
TGAGGTGGTGG
460
GYRB_NC002505_199_225_R
TCCGTCATCGCTG
1042




ATAACTCAATT


ACAGAAACTGAGT




GATGAAGC


T


 


2008
GYRB_NC002505_768_794_F
TATGCAGTGGA
236
GYRB_NC002505_832_860_R
TGGAAACCGGCTA
1262




ACGATGGTTTC


AGTGAGTACCACC




CAAGA


ATC


 


2009
GYRB_NC002505_837_860_F
TGGTACTCACT
603
GYRB_NC002505_937_957_R
TCCTTCACGCGCA
1054




TAGCGGGTTT


TCATCACC




CG


 


2010
GYRB_NC002505_934_956_F
TCGGGTGATGA
377
GYRB_NC002505_982_1007_R
TGGCTTGAGAATT
1283




TGCGCGTGAAG


TAGGATCCGGCAC




G


 


2011
GYRB_NC002505_1161_1190_F
TAAAGCCCGTG
148
GYRB_NC002505_1255_1284_R
TGAGTCACCCTCC
1172




AAATGACTCGT


ACAATGTATAGTT




CGTAAAGG


CAGA


 


2012
OMPU_NC002505_275_110_F
TACGCTGACGG
190
OMPU_NC002505_154_180_R
TGCTTCAGCACGG
1254




AATCAACCAAA


CCACCAACTTCTA




GCGG


G


 


2013
OMPU_NC002505_258_283_F
TGACGGCCTAT
451
OMPU_NC002505_346_369_R
TCCGAGACCAGCG
1033




ACGGTGTTGGT


TAGGTGTAACG




TTCT


 


2014
OMPU_NC002505_431_455_F
TCACCGATATC
266
OMPU_NC002505_544_567_R
TCGGTCAGCAAAA
1094




ATGGCTTACCA


CGGTAGCTTGC




CGG


 


2015
OMPU_NC002505_533_557_F
TAGGCGTGAAA
223
OMPU_NC002505_625_651_R
TAGAGAGTAGCCA
908




GCAAGCTACCG


TCTTCACCGTTGT




TTT


C


 


2016
OMPU_NC002505_689_713_F
TAGGTGCTGGT
224
OMPU_NC002505_725_751_R
TGGGGTAAGACGC
1291




TACGCAGATCA


GGCTAGCATGTAT




AGA


T


 


2017
OMPU_NC002505_727_747_F
TACATGCTAGC
181
OMPU_NC002505_811_835_R
TAGCAGCTAGCTC
911




CGCGTCTTAC


GTAACCAGTGTA


 


2018
OMPU_NC002505_931_953_F
TACTACTTCAA
193
OMPU_NC002505_1033_1053_R
TTAGAAGTCGTAA
1368




GCCGAACTTCC


CGTGGACC




G


 


2019
OMPU_NC002505_927_953_F
TACTTACTACT
197
OMPU_NC002505_1033_1054_R
TGGTTAGAAGTCG
1307




TCAAGCCGAAC


TAACGTGGACC




TTCCG


 


2020
TCPA_NC002505_48_73_F
TCACGATAAGA
269
TCPA_NC002505_148_170_R
TTCTGCGAATCAA
1391




AAACCGGTCAA


TCGCACGCTG




GAGG


 


2021
TDH_NC004605_265_289_F
TGGCTGACATC
574
TDH_NC004605_357_386_R
TGTTGAAGCTGTA
1351




CTACATGACTG


CTTGACCTGATTT




TGA


TACG


 


2022
VVHA_NC004460_772_802_F
TCTTATTCCAA
412
VVHA_NC004460_862_886_R
TACCAAAGCGTGC
887




CTTCAAACCGA


ACGATAGTTGAG




ACTATGACG


 


2023
23S_EC_2643_2667_F
TGCCTGTTCTT
508
23S_EC_2746_2770_R
TGGGTTTCGCGCT
1297




AGTACGAGAGG


AGATGCTTTCA




ACC


 


2024
16S_EC_713_732_TMOD_F
TAGAACACCCG
202
16S_EC_789_811_R
TGCGTGGACTAC
1240




ATGGCGAAGGC


AGGGTATCTA


 


2025
16S_EC_784_806_F
TGGATTAGAGA
560
16S_EC_880_897_TMOD_R
TGGCCGTACTCCC
1278




CCCTGGTAGTC


CAGGCG




C


 


2026
16S_EC_959_981_F
TGTCGATGCAA
634
16S_EC_1052_1074_R
TACGAGCTGACGA
896




CGCGAAGAACC


CAGCCATGCA




T


 


2027
TUFB_EC_956_979_F
TGCACACGCCG
489
TUFB_EC_1034_1058_2_R
TGCATCACCATTT
1204




TTCTTCAACAA


CCTTGTCCTTCG




CT


 


2028
RPOC_EC_2146_2174_TMOD_F
TCAGGAGTCGT
284
RPOC_EC_2227_2249_R
TGCTAGGCCATCA
1244




TCAACTCGATC


GGCCACGCAT




TACATGAT


 


2029
RPOB_EC_1841_1866_F
TGGTTATCGCT
617
RPOB_EC_1909_1929_TMOD_R
TGCTGGATTCGCC
1250




CAGGCGAACTC


TTTGCTACG




CAAC


 


2030
RPLB_EC_650_679_TMOD_F
TGACCTACAGT
449
RPLB_EC_739_763_R
TGCCAAGTGCTGG
1208




AAGAGGTTCTG


TTTACCCCATGG




AATGAACC


 


2031
RPLB_EC_690_710_F
TCCACACGGTG
309
RPLB_EC_737_760_R
TGGGTGCTGGTTT
1295




GTGGTGAAGG


ACCCCATGGAG


 


2032
INFB_EC_1366_1393_F
TCTCGTGGTGC
397
INFB_EC_1439_1469_R
TGTGCTGCTTTCG
1335




ACAAGTAACGG


CATGGTTAATTGC




ATATTA


TTCAA


 


2033
VALS_EC_1105_1124_TMOD_F
TCGTGGCGGCG
385
VALS_EC_1195_1219_R
TGGGTACGAACTG
1292




TGGTTATCGA


GATGTCGCCGTT


 


2034
SSPE_BA_113_137_F
TGCAAGCAAAC
482
SSPE_BA_197_222_TMOD_R
TTGCACGTCTGTT
1402




GCACAATCAGA


TCAGTTGCAAATT




AGC


C


 


2035
RPOC_EC_2218_2241_TMOD_F
TCTGGCAGGTA
405
RPOC_EC_2313_2338_R
TGGCACCGTGGGT
1273




TGCGTTGTCTG


TGAGATGAAGTAC




ATG


 


2056
MECI-NC003923-
TTTACACATAT
698
MECI-NC003923-41798-
TTGTGATATGGAG
1420



41798-41609_33_60_F
CGTGAGCAATG

41609_86_113_R
GTGTAGAAGGTGT




AACTGA


TA


 


2057
AGR-III_NC003923-
TCACCAGTTTG
263
AGR-III_NC003923-
ACCTGCATCCCTA
730



2108074-
CCACGTATCTT

2108074-
AACGTACTTGC



2109508_1_23_F
CAA

2109507_56_79_R


 


2058
AGR-III_NC003923-
TGAGCTTTTAG
457
AGR-III_NC003923-
TACTTCAGCTTCG
906



2108074-
TTGACTTTTTC

2108074-
TCCAATAAAAAAT



2109507_569_596_F
AACAGC

2109507_622_653_R
CACAAT


 


2059
AGR-III_NC003923-
TTTCACACAGC
701
AGR-III_NC003923-
TGTAGGCAAGTGC
1319



2108074-
GTGTTTATAGT

2108074-
ATAAGAAATTGAT



2109507_1024_1052_F
TCTACCA

2109507_1070_1098_R
ACA


 


2060
AGR-
TGGTGACTTCA
610
AGR-
TCCCCATTAATAA
1021



I_AJ617706_622_651_F
TAATGGATGAA

I_AJ617706_694_726_R
TTCCACCTACTAT




GTTGAAGT


CACACT


 


2061
AGR-
TGGGATTTTAA
579
AGR-
TGGTACTTCAACT
1302



I_AJ617706_580_611_F
AAAACATTGGT

I_AJ617706_626_655_R
TCATCCATTATGA




AACATCGCAG


AGTC


 


2062
AGR-II_NC002745-
TCTTGCAGCAG
415
AGR-II_NC002745-
TTGTTTATTGTTT
1424



2079448-
TTTATTTGATG

2079448-
CCATATGCTACAC



2080879_620_651_F
AACCTAAAGT

2080879_700_731_R
ACTTTC


 


2063
AGR-II_NC002745-
TGTACCCGCTG
624
AGR-II_NC002745-
TCGCCATAGCTAA
1077



2079448-
AATTAACGAAT

2079448-
GTTGTTTATTGTT



2080879_649_679_F
TTATACGAC

2080879_715_745_R
TCCAT


 


2064
AGR-
TGGTATTCTAT
606
AGR-
TGCGCTATCAACG
1233



IV_AJ617711_931_961_F
TTTGCTGATAA

IV_AJ617711_1004_1035_R
ATTTTGACAATAT




TGACCTCGC


ATGTGA


 


2065
AGR-
TGGCACTCTTG
562
AGR-
TCCCATACCTATG
1017



IV_AJ617711_250_283_F
CCTTTAATATT

IV_AJ617711_309_335_R
GCGATAACTGTCA




AGTAAACTATC


T




A


 


2066
BLAZ_NC002952
TCCACTTATCG
312
BLAZ_NC002952
TGGCCACTTTTAT
1277



(1913827 . . .
CAAATGGAAAA

(1913827 . . .
CAGCAACCTTACA



1914672)_68_68_F
TTAAGCAA

1914672)_68_68_R
GTC


 


2067
BLAZ_NC002952
TGCACTTATCG
494
BLAZ_NC002952
TAGTCTTTTGGAA
926



(1913827 . . .
CAAATGGAAAA

(1913827 . . .
CACCGTCTTTAAT



1914672)_68_68_2_F

1914672)_68_68_2_R
TAAAGT


 


2068
BLAZ_NC002952
TGATACTTCAA
467
BLAZ_NC002952
TGGAACACCGTCT
1263



(1913827 . . .
CGCCTGCTGCT

(1913827 . . .
TTAATTAAAGTAT



1914672)_68_68_3_F
TTC

1914672_68_68_3_F
CTCC


 


2069
BLAZ_NC002952
TATACTTCAAC
232
BLAZ_NC002952
TCTTTTCTTTGCT
1145



(1913827 . . .
GCCTGCTGCTT

(1913827 . . .
TAATTTTCCATTT



1914672)_68_68_4_F
TC

1914672)_68_68_4_R
GCGAT


 


2070
BLAZ_NC002952
TGCAATTGCTT
487
BLAZ_NC002952
TTACTTCCTTACC
1366



(1913827 . . .
TAGTTTTAAGT

(1913827 . . .
ACTTTTAGTATCT



1914672)_1_33_F
GCATGTAATTC

1914672)_34_67_R
AAAGCATA


 


2071
BLAZ_NC002952
TCCTTGCTTTA
351
BLAZ_NC002952
TGGGGACTTCCTT
1289



(1913827 . . .
GTTTTAAGTGC

(1913827 . . .
ACCACTTTTAGTA



1914672)_3_34_F
ATGTAATTCAA

1914672)_40_68_R
TCTAA


 


2072
BSA-A_NC003923-
TAGCGAATGTG
214
BSA-A_NC003923-
TGCAAGGGAAACC
1197



1304065-
GCTTTACTTCA

1304065-
TAGAATTACAAAC



1303589_99_125_F
CAATT

1303589_165_193_R


 


2073
BSA-A_NC003923-
ATCAATTTGGT
32
BSA-A_NC003923-
TGCATAGGAAGG
1203



1304065-
GGCCAAGAAC

1304065-
TAACACCATAGTT



1303589_194_218_F
CTGG

1303589_253_278_R


 


2074
BSA-A_NC003923-
TTGACTGCGGC
679
BSA-A_NC003923-
TAACAACGTTACC
856



1304065-
ACAACACGGAT

1304065-
TTCGCGATCCACT



1303589_328_349_F


1303589_388_415_R


 


2075
BSA-A_NC003923-
TGCTATGGTGT
519
BSA-A_NC003923-
TGTTGTGCCGCAG
1353



1304065-
TACCTTCCCTA

1304065-
TCAAATATCTAAAT



1303589_253_278_F
TGCA

1303589_317_344_R


 


2076
BSA-B_NC003923-
TAGCAACAAAT
209
BSA-B_NC003923-
TGTGAAGAACTTT
1331



1917149-
ATATCTGAAGC

1917149-
CAAATCTGTGAAT



1914156_953_982_F
AGCGTACT

1914156_1011_1039_R
CCA


 


2077
BSA-B_NC003923-
TGAAAAGTATG
426
BSA-B_NC003923-
TCTTCTTGAAAAA
1138



1917149-
GATTTGAACAA

1917149-
TTGTTGTCCCGAA



1914156_1050_1081_F
CTCGTGAATA

1914156_1109_1136_R
AC


 


2078
BSA-B_NC003923-
TCATTATCATG
300
BSA-B_NC003923-
TGGACTAATAACA
1267



1917149-
CGCCAATGAGT

1917149-
ATGAGCTCATTGT



1914156_1260_1286_F
GCAGA

1914156_1323_1353_R
ACTGA


 


2079
BSA-B_NC003923-
TTTCATCTTAT
703
BSA-B_NC003923-
TGAATATGTAATG
1148



1917149-
CGAGGACCCGA

1917149-
CAAACCAGTCTTT



1914156_2126_2153_F
ATCGA

1914156_2186_2216_R
GTCAT


 


2080
ERMA_NC002952-
TCGCTATCTTA
372
ERMA_NC002952-
TGAGTCTACACTT
1174



55890-
TCGTTGAGAAG

55890-
AGGCTTAGGATGA



56621_366_392_F
GGATT

56621_487_513_R
A


 


2081
ERMA_NC002952-
TAGCTATCTTA
217
ERMA_NC002952-
TGAGCATTTTTAT
1167



55890-
TCGTTGAGAAG

55890-
ATCCATCTCCACC



56621_366_395_F
GGATTTGC

56621_438_465_R
AT


 


2082
ERMA_NC002952-
TGATCGTTGAG
470
ERMA_NC002952-
TCTTGGCTTAGGA
1143



55890-
AAGGGATTTGC

55890-
TGAAAATATAGTG



56621_374_402_F
GAAAAGA

56621_473_504_R
GTGGTA


 


2083
ERMA_NC002952-
TGCAAAATCTG
480
ERMA_NC002952-
TCAATACAGAGTC
964



55890-
CAACGAGCTTT

55890-
TACACTTGGCTTA



56621_404_427_F
GG

56621_491_520_R
GGAT


 


2084
ERMA_NC002952-
TCATCCTAAGC
297
ERMA_NC002952-
TGGACGATATTCA
1266



55890-
CAAGTGTAGAC
55890-
CGGTTTACCCACT



56621_489_516_F
TCTGTA

56621_586_615_R
TATA


 


2085
ERMA_NC002952-
TATAAGTGGGT
231
ERMA_NC002952-
TTGACATTTGCA
1397



55890-
AAACCGTGAAT

55890-
TGCTTCAAAGCCT



56621_586_614_F
ATCGTGT

56621_640_665_R
G


 


2086
ERMC_NC005908-
TCTGAACATGA
399
ERMC_NC005908-
TCCGTAGTTTTG
1041



2004-
TAATATCTTTG

2004-
CATAATTATGGT



2738_85_116_F
AAATCGGCTC

2738_173_206_R
CTATTTCAA


 


2087
ERMC_NC005908-
TCATGATAATA
298
ERMC_NC005908-
TTTATGGTCTAT
1429



2004-
TCTTTGAAATC

2004-
TTCAATGGCAGTT



2738_90_120_F
GGCTCAGGA

2738_460_189_R
ACGAA


 


2088
ERMC_NC005908-
TCAGGAAAAGG
283
ERMC_NC005908-
TATGGTCTATTT
936



2004-
GCATTTTACCC

2004-
CAATGGCAGTTAC



2738_115_139_F
TTG

2738_161_187_R
GA


 


2089
ERMC_NC005908-
TAATCGTGGAA
168
ERMC_NC005908-
TCAACTTCTGCC
956



2004-
TACGGGTTTGC

2004-
ATTAAAAGTAATG



2738_374_397_F
TA

2738_325_452_R
CCA


 


2090
ERMC_NC005908-
TCTTTGAAATC
421
ERMC_NC005908-
TGATGGTCTATT
1185



2004-
GGCTCAGGAAA

2004-
TCAATGGCAGTTA



2738_101_125_F
AGG

2738_159_188_R
CGAAA


 


2091
ERMB_Y13600-625-
TGTTGGGAGTA
644
ERMB_Y13600-625-
TCAACAATCAGA
953



1362_291_321_F
TTCCTTACCAT

1362_352_380_R
TAGATGTCAGACG




TTAAGCACA


CATG


 


2092
ERMB_Y13600-625-
TGGAAAGCCAT
536
ERMB_Y13600-625-
TGCAAGAGCAAC
1196



1362_344_367_F
GCGTCTGACAT

1362_415_437_R
CCTAGTGTTCG


 


2093
ERMB_Y13600-625-
TGGATATTCAC
556
ERMB_Y13600-625-
TAGGATGAAAGC
919



1363_404_429_F
CGAACACTAGG

1362_471_493_R
ATTCCGCTGGC


 


2094
ERMB_Y13600-625-
TAAGCTGCCAG
161
ERMB_Y13600-625-
TCATTCTGTGGTA
989



1362_465_487_F
CGGAATGCTTT

1362_521_545_R
TGGCGGGTAAGTT




C


 


2095
PVLUK_NC003923-
TGAGCTGCATC
456
PVLUK_NC003923-
TGGAAAACTCAT
1261



1529595-
ACTGTATTGGA

1529595-
GAAATTAAAGTGA



1531285_688_713_F
TAG

1531285_775_804_R
AAGGA


 


2096
PVLUK_NC003923-
TGGAACAAAAT
539
PVLUK_NC003923-
TCATTAGGTAAAA
993



1529595-
AGTCTCTCGGA

1529595-
TGTCTGGACATG



1531285_1039_1068_F
TTTTGACT

1531285_1095_1125_R
ATCCAA


 


2097
PVLUK_NC003923-
TGAGTAACATC
461
PVLUK_NC003923-
TCTCATGAAAAAG
1124



1529595-
CATATTTCTGC

1529595-
GCTCAGGAGATAC



1531285_908_936_F
CATACGT

1531285_950_978_R
AAG


 


2098
PVLUK_NC003923-
TCGGAATCTGA
373
PVLUK_NC003923-
TCACACCTGTAAG
968



1529595-
TGTTCAGTTGT

1529595-
TGAGAAAAAGGTT



1531285_610_633_F
TT

1531285_654_682_R
TGAT


 


2099
SA442_NC003923-
TGTCGGTACAC
635
SA442_NC003923-
TTTCCGATCAAC
1433



2538576-
GATATTCTTCA

2538576-
GTAATGAGATTTC



25388311_35_F
CGA

2538831_98_124_R
A


 


2100
SA442_NC003923-
TGAAATCTCAT
427
SA442_NC003923-
TCGTATGACCAGC
1098



2538576-
TACGTTGCATC

2538576-
TTCGGTACTACTA



2538831_98_124_F
G

2538831_163_188_R


 


2101
SA442_NC003923-
TCTCATTACGT
395
SA442_NC003923-
TTTATGACCAGCT
1428



2538576-
TGCATCGGAAA

2538576-
TCGGTACTACTAA



2538831_103_126_F
CA

2538831_161_187_R
A


 


2102
SA442_NC003923-
TAGTACCGAAG
226
SA442_NC003923-
TGATAATGAAGGG
1179



2538576-
CTGGTCATACG

2538576-
AAACCTTTTTCAC



2538831_166_188_F
A


G







2538831_231_257_R


 


2103
SEA_NC003923-
TGCAGGGAACA
495
SEA_NC003923-
TCGATCGTGACTC
1070



2052219-
GCTTTAGGCA

2052219-
TCTTTATTTTCAG



2051456_115_135_F


2051456_173_200_R
TT


 


2104
SEA_NC003923-
TAACTCTGATG
156
SEA_NC003923-
TGTAATTAACCGA
1315



2052219-
TTTTTGATGGG

2052219-
AGGTTCTGTAGA



2051456_572_598_F
AAGGT

2051456_621_651_R
GTATG


 


2105
SEA_NC003923-
TGTATGGTGGT
629
SEA_NC003923-
TAACCGTTTCCA
861



2052219-
GTAACGTTACA

2052219-
AGGTACTGTATTT



2051456_382_414_F
TGATAATAAT

2051456_464_492_R
TGT




C


 


2106
SEA_NC003923-
TTGTATGTATG
695
SEA_NC003923-
TAACCGTTTCCAA
862



2052219-
GTGGTGTAACG

2052219-
AGGTACTGTATTT



2051456_377_406_F
TTACATGA

2051456_459_492_R
TGTTTACC


 


2107
SEB_NC002758-
TTTCACATGTA
702
SEB_NC002758-
TCATCTGGTTTAG
988



2135540-
ATTTTGATATT

2135540-
GATCTGGTTGACT



2135140_208_137_F
CGCACTGA

2135140_273_298_R


 


2108
SEB_NC002758-
TATTTCACATG
244
SEB_NC002758-
TGCAACTCATCTG
1194



2135540-
TAATTTTGATA

2135540-
GTTAGGATCT



2135140_106_235_F
TTCGCACT

2135140_281_304_R


 


2109
SEB_NC002758-
TAACAACTCGC
151
SEB_NC002758-
TGTGCAGGCATCA
1334



2135540-
CTTATGAAACG

2135540-
TGTCATACCAA



2135140_402_402_F
GGATATA

2135140_402_402_R


 


2110
SEB_NC002758-
TTGTATGTATG
696
SEB_NC002758-
TTACCATCTTCAA
1361



2135540-
GTGGTGTAACT

2135540-
ATACCCGAACAG



2135140_402_402_2_F
GAGCA

2135140_402_402_2_R


 


2111
SEC_NC003923-
TTAACATGAAG
648
SEC_NC003923-
TGAGTTTGCACTT
1177



851678-
GAAACCACTTT

851678-
CAAAAGAAATTGT



852768_546_575_F
GATAATGG

852768_620_647_R
GT


 


2112
SEC_NC003923-
TGGAATAACAA
546
SEC_NC003923-
TCAGTTTGCACTT
985



851678-
AACATGAAGGA

851678-
CAAAAGAAATTGT



852768_537_566_F
AACCACTT

852768_619_647_R
GTT


 


2113
SEC_NC003923-
TGAGTTTAACA
466
SEC_NC003923-
TCGCCTGGTGCAG
1078



851678-
GTTCACCATAT

851678-
GCATCATAT



852768_720_749_F
GAAACAGG

852768_794_815_R


 


2114
SEC_NC003923-
TGGTATGATAT
604
SEC_NC003923-
TCTTCACACTTTT
1133



851678-
GATGCCTGCAC

851678-
AGAATCAACCGTT



852768_787_810_F
CA

852768_853_886_R
TTATTGTC


 


2115
SED_M28521_657_
TGGTGGTGAAA
615
SED_M28521_
TGTACACCATTTA
1318



682_F
TAGATAGGACT

741_770_R
TCCACAAATTGAT




GCTT


TGGT


 


2116
SED_M28521_
TGGAGGTGTCA
554
SED_M28521_
TGGGCACCATTTA
1288



690_711_F
ACTCCACACGA

739_770_R
TCCACAAATTGAT




A


TGGTAT


 


2117
SED_M28521_
TTGCACAAGCA
683
SED_M28521_
TCGCGCTGTATTT
1079



833_854_F
AGGCGCTATTT

888_911_R
TTCCTCCGAGA


 


2118
SED_M28521_
TGGATGTTAAG
559
SED_M28521_
TGTCAATATGAAG
1320



962_987_F
GGTGATTTTCC

1022_1048_R
GTGCTCTGTGGAT




CGAA


A


 


2119
SEA-SEE_NC002952-
TTTACACTACT
699
SEA-SEE_NC002952-
TCATTTATTTCTT
994



2131289-
TTTATTCATTG

2131289-
CGCTTTTCTGCT



2130703_16_45_F
CCCTAACG

2130703_71_98_R
AC


 


2120
SEA-SEE_NC002952-
TGATCATCCGT
469
SEA-SEE_NC002952-
TAAGCACCATATA
870



2131289-
GGTATAACGAT

2131289-
AGTCTACTTTTTC



2130703_249_278_F
TTATTAGT

2130703_314_344_R
CCCTT


 


2121
SEE_NC002952-
TGACATGATAA
445
SEE_NC002952-
TCTATAGGTACT
1120



2131289-
TAACCGATTGA

2131289-
GTAGTTTGTTTTC



2130703_409_437_F
CCGAAGA

2130703_485_494_R
CGTCT


 


2122
SEE_NC002952-
TGTTCAAGAGC
640
SEE_NC002952-
TTTGCACCTTAC
1436



2131289-
TAGATCTTCAG

2131289-
CGCCAAAGCT



2130703_525_550_F
GCAA

2130703588_588_R


 


2123
SEE_NC002952-
TGTTCAAGAGC
639
SEE_NC002952-
TACCTTACCGCC
892



2131289-
TAGATCTTCAG

2131289-
AAAGCTGTCT



2130703_525_549_F
GCA

2130703_588_586_2_R


 


2124
SEE_NC002952-
TCTGGAGGCAC
403
SEE_NC002952-
TCCGTCTATCCA
1043



2131289-
ACCAAATAAAA

2131289-
CAAGTTAATTGGT



2130703_381_384_F
CA

2130703_444_471_R
ACT


 


2125
SEG_NC002758-
TGCTCAACCCG
520
SEG_NC002758-
TAACTCCTCTTCC
863



1955100-
ATCCTAAATTA

1955100-
TTCAACAGGTGGA



1954171_225_251_F
GACGA

1954171_321_348_R


 


2126
SEG_NC002758-
TGGACAATAGA
548
SEG_NC002758-
TGCTTTGTAATC
1260



1955100-
CAATCACCTTG

1955100-
AGTTCCTGAATAG



1954171_623_851_F
GATTTACA

1954171_871_702_R
TAACCA


 


2127
SEG_NC002758-
TGGAGGTTGTT
555
SEG_NC002758-
TGTCTATTGTCGA
1329



1955100-
GTATGTATGG

1955100-
ATTGTTACCTGTA



1954171_540_584_F
TGTT

1954171_607_635_R
CAGT


 


2128
SEG_NC002758-
TACAAAGCAAG
173
SEG_NC002758-
TGATTCAAATGCA
1187



1955100-
ACACTGGCTC

1955100-
GAACCATCAAACT



1954171_694_718_F
ACTA

1954171_735_782_R
CG


 


2129
SEH_NC002953-
TTGCAACTGCT
682
SEH_NC002953-
TAGTGTTGTACCT
927



60024-
GATTTAGCTCA

60024-
CCATATAGACATT



60977_449_472_F
GA

60977_547_578_R
CAGA


 


2130
SEH_NC002953-
TAGAAATCAAG
201
SEH_NC002953-
TTCTGAGCTAAAT
1390



60024-
GTGATAGTGGC

60024-
ACAGCAGTTGC



60977_408_434_F
AAATGA

60977_450_473_R


 


2131
SEH_NC002953-
TCTGAARTGTC
400
SEH_NC002953-
TACCATCTACCC
888



60024-
TATATGGAGGT

60024-
AACATTAGCACCA



60977_547_576_F
ACAACACTA

60977_608_634_R
A


 


2132
SEH_NC002953-
TTCTGAATGTC
677
SEH_NC002953-
TAGCACCAATCAC
909



60024-
TATATGGAGGT

60024-
CCTTTCCTGT



60977_546_575_F
ACAACACT

60977_594_616_R


 


2133
SEI_NC002758-
TCAACTCGAAT
253
SEI_NC002758-
TCACAAGGACCAT
966



1957830-
TTTCAACAGGT

1957830-
TATAATCAATGCC



1956949_324_349_F
ACCA

1956949_419_448_R
AA


 


2134
SEI_NC002758-
TTCAACAGGTA
666
SEI_NC002758-
TGTACAAGGACCA
1316



1957830-
CCAATGATTTG

1957830-
TTATAATCAATGC



1956949_336_363_F
ATCTCA

1958949_420_447_R
CA


 


2135
SEI_NC002758-
TGATCTCAGAA
471
SEI_NC002758-
TCTGGCCCCTCCA
1129



1957830-
TCTAATAATTG

1957830-
TACATGTATTTAG



1956949_356_384_F
GGACGAA

1958949_449_474_R


 


2136
SEI_NC002758-
TCTCAAGGTGA
394
SEI_NC002758-
TGGGTAGGTTTTT
1293



1957830-
TATTGGTGTAG

1957830-
ATCTGTGACGCCT



1956949_223_253_F
GTAACTTAA

1958949_290_316_R
T


 


2137
SEJ_AF053140-
TGTGGAGTAAC
637
SEJ_AF053140-
TCTAGCGGAACAA
1118



1307_1332_F
ACTGCATGAAA

1381_1404_R
CAGTTCTGATG




ACAA


 


2138
SEJ_AF053140-
TAGCATCAGAA
211
SEJ_AF053140-
TCCTGAAGATCTA
1049



1378_1403_F
CTGTTGTTCCG

1429_1458_R
GTTCTTGAATGGT




CTAG


TACT


 


2139
SEJ_AF053140-
TAACCATTCAA
153
SEJ_AF053140-
TAGTCCTTTCTGA
925



1431_1459_F
GAACTAGATCT

1500_1531_R
ATTTACCATCAA




TCAGGCA


AGGTAC


 


2140
SEJ_AF053140-
TCATTCAAGAA
301
SEJ_AF053140-
TCAGGTATGAAAC
984



1434_1461_F
CTAGATCTTCA

1521_1549_R
ACGATTAGTCCTT




GGCAAG


TCT


 


2141
TSST_NC002758-
TGGTTTAGATA
619
TSST_NC002758-
TGTAAAAGCAGGG
1312



2137564-
ATTCTTTAGGA

2137564-
CTATAATAAGGAC



2138293_206_236_F
TCTATGCGT

2138293_278_305_R
TC


 


2142
TSST_NC002758-
TGCGTATAAAA
514
TSST_NC002758-
TGCCCTTTTGTAA
1221



2137564-
AACACAGATGG

2137564-
AAGCAGGGCTAT



2138293_232_258_F
CAGCA

2138293_289_313_R


 


2143
TSST_NC002758-
TCCAAATAAGT
304
TSST_NC002758-
TACTTTAAGGGGC
907



2137564-
GGCGTTACAAA

2137564-
TATCTTTACCATG



2138293_382_410_F
TACTGAAA

2138293_448_478_R
AACCT


 


2144
TSST_NC002758-
TCTTTTACAAA
423
TSST_NC002758-
TAAGTTCCTTCGC
874



2137564-
AGGGGAAAAG

2137564-
TAGTATGTTGGCT



2138293_297_325_F
TTGACTT

2138293_347_373_R
T


 


2145
ARCC_NC003923-
TCGCCGGCAAT
368
ARCC_NC003923-
TGAGTTAAAATGC
1175



2725050-
GCCATTGGATA

2725050-
GATTGATTTCAGT



2724595_37_58_F


2724595_97_128_R


 


2146
ARCC_NC003923-
TGAATAGTGAT
437
ARCC_NC003923-
TCTTCTTCTTCG
1137



2725050-
AGAACTGTAGG

2725050-
TATAAAAAGGACC



2724595_131_161_F
CACAATCGT

2724595_214_245_R
AATTGG


 


2147
ARCC_NC003923-
TTGGTCCTTTT
691
ARCC_NC003923-
TGGTGTTCTAGTA
1306



2725050-
TATACGAAAGA

2725050-
TAGATTGAGGTAG



2724595_218_249_F
AGAAGTTGAA

2724595_322_353_R
TGGTGA


 


2148
AROE_NC003923-
TTGCGAATAGA
686
AROE_NC003923-
TCGAATTCAGCTA
1064



1674726-
ACGATGGCTCG

1674726-
AATACTTTTCAGC



1674277_371_393_F
T

1674277_435_464_R
ATCT


 


2149
AROE_NC003923-
TGGGGCTTTAA
590
AROE_NC003923-
TACCTGCATTAAT
891



1674726-
ATATTCCAATT

1674726-
CGCTTGTTCATCA



1674277_30_62_F
GAAGATTTTCA

1674277_155_181_R
A


 


2150
AROE_NC003923-
TGATGGCAAGT
474
AROE_NC003923-
TAAGCAATACCTT
869



1674726-
GGATAGGGTAT

1674726-
TACTTGCACCACC



1674277_204_232_F
AATACAG

1674277_308_335_R
TTG


 


2151
GLPF_NC003923-
TGCACCGGCTA
491
GLPF_NC003923-
TGCAACAATTAAT
1193



1296927-
TTAAGAATTAC

1296927-
GCTCCGACAATTA



297391_270_301_F
TTTGCCAACT

1297391_382_414_R
AAGGATT


 


2152
GLPF_NC003923-
TGGATGGGGAT
558
GLPF_NC003923-
TAAAGACACCGCT
850



1296927-
TAGCGGTTACA

1296927-
GGGTTTAAATGTG



1297391_27_51_F
ATG

1297391_81_108_R
CA


 


2153
GLPF_NC003923-
TAGCTGGCGCG
218
GLPF_NC003923-
TCACCGATAAATA
972



1296927-
AAATTAGGTGT

1296927-
AAATACCTAAAGT



1297391_239_260_F


1297391_323_359_R
TAATGCCATTG


 


2154
GMK_NC003923-
TACTTTTTTAA
200
GMK_NC003923-
TGATATTGAACTG
1180



1190906-
AACTAGGGATG

1190906-
GTGTACCATAATA



119334_91_122_F
CGTTTGAAGC

1191334_166_197_R
GTTGCC


 


2155
GMK_NC003923-
TGAAGTAGAAG
435
GMK_NC003923-
TCGCTCTCTCAAG
1082



1190906-
GTGCAAAGCAA

1190906-
TGATCTAAACTTG



1191334_240_267_F
GTTAGA

1191334_305_333_R
GAG


 


2156
GMK_NC003923-
TCACCTCCAAG
268
GMK_NC003923-
TGGGACGTAATCG
1284



1190906-
TTTAGATCACT

1190906-
TATAAATTCATCA



1191334_301_329_F
TGAGAGA

1191334_403_432_R
TTTC


 


2157
PTA_NC003923-
TCTTGTTTATG
418
PTA_NC003923-
TGGTACACCTGGT
1301



628885-
CTGGTAAAGCA

628885-
TTCGTTTTGATGA



629355_237_263_F
GATGG

629355_314_345_R
TTTGTA


 


2158
PTA_NC003923-
TGAATTAGTTC
439
PTA_NC003923-
TGCATTGTACCGA
1207



628885-
AATCATTTGTT

628885-
AGTAGTTCACATT



629355_141_171_F
GAACGACGT

629355_211_239_R
GTT


 


2159
PTA_NC003923-
TCCAAACCAGG
303
PTA_NC003923-
TGTTCTGGATTGA
1349



628885-
TGTATCAAGAA

628885-
TTGCACAATCACC



629355_328_356_F
CATCAGG

629355_393_422_R
AAAG


 


2160
TPI_NC003923-
TGCAAGTTAAG
486
TPI_NC003923-
TGAGATGTTGATG
1165



830671-
AAAGCTGTTGC

830671-
ATTACCAGTTCC



831072_131_160_F
AGGTTTAT

831072_209_239_R
GATTG


 


2161
TPI_NC003923-
TCCCACGAAAC
318
TPI_NC003923-
TGGTACAACATCG
1300



830671-
AGATGAAGAAA

830671-
TTAGCTTTACCAC



831072_1_34_F
TTAACAAAAAA

831072_97_129_R
TTTCACG




G


 


2162
TPI_NC003923-
TCAAACTGGGC
246
TPI_NC003923-
TGGCAGCAATAGT
1275



830671-
AATCGGAACTG

830671-
TTGACGTACAAAT



831072_199_227_F
GTAAATC

831072_253_286_R
GCACACAT


 


2163
YQI_NC003923-
TGAATTGCTGC
440
YQI_NC003923-
TCGCCAGCTAGCA
1076



378916-
TATGAAAGGTG

378916-
CGATGTCATTTTC



379431_142_167_F
GCTT

379431_259_284_R


 


2164
YQI_NC003923-
TACAACATATT
175
YQI_NC003923-
TTCGTGCTGGATT
1388



378916-
ATTAAAGAGAC

378916-
TTGTCCTTGTCCT



379431_44_77_F
GGGTTTGAATC

379431_120_145_R




C


 


2165
YQI_NC003923-
TCCAGCACGAA
314
YQI_NC003923-
TCCAACCCAGAAC
997



378916-
TTGCTGCTATG

378916-
CACATACTTTATT



379431_135_160_F
AAAG

379431_193_221_R
CAC


 


2166
YQI_NC003923-
TAGCTGGCGGT
219
YQI_NC003923-
TCCATCTGTTAAA
1013



378916-
ATGGAGAATAT

378916-
CCATCATATACCA



379431_275_300_F
GTCT

379431_364_396_R
TGCTATC


 


2167
BLAZ_
TCCACTTATCG
312
BLAZ_
TGGCCACTTTTAT
1277



(1913827 . . .
CAAATGGAAAA

(1913827 . . .
CAGCAACCTTACA



1914672)_546_
TTAAGCAA

1914672)_655_
GTC



575_F


683_R


 


2168
BLAZ_
TGCACTTATCG
494
BLAZ_
TAGTCTTTTGGAA
926



(1913827 . . .
CAAATGGAAAA

(1913827 . . .
CACCGTCTTTAAT



1914672)_546_
TTAAGCAA

1914672)_628_
TAAAGT



575_2_F


659_R


 


2169
BLAZ_
TGATACTTCAA
467
BLAZ_
TGGAACACCGTCT
1263



(1913827 . . .
CGCCTGCTGCT

(1913827 . . .
TTAATTAAAGTAT



1914672)_507_
TTC

1914672)_622_
CTCC



531_F


651_R


 


2170
BLAZ_
TATACTTCAAC
232
BLAZ_
TCTTTTCTTGCT
1145



(1913827 . . .
GCCTGCTGCTT

1913827 . . .
TAATTTTCCATTT



1914672)_508_
TC

1914672)_553_
GCGAT



531_F


583_R


 


2171
BLAZ_
TGCAATTGCTT
487
BLAZ_
TTACTTCCTTACC
1366



(1913827 . . .
TAGTTTTAAGT

(1913827 . . .
CTTTTAGTATCT



1914672)_24_
GCATGTAATTC

1914672)_121_
AAAGCATA



56_F


154_R


 


2172
BLAZ_
TCCTTGCTTTA
351
BLAZ_
TGGGGACTTCCTT
1289



(1913827 . . . 1914672)_
GTTTTAAGTGC

(1913827 . . . 1914672)_
ACCACTTTTAGTA



26_58_F
ATGTAATTCAA

127_157_R
TCTAA


 


2173
BLAZ_NC002952-
TCCACTTATCG
312
BLAZ_NC002952-
TGGCCACTTTTAT
1277



1913827-
CAAATGGAAAA

1913827-
CAGCAACCTTACA



1914672_546_575_F
TTAAGCAA

1914672_655_683_R
GTC


 


2174
BLAZ_NC002952-
TGCACTTATCG
494
BLAZ_NC002952-
TAGTCTTTTGGAA
926



1913827-
CAAATGGAAAA

1913827-
CACCGTCTTTAAT



1914672_546_575_2_F
TTAAGCAA

1914672_628_659_R
TAAAGT


 


2175
BLAZ_NC002952-
TGATACTTCAA
467
BLAZ_NC002952-
TGGAACACCGTCT
1263



1913827-
CGCCTGCTGCT

1913827-
TTAATTAAAGTAT



1914672_507_531_F
TTC

1914672_622_651_R
CTCC


 


2176
BLAZ_NC002952-
TATACTTCAAC
232
BLAZ_NC002952-
TCTTTTCTTTGCT
1145



1913827-
GCCTGCTGCTT

1913827-
TAATTTTCCATTT



1914672_508_531_F
TC

1914672_553_583_R
GCGAT


 


2177
BLAZ_NC002952-
TGCAATTGCTT
487
BLAZ_NC002952-
TTACTTCCTTACC
136



1913827-
TAGTTTTAAGT

1913827-
ACTTTTAGTATCT



1914672_24_56_F
GCATGTAATTC

1914672_121_154_R
AAAGCATA


 


2178
BLAZ_NC002952-
TCCTTGCTTTA
351
BLAZ_NC002952-
TGGGGACTTCCTT
1289



1913827-
GTTTTAAGTGC

1913827-
ACCACTTTTAGTA



1914672_26_58_F
ATGTAATTCAA

1914672_127_157_R
TCTAA


 


2247
TUFB_NC002758-
TGTTGAACGTG
643
TUFB_NC002758-
TGTCACCAGCTTC
1321



615038-
GTCAAATCAAA

615038-
AGCGTAGTCTAA



616222_693_721_F
GTTGGTG

616222_793_820_R
TAA


 


2248
TUFB_NC002758-
TCGTGTTGAAC
386
TUFB_NC002758-
TGTCACCAGCTTC
1321



615038-
GTGGTCAAATC

615038-
AGCGTAGTCTAAT



616222_690_716_F
AAAGT

616222_793_820_R
AA


 


2249
TUFB_NC002758-
TGAACGTGGTC
430
TUFB_NC002758-
TGTCACCAGCTTC
1321



615038-
AAATCAAAGTT

615038-
AGCGTAGTCTAAT



616222_696_725_F
GGTGAAGA

616222_793_820_R
AA


 


2250
TUFB_NC002758-
TCCCAGGTGAC
320
TUFB_NC002758-
TGGTTTGTCAGAA
1311



615038-
GATGTACCTGT

615038-
TCACGTTCTGGAG



616222_488_513_F
AATC

616222_601_630_R
TTGG


 


2251
TUFB_NC002758-
TGAAGGTGGAC
433
TUFB_NC002758-
TAGGCATAACCAT
922



615038-
GTCACACTCCA

615038-
TTCAGTACCTTCT



616222_945_972_F
TTCTTC

616222_1030_1060_R
GGTAA


 


2252
TUFB_NC002758-
TCCAATGCCAC
307
TUFB_NC002758-
TTCCATTTCAACT
1382



615038-
AAACTCGTAA

615038-
AATTCTAATAATT



616222_333_356_F
CA

616222_424_459_R
CTTCATCGTC


 


2253
NUC_NC002758-
TCCTGAAGCAA
342
NUC_NC002758-
TACGCTAAGCCAC
899



894288-
GTGCATTTTAC

894288-
GTCCATATTTATC



894974_402_424_F
GA

894974_483_509_R
A


 


2254
NUC_NC002758-
TCCTTATAGGG
349
NUC_NC002758-
TGTTTGTGATGCA
1354



894288-
ATGGCTATCAG

894288-
TTTGCTGAGCTA



894974_53_81_F
TAATGTT

894974_165_189_R


 


2255
NUC_NC002758-
TCAGCAAATGC
273
NUC_NC002758-
TAGTTGAAGTTGC
928



894288-
ATCACAAACAG

894288-
ACTATATACTGTT



894974_169_194_F
ATAA

894974_222_250_R
GGA


 


2256
NUC_NC002758-
TACAAAGGTCA
174
NUC_NC002758-
TAAATGCACTTGC
853



894288-
ACCAATGACAT

894288-
TTCAGGGCCATAT



894974_316_345_F
TCAGACTA

89474_396_421_R


 


2270
RPOB_EC_3798_3821_1_F
TGGCCAGCGCT
566
RPOB_EC_3868_3895_R
TCACGTCGTCCGA
979




TCGGTGAAATG


CTTCACGGGTCAG




GA


CT


 


2271
RPOB_EC_3789_3812_F
TCAGTTCGGCG
294
RPOB_EC_3860_3890_R
TCGTCGGACTTAA
1107




GTCAGCGCTTC


CGGTCAGCATTTC




GG


CTGCA


 


2272
RPOB_EC_3789_3812_F
TCAGTTCGGCG
294
RPOB_EC_3860_3890_2_R
TCGTCCGACTTAA
1102




GTCAGCGCTTC


CGGTCAGCATTTC




GG


CTGCA


 


2273
RPOB_EC_3789_3812_F
TCAGTTCGGCG
294
RPOB_EC_3862_3890_R
TCGTCGGACTTAA
1106




GTCAGCGCTTC


CGGTCAGCATTTC




GG


CTG


 


2274
RPOB_EC_3789_3812_F
TCAGTTCGGCG
294
RPOB_EC_3862_3890_R
TCGTCCGACTTAA
1101




GTCAGCGCTTC


CGGTCAGCATTTC




GG


CTG


 


2275
RPOB_EC_3793_3812_F
TTCGGCGGTCA
674
RPOB_EC_3865_3890_R
TCGTCGGACTTAA
1105




GCGCTTCGG


CGGTCAGCATTTC


 


2276
RPOB_EC_3793_3812_F
TTCGGCGGTCA
674
RPOB_EC_3865_3890_R
TCGTCCGACTTAA
1100




GCGCTTCGG


CGGTCAGCATTTC


 


2309
MUPR_X75439_1658_1689_F
TCCTTTGATAT
352
MUPR_X75439_1744_1773_R
TCCCTTCCTTAT
1030




ATTATGCGATG


ATGAGAAGGAAAC




GAAGGTTGGT


CACT


 


2310
MUPR_X75439_1330_1353_F
TTCCTCCTTTT
669
MUPR_X75439_1413_1441_R
TGAGCTGGTGCTA
1171




GAAAGCGACGG


TATGAACAATACC




TT


AGT


 


2312
MUPR_X75439_1314_1338_F
TTTCCTCCTTT
704
MUPR_X75439_1381_1409_R
TATATGAACAATA
931




TGAAAGCGAC


CCAGTTCCTTCTG




GGTT


AGT


 


2313
MUPR_X75439_2486_2516_F
TAATTGGGCTC
172
MUPR_X75439_2548_2574_R
TTAATCTGGCGT
1360




TTTCTCGCTTA


GGAAGTGAAATCG




AACACCTTA


T


 


2314
MUPR_X75439_2547_2572_F
TACGATTTCAC
188
MUPR_X75439_2605_2630_R
TCGTCCTCTCGAA
1103




TTCCGCAGCCA


TCTCCGATATACC




GATT


 


2315
MUPR_X75439_2666_2696_F
TGCGTACAATA
513
MUPR_X75439_2711_2740_R
TCAGATATAAATG
981




CGCTTTATGAA


GAACAAATGGAGC




ATTTTAACA


CACT


 


2316
MUPR_X75439_2813_2843_F
TAATCAAGCAT
165
MUPR_X75439_2867_2890_R
TCTGCATTTTTGC
1127




TGGAAGATGAA


GAGCCTGTCTA




ATGCATACC


 


2317
MUPR_X75439_884_914_F
TGACATGGACT
447
MUPR_X75439_977_1007_R
TGTACAATAAGGA
1317




CCCCCTATATA


GTCACCTTATGTC




ACTCTTGAG


CCTTA


 


2318
CTXA_NC002505-
TGGTCTTATGC
608
CTXA_NC002505-
TCGTGCCTAACAA
1109



1568114-
CAAGAGGACAG

1568114-
ATCCCGTCTGAGT



1567341_114_142_F
AGTGAGT

1567341_194_221_R
TC


 


2319
CTXA_NC002505-
TCTTATGCCAA
411
CTXA_NC002505-
TCGTGCCTAACAA
1109



1568114-
GAGGACAGAGT

1568114-
ATCCCGTCTGAGT



1567341_117_145_F
GAGTACT

1567341_194_221_R
TC


 


2320
CTXA_NC002505-
TGGTCTTATGC
608
CTXA_NC002505-
TAACAAATCCCGT
855



1568114-
CAAGAGGACAG

1568114-
CTGAGTTCCTCTT



1567341_114_142_F
AGTGAGT

1567341_186_214_R
GCA


 


2321
CTXA_NC002505-
TCTTATGCCAA
411
CTXA_NC002505-
TAACAAATCCGT
855



1568114-
GAGGACAGAGT

1568114-
CTGAGTTCCTCTT



1567341_117_145_F
GAGTACT

1567341_186_214_R
GCA


 


2322
CTXA_NC002505-
AGGACAGAGTG
27
CTXA_NC002505-
TCCCGTCTGAGTT
1027



1568114-
AGTACTTTGAC

1568114-
CCTCTTGCATGAT



1567341_129_156_F
CGAGGT

1567341_180_207_R
CA


 


2323
CTXA_NC002505-
TGCCAAGAGGA
500
CTXA_NC002505-
TAACAAATCCCGT
855



1568114-
CAGAGTGAGTA

1568114-
CTGAGTTCCTCTT



1567341_122_149_F
CTTTGA

1567341_186_214_R
GCA


 


2324
INV_U22457-74-
TGCTTATTTAC
530
INV_U22457-74-
TGACCCAAAGCT
1154



3772_831_858_F
CTGCACTCCCA

3772_942_966_R
AAAGCTTTACTG




CAACTG


 


2325
INV_U22457-74-
TGAATGCTTAT
438
INV_U22457-74-
TAACTGACCCAAA
864



3772_827_857_F
TTACCTGCACT

3772_942970_R
GCTGAAAGCTTTA




CCCACAACT


CTG


 


2326
INV_U22457-74-
TGCTGGTAACA
526
INV_U22457-74-
TGGGTTGCGTTG
1296



3772_1555_1581_F
GAGCCTTATAG

3772_1619_1647_R
AGATTATCTTTAC




GCGCA


CAA


 


2327
INV_U22457-74-
TGGTAACAGAG
598
INV_U22457-74-
TCATAAGGGTTG
987



3772_1558_1585_F
CCTTATAGGCG

3772_1622_1652_R
GTTGCAGATTATC




CATATG


TTTAC


 


2328
ASD_NC006570-
TGAGGGTTTTA
459
ASD_NC006570-
TGATTCGATCATA
1188



439714-
TGCTTAAAGTT

439714-
CGAGACATTAAA



438608_3_37_F
GGTTTTATTGG

438608_54_84_R
CTGAGT




TT


 


2329
ASD_NC006570-
TAAAGTTGGTT
149
ASD_NC006570-
TCAAAATCTTTTG
948



439714-
TTATTGGTTGG

439714-
ATTCGATCATACG



438608_18_45_F
CGCGGA

438608_66_95_R
AGAC


 


2330
ASD_NC006570-
TTAAAGTTGGT
647
ASD_NC006570-
TCCCAATCTTTTG
1016



439714-
TTTATTGGTTG

439714-
ATTCGATCATACG



438608_17_45_F
GCGCGGA

438608_67_95_R
AGA


 


2331
ASD_NC006570-
TTTTATGCTTA
709
ASD_NC006570-
TCTGCCTGAGATG
1128



439714-
AAGTTGGTTTT

439714-
TCGAAAAAACGT



438608_9_40_F
ATTGGTTGGC

438608_107_134_R
TG


 


2332
GALE_AF513299_171_200_F
TCAGCTAGACC
280
GALE_AF513299_241_271_R
TCTCACCTACAGC
1122




TTTTAGGTAAA


TTTAAAGCCAGCA




GCTAAGCT


AAATG


 


2333
GALE_AF513299_168_199_F
TTATCAGCTAG
658
GALE_AF513299_245_271_R
TCTCACCTACAG
1121




ACCTTTTAGGT


TTTAAAGCCAGCA




AAAGCTAAGC


A


 


2334
GALE_AF513299_168_199_F
TTATCAGCTAG
658
GALE_AF513299_233_264_R
TACAGCTTTAAAG
883




ACCTTTTAGGT


CCAGCAAAATGAA




AAAGCTAAGC


TTACAG


 


2335
GALE_AF513299_169_198_F
TCCCAGCTAGA
319
GALE_AF513299_252_279_R
TTCAACACTCTCA
1374




CCTTTTAGGTA


CCTACAGCTTTAA




AAGCTAAG


AG


 


2236
PLA_AF053945_7371_7403_F
TTGAGAAGACA
680
PLA_AF053945_7434_7468_R
TACGTATGTAAAT
900




TCCGGCTCACG


TCCGCAAAGACTT




TTATTATGGTA


TGGCATTAG


 


2337
PLA_AF053945_7377_7403_F
TGACATCCGGC
443
PLA_AF053945_7428_7455_R
TCCGCAAAGACTT
1035




TCACGTTATTA


TGGCATTAGGTGT




TGGTA


GA


 


2338
PLA_AF053945_7377_7404_F
TGACATCCGGC
444
PLA_AF053945_7430_7460_R
TAAATTCCGCAAA
854




TCACGTTATTA


GACTTTGGCATTA




TGGTAC


GGTGT


 


2339
CAF_AF053947_33412_33441_F
TCCGTTATCGC
329
CAF_AF053947_33498_33523_R
TAAGAGTGATGC
866




CATTGCATTAT


GGCTGGTTCAACA




TTGGAACT


 


2340
CAF_AF053947_33426_33458_F
TGCATTATTTG
499
CAF_AF053947_33483_33507_R
TGGTTCAACAAG
1308




GAACTATTGCA


GTTGCCGTTGCA




ACTGCTAATGC


 


2341
CAF_AF053947_33407_33429_F
TCAGTTCCGTT
291
CAF_AF053947_33483_33504_R
TTCAACAAGAGTT
1373




ATCGCCATTGC


GCCGTTGCA




A


 


2342
CAF_AF053947_33407_33431_F
TCAGTTCCGTT
293
CAF_AF053947_33494_33517_R
TGATGCGGGCTGG
1184




ATCGCCATTG


TTCAACAAGAG




CATT


2344
GAPA_NC_002505_1_28_F_1
TCAATGAACGA
260
GAPA_NC_002505_29_58_R_1
TCCTTTATGCAAC
1060




TCAACAAGTGA


TTGGTATCAACAG




TTGATG


GAAT


 


2472
OMPA_NC000117_68_89_F
TGCCTGTAGGG
507
OMPA_NC000117_145_167_R
TCACACCAAGTAG
967




AATCCTGCTGA


TGCAAGGATC




A


 


2473
OMPA_NC000117_798_821_F
TGATTACCATG
475
OMPA_NC000117_865_893_R
TCAAAACTTGCTC
947




AGTGGCAAGCA


TAGACCATTTAAC




AG


TCC


 


2474
OMPA_NC000117_645_671_F
TGCTCAATCTA
521
OMPA_NC000117_757_777_R
TGTCGCAGCATCT
1328




AACCTAAAGTC


GTTCCTGC




GAAGA


 


2475
OMPA_NC000117_947_973_F
TAACTGCATGG
157
OMPA_NC000117_1011_1040_R
TGACAGGACACAA
1153




AACCCTTCTTT


TCTGCATGAAGTC




ACTAG


TGAG


 


2476
OMPA_NC000117_774_795_F
TACTGGAACAA
196
OMPA_NC000117_871_894_R
TTCAAAAGTTGCT
1371




AGTCTGCGACC


CGAGACCATTG


 


2477
OMPA_NC000117_457_483_F
TTCTATCTCGT
676
OMPA_NC000117_511_534_R
TAAAGAGACGTTT
851




TGGTTTATTCG


GGTAGTTCATTTG




GAGTT


C


 


2478
OMPA_NC000117_687_710_F
TAGCCCAGCAC
212
OMPA_NC000117_787_816_R
TTGCCATTCATGG
1406




AATTTGTGATT


TATTTAAGTGTAG




CA


AGA


 


2479
OMPA_NC000117_540_566_F
TGGCGTAGTAG
571
OMPA_NC000117_649_672_R
TTCTTGAACGCGA
1395




AGCTATTTACA


GGTTTCGATTG




GACAC


 


2480
OMPA_NC000117_338_360_F
TGCACGATGCG
492
OMPA_NC000117_417_444_R
TCCTTTAAAATAA
1058




GAATGGTTCAC


CCGCTAGTAGCTC




A


CT


 


2481
OMP2_NC000117_18_40_F
TATGACCAAAC
234
OMP2_NC000117_71_91_R
TCCCGCTGGCAAA
1025




TCATCAGACGA


TAAACTCG




G


 


2482
OMP2_NC000117_354_382_F
TGCTACGGTAG
516
OMP2_NC000117_445_471_R
TGGATCACTGCTT
1270




GATCTCCTTAT


ACGAACTCAGCTT




CCTATTG


C


 


2483
OMP2_NC000117_1297_1319_F
TGGAAAGGTGT
537
OMP2_NC000117_1396_1419_R
TACGTTTGTATCT
903




TGCAGCTACTC


TCTGCAGAACC




A


 


2484
OMP2_NC000117_1465_1493_F
TCTGGTCCAAC
407
OMP2_NC000117_1541_1569_R
TCCTTTCAATGTT
1062




AAAAGGAACGA


ACAGAAAACTCTA




TTACAGG


CAG


 


2485
OMP2_NC000117_44_66_F
TGACGATCTTC
450
OMP2_NC000117_120_148_R
TGTCAGCTAAGC
1323




GCGGTGACTAG


TAATAACGTTTGT




T


AGAG


 


2486
OMP2_NC000117_166_190_F
TGACAGCGAAG
441
OMP2_NC000117_240_261_R
TTGACATCGTCCC
1396




AAGGTTAGACT


TCTTCACAG




TGTCC


 


2487
GYRA_NC000117_514_536_F
TCAGGCATTGC
287
GYRA_NC000117_640_660_R
TGCTGTAGGGAAA
1251




GGTTGGGATGG


TCAGGGCC


 


2488
GYRA_NC000117_801_827_F
TGTGAATAAAT
636
GYRA_NC000117_871_893_R
TTGTCAGACTCAT
1419




CACGATTGATT


CGCGAACATC




GAGCA


 


2489
GYRA_NC002952_219_242_F
TGTCATGGGTA
632
GYRA_NC002952_319_345_R
TCCATCCATAGAA
1010




AATATCACCCT


CCAAAGTTACCTT




CA


G


 


2490
GYRA_NC002952_964_983_F
TACAAGCACTC
176
GYRA_NC002952_1024_1041_R
TCGCAGCGTGCGT
1073




CCAGCTGCA


GGCAC


 


2491
GYRA_NC002952_1505_1520_F
TCGCCCGCGAG
366
GYRA_NC002952_1546_1562_R
TTGGTGCGCTTGG
1416




GACGT


CGTA


 


2492
GYRA_NC002952_59_81_F
TCAGCTACATC
279
GYRA_NC002952_124_143_R
TGGCGATGCACTG
1279




GACTATGCGAT


GCTTGAG




G


 


2493
GYRA_NC002952_216_239_F
TGACGTCATCG
452
GYRA_NC002952_313_333_R
TCCGAAGTTGCCC
1032




GTAAGTACCAC


TGGCCGTC




CC


 


2494
GYRA_NC002952_219_242_2_F
TGTACTCGGTA
625
GYRA_NC002952_308_330_R
TAAGTTACCTTGC
873




AGTATCACCCG


CCGTCAACCA




CA


2495
GYRA_NC002952_115_141_F
TGAGATGGATT
453
GYRA_NC002952_220_242_R
TGCGGGTGATACT
1236




TAAACCTGTTC


TACCGAGTAC




ACCGC


 


2496
GYRA_NC002952_517_5_39_F
TCAGGCATTGC
287
GYRA_NC002952_643_663_R
TGCTGTAGGGAAA
1251




GGTTGGGATGG


TCAGGGCC




C


 


2497
GYRA_NC002952_273_2_93_F
TCGTATGGCTC
380
GYRA_NC002952_338_360_R
TGCGGCAGCACTA
1234




AATGGTGGAG


TCACCATCCA


 


2498
GYRA_NC000912_257_278_F
TGAGTAAGTT
462
GYRA_NC000912_346_370_R
TCGAGCCGAAGTT
1067




CCACCCGCACG


CCCTGTCCGTC




G


 


2504
ARCC_NC003923-
TAGTpGATpAG
229
ARCC_NC003923-2725050-
TCpTpTpTpCpGT
1116



2725050-
AACpTpGTAGG

2724595_214_239P_R
ATAAAAAGGACpC



2724595_135_161P_F
CpACpAATpCp


pAATpTpGG




GT


 


2505
PTA_NC003923-
TCTTGTpTpTp
417
PTA_NC003923-628885-
TACpACpCpTGGT
904



629355_629355_237_263P_F
ATGCpTpGGTA


pTpTpCpGTpTpT




AAGCAGATGG


pTpGATGATpTpT







pGTA


 


2517
CJMLST_ST1_1852_1883_F
TTTGCGGATGA
708
CJMLST_ST1_1945_1977_R
TGTTTTATGTG
1355




AGTAGGTGCCT


TAGTTGAGCTTAC




ATCTTTTTGC


TTACTACATGAGC


 


2518
CJMLST_ST1_2963_2992_F
TGAAATTGCTA
428
CJMLST_ST1_3073_3097_R
TCCCCATCTCCGCA
1020




CAGGCCCTTTA


AAGACAATAAA




GGACAAGG


 


2519
CJMLST_ST1_2350_2378_F
TGCTTTTGATG
535
CJMLST_ST1_2447_2481_R
TCTACAACACT
1117




GTGATGCAGA


TGATTGTAATTTG




TCGTTTGG


CCTTGTTCTTT


 


2520
CJMLST_ST1_654_684_F
TATGTCCAAGA
240
CJMSLT_ST1_725_756_R
TCGGAAACAAAGA
1084




AGCATAGCAAA


ATTCATTTTCTGG




AAAAGCAAT


TCCAAA


 


2521
CJMSLT_ST1_360_395_F
TCCTGTTATTC
347
CJMLST_ST1_454_457_R
TGCTATATGCTAC
1245




CTGAAGTAGTT


AACTGGTTCAAAA




AATCAAGTTTG


ACATTAAG




TTA


 


2522
CJMSLT_ST1_1231_1258_F
TGGCAGTTTTA
564
CJMSLT_ST1_1312_1340_R
TTTAGCTACTATT
1427




CAAGGTGCTGT


CTAGCTGCCATTT




TTCATC


CCA


 


2523
CJMSLT_ST1_3543_3574_F
TGCTGTAGCTT
529
CJMLST_ST1_3656_3685_R
TCAAAGAACCAGC
1427




ATCGCGAAATG


ACCTAATTCATCA




TCTTTGATTT


TTTA


 


2524
CJMLST_ST1_1_17_F
TAAAACTTTTG
145
CJMSLT_ST1_55_84_R
TGTTCCAATAGCA
1348




CCGTAATGATG


GTTCCGCCCAAAT




GGTGAAGATAT


TGAT


 


2525
CJMSLT_ST1_1312_1342_F
TGGAAATGGCA
538
CJMSLT_ST1_1383_1417_R
TTTCCCCGATC
1432




GCTAGAATAGT


TAAATTTGGATAA




AGCTAAAAT


GCCATAGGAAA


 


2526
CJMSLT_ST1_2254_2286_F
TGGGCCTAATG
582
CJMSLT_ST1_2352_2379_R
TCCAAACGATC
996




GGCTTAATATC


TGCATCACCATCA




AATGAAAATTG


AAAG


 


2527
CJMSLT_ST1_1380_1411_F
TGCTTTCCTAT
534
CJMSLT_ST1_1486_1520_R
TGCATGAAGCATA
1205




GGCTTATCCAA


AAAACTGTATCAA




ATTTAGATCG


GTGCTTTTA


 


2528
CJMLST_ST1_3413_3437_F
TTGTAAATGCC
692
CJMLST_ST1_3511_3542_R
TGCTTGCTCAAAT
1257




GGTGCTTCAGA


CATCATAAACAAT




TCC


TAAAGC


 


2529
CJMSLT_ST1_1130_1156_F
TACGCGTCTTG
189
CJMSLT_ST1_1203_R
TAGGATGAGCATT
920




AAGCGTTTCGTTA


ATCAGGGAAAGAA




TGA


AGAATC


 


2530
CJMSLT_ST1_2840_2872_F
TGGGGCTTTGC
591
CJMSLT_ST1_2940_2973_R
TAGCGATTTCT
917




TTTATAGTTTT


ACTCCTAGAGTTG




TTACATTTAAG


AAATTTCAGG


 


2531
CJMSLT_ST1_2058_2084_F
TATTCAAGGTG
241
CJMSLT_ST1_2131_2162_R
TTGGTTCTTACTT
1417




GTCCTTTGATG


GTTTTGCATAAAC




CATGT


TTTCCA


 


2532
CJMSLT_ST1_553_585_F
TCCTGATGCTC
344
CJMLST_ST1_655_685_R
TATTGCTTTTTTT
942




AAAGTGCTTTT


GCTATGCTTCTTG




TTAGATCCTTT


GACAT


 


2564
GTLA_NC002163-
TCATGTTGAGC
299
GTLA_NC002163-1604930-
TTTTGCTCATGAT
1443



1604930-
TTAAACCTATA

1604529_352_380_R
CTGCATGAAGCAT



1604529_306_338_F
GAAGTAAAAGC


AAA


 


2565
UNCA_NC002163-
TCCCCCACGCT
322
UNCA_NC002163-112166-
TCGACCTGGAGGA
1065



112166-
TTAATTGTTTT

112647_146_171_R
CGACGTAAAATCA



112647_80_113_F
ATGATGATTTG


CGACGTAAAATCA




AG


 


2566
UNCA_NC002163-
TAATGATGAAT
170
UNCA_NC002316-112166-
TGGGATAACAT
1285



112166-
TAGGTGCGGGT

112647_294_329_R
TGGTTGGAATATA



112647_233_305_F
TCTTT


AGCAGAAACATC


 


2567
PGM_NC002163-
TCTTGATACTT
414
PGM_NC002163-327773-
TCCATCGCCAGTT
1012



327773-
GTAATGTGGGC

328270_365_396_R
TTTGCATAATCGC



328270_273_305_F
GATAAATATGT

TAAAAA


 


2568
TKT_NC002163-
TTATGAAGCGT
661
TKT_NC002163-1569415-
TCAAAACGCATTT
946



1569415-
GTTCTTTAGCA

1569873_350_383_R
TTACATCTTCGTT



1569873_255_284_F
GGACTTCA

AAAGGCTA


 


2570
GTLA_NC002163-
TCGTCTTTTTG
381
GLTA_NC002163-1604930-
TGTTCATGTTTAA
1347



1604930-
ATTCTTTCCCT

1604529_109_142_R
ATGATCAGGATAA



1604529_39_68_F
GATAATGC


AAAGCACT


 


2571
TKT_NC002163-
TGATCTTAAAA
472
TKT_NC002163-1569415-
TGCCATAGCAAAG
1214



1569415-
ATTTCCGCCAA

1569903_139_162_R
CCTACAGCATT



1569903_33_62_F
CTTCATTC


CCTACAGCATT


 


2572
TKT_NC002163-
TAAGGTTTATT
164
TKT_NC002163-1569415-
TACATCTCCTTCG
886



1569415-
GTCTTTGTGGA

1569903_313_345_R
ATAGAAATTTCAT



1569903_207_239_F
GATGGGGATTT


TGCTATC


 


2573
TKT_NC002163-
TAGCCTTTAAC
213
TKT_NC002163-1569415-
TAAGACAAGGTTT
865



1569415-
GAAAATGTAAA

1569903_449_481_R
TGTGGATTTTTTA



1569903_350_383_F
AATGCGTTTTG


GCTTGTT




A


 


2574
TKT_NC002163-
TTCAAAAACTC
665
TKT_NC002163-1569415-
TTGCCATAGCAAA
1405



1569415-
CAGGCCATCCT

1569903_139_163_R
GCCTACAGCATT



1569903_60_92_F
GAAATTTCAAC


 


2575
GTLA_NC002163-
TCGTCTTTTTG
382
GLTA_NC002163-1604930-
TGCCATTTCCATG
1216



1604930-
ATTCTTTCCCT

1604529_139_168_R
TACTCTTCTCTAA



1604529_39_70_F
GATAATGCTC


CATT


 


2576
GLYA_NC002163-
TCAGCTATTTT
281
GLYA_NC002163-367572-
ATTGCTTCTTACT
756



367572-
TCCAGGTATCC

368079_476_508_R
TGCTTAGCATAAA



368079_386_414_F
AAGGTGG


TTTTCCA


2577
GLYA_NC002163-
TGGTGCGAGTG
611
GLYA_NC002163-367572-
TGCTCACCTGCTA
1246



367572-
CTTATGCTCGT

368079_242_270_R
CAACAAGTCCAGC



368079_148_174_F
ATTAT


AAT


 


2578
GLYA_NC002163-
TGTAAGCTCTA
622
GLYA_NC002163-367572-
TTCCACCTTGGAT
1381



367572-
CAACCCACAAA

368079_384_416_R
ACCTGGAAAAATA



368079_298_327_F
ACCTTACG


GCTGAAT


 


2579
GLYA_NC002163-
TGGTGGACATT
614
GLYA_NC002163-367572-
TCAAGCTCTACAC
961



367572-
TAACACATGGT

368079_52_81_R
CATAAAAAAAGCT



368079_1_27_F
GCAAA


CTCA


 


2580
PGM_NC002163-
TGAGCAATGGG
455
PGM_NC002163-327746-
TTTGCTCTCCGCC
1438



327746-
GCTTTGAAAGA

328270_356_379_R
AAAGTTTCCAC



328270_254_285_F
AAGAATTTTTA




AAT


 


2581
PGM_NC002163-
TGAAAAGGGTG
425
PGM_NC002163-327746-
TGCCCCATTGCTC
1219



327746-
AAGTAGCAAAT

328270_241_267_R
TCATGATAGTAGT



328270_153_182_F
GGAGATAG


AGCTAC


 


2582
PGM_NC002163-
TGGCCTAATGG
568
PGM_NC002163-327746-
TGCACGCAAACGC
1200



327746-
GCTTAATATCA

328270_79_102_R
TTTACTTCAGC



328270_19_50_F
ATGAAAATTG


 


2583
UNCA_NC002163-
TAAGCATGCTG
160
UNCA_NC002163-112166-
TGCCCTTTCTAAA
1220



112166-
TGGCTTATCGT

112647_196_225_R
AGTCTTGAGTGAA



112647_114_141_F
GAAATG


GATA


 


2584
UNCA_NC002163-
TGCTTCGGATC
532
UBCA_NC002163-112166-
TGCATGCTTACTC
1206



112166-
CAGCAGCACTT
532
112647_88_123_R
AAATCATCATAAA



112647_3_29_F
CAATA


CAATTAAAGC


 


2585
ASPA_NC002163-
TTAATTTGCCA
652
ASPA_NC002163-96692-
TGCAAAAGTAACG
1192



96692-
AAAATGCAACC

97166_403_432_R
GTTACATCTGCTC



97166_308_335_F
AGGTAG


CAAT


 


2586
ASPA_NC002163-
TCGCGTTGCAA
370
ASPA_NC002163-966692-
TCATGATAGAACT
991



96692-
CAAAACTTTCT

97166_316_346_R
ACCTGGTTGCATT



97166_228_258_F
AAAGTATGT


TTTGG


 


2587
GLNA_NC002163-
TGGAATGATGA
547
GLNA_NC002163-658085-
TGAGTTTGAACCA
1176



658085-
TAAAGATTTCG

657609_340_371_R
TTTCAGAGAGCGA



657609_244_275_F
CAGATAGCTA


ATATCTAC


 


2588
TKT_NC002163-
TCGCTACAGGC
371
TKT_NC002163-1569415-
TCCCCATCTCCGC
1020



1569415-
CCTTTAGGACA

1569903_212_236_R
AAAGACAATAAA



1569903_107_130_F
CAGATAGCTA


ATATCTAC


 


2589
TKT_NC002163-
TGTTCTTTAGC
642
TKT_NC002163-1569415-
TCCTTGTGCTTCA
1057



1569415-
AGGACTTCACA

1569903_361_393_R
AAACGCATTTTTA



1569903_265_296_F
AACTTGATAA


CATTTTC


 


2590
GLYA_NC002163-
TGCCTATCTTT
505
GLYA_NC002163-367572-
TCCTCTTGGGCCA
1047



367572-
TTGCTGATATA

368095_317_340_R
CGCAAAGTTTT



368095_214_246_F
GCACATATTGC


CGCAAAGTTTT


 


2591
GLYA_NC002163-
TCCTTTGATGC
353
GLYA_NC002163-367572-
TCTTGAGCATTGG
1141



367572-
ATGTAATTGCT

368095_485_516_R
TTCTTACTTGTTT



368095_415_444_F
GCAAAAGC


TGCATA


 


2592
PGM_NC002163_21_54_F
TCCTAATGGAC
332
PGM_NC002163_116_142_R
TCAAACGATCCGC
949




TTAATATCAAT


ATCACCATCAAAA




GAAAATTGTGG


G




G


 


2593
PGM_NC002163_149_176_F
TAGATGAAAAA
207
PGM_NC002163_247_277_R
TCCCCTTTAAAGC
1023




GGCGAAGTGGC


ACCATTACTCATT




TAATGG


ATAGT


 


2594
GLNA_NC002163-
TGTCCAAGAAG
633
GLNA_NC002163-658085-
TCAAAAACAAAGA



658085-
CATAGCAAAAA

657609_148_179_R
ATTCATTTTCTGG



657609_79_106_F
AAGCAA


TCCAAA


 


2595
ASPA_NC2163-
TCCTGTTATTC
347
ASPA_NC002163-96685-
TCAAGCTATATGC
960



96685-
CTGAAGTAGTT

97196_467_497_R
TACAACTGGTTCA



97196_367_402_F
AATCAAGTTTG


AAAAC




TTA


 


2596
ASPA_NC002163-
TGCCGTAATGA
502
ASPA_NC002163-96685-
TACAACCTTCGGA
880



96685-
TAGGTGAAGAT

97196_95_127_R
TAATCAGGATGAG



97196_1_33_F
ATACAAAGAGT

AATTAAT


 


2597
ASPA_NC002163-
TGGAACAGGAA
540
ASPA_NC002163-96685-
TAAGCTCCCGTAT
872



96685-
TTAATTCTCAT

97196_185_210_R
CTTGAGTCGCCTC



97196_85_117_F
CCTGATTATCC


 


2598
PGM_NC002163-
TGGCAGCTAGA
563
PGM_NC002163-327746-
TCACGATCTAAAT
975



327746-
ATAGTAGCTAA

328270_230_261_R
TTGGATAAGCCAT



328270_165_195_F
AATCCCTAC


AGGAAA


 


2599
PGM_NC002163-
TGGGTCGTGGT
593
PGM_NC002163-327746-
TTTTGCTCATGAT
1443



327746-
TTTACAGAAAA

328270_353_381_R
CTGCATGAAGCAT



328270_252_286_F
TTTCTTATATA


AAA


 


2600
PGM_NC002163-
TGGGATGAAAA
577
PGM_NC002163-327746-
TGATAAAAAGCAC
1178



327746-
AGCGTTCTTTT

328270_95_123_R
TAAGCGATGAAAC



328270_1_30_F
ATCCATGA


AGC


 


2601
PGM_NC002163-
TAAACACGGCT
146
PGM_NC002163-327746-
TCAAGTGCTTTTA
963



327746-
TTCCTATGGCT

328270_314_345_R
CTTCTATAGGTTT



328270_220_250_F
TATCCAAAT


AAGCTC


 


2602
UNCA_NC002163-
TGTAGCTTATC
628
UNCA_NC002163-112166-
TGCTTGCTCTTTC
1258



112166-
GCGAAATGTCT

112647_199_229_R
AAGCAGTCTTGAA



112647_123_152_F
TTGATTTT


TGAAG


 


2603
UBCA_NC002163-
TCCAGATGGAC
313
UNCA_NC002163-112166-
TCCGAAACTTGTT
1031



112166-
AAATTTTCTTA

112647_430_461_R
TGTAGCTTTAATT



112647_333_365_F
GAAACTGATTT


TGAGC


 


2734
GYRA_AY291534_237_264_F
TCACCCTCATG
265
GYRA_AY291534_268_288_R
TTGCGCCATACGT
1407




GTGATTCAGCT


ACCATCGT




GTTTAT


 


2735
GYRA_AY291534_224_252_F
TAATCGGTAAG
167
GYRA_AY291534_256_285_R
TGCCATACGTACC
1213




TATCACCCTCA


ATCGTTTCATAAA




TGGTGAT


CAGC


 


2736
GYRA_AY291534_170_198_F
TAGGAATTACG
221
GYRA_AY291534_268_288_R
TTGCGCCATACGT
1407




GCTGATAAAGC


ACCATCGT




GTATAAA


 


2737
GYRA_AY291534_224_252_F
TAATCGGTAAG
167
GYRA_AY291534_319_346_R
TATCGACAGATCC
935




ATCACCCTCAT


AAAGTTACCATGC




GGTGAT


CC


 


2738
GYRA_NC002953-7005-
TAAGGTATGAC
163
GYRA_NC002953-7005-
TCTTGAGCCATAC
1142



9668_166_195_F
ACCGGATAAAT

9668_265_287_R
GTACCATTGC




CATATAAA


GTACCATTGC


 


2739
GYRA_NC002953-7005-
TAATGGGTAAA
171
GYRA_NC002953-7005-
TATCCATTGAACC
933



9668_221_249_F
TATCACCCTCA

9668_316_343_R
AAAGTTACCTTGG




TGGTGAC


CC


 


2740
GYRA_NC002953-7005-
TAATGGGTAAA
171
GYRA_NC002953-7005-
TAGCCATACGTAC
912



9668_221_249_F
TATCACCCTCA

9668_253_283_R
CATTGCTTCATAA




TGGTGAC


AATAGA


 


2741
GYRA_NC002953-7005-
TCACCCTCATG
264
GYRA_NC002953-7005-
TCTTGAGCCATAC
1142



9668_234_261_F
GTGACTCATCT

9668_265_287_R
GTACCATTGC




ATTTAT


 


2842
CAPC_AF188935-
TGGGATTATTG
578
CAPC_AF188935-56074-
TGGTAACCCTTGT
1299



56074-
TTATCCTGTTA

55628_348_378_R
CTTTGAATTGTAT



55628_271_304_F
TGCCATTTGAG


TTGCA




A


 


2843
CAPC_AF188935-
TGATTATTGTT
476
CAPC_AF188935-56074-
TGTAACCCTTGTC
1344



56074-
ATCCTGTTATG

55628_349_377P_R
TTTGAATpTpGTA



55628_273_303P_F
CpCpATpTpTp


TpTpTpGC




GAG


 


2844
CAPC_AF188935-
TCCGTTGATTA
331
CAPC_AF188935-56074-
TGTTAATGGTAAC
1344



56074-
TTGTTATCCTG

55628_349_384_R
CCTTGTCTTTGAA



55628_268_303_F
TTATGCCATTT


TTGTATTTGC




GAG


 


2845
CAPC_AF188935-
TCCGTTGATTA
331
CAPC_AF188935-56074-
TAACCCTTGTCTT
860



56074-
TTGTTATCCTG

55628_337_375_R
TGAATTGTATTTG



55628_268_303_F
TTATGCCATTT


CAATTAATCCTGG




GAG


 


2846
PARC_X95819_33_58_F
TCAAAAAAAT
302
PARC_X95819_121_153_R
TAAAGGATAGCGG
852




CAGCGCGTACA


TAACTAAATGGCT




GTGG


GAGCCAT


 


2847
PARC_X95819_65_92_F
TACTTGGTAAA
199
PARC_X95819_157_178_R
TACCCCAGTTCCC
889




TACCACCCACA


CTGACCTTC




TGGTGA


 


2848
PARC_X95819_69_93_F
TGGTAAATACC
596
PARC_X95819_97_128_R
TGAGCCATGAGTA
1169




ACCCACACATG


CCATGGCTTCATA




GTGAC


ACATGC


 


2849
PARC_NC003997-
TTCCGTAAGTC
668
PARC_NC003997-3362578-
TCCAAGTTTGACT
1001



3362578-
GGCTAAAACAG

3365001_256_283_R
TAAACGTACCATC



3365001_181_205_F
TCG


GC


 


2850
PARC_NC003997-
TGTAACTATCA
621
PARC_NC003997-3362578-
TCGTCAACACTAC
1099



3362578-
CCCGCACGGTG

3365001_304_335_R
CATTATTACCATG



3365001_217_240_F
AT


CATCTC


 


2851
PARC_NC003997-
TGTAACTATCA
621
PARC_NC003997-3362578-
TGACTTAAACGTA
1162



3362578-
CCCGCACGGTG

3365001_244_275_R
CCATCGCTTCATA



3365001_217_240_F
AT

TCATCTC


 


2852
GYRA_AY642140_1_24_F
TAAATCTGCCC
150
GYRA_AY642140_71_100_R
TGCTAAAGTCTTG
1242




CGTGTCGTTGG


AGCCATACGAACA




TGAC


ATGG


 


2853
GYRA_AY642140_26_54_F
TAATCGGTAAA
166
GYRA_AY642140_121_146_R
TCGATCGAACCGA
1069




TATCACCCGCA


AGTTACCCTGACC




TGGTGAC


 


2854
GYRA_AY642140_26_54_F
TAATCGGTAAA
166
GYRA_AY642140_58_89_R
TGAGCCATACGAA
1168




TATCACCCGCA


CAATGGTTTCATA




TGGTGAC


AACAGC


 


2860
CYA_AF065404_1348_1379_F
TCCAACGAAGT
305
CYA_AF065404_1448_1472_R
TCAGCTGTTAACG
983




ACAATACAAGA


GCTTCAAGACCC




CAAAAGAAGG


 


2861
FEF_BA_AF065404_751_781_F
TCGAAAGCTTT
354
LEF_BA_AF065404_843_881_R
TCTTTAAGTTCTT
1144




TGCATATTATA


CCAAGGATAGATT




TCGAGCCAC


TATTTCTTGTTCG


 


2862
LEF_BA_AF065404_762_788_F
TGCATATTATA
498
LEF_BA_AF065404_843_881_R
TCTTTAAGTTCTT
1144




TCGAGCCACAG


CCAAGGATAGATT




CATCG


TATTTCTTGTTCG


 


2917
MUTS_AY698802_106_125_F
TCCGCTGAATC
326
MUTS_AY698802_172_193_R
TGCGGTCTGGCGC
1237




TGTCGCCGC


ATATAGGTA


 


2918
MUTS_AY698802_172_192_F
TACCTATATGC
187
MUTS_AY698802_228_252_R
TCAATCTCGACTT
965




GCCAGACCGC


TTTGTGCCGGTA


 


2919
MUTS_AY698802_228_252_F
TACCGGCGCAA
186
MUTS_AY698802_314_342_R
TCGGTTTCAGTCA
1097




AAAGTCGAGAT


TCTCCACCATAAA




TGG


GGT


 


2920
MUTS_AY698802_315_342_F
TCTTTATGGTG
419
MUTS_AY698802_413_433_R
TGCCAGCGACAGA
1210




GAGATGACTGAAA


CCATCGTA




CCGA


 


2921
MUTS_AY698802_394_411_F
TGGGCGTGGAA
585
MUTS_AY698802_497_519_R
TCCGGTAACTGGG
1040




CGTCCAC


TCAGCTCGAA


 


2922
AB_MLST-11-
TGGGcGATGCT
583
AB_MLST-11-
TAGTATCACCACG
923



OIF007_991_1018_F
GCgAAATGGTT

OIF007_1110_1137_R
TACACCCGGATCA




AAAAGA


GT


 


2927
GAPA_NC002505_694_721_F
TCAATGAACGA
259
GAPA_NC_002505_29_58_R_1
TCCTTTATGCAAC
1060




CCAACAAGTGA


TTGGTATCAACAG




TTGATG


GAAT


 


2928
GAPA_NC002505_694_721_2_F
TCGATGAACGA
361
GAPA_NC002505_769_798_2_R
TCCTTTATGCAAC
1061




CCAACAAGTGA


TTGGTATCAACCG




TTGATG


GAAT


 


2929
GAPA_NC002505_694_721_2_F
TCGATGAACGA
361
GAPA_NC002505_769_798_3_R
TCCTTTATGCAAC
1059




CCAACAAGTGA


TTAGTATCAACCG




TTGATG


GAAT


 


2932
INFB_EC_1364_1394_F
TTGCTCGTGGT
688
INFB_EC_1439_1468_R
TTGCTGCTTTCGC
1410




GCACAAGTAA


ATGGTTAATCGCT




CGGATATTAC


TCAA


 


2933
INFB_EC_1364_1394_2_F
TTGCTCGTGGT
689
INFB_EC_1439_1468_R
TTGCTGCTTTCGC
1410




GCAIAAGTAA


ATGGTTAATCGCT




CGGATATIAC


TCAA


 


2934
INFB_EC_80_110_F
TTGCCCGCGGT
685
INFB_EC_1439_1468_R
TTGCTGCTTTCGC
1410




GCGGAAGTAAC


ATGGTTAATCGCT




CGATATTAC


TCAA


 


2949
ACS_NC002516-
TCGGCGCCTGC
376
ACS_NC002516-970624-
TGGACCACGCCGA
1265



970624-
CTGATGA

971013_364_383_R
AGAACGG


 


2950
ARO_NC002516_26883-
TCACCGTGCCG
267
ARO_NC002516-26883-
TGTGTTGTCGCCG
1341



27380_4_26_F
TTCAAGGAAGA

27380_111_128_R
CGCAG




G


 


2951
ARO_NC002516-26883-
TTTCGAAGGGC
705
ARO_NC002516-26883-
TCCTTGGCATACA
1056



27380_356_377_F
CTTTCGACCTG

27380_459_484_R
TCATGTCGTAGCA


 


2952
GUA_NC002516-
TGGACTCCTCG
551
GUA_NC002516-4226546-
TCGGCGAACATGG
1091



4226546-
GTGGTCGC

4226174_127_146_R
CCATCAC


 


2953
GUA_NC002516-
TGACCAGGTGA
448
GUA_NC002516-4226546-
TGCTTCTCTTCCG
1256



4226546-
TGGCCATGTTC

4226174_214_233_R
GGTCGGC



4226174_120_142_F
G


 


2954
GUA_NC002516-
TTTTGAAGGTG
710
GUA_NC002516-4226546-
TGCTTGGTGGCTT
1259



4226546-
ATCCGTGCCAA

4226174_265_287_R
CTTCGTCGAA



4226174_155_178_F
CG


 


2955
GUA_NC002516-
TTCCTCGGCCG
670
GUA_NC002516-4226546-
TGCGAGGAACTTC
1229



4226546-
CCTGGC

4226174_288_309_R
ACGTCCTGC



4226174_190_206_F


 


2956
GUA_NC002516-
TCGGCCGCACC
374
GUA_NC002516-4226546-
TCGTGGGCCTTGC
1111



4226546-
TTCATCGAAGT

4226174_355_371_R
CGGT


 


2957
MUT_NC002516-
TGGAAGTCATC
545
MUT_NC002516-5551158-
TCACGGGCCAGCT
978



5551158-
AAGCGCCTGGC

5550717_99_116_R
CGTCT



5550717_5_26_F
AAGCGCCTGGC


 


2958
MUT_NC002516-
TCGAGCAGGC
358
MUT_NC002516-5551158-
TCACCATGCGCCC
971



5551158-
GCTGCCG

5550717_256_277_R
GTTCACATA



5550717_5_26_F


 


2959
NUO_NC002516-
TCAACCTCGGC
249
NUO_NC002516-2984589-
TCGGTGGTGGTAG
1095



2984589-
CCGAACCA

2984954_97_117_R
CCGATCTC


 


2960
NUO_NC002516-
TACTCTCGGTG
195
NUO_NC002516-2984589-
TTCAGGTACAGCA
1376



2984589-
GAGAAGCTCGC

2984954_301_326_R
GGTGGTTCAGGAT


 


2961
PPS_NC002516-
TCCACGGTCAT
311
PPS_NC002516-1915014-
TCCATTTCCGACA
1014



1915014-
GGAGCGCTA

1915383_140_165_R
CGTCGTTGATCAC



1915383_44_63_P


 


3962
PPS_NC002516-
TCGCCATCGTC
365
PPS_NC002516-1915014-
TCCTGGCCATCCT
1052



1915014-
ACCAACCG

1915383_341_360_R
GCAGGAT



1915383_240_258_F


 


2963
TRP_NC002516-
TGCTGGTACGG
527
TRP_NC002516-671831-
TCGATCTCCTTG
1071



671831-
GTCGAGGA

672273_131_150_R
GCGTCCGA



672273_24_42_F


 


2964
TRP_NC002516-
TGCACATCGTG
490
TRP_NC002516-671831-
TGATCTCCATGGC
1182



671831-
TCCAACGTCAC

672273_362_383_R
GCGGATCTT



672273_261_282_F


 


2972
AB_MLST-11-
TGGGIGATGCT
592
AB_MLST-11-
TAGTATCACCACG
924



OIF007_1007_1034_F
GCIAAATGGTT

OIF007_1126_1153_R
TACICCIGGATCA




AAAAGA


GT


 


2993
OMPU_NC002505-
TTCCCACCGAT
667
OMPU_NC002505_544_567_R
TCGGTCAGCAAAA
1094



674828-
ATCATGGCTTA


CGGTAGCTTGC



675880_428_455_F


 


2994
GAPA_NC002505-
TCCTCAATGAA
335
GAPA_NC002505-506780-
TTTTCCCTTTATG
1442



506780
CGAICAACAAG

507937_769_802_R
CAACTTAGTATCA



507937_691_721_F
TGATTGATG


ACIGGAAT


 


2995
GAPA_NC002505-
TCCTCIATGAA
339
GAPA_NC002505-506780-
TCCATACCTTTAT
1008



506780-
ACGAICAACAA

507937_769_803_R
GCAACTTIGTAT



507937_691_721_2_F
GTGATTGATG


CAACIGGAAT


 


2996
GAPA_NC002505-
TCCTCGATGAA
396
GAPA_NC002505-506780-
TCGGAAATATTCT
1085



506780-
CCGACCAACAA

507937_785_817_R
TTCAATACCTT



507937_692_721_F
GTGATTGATTG


TATGCAACT


 


2997
GAPA_NC002505-
TCCTCGATGAA
337
GAPA_NC002505-506780-
TCGGAAATATTCT
1085



506780-
CGAICAACAAG

507937_785_817_R
TTCAATACCTTTA



507937_691_721_3_F
TIATTGATG


TGCAACT


 


2998
GAPA_NC002505-
TCCTCAATGAA
336
GAPA_NC002505-506780-
TCGGAAATATTCT
1087



506780-
TGATCAACAAG

507937_784_817_R
TTCAATICCTTTI



507937_691_721_4_F
TGATTGATG


TGCAACTT


 


2999
GAPA_NC002505-
TCCTCIATGAA
340
GAPA_NC002505-506780-
TCGGAAATATTCT
1086



506780-
IGAICAACAAG

507937_784_817_2_R
TTCAATACCTTTA



507937_691_721_5_F
TIATTGATG


TGCAACTT


 


3000
GAPA_NC002505-
TCCTCGATGAA
338
GAPA_NC002505-506780-
TTTCAATACCTTT
1430



506780-
TGAICAACAAC

507937_769_805_R
GCAACTTIGTATC



507937_691_721_6_F
AAGTIATTGAT


AACIGGAAT




G


 


3001
CTXB_NC002505-
TCAGCATATGC
275
CTXB_NC002505-1566967-
TCCCGGCTAGAGA
1026



1566967-
ACATGGAACAC

1567341_139_163_R
TTCTGTATACGA



1567341_46_71_F
CTCA


TTCTGTATACGA


 


3002
CTXB_NC002505-
TCAGCATATGC
274
CTXB_NC002505-1566967-
TCCGGCTAGAGAT
1038



1566967-
ACATGGAACAC

1567341_132_162_R
TCTGTATACGAAA



1567341_46_70_F
CTC


ATATC


 


3003
CTXB_NC002505-
TCAGCATATGC
274
CTXB_NC002505-1566967-
TGCCGTATACGAA
1225



1566967-
ACATGGAACAC

1567341_118_150_R
AATATCTTATCAT



1567341_46_70_F
CTC


TTAGCGT


 


3004
TUFB_NC002758-
TACAGGCCGTG
180
TUFB_NC002758-615038-
TCAGCGTAGTCTA
982



615038-
TTGAACGTGG

61622_778_809_R
ATAATTTACGGAA



616222_684_704_F



CATTTC


 


3005
TUFB_NC002758-
TGCCGTGTTGA
503
TUFB_NC0027858-615038-
TGCTTCAGCGTAG
1255



615038-
ACGTGGTCAAA

616222_783_813_R
TCTAATAATTTAC



616222_688_710_F
T


GGAAC


 


3006
TUFB_NC0027858-
TGTGGTCAAAT
638
TUFB_NC002758-615038-
TGCGTAGTCTAAT
1238



615038-
CAAAGTTGGTG

616222_778_807_R
AATTTACGGAACA



616222_700_726_F
AAGA


TTTC


 


3007
TUFB_NC002758-
TGGTCAAATCA
607
TUFB_NC002758-615038-
TGCGTAGTCTAAT
1238



615038-
AAGTTGGTGAAG

616222_778_807_R
AATTTACGGAACA



616222_702_726_F
AA


TTTC


 


3008
TUFB_NC002758-
TGAACGTGGTC
431
TUFB_NC002758-615038-
TCACCAGCTTCAG
970



615038-
AAATCAAAGTT

616222_785_818_R
CGTAGTCTAATAA



616222_696_726_F
GGTGAAGAA


TTTACGGA


 


3009
TUFB_NC002758-
TCGTGTTGAAC
386
TUFB_NC002758-615038-
TCTTCAGCGCGTA
1134



615038-
GTGGTCAAATC

616222_778_812_R
GTCTAATAATTTA



616222_690_716_F
AAAGT


CGGAACATTTC


 


3010
MECI-R-NC003923-
TCACATATCGT
261
MECI-R_NC003923-41798-
TGTGATATGGAGG
1332



41798-41609_36_59_F
GAGCAATGAAC

41609_89_112_R
TGTAGAAGGTG




TG


 


3011
MECI-R_NC003923-
TGGGCGTGAGC
584
MECI-R_NC003923-41798-
TGGGATGGAGGTG
1287



41798-41609_40_66_F
AATGAACTGAT

41609_81_110_R
TAGAAGGTGTTAT




TATAC


CATC


 


3012
MECI-R_NC003923-
TGGACACATAT
549
MECI-R_NC003923-41798-
TGGGATGGAGGTG
1286



41798-
CGTGAGCAATG

41609_81_110_R
TAGAAAGGTGTTA



41609_33_60_2_F
AACTGA


TCATC


 


3013
MECI-R_NC003923-
TGGGTTTACAC
595
MECI-R_NC003923-41798-
TGGGGATATGGAG
1290



41798-41609_29_60_F
ATATCGTGAGC

41609_81_113_R
TGTAGAAGGTGTT




AATGAACTGA


ATCATC


 


3014
MUPR_X75439_2409_2513_F
TGGGCTCTTTC
587
MUPR_X75439_2548_2570_R
TCTGGCTGCGGAA
1130




TCGCTTAAACA


GTGAAATCGT




CCT


 


3015
MUPR_X75439_2482_2510_F
TGGGCTCTTTC
586
MUPR_X75439_2547_2568_R
TGGCTGCGGAAGT
1281




TCGCTTAAACA


GAAATCGTA




CC


 


3016
MUPR_X75439_2482_2510_F
TAGATAATTGG
205
MUPR_X75439_2551_2573_R
TAATCTGGCTGCG
876




GCTCTTTCTCG


GAAGTGAAAT




CTTAAAC


 


3017
MUPR_X75439_2490_2514_F
TGGGCTCTTTC
587
MUPR_X75439_2549_2573_R
TAATCTGGCTGCG
877




TCGCTTAAACA


GAAGTGAAATCG




CCT


 


3018
MUPR_X75439_2482_2510_F
TAGATAATTGG
205
MUPR_X75439_2559_2589_R
TGGTATATTCGTT
1303




GCTCTTTCTCG


AATTAATCTGGCT




CTTAAAC


GCGGA


 


3019
MUPR_X75439_2490_2514_F
TGGGCTCTTTC
587
MUPR_X75439_2554_2581_R
TCGTTAATTAATC
1112




GCTTAAACACC


TGGCTGCGGAAGT




T


GA


 


3020
AROE_NC003923-
TGATGGCAAGT
474
AROE_NC003923-1674726-
TAAGCAATACCTT
1378



1674726-
GGATAGGGTAT

1674277_309_335_R
TACTTGCACCACC



1674277_204_232_F
AATACAG


ACCT


 


3021
AROE_NC003923-
TGGCGAGTGGA
570
AROE_NC003923-1674726-
TTCATAAGCAATA
1378



1674726-
TAGGGTATAAT

1674277_311_339_R
CCCTTTACTTGCA



1674277_207_232_F
ACAG


CCAC


 


3022
AROE_NC003923-
TGGCpAAGTpG
572
AROE_NC003923-1674726-
TAAGCAATACCpT
867



1674726-
GATpAGGGTpA

1674277_311_335P_R
pTpTpACTpTpGC



1674277_207_232P_F
TpAATpACpAG


pACpCpAC


 


3023
ARCC_NC003923-
TCTGAAATGAA
398
ARCC_NC003923-2725050-
TCTTCTTCTTTCG
1137



2725050-
TAGTGATAGAA

2724595_214_245_R
TATAAAAAGGACC



2724595_124_155_F
CTGTAGGCAC


AATTGG


 


3024
ARCC_NC003923-
TGAATAGTGAT
437
ARCC_NC003923-2725050-
TCTTCTTTTCGTAT
1139



2725050-
AGAACTGTAGG

2724595_212_242_R
AAAAAGGACCAAT



2724595_131_161_F
CACAATCGT


TGGTT


 


3025
ARCC_NC003923-
TGAATAGTGAT
437
ARCC_NC003923-2725050-
TGCGCTAATTCTT
1232



2725050-
AGAACTGTAGG

2724595_232_260_R
CAACTTCTTCTTT



2724595_131_161_F
CACAATCGT


CGT


 


3026
PTA_NC003923-
TACAATGCTTG
177
PTA_NC003923-628885-
TGTTCTTGATACA
1350



628885-
TTTATGCTGGTA

629355_322_351_R
CCTGGTTTCGTTT



629355_231_259_F
AAGCAG


TGAT


 


3027
PTA_NC003923-
TACAATGCTTG
177
PTA_NC003923-628885-
TGGTACACCTGGT
1301



62885-
TTTATGCTGGT

629355_314_345_R
TTCGTTTTGATGA



629355_231_259_F
AAAGCAG


TTTGTA


 


3028
PTA_NC003923-
TCTTGTTTATG
418
PTA_NC003923-628885-
TGTTCTTGATACA
1350



628885-
CTGGTAAAAGC

629355_322_351_R
CCTGGTTTCGTTT



629355_237_263_F
AGATGG


TGAT









Primer pair name codes and reference sequences are shown in Table 3. The primer name code typically represents the gene to which the given primer pair is targeted. The primer pair name may include specific coordinates with respect to a reference sequence defined by an extraction of a section of sequence or defined by a GenBank gi number, or the corresponding complementary sequence of the extraction, or the entire GenBank gi number as indicated by the label “no extraction.” Where “no extraction” is indicated for a reference sequence, the coordinates of a primer pair named to the reference sequence are with respect to the GenBank gi listing. Gene abbreviations are shown in bold type in the “Gene Name” column.
To determine the exact primer hybridization coordinates of a given pair of primers on a given bioagent nucleic acid sequence and to determine the sequences, molecular masses and base compositions of an amplification product to be obtained upon amplification of nucleic acid of a known bioagent with known sequence information in the region of interest with a given pair of primers, one with ordinary skill in bioinformatics is capable of obtaining alignments of the primers of the present invention with the GenBank gi number of the relevant nucleic acid sequence of the known bioagent. For example, the reference sequence GenBank gi numbers (Table 3) provide the identities of the sequences which can be obtained from GenBank. Alignments can be done using a bioinformatics tool such as BLASTn provided to the public by NCBI (Bethesda, Md.). Alternatively, a relevant GenBank sequence may be downloaded and imported into custom programmed or commercially available bioinformatics programs wherein the alignment can be carried out to determine the primer hybridization coordinates and the sequences, molecular masses and base compositions of the amplification product. For example, to obtain the hybridization coordinates of primer pair number 2095 (SEQ ID NOs: 456:1261), First the forward primer (SEQ ID NO: 456) is subjected to a BLASTn search on the publicly available NCBI BLAST website. “RefSeq_Genomic” is chosen as the BLAST database since the gi numbers refer to genomic sequences. The BLAST query is then performed. Among the top results returned is a match to GenBank gi number 21281729 (Accession Number NC—003923). The result shown below, indicates that the forward primer hybridizes to positions 1530282.1530307 of the genomic sequence of Staphylococcus aureus subsp. aureus MW2 (represented by gi number 21281729).

    Staphylococcus aureus subsp. aureus MW2, complete genome Length=2820462
    Features in this part of subject sequence:
    
        Panton-Valentine leukocidin chain F precursor
    
    
    Score=52.0 bits (26), Expect=2e-05
    Identities=26/26 (100%), Gaps=0/26 (0%)
    Strand=Plus/Plus






The hybridization coordinates of the reverse primer (SEQ ID NO: 1261) can be determined in a similar manner and thus, the bioagent identifying amplicon can be defined in terms of genomic coordinates. The query/subject arrangement of the result would be presented in Strand=Plus/Minus format because the reverse strand hybridizes to the reverse complement of the genomic sequence. The preceding sequence analyses are well known to one with ordinary skill in bioinformatics and thus, Table 3 contains sufficient information to determine the primer hybridization coordinates of any of the primers of Table 2 to the applicable reference sequences described therein.







TABLE 3







Primer Name Codes and Reference Sequence













Reference





GenBank gi


Primer name code
Gene Name
Organism
number













16S_EC
16S rRNA (16S ribosomal RNA gene)
Escherichia coli
16127994


23S_EC
23S rRNA (23S ribosomal RNA gene)
Escherichia coli
16127994


CAPC_BA
capC (capsule biosynthesis gene)
Bacillus anthracis
6470151


CYA_BA
cya (cyclic AMP gene)
Bacillus anthracis
4894216


DNAK_EC
dnaK (chaperone dnaK gene)
Escherichia coli
16127994


GROL_EC
groL (chaperonin groL)
Escherichia coli
16127994


HFLB_EC
hflb (cell division protein peptidase
Escherichia coli
16127994



ftsH)


INFB_EC
infB (protein chain initiation factor
Escherichia coli
16127994



infB gene)


LEF_BA
lef (lethal factor)
Bacillus anthracis
21392688


PAG_BA
pag (protective antigen)
Bacillus anthracis
21392688


RPLB_EC
rplB (50S ribosomal protein L2)
Escherichia coli
16127994


RPOB_EC
rpoB (DNA-directed RNA polymerase beta
Escherichia coli
6127994



chain)


RPOC_EC
rpoC (DNA-directed RNA polymerase
Escherichia coli
16127994



beta' chain)


SP101ET_SPET_11
Artificial Sequence Concatenation
Artificial
15674250



comprising:
Sequence* -



gki (glucose kinase)
partial gene



gtr (glutamine transporter protein)
sequences of



murI (glutamate racemase)
Streptococcus



mutS (DNA mismatch repair protein)
pyogenes



xpt (xanthine phosphoribosyl



transferase)



yqiL (acetyl-CoA-acetyl transferase)



tkt (transketolase)


SSPE_BA
sspE (small acid-soluble spore
Bacillus anthracis
30253828



protein)


TUFB_EC
tufB (Elongation factor Tu)
Escherichia coli
16127994


VALS_EC
valS (Valyl-tRNA synthetase)
Escherichia coli
16127994


ASPS_EC
aspS (Aspartyl-tRNA synthetase)
Escherichia coli
16127994


CAF1_AF053947
caf1 (capsular protein caf1)
Yersinia pestis
2996286


INV_U22457
inv (invasin)
Yersinia pestis
1256565


LL_NC003143
Y. pestis specific chromosomal genes -
Yersinia pestis
16120353



difference region


BONTA_X52066
BoNT/A (neurotoxin type A)
Clostridium
40381




botulinum


MECA_Y14051
mecA methicillin resistance gene
Staphylococcus
2791983




aureus


TRPE_AY094355
trpE (anthranilate synthase (large
Acinetobacter
20853695



component))
baumanii


RECA_AF251469
recA (recombinase A)
Acinetobacter
9965210




baumanii


GYRA_AF100557
gyrA (DNA gyrase subunit A)
Acinetobacter
4240540




baumanii


GYRB_AB008700
gyrB (DNA gyrase subunit B)
Acinetobacter
4514436




baumanii


WAAA_Z96925
waaA (3-deoxy-D-manno-octulosonic-acid
Acinetobacter
2765828



transferase)
baumanii


CJST_CJ
Artificial Sequence Concatenation
Artificial
15791399



comprising:
Sequence* -



tkt (transketolase)
partial gene



glyA (serine hydroxymethyltransferase)
sequences of



gltA (citrate synthase)
Campylobacter



aspA (aspartate ammonia lyase)
jejuni



glnA (glutamine synthase)



pgm (phosphoglycerate mutase)



uncA (ATP synthetase alpha chain)


RNASEP_BDP
RNase P (ribonuclease P)
Bordetella
33591275




pertussis


RNASEP_BKM
RNase P (ribonuclease P)
Burkholderia
53723370




mallei


RNASEP_BS
RNase P (ribonuclease P)
Bacillus subtilis
16077068


RNASEP_CLB
RNase P (ribonuclease P)
Clostridium
18308982




perfringens


RNASEP_EC
RNase P (ribonuclease P)
Escherichia coli
16127994


RNASEP_RKP
RNase P (ribonuclease P)
Rickettsia
15603881




prowazekii


RNASEP_SA
RNase P (ribonuclease P)
Staphylococcus
15922990




aureus


RNASEP_VBC
RNase P (ribonuclease P)
Vibrio cholerae
15640032


ICD_CXB
icd (isocitrate dehydrogenase)
Coxiella burnetii
29732244


IS1111A
multi-locus IS1111A insertion element
Acinetobacter
29732244




baumannii


OMPA_AY485227
ompA (outer membrane protein A)
Rickettsia
40287451




prowazekii


OMPB_RKP
ompB (outer membrane protein B)
Rickettsia
15603881




prowazekii


GLTA_RKP
gltA (citrate synthase)
Vibrio cholerae
15603881


TOXR_VBC
toxR (transcription regulator toxR)
Francisella
15640032




tularensis


ASD_FRT
asd (Aspartate semialdehyde
Francisella
56707187



dehydrogenase)
tularensis


GALE_FRT
galE (UDP-glucose 4-epimerase)
Shigella flexneri
56707187


IPAH_SGF
ipaH (invasion plasmid antigen)
Campylobacter
30061571




jejuni


HUPB_CJ
hupB (DNA-binding protein Hu-beta)
Coxiella burnetii
15791399


AB_MLST
Artificial Sequence Concatenation
Artificial
Sequenced



comprising:
Sequence* -
in-house



trpE (anthranilate synthase component
partial gene
(SEQ ID



I))
sequences of
NO: 1444)



adk (adenylate kinase)
Acinetobacter



mutY (adenine glycosylase)
baumannii



fumC (fumarate hydratase)



efp (elongation factor p)



ppa (pyrophosphate phospho-



hydratase


MUPR_X75439
mupR (mupriocin resistance gene)
Staphylococcus
438226




aureus


PARC_X95819
parC (topoisomerase IV)
Acinetobacter
1212748




baumannii


SED_M28521
sed (enterotoxin D)
Staphylococcus
1492109




aureus


PLA_AF053945
pla (plasminogen activator)
Yersinia pestis
2996216


SEJ_AF053140
sej (enterotoxin J)
Staphylococcus
3372540




aureus


GYRA_NC000912
gyrA (DNA gyrase subunit A)
Mycoplasma
13507739




pneumoniae


ACS_NC002516
acsA (Acetyl CoA Synthase)
Pseudomonas
15595198




aeruginosa


ARO_NC002516
aroE (shikimate 5-dehydrogenase
Pseudomonas
15595198




aeruginosa


GUA_NC002516
guaA (GMP synthase)
Pseudomonas
15595198




aeruginosa


MUT_NC002516
mutL (DNA mismatch repair protein)
Pseudomonas
15595198




aeruginosa


NUO_NC002516
nuoD (NADH dehydrogenase I chain C, D)
Pseudomonas
15595198




aeruginosa


PPS_NC002516
ppsA (Phosphoenolpyruvate synthase)
Pseudomonas
15595198




aeruginosa


TRP_NC002516
trpE (Anthranilate synthetase
Pseudomonas
15595198



component I)
aeruginosa


OMP2_NC000117
ompB (outer membrane protein B)
Chlamydia
15604717




trachomatis


OMPA_NC000117
ompA (outer membrane protein B)
Chlamydia
15604717




trachomatis


GYRA_NC000117
gyrA (DNA gyrase subunit A)
Chlamydia
15604717




trachomatis


CTXA_NC002505
ctxA (Cholera toxin A subunit)
Vibrio cholerae
15640032


CTXB_NC002505
ctxB (Cholera toxin B subunit)
Vibrio cholerae
15640032


FUR_NC002505
fur (ferric uptake regulator protein)
Vibrio cholerae
15640032


GAPA_NC_002505
gapA (glyceraldehyde-3-phosphate
Vibrio cholerae
15640032



dehydrogenase)


GYRB_NC002505
gyrB (DNA gyrase subunit B)
Vibrio cholerae
15640032


OMPU_NC002505
ompU (outer membrane protein)
Vibrio cholerae
15640032


TCPA_NC002505
tcpA (toxin-coregulated pilus)
Vibrio cholerae
15640032


ASPA_NC002163
aspA (aspartate ammonia lyase)
Campylobacter
15791399




jejuni


GLNA_NC002163
glnA (glutamine synthetase)
Campylobacter
15791399




jejuni


GLTA_NC002163
gltA (glutamate synthase)
Campylobacter
15791399




jejuni


GLYA_NC002163
glyA (serine hydroxymethyltransferase)
Campylobacter
15791399




jejuni


PGM_NC002163
pgm (phosphoglyceromutase)
Campylobacter
15791399




jejuni


TKT_NC002163
tkt (transketolase)
Campylobacter
15791399




jejuni


UNCA_NC002163
uncA (ATP synthetase alpha chain)
Campylobacter
15791399




jejuni


AGR-III_NC003923
agr-III (accessory gene regulator-III)
Staphylococcus
21281729




aureus


ARCC_NC003923
arcC (carbamate kinase)
Staphylococcus
21281729




aureus


AROE_NC003923
aroE (shikimate 5-dehydrogenase
Staphylococcus
21281729




aureus


BSA-A_NC003923
bsa-a (glutathione peroxidase)
Staphylococcus
21281729




aureus


BSA-B_NC003923
bsa-b (epidermin biosynthesis protein
Staphylococcus
21281729



EpiB)
aureus


GLPF_NC003923
glpF (glycerol transporter)
Staphylococcus
21281729




aureus


GMK_NC003923
gmk (guanylate kinase)
Staphylococcus
21281729




aureus


MECI-R_NC003923
mecR1 (truncated methicillin
Staphylococcus
21281729



resistance protein)
aureus


PTA_NC003923
pta (phosphate acetyltransferase)
Staphylococcus
21281729




aureus


PVLUK_NC003923
pvluk (Panton-Valentine leukocidin
Staphylococcus
21281729



chain F precursor)
aureus


SA442_NC003923
sa442 gene
Staphylococcus
21281729




aureus


SEA_NC003923
sea (staphylococcal enterotoxin A
Staphylococcus
21281729



precursor)
aureus


SEC_NC003923
sec4 (enterotoxin type C precursor)
Staphylococcus
21281729




aureus


TPI_NC003923
tpi (triosephosphate isomerase)
Staphylococcus
21281729




aureus


YQI_NC003923
yqi (acetyl-CoA C-acetyltransferase
Staphylococcus
21281729



homologue)
aureus


GALE_AF513299
galE (galactose epimerase)
Francisella
23506418




tularensis


VVHA_NC004460
vVhA (cytotoxin, cytolysin precursor)
Vibrio vulnificus
27366463


TDH_NC004605
tdh (thermostable direct hemolysin A)
Vibrio
28899855




parahaemolyticus


AGR-II_NC002745
agr-II (accessory gene regulator-II)
Staphylococcus
29165615




aureus


PARC_NC003997
parC (topoisomerase IV)
Bacillus anthracis
30260195


GYRA_AY291534
gyrA (DNA gyrase subunit A)
Bacillus anthracis
31323274


AGR-I_AJ617706
agr-I (accessory gene regulator-I)
Staphylococcus
46019543




aureus


AGR-IV_AJ617711
agr-IV (accessory gene regulator-III)
Staphylococcus
46019563




aureus


BLAZ_NC002952
blaZ (beta lactamase III)
Staphylococcus
49482253




aureus


ERMA_NC002952
ermA (rRNA methyltransferase A)
Staphylococcus
49482253




aureus


ERMB_Y13600
ermB (rRNA methyltransferase B)
Staphylococcus
49482253




aureus


SEA-SEE_NC002952
sea (staphylococcal enterotoxin A
Staphylococcus
49482253



precursor)
aureus


SEA-SEE_NC002952
sea (staphylococcal enterotoxin A
Staphylococcus
49482253



precursor)
aureus


SEE_NC002952
sea (staphylococcal enterotoxin A
Staphylococcus
49482253



precursor)
aureus


SEH_NC002953
seh (staphylococcal enterotoxin H)
Staphylococcus
49484912




aureus


ERMC_NC005908
ermC (rRNA methyltransferase C)
Staphylococcus
49489772




aureus


MUTS_AY698802
mutS (DNA mismatch repair protein)
Shigella boydii
52698233


NUC_NC002758
nuc (staphylococcal nuclease)
Staphylococcus
57634611




aureus


SEB_NC002758
seb (enterotoxin type B precursor)
Staphylococcus
57634611




aureus


SEG_NC002758
seg (staphylococcal enterotoxin G)
Staphylococcus
57634611




aureus


SEI_NC002758
sei (staphylococcal enterotoxin I)
Staphylococcus
57634611




aureus


TSST_NC002758
tsst (toxic shock syndrome toxin-1)
Staphylococcus
57634611




aureus


TUFB_NC002758
tufB (Elongation factor Tu)
Staphylococcus
57634611




aureus





Note:


artificial reference sequences represent concatenations of partial gene extractions from the indicated reference gi number. Partial sequences were used to create the concatenated sequence because complete gene sequences were not necessary for primer design.






Example 2
Sample Preparation and PCR
Genomic DNA was prepared from samples using the DNeasy Tissue Kit (Qiagen, Valencia, Calif.) according to the manufacturer's protocols.
All PCR reactions were assembled in 50 μL reaction volumes in a 96-well microtiter plate format using a Packard MPII liquid handling robotic platform and M. J. Dyad thermocyclers (MJ research, Waltham, Mass.) or Eppendorf Mastercycler thermocyclers (Eppendorf, Westbury, NY). The PCR reaction mixture consisted of 4 units of Amplitaq Gold, 1× buffer II (Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl2, 0.4 M betaine, 800 μM dNTP mixture and 250 nM of each primer. The following typical PCR conditions were used: 95° C. for 10 min followed by 8 cycles of 95° C. for 30 seconds, 48° C. for 30 seconds, and 72° C. 30 seconds with the 48° C. annealing temperature increasing 0.9° C. with each of the eight cycles. The PCR was then continued for 37 additional cycles of 95° C for 15 seconds, 56° C. for 20 seconds, and 72° C. 20 seconds.
Example 3
Purification of PCR Products for Mass Spectrometry with Ion Exchange Resin-Magnetic Beads
For solution capture of nucleic acids with ion exchange resin linked to magnetic beads, 25 μl of a 2.5 mg/mL suspension of BioClone amine terminated superparamagnetic beads were added to 25 to 50 μl of a PCR (or RT-PCR) reaction containing approximately 10 μM of a typical PCR amplification product. The above suspension was mixed for approximately 5 minutes by vortexing or pipetting, after which the liquid was removed after using a magnetic separator. The beads containing bound PCR amplification product were then washed three times with 50 mM ammonium bicarbonate/50% MeOH or 100 mM ammonium bicarbonate/50% MeOH, followed by three more washes with 50% MEOH. The bound PCR amplicon was eluted with a solution of 25 mM piperidine, 25 mM imidazole, 35% MeOH which included peptide calibration standards.
Example 4
Mass Spectrometry and Base Composition Analysis
The ESI-FTICR mass spectrometer is based on a Bruker Daltonics (Billerica, MA) Apex II 70e electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer that employs an actively shielded 7 Tesla superconducting magnet. The active shielding constrains the majority of the fringing magnetic field from the superconducting magnet to a relatively small volume. Thus, components that might be adversely affected by stray magnetic fields, such as CRT monitors, robotic components, and other electronics, can operate in close proximity to the FTICR spectrometer. All aspects of pulse sequence control and data acquisition were performed on a 600 MHz Pentium II data station running Bruker's Xmass software under Windows NT 4.0 operating system. Sample aliquots, typically 15 μl, were extracted directly from 96-well microtiter plates using a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, N.C.) triggered by the FTICR data station. Samples were injected directly into a 10 μl sample loop integrated with a fluidics handling system that supplies the 100 μl/hr flow rate to the ESI source. Ions were formed via electrospray ionization in a modified Analytica (Branford, Conn.) source employing an off axis, grounded electrospray probe positioned approximately 1.5 cm from the metalized terminus of a glass desolvation capillary. The atmospheric pressure end of the glass capillary was biased at 6000 V relative to the ESI needle during data acquisition. A counter-current flow of dry N2 was employed to assist in the desolvation process. Ions were accumulated in an external ion reservoir comprised of an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode, prior to injection into the trapped ion cell where they were mass analyzed. Ionization duty cycles greater than 99% were achieved by simultaneously accumulating ions in the external ion reservoir during ion detection. Each detection event consisted of 1 M data points digitized over 2.3 s. To improve the signal-to-noise ratio (S/N), 32 scans were co-added for a total data acquisition time of 74 s.
The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOFTM. Ions from the ESI source undergo orthogonal ion extraction and are focused in a reflectron prior to detection. The TOF and FTICR are equipped with the same automated sample handling and fluidics described above. Ions are formed in the standard MicroTOFTM ESI source that is equipped with the same off-axis sprayer and glass capillary as the FTICR ESI source. Consequently, source conditions were the same as those described above. External ion accumulation was also employed to improve ionization duty cycle during data acquisition. Each detection event on the TOF was comprised of 75,000 data points digitized over 75 μs.
The sample delivery scheme allows sample aliquots to be rapidly injected into the electrospray source at high flow rate and subsequently be electrosprayed at a much lower flow rate for improved ESI sensitivity. Prior to injecting a sample, a bolus of buffer was injected at a high flow rate to rinse the transfer line and spray needle to avoid sample contamination/carryover. Following the rinse step, the autosampler injected the next sample and the flow rate was switched to low flow. Following a brief equilibration delay, data acquisition commenced. As spectra were co-added, the autosampler continued rinsing the syringe and picking up buffer to rinse the injector and sample transfer line. In general, two syringe rinses and one injector rinse were required to minimize sample carryover. During a routine screening protocol a new sample mixture was injected every 106 seconds. More recently a fast wash station for the syringe needle has been implemented which, when combined with shorter acquisition times, facilitates the acquisition of mass spectra at a rate of just under one spectrum/minute.
Raw mass spectra were post-calibrated with an internal mass standard and deconvoluted to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides. Quantitative results are obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 500 molecules per well. Calibration methods are commonly owned and disclosed in U.S. Provisional Patent Application Ser. No. 60/545,425 which is incorporated herein by reference in entirety.
Example 5
De Novo Determination of Base Composition of Amplification Products using Molecular Mass Modified Deoxynucleotide Triphosphates
Because the molecular masses of the four natural nucleobases have a relatively narrow molecular mass range (A=313.058, G=329.052, C=289.046, T=304.046—See Table 4), a persistent source of ambiguity in assignment of base composition can occur as follows: two nucleic acid strands having different base composition may have a difference of about 1 Da when the base composition difference between the two strands is G⇄A (−15.994) combined with C⇄T (+15.000). For example, one 99-mer nucleic acid strand having a base composition of A27G30C21T21 has a theoretical molecular mass of 30779.058 while another 99-mer nucleic acid strand having a base composition of A26G31C22T20 has a theoretical molecular mass of 30780.052. A 1 Da difference in molecular mass may be within the experimental error of a molecular mass measurement and thus, the relatively narrow molecular mass range of the four natural nucleobases imposes an uncertainty factor.
The present invention provides for a means for removing this theoretical 1 Da uncertainty factor through amplification of a nucleic acid with one mass-tagged nucleobase and three natural nucleobases. The term “nucleobase” as used herein is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).
Addition of significant mass to one of the 4 nucleobases (dNTPs) in an amplification reaction, or in the primers themselves, will result in a significant difference in mass of the resulting amplification product (significantly greater than 1 Da) arising from ambiguities arising from the G⇄A combined with C ⇄T event (Table 4). Thus, the same the G⇄A (−15.994) event combined with 5-Iodo-C⇄T (−110.900) event would result in a molecular mass difference of 126.894. If the molecular mass of the base composition A27G30 5-Iodo-C21T21 (33422.958) is compared with A26G315-Iodo-C22T20, (33549.852) the theoretical molecular mass difference is +126.894. The experimental error of a molecular mass measurement is not significant with regard to this molecular mass difference. Furthermore, the only base composition consistent with a measured molecular mass of the 99-mer nucleic acid is A27G305-Iodo-C21T21. In contrast, the analogous amplification without the mass tag has 18 possible base compositions.







TABLE 4







Molecular Masses of Natural Nucleobases and the Mass-Modified


Nucleobase 5-Iodo-C and Molecular Mass Differences


Resulting from Transitions










Nucleobase
Molecular Mass
Transition
Molecular Mass













A
313.058
A-->T
−9.012


A
313.058
A-->C
−24.012


A
313.058
A-->5-Iodo-C
101.888


A
313.058
A-->G
15.994


T
304.046
T-->A
9.012


T
304.046
T-->C
−15.000


T
304.046
T-->5-Iodo-C
110.900


T
304.046
T-->G
25.006


C
289.046
C-->A
24.012


C
289.046
C-->T
15.000


C
289.046
C-->G
40.006


5-Iodo-C
414.946
5-Iodo-C-->A
−101.888


5-Iodo-C
414.946
5-Iodo-C-->T
−110.900


5-Iodo-C
414.946
5-Iodo-C-->G
−85.894


G
329.052
G-->A
−15.994


G
329.052
G-->T
−25.006


G
329.052
G-->C
−40.006


G
329.052
G-->5-Iodo-C
85.894









Mass spectra of bioagent-identifying amplicons were analyzed independently using a maximum-likelihood processor, such as is widely used in radar signal processing. This processor, referred to as GenX, first makes maximum likelihood estimates of the input to the mass spectrometer for each primer by running matched filters for each base composition aggregate on the input data. This includes the GenX response to a calibrant for each primer.
The algorithm emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database is used to define the mass base count matched filters. The database contains the sequences of known bacterial bioagents and includes threat organisms as well as benign background organisms. The latter is used to estimate and subtract the spectral signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. The maximum likelihood process is applied to this “cleaned up” data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.
The amplitudes of all base compositions of bioagent-identifying amplicons for each primer are calibrated and a final maximum likelihood amplitude estimate per organism is made based upon the multiple single primer estimates. Models of all system noise are factored into this two-stage maximum likelihood calculation. The processor reports the number of molecules of each base composition contained in the spectra. The quantity of amplification product corresponding to the appropriate primer set is reported as well as the quantities of primers remaining upon completion of the amplification reaction.
Base count blurring can be carried out as follows. “Electronic PCR” can be conducted on nucleotide sequences of the desired bioagents to obtain the different expected base counts that could be obtained for each primer pair. See for example, ncbi.nlm.nih.gov/sutils/e-pcr/; Schuler, Genome Res. 7:541-50, 1997. In one illustrative embodiment, one or more spreadsheets, such as Microsoft Excel workbooks contain a plurality of worksheets. First in this example, there is a worksheet with a name similar to the workbook name; this worksheet contains the raw electronic PCR data. Second, there is a worksheet named “filtered bioagents base count” that contains bioagent name and base count; there is a separate record for each strain after removing sequences that are not identified with a genus and species and removing all sequences for bioagents with less than 10 strains. Third, there is a worksheet, “Sheet1” that contains the frequency of substitutions, insertions, or deletions for this primer pair. This data is generated by first creating a pivot table from the data in the “filtered bioagents base count” worksheet and then executing an Excel VBA macro. The macro creates a table of differences in base counts for bioagents of the same species, but different strains. One of ordinary skill in the art may understand additional pathways for obtaining similar table differences without undo experimentation.
Application of an exemplary script, involves the user defining a threshold that specifies the fraction of the strains that are represented by the reference set of base counts for each bioagent. The reference set of base counts for each bioagent may contain as many different base counts as are needed to meet or exceed the threshold. The set of reference base counts is defined by taking the most abundant strain's base type composition and adding it to the reference set and then the next most abundant strain's base type composition is added until the threshold is met or exceeded. The current set of data was obtained using a threshold of 55%, which was obtained empirically.
For each base count not included in the reference base count set for that bioagent, the script then proceeds to determine the manner in which the current base count differs from each of the base counts in the reference set. This difference may be represented as a combination of substitutions, Si=Xi, and insertions, Ii=Yi, or deletions, Di=Zi. If there is more than one reference base count, then the reported difference is chosen using rules that aim to minimize the number of changes and, in instances with the same number of changes, minimize the number of insertions or deletions. Therefore, the primary rule is to identify the difference with the minimum sum (Xi+Yi) or (Xi+Zi), e.g., one insertion rather than two substitutions. If there are two or more differences with the minimum sum, then the one that will be reported is the one that contains the most substitutions.
Differences between a base count and a reference composition are categorized as one, two, or more substitutions, one, two, or more insertions, one, two, or more deletions, and combinations of substitutions and insertions or deletions. The different classes of nucleobase changes and their probabilities of occurrence have been delineated in U.S. Patent Application Publication No. 2004209260 (U.S. application Ser. No. 10/418,514) which is incorporated herein by reference in entirety.
Example 6
Use of Broad Range Survey and Division Wide Primer Pairs for Identification of Bacteria in an Epidemic Surveillance Investigation
This investigation employed a set of 16 primer pairs which is herein designated the “surveillance primer set” and comprises broad range survey primer pairs, division wide primer pairs and a single Bacillus lade primer pair. The surveillance primer set is shown in Table 5 and consists of primer pairs originally listed in Table 2. This surveillance set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row. Primer pair 449 (non-T modified) has been modified twice. Its predecessors are primer pairs 70 and 357, displayed below in the same row. Primer pair 360 has also been modified twice and its predecessors are primer pairs 17 and 118.







TABLE 5







Bacterial Primer Pairs of the Surveillance Primer Set














Forward

Reverse



Primer

Primer

Primer


Pair

(SEQ ID

(SEQ ID


No.
Forward Primer Name
NO:)
Reverse Primer Name
NO:)
Target Gene















346
16S_EC_713_732_TMOD_F
202
16S_EC_789_809_TMOD_R
1110
16S rRNA


10
16S_EC_713_732_F
21
16S_EC_789_809
798
16S rRNA


347
16S_EC_785_806_TMOD_F
560
16S_EC_880_897_TMOD_R
1278
16S rRNA


11
16S_EC_785_806_F
118
16S_EC_880_897_R
830
16S rRNA


348
16S_EC_960_981_TMOD_F
706
16S_EC_1054_1073_TMOD_R
895
16S rRNA


14
16S_EC_960_981_F
672
16S_EC_1054_1073_R
735
16S rRNA


349
23S_EC_1826_1843_TMOD_F
401
23S_EC_1906_1924_TMOD_R
1156
23S rRNA


16
23S_EC_1826_1843_F
80
23S_EC_1906_1924_R
805
23S rRNA


352
INFB_EC_1365_1393_TMOD_F
687
INFB_EC_1439_1467_TMOD_R
1411
infB


34
INFB_EC_1365_1393_F
524
INFB_EC_1439_1467_R
1248
infB


354
RPOC_EC_2218_2241_TMOD_F
405
RPOC_EC_2313_2337_TMOD_R
1072
rpoC


52
RPOC_EC_2218_2241_F
81
RPOC_EC_2313_2337_R
790
rpoC


355
SSPE_BA_115_137_TMOD_F
255
SSPE_BA_197_222_TMOD_R
1402
sspE


58
SSPE_BA_115_137_F
45
SSPE_BA_197_222_R
1201
sspE


356
RPLB_EC_650_679_TMOD_F
232
RPLB_EC_739_762_TMOD_R
592
rplB


66
RPLB_EC_650_679_F
98
RPLB_EC_739_762_R
999
rplB


358
VALS_EC_1105_1124_TMOD_F
385
VALS_EC_1195_1218_TMOD_R
1093
valS


71
VALS_EC_1105_1124_F
77
VALS_EC_1195_1218_R
795
valS


359
RPOB_EC_1845_1866_TMOD_F
659
RPOB_EC_1909_1929_TMOD_R
1250
rpoB


72
RPOB_EC_1845_1866_F
233
RPOB_EC_1909_1929_R
825
rpoB


360
23S_EC_2646_2667_TMOD_F
409
23S_EC_2745_2765_TMOD_R
1434
23S rRNA


118
23S_EC_2646_2667_F
84
23S_EC_2745_2765_R
1389
23S rRNA


17
23S_EC_2645_2669_F
408
23S_EC_2744_2761_R
1252
23S rRNA


361
16S_EC_1090_1111_2_TMOD_F
697
16S_EC_1175_1196_TMOD_R
1398
16S rRNA


3
16S_EC_1090_1111_2_F
651
16S_EC_1175_1196_R
1159
16S rRNA


362
RPOB_EC_3799_3821_TMOD_F
581
RPOB_EC_3862_3888_TMOD_R
1325
rpoB


289
RPOB_EC_3799_3821_F
124
RPOB_EC_3862_3888_R
840
rpoB


363
RPOC_EC_2146_2174_TMOD_F
284
RPOC_EC_2227_2245_TMOD_R
898
rpoC


290
RPOC_EC_2146_2174_F
52
RPOC_EC_2227_2245_R
736
rpoC


367
TUFB_EC_957_979_TMOD_F
308
TUFB_EC_1034_1058_TMOD_R
1276
tufB


293
TUFB_EC_957_979_F
55
TUFB_EC_1034_1058_R
829
tufB


449
RPLB_EC_690_710_F
309
RPLB_EC_737_758_R
1336
rplB


357
RPLB_EC_688_710_TMOD_F
296
RPLB_EC_736_757_TMOD_R
1337
rplB


67
RPLB_EC_688_710_F
54
RPLB_EC_736_757_R
842
rplB









The 16 primer pairs of the surveillance set are used to produce bioagent identifying amplicons whose base compositions are sufficiently different amongst all known bacteria at the species level to identify, at a reasonable confidence level, any given bacterium at the species level. As shown in Tables 6A-E, common respiratory bacterial pathogens can be distinguished by the base compositions of bioagent identifying amplicons obtained using the 16 primer pairs of the surveillance set. In some cases, triangulation identification improves the confidence level for species assignment. For example, nucleic acid from Streptococcus pyogenes can be amplified by nine of the sixteen surveillance primer pairs and Streptococcus pneumoniae can be amplified by ten of the sixteen surveillance primer pairs. The base compositions of the bioagent identifying amplicons are identical for only one of the analogous bioagent identifying amplicons and differ in all of the remaining analogous bioagent identifying amplicons by up to four bases per bioagent identifying amplicon. The resolving power of the surveillance set was confirmed by determination of base compositions for 120 isolates of respiratory pathogens representing 70 different bacterial species and the results indicated that natural variations (usually only one or two base substitutions per bioagent identifying amplicon) amongst multiple isolates of the same species did not prevent correct identification of major pathogenic organisms at the species level.
Bacillus anthracis is a well known biological warfare agent which has emerged in domestic terrorism in recent years. Since it was envisioned to produce bioagent identifying amplicons for identification of Bacillus anthracis, additional drill-down analysis primers were designed to target genes present on virulence plasmids of Bacillus anthracis so that additional confidence could be reached in positive identification of this pathogenic organism. Three drill-down analysis primers were designed and are listed in Tables 2 and 6. In Table 6, the drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.







TABLE 6







Drill-Down Primer Pairs for Confirmation of Identification of Bacillus anthracis














Forward

Reverse



Primer

Primer

Primer


Pair

(SEQ ID

(SEQ ID


No.
Forward Primer Name
NO:)
Reverse Primer Name
NO:)
Target Gene















350
CAPC_BA_274_303_TMOD_F
476
CAPC_BA_349_376_TMOD_R
1314
capC


24
CAPC_BA_274_303_F
109
CAPC_BA_349_376_R
837
capC


351
CYA_BA_1353_1379_TMOD_F
355
CYA_BA_1448_1467_TMOD_R
1423
cyA


30
CYA_BA_1353_1379_F
64
CYA_BA_1448_1467_R
1342
cyA


353
LEF_BA_756_781_TMOD_F
220
LEF_BA_843_872_TMOD_R
1394
lef


37
LEF_BA_756_781_F
26
LEF_BA_843_872_R
1135
lef









Phylogenetic coverage of bacterial space of the sixteen surveillance primers of Table 5 and the three Bacillus anthracis drill-down primers of Table 6 is shown in FIG. 3 which lists common pathogenic bacteria. FIG. 3 is not meant to be comprehensive in illustrating all species identified by the primers. Only pathogenic bacteria are listed as representative examples of the bacterial species that can be identified by the primers and methods of the present invention. Nucleic acid of groups of bacteria enclosed within the polygons of FIG. 3 can be amplified to obtain bioagent identifying amplicons using the primer pair numbers listed in the upper right hand corner of each polygon. Primer coverage for polygons within polygons is additive. As an illustrative example, bioagent identifying amplicons can be obtained for Chlamydia trachomatis by amplification with, for example, primer pairs 346-349, 360 and 361, but not with any of the remaining primers of the surveillance primer set. On the other hand, bioagent identifying amplicons can be obtained from nucleic acid originating from Bacillus anthracis (located within 5 successive polygons) using, for example, any of the following primer pairs: 346-349, 360, 361 (base polygon), 356, 449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350, 351 and 353 (fifth polygon). Multiple coverage of a given organism with multiple primers provides for increased confidence level in identification of the organism as a result of enabling broad triangulation identification.
In Tables 7A-E, base compositions of respiratory pathogens for primer target regions are shown. Two entries in a cell, represent variation in ribosomal DNA operons. The most predominant base composition is shown first and the minor (frequently a single operon) is indicated by an asterisk (*). Entries with NO DATA mean that the primer would not be expected to prime this species due to mismatches between the primer and target region, as determined by theoretical PCR.







TABLE 7A







Base Compositions of Common Respiratory Pathogens for Bioagent Identifying


Amplicons Corresponding to Primer Pair Nos: 346, 347 and 348













Primer 346
Primer 347
Primer 348


Organism
Strain
[A G C T]
[A G C T]
[A G C T]





Klebsiella
MGH78578
[29 32 25 13]
[23 38 28 26]
[26 32 28 30]


pneumoniae

[29 31 25 13]*
[23 37 28 26]*
[26 31 28 30]*


Yersinia pestis
CO-92 Biovar
[29 32 25 13]
[22 39 28 26]
[29 30 28 29]



Orientalis


[30 30 27 29]*


Yersinia pestis
KIM5 P12 (Biovar
[29 32 25 13]
[22 39 28 26]
[29 30 28 29]



Mediaevalis)


Yersinia pestis
91001
[29 32 25 13]
[22 39 28 26]
[29 30 28 29]






[30 30 27 29]*


Haemophilus
KW20
[28 31 23 17]
[24 37 25 27]
[29 30 28 29]


influenzae


Pseudomonas
PAO1
[30 31 23 15]
[26 36 29 24]
[26 32 29 29]


aeruginosa


[27 36 29 23]*


Pseudomonas
Pf0-1
[30 31 23 15]
[26 35 29 25]
[28 31 28 29]


fluorescens


Pseudomonas
KT2440
[30 31 23 15]
[28 33 27 27]
[27 32 29 28]


putida


Legionella
Philadelphia-1
[30 30 24 15]
[33 33 23 27]
[29 28 28 31]


pneumophila


Francisella
schu 4
[32 29 22 16]
[28 38 26 26]
[25 32 28 31]


tularensis


Bordetella
Tohama I
[30 29 24 16]
[23 37 30 24]
[30 32 30 26]


pertussis


Burkholderia
J2315
[29 29 27 14]
[27 32 26 29]
[27 36 31 24]


cepacia



[20 42 35 19]*


Burkholderia
K96243
[29 29 27 14]
[27 32 26 29]
[27 36 31 24]


pseudomallei


Neisseria
FA 1090, ATCC
[29 28 24 18]
[27 34 26 28]
[24 36 29 27]


gonorrhoeae
700825


Neisseria
MC58 (serogroup B)
[29 28 26 16]
[27 34 27 27]
[25 35 30 26]


meningitidis


Neisseria
serogroup C, FAM18
[29 28 26 16]
[27 34 27 27]
[25 35 30 26]


meningitidis


Neisseria
Z2491 (serogroup A)
[29 28 26 16]
[27 34 27 27]
[25 35 30 26]


meningitidis


Chlamydophila
TW-183
[31 27 22 19]
NO DATA
[32 27 27 29]


pneumoniae


Chlamydophila
AR39
[31 27 22 19]
NO DATA
[32 27 27 29]


pneumoniae


Chlamydophila
CWL029
[31 27 22 19]
NO DATA
[32 27 27 29]


pneumoniae


Chlamydophila
J138
[31 27 22 19]
NO DATA
[32 27 27 29]


pneumoniae


Corynebacterium
NCTC13129
[29 34 21 15]
[22 38 31 25]
[22 33 25 34]


diphtheriae


Mycobacterium
k10
[27 36 21 15]
[22 37 30 28]
[21 36 27 30]


avium


Mycobacterium
104
[27 36 21 15]
[22 37 30 28]
[21 36 27 30]


avium


Mycobacterium
CSU#93
[27 36 21 15]
[22 37 30 28]
[21 36 27 30]


tuberculosis


Mycobacterium
CDC 1551
[27 36 21 15]
[22 37 30 28]
[21 36 27 30]


tuberculosis


Mycobacterium
H37Rv (lab strain)
[27 36 21 15]
[22 37 30 28]
[21 36 27 30]


tuberculosis


Mycoplasma
M129
[31 29 19 20]
NO DATA
NO DATA


pneumoniae


Staphylococcus
MRSA252
[27 30 21 21]
[25 35 30 26]
[30 29 30 29]


aureus



[29 31 30 29]*


Staphylococcus
MSSA476
[27 30 21 21]
[25 35 30 26]
[30 29 30 29]


aureus



[30 29 29 30]*


Staphylococcus
COL
[27 30 21 21]
[25 35 30 26]
[30 29 30 29]


aureus



[30 29 29 30]*


Staphylococcus
Mu50
[27 30 21 21]
[25 35 30 26]
[30 29 30 29]


aureus



[30 29 29 30]*


Staphylococcus
MW2
[27 30 21 21]
[25 35 30 26]
[30 29 30 29]


aureus



[30 29 29 30]*


Staphylococcus
N315
[27 30 21 21]
[25 35 30 26]
[30 29 30 29]


aureus



[30 29 29 30]*


Staphylococcus
NCTC 8325
[27 30 21 21]
[25 35 30 26]
[30 29 30 29]


aureus


[25 35 31 26]*
[30 29 29 30]


Streptococcus
NEM316
[26 32 23 18]
[24 36 31 25]
[25 32 29 30]


agalactiae


[24 36 30 26]*


Streptococcus
NC_002955
[26 32 23 18]
[23 37 31 25]
[29 30 25 32]


equi


Streptococcus
MGAS8232
[26 32 23 18]
[24 37 30 25]
[25 31 29 31]


pyogenes


Streptococcus
MGAS315
[26 32 23 18]
[24 37 30 25]
[25 31 29 31]


pyogenes


Streptococcus
SSI-1
[26 32 23 18]
[24 37 30 25]
[25 31 29 31]


pyogenes


Streptococcus
MGAS10394
[26 32 23 18]
[24 37 30 25]
[25 31 29 31]


pyogenes


Streptococcus
Manfredo (M5)
[26 32 23 18]
[24 37 30 25]
[25 31 29 31]


pyogenes


Streptococcus
SF370 (M1)
[26 32 23 18]
[24 37 30 25]
[25 31 29 31]


pyogenes


Streptococcus
670
[26 32 23 18]
[25 35 28 28]
[25 32 29 30]


pneumoniae


Streptococcus
R6
[26 32 23 18]
[25 35 28 28]
[25 32 29 30]


pneumoniae


Streptococcus
TIGR4
[26 32 23 18]
[25 35 28 28]
[25 32 30 29]


pneumoniae


Streptococcus
NCTC7868
[25 33 23 18]
[24 36 31 25]
[25 31 29 31]


gordonii


Streptococcus
NCTC 12261
[26 32 23 18]
[25 35 30 26]
[25 32 29 30]


mitis



[24 31 35 29]*


Streptococcus
UA159
[24 32 24 19]
[25 37 30 24]
[28 31 26 31]


mutans
















TABLE 7B







Base Compositions of Common Respiratory Pathogens for Bioagent Identifying


Amplicons Corresponding to Primer Pair Nos: 349, 360, and 356













Primer 349
Primer 360
Primer 356


Organism
Strain
[A G C T]
[A G C T]
[A G C T]





Klebsiella
MGH78578
[25 31 25 22]
[33 37 25 27]
NO DATA


pneumoniae


Yersinia pestis
CO-92 Biovar
[25 31 27 20]
[34 35 25 28]
NO DATA



Orientalis
[25 32 26 20]*


Yersinia pestis
KIM5 P12 (Biovar
[25 31 27 20]
[34 35 25 28]
NO DATA



Mediaevalis)
[25 32 26 20]*


Yersinia pestis
91001
[25 31 27 20]
[34 35 25 28]
NO DATA


Haemophilus
KW20
[28 28 25 20]
[32 38 25 27]
NO DATA


influenzae


Pseudomonas
PAO1
[24 31 26 20]
[31 36 27 27]
NO DATA


aeruginosa


[31 36 27 28]*


Pseudomonas
Pf0-1
NO DATA
[30 37 27 28]
NO DATA


fluorescens


[30 37 27 28]


Pseudomonas
KT2440
[24 31 26 20]
[30 37 27 28]
NO DATA


putida


Legionella
Philadelphia-1
[23 30 25 23]
[30 39 29 24]
NO DATA


pneumophila


Francisella
schu 4
[26 31 25 19]
[32 36 27 27]
NO DATA


tularensis


Bordetella
Tohama I
[21 29 24 18]
[33 36 26 27]
NO DATA


pertussis


Burkholderia
J2315
[23 27 22 20]
[31 37 28 26]
NO DATA


cepacia


Burkholderia
K96243
[23 27 22 20]
[31 37 28 26]
NO DATA


pseudomallei


Neisseria
FA 1090, ATCC 700825
[24 27 24 17]
[34 37 25 26]
NO DATA


gonorrhoeae


Neisseria
MC58 (serogroup B)
[25 27 22 18]
[34 37 25 26]
NO DATA


meningitidis


Neisseria
serogroup C, FAM18
[25 26 23 18]
[34 37 25 26]
NO DATA


meningitidis


Neisseria
Z2491 (serogroup A)
[25 26 23 18]
[34 37 25 26]
NO DATA


meningitidis


Chlamydophila
TW-183
[30 28 27 18]
NO DATA
NO DATA


pneumoniae


Chlamydophila
AR39
[30 28 27 18]
NO DATA
NO DATA


pneumoniae


Chlamydophila
CWL029
[30 28 27 18]
NO DATA
NO DATA


pneumoniae


Chlamydophila
J138
[30 28 27 18]
NO DATA
NO DATA


pneumoniae


Corynebacterium
NCTC13129
NO DATA
[29 40 28 25]
NO DATA


diphtheriae


Mycobacterium
k10
NO DATA
[33 35 32 22]
NO DATA


avium


Mycobacterium
104
NO DATA
[33 35 32 22]
NO DATA


avium


Mycobacterium
CSU#93
NO DATA
[30 36 34 22]
NO DATA


tuberculosis


Mycobacterium
CDC 1551
NO DATA
[30 36 34 22]
NO DATA


tuberculosis


Mycobacterium
H37Rv (lab strain)
NO DATA
[30 36 34 22]
NO DATA


tuberculosis


Mycoplasma
M129
[28 30 24 19]
[34 31 29 28]
NO DATA


pneumoniae


Staphylococcus
MRSA252
[26 30 25 20]
[31 38 24 29]
[33 30 31 27]


aureus


Staphylococcus
MSSA476
[26 30 25 20]
[31 38 24 29]
[33 30 31 27]


aureus


Staphylococcus
COL
[26 30 25 20]
[31 38 24 29]
[33 30 31 27]


aureus


Staphylococcus
Mu50
[26 30 25 20]
[31 38 24 29]
[33 30 31 27]


aureus


Staphylococcus
MW2
[26 30 25 20]
[31 38 24 29]
[33 30 31 27]


aureus


Staphylococcus
N315
[26 30 25 20]
[31 38 24 29]
[33 30 31 27]


aureus


Staphylococcus
NCTC 8325
[26 30 25 20]
[31 38 24 29]
[33 30 31 27]


aureus


Streptococcus
NEM316
[28 31 22 20]
[33 37 24 28]
[37 30 28 26]


agalactiae


Streptococcus
NC_002955
[28 31 23 19]
[33 38 24 27]
[37 31 28 25]


equi


Streptococcus
MGAS8232
[28 31 23 19]
[33 37 24 28]
[38 31 29 23]


pyogenes


Streptococcus
MGAS315
[28 31 23 19]
[33 37 24 28]
[38 31 29 23]


pyogenes


Streptococcus
SSI-1
[28 31 23 19]
[33 37 24 28]
[38 31 29 23]


pyogenes


Streptococcus
MGAS10394
[28 31 23 19]
[33 37 24 28]
[38 31 29 23]


pyogenes


Streptococcus
Manfredo (M5)
[28 31 23 19]
[33 37 24 28]
[38 31 29 23]


pyogenes


Streptococcus
SF370 (M1)
[28 31 23 19]
[33 37 24 28]
[38 31 29 23]


pyogenes

[28 31 22 20]*


Streptococcus
670
[28 31 22 20]
[34 36 24 28]
[37 30 29 25]


pneumoniae


Streptococcus
R6
[28 31 22 20]
[34 36 24 28]
[37 30 29 25]


pneumoniae


Streptococcus
TIGR4
[28 31 22 20]
[34 36 24 28]
[37 30 29 25]


pneumoniae


Streptococcus
NCTC7868
[28 32 23 20]
[34 36 24 28]
[36 31 29 25]


gordonii


Streptococcus
NCTC 12261
[28 31 22 20]
[34 36 24 28]
[37 30 29 25]


mitis

[29 30 22 20]*


Streptococcus
UA159
[26 32 23 22]
[34 37 24 27]
NO DATA


mutans
















TABLE 7C







Base Compositions of Common Respiratory Pathogens for Bioagent Identifying


Amplicons Corresponding to Primer Pair Nos: 449, 354, and 352













Primer 449
Primer 354
Primer 352


Organism
Strain
[A G C T]
[A G C T]
[A G C T]





Klebsiella
MGH78578
NO DATA
[27 33 36 26]
NO DATA


pneumoniae


Yersinia pestis
CO-92 Biovar
NO DATA
[29 31 33 29]
[32 28 20 25]



Orientalis


Yersinia pestis
KIM5 P12 (Biovar
NO DATA
[29 31 33 29]
[32 28 20 25]



Mediaevalis)


Yersinia pestis
91001
NO DATA
[29 31 33 29]
NO DATA


Haemophilus
KW20
NO DATA
[30 29 31 32]
NO DATA


influenzae


Pseudomonas
PAO1
NO DATA
[26 33 39 24]
NO DATA


aeruginosa


Pseudomonas
Pf0-1
NO DATA
[26 33 34 29]
NO DATA


fluorescens


Pseudomonas
KT2440
NO DATA
[25 34 36 27]
NO DATA


putida


Legionella
Philadelphia-1
NO DATA
NO DATA
NO DATA


pneumophila


Francisella
schu 4
NO DATA
[33 32 25 32]
NO DATA


tularensis


Bordetella
Tohama I
NO DATA
[26 33 39 24]
NO DATA


pertussis


Burkholderia
J2315
NO DATA
[25 37 33 27]
NO DATA


cepacia


Burkholderia
K96243
NO DATA
[25 37 34 26]
NO DATA


pseudomallei


Neisseria
FA 1090, ATCC 700825
[17 23 22 10]
[29 31 32 30]
NO DATA


gonorrhoeae


Neisseria
MC58 (serogroup B)
NO DATA
[29 30 32 31]
NO DATA


meningitidis


Neisseria
serogroup C, FAM18
NO DATA
[29 30 32 31]
NO DATA


meningitidis


Neisseria
Z2491 (serogroup A)
NO DATA
[29 30 32 31]
NO DATA


meningitidis


Chlamydophila
TW-183
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
AR39
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
CWL029
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
J138
NO DATA
NO DATA
NO DATA


pneumoniae


Corynebacterium
NCTC13129
NO DATA
NO DATA
NO DATA


diphtheriae


Mycobacterium
k10
NO DATA
NO DATA
NO DATA


avium


Mycobacterium
104
NO DATA
NO DATA
NO DATA


avium


Mycobacterium
CSU#93
NO DATA
NO DATA
NO DATA


tuberculosis


Mycobacterium
CDC 1551
NO DATA
NO DATA
NO DATA


tuberculosis


Mycobacterium
H37Rv (lab strain)
NO DATA
NO DATA
NO DATA


tuberculosis


Mycoplasma
M129
NO DATA
NO DATA
NO DATA


pneumoniae


Staphylococcus
MRSA252
[17 20 21 17]
[30 27 30 35]
[36 24 19 26]


aureus


Staphylococcus
MSSA476
[17 20 21 17]
[30 27 30 35]
[36 24 19 26]


aureus


Staphylococcus
COL
[17 20 21 17]
[30 27 30 35]
[35 24 19 27]


aureus


Staphylococcus
Mu50
[17 20 21 17]
[30 27 30 35]
[36 24 19 26]


aureus


Staphylococcus
MW2
[17 20 21 17]
[30 27 30 35]
[36 24 19 26]


aureus


Staphylococcus
N315
[17 20 21 17]
[30 27 30 35]
[36 24 19 26]


aureus


Staphylococcus
NCTC 8325
[17 20 21 17]
[30 27 30 35]
[35 24 19 27]


aureus


Streptococcus
NEM316
[22 20 19 14]
[26 31 27 38]
[29 26 22 28]


agalactiae


Streptococcus
NC_002955
[22 21 19 13]
NO DATA
NO DATA


equi


Streptococcus
MGAS8232
[23 21 19 12]
[24 32 30 36]
NO DATA


pyogenes


Streptococcus
MGAS315
[23 21 19 12]
[24 32 30 36]
NO DATA


pyogenes


Streptococcus
SSI-1
[23 21 19 12]
[24 32 30 36]
NO DATA


pyogenes


Streptococcus
MGAS10394
[23 21 19 12]
[24 32 30 36]
NO DATA


pyogenes


Streptococcus
Manfredo (M5)
[23 21 19 12]
[24 32 30 36]
NO DATA


pyogenes


Streptococcus
SF370 (M1)
[23 21 19 12]
[24 32 30 36]
NO DATA


pyogenes


Streptococcus
670
[22 20 19 14]
[25 33 29 35]
[30 29 21 25]


pneumoniae


Streptococcus
R6
[22 20 19 14]
[25 33 29 35]
[30 29 21 25]


pneumoniae


Streptococcus
TIGR4
[22 20 19 14]
[25 33 29 35]
[30 29 21 25]


pneumoniae


Streptococcus
NCTC7868
[21 21 19 14]
NO DATA
[29 26 22 28]


gordonii


Streptococcus
NCTC 12261
[22 20 19 14]
[26 30 32 34]
NO DATA


mitis


Streptococcus
UA159
NO DATA
NO DATA
NO DATA


mutans
















TABLE 7D







Base Compositions of Common Respiratory Pathogens for Bioagent Identifying


Amplicons Corresponding to Primer Pair Nos: 355, 358, and 359













Primer 355
Primer 358
Primer 359


Organism
Strain
[A G C T]
[A G C T]
[A G C T]





Klebsiella
MGH78578
NO DATA
[24 39 33 20]
[25 21 24 17]


pneumoniae


Yersinia pestis
CO-92 Biovar
NO DATA
[26 34 35 21]
[23 23 19 22]



Orientalis


Yersinia pestis
KIM5 P12 (Biovar
NO DATA
[26 34 35 21]
[23 23 19 22]



Mediaevalis)


Yersinia pestis
91001
NO DATA
[26 34 35 21]
[23 23 19 22]


Haemophilus
KW20
NO DATA
NO DATA
NO DATA


influenzae


Pseudomonas
PAO1
NO DATA
NO DATA
NO DATA


aeruginosa


Pseudomonas
Pf0-1
NO DATA
NO DATA
NO DATA


fluorescens


Pseudomonas
KT2440
NO DATA
[21 37 37 21]
NO DATA


putida


Legionella
Philadelphia-1
NO DATA
NO DATA
NO DATA


pneumophila


Francisella
schu 4
NO DATA
NO DATA
NO DATA


tularensis


Bordetella
Tohama I
NO DATA
NO DATA
NO DATA


pertussis


Burkholderia
J2315
NO DATA
NO DATA
NO DATA


cepacia


Burkholderia
K96243
NO DATA
NO DATA
NO DATA


pseudomallei


Neisseria
FA 1090, ATCC 700825
NO DATA
NO DATA
NO DATA


gonorrhoeae


Neisseria
MC58 (serogroup B)
NO DATA
NO DATA
NO DATA


meningitidis


Neisseria
serogroup C, FAM18
NO DATA
NO DATA
NO DATA


meningitidis


Neisseria
Z2491 (serogroup A)
NO DATA
NO DATA
NO DATA


meningitidis


Chlamydophila
TW-183
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
AR39
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
CWL029
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
J138
NO DATA
NO DATA
NO DATA


pneumoniae


Corynebacterium
NCTC13129
NO DATA
NO DATA
NO DATA


diphtheriae


Mycobacterium
k10
NO DATA
NO DATA
NO DATA


avium


Mycobacterium
104
NO DATA
NO DATA
NO DATA


avium


Mycobacterium
CSU#93
NO DATA
NO DATA
NO DATA


tuberculosis


Mycobacterium
CDC 1551
NO DATA
NO DATA
NO DATA


tuberculosis


Mycobacterium
H37Rv (lab strain)
NO DATA
NO DATA
NO DATA


tuberculosis


Mycoplasma
M129
NO DATA
NO DATA
NO DATA


pneumoniae


Staphylococcus
MRSA252
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
MSSA476
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
COL
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
Mu50
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
MW2
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
N315
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
NCTC 8325
NO DATA
NO DATA
NO DATA


aureus


Streptococcus
NEM316
NO DATA
NO DATA
NO DATA


agalactiae


Streptococcus
NC_002955
NO DATA
NO DATA
NO DATA


equi


Streptococcus
MGAS8232
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
MGAS315
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
SSI-1
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
MGAS10394
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
Manfredo (M5)
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
SF370 (M1)
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
670
NO DATA
NO DATA
NO DATA


pneumoniae


Streptococcus
R6
NO DATA
NO DATA
NO DATA


pneumoniae


Streptococcus
TIGR4
NO DATA
NO DATA
NO DATA


pneumoniae


Streptococcus
NCTC7868
NO DATA
NO DATA
NO DATA


gordonii


Streptococcus
NCTC 12261
NO DATA
NO DATA
NO DATA


mitis


Streptococcus
UA159
NO DATA
NO DATA
NO DATA


mutans
















TABLE 7E







Base Compositions of Common Respiratory Pathogens for Bioagent Identifying


Amplicons Corresponding to Primer Pair Nos: 362, 363, and 367













Primer 362
Primer 363
Primer 367


Organism
Strain
[A G C T]
[A G C T]
[A G C T]





Klebsiella
MGH78578
[21 33 22 16]
[16 34 26 26]
NO DATA


pneumoniae


Yersinia pestis
CO-92 Biovar
[20 34 18 20]
NO DATA
NO DATA



Orientalis


Yersinia pestis
KIM5 P12 (Biovar
[20 34 18 20]
NO DATA
NO DATA



Mediaevalis)


Yersinia pestis
91001
[20 34 18 20]
NO DATA
NO DATA


Haemophilus
KW20
NO DATA
NO DATA
NO DATA


influenzae


Pseudomonas
PAO1
[19 35 21 17]
[16 36 28 22]
NO DATA


aeruginosa


Pseudomonas
Pf0-1
NO DATA
[18 35 26 23]
NO DATA


fluorescens


Pseudomonas
KT2440
NO DATA
[16 35 28 23]
NO DATA


putida


Legionella
Philadelphia-1
NO DATA
NO DATA
NO DATA


pneumophila


Francisella
schu 4
NO DATA
NO DATA
NO DATA


tularensis


Bordetella
Tohama I
[20 31 24 17]
[15 34 32 21]
[26 25 34 19]


pertussis


Burkholderia
J2315
[20 33 21 18]
[15 36 26 25]
[25 27 32 20]


cepacia


Burkholderia
K96243
[19 34 19 20]
[15 37 28 22]
[25 27 32 20]


pseudomallei


Neisseria
FA 1090, ATCC 700825
NO DATA
NO DATA
NO DATA


gonorrhoeae


Neisseria
MC58 (serogroup B)
NO DATA
NO DATA
NO DATA


meningitidis


Neisseria
serogroup C, FAM18
NO DATA
NO DATA
NO DATA


meningitidis


Neisseria
Z2491 (serogroup A)
NO DATA
NO DATA
NO DATA


meningitidis


Chlamydophila
TW-183
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
AR39
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
CWL029
NO DATA
NO DATA
NO DATA


pneumoniae


Chlamydophila
J138
NO DATA
NO DATA
NO DATA


pneumoniae


Corynebacterium
NCTC13129
NO DATA
NO DATA
NO DATA


diphtheriae


Mycobacterium
k10
[19 34 23 16]
NO DATA
[24 26 35 19]


avium


Mycobacterium
104
[19 34 23 16]
NO DATA
[24 26 35 19]


avium


Mycobacterium
CSU#93
[19 31 25 17]
NO DATA
[25 25 34 20]


tuberculosis


Mycobacterium
CDC 1551
[19 31 24 18]
NO DATA
[25 25 34 20]


tuberculosis


Mycobacterium
H37Rv (lab strain)
[19 31 24 18]
NO DATA
[25 25 34 20]


tuberculosis


Mycoplasma
M129
NO DATA
NO DATA
NO DATA


pneumoniae


Staphylococcus
MRSA252
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
MSSA476
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
COL
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
Mu50
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
MW2
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
N315
NO DATA
NO DATA
NO DATA


aureus


Staphylococcus
NCTC 8325
NO DATA
NO DATA
NO DATA


aureus


Streptococcus
NEM316
NO DATA
NO DATA
NO DATA


agalactiae


Streptococcus
NC_002955
NO DATA
NO DATA
NO DATA


equi


Streptococcus
MGAS8232
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
MGAS315
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
SSI-1
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
MGAS10394
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
Manfredo (M5)
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
SF370 (M1)
NO DATA
NO DATA
NO DATA


pyogenes


Streptococcus
670
NO DATA
NO DATA
NO DATA


pneumoniae


Streptococcus
R6
[20 30 19 23]
NO DATA
NO DATA


pneumoniae


Streptococcus
TIGR4
[20 30 19 23]
NO DATA
NO DATA


pneumoniae


Streptococcus
NCTC7868
NO DATA
NO DATA
NO DATA


gordonii


Streptococcus
NCTC 12261
NO DATA
NO DATA
NO DATA


mitis


Streptococcus
UA159
NO DATA
NO DATA
NO DATA


mutans









Four sets of throat samples from military recruits at different military facilities taken at different time points were analyzed using the primers of the present invention. The first set was collected at a military training center from Nov. 1 to Dec. 20, 2002 during one of the most severe outbreaks of pneumonia associated with group A Streptococcus in the United States since 1968. During this outbreak, fifty-one throat swabs were taken from both healthy and hospitalized recruits and plated on blood agar for selection of putative group A Streptococcus colonies. A second set of 15 original patient specimens was taken during the height of this group A Streptococcus—associated respiratory disease outbreak. The third set were historical samples, including twenty-seven isolates of group A Streptococcus, from disease outbreaks at this and other military training facilities during previous years. The fourth set of samples was collected from five geographically separated military facilities in the continental U.S. in the winter immediately following the severe November/December 2002 outbreak.
Pure colonies isolated from group A Streptococcus-selective media from all four collection periods were analyzed with the surveillance primer set. All samples showed base compositions that precisely matched the four completely sequenced strains of Streptococcus pyogenes. Shown in FIG. 4 is a 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.
In addition to the identification of Streptococcus pyogenes, other potentially pathogenic organisms were identified concurrently. Mass spectral analysis of a sample whose nucleic acid was amplified by primer pair number 349 (SEQ ID NOs: 401:1156) exhibited signals of bioagent identifying amplicons with molecular masses that were found to correspond to analogous base compositions of bioagent identifying amplicons of Streptococcus pyogenes (A27 G32 C24 Ti 8), Neisseria meningitidis (A25 G27 C22 T18 Haemophilus influenzae (A28 G28 C25 T20) (see FIG. 5 and Table 7B). These organisms were present in a ratio of 4:5:20 as determined by comparison of peak heights with peak height of an internal PCR calibration standard as described in commonly owned U.S. Patent Application Ser. No: 60/545,425 which is incorporated herein by reference in its entirety.
Since certain division-wide primers that target housekeeping genes are designed to provide coverage of specific divisions of bacteria to increase the confidence level for identification of bacterial species, they are not expected to yield bioagent identifying amplicons for organisms outside of the specific divisions. For example, primer pair number 356 (SEQ ID NOs: 449:1380) primarily amplifies the nucleic acid of members of the classes Bacilli and Clostridia and is not expected to amplify proteobacteria such as Neisseria meningitidis and Haemophilus influenzae. As expected, analysis of the mass spectrum of amplification products obtained with primer pair number 356 does not indicate the presence of Neisseria meningitidis and Haemophilus influenzae but does indicate the presence of Streptococcus pyogenes (FIGS. 3 and 6, Table 7B). Thus, these primers or types of primers can confirm the absence of particular bioagents from a sample.
The 15 throat swabs from military recruits were found to contain a relatively small set of microbes in high abundance. The most common were Haemophilus influenza, Neisseria meningitides, and Streptococcus pyogenes. Staphylococcus epidermidis, Moraxella cattarhalis, Corynebacterium pseudodiphtheriticum, and Staphylococcus aureus were present in fewer samples. An equal number of samples from healthy volunteers from three different geographic locations, were identically analyzed. Results indicated that the healthy volunteers have bacterial flora dominated by multiple, commensal non-beta-hemolytic Streptococcal species, including the viridans group streptococci (S. parasangunis, S. vestibularis, S. mitis, S. oralis and S. pneumoniae; data not shown), and none of the organisms found in the military recruits were found in the healthy controls at concentrations detectable by mass spectrometry. Thus, the military recruits in the midst of a respiratory disease outbreak had a dramatically different microbial population than that experienced by the general population in the absence of epidemic disease.
Example 7
Triangulation Genotyping Analysis for Determination of emm-Type of Streptococcus pyogenes in Epidemic Surveillance
As a continuation of the epidemic surveillance investigation of Example 6, determination of sub-species characteristics (genotyping) of Streptococcus pyogenes, was carried out based on a strategy that generates strain-specific signatures according to the rationale of Multi-Locus Sequence Typing (MLST). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced (Enright et al. Infection and Immunity, 2001, 69, 2416-2427). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced. In the present investigation, bioagent identifying amplicons from housekeeping genes were produced using drill-down primers and analyzed by mass spectrometry. Since mass spectral analysis results in molecular mass, from which base composition can be determined, the challenge was to determine whether resolution of emm classification of strains of Streptococcus pyogenes could be determined.
For the purpose of development of a triangulation genotyping assay, an alignment was constructed of concatenated alleles of seven MLST housekeeping genes (glucose kinase (gki), glutamine transporter protein (gtr), glutamate racemase (murI), DNA mismatch repair protein (mutS), xanthine phosphoribosyl transferase (xpt), and acetyl-CoA acetyl transferase (yqiL)) from each of the 212 previously emm-typed strains of Streptococcus pyogenes. From this alignment, the number and location of primer pairs that would maximize strain identification via base composition was determined. As a result, 6 primer pairs were chosen as standard drill-down primers for determination of emm-type of Streptococcus pyogenes. These six primer pairs are displayed in Table 8. This drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.







TABLE 8







Triangulation Genotyping Analysis Primer Pairs for Group A Streptococcus Drill-Down












Primer

Forward Primer

Reverse Primer



Pair No.
Forward Primer Name
(SEQ ID NO:)
Reverse Primer Name
(SEQ ID NO:)
Target Gene















442
SP101_SPET11_358_387_TMOD_F
588
SP101_SPET11_448_473_TMOD_R
998
gki


80
SP101_SPET11_358_387_F
126
SP101_SPET11_448_473_TMOD_R
766
gki


443
SP101_SPET11_600_629_TMOD_F
348
SP101_SPET11_686_714_TMOD_R
1018
gtr


81
SP101_SPET11_600_629_F
62
SP101_SPET11_686_714_R
772
gtr


426
SP101_SPET11_1314_1336_TMOD_F
363
SP101_SPET11_1403_1431_TMOD_R
849
murI


86
SP101_SPET11_1314_1336_F
68
SP101_SPET11_1403_1431_R
711
murI


430
SP101_SPET11_1807_1835_TMOD_F
235
SP101_SPET11_1901_1927_TMOD_R
1439
mutS


90
SP101_SPET11_1807_1835_F
33
SP101_SPET11_1901_1927_R
1412
mutS


438
SP101_SPET11_3075_3103_TMOD_F
473
SP101_SPET11_3168_3196_TMOD_R
875
xpt


96
SP101_SPET11_3075_3103_F
108
SP101_SPET11_3168_3196_R
715
xpt


441
SP101_SPET11_3511_3535_TMOD_F
531
SP101_SPET11_3605_3629_TMOD_R
1294
yqiL


98
SP101_SPET11_3511_3535_F
116
SP101_SPET11_3605_3629_R
832
yqiL









The primers of Table 8 were used to produce bioagent identifying amplicons from nucleic acid present in the clinical samples. The bioagent identifying amplicons which were subsequently analyzed by mass spectrometry and base compositions corresponding to the molecular masses were calculated.
Of the 51 samples taken during the peak of the November/December 2002 epidemic (Table 9A-C rows 1-3), all except three samples were found to represent emm3, a Group A Streptococcus genotype previously associated with high respiratory virulence. The three outliers were from samples obtained from healthy individuals and probably represent non-epidemic strains. Archived samples (Tables 9A-C rows 5-13) from historical collections showed a greater heterogeneity of base compositions and emm types as would be expected from different epidemics occurring at different places and dates. The results of the mass spectrometry analysis and emm gene sequencing were found to be concordant for the epidemic and historical samples.







TABLE 9A







Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus


samples from Six Military Installations Obtained with Primer Pair Nos. 426 and 430














emm-type by



murI
mutS


# of
Mass
emm-Gene
Location

(Primer Pair
(Primer Pair


Instances
Spectrometry
Sequencing
(sample)
Year
No. 426)
No. 430)





48 
3
 3
MCRD San
2002
A39 G25 C20 T34
A38 G27 C23 T33


2
6
 6
Diego

A40 G24 C20 T34
A38 G27 C23 T33


1
28 
28
(Cultured)

A39 G25 C20 T34
A38 G27 C23 T33


15 
3
ND


A39 G25 C20 T34
A38 G27 C23 T33


6
3
 3
NHRC San
2003
A39 G25 C20 T34
A38 G27 C23 T33


3
5, 58
 5
Diego-

A40 G24 C20 T34
A38 G27 C23 T33


6
6
 6
Archive

A40 G24 C20 T34
A38 G27 C23 T33


1
11 
11
(Cultured)

A39 G25 C20 T34
A38 G27 C23 T33


3
12 
12


A40 G24 C20 T34
A38 G26 C24 T33


1
22 
22


A39 G25 C20 T34
A38 G27 C23 T33


3
25, 75 
75


A39 G25 C20 T34
A38 G27 C23 T33


4
44/61, 82, 9
44/61


A40 G24 C20 T34
A38 G26 C24 T33


2
53, 91
91


A39 G25 C20 T34
A38 G27 C23 T33


1
2
 2
Ft.
2003
A39 G25 C20 T34
A38 G27 C24 T32


2
3
 3
Leonard

A39 G25 C20 T34
A38 G27 C23 T33


1
4
 4
Wood

A39 G25 C20 T34
A38 G27 C23 T33


1
6
 6
(Cultured)

A40 G24 C20 T34
A38 G27 C23 T33


11 
25 or 75
75


A39 G25 C20 T34
A38 G27 C23 T33


1
25, 75, 33,
75


A39 G25 C20 T34
A38 G27 C23 T33



34, 4, 52, 84


1
44/61 or 82
44/61


A40 G24 C20 T34
A38 G26 C24 T33



or 9


2
5 or 58
 5


A40 G24 C20 T34
A38 G27 C23 T33


3
1
 1
Ft. Sill
2003
A40 G24 C20 T34
A38 G27 C23 T33


2
3
 3
(Cultured)

A39 G25 C20 T34
A38 G27 C23 T33


1
4
 4


A39 G25 C20 T34
A38 G27 C23 T33


1
28 
28


A39 G25 C20 T34
A38 G27 C23 T33


1
3
 3
Ft.
2003
A39 G25 C20 T34
A38 G27 C23 T33


1
4
 4
Benning

A39 G25 C20 T34
A38 G27 C23 T33


3
6
 6
(Cultured)

A40 G24 C20 T34
A38 G27 C23 T33


1
11 
11


A39 G25 C20 T34
A38 G27 C23 T33


1
13 
 94**


A40 G24 C20 T34
A38 G27 C23 T33


1
44/61 or 82
82


A40 G24 C20 T34
A38 G26 C24 T33



or 9


1
5 or 58
58


A40 G24 C20 T34
A38 G27 C23 T33


1
78 or 89
89


A39 G25 C20 T34
A38 G27 C23 T33


2
5 or 58
ND
Lackland
2003
A40 G24 C20 T34
A38 G27 C23 T33


1
2

AFB

A39 G25 C20 T34
A38 G27 C24 T32


1
81 or 90

(Throat

A40 G24 C20 T34
A38 G27 C23 T33


1
78 

Swabs)

A38 G26 C20 T34
A38 G27 C23 T33


  3***
No detection



No detection
No detection


7
3
ND
MCRD San
2002
A39 G25 C20 T34
A38 G27 C23 T33


1
3
ND
Diego

No detection
A38 G27 C23 T33


1
3
ND
(Throat

No detection
No detection


1
3
ND
Swabs)

No detection
No detection


2
3
ND


No detection
A38 G27 C23 T33


3
No detection
ND


No detection
No detection
















TABLE 9B







Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus


samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441














emm-type by



xpt
yqiL


# of
Mass
emm-Gene
Location

(Primer Pair
(Primer Pair


Instances
Spectrometry
Sequencing
(sample)
Year
No. 438)
No. 441)





48 
3
 3
MCRD San
2002
A30 G36 C20 T36
A40 G29 C19 T31


2
6
 6
Diego

A30 G36 C20 T36
A40 G29 C19 T31


1
28 
28
(Cultured)

A30 G36 C20 T36
A41 G28 C18 T32


15 
3
ND


A30 G36 C20 T36
A40 G29 C19 T31


6
3
 3
NHRC San
2003
A30 G36 C20 T36
A40 G29 C19 T31


3
5, 58
 5
Diego-

A30 G36 C20 T36
A40 G29 C19 T31


6
6
 6
Archive

A30 G36 C20 T36
A40 G29 C19 T31


1
11 
11
(Cultured)

A30 G36 C20 T36
A40 G29 C19 T31


3
12 
12


A30 G36 C19 T37
A40 G29 C19 T31


1
22 
22


A30 G36 C20 T36
A40 G29 C19 T31


3
25, 75
75


A30 G36 C20 T36
A40 G29 C19 T31


4
44/61, 82, 9
44/61


A30 G36 C20 T36
A41 G28 C19 T31


2
53, 91
91


A30 G36 C19 T37
A40 G29 C19 T31


1
2
 2
Ft.
2003
A30 G36 C20 T36
A40 G29 C19 T31


2
3
 3
Leonard

A30 G36 C20 T36
A40 G29 C19 T31


1
4
 4
Wood

A30 G36 C19 T37
A41 G28 C19 T31


1
6
 6
(Cultured)

A30 G36 C20 T36
A40 G29 C19 T31


11 
25 or 75
75


A30 G36 C20 T36
A40 G29 C19 T31


1
25, 75, 33,
75


A30 G36 C19 T37
A40 G29 C19 T31



34, 4, 52, 84


1
44/61 or 82
44/61


A30 G36 C20 T36
A41 G28 C19 T31



or 9


2
5 or 58
 5


A30 G36 C20 T36
A40 G29 C19 T31


3
1
 1
Ft. Sill
2003
A30 G36 C19 T37
A40 G29 C19 T31


2
3
 3
(Cultured)

A30 G36 C20 T36
A40 G29 C19 T31


1
4
 4


A30 G36 C19 T37
A41 G28 C19 T31


1
28 
28


A30 G36 C20 T36
A41 G28 C18 T32


1
3
 3
Ft.
2003
A30 G36 C20 T36
A40 G29 C19 T31


1
4
 4
Benning

A30 G36 C19 T37
A41 G28 C19 T31


3
6
 6
(Cultured)

A30 G36 C20 T36
A40 G29 C19 T31


1
11 
11


A30 G36 C20 T36
A40 G29 C19 T31


1
13 
 94**


A30 G36 C20 T36
A41 G28 C19 T31


1
44/61 or 82
82


A30 G36 C20 T36
A41 G28 C19 T31



or 9


1
5 or 58
58


A30 G36 C20 T36
A40 G29 C19 T31


1
78 or 89
89


A30 G36 C20 T36
A41 G28 C19 T31


2
5 or 58
ND
Lackland
2003
A30 G36 C20 T36
A40 G29 C19 T31


1
2

AFB

A30 G36 C20 T36
A40 G29 C19 T31


1
81 or 90

(Throat

A30 G36 C20 T36
A40 G29 C19 T31


1
78 

Swabs)

A30 G36 C20 T36
A41 G28 C19 T31


  3***
No detection



No detection
No detection


7
3
ND
MCRD San
2002
A30 G36 C20 T36
A40 G29 C19 T31


1
3
ND
Diego

A30 G36 C20 T36
A40 G29 C19 T31


1
3
ND
(Throat

A30 G36 C20 T36
No detection


1
3
ND
Swabs)

No detection
A40 G29 C19 T31


2
3
ND


A30 G36 C20 T36
A40 G29 C19 T31


3
No detection
ND


No detection
No detection
















TABLE 9C







Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus


samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441














emm-type by



gki
gtr


# of
Mass
emm-Gene
Location

(Primer Pair
((Primer Pair


Instances
Spectrometry
Sequencing
(sample)
Year
No. 442)
No. 443)





48 
3
 3
MCRD San
2002
A32 G35 C17 T32
A39 G28 C16 T32


2
6
 6
Diego

A31 G35 C17 T33
A39 G28 C15 T33


1
28 
28
(Cultured)

A30 G36 C17 T33
A39 G28 C16 T32


15 
3
ND


A32 G35 C17 T32
A39 G28 C16 T32


6
3
 3
NHRC San
2003
A32 G35 C17 T32
A39 G28 C16 T32


3
5, 58
 5
Diego-

A30 G36 C20 T30
A39 G28 C15 T33


6
6
 6
Archive

A31 G35 C17 T33
A39 G28 C15 T33


1
11 
11
(Cultured)

A30 G36 C20 T30
A39 G28 C16 T32


3
12 
12


A31 G35 C17 T33
A39 G28 C15 T33


1
22 
22


A31 G35 C17 T33
A38 G29 C15 T33


3
25, 75
75


A30 G36 C17 T33
A39 G28 C15 T33


4
44/61, 82, 9
44/61


A30 G36 C18 T32
A39 G28 C15 T33


2
53, 91
91


A32 G35 C17 T32
A39 G28 C16 T32


1
2
 2
Ft.
2003
A30 G36 C17 T33
A39 G28 C15 T33


2
3
 3
Leonard

A32 G35 C17 T32
A39 G28 C16 T32


1
4
 4
Wood

A31 G35 C17 T33
A39 G28 C15 T33


1
6
 6
(Cultured)

A31 G35 C17 T33
A39 G28 C15 T33


11 
25 or 75
75


A30 G36 C17 T33
A39 G28 C15 T33


1
25, 75, 33,
75


A30 G36 C17 T33
A39 G28 C15 T33



34, 4, 52, 84


1
44/61 or 82
44/61


A30 G36 C18 T32
A39 G28 C15 T33



or 9


2
5 or 58
 5


A30 G36 C20 T30
A39 G28 C15 T33


3
1
 1
Ft. Sill
2003
A30 G36 C18 T32
A39 G28 C15 T33


2
3
 3
(Cultured)

A32 G35 C17 T32
A39 G28 C16 T32


1
4
 4


A31 G35 C17 T33
A39 G28 C15 T33


1
28 
28


A30 G36 C17 T33
A39 G28 C16 T32


1
3
 3
Ft.
2003
A32 G35 C17 T32
A39 G28 C16 T32


1
4
 4
Benning

A31 G35 C17 T33
A39 G28 C15 T33


3
6
 6
(Cultured)

A31 G35 C17 T33
A39 G28 C15 T33


1
11 
11


A30 G36 C20 T30
A39 G28 C16 T32


1
13 
 94**


A30 G36 C19 T31
A39 G28 C15 T33


1
44/61 or 82
82


A30 G36 C18 T32
A39 G28 C15 T33



or 9


1
5 or 58
58


A30 G36 C20 T30
A39 G28 C15 T33


1
78 or 89
89


A30 G36 C18 T32
A39 G28 C15 T33


2
5 or 58
ND
Lackland
2003
A30 G36 C20 T30
A39 G28 C15 T33


1
2

AFB

A30 G36 C17 T33
A39 G28 C15 T33


1
81 or 90

(Throat

A30 G36 C17 T33
A39 G28 C15 T33


1
78 

Swabs)

A30 G36 C18 T32
A39 G28 C15 T33


  3***
No detection



No detection
No detection


7
3
ND
MCRD San
2002
A32 G35 C17 T32
A39 G28 C16 T32


1
3
ND
Diego

No detection
No detection


1
3
ND
(Throat

A32 G35 C17 T32
A39 G28 C16 T32


1
3
ND
Swabs)

A32 G35 C17 T32
No detection


2
3
ND


A32 G35 C17 T32
No detection


3
No detection
ND


No detection
No detection









Example 8
Design of Calibrant Polynucleotides Based on Bioagent Identifying Amplicons for Identification of Species of Bacteria (Bacterial Bioagent Identifying Amplicons)
This example describes the design of 19 calibrant polynucleotides based on bacterial bioagent identifying amplicons corresponding to the primers of the broad surveillance set (Table 5) and the Bacillus anthracis drill-down set (Table 6).
Calibration sequences were designed to simulate bacterial bioagent identifying amplicons produced by the T modified primer pairs shown in Tables 5 and 6 (primer names have the designation “TMOD”). The calibration sequences were chosen as a representative member of the section of bacterial genome from specific bacterial species which would be amplified by a given primer pair. The model bacterial species upon which the calibration sequences are based are also shown in Table 10. For example, the calibration sequence chosen to correspond to an amplicon produced by primer pair no. 361 is SEQ ID NO: 1445. In Table 10, the forward (_F) or reverse (_R) primer name indicates the coordinates of an extraction representing a gene of a standard reference bacterial genome to which the primer hybridizes e.g.: the forward primer name 16S_EC—713—732_TMOD_F indicates that the forward primer hybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence (in this case, the reference sequence is an extraction consisting of residues 4033120-4034661 of the genomic sequence of E. coli K12 (GenBank gi number 16127994). Additional gene coordinate reference information is shown in Table 11. The designation “TMOD” in the primer names indicates that the 5′ end of the primer has been modified with a non-matched template T residue which prevents the PCR polymerase from adding non-templated adenosine residues to the 5′ end of the amplification product, an occurrence which may result in miscalculation of base composition from molecular mass data (vide supra).
The 19 calibration sequences described in Tables 10 and 11 were combined into a single calibration polynucleotide sequence (SEQ ID NO: 1464—which is herein designated a “combination calibration polynucleotide”) which was then cloned into a pCR®—Blunt vector (Invitrogen, Carlsbad, Calif.). This combination calibration polynucleotide can be used in conjunction with the primers of Tables 5 or 6 as an internal standard to produce calibration amplicons for use in determination of the quantity of any bacterial bioagent. Thus, for example, when the combination calibration polynucleotide vector is present in an amplification reaction mixture, a calibration amplicon based on primer pair 346 (16S rRNA) will be produced in an amplification reaction with primer pair 346 and a calibration amplicon based on primer pair 363 (rpoC) will be produced with primer pair 363. Coordinates of each of the 19 calibration sequences within the calibration polynucleotide (SEQ ID NO: 1464) are indicated in Table 11.







TABLE 10







Bacterial Primer Pairs for Production of Bacterial Bioagent Identifying


Amplicons and Corresponding Representative Calibration Sequences















Forward

Reverse

Calibration




Primer

Primer
Calibration
Sequence


Primer

(SEQ ID

(SEQ ID
Sequence Model
(SEQ ID


Pair No.
Forward Primer Name
NO:)
Reverse Primer Name
NO:)
Species
NO:)
















361
16S_EC_1090_1111_2_TMOD_F
697
16S_EC_1175_1196_TMOD_R
1398
Bacillus
1445







anthracis


346
16S_EC_713_732_TMOD_F
202
16S_EC_789_809_TMOD_R
1110
Bacillus
1446







anthracis


347
16S_EC_785_806_TMOD_F
560
16S_EC_880_897_TMOD_R
1278
Bacillus
1447







anthracis


348
16S_EC_960_981_TMOD_F
706
16S_EC_1054_1073_TMOD_R
895
Bacillus
1448







anthracis


349
23S_EC_1826_1843_TMOD_F
401
23S_EC_1906_1924_TMOD_R
1156
Bacillus
1449







anthracis


360
23S_EC_2646_2667_TMOD_F
409
23S_EC_2745_2765_TMOD_R
1434
Bacillus
1450







anthracis


350
CAPC_BA_274_303_TMOD_F
476
CAPC_BA_349_376_TMOD_R
1314
Bacillus
1451







anthracis


351
CYA_BA_1353_1379_TMOD_F
355
CYA_BA_1448_1467_TMOD_R
1423
Bacillus
1452







anthracis


352
INFB_EC_1365_1393_TMOD_F
687
INFB_EC_1439_1467_TMOD_R
1411
Bacillus
1453







anthracis


353
LEF_BA_756_781_TMOD_F
220
LEF_BA_843_872_TMOD_R
1394
Bacillus
1454







anthracis


356
RPLB_EC_650_679_TMOD_F
449
RPLB_EC_739_762_TMOD_R
1380
Clostridium
1455







botulinum


449
RPLB_EC_690_710_F
309
RPLB_EC_737_758_R
1336
Clostridium
1456







botulinum


359
RPOB_EC_1845_1866_TMOD_F
659
RPOB_EC_1909_1929_TMOD_R
1250
Yersinia
1457







Pestis


362
RPOB_EC_3799_3821_TMOD_F
581
RPOB_EC_3862_3888_TMOD_R
1325
Burkholderia
1458







mallei


363
RPOC_EC_2146_2174_TMOD_F
284
RPOC_EC_2227_2245_TMOD_R
898
Burkholderia
1459







mallei


354
RPOC_EC_2218_2241_TMOD_F
405
RPOC_EC_2313_2337_TMOD_R
1072
Bacillus
1460







anthracis


355
SSPE_BA_115_137_TMOD_F
255
SSPE_BA_197_222_TMOD_R
1402
Bacillus
1461







anthracis


367
TUFB_EC_957_979_TMOD_F
308
TUFB_EC_1034_1058_TMOD_R
1276
Burkholderia
1462







mallei


358
VALS_EC_1105_1124_TMOD_F
385
VALS_EC_1195_1218_TMOD_R
1093
Yersinia
1463







Pestis
















TABLE 11







Primer Pair Gene Coordinate References and Calibration Polynucleotide Sequence


Coordinates within the Combination Calibration Polynucleotide















Coordinates of



Gene Extraction


Calibration Sequence in


Bacterial
Coordinates
Reference GenBank GI

Combination Calibration


Gene and
of Genomic or Plasmid
No. of Genomic (G) or
Primer
Polynucleotide (SEQ ID


Species
Sequence
Plasmid (P) Sequence
Pair No.
NO: 1464)





16S E. coli
4033120 . . . 4034661
16127994 (G)
346
 16 . . . 109


16S E. coli
4033120 . . . 4034661
16127994 (G)
347
 83 . . . 190


16S E. coli
4033120 . . . 4034661
16127994 (G)
348
246 . . . 353


16S E. coli
4033120 . . . 4034661
16127994 (G)
361
368 . . . 469


23S E. coli
4166220 . . . 4169123
16127994 (G)
349
743 . . . 837


23S E. coli
4166220 . . . 4169123
16127994 (G)
360
865 . . . 981


rpoB E. coli.
4178823 . . . 4182851
16127994 (G)
359
1591 . . . 1672



(complement strand)


rpoB E. coli
4178823 . . . 4182851
16127994 (G)
362
2081 . . . 2167



(complement strand)


rpoC E. coli
4182928 . . . 4187151
16127994 (G)
354
1810 . . . 1926


rpoC E. coli
4182928 . . . 4187151
16127994 (G)
363
2183 . . . 2279


infB E. coli
3313655 . . . 3310983
16127994 (G)
352
1692 . . . 1791



(complement strand)


tufB E. coli
4173523 . . . 4174707
16127994 (G)
367
2400 . . . 2498


rplB E. coli
3449001 . . . 3448180
16127994 (G)
356
1945 . . . 2060


rplB E. coli
3449001 . . . 3448180
16127994 (G)
449
1986 . . . 2055


valS E. coli
4481405 . . . 4478550
16127994 (G)
358
1462 . . . 1572



(complement strand)


capC
56074 . . . 55628
 6470151 (P)
350
2517 . . . 2616


B. anthracis
(complement strand)


cya
156626 . . . 154288
 4894216 (P)
351
1338 . . . 1449


B. anthracis
(complement strand)


lef
127442 . . . 129921
 4894216 (P)
353
1121 . . . 1234


B. anthracis


sspE
226496 . . . 226783
30253828 (G)
355
1007-1104


B. anthracis









Example 9
Use of a Calibration Polynucleotide for Determining the Quantity of Bacillus Anthracis in a Sample Containing a Mixture of Microbes
The process described in this example is shown in FIG. 2. The capC gene is a gene involved in capsule synthesis which resides on the pX02 plasmid of Bacillus anthracis. Primer pair number 350 (see Tables 10 and 11) was designed to identify Bacillus anthracis via production of a bacterial bioagent identifying amplicon. Known quantities of the combination calibration polynucleotide vector described in Example 8 were added to amplification mixtures containing bacterial bioagent nucleic acid from a mixture of microbes which included the Ames strain of Bacillus anthracis. Upon amplification of the bacterial bioagent nucleic acid and the combination calibration polynucleotide vector with primer pair no. 350, bacterial bioagent identifying amplicons and calibration amplicons were obtained and characterized by mass spectrometry. A mass spectrum measured for the amplification reaction is shown in FIG. 7. The molecular masses of the bioagent identifying amplicons provided the means for identification of the bioagent from which they were obtained (Ames strain of Bacillus anthracis) and the molecular masses of the calibration amplicons provided the means for their identification as well. The relationship between the abundance (peak height) of the calibration amplicon signals and the bacterial bioagent identifying amplicon signals provides the means of calculation of the copies of the pX02 plasmid of the Ames strain of Bacillus anthracis. Methods of calculating quantities of molecules based on internal calibration procedures are well known to those of ordinary skill in the art.
Averaging the results of 10 repetitions of the experiment described above, enabled a calculation that indicated that the quantity of Ames strain of Bacillus anthracis present in the sample corresponds to approximately 10 copies of pX02 plasmid.
Example 10
Triangulation Genotyping Analysis of Campylobacter Species
A series of triangulation genotyping analysis primers were designed as described in Example 1 with the objective of identification of different strains of Campylobacter jejuni. The primers are listed in Table 12 with the designation “CJST_CJ.” Housekeeping genes to which the primers hybridize and produce bioagent identifying amplicons include: tkt (transketolase), glyA (serine hydroxymethyltransferase), gltA (citrate synthase), aspA (aspartate ammonia lyase), glnA (glutamine synthase), pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).







TABLE 12







Campylobacter Genotyping Primer Pairs












Primer







Pair

Forward Primer

Reverse Primer


No.
Forward Primer Name
(SEQ ID NO:)
Reverse Primer Name
(SEQ ID NO:)
Target Gene















1053
CJST_CJ_1080_1110_F
681
CJST_CJ_1166_1198_R
1022
gltA


1047
CJST_CJ_584_616_F
315
CJST_CJ_663_692_R
1379
glnA


1048
CJST_CJ_360_394_F
346
CJST_CJ_442_476_R
955
aspA


1049
CJST_CJ_2636_2668_F
504
CJST_CJ_2753_2777_R
1409
tkt


1054
CJST_CJ_2060_2090_F
323
CJST_CJ_2148_2174_R
1068
pgm


1064
CJST_CJ_1680_1713_F
479
CJST_CJ_1795_1822_R
938
glyA









The primers were used to amplify nucleic acid from 50 food product samples provided by the USDA, 25 of which contained Campylobacter jejuni and 25 of which contained Campylobacter coli. Primers used in this study were developed primarily for the discrimination of Campylobacter jejuni clonal complexes and for distinguishing Campylobacter jejuni from Campylobacter coli. Finer discrimination between Campylobacter coli types is also possible by using specific primers targeted to loci where closely-related Campylobacter coli isolates demonstrate polymorphisms between strains. The conclusions of the comparison of base composition analysis with sequence analysis are shown in Tables 1 3A-C.







TABLE 13A







Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-down


MLST Primer Pair Nos: 1048 and 1047




















Base
Base








Composition of
Composition of





MLST type or


Bioagent
Bioagent





Clonal
MLST Type

Identifying
Identifying





Complex by
or Clonal

Amplicon
Amplicon





Base
Complex by

Obtained with
Obtained with




Isolate
Composition
Sequence

Primer Pair No:
Primer Pair


Group
Species
origin
analysis
analysis
Strain
1048 (aspA)
No: 1047 (glnA)





J-1
C. jejuni
Goose
ST 690/
ST 991
RM3673
A30 G25 C16 T46
A47 G21 C16 T25





692/707/991


J-2
C. jejuni
Human
Complex
ST 356,
RM4192
A30 G25 C16 T46
A48 G21 C17 T23





206/48/353
complex






353


J-3
C. jejuni
Human
Complex
ST 436
RM4194
A30 G25 C15 T47
A48 G21 C18 T22





354/179


J-4
C. jejuni
Human
Complex 257
ST 257,
RM4197
A30 G25 C16 T46
A48 G21 C18 T22






complex






257


J-5
C. jejuni
Human
Complex 52
ST 52,
RM4277
A30 G25 C16 T46
A48 G21 C17 T23






complex 52


J-6
C. jejuni
Human
Complex 443
ST 51,
RM4275
A30 G25 C15 T47
A48 G21 C17 T23






complex
RM4279
A30 G25 C15 T47
A48 G21 C17 T23






443


J-7
C. jejuni
Human
Complex 42
ST 604,
RM1864
A30 G25 C15 T47
A48 G21 C18 T22






complex 42


J-8
C. jejuni
Human
Complex
ST 362,
RM3193
A30 G25 C15 T47
A48 G21 C18 T22





42/49/362
complex






362


J-9
C. jejuni
Human
Complex
ST 147,
RM3203
A30 G25 C15 T47
A47 G21 C18 T23





45/283
Complex 45



C. jejuni
Human
Consistent
ST 828
RM4183
A31 G27 C20 T39
A48 G21 C16 T24


C-1
C. coli

with 74
ST 832
RM1169
A31 G27 C20 T39
A48 G21 C16 T24





closely
ST 1056
RM1857
A31 G27 C20 T39
A48 G21 C16 T24




Poultry
related
ST 889
RM1166
A31 G27 C20 T39
A48 G21 C16 T24





sequence
ST 829
RM1182
A31 G27 C20 T39
A48 G21 C16 T24





types (none
ST 1050
RM1518
A31 G27 C20 T39
A48 G21 C16 T24





belong to a
ST 1051
RM1521
A31 G27 C20 T39
A48 G21 C16 T24





clonal
ST 1053
RM1523
A31 G27 C20 T39
A48 G21 C16 T24





complex)
ST 1055
RM1527
A31 G27 C20 T39
A48 G21 C16 T24






ST 1017
RM1529
A31 G27 C20 T39
A48 G21 C16 T24






ST 860
RM1840
A31 G27 C20 T39
A48 G21 C16 T24






ST 1063
RM2219
A31 G27 C20 T39
A48 G21 C16 T24






ST 1066
RM2241
A31 G27 C20 T39
A48 G21 C16 T24






ST 1067
RM2243
A31 G27 C20 T39
A48 G21 C16 T24






ST 1068
RM2439
A31 G27 C20 T39
A48 G21 C16 T24




Swine

ST 1016
RM3230
A31 G27 C20 T39
A48 G21 C16 T24






ST 1069
RM3231
A31 G27 C20 T39
A48 G21 C16 T24






ST 1061
RM1904
A31 G27 C20 T39
A48 G21 C16 T24




Unknown

ST 825
RM1534
A31 G27 C20 T39
A48 G21 C16 T24






ST 901
RM1505
A31 G27 C20 T39
A48 G21 C16 T24


C-2
C. coli
Human
ST 895
ST 895
RM1532
A31 G27 C19 T40
A48 G21 C16 T24


C-3
C. coli
Poultry
Consistent
ST 1064
RM2223
A31 G27 C20 T39
A48 G21 C16 T24





with 63
ST 1082
RM1178
A31 G27 C20 T39
A48 G21 C16 T24





closely
ST 1054
RM1525
A31 G27 C20 T39
A48 G21 C16 T24





related
ST 1049
RM1517
A31 G27 C20 T39
A48 G21 C16 T24




Marmoset
sequence
ST 891
RM1531
A31 G27 C20 T39
A48 G21 C16 T24





types (none





belong to a





clonal





complex)
















TABLE 13B







Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-


down MLST Primer Pair Nos: 1053 and 1064




















Base
Base








Composition of
Composition of





MLST type or


Bioagent
Bioagent





Clonal
MLST Type

Identifying
Identifying





Complex by
or Clonal

Amplicon
Amplicon





Base
Complex by

Obtained with
Obtained with




Isolate
Composition
Sequence

Primer Pair
Primer Pair


Group
Species
origin
analysis
analysis
Strain
No: 1053 (gltA)
No: 1064 (glyA)





J-1
C. jejuni
Goose
ST 690/
ST 991
RM3673
A24 G25 C23 T47
A40 G29 C29 T45





692/707/991


J-2
C. jejuni
Human
Complex
ST 356,
RM4192
A24 G25 C23 T47
A40 G29 C29 T45





206/48/353
complex






353


J-3
C. jejuni
Human
Complex
ST 436
RM4194
A24 G25 C23 T47
A40 G29 C29 T45





354/179


J-4
C. jejuni
Human
Complex 257
ST 257,
RM4197
A24 G25 C23 T47
A40 G29 C29 T45






complex






257


J-5
C. jejuni
Human
Complex 52
ST 52,
RM4277
A24 G25 C23 T47
A39 G30 C26 T48






complex 52


J-6
C. jejuni
Human
Complex 443
ST 51,
RM4275
A24 G25 C23 T47
A39 G30 C28 T46






complex
RM4279
A24 G25 C23 T47
A39 G30 C28 T46






443


J-7
C. jejuni
Human
Complex 42
ST 604,
RM1864
A24 G25 C23 T47
A39 G30 C26 T48






complex 42


J-8
C. jejuni
Human
Complex
ST 362,
RM3193
A24 G25 C23 T47
A38 G31 C28 T46





42/49/362
complex






362


J-9
C. jejuni
Human
Complex
ST 147,
RM3203
A24 G25 C23 T47
A38 G31 C28 T46





45/283
Complex 45



C. jejuni
Human
Consistent
ST 828
RM4183
A23 G24 C26 T46
A39 G30 C27 T47


C-1
C. coli

with 74
ST 832
RM1169
A23 G24 C26 T46
A39 G30 C27 T47





closely
ST 1056
RM1857
A23 G24 C26 T46
A39 G30 C27 T47




Poultry
related
ST 889
RM1166
A23 G24 C26 T46
A39 G30 C27 T47





sequence
ST 829
RM1182
A23 G24 C26 T46
A39 G30 C27 T47





types (none
ST 1050
RM1518
A23 G24 C26 T46
A39 G30 C27 T47





belong to a
ST 1051
RM1521
A23 G24 C26 T46
A39 G30 C27 T47





clonal
ST 1053
RM1523
A23 G24 C26 T46
A39 G30 C27 T47





complex)
ST 1055
RM1527
A23 G24 C26 T46
A39 G30 C27 T47






ST 1017
RM1529
A23 G24 C26 T46
A39 G30 C27 T47






ST 860
RM1840
A23 G24 C26 T46
A39 G30 C27 T47






ST 1063
RM2219
A23 G24 C26 T46
A39 G30 C27 T47






ST 1066
RM2241
A23 G24 C26 T46
A39 G30 C27 T47






ST 1067
RM2243
A23 G24 C26 T46
A39 G30 C27 T47






ST 1068
RM2439
A23 G24 C26 T46
A39 G30 C27 T47




Swine

ST 1016
RM3230
A23 G24 C26 T46
A39 G30 C27 T47






ST 1069
RM3231
A23 G24 C26 T46
NO DATA






ST 1061
RM1904
A23 G24 C26 T46
A39 G30 C27 T47




Unknown

ST 825
RM1534
A23 G24 C26 T46
A39 G30 C27 T47






ST 901
RM1505
A23 G24 C26 T46
A39 G30 C27 T47


C-2
C. coli
Human
ST 895
ST 895
RM1532
A23 G24 C26 T46
A39 G30 C27 T47


C-3
C. coli
Poultry
Consistent
ST 1064
RM2223
A23 G24 C26 T46
A39 G30 C27 T47





with 63
ST 1082
RM1178
A23 G24 C26 T46
A39 G30 C27 T47





closely
ST 1054
RM1525
A23 G24 C25 T47
A39 G30 C27 T47





related
ST 1049
RM1517
A23 G24 C26 T46
A39 G30 C27 T47




Marmoset
sequence
ST 891
RM1531
A23 G24 C26 T46
A39 G30 C27 T47





types (none





belong to a





clonal





complex)
















TABLE 13C







Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-


down MLST Primer Pair Nos: 1054 and 1049




















Base
Base








Composition of
Composition of





MLST type or


Bioagent
Bioagent





Clonal
MLST Type

Identifying
Identifying





Complex by
or Clonal

Amplicon
Amplicon





Base
Complex by

Obtained with
Obtained with




Isolate
Composition
Sequence

Primer Pair No:
Primer Pair


Group
Species
origin
analysis
analysis
Strain
1054 (pgm)
No: 1049 (tkt)





J-1
C. jejuni
Goose
ST 690/
ST 991
RM3673
A26 G33 C18 T38
A41 G28 C35 T38





692/707/991


J-2
C. jejuni
Human
Complex
ST 356,
RM4192
A26 G33 C19 T37
A41 G28 C36 T37





206/48/353
complex






353


J-3
C. jejuni
Human
Complex
ST 436
RM4194
A27 G32 C19 T37
A42 G28 C36 T36





354/179


J-4
C. jejuni
Human
Complex 257
ST 257,
RM4197
A27 G32 C19 T37
A41 G29 C35 T37






complex






257


J-5
C. jejuni
Human
complex 52
ST 52,
RM4277
A26 G33 C18 T38
A41 G28 C36 T37






complex 52


J-6
C. jejuni
Human
Complex 443
ST 51,
RM4275
A27 G31 C19 T38
A41 G28 C36 T37






complex
RM4279
A27 G31 C19 T38
A41 G28 C36 T37






443


J-7
C. jejuni
Human
Complex 42
ST 604,
RM1864
A27 G32 C19 T37
A42 G28 C35 T37






complex 42


J-8
C. jejuni
Human
Complex
ST 362,
RM3193
A26 G33 C19 T37
A42 G28 C35 T37





42/49/362
complex






362


J-9
C. jejuni
Human
Complex
ST 147,
RM3203
A28 G31 C19 T37
A43 G28 C36 T35





45/283
Complex 45



C. jejuni
Human
Consistent
ST 828
RM4183
A27 G30 C19 T39
A46 G28 C32 T36


C-1
C. coli

with 74
ST 832
RM1169
A27 G30 C19 T39
A46 G28 C32 T36





closely
ST 1056
RM1857
A27 G30 C19 T39
A46 G28 C32 T36




Poultry
related
ST 889
RM1166
A27 G30 C19 T39
A46 G28 C32 T36





sequence
ST 829
RM1182
A27 G30 C19 T39
A46 G28 C32 T36





types (none
ST 1050
RM1518
A27 G30 C19 T39
A46 G28 C32 T36





belong to a
ST 1051
RM1521
A27 G30 C19 T39
A46 G28 C32 T36





clonal
ST 1053
RM1523
A27 G30 C19 T39
A46 G28 C32 T36





complex)
ST 1055
RM1527
A27 G30 C19 T39
A46 G28 C32 T36






ST 1017
RM1529
A27 G30 C19 T39
A46 G28 C32 T36






ST 860
RM1840
A27 G30 C19 T39
A46 G28 C32 T36






ST 1063
RM2219
A27 G30 C19 T39
A46 G28 C32 T36






ST 1066
RM2241
A27 G30 C19 T39
A46 G28 C32 T36






ST 1067
RM2243
A27 G30 C19 T39
A46 G28 C32 T36






ST 1068
RM2439
A27 G30 C19 T39
A46 G28 C32 T36




Swine

ST 1016
RM3230
A27 G30 C19 T39
A39 G30 C27 T47






ST 1069
RM3231
A27 G30 C19 T39
A46 G28 C32 T36






ST 1061
RM1904
A27 G30 C19 T39
A46 G28 C32 T36




Unknown

ST 825
RM1534
A27 G30 C19 T39
A46 G28 C32 T36






ST 901
RM1505
A27 G30 C19 T39
A46 G28 C32 T36


C-2
C. coli
Human
ST 895
ST 895
RM1532
A27 G30 C19 T39
A46 G28 C32 T36


C-3
C. coli
Poultry
Consistent
ST 1064
RM2223
A27 G30 C19 T39
A46 G28 C32 T36





with 63
ST 1082
RM1178
A27 G30 C19 T39
A46 G28 C32 T36





closely
ST 1054
RM1525
A27 G30 C19 T39
A46 G28 C32 T36





related
ST 1049
RM1517
A27 G30 C19 T39
A46 G28 C32 T36




Marmoset
sequence
ST 891
RM1531
A27 G30 C19 T39
A46 G28 C32 T36





types (none





belong to a





clonal





complex)









The base composition analysis method was successful in identification of 12 different strain groups. Campylobacter jejuni and Campylobacter coli are generally differentiated by all loci. Ten clearly differentiated Campylobacter jejuni isolates and 2 major Campylobacter coli groups were identified even though the primers were designed for strain typing of Campylobacter jejuni. One isolate (RM4183) which was designated as Campylobacter jejuni was found to group with Campylobacter coli and also appears to actually be Campylobacter coli by full MLST sequencing.
Example 11
Identification of Acinetobacter baumannii Using Broad Range Survey and Division-Wide Primers in Epidemiological Surveillance
To test the capability of the broad range survey and division-wide primer sets of Table 5 in identification of Acinetobacter species, 183 clinical samples were obtained from individuals participating in, or in contact with individuals participating in Operation Iraqi Freedom (including US service personnel, US civilian patients at the Walter Reed Army Institute of Research (WRAIR), medical staff, Iraqi civilians and enemy prisoners. In addition, 34 environmental samples were obtained from hospitals in Iraq, Kuwait, Germany, the United States and the USNS Comfort, a hospital ship.
Upon amplification of nucleic acid obtained from the clinical samples, primer pairs 346-349, 360, 361, 354, 362 and 363 (Table 5) all produced bacterial bioagent amplicons which identified Acinetobacter baumannii in 215 of 217 samples. The organism Klebsiella pneumoniae was identified in the remaining two samples. In addition, 14 different strain types (containing single nucleotide polymorphisms relative to a reference strain of Acinetobacter baumannii) were identified and assigned arbitrary numbers from 1 to 14. Strain type 1 was found in 134 of the sample isolates and strains 3 and 7 were found in 46 and 9 of the isolates respectively.
The epidemiology of strain type 7 of Acinetobacter baumannii was investigated. Strain 7 was found in 4 patients and 5 environmental samples (from field hospitals in Iraq and Kuwait). The index patient infected with strain 7 was a pre-war patient who had a traumatic amputation in March of 2003 and was treated at a Kuwaiti hospital. The patient was subsequently transferred to a hospital in Germany and then to WRAIR. Two other patients from Kuwait infected with strain 7 were found to be non-infectious and were not further monitored. The fourth patient was diagnosed with a strain 7 infection in September of 2003 at WRAIR. Since the fourth patient was not related involved in Operation Iraqi Freedom, it was inferred that the fourth patient was the subject of a nosocomial infection acquired at WRAIk as a result of the spread of strain 7 from the index patient.
The epidemiology of strain type 3 of Acinetobacter baumannii was also investigated. Strain type 3 was found in 46 samples, all of which were from patients (US service members, Iraqi civilians and enemy prisoners) who were treated on the USNS Comfort hospital ship and subsequently returned to Iraq or Kuwait. The occurrence of strain type 3 in a single locale may provide evidence that at least some of the infections at that locale were a result of nosocomial infections.
This example thus illustrates an embodiment of the present invention wherein the methods of analysis of bacterial bioagent identifying amplicons provide the means for epidemiological surveillance.
Example 12
Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Acinetobacter baumanii 
To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, an additional 21 primer pairs were selected based on analysis of housekeeping genes of the genus Acinetobacter. Genes to which the drill-down triangulation genotyping analysis primers hybridize for production of bacterial bioagent identifying amplicons include anthranilate synthase component I (trpE), adenylate kinase (adk), adenine glycosylase (mutY), fumarate hydratase (fumC), and pyrophosphate phospho-hydratase (ppa). These 21 primer pairs are indicated with reference to sequence listings in Table 14. Primer pair numbers 1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers 1155-1157 hybridize to and amplify segments of adk. Primer pair numbers 1158-1164 hybridize to and amplify segments of mutY. Primer pair numbers 1165-1170 hybridize to and amplify segments of fumC. Primer pair number 1171 hybridizes to and amplifies a segment of ppa. Primer pair numbers: 2846-2848 hybridize to and amplify segments of the parC gene of DNA topoisomerase which include a codon known to confer quinolone drug resistance upon sub-types of Acinetobacter baumannii. Primer pair numbers 2852-2854 hybridize to and amplify segments of the gyrA gene of DNA gyrase which include a codon known to confer quinolone drug resistance upon sub-types of Acinetobacter baumannii. Primer pair numbers 2922 and 2972 are speciating primers which are useful for identifying different species members of the genus Acinetobacter. The primer names given in Table 14A (with the exception of primer pair numbers 2846-2848, 2852-2854) indicate the coordinates to which the primers hybridize to a reference sequence which comprises a concatenation of the genes TrpE, efp (elongation factor p), adk, mutT, fumC, and ppa. For example, the forward primer of primer pair 1151 is named AB_MLST-11-OIF007—62—91_F because it hybridizes to the Acinetobacter primer reference sequence of strain type 11 in sample 007 of Operation Iraqi Freedom (OIF) at positions 62 to 91. DNA was sequenced from strain type 11 and from this sequence data and an artificial concatenated sequence of partial gene extractions was assembled for use in design of the triangulation genotyping analysis primers. The stretches of arbitrary residues “N”s in the concatenated sequence were added for the convenience of separation of the partial gene extractions (40N for AB_MLST (SEQ ID NO: 1444)).
The hybridization coordinates of primer pair numbers 2846-2848 are with respect to GenBank Accession number X95819. The hybridization coordinates of primer pair numbers 2852-2854 are with respect to GenBank Accession number AY642140. Sequence residue “I” appearing in the forward and reverse primers of primer pair number 2972 represents inosine.







TABLE 14A







Triangulation Genotyping Analysis Primer Pairs for Identification of Sub-species


characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter











Primer

Forward Primer

Reverse Primer


Pair No.
Forward Primer Name
(SEQ ID NO:)
Reverse Primer Name
(SEQ ID NO:)














1151
AB_MLST-11-OIF007_62_91_F
454
AB_MLST-11-OIF007_169_203_R
1418


1152
AB_MLST-11-OIF007_185_214_F
243
AB_MLST-11-OIF007_291_324_R
969


1153
AB_MLST-11-OIF007_260_289_F
541
AB_MLST-11-OIF007_364_393_R
1400


1154
AB_MLST-11-OIF007_206_239_F
436
AB_MLST-11-OIF007_318_344_R
1036


1155
AB_MLST-11-OIF007_522_552_F
378
AB_MLST-11-OIF007_587_610_R
1392


1156
AB_MLST-11-OIF007_547_571_F
250
AB_MLST-11-OIF007_656_686_R
902


1157
AB_MLST-11-OIF007_601_627_F
256
AB_MLST-11-OIF007_710_736_R
881


1158
AB_MLST-11-OIF007_1202_1225_F
384
AB_MLST-11-OIF007_1266_1296_R
878


1159
AB_MLST-11-OIF007_1202_1225_F
384
AB_MLST-11-OIF007_1299_1316_R
1199


1160
AB_MLST-11-OIF007_1234_1264_F
694
AB_MLST-11-OIF007_1335_1362_R
1215


1161
AB_MLST-11-OIF007_1327_1356_F
225
AB_MLST-11-OIF007_1422_1448_R
1212


1162
AB_MLST-11-OIF007_1345_1369_F
383
AB_MLST-11-OIF007_1470_1494_R
1083


1163
AB_MLST-11-OIF007_1351_1375_F
662
AB_MLST-11-OIF007_1470_1494_R
1083


1164
AB_MLST-11-OIF007_1387_1412_F
422
AB_MLST-11-OIF007_1470_1494_R
1083


1165
AB_MLST-11-OIF007_1542_1569_F
194
AB_MLST-11-OIF007_1656_1680_R
1173


1166
AB_MLST-11-OIF007_1566_1593_F
684
AB_MLST-11-OIF007_1656_1680_R
1173


1167
AB_MLST-11-OIF007_1611_1638_F
375
AB_MLST-11-OIF007_1731_1757_R
890


1168
AB_MLST-11-OIF007_1726_1752_F
182
AB_MLST-11-OIF007_1790_1821_R
1195


1169
AB_MLST-11-OIF007_1792_1826_F
656
AB_MLST-11-OIF007_1876_1909_R
1151


1170
AB_MLST-11-OIF007_1792_1826_F
656
AB_MLST-11-OIF007_1895_1927_R
1224


1171
AB_MLST-11-OIF007_1970_2002_F
618
AB_MLST-11-OIF007_2097_2118_R
1157


2846
PARC_X95819_33_58_F
302
PARC_X95819_121_153_R
852


2847
PARC_X95819_33_58_F
199
PARC_X95819_157_178_R
889


2848
PARC_X95819_33_58_F
596
PARC_X95819_97_128_R
1169


2852
GYRA_AY642140_−1_24_F
150
GYRA_AY642140_71_100_R
1242


2853
GYRA_AY642140_26_54_F
166
GYRA_AY642140_121_146_R
1069


2854
GYRA_AY642140_26_54_F
166
GYRA_AY642140_58_89_R
1168


2922
AB_MLST-11-OIF007_991_1018_F
583
AB_MLST-11-OIF007_1110_1137_R
923


2972
AB_MLST-11-OIF007_1007_1034_F
592
AB_MLST-11-OIF007_1126_1153_R
924

















TABLE 14B







Triangulation Genotyping Analysis Primer Pairs for Identification of Sub-species



characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter













Forward

Reverse




Primer
Primer

Primer


Pair No.
(SEQ ID NO:)
SEQUENCE
(SEQ ID NO:
SEQUENCE





1151
454
TGAGATTGCTGAACATTTAATGCTGATTGA
1418
TTGTACATTTGAAACAATATGCATGACATGTGAAT



 


1152
243
TATTGTTTCAAATGTACAAGGTGAAGTGCG
 969
TCACAGGTTCACTTCATCAATAATTTCCATTGC


 


1153
541
TGGAACGTTATCAGGTGCCCCAAAAATTCG
1400
TTGCAATCGACATATCCATTTCACCATGCC


 


1154
436
TGAAGTGCGTGATGATATCGATGCACTTGATGTA
1036
TCCGCCAAAAACTCCCCTTTTCACAGG


 


1155
378
TCGGTTTAGTAAAAGAACGTATTGCTCAACC
1392
TTCTGCTTGAGGAATAGTGCGTGG


 


1156
250
TCAACCTGACTGCGTGAATGGTTGT
 902
TACGTTCTACGATTTCTTCATCAGGTACATC


 


1157
256
TCAAGCAGAAGCTTTGGAAGAAGAAGG
 881
TACAACGTGATAAACACGACCAGAAGC


 


1158
384
TCGTGCCCGCAATTTGCATAAAGC
 878
TAATGCCGGGTAGTGCAATCCATTCTTCTAG


 


1159
384
TCGTGCCCGCAATTTGCATAAAGC
1199
TGCACCTGCGGTCGAGCG


 


1160
694
TTGTAGCACAGCAAGGCAAATTTCCTCAAAC
1215
TGCCATCCATAATCACGCCATACTGACG


 


1161
225
TAGGTTTACGTCAGTATGGCGTGATTATGG
1212
TGCCAGTTTCCACATTTCACGTTCGTG


 


1162
383
TCGTGATTATGGATGGCAACGTGAA
1083
TCGCTTGAGTGTACTGATGATTGCG


 


1163
662
TTATGGATGGCAACGTGAAACGCGT
1083
TCGCTTGAGTGTAGTCATGATTGCG


 


1164
422
TCTTTGCCATTGAAGATGACTTAAGC
1083
TCGCTTGAGTGTAGTCATGATTGCG


 


1165
194
TACTAGCGGTAAGCTTAAACAAGATTGC
1173
TGAGTCGGGTTCACTTTACCTGGCA


 


1166
684
TTGCCAATGATATTCGTTGGTTAGCAAG
1173
TGAGTCGGGTTCACTTTACCTGGCA


 


1167
375
TCGGCGAAATCCGTATTCCTGAAAATGA
 890
TACCGGAAGCACCAGCGACATTAATAG


 


1168
182
TACCACTATTAATGTCGCTGGTGCTTC
1195
TGCAACTGAATAGATTGCAGTAAGTTATAAGC


 


1169
656
TTATAACTTACTGCAATCTATTCAGTTGCTTGGT
1151
TGAATTATGCAAGAAGTGATCAATTTTCTCACGA




G


 


1170
656
TTATAACTTACTGCAATCTATTCAGTTGCTTGGT
1224
TGCCGTAACTAACATAAGAGAATTATGCAAGAA




G


 


1171
618
TGGTTATGTACCAAATACTTTGTCTGAAGATGG
1157
TGACGGCATCGATACCACCGTC


 


2846
302
TCCAAAAAAATCAGCGCGTACAGTGG
 852
TAAAGGATAGCGGTAACTAAATGGCTGAGCCAT


 


2847
199
TACTTGGTAAATACCACCCACATGGTGA
 889
TACCCCAGTTCCCCTGACCTTC


 


2848
596
TGGTAAATACCACCCACATGGTGAC
1169
TGAGCCATGAGTACCATGGCTTCATAACATGC


 


2852
150
TAAATCTGCCCGTGTCGTCGGTGAC
1242
TGCTAAAGTCTTGAGCCATACGAACAATGG


 


2853
166
TAATCGGTAAATATCACCCGCATGGTGAC
1069
TCGATCGAACCGAAGTTACCCTGACC


 


2854
166
TAATCGGTAAATATCACCCGCATGGTGAC
1168
TGAGCCATACGAACAATGGTTTCATAAACAGC


 


2922
583
TGGGCGATGCTGCGAAATGGTTAAAAGA
 923
TAGTATCACCACGTACACCCGCATCAGT


 


2972
592
TGGGIGATGCTGCIAAATGGTTAAAAGA
 924
TAGTATCACCACGTACICCIGGATCAGT









Analysis of bioagent identifying amplicons obtained using the primers of Table 14B for over 200 samples from Operation Iraqi Freedom resulted in the identification of 50 distinct strain type clusters. The largest cluster, designated strain type 11 (ST11) includes 42 sample isolates, all of which were obtained from US service personnel and Iraqi civilians treated at the 28th Combat Support Hospital in Baghdad. Several of these individuals were also treated on the hospital ship USNS Comfort. These observations are indicative of significant epidemiological correlation/linkage.
All of the sample isolates were tested against a broad panel of antibiotics to characterize their antibodies resistance profiles. As an example of a representative result from antibiotic susceptibility testing, ST11 was found to consist of four different clusters of isolates, each with a varying degree of sensitivity/resistance to the various antibiotics tested which included penicillins, extended spectrum penicillins, cephalosporins, carbepenem, protein synthesis inhibitors, nucleic acid synthesis inhibitors, anti-metabolites, and anti-cell membrane antibiotics. Thus, the genotyping power of bacterial bioagent identifying amplicons, particularly drill-down bacterial bioagent identifying amplicons, has the potential to increase the understanding of the transmission of infections in combat casualties, to identify the source of infection in the environment, to track hospital transmission of nosocomial infections, and to rapidly characterize drug-resistance profiles which enable development of effective infection control measures on a time-scale previously not achievable.
Example 13
Triangulation Genotyping Analysis and Codon Analysis of Acinetobacter baumannii Samples from Two Health Care Facilities
In this investigation, 88 clinical samples were obtained from Walter Reed Hospital and 95 clinical samples were obtained from Northwestern Medical Center. All samples from both healthcare facilities were suspected of containing sub-types of Acinetobacter baumannii, at least some of which were expected to be resistant to quinolone drugs. Each of the 183 samples was analyzed by the method of the present invention. DNA was extracted from each of the samples and amplified with eight triangulation genotyping analysis primer pairs represented by primer pair numbers: 1151, 1156, 1158, 1160, 1165, 1167, 1170, and 1171. The DNA was also amplified with speciating primer pair number 2922 and codon analysis primer pair numbers 2846-2848 which interrogate a codon present in the parC gene, and primer pair numbers 2852-2854 which bracket a codon present in the gyrA gene. The parC and gyrA codon mutations are both responsible for causing drug resistance in Acinetobacter baumannii. During evolution of drug resistant strains, the gyrA mutation usually occurs before the parC mutation. Amplification products were measured by ESI-TOF mass spectrometry as indicated in Example 4. The base compositions of the amplification products were calculated from the average molecular masses of the amplification products and are shown in Tables 15-18. The entries in each of the tables are grouped according to strain type number, which is an arbitrary number assigned to Acinetobacter baumannii strains in the order of observance beginning from the triangulation genotyping analysis OIF genotyping study described in Example 12. For example, strain type 11 which appears in samples from the Walter Reed Hospital is the same strain as the strain type 11 mentioned in Example 12. Ibis# refers to the order in which each sample was analyzed. Isolate refers to the original sample isolate numbering system used at the location from which the samples were obtained (either Walter Reed Hospital or Northwestern Medical Center). ST=strain type. ND=not detected. Base compositions highlighted with bold type indicate that the base composition is a unique base composition for the amplification product obtained with the pair of primers indicated.







TABLE 15A







Base Compositions of Amplification Products of 88 A. baumannii Samples Obtained from


Walter Reed Hospital and Amplified with Codon Analysis Primer Pairs Targeting the gyrA Gene

















PP No: 2852
PP No: 2853
PP No: 2854


Species
Ibis#
Isolate
ST
gyrA
gyrA
gyrA
















A. baumannii
20
1082
1
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. baumannii
13
 854
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
22
1162
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
27
1230
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
31
1367
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
37
1459
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
55
1700
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
64
1777
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
73
1861
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
74
1877
10
ND
A29G28C21T43
A17G13C13T21


A. baumannii
86
1972
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
 3
 684
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
 6
 720
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
 7
 726
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
19
1079
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
21
1123
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
23
1188
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
33
1417
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
34
1431
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
38
1496
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
40
1523
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
42
1640
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
50
1666
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
51
1668
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
52
1695
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
65
1781
11
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
44
1649
12
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
  49A
  1658.1
12
A25G23C22T31
A29G28C21T43
A17G13C13T21


A. baumannii
  49B
  1658.2
12
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
56
1707
12
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
80
1893
12
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
 5
 693
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
 8
 749
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
10
 839
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
14
 865
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
16
 888
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
29
1326
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
35
1440
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
41
1524
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
46
1652
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
47
1653
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
48
1657
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
57
1709
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
61
1727
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
63
1762
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
67
1806
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
75
1881
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
77
1886
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
 1
 649
46
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
 2
 653
46
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
39
1497
16
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
24
1198
15
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
28
1243
15
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
43
1648
15
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
62
1746
15
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
 4
 689
15
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
68
1822
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
69
  1823A
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
70
  1823B
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
71
1826
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
72
1860
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
81
1924
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
82
1929
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
85
1966
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
11
 841
3
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. baumannii
32
1415
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
45
1651
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
54
1697
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
58
1712
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
60
1725
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
66
1802
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
76
1883
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
78
1891
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
79
1892
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
83
1947
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
84
1964
24
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
53
1696
24
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. baumannii
36
1458
49
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
59
1716
9
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. baumannii
 9
 805
30
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. baumannii
18
 967
39
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. baumannii
30
1322
48
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. baumannii
26
1218
50
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. sp. 13TU
15
 875
A1
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. sp. 13TU
17
 895
A1
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. sp. 3
12
 853
B7
A25G22C22T32
A30G29C22T40
A17G13C14T20


A. johnsonii
25
1202
NEW1
A25G22C22T32
A30G29C22T40
A17G13C14T20


A. sp. 2082
87
2082
NEW2
A25G22C22T32
A31G28C22T40
A17G13C14T20
















TABLE 15B







Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter


Reed Hospital and Amplified with Codon Analysis Primer Pairs Targeting the parC Gene

















PP No: 2846
PP No: 2847
PP No: 2848


Species
Ibis#
Isolate
ST
parC
parC
parC
















A. baumannii
20
1082
1
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
13
 854
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
22
1162
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
27
1230
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
31
1367
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
37
1459
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
55
1700
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
64
1777
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
73
1861
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
74
1877
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
86
1972
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
 3
 684
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
 6
 720
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
 7
 726
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
19
1079
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
21
1123
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
23
1188
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
33
1417
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
34
1431
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
38
1496
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
40
1523
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
42
1640
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
50
1666
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
51
1668
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
52
1695
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
65
1781
11
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
44
1649
12
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
  49A
  1658.1
12
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
  49B
  1658.2
12
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
56
1707
12
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
80
1893
12
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
 5
 693
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
 8
 749
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
10
 839
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
14
 865
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
16
 888
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
29
1326
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
35
1440
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
41
1524
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
46
1652
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
47
1653
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
48
1657
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
57
1709
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
61
1727
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
63
1762
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
67
1806
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
75
1881
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
77
1886
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
 1
 649
46
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
 2
 653
46
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
39
1497
16
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
24
1198
15
A33G26C28T34
A29G29C23T33
A16G14C14T16


A. baumannii
28
1243
15
A33G26C28T34
A29G29C23T33
A16G14C14T16


A. baumannii
43
1648
15
A33G26C28T34
A29G29C23T33
A16G14C14T16


A. baumannii
62
1746
15
A33G26C28T34
A29G29C23T33
A16G14C14T16


A. baumannii
 4
 689
15
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
68
1822
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
69
  1823A
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
70
  1823B
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
71
1826
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
72
1860
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
81
1924
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
82
1929
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
85
1966
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
11
 841
3
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
32
1415
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
45
1651
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
54
1697
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
58
1712
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
60
1725
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
66
1802
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
76
1883
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
78
1891
24
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
79
1892
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
83
1947
24
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
84
1964
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
53
1696
24
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
36
1458
49
A34G26C29T32
A30G28C24T32
A16G14C15T15


A. baumannii
59
1716
9
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
 9
 805
30
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
18
 967
39
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
30
1322
48
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
26
1218
50
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. sp. 13TU
15
 875
A1
A32G26C28T35
A28G28C24T34
A16G14C15T15


A. sp. 13TU
17
 895
A1
A32G26C28T35
A28G28C24T34
A16G14C15T15


A. sp. 3
12
 853
B7
A29G26C27T39
A26G32C21T35
A16G14C15T15


A. johnsonii
25
1202
NEW1
A32G28C26T35
A29G29C22T34
A16G14C15T15


A. sp. 2082
87
2082
NEW2
A33G27C26T35
A31G28C20T35
A16G14C15T15
















TABLE 16A







Base Compositions Determined from A. baumannii DNA Samples Obtained from


Northwestern Medical Center and Amplified with Codon Analysis Primer Pairs Targeting


the gyrA Gene

















PP No: 2852
PP No: 2853
PP No: 2854


Species
Ibis#
Isolate
ST
gyrA
gyrA
gyrA
















A. baumannii
54
536
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
87
665
3
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
8
80
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
9
91
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
10
92
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
11
131
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
12
137
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
21
218
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
26
242
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
94
678
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
1
9
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
2
13
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
3
19
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
4
24
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
5
36
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
6
39
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
13
139
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
15
165
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
16
170
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
17
186
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
20
202
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
22
221
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
24
234
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
25
239
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
33
370
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
34
389
10
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
19
201
14
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
27
257
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
29
301
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
31
354
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
36
422
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
37
424
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
38
434
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
39
473
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
40
482
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
44
512
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
45
516
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
47
522
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
48
526
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
50
528
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
52
531
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
53
533
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
56
542
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
59
550
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
62
556
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
64
557
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
70
588
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
73
603
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
74
605
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
75
606
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
77
611
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
79
622
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
83
643
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
85
653
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
89
669
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
93
674
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
23
228
51
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
32
369
52
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
35
393
52
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
30
339
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
41
485
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
42
493
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
43
502
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
46
520
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
49
527
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
51
529
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
65
562
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
68
579
53
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
57
546
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
58
548
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
60
552
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
61
555
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
63
557
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
66
570
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
67
578
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
69
584
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
71
593
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
72
602
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
76
609
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
78
621
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
80
625
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
81
628
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
82
632
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
84
649
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
86
655
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
88
668
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
90
671
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
91
672
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
92
673
54
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
18
196
55
A25G23C22T31
A29G28C21T43
A17G13C13T21


A. baumannii
55
537
27
A25G23C21T32
A29G28C21T43
A17G13C13T21


A. baumannii
28
263
27
A25G23C22T31
A29G28C22T42
A17G13C14T20


A. sp. 3
14
164
B7
A25G22C22T32
A30G29C22T40
A17G13C14T20


mixture
7
71
—
ND
ND
A17G13C15T19
















TABLE 16B







Base Compositions Determined from A. baumannii DNA Samples Obtained from


Northwestern Medical Center and Amplified with Codon Analysis Primer Pairs Targeting


the parC Gene

















PP No: 2846
PP No: 2847
PP No: 2848


Species
Ibis#
Isolate
ST
parC
parC
parC
















A. baumannii
54
536
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
87
665
3
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
8
80
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
9
91
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
10
92
10
A33G26C28T34
A29G28C25T32
ND


A. baumannii
11
131
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
12
137
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
21
218
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
26
242
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
94
678
10
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
1
9
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
2
13
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
3
19
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
4
24
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
5
36
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
6
39
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
13
139
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
15
165
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
16
170
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
17
186
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
20
202
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
22
221
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
24
234
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
25
239
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
33
370
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
34
389
10
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
19
201
14
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
27
257
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
29
301
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
31
354
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
36
422
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
37
424
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
38
434
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
39
473
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
40
482
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
44
512
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
45
516
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
47
522
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
48
526
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
50
528
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
52
531
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
53
533
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
56
542
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
59
550
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
62
556
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
64
557
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
70
588
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
73
603
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
74
605
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
75
606
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
77
611
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
79
622
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
83
643
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
85
653
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
89
669
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
93
674
51
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
23
228
51
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
32
369
52
A34G25C28T34
A30G27C25T32
A16G14C14T16


A. baumannii
35
393
52
A34G25C28T34
A30G27C25T32
A16G14C14T16


A. baumannii
30
339
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
41
485
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
42
493
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
43
502
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
46
520
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
49
527
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
51
529
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
65
562
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
68
579
53
A34G25C29T33
A30G27C26T31
A16G14C15T15


A. baumannii
57
546
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
58
548
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
60
552
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
61
555
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
63
557
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
66
570
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
67
578
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
69
584
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
71
593
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
72
602
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
76
609
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
78
621
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
80
625
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
81
628
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
82
632
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
84
649
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
86
655
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
88
668
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
90
671
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
91
672
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
92
673
54
A33G26C28T34
A29G28C25T32
A16G14C14T16


A. baumannii
18
196
55
A33G27C28T33
A29G28C25T31
A15G14C15T16


A. baumannii
55
537
27
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. baumannii
28
263
27
A33G26C29T33
A29G28C26T31
A16G14C15T15


A. sp. 3
14
164
B7
A35G25C29T32
A30G28C17T39
A16G14C15T15


mixture
7
71
—
ND
ND
A17G14C15T14
















TABLE 17A







Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter


Reed Hospital and Amplified with Speciating Primer Pair No. 2922 and Triangulation Genotyping


Analysis Primer Pair Nos. 1151 and 1156

















PP No: 2922
PP No: 1151
PP No: 1156


Species
Ibis#
Isolate
ST
efp
trpE
Adk
















A. baumannii
20
1082
1
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
13
 854
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
22
1162
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
27
1230
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
31
1367
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
37
1459
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
55
1700
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
64
1777
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
73
1861
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
74
1877
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
86
1972
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
 3
 684
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
 6
 720
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
 7
 726
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
19
1079
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
21
1123
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
23
1188
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
33
1417
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
34
1431
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
38
1496
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
40
1523
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
42
1640
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
50
1666
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
51
1668
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
52
1695
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
65
1781
11
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
44
1649
12
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
  49A
  1658.1
12
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
  49B
  1658.2
12
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
56
1707
12
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
80
1893
12
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
 5
 693
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
 8
 749
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
10
 839
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
14
 865
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
16
 888
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
29
1326
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
35
1440
14
A44G35C25T43
ND
A44G32C27T37


A. baumannii
41
1524
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
46
1652
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
47
1653
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
48
1657
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
57
1709
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
61
1727
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
63
1762
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
67
1806
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
75
1881
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
77
1886
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
 1
 649
46
A44G35C25T43
A44G35C22T41
A44G32C26T38


A. baumannii
 2
 653
46
A44G35C25T43
A44G35C22T41
A44G32C26T38


A. baumannii
39
1497
16
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
24
1198
15
A44G35C25T43
A44G35C22T41
A44G32C26T38


A. baumannii
28
1243
15
A44G35C25T43
A44G35C22T41
A44G32C26T38


A. baumannii
43
1648
15
A44G35C25T43
A44G35C22T41
A44G32C26T38


A. baumannii
62
1746
15
A44G35C25T43
A44G35C22T41
A44G32C26T38


A. baumannii
 4
 689
15
A44G35C25T43
A44G35C22T41
A44G32C26T38


A. baumannii
68
1822
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
69
  1823A
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
70
  1823B
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
71
1826
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
72
1860
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
81
1924
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
82
1929
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
85
1966
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
11
 841
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
32
1415
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
45
1651
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
54
1697
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
58
1712
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
60
1725
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
66
1802
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
76
1883
24
ND
A43G36C20T43
A44G32C27T37


A. baumannii
78
1891
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
79
1892
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
83
1947
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
84
1964
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
53
1696
24
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
36
1458
49
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
59
1716
9
A44G35C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
 9
 805
30
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
18
 967
39
A45G34C25T43
A44G35C22T41
A44G32C26T38


A. baumannii
30
1322
48
A44G35C25T43
A43G36C20T43
A44G32C27T37


A. baumannii
26
1218
50
A44G35C25T43
A44G35C21T42
A44G32C26T38


A. sp. 13TU
15
 875
A1
A47G33C24T43
A46G32C20T44
A44G33C27T36


A. sp. 13TU
17
 895
A1
A47G33C24T43
A46G32C20T44
A44G33C27T36


A. sp. 3
12
 853
B7
A46G35C24T42
A42G34C20T46
A43G33C24T40


A. johnsonii
25
1202
NEW1
A46G35C23T43
A42G35C21T44
A43G33C23T41


A. sp. 2082
87
2082
NEW2
A46G36C22T43
A42G32C20T48
A42G34C23T41
















TABLE 17B







Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter


Reed Hospital and Amplified with Triangulation Genotyping Analysis Primer Pair Nos. 1158 and


1160 and 1165

















PP No: 1158
PP No: 1160
PP No: 1165


Species
Ibis#
Isolate
ST
mutY
mutY
fumC
















A. baumannii
20
1082
1
A27G21C25T22
A32G35C29T33
A40G33C30T36


A. baumannii
13
 854
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
22
1162
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
27
1230
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
31
1367
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
37
1459
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
55
1700
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
64
1777
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
73
1861
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
74
1877
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
86
1972
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
 3
 684
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
 6
 720
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
 7
 726
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
19
1079
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
21
1123
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
23
1188
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
33
1417
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
34
1431
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
38
1496
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
40
1523
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
42
1640
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
50
1666
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
51
1668
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
52
1695
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
65
1781
11
A27G21C25T22
A32G34C28T35
A40G33C30T36


A. baumannii
44
1649
12
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
  49A
  1658.1
12
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
  49B
  1658.2
12
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
56
1707
12
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
80
1893
12
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
 5
 693
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
 8
 749
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
10
 839
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
14
 865
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
16
 888
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
29
1326
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
35
1440
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
41
1524
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
46
1652
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
47
1653
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
48
1657
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
57
1709
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
61
1727
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
63
1762
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
67
1806
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
75
1881
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
77
1886
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
 1
 649
46
A29G19C26T21
A31G35C29T34
A40G33C29T37


A. baumannii
 2
 653
46
A29G19C26T21
A31G35C29T34
A40G33C29T37


A. baumannii
39
1497
16
A29G19C26T21
A31G35C29T34
A40G34C29T36


A. baumannii
24
1198
15
A29G19C26T21
A31G35C29T34
A40G33C29T37


A. baumannii
28
1243
15
A29G19C26T21
A31G35C29T34
A40G33C29T37


A. baumannii
43
1648
15
A29G19C26T21
A31G35C29T34
A40G33C29T37


A. baumannii
62
1746
15
A29G19C26T21
A31G35C29T34
A40G33C29T37


A. baumannii
 4
 689
15
A29G19C26T21
A31G35C29T34
A40G33C29T37


A. baumannii
68
1822
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
69
  1823A
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
70
  1823B
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
71
1826
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
72
1860
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
81
1924
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
82
1929
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
85
1966
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
11
 841
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
32
1415
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
45
1651
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
54
1697
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
58
1712
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
60
1725
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
66
1802
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
76
1883
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
78
1891
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
79
1892
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
83
1947
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
84
1964
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
53
1696
24
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
36
1458
49
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
59
1716
9
A27G21C25T22
A32G35C28T34
A39G33C30T37


A. baumannii
 9
 805
30
A27G21C25T22
A32G35C28T34
A39G33C30T37


A. baumannii
18
 967
39
A27G21C26T21
A32G35C28T34
A39G33C30T37


A. baumannii
30
1322
48
A28G21C24T22
A32G35C29T33
A40G33C30T36


A. baumannii
26
1218
50
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. sp. 13TU
15
 875
A1
A27G21C25T22
A30G36C26T37
A41G34C28T36


A. sp. 13TU
17
 895
A1
A27G21C25T22
A30G36C26T37
A41G34C28T36


A. sp. 3
12
 853
B7
A26G23C23T23
A30G36C27T36
A39G37C26T37


A. johnsonii
25
1202
NEW1
A25G23C24T23
A30G35C30T34
A38G37C26T38


A. sp. 2082
87
2082
NEW2
A26G22C24T23
A31G35C28T35
A42G34C27T36
















TABLE 17C







Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter


Reed Hospital and Amplified with Triangulation Genotyping Analysis Primer Pair Nos. 1167 and


1170 and 1171

















PP No: 1167
PP No: 1170
PP No: 1171


Species
Ibis#
Isolate
ST
fumC
fumC
ppa
















A. baumannii
20
1082
1
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
13
 854
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
22
1162
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
27
1230
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
31
1367
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
37
1459
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
55
1700
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
64
1777
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
73
1861
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
74
1877
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
86
1972
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
 3
 684
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
 6
 720
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
 7
 726
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
19
1079
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
21
1123
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
23
1188
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
33
1417
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
34
1431
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
38
1496
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
40
1523
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
42
1640
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
50
1666
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
51
1668
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
52
1695
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
65
1781
11
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
44
1649
12
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
  49A
  1658.1
12
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
  49B
  1658.2
12
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
56
1707
12
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
80
1893
12
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
 5
 693
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
 8
 749
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
10
 839
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
14
 865
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
16
 888
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
29
1326
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
35
1440
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
41
1524
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
46
1652
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
47
1653
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
48
1657
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
57
1709
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
61
1727
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
63
1762
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
67
1806
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
75
1881
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
77
1886
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
 1
 649
46
A41G35C32T39
A37G28C20T51
A35G37C32T45


A. baumannii
 2
 653
46
A41G35C32T39
A37G28C20T51
A35G37C32T45


A. baumannii
39
1497
16
A41G35C32T39
A37G28C20T51
A35G37C30T47


A. baumannii
24
1198
15
A41G35C32T39
A37G28C20T51
A35G37C30T47


A. baumannii
28
1243
15
A41G35C32T39
A37G28C20T51
A35G37C30T47


A. baumannii
43
1648
15
A41G35C32T39
A37G28C20T51
A35G37C30T47


A. baumannii
62
1746
15
A41G35C32T39
A37G28C20T51
A35G37C30T47


A. baumannii
 4
 689
15
A41G35C32T39
A37G28C20T51
A35G37C30T47


A. baumannii
68
1822
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
69
  1823A
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
70
  1823B
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
71
1826
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
72
1860
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
81
1924
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
82
1929
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
85
1966
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
11
 841
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
32
1415
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
45
1651
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
54
1697
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
58
1712
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
60
1725
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
66
1802
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
76
1883
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
78
1891
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
79
1892
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
83
1947
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
84
1964
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
53
1696
24
A40G35C34T38
A39G26C22T49
A35G37C33T44


A. baumannii
36
1458
49
A40G35C34T38
A39G26C22T49
A35G37C30T47


A. baumannii
59
1716
9
A40G35C32T40
A38G27C20T51
A36G35C31T47


A. baumannii
 9
 805
30
A40G35C32T40
A38G27C21T50
A35G36C29T49


A. baumannii
18
 967
39
A40G35C33T39
A38G27C20T51
A35G37C30T47


A. baumannii
30
1322
48
A40G35C35T37
A38G27C21T50
A35G37C30T47


A. baumannii
26
1218
50
A40G35C34T38
A38G27C21T50
A35G37C33T44


A. sp. 13TU
15
 875
A1
A41G39C31T36
A37G26C24T49
A34G38C31T46


A. sp. 13TU
17
 895
A1
A41G39C31T36
A37G26C24T49
A34G38C31T46


A. sp. 3
12
 853
B7
A43G37C30T37
A36G27C24T49
A34G37C31T47


A. johnsonii
25
1202
NEW1
A42G38C31T36
A40G27C19T50
A35G37C32T45


A. sp. 2082
87
2082
NEW2
A43G37C32T35
A37G26C21T52
A35G38C31T45
















TABLE 18A







Base Compositions Determined from A. baumannii DNA Samples Obtained from


Northwestern Medical Center and Amplified with Speciating Primer Pair No. 2922 and


Triangulation Genotyping Analysis Primer Pair Nos. 1151 and 1156

















PP No: 2922
PP No: 1151
PP No: 1156


Species
Ibis#
Isolate
ST
efp
trpE
adk
















A. baumannii
54
536
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
87
665
3
A44G35C24T44
A44G35C22T41
A44G32C26T38


A. baumannii
8
80
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
9
91
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
10
92
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
11
131
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
12
137
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
21
218
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
26
242
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
94
678
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
1
9
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
2
13
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
3
19
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
4
24
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
5
36
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
6
39
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
13
139
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
15
165
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
16
170
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
17
186
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
20
202
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
22
221
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
24
234
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
25
239
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
33
370
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
34
389
10
A45G34C25T43
A44G35C21T42
A44G32C26T38


A. baumannii
19
201
14
A44G35C25T43
A44G35C22T41
A44G32C27T37


A. baumannii
27
257
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
29
301
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
31
354
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
36
422
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
37
424
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
38
434
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
39
473
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
40
482
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
44
512
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
45
516
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
47
522
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
48
526
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
50
528
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
52
531
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
53
533
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
56
542
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
59
550
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
62
556
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
64
557
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
70
588
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
73
603
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
74
605
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
75
606
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
77
611
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
79
622
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
83
643
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
85
653
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
89
669
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
93
674
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
23
228
51
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
32
369
52
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
35
393
52
A44G35C25T43
A43G36C20T43
A44G32C26T38


A. baumannii
30
339
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
41
485
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
42
493
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
43
502
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
46
520
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
49
527
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
51
529
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
65
562
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
68
579
53
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
57
546
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
58
548
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
60
552
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
61
555
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
63
557
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
66
570
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
67
578
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
69
584
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
71
593
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
72
602
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
76
609
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
78
621
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
80
625
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
81
628
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
82
632
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
84
649
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
86
655
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
88
668
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
90
671
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
91
672
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
92
673
54
A44G35C25T43
A44G35C20T43
A44G32C26T38


A. baumannii
18
196
55
A44G35C25T43
A44G35C20T43
A44G32C27T37


A. baumannii
55
537
27
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. baumannii
28
263
27
A44G35C25T43
A44G35C19T44
A44G32C27T37


A. sp. 3
14
164
B7
A46G35C24T42
A42G34C20T46
A43G33C24T40


mixture
7
71
?
mixture
ND
ND
















TABLE 18B







Base Compositions Determined from A. baumannii DNA Samples Obtained from


Northwestern Medical Center and Amplified with Triangulation Genotyping Analysis


Primer Pair Nos. 1158, 1160 and 1165

















PP No: 1158
PP No: 1160
PP No: 1165


Species
Ibis#
Isolate
ST
mutY
mutY
fumC
















A. baumannii
54
536
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
87
665
3
A27G20C27T21
A32G35C28T34
A40G33C30T36


A. baumannii
8
80
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
9
91
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
10
92
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
11
131
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
12
137
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
21
218
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
26
242
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
94
678
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
1
9
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
2
13
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
3
19
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
4
24
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
5
36
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
6
39
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
13
139
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
15
165
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
16
170
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
17
186
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
20
202
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
22
221
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
24
234
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
25
239
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
33
370
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
34
389
10
A27G21C26T21
A32G35C28T34
A40G33C30T36


A. baumannii
19
201
14
A27G21C25T22
A31G36C28T34
A40G33C29T37


A. baumannii
27
257
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
29
301
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
31
354
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
36
422
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
37
424
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
38
434
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
39
473
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
40
482
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
44
512
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
45
516
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
47
522
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
48
526
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
50
528
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
52
531
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
53
533
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
56
542
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
59
550
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
62
556
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
64
557
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
70
588
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
73
603
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
74
605
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
75
606
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
77
611
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
79
622
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
83
643
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
85
653
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
89
669
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
93
674
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
23
228
51
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
32
369
52
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
35
393
52
A27G21C25T22
A32G35C28T34
A40G33C29T37


A. baumannii
30
339
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
41
485
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
42
493
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
43
502
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
46
520
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
49
527
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
51
529
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
65
562
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
68
579
53
A28G20C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
57
546
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
58
548
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
60
552
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
61
555
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
63
557
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
66
570
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
67
578
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
69
584
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
71
593
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
72
602
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
76
609
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
78
621
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
80
625
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
81
628
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
82
632
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
84
649
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
86
655
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
88
668
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
90
671
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
91
672
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
92
673
54
A27G21C26T21
A32G34C29T34
A40G33C30T36


A. baumannii
18
196
55
A27G21C25T22
A31G36C27T35
A40G33C29T37


A. baumannii
55
537
27
A27G21C25T22
A32G35C28T34
A40G33C30T36


A. baumannii
28
263
27
A27G21C25T22
A32G35C28T34
A40G33C30T36


A. sp. 3
14
164
B7
A26G23C23T23
A30G36C27T36
A39G37C26T37


mixture
7
71
?
ND
ND
ND
















TABLE 18C







Base Compositions Determined from A. baumannii DNA Samples Obtained from


Northwestern Medical Center and Amplified with Triangulation Genotyping Analysis


Primer Pair Nos. 1167, 1170 and 1171

















PP No: 1167
PP No: 1170
PP No: 1171


Species
Ibis#
Isolate
ST
fumC
fumC
ppa
















A. baumannii
54
536
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
87
665
3
A41G34C35T37
A38G27C20T51
A35G37C31T46


A. baumannii
8
80
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
9
91
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
10
92
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
11
131
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
12
137
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
21
218
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
26
242
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
94
678
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
1
9
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
2
13
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
3
19
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
4
24
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
5
36
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
6
39
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
13
139
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
15
165
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
16
170
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
17
186
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
20
202
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
22
221
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
24
234
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
25
239
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
33
370
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
34
389
10
A41G34C34T38
A38G27C21T50
A35G37C33T44


A. baumannii
19
201
14
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
27
257
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
29
301
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
31
354
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
36
422
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
37
424
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
38
434
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
39
473
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
40
482
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
44
512
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
45
516
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
47
522
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
48
526
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
50
528
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
52
531
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
53
533
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
56
542
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
59
550
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
62
556
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
64
557
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
70
588
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
73
603
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
74
605
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
75
606
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
77
611
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
79
622
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
83
643
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
85
653
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
89
669
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
93
674
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
23
228
51
A40G35C34T38
A38G27C21T50
A35G37C30T47


A. baumannii
32
369
52
A40G35C34T38
A38G27C21T50
A35G37C31T46


A. baumannii
35
393
52
A40G35C34T38
A38G27C21T50
A35G37C31T46


A. baumannii
30
339
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
41
485
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
42
493
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
43
502
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
46
520
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
49
527
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
51
529
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
65
562
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
68
579
53
A40G35C35T37
A38G27C21T50
A35G37C31T46


A. baumannii
57
546
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
58
548
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
60
552
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
61
555
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
63
557
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
66
570
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
67
578
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
69
584
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
71
593
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
72
602
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
76
609
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
78
621
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
80
625
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
81
628
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
82
632
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
84
649
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
86
655
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
88
668
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
90
671
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
91
672
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
92
673
54
A40G35C34T38
A39G26C22T49
A35G37C31T46


A. baumannii
18
196
55
A42G34C33T38
A38G27C20T51
A35G37C31T46


A. baumannii
55
537
27
A40G35C33T39
A38G27C20T51
A35G37C33T44


A. baumannii
28
263
27
A40G35C33T39
A38G27C20T51
A35G37C33T44


A. sp. 3
14
164
B7
A43G37C30T37
A36G27C24T49
A34G37C31T47


mixture
7
71
—
ND
ND
ND









Base composition analysis of the samples obtained from Walter Reed hospital indicated that a majority of the strain types identified were the same strain types already characterized by the OIF study of Example 12. This is not surprising since at least some patients from which clinical samples were obtained in OIF were transferred to the Walter Reed Hospital (WRAIR). Examples of these common strain types include: ST10, ST11, ST12, ST14, ST15, ST16 and ST46. A strong correlation was noted between these strain types and the presence of mutations in the gyrA and parC which confer quinolone drug resistance.
In contrast, the results of base composition analysis of samples obtained from Northwestern Medical Center indicate the presence of 4 major strain types: ST10, ST51, ST53 and ST54. All of these strain types have the gyrA quinolone resistance mutation and most also have the parC quinolone resistance mutation, with the exception of ST35. This observation is consistent with the current understanding that the gyrA mutation generally appears before the parC mutation and suggests that the acquisition of these drug resistance mutations is rather recent and that resistant isolates are taking over the wild-type isolates. Another interesting observation was that a single isolate of ST3 (isolate 841) displays a triangulation genotyping analysis pattern similar to other isolates of ST3, but the codon analysis amplification product base compositions indicate that this isolate has not yet undergone the quinolone resistance mutations in gyrA and parC.
The six isolates that represent species other than Acinetobacter baumannii in the samples obtained from the Walter Reed Hospital were each found to not carry the drug resistance mutations.
The results described above involved analysis of 183 samples using the methods and compositions of the present invention. Results were provided to collaborators at the Walter Reed hospital and Northwestern Medical center within a week of obtaining samples. This example highlights the rapid throughput characteristics of the analysis platform and the resolving power of triangulation genotyping analysis and codon analysis for identification of and determination of drug resistance in bacteria.
Example 14
Identification of Drug Resistance Genes and Virulence Factors in Staphylococcus aureus 
An eight primer pair panel was designed for identification of drug resistance genes and virulence factors of Staphylococcus aureus and is shown in Table 19. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 19.







TABLE 19







Primer Pairs for Identification of Drug Resistance Genes and Virulence Factors in Staphylococcus


aureus














Forward

Reverse



Primer

Primer

Primer


Pair

(SEQ ID

(SEQ ID
Target


No.
Forward Primer Name
NO:)
Reverse Primer Name
NO:)
Gene















879
MECA_Y14051_4507_4530_F
288
MECA_Y14051_4555_4581_R
1269
mecA


2056
MECI-R_NC003923-41798-
698
MECI-R_NC003923-41798-
1420
MecI-R



41609_33_60_F

41609_86_113_R


2081
ERMA_NC002952-55890-
217
ERMA_NC002952-55890-
1167
ermA



56621_366_395_F

56621_438_465_R


2086
ERMC_NC005908-2004-
399
ERMC_NC005908-2004-
1041
ermC



2738_85_116_F

2738_173_206_R


2095
PVLUK_NC003923-1529595-
456
PVLUK_NC003923-1529595-
1261
Pv-luk



1531285_688_713_F

1531285_775_804_R


2249
TUFB_NC002758-615038-
430
TUFB_NC002758-615038-
1321
tufB



616222_696_725_F

616222_793_820_R


2256
NUC_NC002758-894288-
174
NUC_NC002758-894288-
853
Nuc



894974_316_345_F

894974_396_421_R


2313
MUPR_X75439_2486_2516_F
172
MUPR_X75439_2548_2574_R
1360
mupR









Primer pair numbers 2256 and 2249 are confirmation primers designed with the aim of high level identification of Staphylococcus aureus. The nuc gene is a Staphylococcus aureus-specific marker gene. The tufB gene is a universal housekeeping gene but the bioagent identifying amplicon defined by primer pair number 2249 provides a unique base composition (A43 G28 C19 T35) which distinguishes Staphylococcus aureus from other members of the genus Staphylococcus. 
High level methicillin resistance in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair numbers 879 and 2056. Analyses have indicated that primer pair number 879 is not expected to prime S. sciuri homolog or Enterococcus faecalis/faciem ampicillin-resistant PBP5 homologs.
Macrolide and erythromycin resistance in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair numbers 2081 and 2086.
Resistance to mupriocin in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair number 2313.
Virulence in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair number 2095. This primer pair can simultaneously and identify the pvl (lukS-PV) gene and the lukD gene which encodes a homologous enterotoxin. A bioagent identifying amplicon of the lukD gene has a six nucleobase length difference relative to the lukS-PV gene.
A total of 32 blinded samples of different strains of Staphylococcus aureus were provided by the Center for Disease Control (CDC). Each sample was analyzed by PCR amplification with the eight primer pair panel, followed by purification and measurement of molecular masses of the amplification products by mass spectrometry. Base compositions for the amplification products were calculated. The base compositions provide the information summarized above for each primer pair. The results are shown in Tables 20A and B. One result noted upon un-blinding of the samples is that each of the PVL+identifications agreed with PVL+identified in the same samples by standard PCR assays. These results indicate that the panel of eight primer pairs is useful for identification of drug resistance and virulence sub-species characteristics for Staphylococcus aureus. It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.







TABLE 20A







Drug Resistance and Virulence Identified in Blinded Samples


of Various Strains of Staphylococcus aureus with


Primer Pair Nos. 2081, 2086, 2095 and 2256












Primer
Primer

Primer


Sample
Pair No.
Pair No.
Primer Pair No.
Pair No.


Index No.
2081 (ermA)
2086 (ermC)
2095 (pv-luk)
2256 (nuc)





CDC0010
−
−
PVL−/lukD+
+


CDC0015
−
−
PVL+/lukD+
+


CDC0019
−
+
PVL−/lukD+
+


CDC0026
+
−
PVL−/lukD+
+


CDC0030
+
−
PVL−/lukD+
+


CDC004
−
−
PVL+/lukD+
+


CDC0014
−
+
PVL+/lukD+
+


CDC008
−
−
PVL−/lukD+
+


CDC001
+
−
PVL−/lukD+
+


CDC0022
+
−
PVL−/lukD+
+


CDC006
+
−
PVL−/lukD+
+


CDC007
−
−
PVL−/lukD+
+


CDCVRSA1
+
−
PVL−/lukD+
+


CDCVRSA2
+
+
PVL−/lukD+
+


CDC0011
+
−
PVL−/lukD+
+


CDC0012
−
−
PVL+/lukD−
+


CDC0021
+
−
PVL−/lukD+
+


CDC0023
+
−
PVL−/lukD+
+


CDC0025
+
−
PVL−/lukD+
+


CDC005
−
−
PVL−/lukD+
+


CDC0018
+
−
PVL+/lukD−
+


CDC002
−
−
PVL−/lukD+
+


CDC0028
+
−
PVL−/lukD+
+


CDC003
−
−
PVL−/lukD+
+


CDC0013
−
−
PVL+/lukD+
+


CDC0016
−
−
PVL−/lukD+
+


CDC0027
+
−
PVL−/lukD+
+


CDC0029
−
−
PVL+/lukD+
+


CDC0020
−
+
PVL−/lukD+
+


CDC0024
−
−
PVL−/lukD+
+


CDC0031
−
−
PVL−/lukD+
+
















TABLE 20B







Drug Resistance and Virulence Identified in Blinded Samples of Various


Strains of Staphylococcus aureus with Primer Pair Nos. 2249,


879, 2056, and 2313













Primer
Primer
Primer




Pair No.
Pair No.
Pair No.


Sample
Primer Pair No. 2249
879
2056
2313


Index No.
(tufB)
(mecA)
(mecI-R)
(mupR)





CDC0010
Staphylococcus aureus
+
+
−


CDC0015
Staphylococcus aureus
−
−
−


CDC0019
Staphylococcus aureus
+
+
−


CDC0026
Staphylococcus aureus
+
+
−


CDC0030
Staphylococcus aureus
+
+
−


CDC004
Staphylococcus aureus
+
+
−


CDC0014
Staphylococcus aureus
+
+
−


CDC008
Staphylococcus aureus
+
+
−


CDC001
Staphylococcus aureus
+
+
−


CDC0022
Staphylococcus aureus
+
+
−


CDC006
Staphylococcus aureus
+
+
+


CDC007
Staphylococcus aureus
+
+
−


CDCVRSA1
Staphylococcus aureus
+
+
−


CDCVRSA2
Staphylococcus aureus
+
+
−


CDC0011
Staphylococcus aureus
−
−
−


CDC0012
Staphylococcus aureus
+
+
−


CDC0021
Staphylococcus aureus
+
+
−


CDC0023
Staphylococcus aureus
+
+
−


CDC0025
Staphylococcus aureus
+
+
−


CDC005
Staphylococcus aureus
+
+
−


CDC0018
Staphylococcus aureus
+
+
−


CDC002
Staphylococcus aureus
+
+
−


CDC0028
Staphylococcus aureus
+
+
−


CDC003
Staphylococcus aureus
+
+
−


CDC0013
Staphylococcus aureus
+
+
−


CDC0016
Staphylococcus aureus
+
+
−


CDC0027
Staphylococcus aureus
+
+
−


CDC0029
Staphylococcus aureus
+
+
−


CDC0020
Staphylococcus aureus
−
−
−


CDC0024
Staphylococcus aureus
+
+
−


CDC0031
Staphylococcus scleiferi
−
−
−









Example 15
Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Staphylococcus aureus 
To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of eight triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within six different housekeeping genes which are listed in Table 21. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 21.







TABLE 21







Primer Pairs for Triangulation Genotyping Analysis of Staphylococcus aureus














Forward

Reverse



Primer

Primer

Primer


Pair

(SEQ ID

(SEQ ID
Target


No.
Forward Primer Name
NO:)
Reverse Primer Name
NO:)
Gene















2146
ARCC_NC003923-2725050-
437
ARCC_NC003923-2725050-
1137
arcC



2724595_131_161_F

2724595_214_245_R


2149
AROE_NC003923-1674726-
530
AROE_NC003923-1674726-
891
aroE



1674277_30_62_F

1674277_155_181_R


2150
AROE_NC003923-1674726-
474
AROE_NC003923-1674726-
869
aroE



1674277_204_232_F

1674277_308_335_R


2156
GMK_NC003923-1190906-
268
GMK_NC003923-1190906-
1284
gmk



1191334_301_329_F

1191334_403_432_R


2157
PTA_NC003923-628885-
418
PTA_NC003923-628885-
1301
pta



629355_237_263_F

629355_314_345_R


2161
TPI_NC003923-830671-
318
TPI_NC003923-830671-
1300
tpi



831072_1_34_F

831072_97_129_R


2163
YQI_NC003923-378916-
440
YQI_NC003923-378916-
1076
yqi



379431_142_167_F

379431_259_284_R


2166
YQI_NC003923-378916-
219
YQI_NC003923-378916-
1013
yqi



379431_275_300_F

379431_364_396_R









The same samples analyzed for drug resistance and virulence in Example 14 were subjected to triangulation genotyping analysis. The primer pairs of Table 21 were used to produce amplification products by PCR, which were subsequently purified and measured by mass spectrometry. Base compositions were calculated from the molecular masses and are shown in Tables 22A and 22B.







TABLE 22A







Triangulation Genotyping Analysis of Blinded Samples of Various Strains of


Staphylococcus aureus with Primer Pair Nos. 2146, 2149, 2150 and 2156












Sample

Primer Pair No.
Primer Pair No.
Primer Pair No.
Primer Pair No.


Index No.
Strain
2146 (arcC)
2149(aroE)
2150 (aroE)
2156 (gmk)





CDC0010
COL
A44 G24 C18 T29
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC0015
COL
A44 G24 C18 T29
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC0019
COL
A44 G24 C18 T29
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC0026
COL
A44 G24 C18 T29
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC0030
COL
A44 G24 C18 T29
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC004
COL
A44 G24 C18 T29
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC0014
COL
A44 G24 C18 T29
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC008
????
A44 G24 C18 T29
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC001
Mu50
A45 G23 C20 T27
A58 G24 C18 T52
A40 G36 C13 T43
A51 G29 C21 T31


CDC0022
Mu50
A45 G23 C20 T27
A58 G24 C18 T52
A40 G36 C13 T43
A51 G29 C21 T31


CDC006
Mu50
A45 G23 C20 T27
A58 G24 C18 T52
A40 G36 C13 T43
A51 G29 C21 T31


CDC0011
MRSA252
A45 G24 C18 T28
A58 G24 C19 T51
A41 G36 C12 T43
A51 G29 C21 T31


CDC0012
MRSA252
A45 G24 C18 T28
A58 G24 C19 T51
A41 G36 C12 T43
A51 G29 C21 T31


CDC0021
MRSA252
A45 G24 C18 T28
A58 G24 C19 T51
A41 G36 C12 T43
A51 G29 C21 T31


CDC0023
ST:110
A45 G24 C18 T28
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC0025
ST:110
A45 G24 C18 T28
A59 G24 C18 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC005
ST:338
A44 G24 C18 T29
A59 G23 C19 T51
A40 G36 C14 T42
A51 G29 C21 T31


CDC0018
ST:338
A44 G24 C18 T29
A59 G23 C19 T51
A40 G36 C14 T42
A51 G29 C21 T31


CDC002
ST:108
A46 G23 C20 T26
A58 G24 C19 T51
A42 G36 C12 T42
A51 G29 C20 T32


CDC0028
ST:108
A46 G23 C20 T26
A58 G24 C19 T51
A42 G36 C12 T42
A51 G29 C20 T32


CDC003
ST:107
A45 G23 C20 T27
A58 G24 C18 T52
A40 G36 C13 T43
A51 G29 C21 T31


CDC0013
ST:12
ND
A59 G24 C18 T51
A40 G36 C13 T43
A51 G29 C21 T31


CDC0016
ST:120
A45 G23 C18 T29
A58 G24 C19 T51
A40 G37 C13 T42
A51 G29 C21 T31


CDC0027
ST:105
A45 G23 C20 T27
A58 G24 C18 T52
A40 G36 C13 T43
A51 G29 C21 T31


CDC0029
MSSA476
A45 G23 C20 T27
A58 G24 C19 T51
A40 G36 C13 T43
A50 G30 C20 T32


CDC0020
ST:15
A44 G23 C21 T27
A59 G23 C18 T52
A40 G36 C13 T43
A50 G30 C20 T32


CDC0024
ST:137
A45 G23 C20 T27
A57 G25 C19 T51
A40 G36 C13 T43
A51 G29 C22 T30


CDC0031
***
No product
No product
No product
No product
















TABLE 22B







Triangulation Genotyping Analysis of Blinded Samples of Various Strains of


Staphylococcus aureus with Primer Pair Nos. 2146, 2149, 2150 and 2156












Sample

Primer Pair No.
Primer Pair No.
Primer Pair No.
Primer Pair No.


Index No.
Strain
2157 (pta)
2161 (tpi)
2163 (yqi)
2166 (yqi)





CDC0010
COL
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC0015
COL
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC0019
COL
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC0026
COL
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC0030
COL
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC004
COL
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC0014
COL
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC008
unknown
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC001
Mu50
A33 G25 C22 T29
A50 G28 C22 T29
A42 G36 C22 T43
A36 G31 C19 T36


CDC0022
Mu50
A33 G25 C22 T29
A50 G28 C22 T29
A42 G36 C22 T43
A36 G31 C19 T36


CDC006
Mu50
A33 G25 C22 T29
A50 G28 C22 T29
A42 G36 C22 T43
A36 G31 C19 T36


CDC0011
MRSA252
A32 G25 C23 T29
A50 G28 C22 T29
A42 G36 C22 T43
A37 G30 C18 T37


CDC0012
MRSA252
A32 G25 C23 T29
A50 G28 C22 T29
A42 G36 C22 T43
A37 G30 C18 T37


CDC0021
MRSA252
A32 G25 C23 T29
A50 G28 C22 T29
A42 G36 C22 T43
A37 G30 C18 T37


CDC0023
ST:110
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC0025
ST:110
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC005
ST:338
A32 G25 C24 T28
A51 G27 C21 T30
A42 G36 C22 T43
A37 G30 C18 T37


CDC0018
ST:338
A32 G25 C24 T28
A51 G27 C21 T30
A42 G36 C22 T43
A37 G30 C18 T37


CDC002
ST:108
A33 G25 C23 T28
A50 G28 C22 T29
A42 G36 C22 T43
A37 G30 C18 T37


CDC0028
ST:108
A33 G25 C23 T28
A50 G28 C22 T29
A42 G36 C22 T43
A37 G30 C18 T37


CDC003
ST:107
A32 G25 C23 T29
A51 G28 C22 T28
A41 G37 C22 T43
A37 G30 C18 T37


CDC0013
ST:12
A32 G25 C23 T29
A51 G28 C22 T28
A42 G36 C22 T43
A37 G30 C18 T37


CDC0016
ST:120
A32 G25 C24 T28
A50 G28 C21 T30
A42 G36 C22 T43
A37 G30 C18 T37


CDC0027
ST:105
A33 G25 C22 T29
A50 G28 C22 T29
A43 G36 C21 T43
A36 G31 C19 T36


CDC0029
MSSA476
A33 G25 C22 T29
A50 G28 C22 T29
A42 G36 C22 T43
A36 G31 C19 T36


CDC0020
ST:15
A33 G25 C22 T29
A50 G28 C21 T30
A42 G36 C22 T43
A36 G31 C18 T37


CDC0024
ST:137
A33 G25 C22 T29
A51 G28 C22 T28
A42 G36 C22 T43
A37 G30 C18 T37


CDC0031
***
A34 G25 C25 T25
A51 G27 C24 T27
No product
No product





Note:


*** The sample CDC0031 was identified as Staphylococcus scleiferi as indicated in Example 14. Thus, the triangulation genotyping primers designed for Staphylococcus aureus would generally not be expected to prime and produce amplification products of this organism. Tables 22A and 22B indicate that amplification products are obtained for this organism only with primer pair numbers 2157 and 2161.






A total of thirteen different genotypes of Staphylococcus aureus were identified according to the unique combinations of base compositions across the eight different bioagent identifying amplicons obtained with the eight primer pairs. These results indicate that this eight primer pair panel is useful for analysis of unknown or newly emerging strains of Staphylococcus aureus. It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.
Example 16
Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Members of the Bacterial Genus Vibrio 
To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of eight triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within seven different housekeeping genes which are listed in Table 23. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 23.







TABLE 23







Primer Pairs for Triangulation Genotyping Analysis of Members of the Bacterial Genus


Vibrio














Forward

Reverse



Primer

Primer

Primer


Pair

(SEQ ID

(SEQ ID
Target


No.
Forward Primer Name
NO:)
Reverse Primer Name
NO:)
Gene















1098
RNASEP_VBC_331_349_F
325
RNASEP_VBC_388_414_R
1163
RNAse P


2000
CTXB_NC002505_46_70_F
278
CTXB_NC002505_132_162_R
1039
ctxB


2001
FUR_NC002505_87_113_F
465
FUR_NC002505_205_228_R
1037
fur


2011
GYRB_NC002505_1161_1190_F
148
GYRB_NC002505_1255_1284_R
1172
gyrB


2012
OMPU_NC002505_85_110_F
190
OMPU_NC002505_154_180_R
1254
ompU


2014
OMPU_NC002505_431_455_F
266
OMPU_NC002505_544_567_R
1094
ompU


2323
CTXA_NC002505-1568114-
508
CTXA_NC002505-1568114-
1297
ctxA



1567341_122_149_F

1567341_186_214_R


2927
GAPA_NC002505_694_721_F
259
GAPA_NC_002505_29_58_R
1060
gapA









A group of 50 bacterial isolates containing multiple strains of both environmental and clinical isolates of Vibrio cholerae, 9 other Vibrio species, and 3 species of Photobacteria were tested using this panel of primer pairs. Base compositions of amplification products obtained with these 8 primer pairs were used to distinguish amongst various species tested, including sub-species differentiation within Vibrio cholerae isolates. For instance, the non-O1/non-O139 isolates were clearly resolved from the O1 and the O139 isolates, as were several of the environmental isolates of Vibrio cholerae from the clinical isolates.
It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.
Example 17
Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Members of the Bacterial Genus Pseudomonas 
To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of twelve triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within seven different housekeeping genes which are listed in Table 24. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 24.







TABLE 24







Primer Pairs for Triangulation Genotyping Analysis of Members of the Bacterial Genus


Pseudomonas














Forward

Reverse



Primer

Primer

Primer


Pair

(SEQ ID

(SEQ ID
Target


No.
Forward Primer Name
NO:)
Reverse Primer Name
NO:)
Gene















2949
ACS_NC002516-970624-
376
ACS_NC002516-970624-
1265
acsA



971013_299_316_F

971013_364_383_R


2950
ARO_NC002516-26883-
267
ARO_NC002516-26883-
1341
aroE



27380_4_26_F

27380_111_128_R


2951
ARO_NC002516-26883-
705
ARO_NC002516-26883-
1056
aroE



27380_356_377_F

27380_459_484_R


2954
GUA_NC002516-4226546-
710
GUA_NC002516-4226546-
1259
guaA



4226174_155_178_F

4226174_265_287_R


2956
GUA_NC002516-4226546-
374
GUA_NC002516-4226546-
1111
guaA



4226174_242_263_F

4226174_355_371_R


2957
MUT_NC002516-5551158-
545
MUT_NC002516-5551158-
978
mutL



5550717_5_26_F

5550717_99_116_R


2959
NUO_NC002516-2984589-
249
NUO_NC002516-2984589-
1095
nuoD



2984954_8_26_F

2984954_97_117_R


2960
NUO_NC002516-2984589-
195
NUO_NC002516-2984589-
1376
nuoD



2984954_218_239_F

2984954_301_326_R


2961
PPS_NC002516-1915014-
311
PPS_NC002516-1915014-
1014
pps



1915383_44_63_F

1915383_140_165_R


2962
PPS_NC002516-1915014-
365
PPS_NC002516-1915014-
1052
pps



1915383_240_258_F

1915383_341_360_R


2963
TRP_NC002516-671831-
527
TRP_NC002516-671831-
1071
trpE



672273_24_42_F

672273_131_150_R


2964
TRP_NC002516-671831-
490
TRP_NC002516-671831-
1182
trpE



672273_261_282_F

672273_362_383_R









It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.
The present invention includes any combination of the various species and subgeneric groupings falling within the generic disclosure. This invention therefore includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
While in accordance with the patent statutes, description of the various embodiments and examples have been provided, the scope of the invention is not to be limited thereto or thereby. Modifications and alterations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.
Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims, rather than by the specific examples which have been presented by way of example.
Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank gi or accession numbers, internet web sites, and the like) cited in the present application is incorporated herein by reference in its entirety.

## Claims

1. An oligonucleotide primer pair comprising a forward primer and a reverse primer, wherein said forward primer comprises the sequence of SEQ ID NO:318, and wherein said reverse primer comprises a sequence 95% identical to SEQ ID NO:1300.

2. The oligonucleotide primer pair of claim 1 wherein said forward primer is SEQ ID NO: 318.

3. The oligonucleotide primer pair of claim 1, wherein said reverse primer comprises the sequence of SEQ ID NO:1300.

4. The oligonucleotide primer pair of claim 1 wherein said reverse primer is SEQ ID NO: 1300.

5. The oligonucleotide primer pair of claim 1 wherein at least one of said forward primer and said reverse primer comprises at least one modified nucleobase.

6. The oligonucleotide primer pair of claim 5 wherein at least one of said at least one modified nucleobase is a mass modified nucleobase.

7. The oligonucleotide primer pair of claim 6 wherein said mass modified nucleobase is 5-Iodo-C.

8. The composition of claim 6 wherein said mass modified nucleobase comprises a molecular mass modifying tag.

9. The oligonucleotide primer pair of claim 5 wherein at least one of said at least one modified nucleobase is a universal nucleobase.

10. The oligonucleotide primer pair of claim 9 wherein said universal nucleobase is inosine.

11. The oligonucleotide primer pair of claim 1, wherein at least one of said forward primer and said reverse primer comprises a T residue at its 5′ end.

12. A kit for identifying, determining one or more characteristics of, or detecting a Staphylococcus aureus bioagent comprising the oligonucleotide primer pair of claim 1 and at least one additional primer pair designed to hybridize to a Staphylococcus aureus gene encoding arcC, aroE, gmk, pta, yqi, or a combination thereof.

13. The kit of claim 12 further comprising at least one other additional primer pair designed to hybridize to a Staphylococcus aureus gene encoding mecA, mecR1, pvluk, or a combination thereof.

14. The kit of claim 12 wherein said at least one additional primer pair comprises SEQ ID NOs: 437:1232, SEQ ID NOs: 590:891, SEQ ID NOs: 474:869, SEQ ID NOs: 268:1284, SEQ ID NOs: 418:1301, SEQ ID NOs: 440:1076, SEQ ID NOs: 219:1013, or a combination thereof.

15. The kit of claim 12 wherein each primer of said at least one additional primer pair has at least 70% sequence identity with one of SEQ ID NOs: 437, 1232, 590, 891, 474, 869, 268, 1284, 418, 1301, 318, 1300, 440, 1076, 219 and 1013.