Mycobacterium primers and probes

Primers and probes can be used to detect nucleic acid from Mycobacterium in a sample and determine the species from which the nucleic acid originates. The primers amplify regions of the 16S ribosomal RNA gene and hybridize to regions conserved among species. Genus specific probes hybridize to sequences within the amplified region conserved among mycobacterial species, whereas the species specific probes hybridize to a variable region, so that the species identity can be uniquely determined. Consensus probes for detecting mycobacteria nucleic acids are provided which probes are not identical to any of the sequences of mycobacterial species.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to methods and reagents for detecting the 
presence of mycobacterial nucleic acid and identifying the mycobacterial 
species from which a mycobacterial nucleic acid in a sample originates. 
2. Description of Related Art 
Mycobacteria are slow growing, acid-fast, aerobic bacilli. At least 
nineteen Mycobacterium species have so far been associated with disease in 
humans, most notably M. tuberculosis, M. bovis, and M. leprae. Some 
species, such as M. avium, M. intracellulare, and M. kansasii, though not 
normally pathogenic to healthy individuals, may cause disease in 
immunocompromised individuals, such as those infected with the ADS virus. 
In addition, several species rarely cause disease in humans but may occur 
in clinical specimens as saprophytes. Methods for the detection and 
identification of Mycobacterium species include bacterial culture, 
antibody detection, and, more recently, detection of rRNA by hybridization 
with a radioactively labelled nucleic acid probe. Each of these methods 
has considerable problems. 
Detection by culturing the bacilli is slow, requiring up to two months, and 
typically requires additional biochemical testing for species 
identification. Antibody detection lacks specificity because of 
cross-reactivity between mycobacteria species and also lacks sensitivity. 
Furthermore, differentiation between current and past infections is 
difficult. Detection using radioactively labelled DNA fragments as probes 
that hybridize to the small subunit ribosomal RNA (16S rRNA) lacks 
sensitivity and still requires at least a several-day culturing period 
(see PCT/WO 84/02721). 
The invention of the polymerase chain reaction (PCR), a method for 
amplifying specific sequences of nucleic acids, makes possible the rapid 
detection of nucleic acids present in a cell in what was previously an 
undetectably low quantity. Using PCR amplification, one can detect even a 
single copy of the target nucleic acid. Direct detection by hybridization 
with a sequence-specific oligonucleotide probe of a nucleic acid sequence 
amplified to a detectable level makes possible diagnostic tests that are 
specific enough to detect single nucleotide changes in sequence. However, 
not all primer pairs and probes are useful. The choice of primers and, 
hence, the region to be amplified, along with the choice of probes largely 
determines the specificity and sensitivity obtainable. 
Amplification by the PCR hits been used in the sequencing of mycobacterial 
nucleic acid, detection of mycobacterial nucleic acids in a sample, and 
identification of mycobacteria species. Various regions of the bacterial 
genome have been used to detect and identify mycobacterial nucleic acids 
in samples. Most of these diagnostic tests were designed to detect only 
one or a small number of species, and limited specificity checks, if any, 
were performed against non-mycobacterial DNA. 
Detection of a region of the gene that encodes the 65 kilodalton antigen 
was described in Chia et al., 1990, J. Clin. Microbiol. 28(9):1877-1880; 
Brisson-Noel et al., 1989, Lancet 334:1069-1071; Hackel et al., 1990, 
Molecular and Cellular Probes 4:205-210; Woods and Cole, 1989, FEMS 
Microbiology Letters 65:305-310; and Hance et al., 1989, Molecular 
Microbiology 3(7):843-849. No more than three sets of mycobacteria species 
were distinguished in any one test based on the 65 kilodalton antigen 
gene. 
Amplification of the repetitive DNA element, IS6110, was reported in 
Thierry et al., 1990, J. Clin. Microbiol. 28(12):2668-2673, and Eisenach 
et al., 1990, J. Infectious Disease 161:977-981. Amplification of IS6110 
basically serves only to test for the presence of particular species of 
mycobacteria, although M. tuberculosis and M. bovis can be distinguished 
by copy number (Plikaytis et al., 1991, Molecular and Cellular Probes 
5:215-219). 
The 36 kilodalton antigen of M. leprae was used in a diagnostic test in 
Hartskeerl et al., 1989, J. Gen. Microbiol. 135:2357-2364. Though the test 
was meant to be specific for M. leprae, weak to moderate hybridization to 
DNA from other mycobacteria was observed. 
The gene sequence coding for protein antigen b was used in Sjobring et al., 
1990, J. Clin. Microbiol. 28(10):2200-2204, to produce a test for M. 
tuberculosis/bovis based on the presence or absence of an amplified 
product. 
A test solely for the presence of M. tuberculosis based on the gene 
sequence encoding the MPB 64 protein was described in Shankar et al., 
1990, Lancet 335:423. 
Probes constructed from cloned DNA fragments were described in Patel et 
al., 1990, J. Clin. Microbiol. 28(3):513-518, and Fries et al., 1990, 
Molecular and Cellular Probes 4:87-105. Probe specificity was obtained 
through a selection process rather than by sequence analysis during the 
probe design. 
One of the regions of the mycobacterial genome that has been analyzed and 
targeted for use in a diagnostic test is the small subunit ribosomal RNA 
(16S rRNA). In Bottger, 1989, FEMS Microbiology Letters 65:171-176, the 
16S rRNA genes from a variety of organisms were amplified using 
"universal" primers designed to amplify nucleic acid from a wide range of 
organisms and then directly sequenced. The phylogenetic relationship of 
mycobacterial species was studied by comparing 16S rRNA germ sequences in 
Rogall et al. 1990, J. Gen. Micro. 136:1915-1920. In Boddinghaus et al., 
1990, J. Clin. Microbiol. 28(8): 1751-1759, evidence was presented 
regarding determinations that can be made using sequence specific 
oligonucleotides for amplification and hybridization to regions of the 16S 
rRNA sequence. A highly variable region of the 16S rRNA sequence was 
studied with respect to three mycobacteria species. Genus specific primers 
were used to amplify a region containing the variable region used for 
species specific probe hybridization. 
The small subunit rRNA from a large number of organisms, both closely and 
distantly related to mycobacteria, has been studied and sequenced. A 
compilation of small subunit rRNA sequences from a large number of 
organisms is provided by Neefs et al., 1990, Nuc. Acids Res. Supplement 
18:2237-2317. 
There is still a need for a rapid and sensitive test to identify the 
presence of mycobacterial DNA and the species from which the DNA 
originates. 
SUMMARY OF THE INVENTION 
The present invention provides a rapid and sensitive PCR based assay for 
the detection and species identification of mycobacteria. Primers and 
probes specific for 16S ribosomal RNA gene sequences are provided. 
Mycobacteria detection is accomplished by amplification with genus 
specific primers followed by screening with genus specific probes in a dot 
blot hybridization assay. If mycobacteria are detected, species 
identification is determined from amplified DNA, normally from the same 
amplification reaction, using the species specific probes in a reverse dot 
blot assay. 
The amplification of sequences encoding the 16S ribosomal RNA (rRNA) has 
several advantages. The present invention can be used to detect and 
distinguish between more than 30 mycobacterial species and numerous other 
organisms that might be present in a clinical sample. The probes and 
primers of the present invention provide the maximum specificity possible, 
thereby minimizing the probability of a false positive caused by the 
presence of a related organism with a similar sequence. The 16S rRNA gene 
contains highly conserved regions. The genus specific primers and probes 
of the present invention hybridize to such conserved regions and are able 
to hybridize to sequences from almost all species in the genus; the 
primers amplify nucleic acid from 14 of the 15 mycobacterial species 
tested, and of these 14 amplified mycobacterial DNA sequences, the genus 
specific probes hybridize to 12. The 16S rRNA also contains highly 
variable regions within the amplified region. The species specific probes 
of the present invention hybridize in a variable region where each species 
of interest has a unique sequence. 
An additional advantage of choosing primers and probes from the 16S rRNA is 
that the rRNA is present in a growing cell in large copy numbers (10.sup.3 
to 10.sup.4). The number of gene sequences in the form of RNA in a given 
clinical sample would be, therefore, up to 10.sup.4 times greater than the 
number of the corresponding DNA sequences. If additional detection 
sensitivity is desired, the RNA itself can be used as the amplification 
target. 
In another aspect of the invention, a second amplification reaction is 
carried out as a confirmatory test. The second amplification reaction 
relies on the presence of target sequences not directly related to the 
first target, i.e., the 16S ribosomal RNA nucleic acids. Suitable target 
sequences are preferably conserved among Mycobacterium species and are not 
related to non-Mycobacterium species. A suitable target gene may be, for 
example, the gene encoding the 65 kDa protein gene. Pao et al., 1989, FEMS 
Micro. Letters 65:305-310; Hartskeerl et al., 1989, J. Gen. Micro. 
135:2357-2364; and Hackel et al., 1990; Mol. Cel. Probes 4:205-210. While 
useful for confirming the results of a first amplification reaction, the 
amplification of a second target sequence is particularly meaningful for 
resolving discordant results that may arise from comparative studies, 
notably the comparison of PCR and culture methods. 
An additional aspect of the present invention relates to novel compositions 
for use as positive controls for detecting Mycobacterium. The invention 
provides a novel composition for confirming the results of an assay using 
genus specific probes as well as species specific Mycobacterium probes. 
One aspect of the invention relates to probes capable of detecting the 
presence of Mycobacterium nucleic acid (genus specific probes) and 
determining the identity of the species from which the nucleic acid 
originates (species specific probes). 
Another aspect of the invention relates to consensus probes. In the 
preferred embodiment the invention provides consensus oligonucleotides for 
the amplification and detection of disparate species of Mycobacterium 
isolates. The consensus oligonucleotide probes do not hybridize to 
non-Mycobacterium species that are closely related to mycobacteria. 
Consensus probes are suitable for a broad range of target-specific 
detection using a single oligonucleotide probe. A consensus probe, as used 
herein, is an oligonucleotide probe which is not identical in sequence to 
any of the mycobacteria nucleic acid sequences to be detected. The 
consensus probes are hybrid oligonucleotide compositions comprising 
non-native nucleic acid sequences. Consensus probes as described in the 
present invention can be used to exclude as well as to include selected 
species in a detection assay. In one embodiment, the invention provides 
oligonucleotide probes comprising novel sequences. While these probes 
broadly detect mycobacterial species, they do not detect closely related 
non-mycobacterial species, for example, Corynebacter. 
Another aspect of the invention relates to primers for amplifying a 
specific region of mycobacteria nucleic acid. This region contains both a 
region conserved among Mycobacterium species and a variable region with 
sufficient heterogeneity among species to enable the origin of the target 
nucleic acid to be determined using sequence specific oligonucleotide 
probes. 
Another aspect of the invention relates to detection and species 
identification methods. Amplification of the target nucleic acid by PCR, 
using the primers of the invention, allows one to detect the presence of 
mycobacterial nucleic acid by mixing the amplified nucleic acid with the 
genus specific probes and detecting if hybridization occurs, whereas 
species identification is carried out by determining the pattern of 
hybridization to the species specific probes. 
A fourth aspect of the invention relates to kits. These kits take a variety 
of forms and comprise one or more probes and, in one embodiment, comprise 
a panel of probes sufficient to determine the identity of an infecting 
Mycobacterium at the species level and instructions for using the kit 
ingredients. The kits can also comprise one or more amplification 
reagents, e.g., genus specific primers, polymerase, buffers, and 
nucleoside triphosphates. 
In a further embodiment, the kit may also comprise positive and negative 
controls. A preferred positive control is described herein. 
To aid in understanding the invention, several terms are defined below. 
The term "oligonucleotide" refers to a molecule comprised of two or usually 
more deoxyribonucleotides or ribonucleotides, such as primers, probes, 
nucleic acid fragments to be detected, and nucleic acid controls. The 
exact size of an oligonucleotide depends on many factors and the ultimate 
function or use of the oligonucleotide. Oligonucleotides can be prepared 
by any suitable method, including, for example, cloning and restriction of 
appropriate sequences and direct chemical synthesis by a method such as 
the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 
68:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 
68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981, 
Tetrahedron Lett. 22:1859-1862; and the solid support method of U.S. Pat. 
No. 4,458,066. 
The term "primer" refers to an oligonucleotide, whether natural or 
synthetic, capable of acting as a point of initiation of DNA synthesis 
under conditions in which synthesis of a primer extension product 
complementary to a nucleic acid strand is induced, i.e., in the presence 
of four different deoxyribonucleoside triphosphates and an agent for 
polymerization (i.e., DNA polymerase or reverse transcriptase) in an 
appropriate buffer and at a suitable temperature. A primer is preferably a 
single-stranded oligodeoxyribonucleotide. The appropriate length of a 
primer depends on the intended use of the primer but typically ranges from 
15 to 25 nucleotides. Short primer molecules generally require cooler 
temperatures to form sufficiently stable hybrid complexes with the 
template. A primer need not reflect the exact sequence of the template but 
must be sufficiently complementary to hybridize with a template and serve 
to initiate DNA synthesis. 
In the disclosed embodiments of the invention, specific sequence primers 
and probes are provided. It will be apparent to those of skill in the art 
that, provided with those embodiments, specific sequence primers and 
probes can be modified by, for example, the addition of nucleotides to 
either the 5' or 3' ends, which nucleotides are complementary to the 
target sequence or are uncomplementary to the target sequence. So long as 
primer compositions serve as a point of initiation for extension on the 
target sequences, and the primers and probes comprise at least 14 
consecutive nucleotides contained within those exemplified embodiments, 
such compositions are within the scope of the invention. 
The term "primer" may refer to more than one primer, particularly in the 
case where there is some ambiguity in the information regarding one or 
both ends of the target region to be amplified. If a "conserved" region 
shows significant levels of polymorphism in a population, mixtures of 
primers can be prepared that will amplify such sequences, or the primers 
can be designed to amplify even mismatched sequences. A primer can be 
labeled, if desired, by incorporating a label detectable by spectroscopic, 
photochemical, biochemical, immunochemical, or chemical means. For 
example, useful labels include .sup.32 P, fluorescent dyes, electron-dense 
reagents, enzymes (as commonly used in ELISAs), biotin, or haptens and 
proteins for which antisera or monoclonal antibodies are available. A 
label can also be used to "capture" the primer, so as to facilitate the 
immobilization of either the primer or a primer extension product, such as 
amplified DNA, on a solid support. 
The terms "sequence specific oligonucleotide" and "SSO" refer to 
oligonucleotides that have a sequence, called a "hybridizing region", 
complementary to the sequence to be detected, which, under "sequence 
specific, stringent hybridization conditions", will hybridize only to that 
exactly complementary target sequence. Relaxing the stringency of the 
hybridization conditions will allow sequence mismatches to be tolerated; 
the degree of mismatch tolerated can be controlled by suitable adjustment 
of the hybridization conditions. The terms "probe" and "SSO probe" are 
used interchangeably with SSO. 
The term "target region" refers to a region of a nucleic acid to be 
analyzed. 
The term "thermostable polymerase enzyme" refers to an enzyme that is 
relatively stable to heat and catalyzes the polymerization of nucleoside 
triphosphates to form primer extension products that are complementary to 
one of the nucleic acid strands of the target sequence. The enzyme 
initiates synthesis at the 3'-end of the primer and proceeds in the 
direction toward the 5'-end of the template until synthesis terminates. A 
purified thermostable polymerase enzyme is described more fully in U.S. 
Pat. No. 4,889,818, incorporated herein by reference, and is commercially 
available from Perkin Elmer (Norwalk, Conn.). 
The term "reverse transcriptase" refers to an enzyme that catalyzes the 
polymerization of nucleoside triphosphates to form primer extension 
products that are complementary to a ribonucleic acid template. The enzyme 
initiates synthesis at the 3'-end of the primer and proceeds in the 
direction toward the 5' end of the template until synthesis terminates. 
Examples of suitable polymerizing agents that convert the RNA target 
sequence into a complementary, copy DNA (cDNA) sequence are avian 
myeloblastosis virus reverse transcriptase and Thermus thermophilus DNA 
polymerase, a thermostable DNA polymerase with reverse transcriptase 
activity marketed by Perkin Elmer.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a rapid and sensitive PCR based assay for 
the detection and species identification of Mycobacterium. Primers and 
probes specific for mycobacterial 16S ribosomal RNA gene sequences are 
provided. Mycobacterial detection is accomplished by amplification with 
genus specific primers followed by screening with genus specific probes in 
a dot blot hybridization assay. If mycobacteria are detected, species 
identification is determined from DNA from the same amplification reaction 
using the species-specific probes in a reverse dot-blot assay. Both the 
forward and reverse dot blot assays can be carried out very conveniently 
in a microtiter plate; see U.S. patent application Ser. No. 414,542, filed 
Sep. 29, 1989, incorporated herein by reference. 
The genus specific primers and probes hybridize to conserved regions of the 
16S rRNA gene, and the species specific probes hybridize to variable 
regions of the 16S rRNA gene. Because synthesis starts at the 3' end of 
the primer, mismatches at the 3' end are more critical. Thymidine is more 
tolerant than the other bases of a mismatch; so primers were designed to 
avoid a thymidine base at the 3' end. The base content of an 
oligonucleotide affects the denaturation temperature. The stringency and 
specificity of primer or probe binding increases with increasing 
temperature. However, because all probes are hybridized simultaneously in 
the reverse dot-blot format, optimal probe hybridization conditions are 
similar for all the probes. 
The primer pairs of the invention function efficiently in the amplification 
of a sequence of the 16S rRNA gene from all the Mycobacterium species of 
interest but do not amplify the corresponding DNA from most other sources. 
Furthermore, the amplification conditions and efficiency for these primers 
are fairly uniform across species so that nearly all Mycobacterium species 
are detectable using a single test. Table 1 shows the hybridizing 
sequences of the primers of the present invention. 
TABLE I 
______________________________________ 
Sequence 
Primer 
Listing Hybridizing Sequence 
______________________________________ 
KY18 SEQ ID 5' CACATGCAAGTCGAACGGAAAGG 3' 
NO: 1 
KY75 SEQ ID 5' GCCCGTATCGCCCGCACGCTCACA 3' 
NO: 2 
______________________________________ 
Using the E. coli numbering system, the upstream primer, KY18 (SEQ ID NO: 
1), spans bases 52-74, and the downstream primer, KY75 (SEQ ID NO: 2), 
spans bases 624-647 of the 16S rRNA gene. Together, these primers specify 
the synthesis of a product approximately 583 base-pairs in length; the 
exact size is species dependent. 
The initial screening for the presence of mycobacterial DNA is accomplished 
with two genus specific probes that are used simultaneously as a mixture. 
TABLE 2 
__________________________________________________________________________ 
Probe 
Sequence Listing 
Hybridizing Sequence 
__________________________________________________________________________ 
KY101 
SEQ ID NO: 3 
5' TCGCGTTGTTCGTG .sub.-- AAATCTCAC -gGCTTAA 3' 
KY102 
SEQ ID NO: 4 
5' TCGCGTTGTTCGTG .sub.-- AAA -aCTCACAGCTTAA 3' 
KY165 
SEQ ID NO. 13 
5' TCGCGTTGTTCGTG .sub.-- AAATCTCACAGCTTAA 3' 
KY166 
SEQ ID NO. 14 
5' TCGCGTTGTTCGTGgAATCTCACAGCTTAA 3' 
M. xenopi 
SEQ ID NO. 15 
5' TCGCGTTGTTCGTGgAAT --gcCACAGCTTAA 3' 
__________________________________________________________________________ 
The reason for the mixed probes is that most mycobacteria species can be 
divided into two groups with respect to the sequences in the region of 
KY101 (SEQ ID NO. 3) and KY102 (SEQ ID NO. 4). These two probes detect DNA 
from 12 out of 14 species of the genus Mycobacterium tested. 
In an alternative embodiment, KY165 (SEQ ID NO. 13) replaces probes KY101 
(SEQ ID NO. 3) and KY102 (SEQ ID NO. 4). The sequence of probes KY101 (SEQ 
ID NO. 3), KY102 (SEQ ID NO. 4), KY165 (SEQ ID NO. 13), and KY166 (SEQ ID 
NO. 14) are given in Table 2. KY165 (SEQ ID NO. 13) is a consensus probe 
encompassing the sequences of both KY101 (SEQ ID NO. 3) and KY102 (SEQ ID 
NO. 4). KY101 (SEQ ID NO. 3) and KY102 (SEQ ID NO. 4) differ from each 
other by two bases. KY165 (SEQ ID NO. 13) is not identical to either KY101 
(SEQ ID NO. 3) or KY102 (SEQ ID NO. 4), but differs from each by a single 
base. This consensus was arrived at by "favoring" KY101 (SEQ ID NO. 3) in 
one of the mismatched positions and KY102 (SEQ ID NO. 4) in the other 
mismatched positions. KY165 (SEQ ID NO. 13) is able to hybridize 
sufficiently to all KY101 (SEQ ID NO. 3) and KY102-specific (SEQ ID NO. 4) 
mycobacterial species. KY165 (SEQ ID NO. 13) does not hybridize to M. 
xenopi (SEQ ID NO. 15) under conditions of high stringency due to the 
presence of additional mismatches. 
KY166 (SEQ ID NO. 14) is a broader consensus probe for detecting 
mycobacterial species including M. xenopi (SEQ ID NO. 15). The sequence of 
KY166 (SEQ ID NO. 14), like the sequence of KY165 (SEQ ID NO. 13), does 
not correspond to any non-mycobacterial species. The probe is designed to 
be equally dissimilar to KY101 (SEQ ID NO. 3), KY102 (SEQ ID NO. 4), and 
the corresponding sequence in M. xenopi (SEQ ID NO. 15) (GenBank accession 
No. X52929, available through Intelligenetics) KY166 (SEQ ID NO. 14) 
differs from KY101 (SEQ ID NO. 3), KY102 (SEQ ID NO. 4), and M. xenopi 
(SEQ ID NO. 15) by two bases each. KY166 (SEQ ID NO. 14) efficiently 
hybridizes to all KY101 (SEQ ID NO. 3) and KY102 (SEQ ID NO. 4) specific 
species and M. xenopi (SEQ ID NO. 15). In addition, KY166 (SEQ ID NO. 14) 
does not hybridize to Corynebacter pseudodiphtheriticum or C. diphtheriae, 
two non-mycobacterial species that are closely related to Mycobacterium. 
The corresponding sequence of M. xenopi (SEQ ID NO. 15) is included in 
Table 2. In the Table, the mismatches relative to KY166 (SEQ ID NO. 14) 
are underlined. Mismatches relative to KY165 (SEQ ID NO. 13) are in lower 
case. 
If mycobacterial nucleic acid is present in the sample, the species from 
which the nucleic acid originates can be determined by hybridization to a 
panel of species specific probes. The probes used in the species 
identification step are shown in Table 3. 
TABLE 3 
__________________________________________________________________________ 
Probe Sequence Listing 
Specificity 
__________________________________________________________________________ 
KY21 SEQ ID NO: 5 M. tuberculosilis/bovis/tb 
5' ACGGGATGCATGTCTTGTGGTGGAAAGCGCTTTAGC 3' 
KY25 SEQ ID NO: 6 M. kansasii/gastri/scrofulaceum 
5' ACTTGGCGCATGCCTTGTGGTGGAAAGCTT 3' 
KY26 SEQ ID NO: 7 M. intacellulare 
5' TTTAGGCGCATGTCTTTAGGTGGAAAGCTT 3' 
KY63 SEQ ID NO: 8 M. avium/maringum/microti 
5' TCAAGACGCATGTTCTTCTGGTGGAAAGCTTTTGC 3' 
KY151 SEQ ID NO: 9 M. marinum/M. microti 
5' TCCCGAAGTGCAGGCCAGATTGCCCACGTG 3' 
KY106 SEQ ID NO: 10 M. scrofulaceum 
5' GAAGGCTCACTTTGTGGGTTGACGGTAGGT 3' 
KY126 SEQ ID NO: 11 M. kansasii/gastri 
5' GCAATCTGCCTGCACACCGGGATAAGCCTG 3' 
KY139 SEQ ID NO: 12 M. gordonae 
5' GGGTCTAATACCGAATAGGACCACAGGACACATG 3' 
KY157 SEQ ID NO: 16 M. xenopi 
5' ATAGGACCATTCTGCGCATGTGGTGTGGTG 3' 
KY167 SEQ ID NO: 17 M. avium/marinun/microti 
5' ACCTCAAGACGCATGTCTTCTGGT 3' 
KY168 SEQ ID NO: 18 M. gordonae 
5' CCGAATAGGACCACAGGACACATG 3' 
KY169 SEQ ID NO: 19 M. intracellulare 
5' ACCTTTAGGCGCATGTCTTTAGGT 3' 
KY170 SEQ ID NO: 20 M. kansasii/gastri/scrofulaceum 
5' AACACTTGGCGCATGCCTTGTGGT 3' 
KY171 SEQ ID NO: 21 M. scrofulaceum 
5' GAAGGCTCACTTTGTGGGTTGACG 3' 
KY172 SEQ ID NO: 22 M. bovis/tb 
5' TGTGGTGGAAAGCGCTTTAGCGGT 3' 
KY173 SEQ ID NO: 23 M. xenopi 
5' AGGACCATTCTGCGCATGTGGTGT 3' 
__________________________________________________________________________ 
The species of greatest clinical interest are M. tuberculosis, M. kansasii, 
M. xenopi, M. intracellulare, and M. avium, M. gordonae is not ordinarily 
associated with disease but frequently occurs in human samples. 
Consequently, detection of mycobacterial nucleic acid by the genus 
specific probes is expected frequently to be due to the clinically 
unimportant M. gordonae. Example 6 contains additional information 
regarding the specificity of the species specific probes. 
An important aspect of the present invention is the amplification of a 
region of the 16S rRNA gene. Those practicing the present invention should 
note that, although the polymerase chain reaction is the preferred 
amplification method, amplification of target sequences in a sample may be 
accomplished by any known method, such as ligase chain reaction (LCR), 
transcription amplification, and self-sustained sequence replication, each 
of which provides sufficient amplification so that the target sequence can 
be detected by nucleic acid hybridization to an SSO probe. Alternatively, 
methods that amplify the probe to detectable levels can be used, such as 
Q.beta.-replicase amplification. The term "probe" encompasses the sequence 
specific oligonucleotides used in the above procedures; for instance, the 
two or more oligonucleotides used in LCR are "probes" for purposes of the 
present invention, even though LCR only requires ligation of the probes to 
indicate the presence of the sequence. 
Although the PCR process is well known in the art (see U.S. Pat. Nos. 
4,683,195; 4,683,202; and 4,965,188, each of which is incorporated herein 
by reference) and although commercial vendors, such as Perkin Elmer, sell 
PCR reagents and publish PCR protocols, some general PCR information is 
provided below for purposes of clarity and full understanding of the 
invention to those unfamiliar with the PCR process. 
To amplify a target nucleic acid sequence in a sample by PCR, the sequence 
must be accessible to the components of the amplification system. In 
general, this accessibility is ensured by isolating the nucleic acids from 
the sample. A variety of techniques for extracting nucleic acids from 
biological samples are known in the art. For example, see those described 
in Higuchi et al., 1989, in PCR Technology (Erlich ed., Stockton Press, 
New York). Alternatively, if the sample is fairly readily disruptable, the 
nucleic acid need not be purified prior to amplification by the PCR 
technique, i.e., if the sample is comprised of cells, particularly 
peripheral blood lymphocytes or aminiocytes, lysis and dispersion of the 
intracellular components can be accomplished merely by suspending the 
cells in hypotonic buffer. 
Each cycle of the PCR involves the separation of the nucleic acid duplex 
formed by primer extension. In a preferred embodiment of the PCR process, 
strand separation is achieved by heating the reaction to a sufficiently 
high temperature for an effective time to cause the denaturation of the 
duplex, but not to cause an irreversible denaturation of the polymerase 
(see U.S. Pat. No. 4,965,188). Typical heat denaturation involves 
temperatures ranging from about 80.degree. C. to 105.degree. C. for times 
ranging from seconds to minutes. Strand separation, however, can be 
accomplished by any suitable denaturing method including physical, 
chemical, or enzymatic means. Strand separation can be induced by a 
helicase, for example, or an enzyme capable of exhibiting helicase 
activity. For example, the enzyme RecA has helicase activity in the 
presence of ATP. The reaction conditions suitable for strand separation by 
helicases are known in the art (see Kuhn Hoffman-Berling, 1978, 
CSH-Quantitative Biology 43:63-67; and Radding, 1982, Ann. Rev. Genetics 
16:405-436). 
No matter how strand separation is achieved, however, once the strands are 
separated, the next step in PCR involves hybridizing the separated strands 
with primers that flank the target sequence. The primers are then extended 
to form complementary copies of file target strands. For successful PCR 
amplification, the primers are designed so that the position at which each 
primer hybridizes along a duplex sequence is such that an extension 
product synthesized from one primer, when separated from the template 
(complement), serves as a template for the extension of the other primer. 
The cycle of denaturation, hybridization, and extension is repeated as 
many times as necessary to obtain the desired amount of amplified nucleic 
acid. 
Template-dependent extension of primers in PCR is catalyzed by a 
polymerizing agent in the presence of adequate amounts of four 
deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP; dUTP is 
used in place of or in addition to dTTP if the UNG sterilization system 
described below is incorporated) in a reaction medium comprised of the 
appropriate salts, metal cations, and pH buffering system. Suitable 
polymerizing agents are enzymes known to catalyze template-dependent DNA 
synthesis. Examples of polymerases suitable for use with a DNA template 
include E. coli DNA polymerase I or the Klenow fragment of that enzyme, 
T.sub.4 DNA polymerase, and Taq polymerase, a heat stable DNA polymerase 
isolated from Thermus aquaticus and commercially available from Perkin 
Elmer. The latter enzyme is widely used in the amplification and 
sequencing of nucleic acids. The reaction conditions for using Taq 
polymerases are known in the art and are described in Gelfand, 1989, in 
PCR Technology, supra. Polymerizing agents suitable for synthesizing a 
complementary, copy DNA (cDNA) sequence from the RNA template are reverse 
transcriptase (RT), such as avian myeloblastosis virus RT, or Thermus 
thermophilus DNA polymerase, a thermostable DNA polymerase with reverse 
transcriptase activity marketed by Perkin Elmer. Typically, the RNA 
template is heat degraded during the first denaturation step after the 
initial reverse transcription step leaving only DNA template for 
subsequent amplification. 
If 16S rRNA is to be amplified, an initial reverse transcription (RT) step 
is carried out to create a DNA copy (cDNA) of the RNA. PCT patent 
publication No. WO 91/09944, published Jul. 11, 1991, incorporated herein 
by reference, describes high temperature reverse transcription by a 
thermostable polymerase that also functions in PCR amplification. High 
temperature RT provides greater primer specificity and improved 
efficiency. Copending U.S. patent application Ser. No. 746,121, Attorney 
Docket No. 2532.3, inventors Gelfand and Myers, filed Aug. 15, 1991, 
incorporated herein by reference, describes a "homogeneous RT-PCR" in 
which the same primers and polymerase suffice for both the reverse 
transcription and the PCR amplification steps, and the reaction conditions 
are optimized so that both reactions occur without a change of reagents. 
Thermus thermophilus DNA polymerase, a thermostable DNA polymerase that 
can function as a reverse transcriptase, is used for all primer extension 
steps, regardless of template. Both processes can be done without having 
to open the tube to change or add reagents; only the temperature profile 
is adjusted between the first cycle (RNA template) and the rest of the 
amplification cycles (DNA template). 
The PCR method can be performed in a step wise fashion, where after each 
step new reagents are added, or in a fashion where all of the reagents are 
added simultaneously, or in a partial step wise fashion, where fresh or 
different reagents are added after a given number of steps. For example, 
if strand separation is induced by heat, and the polymerase is heat 
sensitive, then the polymerase has to be added after every round of strand 
separation. However, if, for example, a helicase is used for denaturation, 
or if a thermostable polymerase is used for extension, then all of the 
reagents may be added initially, or, alternatively, if molar ratios of 
reagents are of consequence to the reaction, the reagents may be 
replenished periodically as they are depleted by the synthetic reaction. 
Those skilled in the art will know that the PCR process is most usually 
carried out as an automated process with a thermostable enzyme. In this 
process, the temperature of the reaction mixture is cycled through a 
denaturing region, a primer annealing region, and a reaction region. A 
machine specifically adapted for use with a thermostable enzyme is 
commercially available from Perkin Elmer. 
Those skilled in the art will also be aware of the problem of contamination 
of a PCR by the amplified nucleic acid from previous reactions. Methods to 
reduce this problem are provided in PCT patent application Ser. No. US 
91/05210, filed Jul. 23, 1991, and U.S. patent application Ser. No. 
609,157, filed Nov. 2, 1990, each of which is incorporated herein by 
reference. The methods allow the enzymatic degradation of any amplified 
DNA from previous reactions. The PCR amplification is carried out in the 
presence of dUTP instead of dTTP. The resulting double stranded uracil 
containing product is subject to degradation by uracil N-glycosylase 
(UNG), whereas normal thymine-containing DNA is not degraded by UNG. 
Adding UNG to the amplification reaction mixture before the amplification 
is started degrades all uracil containing DNA that might serve as target. 
Because the only source of uracil containing DNA is the amplified product 
of a previous reaction, this method effectively sterilizes the reaction 
mixture, eliminating the problem of contamination from previous reactions 
(carryover). UNG is rendered temporarily inactive by heat, so the 
denaturation steps in the amplification procedure also serve to inactivate 
the UNG. New amplification products, therefore, though incorporating 
uracil, are formed in an UNG-free environment and are not degraded. 
Sequence specific probe hybridization is an important step in successful 
performance of the present methods. The sequence specific oligonucleotide 
probes of the present invention hybridize specifically with a particular 
segment of the mycobacterial genome and have destabilizing mismatches with 
the sequences from other organisms in the case of genus specific probes, 
and other mycobacteria species, in the case of species-specific probes. 
Stringent hybridization conditions may be chosen so that the probes 
hybridize specifically only to exactly complementary sequences. Detection 
of the amplified product utilizes this sequence specific hybridization to 
insure that only the correct amplified target is detected, decreasing the 
chance of a false positive caused by the presence of homologous sequences 
from related organisms. 
The assay methods for detecting hybrids formed between SSO probes and 
nucleic acid sequences can require that the probes contain additional 
features in addition to the hybridizing region. In the dot blot format, 
for example, the probes are typically labelled. If the probe is first 
immobilized, as in the "reverse" dot blot format described below, the 
probe can also contain long stretches of poly-dT that can be fixed to a 
nylon support by irradiation, a technique described in more detail in PCT 
Patent Publication No. 89/11548, incorporated herein by reference. 
The probes of the invention can be synthesized and labeled using the 
techniques described above for synthesizing oligonucleotides. For example, 
the probe may be labeled at the 5'-end with .sup.32 P by incubating the 
probe with .sup.32 P-ATP and kinase. A suitable non-radioactive label for 
SSO probes is horseradish peroxidase (HRP). Methods for preparing and 
detecting probes containing this label are described in U.S. Pat. Nos. 
4,914,210 and 4,962,029, each of which is incorporated herein by 
reference. For additional information on the use of such labeled probes, 
see U.S. Pat. No. 4,789,630; Saiki et al., 1988, N. Eng. J. Med. 
319:537-541; and Bugawan et al., 1988, Bio/Technology 6:943-947, each of 
which is incorporated herein by reference. Useful chromogens include red 
leuco dye and 3,3',5,5'-tetramethylbenzidine (TMB). Helmuth, PCR 
Protocols, San Diego, Calif., Academic Press, Inc., 1990, pp. 119-128, 
describes procedures for non-isotopic detection of PCR products and is 
incorporated herein by reference. 
The probes of the invention can be used to determine if nucleic acid 
sequences are present in a sample by determining if the probes bind to the 
sequences present in the sample. Suitable assay methods for purposes of 
the present invention to detect hybrids formed between probes and nucleic 
acid sequences in a sample are known in the art. For example, the 
detection can be accomplished using a dot blot format, as described in the 
Example 4. In the dot blot format, the unlabeled amplified sample is bound 
to a solid support, such as a membrane, the membrane incubated with 
labeled probe under suitable hybridization conditions, the unhybridized 
probe removed by washing, and the filter monitored for the presence of 
bound probe. When multiple samples are analyzed with few probes, such as 
the case when samples are screened for the presence of mycobacterial 
nucleic acid using genus specific probes, the dot blot format is quite 
useful. 
An alternate method is quite useful when large numbers of different probes 
are to be used. This method is a "reverse" dot blot, in which the 
amplified sequence contains a label, and the probe is bound to the solid 
support. In this format, the unlabeled probes are bound to the membrane 
and exposed to the labeled sample under appropriately stringent 
hybridization conditions. Unhybridized labeled sample is then removed by 
washing under suitably stringent conditions, and the filter is then 
monitored for the presence of bound sequences. Because species 
determination requires the use of multiple species specific probes for 
each amplified sample, the reverse dot blot format is the preferred test 
format for this step. 
Alternatively, it may be desirable to use a detection method having a 
plurality of probe hybridization sites or wells. For example, a solid 
support such as a microtiter plate is particularly useful in large scale 
clinical applications of the present methods. Copending U.S. patent 
application Ser. Nos. 414,542 and 695,072, filed Nov. 20, 1991, May 3, 
1991, respectively, and incorporated herein by reference, describe 
preferred methods for hybridization/capture of PCR amplified DNA or solid 
supports. In one embodiment of those methods the amplified target DNA is 
labeled (e.g., with biotin) during amplification in the PCR reaction. The 
labeled DNA is specifically captured by hybridization of PCR product to a 
target-specific oligonucleotide capture probe that has been bound to the 
microtiter plate well. The bound product is suitably detected according to 
the type of label used. For example, if biotin is used as a label, avidin 
HRP complex is added and is reacted with either (a) hydrogen peroxide 
substrate and O-phenylene diamine (OPD) chromogen or (b) hydrogen peroxide 
substrate and tetramethylbenzidine chromogen (TMB). A color metric signal 
develops, allowing for the quantitative detection of the PCR amplified 
DNA. 
As practiced in clinical biomedical labs, detection procedures using 
microtiter plate assays can be standardized for a wide range of targets. 
It may be preferable to have detection probes less than 25 nucleotides in 
length. Shorter probes minimize opportunity for cross reactivity and are 
particularly helpful in large scale screening procedures. Accordingly, 
Example 8 a describes preferred method for detecting Mycobacterium species 
in a microtiter plate format. One skilled in the art would recognize that 
probes longer than 25 nucleotides are equally suitable for microtiter 
plate detection schemes; however, it may be necessary to individually 
determine the appropriate hybridization and stringency conditions to 
insure the maximum specificity. 
Another suitable assay system is described in U.S. patent application Ser. 
No. 563,758, filed Aug. 6, 1990, incorporated herein by reference, in 
which a labeled probe is added during the PCR amplification process. Any 
probe that hybridizes to target DNA during each synthesis step is degraded 
by the 5' to 3' exonuclease activity of the polymerase used to catalyze 
primer extension. The degradation product from the probe is then detected. 
Thus, the presence of the degradation product indicates that the 
hybridization between the probe and the target DNA occurred. 
The present invention also relates to kits, multicontainer units comprising 
the primers and probes of the invention. A useful kit can contain SSO 
probes for detecting mycobacterial nucleic acid. In some cases, the SSO 
probes may be fixed to an appropriate support membrane. The kit can also 
contain primers for PCR amplification. Other optional components of the 
kit include, for example, reverse-transcriptase or polymerase, the 
substrate nucleoside triphosphates, means used to label (for example, an 
avidin-enzyme conjugate and enzyme substrate and chromogen if the label is 
biotin) or detect label, and the appropriate buffers for PCR, reverse 
transcription, or hybridization reactions. In addition to the above 
components, the kit can also contain instructions for carrying out 
amplification and detection methods of the invention. 
In a preferred embodiment of the invention, kits for detecting mycobacteria 
may also include positive and negative controls. Preferably a positive 
control includes a nucleic acid sequence that is amplifiable using the 
same primer pair used to amplify mycobacterial nucleic acids in a test 
sample. Methods for using a positive control, wherein both the target that 
may or may not be present, and the positive control, use the same primer 
pair are described in copending U.S. Pat. No. 5,219,727, filed Sep. 28, 
1989, incorporated herein by reference. Preferably the positive control is 
designed so that the product DNA is of a discrete size readily 
distinguishable from the size of the target. 
In another aspect, the present invention provides a positive control that 
is capable of hybridizing to probes for detecting genus-specific 
mycobacterium probes as well as species-specific mycobacterium probes. 
Example 9 describes the construction of a positive control nucleic acid. 
As described herein, it may be desirable to utilize a second amplification 
target, particularly for resolving discordant PCR and culture data. Given 
the teaching of the present invention for providing an internal positive 
control vector, one of ordinary skill in the art would readily appreciate 
that additional internal positive controls could be constructed. For 
example, a positive control could incorporate primer sites for both the 
primary (16S rRNA) and secondary target (e.g., 65 -kDa protein gene) to 
hybridize and subsequently amplify a discrete segment of positive control 
DNA. 
The examples of the present invention presented below are provided only for 
illustrative purposes and not to limit the scope of the invention. 
Numerous embodiments of the invention within the scope of the claims that 
follow the examples will be apparent to those of ordinary skill in the art 
from reading the foregoing text and following examples. 
EXAMPLE 1 
Sample Preparation 
Nucleic acids are isolated from sputum samples using the IsoQuick.TM. 
system commercially available from MicroProbe. About 10 ml of a sputum 
sample is liquified/disinfected, pelleted by centrifugation, and 
resuspended in about 1 ml of buffer with BSA. From this sample, 200 to 500 
.mu.l are centrifuged to pellet the bacteria. The pellets are resuspended 
in 100 .mu.l of sample buffer A, then lysed with 100 .mu.l of lysis 
Reagent 1. Lysates are then extracted with 7 volumes of Reagents 2 and 4 
volumes of Reagent 3 (Reagents 1, 2, and 3, along with sample buffer A, 
are supplied with the IsoQuick.TM. system). The sample is centrifuged and, 
afterwards, 1/3 volume of 10 M NH.sub.4 Ac is added to the aqueous phase 
and the DNA precipitated with an equal volume of isopropanol. The pelleted 
DNA is washed with 70% EtOH, air dried, and resuspended in 100 .mu.l TE, 
pH 8.0. A 50 .mu.l volume of each DNA preparation is used in the 
amplification reaction. 
EXAMPLE 2 
Amplification of Mycobacterial DNA 
A master reagent mixture is prepared so that each reaction contains the 
following reagents: 25 pmol of each primer, 10 nmol of each dNTP, PCR 
buffer at 233 (10.times. buffer=500 mM KCL, 500 mM Tris-HCL, pH 8.9, 20 
mM MgCl.sub.2), 3 units of Taq polymerase (Perkin Elmer), 2 units of UNG 
(Perkin Elmer), and H.sub.2 O to make 50 .mu.l reaction mixture per 
reaction. This master mix is overlayed with 50 .mu.l of mineral oil, and 
the DNA sample is added to the reaction mixture under the oil layer. As 
necessary, H.sub.2 O is added to make up a total reaction volume of 100 
.mu.l. 
The DNA is amplified in a Perkin Elmer Thermal Cycler. The Thermal Cycler 
is programmed to go through 37 cycles of denaturation, primer annealing, 
and primer extension; two cycles of 98.degree. C., 62.degree. C., and 
72.degree. C. for one minute each, followed by 35 cycles of 94.degree. C., 
62.degree. C., and 72.degree. C. for one minute each. The Perkin Elmer 
Thermal Cycler is programmed to soak the samples at 72.degree. C. for an 
indeterminate time after the last cycle to ensure that the final extension 
is complete and to keep the UNG enzyme inactive, if the UNG sterilization 
system is used. The amplification products can then be analyzed by gel 
electrophoresis and/or dot blot hybridization. If analysis by gel 
electrophoresis is to be done, about 10 .mu.l of 10.times. sample buffer 
(0.25% xylene cyanol, 0.25% bromophenol blue, 25% Ficoll) are added, and 
the mineral oil is extracted, and Taq polymerase is inactivated, with 100 
.mu.l of chloroform. 
EXAMPLE 3 
Dot Blot Format 
The initial screening of the amplified sample detects the presence of 
Mycobacterium nucleic acid. In the dot blot format, a small portion of the 
amplified DNA is denatured, applied to a nylon filter, and immobilized as 
described below. The filter is then immersed in a probe solution to allow 
hybridization to one of the labelled probes. Each of the probes can be 
radioactively labelled, but probes covalently conjugated to horseradish 
peroxidase (HRP) can also be used to provide a means of nonisotopic 
detection in the presence of a chromogenic or chemiluminescent substrate. 
Immobilized target DNA is hybridized to a mixture of the two genus 
specific probes KY101 (SEQ ID NO: 3) and KY102 (SEQ ID NO: 4). Because the 
number of samples examined is expected to exceed greatly the number of 
probes (one mixture of two probes), the dot blot format is most convenient 
for this initial screening. A large number of different samples can be 
hybridized onto discrete locations of a single solid support and exposed 
to the labelled probes simultaneously by immersion of the support in a 
probe solution. 
The amplification is carried out as in Example 2. The PCR product is then 
denatured by treatment with alkali. To 5 .mu.l of PCR product is added 5 
.mu.l of 0.5M EDTA, pH 8.0, 8 .mu.l of 5N NaOH, and 82 .mu.l of H.sub.2 O. 
The mixture is allowed to stand at room temperature for 10 minutes to 
complete denaturation. 
BioDyne.TM. B nylon filters (Pall Corp., Glen Cove, N.Y.) are prepared by 
soaking in H.sub.2 O for 5 to 10 minutes and further rinsing with 200 
.mu.l of H.sub.2 O after the dot-blot manifold (Bio-Dot.TM. from Bio Rad, 
Richmond, Calif.) has been set up. Following denaturation, 100 .mu.l of 
the sample mixture is applied under vacuum to the nylon membrane using the 
dot blot apparatus. Each well is then rinsed with 200 .mu.l of 0.4N NaOH, 
then rinsed briefly with 2.times. SSC, and air dried until no pools of 
liquid are left. The DNA is immobilized and crosslinked to the nylon 
filter by ultraviolet irradiation at a flux of 1200 mJ/cm.sup.2 with a 
Stratalinker.TM. (Stratagene, La Jolla, Calif.) UV light box (at the 
"autocrosslink" setting). 
Filters are "prehybridized" by soaking in the hybridization buffer 
(0.5.times. SSC, 5.times. Denhardt's solution, 0.1% SDS, 50 .mu.g/ml of 
herring sperm DNA) in heat sealable bags at 60.degree. C. (air shaker) for 
at least 30 minutes. If radioactively labeled probes are used, the buffer 
is then replaced with an equal amount of the same solution containing 
1.times.10.sup.6 cpm probe, and the filter is allowed to hybridize between 
2 hours and overnight at 60.degree. C. 
After hybridization, filters are washed three times in 2.times. SSC/0.1% 
SDS; twice for 20 minutes at room temperature, and then once for twenty 
minutes at the high stringency temperature of 71.degree. C. in a shaking 
water bath. The filters are then blotted dry, wrapped in plastic wrap, and 
exposed to X-ray film at -70.degree. C. with one or two intensifying 
screens. 
An alternate method of visualization is to hybridize with horseradish 
peroxidase conjugated oligonucleotide probes, prepared as described by 
Levenson and Chang, 1989, in PCR Protocols: A Guide to Methods and 
Applications, (Innis et at., eds., Academic Press. San Diego) pages 
92-112, incorporated herein by reference, and Saiki et al., 1988, N. Eng. 
J. Med. 319:537-541. Hybridization is carried out with 2 pmoles of HRP-SSO 
probe per 5 ml of hybridization solution. 
After washing, filters to be developed with a chromogenic dye substrate are 
rinsed in 100 mM sodium citrate, pH 5.0, then placed in 100 mM sodium 
citrate, pH 5.0, containing 0.1 mg/ml of 3,3',5,5'-tetramethylbenzidine 
per milliliter (Fluka) and 0.0015 percent hydrogen peroxide, and incubated 
with gentle agitation for 10 to 30 minutes at room temperature. Developed 
filters are rinsed in water and immediately photographed. The TMB 
detection system is prepared and used substantially as described in 
AmpliType.RTM. DQalpha DNA typing kit developed and manufactured by 
Hoffmann-La Roche and available through Perkin Elmer. In another 
embodiment, filters are developed with the chemiluminescent detection 
system (ECL; Amersham, Arlington Heights, Ill.). Filters are rinsed in PBS 
for 5 minutes and placed in the ECL solution for 1 minute with gentle 
agitation. Filters are then exposed to X-ray film at room temperature for 
1 to 5 minutes. 
EXAMPLE 4 
Reverse Dot Blot Format 
Species identification requires that each sample be exposed to a variety of 
species specific probes: the identity is indicated by which of the probes 
bind to the sample DNA. Because each sample is exposed to multiple probes, 
the reverse dot blot format is more convenient. The probes are fixed to 
discrete locations on a membrane and then the entire membrane is immersed 
in a solution containing the amplified target DNA to allow hybridization 
to the membrane-bound probes. The reverse dot blot process is described in 
copending application Ser. Nos. 197,000 and 347,495; in Saiki et al., 
1989, Proc. Natl. Acad. Sci. 86:6230-6234; and in the AmpliType.RTM. 
DQalpha DNA typing kit developed and manufactured by Hoffmann-La Roche and 
available through Perkin Elmer, each of which is incorporated herein by 
reference. The amplification primers are biotinylated, as described in 
Levenson and Chang, 1989, supra, so that any amplified DNA that hybridizes 
to the membrane bound probes can be easily detected. 
In one embodiment, detection is carried out by reacting streptavidin 
conjugated horseradish peroxidase (SA-HRP) with any biotinylated (through 
the primers), amplified DNA hybridized to the membrane-bound probe. The 
HRP thus becomes bound, through the SA-biotin interaction, to the 
amplified DNA and can be used to generate a signal by a variety of well 
known means, such as the generation of a colored compound, e.g., by the 
oxidation of tetramethylbenzidine (see U.S. Pat. No. 4,789,630, 
incorporated herein by reference). 
Although the probe can be fixed to the membrane by any means, a preferred 
method involves "tailing" all oligonucleotide probe's hybridizing region 
with a much longer sequence of poly-dT. The resulting poly-dT "tail" can 
then be reacted with amine groups on a nylon membrane to fix the probe 
covalently to the membrane. This reaction can be facilitated by UV 
irradiation. 
Terminal deoxyribonucleotidyl transferase (TdT, Ratliff Biochemicals; for 
the reactions below assume a concentration of abut 120 Units/.mu.l, which 
is 100 pmole/.mu.l) can be used to create a poly-dT tail on a probe, 
although one can also synthesize the tailed probe on a commercially 
available DNA synthesizer. When one uses a DNA synthesizer to make the 
tailed probe, however, one should place the tail on the 5' end of the 
probe, so that undesired premature chain termination occurs primarily in 
the tail region. 
TdT reactions should be carried out in a volume of about 100 .mu.l 
containing 1.times. TdT salts, 200 pmole of oligonucleotide, 800 .mu.M 
DTT, and 60 units of TdT. 10.times. TdT salts is 1,000 mM K-cacodylate, 10 
mM COCl.sub.2, 2 mM dithiothreitol, 250 mM Tris-Cl, pH 7.6, and is 
prepared as described by Roychoudhury and Wu, Meth. Enzymol. 65: 43-62, 
incorporated herein by reference. A 10.times. stock solution of 8 mM dTTP 
can be prepared (neutralized to pH 7 with NaOH) for convenience. 
The TdT reaction should be carried out at 37.degree. C. for two hours and 
then stopped by the addition of 100 .mu.l of 10 mM EDTA, pH 8. The final 
concentration of tailed oligonucleotide is 1 .mu.M (1 pmole/.mu.l), and 
the length of the homopolymer tail is about 400 residues. Tail length can 
be changed by adjusting the molar ratio of dTTP to oligonucleotide. The 
tailed probes can be stored at -20.degree. C. until use. 
The nylon membrane preferred for the reverse dot blot format is the 
Biodyne.TM. B nylon membrane, 0.45 micron pore size, manufactured by Pall 
and also marketed by ICN as the BioTrans.TM. nylon membrane. The probes 
can be spotted onto the membrane very conveniently with the Bio-Dot.TM. 
dot blot apparatus manufactured by BioRad. Each probe is spotted onto a 
unique, discrete location on the membrane. About 2 to 10 picomoles of each 
tailed probe is premixed with 50 to 100 .mu.l of TE buffer before 
application to the dot blot apparatus. After dot blotting, the membrane is 
briefly placed on absorbent paper to draw off excess liquid. The membrane 
is then placed inside a UV light box, such as the Stratalinker.TM. light 
box manufactured by Stratagene, and exposed to 50 to 60 
millijoules/cm.sup.2 of flux at 254 nm to fix the tailed probe to the 
nylon membrane. After a brief rinse (for about 15 minutes in hybridization 
solution) to remove unbound probe, the membrane is then ready for 
hybridization with biotinylated PCR product. 
Amplified PCR products are denatured by heating to 95.degree. C. for 3 to 
10 minutes, and 40 .mu.l of the denatured PCR product are added to each 
probe panel for hybridization. Hybridization is carried out at 57.degree. 
C. for 20 minutes in a shaking water bath in a hybridization buffer 
composed of 0.5.times. SSC, 0.25% SDS, and 5.times. Denhardt's solution. 
The hybridization buffer is replaced with 3 ml of a solution consisting of 
25 .mu.l of SA-HRP, commercially available from Perkin Elmer, in 3.1 ml of 
hybridization buffer, and incubated for 20 minutes at 57.degree. C. in a 
shaking water bath. 
Washing is carried out in a wash buffer of 2.times. SSC and 0.1% SDS. After 
a brief rinse of the membrane in 10 ml of wash buffer, a 12 minute 
stringent wash in 10 ml of buffer is done at 57.degree. C. Another 5 
minute room temperature wash is then carried out, followed by a 5 minute 
wash in 10 ml of 0.1M sodium citrate, pH 5.0. 
Chromogen binding is carried out in 5 ml of chromogen solution consisting 
of 5 ml of 0.1M sodium citrate, 5 .mu.l of 3% hydrogen peroxide, and 0.25 
ml of chromogen (TMB from Perkin Elmer) for 25-30 minutes at room 
temperature. Three 10 minute washes in distilled water are carried out at 
room temperature. A post-wash of 1.times. PBS at room temperature for 30 
minutes can enhance signal quality. During steps in which chromogen is 
present, the membrane should be shielded from light by an aluminum foil 
covering. The developed membrane should be photographed for a permanent 
record. 
EXAMPLE 5 
Mycobacterial DNA Detection 
Detection of mycobacterial DNA was accomplished by amplification with 
biotinylated forms of the genus specific primers KY18 (SEQ ID NO: 1) and 
KY75 (SEQ ID NO: 2), using the protocol described in Example 2, above, 
followed by hybridization to the genus specific probes KY101 (SEQ ID NO: 
3) and KY102 (SEQ ID NO: 4), using the dot blot assay described in Example 
3, above. The sequences of the hybridizing regions of the upstream primer 
KY18 (SEQ ID NO: 1) and the downstream primer KY75 (SEQ ID NO: 2) are 
given in Table 1, above. The sequences of the hybridizing regions of the 
genus-specific probes KY101 (SEQ ID NO: 3) and KY102 (SEQ ID NO: 4) are 
given in Table 2, above. 
The genus specific primers KY18 (SEQ ID NO. 1) and KY75 (SEQ ID NO. 2) were 
used in polymerase chain reaction (PCR) amplifications to amplify nucleic 
acid from 15 Mycobacterium species. The results are shown in Table 4. As 
expected, KY18 (SEQ ID NO. 1)/KY75 (SEQ ID NO. 2) amplified DNA from all 
Mycobacterium species except M. simiae. Amplification of M. simiae or M. 
chitae DNA was not expected because KY75 (SEQ ID NO. 2) differs in four of 
the five 3'-terminal bases from M. simiae and in two of the 3'-terminal 
bases from M. chitae. However, because the association of M. simiae with 
human disease has rarely been reported, detection is not clinically 
important. With the exception of DNA from M. xenopi and M. terrae, all 
amplified mycobacterial DNA hybridized was detected by hybridization to 
the genus specific probes KY101 (SEQ ID NO. 3) and KY102 (SEQ ID NO. 4). 
TABLE 4 
______________________________________ 
Amplificafion of DNA from Different Mycobacterial Species 
and Hybridization to Genus SDecific Probes 
Mycobacteria Amplification 
Hybridization 
______________________________________ 
M. tuberculosis 
+ + 
M. scrofulaceum 
+ + 
M. fortuitum + + 
M. avium + + 
M. kansasii + + 
M. intracellulare 
+ + 
M. phlei + + 
M. smegmatis + + 
M. marinum + + 
M. favescens + + 
M. xenopi + - 
M. simiae - - 
M. chelonae + + 
M. gordonae + + 
M. terrae + - 
______________________________________ 
The specificity of these primers was tested by attempting to amplify DNA 
from 22 different non-mycobacterial species. Amplification products 
resulted only from the DNA of Corynebacter diptheriae and Corynebacter 
xerosis, Nisseria sicca, and Propionibacterium acnes. However, these 
amplification products failed to hybridize with the genus specific probes, 
so no false positives resulted. The organisms tested are listed in Table 
5, below. 
TABLE 5 
______________________________________ 
Amplification of DNA from Non-Mycobacterial Organisms 
Organism amplification 
Hybridization 
______________________________________ 
Bordatella pertussis 
- - 
Borrelia burgdorferi 
- - 
Corynebacter diphtheriae 
+ - 
Corynebacter xerosis 
+ - 
Enterobacter aerogenes 
- - 
Escherichia coli - - 
Haemophilus influenzae 
- - 
Klebsiella pneumoniae 
- - 
Legionella pneumophila 
- - 
Neisseria gonorrhea 
- - 
Neisseria meningitidis 
- - 
Nisseria sicca + - 
Propionibacterium acnes 
+ - 
Psuedomonas aeruginosa 
- - 
Salmonella typhimurium 
- - 
Serratia marcescens 
- - 
Staphylococcus aureus 
- - 
Streptococcus agalactiae 
- - 
Streptococcus pyogenes 
- - 
Streptomyces hygrocopicus 
- - 
Streptomyces rubiginosis 
- 
Treponema pallidum 
- - 
______________________________________ 
EXAMPLE 6 
Species Identification 
Once mycobacterial nucleic acid has been detected in a clinical sample, the 
species from which the nucleic acid originates can be determined by the 
pattern of hybridization with the species specific probes using the 
reverse dot blot format of Example 4. The species of clinical interest to 
be detected by the present system are M. avium, M. intracellulare, M. 
kansasii, and M. tuberculosis. In addition, detection of M. gordonae is 
desired because that organism is frequently found in clinical samples. 
FIG. 1 shows the results of a test of the specificity of species specific 
probes selected from the probes listed in Table 3. The sequence of the 
hybridizing region of each probe, along with the expected specificity, is 
shown in Table 3, above. Amplified products from purified DNA from 
thirteen different species of Mycobacterium were used to test the 
specificity of both the genus specific and species specific probes. For 
each species, 1 pg of DNA purified from cultured bacteria (the equivalent 
of about 300 bacterial genomes) was amplified as in Example 2 using 
biotinylated primers. Detection of probe hybridization was done using the 
reverse dot-blot format of Example 4. As a positive control for the 
presence of amplified DNA, the genus specific probes were included on the 
test strips along with the species specific probes. 
EXAMPLE 7 
Amplification of Mycobacterial 16S rRNA 
The 16S rRNA can be amplified by first creating cDNA by reverse 
transcription and amplifying the cDNA. The same primers are used as in 
Example 2, above. In this example, both the high temperature reverse 
transcription and the PCR amplification are carried out with the 
thermostable Tth polymerase. 
The reverse transcription is carried out in a volume of 20 .mu.l containing 
the following components: 8 .mu.l of H.sub.2 O, 2 .mu.l of 10.times. RT 
reaction buffer (100 mM Tris-HCL, pH 8.3, and 900 mM KCL), 2 .mu.l of 10 
mM MnCl.sub.2, 2 .mu.l of dNTP solution (2 mM each of dATP, dCTP, dGTP, 
and dTTP in H.sub.2 O, pH 7.0), 2 .mu.l of the "downstream" primer (7.5 mM 
in H.sub.2 O), 2 .mu.l of 0.18 .mu.M Tth polymerase in 1.times. storage 
buffer (20 mM Tris-HCL, pH 7.5, 100 mM KCL, 0.1 mM EDTA, 1 mM DTT, 0.2% 
Tween 20 (Pierce Surfactants), 50% (volume/volume) glycerol), and 2 .mu.l 
of template RNA solution (&lt;250 ng in 10 mM Tris-HCL and 1 mM EDTA). All 
solutions not containing Tris are treated with diethylpyrocarbonate (DEPC) 
to remove any contaminating ribonuclease as described on page 190 of 
Maniatus et al., 1982, Molecular Cloning, a Laboratory Manual (Cold 
Springs Harbor Laboratory, New York). The reverse transcription is carried 
out at 72.degree. C. for 5 minutes in a thermocycler. The reaction is 
stopped by cooling the reaction to 4.degree. C. with ice. 
The PCR amplification is performed with the following reagents added: 2 
.mu.l of the remaining primer (7.5 mM in H.sub.2 O), 2 .mu.l of dNTP 
solution (10 mM each of dATP, dCTP, dGTP, and dTTP in H.sub.2 O, pH 7.0), 
8 .mu.l of 10.times. PCR reaction buffer (100 mM Tris-HCL, pH 8.3, 1 mM 
KCL, 18.75 mM MgCl.sub.2, 7.5 mM EGTA, and 50% (volume/volume) glycerol), 
and 68 .mu.l DEPC treated H.sub.2 O. The nucleic acid is amplified in a 
Perkin Elmer Thermal Cycler with the same thermal profile as in Example 2. 
The amplified product is analyzed as in the prior examples. 
EXAMPLE 8 
Microtiter Plate Assay for the Detection of Mycobacterium 
In this embodiment of the invention, the probe is fixed to a well of a 
microtiter plate. The amplified target DNA is hybridized to the bound 
probe as described above. As in the previous example, the amplification 
primers are biotinylated to allow detection of amplified DNA that 
hybridizes to the bound probes. 
The desired probes, conjugated to BSA, were first allowed to adsorb to the 
plastic surface of the individual wells. The wells were then blocked with 
protein, such as bovine serum albumin. Preferably, 96 well plates 
available from Corning are used. 
Once the amplification has been completed, the PCR tubes were removed from 
the thermocycler (available through Perkin Elmer). One hundred microliters 
of denaturation solution were added to each PCR tube. A new pipette tip is 
used for each tube. In one embodiment, detection may not be preformed 
immediately. In that case, the PCR tubes were storied overnight at 
2.degree. C. to 8.degree. C. Denatured amplification reactions become 
viscus upon storage at 2.degree. C. to 8.degree. C. Tubes were briefly 
warmed at 25.degree. C. to 30.degree. C. prior to opening tubes to make 
pipette easy. 
The appropriate number of eight well microtiter plate strips (minimally 2 
strips) were removed and set into the microtiter plate frame. One hundred 
microliters of hybridization buffer was pipetted into each well of the 
microtiter plate. 
The denaturation solution contains 0.4M NaOH; 80 mM EDTA and 0.005% Thymol 
blue. Hybridization/neutralization buffer contains: 3M NaSCN; 80 mM 
NaH.sub.2 PO.sub.4 ; 10 mM NaH.sub.2 PO.sub.4 ; and 0.125% Tween 20. 
Before use the pH is checked to be 5.0.+-.0.2. 
Using plugged tips with a multi channel pipetter, 25 .mu.l of the denatured 
amplification reaction from each PCR tube in the tray was pipetted to the 
corresponding well position in the microtiter plate. The plate was covered 
with the microtiter plate lid and gently tapped on the side 10 to 15 
times. Wells in which proper reagent pipeting has been done will turn 
light yellow in color. If no or only a single change in blue color is 
noted, excess amplicon has been added. The test is continued as positive 
OD values will increase but negative OD values are not affected. The plate 
was incubated for 60 minutes at 37.degree. C. After the initial 
hybridization at 37.degree. C. for one hour, the 
hybridization/neutralization buffer was removed and replaced with the same 
buffer and the plate was incubated for an additional 15 minutes at 
37.degree. C. 
Following incubation the plate was washed five times with wash solution. 
Washing of the plate may be preformed manually or with an automated 
microtiter plate washer programmed accordingly. For washing, a 1.times. 
PCR wash buffer was used. A 10.times. concentrate of PCR washed buffer was 
prepared as follows: 9.94 grams per liter of sodium phosphate dibasic; 
4.41 grams per liter sodium phosphate (monobasic); 3.722 grams per liter 
EDTA; 87.66 grams per liter sodium chloride; 13.7 grams per liter of Tween 
20; and 10 grams per liter of Pro Clin 300 (Rohm and Haas, Philadelphia, 
Pa.). The solution is pit with phosphoric acid (pH 6.5-7.1 is preferred). 
For manual washing the contents of the plate were emptied and tapped dry. 
Three hundred microliters of wash solution was added to each well in the 
plate being tested, and the plate was allowed to dry for 15 to 30 seconds. 
The plate was again emptied and tapped dry. This wash process was repeated 
four additional times. 
For an automated microplate washer, the following procedure was used. The 
contents of the wells was aspirated. The washer was programmed to add 350 
microliters of working wash solution to each well in the plate being 
tested and soaked for 30 seconds and aspirated. The steps were repeated 
four additional times. The plate was then tapped dry. 
One hundred microliters of conjugate was added to each well in the plate 
being tested. The avidin-HRP conjugate is prepared as follows. The diluent 
contains 0.1 molar; 0.25% Emulsit 25 (DKS International, Inc., Tokyo, 
Japan); 1.0% Kathon CG (Rohm and Haas, Philadelphia, Pa.); 0.1% phenol; 
1.0% bovine gamma globulin. The solution was pH to 7.3 with concentrated 
HCl. To this diluent 10 nM of conjugated avidin (Vector Labs, Burlingame, 
Calif.) was added. The plate was then covered and then incubated 50 
minutes at 37.degree. C. and again washed as described above. The working 
substrate was prepared by mixing 2.0 ml of Substrate A and 0.5 ml of 
Substrate B for each multiple of two 8 well microtiter plate strips (16 
tests). Substrate A contains 3 mM hydrogen peroxide, 6.5 mM citrate and 
0.1% Kathon CG. Substrate B contains 4.2 mM 3,3',5,5' tetramethylbenzidine 
and 40% dimethylformamide. The working substrate was prepared no more than 
three hours before use and was stored away from direct sunlight. 
One hundred microliters of working substrate (substrate A and B mixture) 
was added to each well of the plate being tested. The plate was then 
covered and incubated in the dark for 10 minutes at room temperature 
(20.degree. C. to 25.degree. C.). One hundred microliters of Stop Reagent 
(5% H.sub.2 SO4) was added to each well being tested. The absorbance of 
each well of 450 nM was read within one hour of adding the Stop Reagent. 
The absorbance value was recorded for specimen and control. 
EXAMPLE 9 
The Construction of a Positive Control Vector Useful in Methods for the 
Amplification and Detection of Mycrobacterial Nucleic Acids 
Oligonucleotides which contain the species-specific probe binding sequences 
as well as their complements (KY178 [SEQ ID NO. 24]-KY181 [SEQ ID NO. 27] 
below) were synthesized. (These oligos contain recognition sites for 
restriction enzymes at both termini to facilitate cloning.) One microgram 
each of KY178 (SEQ ID NO. 24) and KY179 (SEQ ID NO. 25) or KY180 (SEQ ID 
NO. 26) and KY181 (SEQ ID NO. 27) were combined, heated for 5 minutes at 
98.degree. C. then incubated for one hour at 75.degree. C. to allow 
annealing of the complementary strands. Annealed products were separated 
from residual single-stranded oligos by electrophoresis through 3% Nusieve 
(FMC Products)/1% agarose gel. The bands of double-stranded products are 
cut out and the DNA eluted. The DNA fragments are then cut with the 
appropriate restriction enzymes and ligated to each other. The ligation 
products are isolated from Nusieve/agarose gel as above. 
The recipient vector was prepared. The recipient vector was a plasmid into 
which a fragment of the M. tb 16S rRNA gene containing the primer and 
probe binding sites have been inserted and was prepared as follows. 
Fifty picograms of M. tuberculosis DNA was amplified using primers KY70 
(SEQ ID NO. 28) and CR01 (SEQ ID NO. 29) in the presence of 50 pmol CR01 
(SEQ ID NO. 29), 80 pmol KY70 (SEQ ID NO. 28), 20 nmol of each dNTP, 2.5 
units Taq polymerase, and 1.times. PCR buffer (50 mM Tris-HCl, pH 8.9; 50 
mM KCl; 1.5 mM MgCl.sub.2) in a total reaction volume of 100 microliters. 
Thermal cycling conditions are as outlined in Example 2. The amplification 
products were extracted with 100 microliters chloroform. 
The amplification products and vector pBS(+) (Stratagene) were both 
digested with restriction endonuclease Pst I, extracted once with 
phenol/chloroform, and then precipitated with ethanol. (CRO1 contains a 
Pst I site at the 5'-end and the amplification product contains an 
internal Pst I site downstream of the binding sites for the 
mycobacteria-specific primers and probes.) The Pst I cut vector was 
dephosphorylated by treatment with calf intestine phosphatase (Maniatis), 
extracted with phenol/chloroform, and precipitated with ethanol. The 
prepared amplification products were ligated to the vector under standard 
condition (Maniatis). 
The ligated DNA were transformed into competent E. coli. Colonies carrying 
plasmids that contain the desired insert were identified by colony blot 
hybridization to the tb-specific probe KY21 (SEQ ID NO. 5) as follows. 
Bacteria were streaked onto a nitrocellulose filter disk overlaid onto a 
nutrient agar plate and allowed to grow overnight. The filter was removed 
and successively overlaid (bacteria-side up) onto 3 MM filter papers 
soaked with 10% SDS (3 minutes), 0.5M NaOH/1.5M NaCl (5 minutes), 0.5M 
Tris-HCl, pH8/1.5M NaCl (5 minutes), and 2.times. SSC (5 minutes). The 
filters were air-dried. The DNA were cross-linked onto the filter by UV 
irradiation and then hybridized to KY21 (SEQ ID NO. 5) and washed as 
outlined in Example 3. 
Oligonucleotide Sequences: 
KY70 SEQ ID NO. 28 
GCGGTACCTG CACACAGGCC ACAAGGGAA 
CR01 SEQ ID NO. 29 
CGCCTGCAGT TAACACATGC AAGTCGAACG G 
This vector, designated pKY5, was cut with the restriction enzymes Sty I 
and Xho I to remove a 174 bp fragment containing the species-specific 
probe binding site but leaving intact the primer and genus-specific probe 
binding sites. The cut plasmid was separated from the 174 bp fragment by 
electrophoresis through 1.5% low melting temperature agarose gel. The band 
containing the vector was cut from the gel and purified by chromatography 
through a NACS column (Bethesda Research Lab) and ethanol precipitation. 
The insert fragment containing recognition sites for the species-specific 
probes is ligated to the prepared vector. The ligation products are 
transformed into competent host bacteria. 
Transformants containing the appropriate inserts are identified by PCR 
amplification. Transformant bacterial colonies are resuspended in 0.5 ml 
TE buffer. Fifty microliters of the bacterial suspension are placed in PCR 
reaction tubes containing components necessary for amplification of 
mycobacterial DNA and amplification is carried as discussed above. 
Bacteria which carry plasmids containing the desired insert will generate 
PCR products of 640 bp using primer pair KY18 (SEQ ID NO. 1) and KY75 (SEQ 
ID NO. 2). Amplification of bacteria containing the original pKY5 plasmid 
generates PCR products of 584 bp. 
The amplicons thus generated can be hybridized to mycobacterial 
genus-specific and species-specific probes by reverse dot-blot 
hybridization as outlined in Example 4 to confirm the presence of the 
hybridization sites for the genus-specific and species-specific probes 
described in the Examples. Positive control plasmid can be similarly 
prepared for hybridizing to genus probes and a select subset of species 
specific probes. In a kit format, for example, it may be desirable to 
include a positive control plasmid for distinguishing tuberculosis from 
other species, in addition to including a positive control plasmid 
containing sequences KY178-KY181 (SEQ ID NOS. 24-27). 
Oligonucleotide sequences: 
__________________________________________________________________________ 
KY178 - SEQ. ID NO. 24 
5' 
CCATCGATAG 
GACCATTCTG 
CGCATGTGGT 
GTGGTGGGTC 
TAATACCGAA 
TAGGACCACA 
GGACACATGA 
AGGCTCACTT 
TGTGGGTTGA 
CGGTAGGTAA 
CACTTGGCGC 
ATGCCTTGTG 
GTGGAAAGCT 
TCCAAGGCA 3' 
KY179 - SEQ. ID NO. 25 
5' 
TGCCTTGGAA 
GCTTTCCACC 
ACAAGGCATG 
CGCCAAGTGT 
TACCTACCGT 
CAACCCACAA 
AGTGAGCCIT 
CATGTGTCCT 
GTGGTCCTAT 
TCGGTATTAG 
ACCCACCACA 
CCACATGCGC 
AGAATGGTCC 
TATCGATGG 3' 
KY180 - SEQ. ID NO. 26 
5' 
CCGCTCGAGA 
CGGGATGCAT 
GTCTTGTGGT 
GGAAAGCGCT 
TTAGCGGTAA 
CTTTAGGCGC 
ATGTCTTTAG 
GTGGAAAGCT 
TAACTCAAGA 
CGCATGTCTT 
CTGGTGGAAA 
GCTTTTGCAT 
CGATGG 3' 
KY181 - SEQ. ID NO. 27 
5' 
CCATCGATGC 
AAAAGCTTTC 
CACCAGAAGA 
CATGCGTCTT 
GAGTTAAGCT 
TTCCACCTAA 
AGACATGCGC 
CTAAAGTTAC 
CGCTAAAGCG 
CTTTCCACCA 
CAAGACATGC 
ATCCCGTCTC 
GAGCGG 3' 
__________________________________________________________________________ 
EXAMPLE 10 
The Use of Positive Control Plasmid 
One use of the positive control plasmid is to monitor the efficiency of 
amplification in any specific experiment. In such applications, serial 
dilutions of the positive control plasmid are made. Known copy numbers of 
the plasmid can be used as templates in amplification reactions. The 
lowest number of plasmid DNA molecules that can be amplified gives a 
measurement of the efficiency of the amplification reaction. Another use 
of the positive control plasmid is to generate products which can be used 
to monitor the efficiency with which the genus and species-specific probes 
detects mycobacterial DNA. Amplification products generated as above can 
serve as substrate in hybridization reaction. Generation of the 
appropriate hybridization signals allows for an assessment of how well the 
probes are able to detect mycobacterial DNA. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 29 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CACATGC AAGTCGAACGGAAAGG23 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GCCCG TATCGCCCGCACGCTCACA24 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TCG CGTTGTTCGTGAAATCTCACGGCTTAA30 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
T CGCGTTGTTCGTGAAAACTCACAGCTTAA30 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ACGGGATGCATGTCTTGTGGTGGAAAGCGCTTTAGC36 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ACTTGGCGCATGCCTTGTGGTGGAAAGCTT30 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
TTTAGGCGCATGTCTTTAGGTGGAAAGCTT30 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TCAAGACGCATGTCTTCTGGTGGAAAGCTTTTGC34 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TCCCGAAGTGCAGGCCAGATTGCCCACGTG30 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
( xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GAAGGCTCACTTTGTGGGTTGACGGTAGGT30 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GCAATCTGCCTGCACACCGGGATAAGCCTG30 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GGGTCTAATACCGAATAGGACCACAGGACACATG34 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
TCGCGTTGTTCGTGAAATCTCACAGCTTAA30 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
TCGCGTTGTTCGTGGAATCTCACAGCTTAA30 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
TCGCGTTGTTCGTGGAATGCCACAGCTTAA30 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
ATAGGACCATTCTGCGCATGTGGTGTGGTG30 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(i i) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
ACCTCAAGACGCATGTCTTCTGGT24 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
CCGAATAGGACCACAGGACACATG24 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
ACCTTTAGGCGCATGTCTTTAGGT24 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
AACACTTGGCGCATGCCTTGTGGT24 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GAAGGCTCACTTTGTGGGTTGACG24 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
TGTGGTGGAAAGCGCTTTAGCGGT24 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D ) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
AGGACCATTCTGCGCATGTGGTGT24 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 139 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
CCATCGATAGGACCATTCTGCGCATGTGGTGTGGTGGGTCTAATACCGAATAGGACCACA60 
GGACACATGAAGGCTCACTTTGTGGGTTGACGGTAGGTAACACTTGGCGCATGCCTTGTG120 
GTGGAAAGCT TCCAAGGCA139 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 139 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
TGCCTTGGA AGCTTTCCACCACAAGGCATGCGCCAAGTGTTACCTACCGTCAACCCACAA60 
AGTGAGCCTTCATGTGTCCTGTGGTCCTATTCGGTATTAGACCCACCACACCACATGCGC120 
AGAATGGTCCTATCGATGG 139 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 126 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
CCGCTCGAGACGGGATGCATGTCTTGTGGTGGAAAGCGCTTTAGCGGTAACT TTAGGCGC60 
ATGTCTTTAGGTGGAAAGCTTAACTCAAGACGCATGTCTTCTGGTGGAAAGCTTTTGCAT120 
CGATGG126 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 126 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
CCATCGATGCAAAAGCTTTCCACCAGAAGACATGCGTCTTGAGTTAAGCTTTCCACCTAA60 
AGACATGCGCCTAAAGTTACCGCTAAAG CGCTTTCCACCACAAGACATGCATCCCGTCTC120 
GAGCGG126 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
GCGGTACCTGCACACAGGCCACAAGGGAA29 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
CGCCTGCAGTTAACACATGCAAGTCGAACGG31