# Patent

## Title
Nucleic acid compositions and nucleic acid amplification based methods for detecting E. coli O157:H7

## Abstract

Disclosed are primer and probe compositions, PCR assays, methods and kits for the specific detection of E. coli O157:H7 and not E. coli O55:H7 from a variety of samples. In some embodiments, methods for specifically detecting E. coli O157:H7 comprise: hybridizing at least a first pair of polynucleotide primers to at least a first target polynucleotide sequence, hybridizing at least a second pair of polynucleotide primers to at least a second target polynucleotide sequence, amplifying said at least first and said at least second target polynucleotide sequences, and detecting said at least first and said at least second amplified target polynucleotide sequence products, wherein the detection of both the first amplified target polynucleotide sequence product and the second amplified target polynucleotide sequence product is indicative of the presence of E. coli O157:H7 in a sample and not E. coli O55:H7.

## Background
CROSS-REFERENCE(S) TO RELATED APPLICATION(S)
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/178,931, filed May 15, 2009, the contents of which is incorporated herein by reference.


FIELD OF THE INVENTION
The present teachings relate to assays and methods for the specific detection and differentiation of pathogenic organisms.
BACKGROUND
Identification of bacterial contamination in food often occurs subsequent to an outbreak of a foodborne illness. The bacterium Escherichia coli is frequently identified as the food contaminant of many foodborne illnesses. The serotype known as E. coli O157:H7 causes enterohemorrhagic colitis and possibly kidney failure. It often results in hospitalization of the infected patient and can be particularly lethal in young children and the elderly. O157:H7 is most often associated with outbreaks of foodborne illness in the United States and elsewhere in the world.
Detection of pathogenic E. coli, particularly serotypes causative of hemorrhagic colitis has become a public health priority. O157:H7 is frequently isolated from cattle, including healthy animals and has also been associated with illness in contaminated produce. The presence of O157:H7 in a food product released to consumers is considered as evidence of adulteration of the product. Regulations by the United States government require meat processors to screen for the presence of O157:H7 in their finished products and more stringent guidelines are being considered in a number of states for the identification of O157:H7 in other commodities and food stuffs. An assay for the rapid, sensitive and specific detection of infectious pathogens is extremely important from both a public health and economic perspective.
Many strains of genetically similar E. coli exist that vary dramatically in their pathogenicity. Genomic comparisons are revealing the consequences of genetic changes often underlie the emergence of new pathogenic bacteria. E. coli O157:H7 has been determined to have evolved stepwise from the O55:H7 which is associated with infantile diarrhea. These two serotypes are more closely related at the nucleotide level while divergence was markedly different at the gene level. Likewise, other pathogenic serotypes have been shown to be less divergent at the nucleotide level making identification of pathogenic strains difficult.
An assay utilizing molecular methods such as sequence specific amplification and detection offer significant improvements in speed, sensitivity and specificity over traditional microbiological methods. Design and development of a molecular detection assay that requires the identification of a target sequence that is present in all organisms to be detected and absent or divergent in organisms not to be detected is an unmet need for the definitive detection of the O157:H7 serotype of E. coli. 
SUMMARY
In accordance with the embodiments, there is disclosed a method of detecting the presence of E. coli O157:H7 in a sample, comprising: detecting the presence of SEQ ID NO:111 or complement thereof; and detecting the presence of a sequence selected from SEQ ID NO:91-110 and complements thereof; wherein detection of SEQ ID NO:111 and detection of a sequence selected from SEQ ID NO:91-110 confirms the presence of E. coli O157:H7 in a sample and not E. coli O55:H7. The detection is by a nucleic acid amplification reaction, the amplification reaction is an end-point determination, the amplification reaction is quantitative, the quantification is a real-time PCR, the real-time PCR is a SYBR® Green Assay or the real-time PCR is a TaqMan® Assay.
In another embodiment, disclosed is an assay for the detection of E. coli O157:H7 in a sample comprising a) hybridizing a first pair of PCR primers selected from the group consisting of: SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQ ID NO:1 and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6 and complements thereof to at least a first target polynucleotide sequence; b) hybridizing a second pair of PCR primers selected from SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ ID NO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ ID NO:49-50, SEQ ID NO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ ID NO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ ID NO:82-83, SEQ ID NO:85-86, and SEQ ID NO:88-89 and complements thereof to at least a second target polynucleotide sequence; c) amplifying said at least first and said at least second target polynucleotide sequences; and d) detecting said at least first and said at least second amplified target polynucleotide sequence products; wherein the detection of the at least first amplified target polynucleotide sequence product and the detection of the at least second amplified target polynucleotide sequence product is indicative of the presence of E. coli O157:H7 in the sample and not E. coli O55:H7.
In further embodiments, the assay further comprises using a first probe of SEQ ID NO:3 and a second probe selected from SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:90, the first probe further comprises a first label and said second probe further comprises a second label, wherein both labels are selected from a dye, a radioactive isotope, a chemiluminescent label, and an enzyme, the dye comprises a fluorescein dye, a rhodamine dye, or a cyanine dye, the dye is a fluorescein dye and first probe is labeled with FAMTM dye and said second probe is labeled with VIC® dye. The assay, in some embodiments, further comprises preparing the sample for PCR amplification prior to hybridizing comprising steps, for example, but not limited to (1) bacterial enrichment, (2) separation of bacterial cells from the sample, (3) cell lysis, and (4) total DNA extraction, the sample can be for or a water sample, an environmental sample, and so on and the food sample comprises a selectively enriched food matrix. The assay can be by polymerase chain reaction, wherein hybridizing and amplifying of said first pair of polynucleotide primers occurs in a first vessel and said hybridizing and amplifying of said second pair of polynucleotide primers occurs in a second vessel, or hybridizing and amplifying of said first pair of polynucleotide primers and said hybridizing and amplifying of said second pair of polynucleotide primers occurs in a single vessel, the detection is a real-time assay, the real-time assay is a SYBR® Green dye assay or a TaqMan® assay.
In still another embodiment, the invention teaches a assay for the detection of E. coli O157:H7 in a sample comprising: a) hybridizing a first pair of PCR primers to a first target polynucleotide sequence within SEQ ID NO:111; b) hybridizing a second pair of PCR primers to a second target polynucleotide sequence within a sequence selected from SEQ ID NO:91-110; c) amplifying said at least first and said at least second target polynucleotide sequences; and d) detecting said at least first and said at least second amplified target polynucleotide sequence products; wherein the detection of the at least first amplified target polynucleotide sequence product and the detection of the at least second amplified target polynucleotide sequence product is indicative of the presence of E. coli O157:H7 in the sample and not E. coli O55:H7. The assay can further have a first probe of SEQ ID NO:3 and a second probe selected from SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:90, the first probe further comprises a first label and said second probe further comprises a second label, wherein both labels are selected from a dye, a radioactive isotope, a chemiluminescent label, and an enzyme, the dye comprises a fluorescein dye, a rhodamine dye, or a cyanine dye, the dye is a fluorescein dye and first probe is labeled with FAMTM dye and said second probe is labeled with VIC® dye. The assay further has preparing the sample for PCR amplification prior to hybridizing, for example, but not limited to (1) bacterial enrichment, (2) separation of bacterial cells from the sample, (3) cell lysis, and (4) total DNA extraction, the sample can be for or a water sample, an environmental sample, and so on and the food sample comprises a selectively enriched food matrix. The assay can be by polymerase chain reaction, wherein hybridizing and amplifying of said first pair of polynucleotide primers occurs in a first vessel and said hybridizing and amplifying of said second pair of polynucleotide primers occurs in a second vessel, or hybridizing and amplifying of said first pair of polynucleotide primers and said hybridizing and amplifying of said second pair of polynucleotide primers occurs in a single vessel, the detection is a real-time assay, the real-time assay is a SYBR® Green dye assay or a TaqMan® assay.
In one embodiment, disclosed is a method for specifically detecting E. coli O157:H7, comprising: hybridizing at least a first pair of polynucleotide primers to at least a first target polynucleotide sequence, hybridizing at least a second pair of polynucleotide primers to at least a second target polynucleotide sequence, amplifying said at least first and said at least second target polynucleotide sequences, and
detecting said at least first and said at least second amplified target polynucleotide sequence products, wherein the detection of the at least first amplified target polynucleotide sequence product and the detection of the at least second amplified target polynucleotide sequence product is indicative of the presence of E. coli O157:H7 in a sample and not E. coli O55:H7. The method further has preparing the sample for PCR amplification prior to hybridizing, for example, but not limited to (1) bacterial enrichment, (2) separation of bacterial cells from the sample, (3) cell lysis, and (4) total DNA extraction, the sample can be for or a water sample, an environmental sample, and so on and the food sample comprises a selectively enriched food matrix. The method can be by polymerase chain reaction, having at least a first probe and at least a second probe, said first probe further comprises a first label and said second probe further comprises a second label, both labels are selected from a dye, a radioactive isotope, a chemiluminescent label, and an enzyme, the dye comprises a fluorescein dye, a rhodamine dye, or a cyanine dye, the first probe is labeled with FAMTM dye and said second probe is labeled with VIC® dye, wherein hybridizing and amplifying of said first pair of polynucleotide primers occurs in a first vessel and said hybridizing and amplifying of said second pair of polynucleotide primers occurs in a second vessel, or hybridizing and amplifying of said first pair of polynucleotide primers and said hybridizing and amplifying of said second pair of polynucleotide primers occurs in a single vessel, the detection is a real-time assay, the real-time assay is a SYBR® Green dye assay or a TaqMan® assay. The method includes a first primer pair is selected from SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQ ID NO:1 and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6 and said second primer pair is selected from SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ ID NO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ ID NO:49-50, SEQ ID NO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ ID NO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ ID NO:82-83, SEQ ID NO:85-86, and SEQ ID NO:88-89, and said first probe is SEQ ID NO:3 for use for said first primer pair and said second probe is selected according to FIG. 3 for use with said second primer pair from SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:90, wherein said first target polynucleotide sequence is within SEQ ID NO:111 and said second target polynucleotide sequence is within the group selected from SEQ ID NO:91-110.
In another embodiment, disclosed is a polynucleotide sequence or its complement for the detection of E. coli O157:H7 and not E. coli O55:H7 identical to at least 16 contiguous polynucleotides from a first sequence selected from the group consisting of SEQ ID NO:91-110 and a second sequence of SEQ ID NO:111, the contiguous polynucleotide is a primer or a probe, the has a label selected from a dye, a radioactive isotope, a chemiluminescent label, and an enzyme, the dye comprises a fluorescein dye, a rhodamine dye, or a cyanine dye, including but not limited to FAMTM dye and VIC® dye, as exemplary dyes.
In another embodiment, disclosed is a kit for the detection of E. coli O157:H7 in a sample comprising a first pair of PCR primers selected from the group consisting of: SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQ ID NO:1 and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6; b) a second pair of PCR primers selected from SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ ID NO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ ID NO:49-50, SEQ ID NO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ ID NO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ ID NO:82-83, SEQ ID NO:85-86, and SEQ ID NO:88-89; c) a polymerase; and optionally a first probe of SEQ ID NO:3 for use with selected said first pair of PCR primers and a second probe according to FIG. 3 for use with selected said second pair of PCR primers selected from SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:90. The first probe further comprises a first label and said second probe further comprises a second label, both labels are selected from a dye, a radioactive isotope, a chemiluminescent label, and an enzyme, the dye comprises a fluorescein dye, a rhodamine dye, or a cyanine dye, and the first probe is labeled with FAMTM dye and said second probe is labeled with VIC® dye.
In the following description, certain aspects and embodiments will become evident. It should be understood that a given embodiment need not have all aspects and features described herein. It should be understood that these aspects and embodiments are merely exemplary and explanatory and are not restrictive of the invention.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several exemplary embodiments of the disclosure and together with the description, serve to explain certain teachings.
There still exists a need for improved assays and methods for detecting and differentiating pathogenic organisms from non-pathogenic organisms which can be complicated by globally regional differences as well as by the need for improved sensitivity and specificity of detection.
These and other features of the present teachings are set forth herein.



DESCRIPTION OF THE DRAWINGS
The skilled artisan will understand that the drawings described below are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
FIG. 1 illustrates primer and probe sets useful in detecting SEQ ID NO:111.
FIG. 2 illustrates TaqMan® assay results for assays designed from SEQ ID NO:1-6 to detect SEQ ID NO:111, in accordance with various embodiments.
FIG. 3 illustrates a seven by nucleotide insertion unique to SEQ ID NO:111.
FIG. 4 illustrates primer and probe sets useful in detecting SEQ ID NO:90-110.
FIG. 5 illustrates the results of assays used for the specific detection of E. coli O157:H7 and not O55:H7.



DETAILED DESCRIPTION
For the purposes of interpreting of this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” include plural referents unless expressly and unequivocally limited to one referent. The use of “or” means “and/or” unless stated otherwise. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of” and/or “consisting of.”
DEFINITIONS
As used herein, the phrase “nucleic acid,” “oligonucleotide”, and polynucleotide(s)” are interchangeable and not intended to be limiting.
Reference will now be made to various embodiments, examples of which are illustrated in the accompanying drawings.
As used herein, the phrase “stringent hybridization conditions” refers to hybridization conditions which can take place under a number of pH, salt and temperature conditions. The pH can vary from 6 to 9, preferably 6.8 to 8.5. The salt concentration can vary from 0.15 M sodium to 0.9 M sodium, and other cations can be used as long as the ionic strength is equivalent to that specified for sodium. The temperature of the hybridization reaction can vary from 30° C. to 80° C., preferably from 45° C. to 70° C. Additionally, other compounds can be added to a hybridization reaction to promote specific hybridization at lower temperatures, such as at or approaching room temperature. Among the compounds contemplated for lowering the temperature requirements is formamide. Thus, a polynucleotide is typically “substantially complementary” to a second polynucleotide if hybridization occurs between the polynucleotide and the second polynucleotide. As used herein, “specific hybridization” refers to hybridization between two polynucleotides under stringent hybridization conditions.
As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxynucleotides, or peptide nucleic acids (PNA), and includes both double- and single-stranded RNA, DNA, and PNA. A polynucleotide may include nucleotide sequences having different functions, including, for instance, coding regions, and non-coding regions such as regulatory regions. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. An “oligonucleotide” refers to a polynucleotide of the present invention, typically a primer and/or a probe.
As used herein a “target-specific polynucleotide” refers to a polynucleotide having a target-binding segment that is perfectly or substantially complementary to a target sequence, such that the polynucleotide binds specifically to an intended target without significant binding to non-target sequences under sufficiently stringent hybridization conditions. The target-specific polynucleotide can be e.g., a primer or probe and the subject of hybridization with its complementary target sequence.
The term “target sequence”, “target nucleic acid”, “target” or “target polynucleotide sequence” refers to a nucleic acid of interest. The target sequence can be a polynucleotide sequence that is the subject of hybridization with a complementary polynucleotide, e.g. a primer or probe. The target sequence can be composed of DNA, RNA, an analog thereof, and including combinations thereof. The target sequence may be known or not known, in terms of its actual sequence and its amplification can be desired. The target sequence may or may not be of biological significance. Typically, though not always, it is the significance of the target sequence which is being studied in a particular experiment. As non-limiting examples, target sequences may include regions of genomic DNA, regions of genomic DNA which are believed to contain one or more polymorphic sites, DNA encoding or believed to encode genes or portions of genes of known or unknown function, DNA encoding or believed to encode proteins or portions of proteins of known or unknown function, DNA encoding or believed to encode regulatory regions such as promoter sequences, splicing signals, polyadenylation signals, etc.
As used herein an “amplified target polynucleotide sequence product” refers to the resulting amplicon from an amplification reaction such as a polymerase chain reaction. The resulting amplicon product arises from hybridization of complementary primers to a target polynucleotide sequence under suitable hybridization conditions and the repeating in a cyclic manner the polymerase chain reaction as catalyzed by DNA polymerase for DNA amplification or RNA polymerase for RNA amplification.
As used herein, the “polymerase chain reaction” or PCR is a an amplification of nucleic acid consisting of an initial denaturation step which separates the strands of a double stranded nucleic acid sample, followed by repetition of (i) an annealing step, which allows amplification primers to anneal specifically to positions flanking a target sequence; (ii) an extension step which extends the primers in a 5′ to 3′ direction thereby forming an amplicon polynucleotide complementary to the target sequence, and (iii) a denaturation step which causes the separation of the amplicon from the target sequence (Mullis et al., eds, The Polymerase Chain Reaction, BirkHauser, Boston, Mass. (1994). Each of the above steps may be conducted at a different temperature, preferably using an automated thermocycler (Applied Biosystems LLC, a division of Life Technologies Corporation, Foster City, Calif.). If desired, RNA samples can be converted to DNA/RNA heteroduplexes or to duplex cDNA by methods known to one of skill in the art.
As used herein, “amplifying” and “amplification” refers to a broad range of techniques for increasing polynucleotide sequences, either linearly or exponentially. Exemplary amplification techniques include, but are not limited to, PCR or any other method employing a primer extension step. Other nonlimiting examples of amplification include, but are not limited to, ligase detection reaction (LDR) and ligase chain reaction (LCR). Amplification methods may comprise thermal-cycling or may be performed isothermally. In various embodiments, the term “amplification product” includes products from any number of cycles of amplification reactions.
In certain embodiments, amplification methods comprise at least one cycle of amplification, for example, but not limited to, the sequential procedures of: hybridizing primers to primer-specific portions of target sequence or amplification products from any number of cycles of an amplification reaction; synthesizing a strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated.
Descriptions of certain amplification techniques can be found, among other places, in H. Ehrlich et al., Science, 252:1643-50 (1991), M. Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, N.Y. (1990), R. Favis et al., Nature Biotechnology 18:561-64 (2000), and H. F. Rabenau et al., Infection 28:97-102 (2000); Sambrook and Russell, Molecular Cloning, Third Edition, Cold Spring Harbor Press (2000) (hereinafter “Sambrook and Russell”), Ausubel et al., Current Protocols in Molecular Biology (1993) including supplements through September 2005, John Wiley & Sons (hereinafter “Ausubel et al.”).
The term “label” refers to any moiety which can be attached to a molecule and: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g. FRET; (iii) stabilizes hybridization, i.e. duplex formation; or (iv) provides a capture moiety, i.e. affinity, antibody/antigen, ionic complexation. Labelling can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods. Labels include light-emitting compounds which generate a detectable signal by fluorescence, chemiluminescence, or bioluminescence (Kricka, L. in Nonisotopic DNA Probe Techniques (1992), Academic Press, San Diego, pp. 3-28). Another class of labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g. intercalators, minor-groove binders, and cross-linking functional groups (Blackburn, G. and Gait, M. Eds. “DNA and RNA structure” in Nucleic Acids in Chemistry and Biology, 2.sup.nd Edition, (1996) Oxford University Press, pp. 15-81). Yet another class of labels effect the separation or immobilization of a molecule by specific or non-specific capture, for example biotin, digoxigenin, and other haptens (Andrus, A. “Chemical methods for 5′ non-isotopic labelling of PCR probes and primers” (1995) in PCR 2: A Practical Approach, Oxford University Press, Oxford, pp. 39-54).
The terms “annealing” and “hybridization” are used interchangeably and mean the base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex or other higher-ordered structure. The primary interaction is base specific, i.e. A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.
The term “end-point analysis” refers to a method where data collection occurs only when a reaction is substantially complete.
The term “real-time analysis” refers to periodic monitoring during PCR. Certain systems such as the ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.) conduct monitoring during each thermal cycle at a pre-determined or user-defined point. Real-time analysis of PCR with FRET probes measures fluorescent dye signal changes from cycle-to-cycle, preferably minus any internal control signals.
The term “quenching” refers to a decrease in fluorescence of a first moiety (reporter dye) caused by a second moiety (quencher) regardless of the mechanism.
A “primer,” as used herein, is an oligonucleotide that is complementary to a portion of target polynucleotide and, after hybridization to the target polynucleotide, may serve as a starting-point for an amplification reaction and the synthesis of an amplification product. Primers include, but are not limited to, spanning primers. A “primer pair” refers to two primers that can be used together for an amplification reaction. A “PCR primer” refers to a primer in a set of at least two primers that are capable of exponentially amplifying a target nucleic acid sequence in the polymerase chain reaction.
The term “probe” comprises a polynucleotide that comprises a specific portion designed to hybridize in a sequence-specific manner with a complementary region of a specific nucleic acid sequence, e.g., a target nucleic acid sequence. In certain embodiments, the specific portion of the probe may be specific for a particular sequence, or alternatively, may be degenerate, e.g., specific for a set of sequences. In certain embodiments, the probe is labeled. The probe can be an oligonucleotide that is complementary to at least a portion of an amplification product formed using two primers.
The terms “complement” and “complementary” as used herein, refer to the ability of two single stranded polynucleotides (for instance, a primer and a target polynucleotide) to base pair with each other, where an adenine on one strand of a polynucleotide will base pair to a thymine or uracil on a strand of a second polynucleotide and a cytosine on one strand of a polynucleotide will base pair to a guanine on a strand of a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For instance, 5′-ATGC and 5′-GCAT are complementary.
A “label” refers to a moiety attached (covalently or non-covalently), or capable of being attached, to an oligonucleotide, which provides or is capable of providing information about the oligonucleotide (e.g., descriptive or identifying information about the oligonucleotide) or another polynucleotide with which the labeled oligonucleotide interacts (e.g., hybridizes). Labels can be used to provide a detectable (and optionally quantifiable) signal. Labels can also be used to attach an oligonucleotide to a surface.
A “fluorophore” is a moiety that can emit light of a particular wavelength following absorbance of light of shorter wavelength. The wavelength of the light emitted by a particular fluorophore is characteristic of that fluorophore. Thus, a particular fluorophore can be detected by detecting light of an appropriate wavelength following excitation of the fluorophore with light of shorter wavelength.
The term “quencher” as used herein refers to a moiety that absorbs energy emitted from a fluorophore, or otherwise interferes with the ability of the fluorescent dye to emit light. A quencher can re-emit the energy absorbed from a fluorophore in a signal characteristic for that quencher, and thus a quencher can also act as a fluorophore (a fluorescent quencher). This phenomenon is generally known as fluorescent resonance energy transfer (FRET). Alternatively, a quencher can dissipate the energy absorbed from a fluorophore as heat (a non-fluorescent quencher).
As used herein, a “sample” refers to any substance comprising nucleic acid material. A sample to be tested and can include food intended for human or animal consumption such as meat, nuts, legumes, fruit, and vegetables, a beverage sample, a fermentation broth, a forensic sample, an environmental sample (e.g., soil, dirt, garbage, sewage, air, or water), including food processing and manufacturing surfaces, or a biological sample. A “biological sample” refers to a sample obtained from eukaryotic or prokaryotic sources. Examples of eukaryotic sources include mammals, such as a human or a cow or a member of the family Muridae (a murine animal such as rat or mouse). Alternatively, the sample may include blood, urine, feces, or other materials from a human or a livestock animal. The sample may be tested directly, or may be treated in some manner prior to testing. For example, the sample may be subjected to PCR amplification using appropriate oligonucleotide primers. Examples of prokaryotic sources include enterococci. The biological sample can be, for instance, in the form of a single cell, in the form of a tissue, or in the form of a fluid.
As used herein, “detecting” or “detection” refers to the disclosure or revelation of the presence or absence in a sample of a target polynucleotide sequence or amplified target polynucleotide sequence product. The detecting can be by end point, real-time, enzymatic, and by resolving the amplification product on a gel and determining whether the expected amplification product is present, or other methods known to one of skill in the art.
The presence or absence of an amplified product can be determined or its amount measured. Detecting an amplified product can be conducted by standard methods well known in the art and used routinely. The detecting may occur, for instance, after multiple amplification cycles have been run (typically referred to an end-point analysis), or during each amplification cycle (typically referred to as real-time). Detecting an amplification product after multiple amplification cycles have been run is easily accomplished by, for instance, resolving the amplification product on a gel and determining whether the expected amplification product is present. In order to facilitate real-time detection or quantification of the amplification products, one or more of the primers and/or probes used in the amplification reaction can be labeled, and various formats are available for generating a detectable signal that indicates an amplification product is present. For example, a convenient label is typically a label that is fluorescent, which may be used in various formats including, but are not limited to, the use of donor fluorophore labels, acceptor fluorophore labels, fluorophores, quenchers, and combinations thereof. Assays using these various formats may include the use of one or more primers that are labeled (for instance, scorpions primers, amplifluor primers), one or more probes that are labeled (for instance, adjacent probes, TaqMan® probes, light-up probes, molecular beacons), or a combination thereof. The skilled person will understand that in addition to these known formats, new types of formats are routinely disclosed. The present invention is not limited by the type of method or the types of probes and/or primers used to detect an amplified product. Using appropriate labels (for example, different fluorophores) it is possible to combine (multiplex) the results of several different primer pairs (and, optionally, probes if they are present) in a single reaction. As an alternative to detection using a labeled primer and/or probe, an amplification product can be detected using a polynucleotide binding dye such as a fluorescent DNA binding dye. Examples include, for instance, SYBR® Green dye or SYBR® Gold dye (Molecular Probes). Upon interaction with the double-stranded amplification product, such polynucleotide binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength. A polynucleotide binding dye such as a polynucleotide intercalating dye also can be used.
As used herein, an “E. coli O157:H7-specific nucleotide probe” refers to a sequence that is able to specifically hybridize to an E. coli O157:H7 target sequence present in a sample containing E. coli O157:H7 under suitable hybridization conditions and which does not hybridize with DNA from other E. coli strains or from other bacterial species. It is well within the ability of one skilled in the art to determine suitable hybridization conditions based on probe length, G+C content, and the degree of stringency required for a particular application.
It is expected that minor sequence variations in E. coli O157:H7-specific nucleotide sequences associated with nucleotide additions, deletions and mutations, whether naturally occurring or introduced in vitro, would not interfere with the usefulness of SEQ ID NO:1-111 in the detection of enterohemorrhagic E. coli (EHEC), in methods for preventing EHEC infection, and in methods for treating EHEC infection, as would be understood by one of skill in the art. Therefore, the scope of the present invention as claimed is intended to encompass minor variations in the sequences of SEQ ID NO:1-111 and sequences having at least 90% homology to the SEQ ID NO:1-111 sequences.
The probe may be RNA or DNA. Depending on the detection means employed, the probe may be unlabeled, radiolabeled, chemiluminescent labeled, enzyme labeled, or labeled with a dye. The probe may be hybridized with a sample in solution or immobilized on a solid support such as nitrocellulose, a microarray or a nylon membrane, or the probe may be immobilized on a solid support, such as a silicon chip or a microarray.
Conditions that “allow” an event to occur or conditions that are “suitable” for an event to occur, such as hybridization, strand extension, and the like, or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. Such conditions, known in the art and described herein, may depend upon, for example, the nature of the nucleotide sequence, temperature, and buffer conditions. These conditions may also depend on what event is desired, such as hybridization, cleavage, or strand extension. An “isolated” polynucleotide refers to a polynucleotide that has been removed from its natural environment. A “purified” polynucleotide is one that is at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
There are many known methods of amplifying nucleic acid sequences including e.g., PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188 and 5,333,675 each of which is incorporated herein by reference in their entireties for all purposes.
Nucleic acid amplification techniques are traditionally classified according to the temperature requirements of the amplification process. Isothermal amplifications are conducted at a constant temperature, in contrast to amplifications that require cycling between high and low temperatures. Examples of isothermal amplification techniques are: Strand Displacement Amplification (SDA; Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392 396; Walker et al., 1992, Nuc. Acids. Res. 20:1691 1696; and EP 0 497 272, all of which are incorporated herein by reference), self-sustained sequence replication (3SR; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874 1878), the Q.beta. replicase system (Lizardi et al., 1988, BioTechnology 6:1197 1202), and the techniques disclosed in WO 90/10064 and WO 91/03573.
Examples of techniques that require temperature cycling are: polymerase chain reaction (PCR; Saiki et al., 1985, Science 230:1350 1354), ligase chain reaction (LCR; Wu et al., 1989, Genomics 4:560 569; Barringer et al., 1990, Gene 89:117 122; Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189 193), transcription-based amplification (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173 1177) and restriction amplification (U.S. Pat. No. 5,102,784).
Other exemplary techniques include Nucleic Acid Sequence-Based Amplification (“NASBA”; see U.S. Pat. No. 5,130,238), Q.beta. replicase system (see Lizardi et al., BioTechnology 6:1197 (1988)), and Rolling Circle Amplification (see Lizardi et al., Nat Genet. 19:225 232 (1998)). The amplification primers of the present invention may be used to carry out, for example, but not limited to, PCR, SDA or tSDA. Any of the amplification techniques and methods disclosed herein can be used to practice the claimed invention as would be understood by one of ordinary skill in the art.
PCR is an extremely powerful technique for amplifying specific polynucleotide sequences, including genomic DNA, single-stranded cDNA, and mRNA among others. Various methods of conducting PCR amplification and primer design and construction for PCR amplification will be known to those of skill in the art. Generally, in PCR a double-stranded DNA to be amplified is denatured by heating the sample. New DNA synthesis is then primed by hybridizing primers to the target sequence in the presence of DNA polymerase and excess dNTPs. In subsequent cycles, the primers hybridize to the newly synthesized DNA to produce discreet products with the primer sequences at either end. The products accumulate exponentially with each successive round of amplification.
The DNA polymerase used in PCR is often a thermostable polymerase. This allows the enzyme to continue functioning after repeated cycles of heating necessary to denature the double-stranded DNA. Polymerases that are useful for PCR include, for example, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tma DNA polymerase, Tli DNA polymerase, and Pfu DNA polymerase. There are many commercially available modified forms of these enzymes including: AmpliTaq® and AmpliTaq Gold® both available from Applied Biosystems. Many are available with or without a 3- to 5′ proofreading exonuclease activity. See, for example, Vent® and Vent®. (exo-) available from New England Biolabs.
Other suitable amplification methods include the ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989) and Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)) and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603). The latter two amplification methods include isothermal reactions based on isothermal transcription, which produce both single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
Amplicon Selection:
Detection of E. coli O157:H7 by the use of the polymerase chain reaction provides a rapid method for detection. Moreover, the primer(s) (and probe used in a real-time PCR reaction) is desired to be specific and sensitive for only the organism of interest, i.e., E. coli O157:H7. However, the genome of E. coli O157:H7 is very similar to other E. coli genomes, both those which are disease causing (i.e., infectious) and pathogenic (ability to cause damage) e.g., serotype O55:H7 and those which are not pathogenic, e.g., serotype K12. Therefore, in one embodiment, the identification and selection of genomic sequence from E. coli O157:H7 (e.g., strain EDL993, GenBank Accession No. AE005174, Perna, N. T., et al., (2001) Nature 409(25):529-533) for the design of real-time PCR assays is based on the differential identification of E. coli O157:H7 genomic sequence (I, inclusion set) not found in closely related strains of E. coli (E, exclusion set). By identifying sequences not found in both infections and non-infectious strains of E. coli, target sequences specific to E. coli O157:H7 can be identified for primer design that do not, or only with rare exception, detect closely related E. coli strains and therefore identify E. coli O157:H7 with specificity and sensitivity.
Identification of E. coli O157:H7 Unique Sequence Regions:
The E. coli O157:H7 strain EDL933 genome has about 5.6 million base pairs (Mb) of DNA in the backbone sequence. When compared to a common, non-pathogenic laboratory strain, E. coli K12 strain MG1655, having about 4.6 Mb of DNA. Each genome has regions of DNA unique to one strain or the other, numbering in the hundreds of regions.
Prior to the claimed invention ‘O-islands’ where described as nucleic acid sequence regions unique to and found only in the O157:H7 serotype. O-islands regions total 1.34 megabases and 1,387 genes whereas ‘K-islands’ are described as being unique to serotype K12, totaling 0.53 megabases and 528 genes. (Perna, N. T., et al., supra). The design of assays specific for the O157:H7 serotype as described herein refutes the uniqueness of O-islands to only the O157:H7 serotype. Comparison of the genome of O157:H7 with that of O55:H7 unexpectedly revealed that the O55:H7 serotype also contained O-islands virtually identical to those of O157:H7. Therefore, prior to the claimed invention, a definitive identification of O157:H7 was difficult due to genome sequence similarity, even in supposedly serotype specific regions. The present invention as claimed has identified serotype unique DNA sequences for E. coli O157:H7 which were utilized for assay design and the subsequent detection of E. coli O157:H7 and not O55:H7 by PCR, hybridization and other molecular biology techniques as known to one skilled in the art.
The sequence of the E. coli O157:H7 genome is represented in public databases such as GenBank (NCBI, National Library of Medicine, The National Institutes of Health, Bethesda, Md.) EMBL, and DDBJ. One of skill in the art quickly recognizes that the genomic sequence of O157:H7 serotype is representative of a variety of non-O157:H7, yet pathogenic serotypes such as O111:H7, O26:H11 and O103:H2. A bioinformatic approach to identify E. coli O157:H7 unique sequence regions was undertaken and evaluated by Sanger sequencing and real-time PCR assays.
In one embodiment of the current teachings bioinformatic and direct DNA sequencing comparisons were conducted in an effort to identify E. coli O157:H7 serotype-specific sequences. 28 separate E. coli O157:H7 serotype EDL933 (GenBank Acc No. AE005174) O-island sequence regions were downloaded from the NCBI database GenBank, release 160). These O-islands were known to be present, absent in some or variants of O-islands in E. coli O157:H7. Additional O-island and K-island regions were sequenced by the Sanger/capillary electrophoresis method known to one of skill in the art from the Applied Biosystems microbial DNA collection of E. coli serotypes O157:H7, K12 and others. Alignment of the sequenced regions using algorithms known to one of ordinary skill in the art identified no O157:H7 “unique” regions. PCR primer pairs were designed for each of the 28 regions to specifically amplify the unique sequences within the O-islands against both inclusion (organism to be detected, i.e., E. coli O157:H7) and exclusion genomes (organisms not to be detected, i.e., E. coli non-O157:H7 serotypes and Shigella spp.) within the Applied Biosystems microbial DNA collection. The resulting amplification products were then sequenced and aligned to the EDL933 genome. Comparison of the alignments of the O157:H7 specific regions to non-O157:H7 sequences identified an E. coli O157:H7 sequence region (SEQ ID NO:111) that was used for the identification of E. coli O157:H7 antigen specific strains. PCR primer pairs (and probes for use in real-time PCR assays) were designed to the O157:H7 specific region and screened against ground beef. Any assay having a positive result indicated the assay cross-reacted with ground beef and was removed from further analyses. As shown in FIG. 1, the remaining PCR primer pairs and probes (SEQ ID NOs:1-6) were used in real-time PCR assays and evaluated against the Applied Biosystems microbial DNA collection (FIG. 2). For example, primer pairs SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQ ID NO:1 and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6 can be used for end-point analysis and when used in conjunction with SEQ ID NO:3 as the probe comprised the real-time PCR assays as listed in FIG. 1.
The primers and probes from FIG. 1 were used in real-time PCR assays on DNA extracts from various strains from the Applied Biosystems microbial DNA collection and have experimentally demonstrated the specificity of each assay for identification of E. coli O157:H7 and a weak signal for E. coli O55:H7 (FIG. 2), an unexpected result. A Ct value >35 was consider a “weak positive” result. When a Ct value is present a signal was detected, a positive result and ‘no signal’ indicates no detectable signal, and so a negative result. In all selected serotypes shown, the internal positive control had a detectable signal/positive result (data not shown).
As shown in FIG. 3, this region of the O157:H7 genome has a seven basepair insertion (a direct repeat) that is not present in O55:H7. This seven basepair insertion in O157:H7 relative to O55:H7 is the only significant sequence difference found between the two serotypes in SEQ ID NO:111. The forward primer of assay 18158 overlaps this insertion, which provides some specificity for O157:H7. Analysis of GenBank sequences for the SEQ ID NO:111 region indicted that the full-length sequence of SEQ ID 111 is found only in E. coli O157:H7, Shigella sonnei, and Shigella boydii. In all cases except O157:H7, the seven nucleotide O157:H7-specific insertion is absent. Other fragments of SEQ ID 111 that are more widely conserved among E. coli are nucleotide sequence positions 355 to 456, and 156 to 238. The 355-456 fragment overlaps the reverse primer of assay 18158, and partially overlaps the probe, but not the forward primer, which can account for the 18158 assay not detecting more serotypes.
These assays have been shown to be very specific for the rapid detection of E. coli O157:H7 and weakly positive for E. coli O55:H7 from isolated DNA and from ground beef samples. Experimental results confirmed that these assays have 100% sequence identity to the O157:H7 genome. Bioinformatic evidence indicated that the assays were specific for O157:H7 EDL933 and O157:H7 Sakai genomes, GenBank Accession Nos. AE005174 and NC—002695.1, respectively.
In another embodiment of the current teachings bioinformatic and direct DNA sequencing comparisons were conducted in an effort to identify sequences common to E. coli O157:H7 strains but absent or highly divergent in E. coli strains within the exclusion set. 14 E. coli O157:H7 genome sequences and 30 non-O157:H7 E. coli and Shigella spp. were downloaded from public databases (NCBI database GenBank, release 161.0, or the Sanger Center's public FTP site (Wellcome Trust Sanger Institute). The bioinformatic approach entailed aligning each of the 45 sequences against an E. coli O157:H7 reference genome (GenBank Accession No. NC—002695.1). The alignments and parsed results from the initial genome comparisons resulted in identifying 157 O157:H7 unique regions. These regions were then analyzed by BLASTN against the GenBank non-redundant database (nr). Sequences with at least 80% similarity over 50 or more contiguous nucleotides were removed from further analysis. Of those removed about two thirds of hits with more than 85% sequence identity were from strains of E. coli, Shigella or E. fergusonii for which whole genome sequence was not available. The remain 1⁄3 were to Enterobacter, Salmonella or Erwina. The resulting set of 118 O157:H7 unique regions were considered target sequences totaling 117 kb. Assays were designed from unique E. coli O157:H7 sequence regions which were identified by alignment of genomes.
The nearest neighbor of E. coli O157:H7 is E. coli O55:H7. Our sequencing in E. coli O55:H7 strains for known E. coli O157:H7 O-islands further supported the close relationship between the two serotypes as most O-islands were conserved between the two serotypes. Further genomic sequencing of the O55:H7 genome (PE704 isolate) in parallel with the sequencing of an O157:H7 strain (PE30 isolate) also supported the very close relationship of the two serotypes as single nucleotide polymorphisms (SNPs) in O55:H7 verse O157:H7 was only 0.28% (data not shown).
The 118 target sequences for O157:H7 were screened against the O55:H7 consensus genome assembly using BLASTN. Only 9.9 kb or 8.5% of the candidate O157:H7-specific sequence identified by genome comparison was found to be absent from the O55:H7 genome. 20 target sequences were found to be absent from the O55:H7 genome and all 20 had at least 98.85% sequence identity between any pair of the 15 publicly available O157:H7 genomes indicating each target sequence was highly conserved and assays designed from these regions would be highly indicative of E. coli O157:H7 strains and not O55:H7.
The 20 target sequences corresponded to seven regions identified in U.S. Pat. No. 6,365,723, incorporated herein by reference in its entirety. As indicated in Table 1, these regions are associated with CP933-M,N,X and P, cryptic prophages, each having a collection of open reading frames, and fimbrial proteins. For example, primer pairs SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ ID NO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ ID NO:49-50, SEQ ID NO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ ID NO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ ID NO:82-83, SEQ ID NO:85-86, and SEQ ID NO:88-89 can be used for end-point analysis and when used in conjunction with a probe of SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, or SEQ ID NO:90 as listed in FIG. 4, comprise real-time PCR assays. These 20 target sequences (SEQ ID NO:91-110) were used in the design of O157:H7-specific real-time PCR assays using algorithms known by one of skill in the art (FIG. 4). The 29 assays map to the seven regions as indicated in Table 1.







TABLE 1







E. coli O157:H7-specific genomic regionsa












Region
EDL933
EDL933
Length




number
start
end
(nt)
ORFs
Features















1
1255352
1256121
770
Z1328, Z1329
CP933-M


2
1256573
1257839
1267
Z1332-Z1334
CP933-M


3
1262147
1263665
1519
Z1341
CP933-M


 4*
1635627
1635757
131
Z1781
CP933-N


5
1744789
1747582
2794
Z1921-Z1924
CP933-X


 6*
2319451
2319578
128
Z6065
CP933-P


7
2933425
2934070
646
Z3276
Fimbrial







protein





aas mapped to GenBank Acc. No. AE005174


*Regions 4 and 6 are duplicate copies of the same sequence.






Real-time PCR assays were designed from seven unique and specific E. coli O157:H7 sequence regions. The assays were then tested against the Applied Biosystems microbial DNA collection of E. coli strains and serotypes to confirm the specificity, sensitivity and unambiguous detection of only E. coli O157:H7 nucleic acid. However, unexpectedly, the selected assays also amplified nucleic acid from one strain of E. coli O103:H2 serotype and only one of four strains of the E. coli O26:H11 serotype. Subsequent testing revealed that the positive results for only one E. coli O26:H11 sample indicated mistyping of the sample.
In one embodiment, combining the results of an assay that detects a target polynucleotide sequence of SEQ ID NO:111 with the results of an assay that detects a target polynucleotide sequence selected from SEQ ID NO:91-110 provides specific and definitive detection for E. coli O157:H7 and not O55:H7. As shown in Table 2, the combination of assay Nos. 18158 and 19055 whether in single or duplex assay format provides an unambiguous positive test for E. coli O157:H7, i.e., a positive test for O157:H7 in each assay confirms a positive test for E. coli O157:H7.












TABLE 2








Assay No.
Assay No.
Interpreted



Serotype
18158
19055
result









O157:H7 (all)
+
+
Positive



O55:H7 (all)
weak+
−
−



O103:H2 (1/1)
−
+
−



O26:H11 (1/4)
−
+
−










Clearly, neither assay alone is definitive for a single serotype of E. coli due to genomic similarity between the genomic regions of other E. coli serotypes. Yet, when two assays, as example, but not limited to the assays of Table 2, are used either in parallel or as a multiplex assay, e.g., in a real-time TaqMan® assay, for example, where each probe in each of the two assays has a different label for distinguishing results on a real-time PCR instrument, e.g., a 7500 Fast Real-Time PCR System (Applied Biosystems), a positive result from each assay is indicative of only E. coli O157:H7. Thus, without knowledge of genomic regions shared by serotype O157:H7 and O55:H7, design of an unambiguous, specific and sensitive test for E. coli O157:H7 would not be possible.
The claimed method to identify E. coli O157:H7 results from a two-pronged approach to identify target sequences for O157:H7 that do not also detect closely related either pathogenic or non-pathogenic E. coli. The identified E. coli O157:H7 target sequences were used to design primers and probes for real-time PCR assays. Programs known to one of skill in the art for assay design include Primer3 (Steve Rozen and Helen J. Skaletsky (2000) “Primer3” on the World Wide Web for general users and for biologist programmers as published in: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386), Primer Express® software (Applied Biosystems), and OLIGO 7 (Wojciech Rychlik (2007). “OLIGO 7 Primer Analysis Software”. Methods Mol. Biol. 402: 35-60). The subsequently designed PCR primers and probes for use in assays by real-time PCR detected unambiguously, specifically and with great sensitivity E. coli O157:H7 and not O55:H7.
An assay from FIG. 2 along with an assay from FIG. 3 either in the same reaction vessel as a duplex (multiplex) assay or in separate reaction vessels have experimentally demonstrated the specificity of the dual-assay approach for identification of E. coli O157:H7 and the ability to distinguish O157:H7 from E. coli O55:H7 (Example 2). The results were also confirmed by sequencing various isolates of both serotypes O157:H7 and O55:H7. As indicated in FIG. 5, the results of real-time PCR analyses on DNA extracts from various matrix samples and various strains from the Applied Biosystems microbial DNA collection. When a Ct value is present a signal was detected, a positive result and ‘no result’ indicates no signal detected, a negative result. A Ct value >35 was determined to be a “weak positive”. Both assays having a detected signal for O157:H7 were a positive identification of E. coli O157:H7. When at least one or both assays have no signal for O157:H7, the result is considered negative for E. coli O157:H7. In all selected serotypes shown, the internal positive control had a detectable signal/positive result.
The rare signal detected from a serotype other than E. coli O157:H7 was unexpected for the dual/multiplex assay. The likelihood of the simultaneous presence of a rare serotype being mistaken for E. coli O157:H7 is extremely remote. Requiring a positive result for each real-time assay as seen by a different detectable label on each probe for discrimination between each assay's amplification reaction provides a result that would be understood by the skilled artisan to be unambiguous, specific and sensitive for the detection of E. coli O157:H7.
The dual or multiplex (more than 2 assay sets) assay approach can be used to detect and distinguish other regional pathogenic E. coli. EHEC is identified as being caused by two classes of E. coli. Class 1 is predominant in the United States, caused by pathogenic strains of E. coli O157:H7 and Class 2 is more geographically dispersed and is caused by E. coli O26 and E. coli O111. As provided in Table 3, depending on the local beef population's indigenous E. coli and possible imported beef source, various pathogenic strains of E. coli are a public health threat.











TABLE 3







Country/Continent
E. coli Serotye (s)
Class









United States
O157:H7
1



South America
O26 and O111
2



Scotland
O26 and O111
2



Germany
O157:H7 and O26
1 & 2



Australia
O26 and O111
2










The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
The materials for use in the present invention are ideally suited for the preparation of a kit suitable for identifying the presence of E. coli O157:H7 and not E. coli O55:H7. Such a kit may comprise various reagents utilized in the methods, preferably in concentrated form. The reagents of this kit may comprise, but are not limited to, buffer, appropriate nucleotide triphosphates, DNA polymerases, intercalating dye, primers, probes, salt, and instructions for the use of the kit.
Those having ordinary skill in the art will understand that many modifications, alternatives, and equivalents are possible. All such modifications, alternatives, and equivalents are intended to be encompassed herein.
EXAMPLES
The following procedures are representative of procedures that can be employed for the detection of E. coli O157:H7.
Example 1
PCR Reaction Conditions to Evaluate O-Island Assays
All amplifications are done with “TrueAllele” PCR Master Mix
Primer pairs prepped at a [5 μM] each
Template is 1 μL, of a 1→100 dilution of the bacterial lysate
Reaction Mix Preparation:














Per reaction
for 50 reactions




















True Allele MM
18 μl 
900 μl



Primer mix 5 μM
2 μl
100 μl



ddH2O
9 μl
450 μl







vortex briefly to mix components






Plate Preparation:

    
    
        Pipette 1 μl of template from the template plate to reaction plate conserving well position.
        Add 29 μl of Reaction Mix
        Seal with adhesive cover, staking cover very well
        Vortex briefly to mix components
        Spin down plates and thermalcycle
    
    


Thermal cycler profile: 95° C. 10′→95° C. 15′′−60° C. 60′′ (×30 cycles)→4° C.
Example 2
Sequencing Reaction Protocol
Sequencing Reaction Mix:














Per reaction
for 100 reactions




















5x sequencing buffer
3.2
320



BDT v.3.1 RR mix
1.6
160



Seq. primer 1 μM
1.0
100



ddH2O
7.2
720



total
13
1300










    1. Add 13 μl of above reaction mix to 7 μl of Exo-SAPped PCR amplicon, vortex, spin down and thermal cycle


Dye Exterminator Clean-Up of Sequencing Reactions Protocol


    1. Prepare Dye Exterminator Mix:
    
        82% SAM buffer
        18% Dye Exterminators beads (agitate before pipetting)
    
    


For 96 sequences prepare 10 ml: 8.2 ml of SAM+1.8 ml of beads

    2. For each 10 μl of sequencing reactions add 50 μl of Dye Exterminator mix: to each 20 μl reaction add 100 μl
    3. Heat seal plates at 160° C. for 2 seconds
    4. Vortex at  ̃1800 rpm for 30 minutes
    5. Spin down in centrifuge at  ̃1000 rpm for 2 minutes
    6. Load plates on sequencer and make sure to use appropriate Dye Exterminator module for injection


While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be appreciated by one skilled in the art from reading this disclosure that various changes in form and detail can be made without departing from the spirit and scope of the invention. These methods are not limited to any particular type of nucleic acid sample: plant, bacterial, animal (including human) total genome DNA, RNA, cDNA and the like may be analyzed using some or all of the methods disclosed in this invention. This invention provides a powerful tool for analysis of complex nucleic acid samples. From experiment design to detection of E. coli O157:H7 assay results, the above invention provides for fast, efficient and inexpensive methods for detection of pathogenic E. coli O157:H7.
All publications and patent applications cited above are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application were specifically and individually indicated to be so incorporated by reference. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. A method of detecting the presence of E. coli O157:H7 in a sample, comprising:
a.) detecting the presence of SEQ ID NO:111 or full complements complement thereof; and
b.) detecting the presence of a sequence selected from SEQ ID NO:91-110 and full complements thereof;
wherein detection of SEQ ID NO:111 and detection of a sequence selected from SEQ ID NO:91-110 confirms the presence of E. coli O157:H7 in a sample and not E. coli O55:H7.

2. The method of claim 1, wherein the detection is by a nucleic acid amplification reaction.

3. The method of claim 2, wherein the amplification reaction is an end-point determination.

4. The method of claim 2, wherein the amplification reaction is quantitative.

5. The method of claim 4, wherein the quantification is a real-time PCR.

6. The method of claim 5, wherein the real-time PCR is a SYBR® Green Assay.

7. The method of claim 5, wherein the real-time PCR is a TaqMan® Assay.

8. An assay for the detection of E. coli O157:H7 in a sample comprising:
a) hybridizing a first pair of PCR primers selected from the group consisting of:
SEQ ID NO:1-2, SEQ ID NO:1 and SEQ ID NO:4, SEQ ID NO:1 and SEQ ID NO:5, and SEQ ID NO:1 and SEQ ID NO:6 and full complements thereof to at least a first target polynucleotide sequence;
b) hybridizing a second pair of PCR primers selected from SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ ID NO:40-41, SEQ ID NO:43-44, SEQ ID NO:46-47, SEQ ID NO:49-50, SEQ ID NO:52-53, SEQ ID NO:55-56, SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:61-62, SEQ ID NO:64-65, SEQ ID NO:67-68, SEQ ID NO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ ID NO:82-83, SEQ ID NO:85-86, and SEQ ID NO:88-89 and full complements thereof to at least a second target polynucleotide sequence;
c) amplifying said at least first and said at least second target polynucleotide sequences; and
d) detecting at least a first and at least a second amplified target polynucleotide sequence products;
wherein the detection of the at least first amplified target polynucleotide sequence product and the detection of the at least second amplified target polynucleotide sequence product is indicative of the presence of E. coli O157:H7 in the sample and not E. coli O55:H7.

9. The assay of claim 8, further comprising using a first probe of SEQ ID NO:3 to detect the first amplification product and a second probe selected from SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27 SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:90 to detect the second amplification product.

10. The assay of claim 9 wherein said first probe further comprises a first label and said second probe further comprises a second label.

11. The assay of claim 10, wherein both labels are selected from a dye, a radioactive isotope, a chemiluminescent label, and an enzyme.

12. The assay of claim 11, wherein the dye comprises a fluorescein dye, a rhodamine dye, or a cyanine dye.

13. The assay of claim 12, wherein the dye is a fluorescein dye.

14. The assay of claim 13, wherein said first probe is labeled with FAMTM dye and said second probe is labeled with VIC® dye.

15. The assay of claim 8, further comprising preparing the sample for PCR amplification prior to hybridizing, wherein said preparing comprises at least one of the following processes: (1) bacterial enrichment, (2) separation of bacterial cells from the sample, (3) cell lysis, and (4) total DNA extraction.

16. The assay of claim 8, wherein the sample comprises a food or a water sample.

17. The assay of claim 16, wherein the food sample comprises a selectively enriched food matrix.

18. The assay of claim 8, wherein said amplifying is by polymerase chain reaction.

19. The assay of claim 8, wherein said hybridizing and amplifying of said first pair of PCR primers occurs in a first vessel and said hybridizing and amplifying of said second pair of PCR primers occurs in a second vessel.

20. The assay of claim 8, wherein said hybridizing and amplifying of said first pair of PCR primers and said hybridizing and amplifying of said second pair of PCR primers occurs in a single vessel.

21. The assay of claim 18, wherein said detection is a real-time assay.

22. The assay of claim 21, wherein said real-time assay is a SYBR® Green dye assay.

23. The assay of claim 18, wherein said detection is a TaqMan® assay.