TREA

SEQUENCES AND THEIR USE FOR DETECTION AND CHARACTERIZATION OF ESCHERICHIA COLI SEROTYPE O157:H7

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Information

  • Patent Application
  • 20210324452
  • Publication Number
    20210324452
  • Date Filed
    September 05, 2019
    5 years ago
  • Date Published
    October 21, 2021
    3 years ago
  • Inventors
    • Kalburge; Sai Siddarth (Claymont, DE, US)
    • Wang; YangYang (Bear, DE, US)
    • Padmalayam; Indira (Wilmington, DE, US)
    • Easter; Martin
  • Original Assignees
Information
Abstract
This disclosure relates to a rapid method for detection and characterization of Escherichia coli bacteria serotype O157:H7 based on the presence of nucleic acid sequences, in particular, to a PCR-based method for detection, and to oligonucleotide molecules and reagents and kits useful therefore. This method can be employed to detect E. coli O157:H7 in a food or water sample, such as a beef enrichment. The present disclosure further relates to replication compositions and kits for carrying out methods disclosed herein.
Description
FIELD

The field relates to methods for detection and characterization of Escherichia coli bacteria serotype O157:H7 based on the presence of nucleic acid sequences, for example PCR-based methods for detection, and to oligonucleotide molecules and reagents and kits useful therefor.


BACKGROUND


Escherichia coli (E. coli) is a gram-negative, rod-shaped bacterium. Most E. coli strains are benign and are part of the normal intestinal flora of humans and other animal. However, some strains of E. coli have acquired virulence factors and evolved into pathogens that can cause severe and sometimes-fatal disease. Pathogenic strains of E. coli are categorized based on virulence factors, mechanism of pathogenesis, clinical symptoms and/or serogroups, as defined by the 0 and H antigens.


Enterohemorrhagic E. coli (EHEC) are a category of E. coli strains that produce shiga toxins and cause hemorrhagic colitis or bloody diarrhea that can progress to life-threatening sequalae such as hemolytic uremic syndrome in humans. Of the several pathogenic serotypes of EHEC, the O157:H7 serotype is one of the most frequently isolated EHEC strains from clinical cases.


The E. coli O157:H7 serotype has been associated with several food and water borne outbreaks worldwide. Cattle are the major reservoir of E. coli O157:H7, and it is hence regulated as an adulterant in ground beef by the U.S. Department of Agriculture (USDA) with a zero-tolerance standard. Additionally, E. coli O157:H7 has also been isolated from fecal samples of sheep, goat, pigs and turkeys. Other contaminated foods that have been associated with outbreaks include unpasteurized milk, drinking water, salami, beef jerky, and fresh produce such as lettuce, radish sprouts, fresh spinach, and apple cider.


Considering the ubiquitous nature of E. coli, it is critical to be able to specifically detect the highly pathogenic and tightly regulated E. coli serotype O157:H7 in food and environmental samples, even in the presence of other E. coli serotypes. Published U.S. patent application No. 2011/0020823 and Sharma (Mol. Cell. Probes 20:298-306 (2006)) describe a method for detecting E. coli O157:H7 through simultaneous amplification of two sequences which when amplified together in combination indicate the presence of E. coli O157:H7. However, while E. coli O157:H7 strains are the only group that contain both sequences, there are other strains of E. coli that contain one or the other of the two sequences. In a sample containing a mixture of two different E. coli strains that contain the two sequences, there is a potential to obtain erroneous results that can be falsely interpreted as being positive for the presence of E. coli O157:H7.


Therefore, it is desirable to have an assay that reduces the potential for false-positive results and accurately detects E. coli O157:H7 in a sample using a fast and easy-to-use detection system.


SUMMARY

One aspect is for a method for detecting the presence of E. coli O157:H7 in a sample, said sample comprising nucleic acids, said method comprising:

    • (a) providing a reaction mixture comprising a primer pair selected from group consisting of primer pair SEQ ID NO:1 and SEQ ID NO:2, primer pair SEQ ID NO:3 and SEQ ID NO:4, primer pair SEQ ID NO:5 and SEQ ID NO:6, primer pair SEQ ID NO:7 and SEQ ID NO:8, and a combination thereof;
    • (b) performing PCR amplification of said nucleic acids of said sample using the reaction mixture of step (a); and
    • (c) detecting the amplification of step (b), whereby a positive detection of amplification indicates the presence of E. coli O157:H7 in the sample.


Another aspect is for an isolated polynucleotide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.


A further aspect is for a replication composition for use in performance of PCR, comprising:

    • (a) a primer pair selected from the group consisting of primer pair SEQ ID NO:1 and SEQ ID NO:2, primer pair SEQ ID NO:3 and SEQ ID NO:4, primer pair SEQ ID NO:5 and SEQ ID NO:6, primer pair SEQ ID NO:7 and SEQ ID NO:8, and a combination thereof; and
    • (b) thermostable DNA polymerase.


An additional aspect is for a kit for detection of E. coli O157:H7, inclusive of all serotypes, in a sample, comprising the aforementioned replication composition.


A further aspect is for a tablet comprising the aforementioned replication composition.


Another aspect is for a method for detecting the presence of E. coli O157:H7 in a sample, said sample comprising nucleic acids, said method comprising:

    • (a) providing a reaction mixture comprising a suitable primer pair for amplification of a polynucleotide sequence comprising SEQ ID NO:13;
    • (b) performing PCR amplification of said nucleic acids of said sample using the reaction mixture of step (a); and
    • (c) detecting the amplification of step (b), whereby a positive detection of amplification indicates the presence of E. coli O157:H7 in the sample.


Other objects and advantages will become apparent to those skilled in the art upon reference to the detailed description that hereinafter follows.


SUMMARY OF THE SEQUENCES

SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, and 8 are primer sequences for use in the detection of E. coli O157:H7.


SEQ ID NOs:9, 10, 11, and 12 are probe sequences for use in the detection of E. coli O157:H7. In some embodiments, the probe is 5′-labeled with a fluorescent dye and 3′-termini is composed of a quencher dye. In some embodiments, the 3′ termini are comprised of one of the primers listed above, for example SEQ ID NO:5, a suitable linker moiety, such as a spacer consisting of 6 polyethylene glycol units and a quencher dye.


SEQ ID NO:13 is an Escherichia coli sequence for detection.







DETAILED DESCRIPTION

Applicants specifically incorporate the entire contents of all cited references in this disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range or a list of upper values and lower values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or value and any lower range limit or value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the present disclosure be limited to the specific values recited when defining a range.


Definitions

In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.


As used herein, the term “about” or “approximately” means within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%, or less of a given value or range.


The term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of”. Similarly, the term “consisting essentially of” is intended to include embodiments encompassed by the term “consisting of”.


“Polymerase chain reaction” is abbreviated PCR.


The term “isolated” refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.


The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural, or altered nucleotide bases. A polynucleotide may also consist of nucleotide sequences joined at the 3′ end of one nucleotide sequence to the 5′ end of another nucleotide sequence by a linker such as a 3 or 6 carbon (propandiol or hexandiol, respectively) moiety, or a linker arm of either 3 or 6 polyethylene glycol subunits (triethylene glycol or hexaethylene glycol, respectively). Any suitable linkers or spacers that are known in the art will work for this application. A polynucleotide in the form of a polymer of DNA may be comprised of one or more strands of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.


The term “amplification product” refers to nucleic acid fragments produced during a primer-directed amplification reaction. Typical methods of primer-directed amplification include polymerase chain reaction (PCR), ligase chain reaction (LCR), or strand displacement amplification (SDA). If PCR methodology is selected, the replication composition may comprise the components for nucleic acid replication, for example: nucleotide triphosphates, two (or more) primers with appropriate sequences, thermostable polymerase, buffers, solutes, and proteins. These reagents and details describing procedures for their use in amplifying nucleic acids are provided in, e.g., U.S. Pat. No. 4,683,202 (1987, Mullis et al.) and U.S. Pat. No. 4,683,195 (1986, Mullis et al.), each of which is incorporated by reference in their entireties. If LCR methodology is selected, then the nucleic acid replication compositions may comprise, for example: a thermostable ligase (e.g., Thermus aquaticus ligase), two sets of adjacent oligonucleotides (wherein one member of each set is complementary to each of the target strands), Tris-HCl buffer, KCl, EDTA, NAD, dithiothreitol, and salmon sperm DNA. See, for example, Tabor et al., Proc. Natl. Acad. Sci. U.S.A. 82:1074-1078 (1985).


The term “primer” refers to an oligonucleotide (synthetic or occurring naturally) that is capable of acting as a point of initiation of nucleic acid synthesis or replication along a complementary strand when placed under conditions in which synthesis of a complementary strand is catalyzed by a polymerase. A primer can further contain a detectable label, for example a 5′ end label.


The term “probe” refers to an oligonucleotide (synthetic or occurring naturally) that is complementary (though not necessarily fully complementary) to a polynucleotide of interest and forms a duplexed structure by hybridization with at least one strand of the polynucleotide of interest. A probe or primer-probe complex can further contain a detectable label.


A probe can either be an independent entity or complexed with or otherwise attached to a primer, such as where a probe is connected via its 3′ terminus to a primer's 5′ terminus through a linker, which may be a nucleotide or non-nucleotide linker and which may be a non-amplifiable linker, such as a hexaethylene glycol (HEG) or 18-carbon linker. In such a case, this would be termed a “primer-probe complex.” One example of such a primer-probe complex can be found in U.S. Pat. No. 6,326,145, incorporated herein by reference in its entirety, which are frequently referred to as “Scorpion® probes” or “Scorpion® primers.”


As used herein, the terms “label” and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, semiconductor nanocrystals, ligands (e.g., biotin, avidin, streptavidin, or haptens), and the like. A detectable label can also include a combination of a reporter and a quencher.


The term “reporter” refers to a substance or a portion thereof which is capable of exhibiting a detectable signal, which signal can be suppressed by a quencher. The detectable signal of the reporter is, e.g., fluorescence in the detectable range. The term “quencher” refers to a substance or portion thereof which is capable of suppressing, reducing, inhibiting, etc., the detectable signal produced by the reporter.


As used herein, the terms “quenching” and “fluorescence energy transfer” refer to the process whereby, when a reporter and a quencher are in close proximity, and the reporter is excited by an energy source, a substantial portion of the energy of the excited state non-radioactively transfers to the quencher where it either dissipates non-radioactively or is emitted at a different emission wavelength than that of the reporter.


In some embodiments, the reporter may be selected from fluorescent organic dyes modified with a suitable linking group for attachment to the oligonucleotide, such as to the terminal 3′ carbon or terminal 5′ carbon. The quencher may also be selected from organic dyes, which may or may not be fluorescent, depending on the embodiment. Generally, whether the quencher is fluorescent or simply releases the transferred energy from the reporter by non-radiative decay, the absorption band of the quencher should at least substantially overlap the fluorescent emission band of the reporter to optimize the quenching. Non-fluorescent quenchers or dark quenchers typically function by absorbing energy from excited reporters, but do not release the energy radiatively.


Selection of appropriate reporter-quencher pairs for particular probes may be undertaken in accordance with known techniques. Fluorescent and dark quenchers and their relevant optical properties from which exemplary reporter-quencher pairs may be selected are listed and described, for example, in Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, 1971, the content of which is incorporated herein by reference. Examples of modifying reporters and quenchers for covalent attachment via common reactive groups that can be added to an oligonucleotide in the present disclosure may be found, for example, in Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes of Eugene, Oreg., 1992, the content of which is incorporated herein by reference.


In some embodiments, reporter-quencher pairs may be selected from xanthene dyes including fluoresceins and rhodamine dyes. Many suitable forms of these compounds are available commercially with substituents on the phenyl groups, which can be used as the site for bonding or as the bonding functionality for attachment to an oligonucleotide. In some embodiments, fluorescent compounds for use as reporters are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are 1-dimethylaminonaphthyl-5 sulfonate, 1-anilino-8-naphthalene sulfonate and 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin; acridines such as 9-isothiocyanatoacridine; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles; stilbenes; pyrenes and the like.


In some embodiments, the reporters and quenchers are selected from fluorescein and rhodamine dyes. These dyes and appropriate linking methodologies for attachment to oligonucleotides are well known in the art.


Suitable examples of quenchers may be selected from 6-carboxy-tetramethyl-rhodamine, 4-(4-dimethylaminophenylazo) benzoic acid (DABYL), tetramethylrhodamine (TAMRA), BHQ-0™, BHQ-1™, BHQ-2™, and BHQ-3™, each of which are available from LGC Biosearch Technologies, Inc. of Novato, Calif., QSY-7™, QSY-9™, QSY-21™ and QSY-35™, each of which are available from ThermoFisher Scientific (Waltham, Mass.), and the like.


Suitable examples of reporters may be selected from dyes such as SYBR® green, 5-carboxyfluorescein (5-FAM™ available from Applied Biosystems of Foster City, Calif.), 6-carboxyfluorescein (6-FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, hexachloro-6-carboxyfluorescein (HEX), 6-carboxy-2′,4,7,7′-tetrachlorofluorescein (6-TET™ available from Applied Biosystems), carboxy-X-rhodamine (ROX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (6-JOE™ available from Applied Biosystems), VIC™ dye products available from ThermoFisher Scientific, NED™ dye products available from available from Applied Biosystems, Cal Fluor® dye products (such as, e.g., Cal Fluor® Gold 540, Orange 560, Red 590, Red 610, Red 635) available from LGC Biosearch Technologies, Quasar dye products (such as, e.g., Quasar 570, 670, 705) available from LGC Biosearch Technologies, and the like.


One example of a probe which contains a reporter and a quencher is a probe that is to be used in a 5′-exonuclease assay, such as the Taqman® real-time PCR technique. In this context, the oligonucleotide probe will have a sufficient number of phosphodiester linkages adjacent to its 5′ end so that the 5′ to 3′ nuclease activity employed can efficiently degrade the bound probe to separate the reporters and quenchers.


Another example of a probe which contains a reporter and a quencher is a Scorpion® probe in either a unimolecular or bimolecular conformation. In a unimolecular Scorpion®, the probe portion of the primer-probe complex is flanked by self-complementary regions which allow the probe to form into a stem-loop structure when the probe is unbound from its target DNA. Further, in a unimolecular Scorpion®, a reporter is typically attached at or near one of the self-complementary regions, such as at the 5′ terminus of the Scorpion® probe, and a quencher is attached at or near the other self-complementary region, such as at the 3′ end of the complementary sequence and adjacent to the non-amplifiable linker, such that the quencher is in sufficiently close proximity to the reporter to cause quenching when the probe is in its stem-loop conformation. In a bimolecular Scorpion®, self-complementary flanking regions are not typically employed, but rather a separate “blocking oligonucleotide” is employed in conjunction with the Scorpion® probe. This blocking oligonucleotide is capable of hybridizing to the probe region of the Scorpion® probe when the probe is unbound from its target DNA. Further, in a bimolecular Scorpion®, the reporter is typically attached to the probe region of the Scorpion® probe, such as at the 5′ terminus of the Scorpion® probe, while the quencher is attached to the blocking oligonucleotide, such as at the 3′ terminus of the blocking oligonucleotide, such that the quencher is in sufficiently close proximity to the reporter to cause quenching when the probe is unbound from its target DNA and is instead hybridized to the blocking oligonucleotide.


Yet another example of a probe which contains a reporter and quencher is a Molecular Beacon type probe, which contains a probe region flanked by self-complementary regions that allow the probe to form a stem-loop structure when unbound from the probe's target sequence. Such probes typically have a reporter attached at or near one terminus and a quencher attached at or near the other terminus such that the quencher is in sufficiently close proximity to the reporter to cause quenching when the probe is in its unbound, and thus stem-loop, form.


The term “replication inhibitor moiety” refers to any atom, molecule or chemical group that is attached to the 3′ terminal hydroxyl group of an oligonucleotide that will block the initiation of chain extension for replication of a nucleic acid strand. Examples include, but are not limited to: 3′-deoxynucleotides (e.g., cordycepin), dideoxynucleotides, phosphate, ligands (e.g., biotin and dinitrophenol), reporter molecules (e.g., fluorescein and rhodamine), carbon chains (e.g., propanol), a mismatched nucleotide or polynucleotide, or peptide nucleic acid units. The term “non-participatory” refers to the lack of participation of a probe or primer in a reaction for the amplification of a nucleic acid molecule. Specifically, a non-participatory probe or primer is one that will not serve as a substrate for, or be extended by, a DNA or RNA polymerase. A “non-participatory probe” is inherently incapable of being chain extended by a polymerase. It may or may not have a replication inhibitor moiety.


A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified, for example, in Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Examples of salt concentration ranges and temperature ranges for different hybridization conditions are as follows: high stringency, approximately 0.01 M to approximately 0.05 M salt, hybridization temperature 5° C. to 10° C. below Tm; moderate stringency, approximately 0.16 M to approximately 0.33 M salt, hybridization temperature 20° C. to 29° C. below Tm; low stringency, approximately 0.33 M to approximately 0.82 M salt, hybridization temperature 40° C. to 48° C. below Tm. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55° C., can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6×SSC. Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). Algorithm prediction tools to estimate Tm are also widely available. For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In some embodiments, the length for a hybridizable nucleic acid is at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, or, at least about 30 nucleotides or more. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.


Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by, e.g., Sambrook et al. (supra); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).


Genome Detection Regions

Applicants have solved the stated problem through a method that uses a pair of phage protein sequences the amplification of which is only detectable in E. coli O157:H7. The use of this target eliminates the potential for false positive results in mixed cultures of E. coli.


The present detection method finds utility in detection of E. coli O157:H7 in any type of sample, for example in appropriate samples for food testing, environmental testing, or human or animal diagnostic testing. While examples of suitable methods for detecting these regions are included herein, it is to be understood that use of the presently disclosed sequences is not limited to the methods described herein. Rather any suitable method can be employed to detect these DNA regions and subsequently the E. coli itself.


Oligonucleotides

Oligonucleotides of the instant disclosure are set forth in SEQ ID NOs: 1-12.


Oligonucleotides of the instant disclosure may be used as primers for PCR amplification. Exemplary primer pairs and their corresponding probes are shown in Table 1.











TABLE 1





5′ (Forward)
3′ (Reverse)



Primer
Primer
Probe







SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID NO: 9


SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 10


SEQ ID NO: 5
SEQ ID NO: 6
SEQ ID NO: 11


SEQ ID NO: 7
SEQ ID NO: 8
SEQ ID NO: 12









These oligonucleotide primers may also be useful for other nucleic acid amplification methods such as the ligase chain reaction (LCR) (Backman et al., 1989, EP 0 320 308; Carrino et al., 1995, J. Microbiol. Methods 23: 3-20); nucleic acid sequence-based amplification (NASBA) (Carrino et al., 1995, supra); and self-sustained sequence replication (3SR) and ‘Q replicase amplification’ (Pfeffer et al., 1995 Veterinary Res. Comm. 19: 375-407).


In some embodiments, the oligonucleotide primers can also contain a detectable label, for example a 5′ end label.


In addition, oligonucleotides also may be used in some embodiments as hybridization probes. In some embodiments, hybridization probes can be SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Hybridization using DNA probes has been frequently used for the detection of pathogens in food, clinical and environmental samples, and the methodologies are generally known to one skilled in the art. It is generally recognized that the degree of sensitivity and specificity of probe hybridization is lower than that achieved through the previously described amplification techniques. The nucleic acid probes can also possess a detectable label, such as a reporter-quencher combination as are employed in 5′-exonuclease detection assays, such as the Taqman® assay or in Scorpion® probe assays.


The 3′ terminal nucleotide of the nucleic acid probe may be rendered incapable of extension by a nucleic acid polymerase in some embodiments. Such blocking may be carried out, for example by the attachment of a replication inhibitor moiety, such as a reporter or quencher, to the terminal 3′ carbon of the nucleic acid probe by a linking moiety, or by making the 3′-terminal nucleotide a dideoxynucleotide. Alternatively, the 3′ end of the nucleic acid probe may be rendered impervious to the 3′ to 5′ extension activity of a polymerase by incorporating one or more modified internucleotide linkages onto the 3′ end of the oligonucleotide. Minimally, the 3′ terminal internucleotide linkage must be modified; however, additional internucleotide linkages may be modified. Internucleotide modifications which prevent elongation from the 3′ end of the nucleic acid probe and/or which block the 3′ to 5′ exonuclease activity of the DNA polymerase during PCR may include phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, and other similar polymerase-resistant internucleotide linkages. An alternative method to block 3′ extension of the probe is to form an adduct at the 3′ end of the probe using mitomycin C or other like antitumor antibiotics such as described in Basu et al., Biochemistry 32:4708-4718, 1993. Thus, the precise mechanism by which the 3′ end of the nucleic acid probe is protected from cleavage is not essential so long as the quencher is not cleaved from the nucleic acid probe.


A nucleic acid probe sequence can also optionally be employed with the primer sequence pairs of the present disclosure in an amplification-based detection technique, such as in the 3′-exonuclease assay. Exemplary primer/probe combinations are indicated in Table 1.


Assay Methods

Detection of the presence of E. coli O157:H7 itself may be accomplished in any suitable manner. Exemplary methods are primer-directed amplification methods and nucleic acid hybridization methods. These methods may be used to detect E. coli O157:H7 in a sample that is either a complex matrix or a purified culture, e.g., from an animal, environmental, or food source suspected of contamination.


In some embodiments, the assay method comprises (1) culturing a complex sample mixture in a non-selective growth media to resuscitate the target bacteria, (2) releasing total target bacterial DNA, and (3) subjecting the total DNA to an amplification protocol with a primer pair, such as a primer pair disclosed herein, and optionally with a nucleic acid probe comprising a detectable label.


Primer-Directed Amplification Assay Methods


A variety of primer-directed nucleic acid amplification methods are known in the art which can be employed, including thermal cycling methods (e.g., PCR, RT-PCR, and LCR), as well as isothermal methods and strand displacement amplification (SDA). In some embodiments, the method is PCR. In some embodiments, the primer pairs listed in Table 1 may be used as primers for use in primer-directed nucleic acid amplification for the detection of SEQ ID NOs: 1-3 and subsequently detection and identification of E. coli O157:H7.


Sample Preparation:


The oligonucleotides and methods disclosed herein may be used directly with any suitable clinical or environmental samples, without any need for sample preparation. In order to achieve higher sensitivity, and in situations where time is not a limiting factor, the samples can be pre-treated and then pre-amplification enrichment is performed.


The minimum industry standard for the detection of food-borne bacterial pathogens is a method that will reliably detect the presence of one pathogen cell in 25 g of food matrix as described in Andrews et al., 1984, “Food Sample and Preparation of Sample Homogenate”, Chapter 1 in Bacteriological Analytical Manual, 8th Edition, Revision A, Association of Official Analytical Chemists, Arlington, Va. In order to satisfy this stringent criterion, enrichment methods and media have been developed to enhance the growth of the target pathogen cell in order to facilitate its detection by biochemical, immunological or nucleic acid hybridization means. Typical enrichment procedures employ media that will enhance the growth and health of the target bacteria and also inhibit the growth of any background or non-target microorganisms present. For example, the USDA has set forth a protocol for enrichment of samples of ground beef to be tested for pathogenic E. coli (FDA Bacteriological Analytical Manual. (1998) published and distributed by the Association of Analytical Chemists, Suite 400, 2200 Wilson Blvd, Arlington, Va. 22201-3301; available on the U.S. Food and Drug Administration website, https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm2006949. htm).


Selective media have been developed for a variety of bacterial pathogens and one of skill in the art will know to select a medium appropriate for the particular organism to be enriched, e.g. E. coli O157:H7. A general discussion and recipes of non-selective media are described in the FDA Bacteriological Analytical Manual (supra).


After selective growth, a sample of the complex mixtures is removed for further analysis. This sampling procedure may be accomplished by a variety of means well known to those skilled in the art. In some embodiments, 5 μl of the enrichment culture is removed and added to 200 μl of lysis solution containing protease. The lysis solution is heated at 37° C. for 20 min followed by protease inactivation at 95° C. for 10 min as described in the BAX® System User's Guide, Hygiena Qualicon, Inc., Wilmington, Del. (available on the Hygiena website, https://www.hygiena.com/index.php?option=com_docman&view=document&alias=945-ins2025-e-coli-o157h7-mp-rev-03&category_slug=qualicon-instructions&Itemid=1134).


PCR Assay Methods:


In some embodiments, a method for detecting the presence of E. coli O157:H7 in a sample comprises (a) performing PCR amplification using primer pairs listed in Table 1 to produce a PCR amplification result; and (b) detecting the amplification, whereby a positive detection of the amplification indicates the presence of E. coli O157:H7 in the sample.


In some embodiments, prior to performing PCR amplification, a step of preparing the sample may be carried out. The preparing step may comprise 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.


Amplification Conditions:


A skilled person will understand that any generally acceptable PCR condition may be used for successfully detecting E. coli O157:H7 bacteria using the oligonucleotides of the instant disclosure, and depending on the sample to be tested and other laboratory conditions, routine optimization for the PCR conditions may be necessary to achieve optimal sensitivity and specificity. Optimally, PCR amplification results may be achieved from all of the intended specific targets while giving no PCR results for other, non-target species.


Detection/Examination/Analysis:


Primer-directed amplification products can be analyzed using various methods.


“Homogenous detection” refers to a method for the detection of amplification products where no separation (such as by gel electrophoresis) of amplification products from template or primers is necessary. Homogeneous detection is typically accomplished by measuring the level of fluorescence of the reaction mixture during or immediately following amplification. In addition, heterogeneous detection methods, which involve separation of amplification products during or prior to detection, can be employed in the present methods.


Homogenous detection may be employed to carry out “real-time” primer-directed nucleic acid amplification and detection, using primer pairs of the instant disclosure (e.g., “real-time” PCR and “real-time” RT-PCR). Exemplary “real-time” methods are set forth in U.S. Pat. Nos. 6,171,785, 5,994,056, 6,326,145, 5,804,375, 5,538,848, 5,487,972, and 5,210,015, each of which is hereby incorporated by reference in its entirety.


In some embodiments, the “real-time” detection method is the 5′-exonuclease detection method, as set forth in U.S. Pat. Nos. 5,804,375, 5,538,848, 5,487,972, and 5,210,015, each of which is hereby incorporated by reference in its entirety. In the 5′-exonuclease detection assay, a modified probe is employed during PCR which binds intermediate to or between the two members of the amplification primer pair. The modified probe possesses a reporter and a quencher and is designed to generate a detectable signal to indicate that it has hybridized with the target nucleic acid sequence during PCR. As long as both the reporter and the quencher are on the probe, the quencher stops the reporter from emitting a detectable signal. However, as the polymerase extends the primer during amplification, the intrinsic 5′ to 3′ nuclease activity of the polymerase degrades the probe, separating the reporter from the quencher, and enabling the detectable signal to be emitted. Generally, the amount of detectable signal generated during the amplification cycle is proportional to the amount of product generated in each cycle.


It is well known that the efficiency of quenching is a strong function of the proximity of the reporter and the quencher, i.e., as the two molecules get closer, the quenching efficiency increases. As quenching is strongly dependent on the physical proximity of the reporter and quencher, the reporter and the quencher are, in some embodiments, attached to the probe within a few nucleotides of one another, usually within 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 nucleotides of one another. Typically, this separation is achieved by attaching one member of a reporter-quencher pair to the 5′ end of the probe and the other member to a nucleotide about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 nucleotides away.


In some embodiments, amplification and detection is performed using labeled Taqman® probes. SEQ ID NOs: 9-12 possess, in some embodiments, a CAL Fluor 610 reporter attached at the 5′ terminus and a BHQ2 quencher attached at the 3′ terminus.


Another “real-time” detection method is the Scorpion® probe assay as set forth in U.S. Pat. No. 6,326,145, which is hereby incorporated by reference in its entirety. In the Scorpion® probe assay, PCR amplification is performed using a Scorpion® probe (either unimolecular or bimolecular) as a primer-probe complex, the Scorpion® probe possessing an appropriate reporter-quencher pair to allow the detectable signal of the reporter to be quenched prior to elongation of the primer. Post-elongation, the quenching effect is eliminated, and the amount of signal present is quantitated. As the amount of amplification product increases, an equivalent increase in detectable signal will be observed, thus allowing the amount of amplification product present to be determined as a function of the amount of detectable signal measured. When more than one Scorpion® probe is employed in a Scorpion® probe assay, such as one directed to more than one DNA region of interest (such as, e.g., one probe directed to SEQ ID NO:13 and a second probe directed to a different target region), each probe can have a different detectable label (e.g., reporter-quencher pair) attached, thus allowing each probe to be detected independently of the other probes.


Another method of homogenous detection involves the use of DNA melting curve analysis, particularly with the BAX® System hardware and reagent tablets from Hygiena Qualicon Inc. (Wilmington, Del.). The details of the system are given in U.S. Pat. No. 6,312,930 and PCT Publication Nos. WO 97/11197 and WO 00/66777, each of which is hereby incorporated by reference in its entirety.


Melting curve analysis detects and quantifies double stranded nucleic acid molecule (“dsDNA” or “target”) by monitoring the fluorescence of the target amplification product (“target amplicon”) during each amplification cycle at selected time points.


As is well known to the skilled artisan, the two strands of a dsDNA separate or melt, when the temperature is higher than its melting temperature. Melting of a dsDNA molecule is a process, and under a given solution condition, melting starts at a temperature (designated TMs hereinafter), and completes at another temperature (designated TME hereinafter). The familiar term, Tm, designates the temperature at which melting is 50% complete.


A typical PCR cycle involves a denaturing phase where the target dsDNA is melted, a primer annealing phase where the temperature optimal for the primers to bind to the now-single-stranded target, and a chain elongation phase (at a temperature TE) where the temperature is optimal for DNA polymerase to function.


In a melt curve analysis, TMs should be higher than TE, and TME should be lower (often substantially lower) than the temperature at which the DNA polymerase is heat-inactivated. Melting characteristics are affected by the intrinsic properties of a given dsDNA molecule, such as deoxynucleotide composition and the length of the dsDNA.


Intercalating dyes will bind to double stranded DNA. The dye/dsDNA complex will fluoresce when exposed to the appropriate excitation wavelength of light, which is dye dependent, and the intensity of the fluorescence may be proportionate to concentration of the dsDNA. Methods taking advantage of the use of DNA intercalating dyes to detect and quantify dsDNA are known in the art. Many dyes are known and used in the art for these purposes. The instant methods also take advantage of such relationship.


Examples of such intercalating dyes include, but are not limited to, SYBR® Green-I, ethidium bromide, propidium iodide, TOTO®-1 {Quinolinium, 1-1′-[1,3-propanediylbis [(dimethyliminio)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzothiazolylidene) methyl]]-, tetraiodide}, and YoPro® {Quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)-propyl]-diiodide}. In some embodiments, a non-asymmetrical cyanide dye such as SYBR® Green-I, manufactured by ThermoFisher Scientific, is the intercalating dye.


Melting curve analysis is achieved by monitoring the change in fluorescence while the temperature is increased. When the temperature reaches the TMs specific for the target amplicon, the dsDNA begins to denature. When the dsDNA denatures, the intercalating dye dissociates from the DNA and fluorescence decreases. Mathematical analysis of the negative of the change of the log of fluorescence divided by the change in temperature plotted against the temperature results in the graphical peak known as a melting curve.


It should be understood that the present methods could be operated using a combination of techniques, such as by having a Scorpion® probe directed to one target region and a Taqman® probe directed to a second target region. It should also be understood that the methods are not limited to the above described techniques. Rather, one skilled in the art would recognize that other techniques for detecting amplification as known in the art may also be used. For example, techniques such as PCR-based quantitative sequence detection (QSD) may be performed using nucleic acid probes which, when present in the single-stranded state in solution, are configured such that the reporter and quencher are sufficiently close to substantially quench the reporter's emission. However, upon hybridization of the intact reporter-quencher nucleic acid probe with the amplified target nucleic acid sequence, the reporter and quenchers become sufficiently distant from each other. As a result, the quenching is substantially abated causing an increase in the fluorescence emission detected.


In addition to homogenous detection methods, a variety of other heterogeneous detection methods are known in the art which can be employed in the present methods, including standard non-denaturing gel electrophoresis (e.g., acrylamide or agarose), denaturing gradient gel electrophoresis, and temperature gradient gel electrophoresis. Standard non-denaturing gel electrophoresis is a simple and quick method of PCR detection but may not be suitable for all applications.


Denaturing Gradient Gel Electrophoresis (DGGE) is a separation method that detects differences in the denaturing behavior of small DNA fragments (200-700 bp). The principle of the separation is based on both fragment length and nucleotide sequence. In fragments that are the same length, a difference as little as one base pair can be detected. This is in contrast to non-denaturing gel electrophoresis, where DNA fragments are separated only by size. This limitation of non-denaturing gel electrophoresis results because the difference in charge density between DNA molecules is near neutral and plays little role in their separation. As the size of the DNA fragment increases, its velocity through the gel decreases.


DGGE is primarily used to separate DNA fragments of the same size based on their denaturing profiles and sequence. Using DGGE, two strands of a DNA molecule separate, or melt, when heat or a chemical denaturant is applied. The denaturation of a DNA duplex is influenced by two factors: 1) the hydrogen bonds formed between complimentary base pairs (since GC rich regions melt at higher denaturing conditions than regions that are AT rich); and 2) the attraction between neighboring bases of the same strand, or “stacking”. Consequently, a DNA molecule may have several melting domains with each of their individual characteristic denaturing conditions determined by their nucleotide sequence. DGGE exploits the fact that otherwise identical DNA molecules having the same length and DNA sequence, with the exception of only one nucleotide within a specific denaturing domain, will denature at different temperatures or Tm. Thus, when the double-stranded (ds) DNA fragment is electrophoresed through a gradient of increasing chemical denaturant, it begins to denature and undergoes both a conformational and mobility change. The dsDNA fragment will travel faster than a denatured single-stranded (ss) DNA fragment, since the branched structure of the single-stranded moiety of the molecule becomes entangled in the gel matrix. As the denaturing environment increases, the dsDNA fragment will completely dissociate and mobility of the molecule through the gel is retarded at the denaturant concentration at which the particular low denaturing domains of the DNA strand dissociate. In practice, the electrophoresis is conducted at a constant temperature (around 60° C.) and chemical denaturants are used at concentrations that will result in 100% of the DNA molecules being denatured (e.g., 40% formamide and 7M urea). This variable denaturing gradient is created using a gradient maker, such that the composition of each DGGE gel gradually changes from 0% denaturant up to 100% denaturant. Of course, gradients containing a reduced range of denaturant (e.g., 35% to 60%) may also be poured for increased separation of DNA.


The principle used in DGGE can also be applied to a second method that uses a temperature gradient instead of a chemical denaturant gradient. This method is known as Temperature Gradient Gel Electrophoresis (TGGE). This method makes use of a temperature gradient to induce the conformational change of dsDNA to ssDNA to separate fragments of equal size with different sequences. As in DGGE, DNA fragments with different nucleotide sequences will become immobile at different positions in the gel. Variations in primer design can be used to advantage in increasing the usefulness of DGGE for characterization and identification of the PCR products. These methods and principles of using primer design variations are described in PCR Technology Principles and Applications, Henry A. Erlich Ed., M. Stockton Press, NY, pages 71 to 88 (1988).


Instrumentation:


When homogenous detection is employed, the level of fluorescence is, in some embodiments, measured using a laser fluorometer such as, for example, an ABI Prism Model 7500 Fast Sequence Detector. However, similar detection systems for measuring the level of fluorescence in a sample can be used in the methods disclosed herein.


Reagents and Kits:


Any suitable nucleic acid replication composition (“replication composition”) in any format can be used. A typical replication composition for PCR amplification may comprise, for example, dATP, dCTP, dGTP, dTTP, target specific primers and a suitable polymerase.


If the replication composition is in liquid form, suitable buffers known in the art may be used (Sambrook, J. et al., supra).


Alternatively, if the replication composition is contained in a tablet form, then typical tabletization reagents may be included such as stabilizers and binding agents. Exemplary tabletization technology is set forth in U.S. Pat. Nos. 4,762,857 and 4,678,812, each of which is hereby incorporated by reference in its entirety.


In some embodiments, the replication composition can comprise (a) at least one primer pair selected from Table 1, and (b) a thermostable DNA polymerase. In some embodiments, the replication composition can comprise (a) at least two primer pairs selected from Table 1, each directed toward a different target DNA region; and (b) a thermostable DNA polymerase. In some embodiments, at least one primer pair is directed to SEQ ID NO:13.


In some embodiments, the replication composition can comprise (a) at least two primer pairs and any corresponding probe or blocking oligonucleotide selected from Table 1, wherein each nucleic acid probe or primer-probe complex employed comprises a detectable label; and (b) a thermostable DNA polymerase. The detectable label can comprise, in some embodiments, a reporter capable of emitting a detectable signal and a quencher capable of substantially quenching the reporter and preventing the emission of the detectable signal when the reporter and quencher are in sufficiently close proximity to one another.


Kits disclosed herein can comprise any one of the above replication compositions. Tablets disclosed herein can comprise any one of the above replication compositions. In some embodiments, a kit can comprise tablet comprising any one of the above replication compositions.


In some instances, an internal positive control can be included in the reaction. The internal positive control can include control template nucleic acids (e.g. DNA or RNA), control primers, and control nucleic acid probe. The advantages of an internal positive control contained within a PCR reaction have been previously described (U.S. Pat. No. 6,312,930 and PCT Application No. WO 97/11197, each of which is hereby incorporated by reference in its entirety), and include: (i) the control may be amplified using a single primer; (ii) the amount of the control amplification product is independent of any target DNA or RNA contained in the sample; (iii) the control DNA can be tableted with other amplification reagents for ease of use and high degree of reproducibility in both manual and automated test procedures; (iv) the control can be used with homogeneous detection, i.e., without separation of product DNA from reactants; and (v) the internal control has a melting profile that is distinct from other potential amplification products in the reaction and/or a detectable label on the control nucleic acid that is distinct from the detectable label on the nucleic acid probe directed to the target.


Control DNA will be of appropriate size and base composition to permit amplification in a primer-directed amplification reaction. The control template DNA sequence may be obtained from the E. coli genome, or from another source, but must be reproducibly amplified under the same conditions that permit the amplification of the target amplification product.


Control sequences can include, for example, those found in SV40. The concentration range of SV40, when used, can be for example 101, 102, 103, 104, 105, 106, or 107 copies per PCR reaction.


The control reaction is useful to validate the amplification reaction. Amplification of the control DNA occurs within the same reaction tube as the sample that is being tested, and therefore indicates a successful amplification reaction when samples are target negative, i.e. no target amplification product is produced. In order to achieve significant validation of the amplification reaction, a suitable number of copies of the control DNA template must be included in each amplification reaction.


In some instances, it may be useful to include an additional negative control replication composition. The negative control replication composition will contain the same reagents as the replication composition but without the polymerase. The primary function of such a control is to monitor spurious background fluorescence in a homogeneous format when the method employs a fluorescent means of detection.


Replication compositions may be modified depending on whether they are designed to be used to amplify target DNA or the control DNA. Replication compositions that will amplify the target DNA (test replication compositions) may include (i) a polymerase (generally thermostable), (ii) a primer pair capable of hybridizing to the target DNA and (iii) necessary buffers for the amplification reaction to proceed. Replication compositions that will amplify the control DNA (positive control, or positive replication composition) may include (i) a polymerase (generally thermostable) (ii) the control DNA; (iii) at least one primer capable of hybridizing to the control DNA; and (iv) necessary buffers for the amplification reaction to proceed. In addition, the replication composition for either target DNA or control DNA amplification can contain a nucleic acid probe, in some embodiments possessing a detectable label.


Nucleic Acid Hybridization Methods


In addition to primer-directed amplification assay methods, nucleic acid hybridization assay methods can be employed for detection of E. coli O157:H7. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing E. coli O157:H7, and a specific hybridization method. Typically, the probe length can vary from as few as 5 bases to the full length of the E. coli diagnostic sequence and will depend upon the specific test to be done. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.


Probes particularly useful in nucleic acid hybridization methods are any of SEQ ID NOs:1-12, or sequences derived therefrom.


The sample may or may not contain E. coli O157:H7. The sample may take a variety of forms; however, the sample will generally be extracted from an animal, environmental or food source suspected of contamination. The DNA may be detected directly but, in some embodiments, the sample nucleic acid must be made available to contact the probe before any hybridization of probe and target molecule can occur. Thus the organism's DNA is in some embodiments free from the cell and placed under the proper conditions before hybridization can occur. Methods of in-solution hybridization necessitate the purification of the DNA in order to be able to obtain hybridization of the sample DNA with the probe. This has meant that utilization of the in-solution method for detection of target sequences in a sample requires that the nucleic acids of the sample must first be purified to eliminate protein, lipids, and other cell components, and then contacted with the probe under hybridization conditions. Methods for the purification of the sample nucleic acid are common and well known in the art (Sambrook et al., supra).


In some embodiments, hybridization assays may be conducted directly on cell lysates, without the need to extract the nucleic acids. This eliminates several steps from the sample-handling process and speeds up the assay. To perform such assays on crude cell lysates, a chaotropic agent is typically added to the cell lysates prepared as described above. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes to DNA at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).


Alternatively, one can purify the sample nucleic acids prior to probe hybridization. A variety of methods are known to one of skill in the art (e.g., phenol-chloroform extraction, IsoQuick extraction (MicroProbe Corp., Bothell, Wash.), and others). Pre-hybridization purification is particularly useful for standard filter hybridization assays. Furthermore, purification facilitates measures to increase the assay sensitivity by incorporating in vitro RNA amplification methods such as self-sustained sequence replication (see for example Fahy et al., In PCR Methods and Applications, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1991), pp. 25-33) or reverse transcriptase PCR (Kawasaki, In PCR Protocols: A Guide to Methods and Applications, M. A. Innis et al., Eds., (1990), pp. 21-27).


Once the DNA is released, it can be detected by any of a variety of methods. However, the most useful embodiments have at least some characteristics of speed, convenience, sensitivity, and specificity.


Hybridization methods are well known in the art. Typically the probe and sample must be mixed under conditions which will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed.


Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, e.g., about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%, of a polar organic solvent. A common hybridization solution employs about 30-50% (e.g., about 30%, about 35%, about 40%, about 45%, or about 50%) v/v formamide, about 0.15 M to 1 M (e.g., about 0.15 M, about 0.20 M, about 0.25 M, about 0.30 M, about 0.35 M, about 0.35 M, about 0.40 M, about 0.45 M, about 0.50 M, about 0.55 M, about 0.60 M, about 0.65 M, about 0.70 M, about 0.75 M, about 0.80 M, about 0.85 M, about 0.90 M, about 0.95 M, or about 1 M) sodium chloride, about 0.05 M to 0.1 M (e.g., about 0.05 M, about 0.10 M, about 0.15 M, about 0.20 M, about 0.25 M, about 0.30 M, about 0.35 M, about 0.35 M, about 0.40 M, about 0.45 M, about 0.50 M, about 0.55 M, about 0.60 M, about 0.65 M, about 0.70 M, about 0.75 M, about 0.80 M, about 0.85 M, about 0.90 M, about 0.95 M, or about 1 M) buffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9 (e.g., about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, or about pH 9.0), about 0.05% to 0.2% (e.g., about 0.05%, about 0.10%, about 0.15%, or about 0.20%) detergent, such as sodium dodecylsulfate, or between 0.5 mM-20 mM (e.g., about 0.5 mM, about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, about 14.0 mM, about 14.5 mM, about 15.0 mM, about 15.5 mM, about 16.0 mM, about 16.5 mM, about 17.0 mM, about 17.5 mM, about 18.0 mM, about 18.5 mM, about 19.0 mM, about 19.5 mM, or about 20.0 mM) EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons), polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 mg/ml to 5 mg/ml (e.g., about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml, about 1.0 mg/ml, about 1.5 mg/ml, about 2.0 mg/ml, about 2.5 mg/ml, about 3.0 mg/ml, about 3.5 mg/ml, about 4.0 mg/ml, about 4.5 mg/ml, or about 5.0 mg/ml), fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5% to 2% (e.g., about 0.05%, about 0.10%, about 0.15%, about 0.20%, about 0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, about 0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, about 0.90%, about 0.95%, about 1.00%, about 1.05%, about 1.10%, about 1.15%, about 1.20%, about 1.25%, about 1.30%, about 1.35%, about 1.40%, about 1.45%, about 1.50%, about 1.55%, about 1.60%, about 1.65%, about 1.70%, about 1.75%, about 1.80%, about 1.85%, about 1.90%, about 1.95%, or about 2.00%) wt/vol glycine. Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate), and anionic saccharidic polymers (e.g., dextran sulfate).


Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the DNA sequence.


The sandwich assay may be encompassed in an assay kit. This kit would include a first component for the collection of samples suspected of contamination and buffers for the disbursement and lysis of the sample. A second component would include media in either dry or liquid form for the hybridization of target and probe polynucleotides, as well as for the removal of undesirable and nonduplexed forms by washing. A third component includes a solid support (dipstick) upon which is fixed (or to which is conjugated) unlabeled nucleic acid probe(s) that is (are) complementary to the target sequences. A fourth component would contain labeled probe that is complementary to a second and different region of the same DNA strand to which the immobilized, unlabeled nucleic acid probe of the third component is hybridized.


In some embodiments, SEQ ID NOs: 1-12 or derivations thereof may be used as 3′ blocked detection probes in either a homogeneous or heterogeneous assay format. For example, a probe generated from these sequences may be 3′ blocked or non-participatory and will not be extended by, or participate in, a nucleic acid amplification reaction. Additionally, the probe, in some embodiments, incorporates a label that can serve as a reactive ligand that acts as a point of attachment for the immobilization of the probe/analyte hybrid or as a reporter to produce detectable signal. Accordingly, genomic or cDNA isolated from a sample suspected of E. coli contamination is amplified by standard primer-directed amplification protocols in the presence of an excess of the 3′ blocked detection probe to produce amplification products. Because the probe is 3′ blocked, it does not participate or interfere with the amplification of the target. After the final amplification cycle, the detection probe anneals to the relevant portion of the amplified DNA and the annealed complex is then captured on a support through the reactive ligand.


In some instances, it is desirable to incorporate a ligand labeled dNTP with the labeled probe in the replication composition to facilitate immobilization of the PCR reaction product on a support and then detection of the immobilized product by means of the labeled probe reagent. For example, a biotin, digoxigenin, or digoxin labeled dNTP could be added to PCR reaction composition. The biotin, digoxigenin, or digoxin incorporated in the PCR product could then be immobilized respectively on to a strepavidin, anti-dixogin or antidigoxigenin antibody support. The immobilized PCR product could then be detected by the presence of the probe label.


EXAMPLES

General Methods and Materials Used in the Examples


Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following Examples may be found in Manual of Methods for Genus Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994) or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass. or Bacteriological Analytical Manual. 6th Edition, Association of Official Analytical Chemists, Arlington, Va. (1984).


The medium used to grow the pathogenic E. coli strains and comparative non-target strains was Brain Heart Infusion broth (BHI) obtained from BBL (Becton-Dickenson). Samples of pathogenic E. coli strains were obtained from cultures grown overnight in BHI broth to approximately 109 cfu/ml. Samples of the comparative non-target strains were enriched in BHI at approximately 109 cfu/ml.


Primers and probes (SEQ ID NOs: 1-12) were prepared by LGC Biosearch Technologies, Inc., 2199 S. McDowell Blvd., Petaluma, Calif. 94954 USA.


All PCR reactions were carried out using a standard BAX® System (Hygiena Qualicon, Wilmington, Del.).


The meaning of abbreviations is as follows: “h” means hour(s), “min” means minute(s), “sec” means second(s), “d” means day(s), “ml” means milliliter(s), “μl” means microliter(s), “cfu” means colony forming unit(s).


Example 1
Determination of Inclusivity/Exclusivity of the Individual Targets Via Taqman® Assay

Samples of organisms were analyzed to establish inclusivity and exclusivity of individual Taqman® probes disclosed herein. Pure cultures grown overnight achieved cell densities of approximately 1×109 cfu/ml. For inclusivity, independent, bona fide E. coli O157:H7 isolates were used; for exclusivity non O157:H7 E. coli and other closely related non-target organisms were used to ensure that the assay would discriminate the target organism (O157:H7) from other E. coli and non-target organisms.


DNA Lysate Preparation

Material tested was either food enrichment (ground beef enrichment prepared as described in the BAX® system user guide for the BAX® MP assay) or overnight growth of E. coli O157:H7 isolates at 37° C. in BHI media. 5 μl of the material to be tested was added to 200 μl of BAX® lysis reagent (Hygiena Qualicon, Wilmington, Del.). The mixture was incubated at 37° C. for 20 minutes, then further incubated at 95° C. for 10 minutes, and finally cooled to 4° C.


PCR Conditions

2 μl of the DNA lysate as prepared above was used in a PCR reaction that included other reaction components including the primers and probes as listed in TABLE 2.














TABLE 2








Per





Primers/ Probe
reaction
Dye
Quencher






















Forward primer
150
nM





Reverse primer
175
nM





Probe
66.7
nM
CALFluor610
BHQ-2











The reagents that were used in the PCR amplification reaction were custom made BAX® System No-Taq Reagent Tablet Kits (Hygiena Qualicon, Wilmington, Del.) that contained deoxynucleotides (Roche Diagnostics, Indianapolis, Ind., USA), BSA and Surfact-amps (Sigma-Aldrich, ST. Louis, Mo., USA). Additionally the reaction included Go Taq DNA Polymerase (Promega, Madison, Wis., USA) and PCR buffer (Hygiena Qualicon, Wilmington, Del.).


Amplification and testing was performed on the BAX® Q7 machine (Hygiena Qualicon, Wilmington, Del.). The thermal cycling conditions were: 2 minutes at 94° C., followed by 43 cycles of 94° C. for 10 seconds and 63° C. for 40 seconds, with the fluorescent signal captured during the 63° C. step at each cycle.


Results

As can be seen in Tables 3-5, below, using individual Taqman® probes, the present methods were able to correctly detect all O157:H7 isolates and did not detect any non-O157:H7 E. coli strains or other non-target organisms.









TABLE 3







Inclusivity demonstrated towards 242 strains of



E. coli O157:H7












Qualicon






Culture






Collection


BAX ®



Strain #
Strain Name
Serovar
Result







DD640

Escherichia
coli

O157:H7
Positive



DD641

Escherichia
coli

O157:H7
Positive



DD642

Escherichia
coli

O157:H7
Positive



DD914

Escherichia
coli

O157:H7
Positive



DD915

Escherichia
coli

O157:H7
Positive



DD916

Escherichia
coli

O157:H7
Positive



DD935

Escherichia
coli

O157:H7
Positive



DD1449

Escherichia
coli

O157:H7
Positive



DD1450

Escherichia
coli

O157:H7
Positive



DD1451

Escherichia
coli

O157:H7
Positive



DD1452

Escherichia
coli

O157:H7
Positive



DD1453

Escherichia
coli

O157:H7
Positive



DD1454

Escherichia
coli

O157:H7
Positive



DD1455

Escherichia
coli

O157:H7
Positive



DD1456

Escherichia
coli

O157:H7
Positive



DD1457

Escherichia
coli

O157:H7
Positive



DD1458

Escherichia
coli

O157:H7
Positive



DD1459

Escherichia
coli

O157:H7
Positive



DD1460

Escherichia
coli

O157:H7
Positive



DD1461

Escherichia
coli

O157:H7
Positive



DD1462

Escherichia
coli

O157:H7
Positive



DD1463

Escherichia
coli

O157:H7
Positive



DD1972

Escherichia
coli

O157:H7
Positive



DD1973

Escherichia
coli

O157:H7
Positive



DD1974

Escherichia
coli

O157:H7
Positive



DD1975

Escherichia
coli

O157:H7
Positive



DD1976

Escherichia
coli

O157:H7
Positive



DD1977

Escherichia
coli

O157:H7
Positive



DD1978

Escherichia
coli

O157:H7
Positive



DD1979

Escherichia
coli

O157:H7
Positive



DD1980

Escherichia
coli

O157:H7
Positive



DD1981

Escherichia
coli

O157:H7
Positive



DD1982

Escherichia
coli

O157:H7
Positive



DD1983

Escherichia
coli

O157:H7
Positive



DD1984

Escherichia
coli

O157:H7
Positive



DD1985

Escherichia
coli

O157:H7
Positive



DD1986

Escherichia
coli

O157:H7
Positive



DD1987

Escherichia
coli

O157:H7
Positive



DD1988

Escherichia
coli

O157:H7
Positive



DD1989

Escherichia
coli

O157:H7
Positive



DD1990

Escherichia
coli

O157:H7
Positive



DD1991

Escherichia
coli

O157:H7
Positive



DD5892

Escherichia
coli

O157:H7
Positive



DD5893

Escherichia
coli

O157:H7
Positive



DD5894

Escherichia
coli

O157:H7
Positive



DD5895

Escherichia
coli

O157:H7
Positive



DD5896

Escherichia
coli

O157:H7
Positive



DD5897

Escherichia
coli

O157:H7
Positive



DD5898

Escherichia
coli

O157:H7
Positive



DD6972

Escherichia
coli

O157:H7
Positive



DD6973

Escherichia
coli

O157:H7
Positive



DD7101

Escherichia
coli

O157:H7
Positive



DD8295

Escherichia
coli

O157:H7
Positive



DD8296

Escherichia
coli

O157:H7
Positive



DD8297

Escherichia
coli

O157:H7
Positive



DD8298

Escherichia
coli

O157:H7
Positive



DD8299

Escherichia
coli

O157:H7
Positive



DD8300

Escherichia
coli

O157:H7
Positive



DD8301

Escherichia
coli

O157:H7
Positive



DD8302

Escherichia
coli

O157:H7
Positive



DD8303

Escherichia
coli

O157:H7
Positive



DD8856

Escherichia
coli

O157:H7
Positive



DD8857

Escherichia
coli

O157:H7
Positive



DD8858

Escherichia
coli

O157:H7
Positive



DD8859

Escherichia
coli

O157:H7
Positive



DD8860

Escherichia
coli

O157:H7
Positive



DD8861

Escherichia
coli

O157:H7
Positive



DD8862

Escherichia
coli

O157:H7
Positive



DD8863

Escherichia
coli

O157:H7
Positive



DD8864

Escherichia
coli

O157:H7
Positive



DD8865

Escherichia
coli

O157:H7
Positive



DD8866

Escherichia
coli

O157:H7
Positive



DD8867

Escherichia
coli

O157:H7
Positive



DD8868

Escherichia
coli

O157:H7
Positive



DD8869

Escherichia
coli

O157:H7
Positive



DD8870

Escherichia
coli

O157:H7
Positive



DD8871

Escherichia
coli

O157:H7
Positive



DD8872

Escherichia
coli

O157:H7
Positive



DD8873

Escherichia
coli

O157:H7
Positive



DD8874

Escherichia
coli

O157:H7
Positive



DD9047

Escherichia
coli

O157:H7
Positive



DD9048

Escherichia
coli

O157:H7
Positive



DD9711

Escherichia
coli

O157:H7
Positive



DD9712

Escherichia
coli

O157:H7
Positive



DD9713

Escherichia
coli

O157:H7
Positive



DD9714

Escherichia
coli

O157:H7
Positive



DD9715

Escherichia
coli

O157:H7
Positive



DD10133

Escherichia
coli

O157:H7
Positive



DD10134

Escherichia
coli

O157:H7
Positive



DD10135

Escherichia
coli

O157:H7
Positive



DD10136

Escherichia
coli

O157:H7
Positive



DD10901

Escherichia
coli

O157:H7
Positive



DD10902

Escherichia
coli

O157:H7
Positive



DD10903

Escherichia
coli

O157:H7
Positive



DD10904

Escherichia
coli

O157:H7
Positive



DD10905

Escherichia
coli

O157:H7
Positive



DD10906

Escherichia
coli

O157:H7
Positive



DD10907

Escherichia
coli

O157:H7
Positive



DD10908

Escherichia
coli

O157:H7
Positive



DD10909

Escherichia
coli

O157:H7
Positive



DD10910

Escherichia
coli

O157:H7
Positive



DD10911

Escherichia
coli

O157:H7
Positive



DD10912

Escherichia
coli

O157:H7
Positive



DD10913

Escherichia
coli

O157:H7
Positive



DD10914

Escherichia
coli

O157:H7
Positive



DD10915

Escherichia
coli

O157:H7
Positive



DD10916

Escherichia
coli

O157:H7
Positive



DD10917

Escherichia
coli

O157:H7
Positive



DD10918

Escherichia
coli

O157:H7
Positive



DD10919

Escherichia
coli

O157:H7
Positive



DD10920

Escherichia
coli

O157:H7
Positive



DD10921

Escherichia
coli

O157:H7
Positive



DD12786

Escherichia
coli

O157:H7
Positive



DD12787

Escherichia
coli

O157:H7
Positive



DD12788

Escherichia
coli

O157:H7
Positive



DD12789

Escherichia
coli

O157:H7
Positive



DD12790

Escherichia
coli

O157:H7
Positive



DD12791

Escherichia
coli

O157:H7
Positive



DD12792

Escherichia
coli

O157:H7
Positive



DD12793

Escherichia
coli

O157:H7
Positive



DD12794

Escherichia
coli

O157:H7
Positive



DD12796

Escherichia
coli

O157:H7
Positive



DD12797

Escherichia
coli

O157:H7
Positive



DD12798

Escherichia
coli

O157:H7
Positive



DD12799

Escherichia
coli

O157:H7
Positive



DD12800

Escherichia
coli

O157:H7
Positive



DD12801

Escherichia
coli

O157:H7
Positive



DD12802

Escherichia
coli

O157:H7
Positive



DD12803

Escherichia
coli

O157:H7
Positive



DD12805

Escherichia
coli

O157:H7
Positive



DD12806

Escherichia
coli

O157:H7
Positive



DD12807

Escherichia
coli

O157:H7
Positive



DD12808

Escherichia
coli

O157:H7
Positive



DD12809

Escherichia
coli

O157:H7
Positive



DD12810

Escherichia
coli

O157:H7
Positive



DD12811

Escherichia
coli

O157:H7
Positive



DD12812

Escherichia
coli

O157:H7
Positive



DD12813

Escherichia
coli

O157:H7
Positive



DD12814

Escherichia
coli

O157:H7
Positive



DD12815

Escherichia
coli

O157:H7
Positive



DD12816

Escherichia
coli

O157:H7
Positive



DD12817

Escherichia
coli

O157:H7
Positive



DD12818

Escherichia
coli

O157:H7
Positive



DD12819

Escherichia
coli

O157:H7
Positive



DD12820

Escherichia
coli

O157:H7
Positive



DD12824

Escherichia
coli

O157:H7
Positive



DD12825

Escherichia
coli

O157:H7
Positive



DD12826

Escherichia
coli

O157:H7
Positive



DD12827

Escherichia
coli

O157:H7
Positive



DD12828

Escherichia
coli

O157:H7
Positive



DD12829

Escherichia
coli

O157:H7
Positive



DD12830

Escherichia
coli

O157:H7
Positive



DD12831

Escherichia
coli

O157:H7
Positive



DD12832

Escherichia
coli

O157:H7
Positive



DD12833

Escherichia
coli

O157:H7
Positive



DD12834

Escherichia
coli

O157:H7
Positive



DD12835

Escherichia
coli

O157:H7
Positive



DD12836

Escherichia
coli

O157:H7
Positive



DD12837

Escherichia
coli

O157:H7
Positive



DD12838

Escherichia
coli

O157:H7
Positive



DD12839

Escherichia
coli

O157:H7
Positive



DD12840

Escherichia
coli

O157:H7
Positive



DD12841

Escherichia
coli

O157:H7
Positive



DD12842

Escherichia
coli

O157:H7
Positive



DD12843

Escherichia
coli

O157:H7
Positive



DD12844

Escherichia
coli

O157:H7
Positive



DD12845

Escherichia
coli

O157:H7
Positive



DD12846

Escherichia
coli

O157:H7
Positive



DD12847

Escherichia
coli

O157:H7
Positive



DD12848

Escherichia
coli

O157:H7
Positive



DD12852

Escherichia
coli

O157:H7
Positive



DD12853

Escherichia
coli

O157:H7
Positive



DD12854

Escherichia
coli

O157:H7
Positive



DD12855

Escherichia
coli

O157:H7
Positive



DD12856

Escherichia
coli

O157:H7
Positive



DD12857

Escherichia
coli

O157:H7
Positive



DD12858

Escherichia
coli

O157:H7
Positive



DD12860

Escherichia
coli

O157:H7
Positive



DD12861

Escherichia
coli

O157:H7
Positive



DD12862

Escherichia
coli

O157:H7
Positive



DD12863

Escherichia
coli

O157:H7
Positive



DD12864

Escherichia
coli

O157:H7
Positive



DD12865

Escherichia
coli

O157:H7
Positive



DD12866

Escherichia
coli

O157:H7
Positive



DD12867

Escherichia
coli

O157:H7
Positive



DD12868

Escherichia
coli

O157:H7
Positive



DD12869

Escherichia
coli

O157:H7
Positive



DD12870

Escherichia
coli

O157:H7
Positive



DD12871

Escherichia
coli

O157:H7
Positive



DD12872

Escherichia
coli

O157:H7
Positive



DD12873

Escherichia
coli

O157:H7
Positive



DD12874

Escherichia
coli

O157:H7
Positive



DD12875

Escherichia
coli

O157:H7
Positive



DD12876

Escherichia
coli

O157:H7
Positive



DD12877

Escherichia
coli

O157:H7
Positive



DD12878

Escherichia
coli

O157:H7
Positive



DD12879

Escherichia
coli

O157:H7
Positive



DD12880

Escherichia
coli

O157:H7
Positive



DD12881

Escherichia
coli

O157:H7
Positive



DD12882

Escherichia
coli

O157:H7
Positive



DD12883

Escherichia
coli

O157:H7
Positive



DD12885

Escherichia
coli

O157:H7
Positive



DD12905

Escherichia
coli

O157:H7
Positive



DD13038

Escherichia
coli

O157:H7
Positive



DD13040

Escherichia
coli

O157:H7
Positive



DD13054

Escherichia
coli

O157:H7
Positive



DD13055

Escherichia
coli

O157:H7
Positive



DD13072

Escherichia
coli

O157:H7
Positive



DD13077

Escherichia
coli

O157:H7
Positive



DD13078

Escherichia
coli

O157:H7
Positive



DD13085

Escherichia
coli

O157:H7
Positive



DD13174

Escherichia
coli

O157:H7
Positive



DD13175

Escherichia
coli

O157:H7
Positive



DD13176

Escherichia
coli

O157:H7
Positive



DD13182

Escherichia
coli

O157:H7
Positive



DD13189

Escherichia
coli

O157:H7
Positive



DD13190

Escherichia
coli

O157:H7
Positive



DD13197

Escherichia
coli

O157:H7
Positive



DD13199

Escherichia
coli

O157:H7
Positive



DD13241

Escherichia
coli

O157:H7
Positive



DD13262

Escherichia
coli

O157:H7
Positive



DD13289

Escherichia
coli

O157:H7
Positive



DD13290

Escherichia
coli

O157:H7
Positive



DD13291

Escherichia
coli

O157:H7
Positive



DD13405

Escherichia
coli

O157:H7
Positive



DD13406

Escherichia
coli

O157:H7
Positive



DD13407

Escherichia
coli

O157:H7
Positive



DD13480

Escherichia
coli

O157:H7
Positive



DD13482

Escherichia
coli

O157:H7
Positive



DD13483

Escherichia
coli

O157:H7
Positive



DD13484

Escherichia
coli

O157:H7
Positive



DD13485

Escherichia
coli

O157:H7
Positive



DD13486

Escherichia
coli

O157:H7
Positive



DD13487

Escherichia
coli

O157:H7
Positive



DD13488

Escherichia
coli

O157:H7
Positive



DD13489

Escherichia
coli

O157:H7
Positive



DD13490

Escherichia
coli

O157:H7
Positive



DD13491

Escherichia
coli

O157:H7
Positive



DD13492

Escherichia
coli

O157:H7
Positive

















TABLE 4







Exclusivity demonstrated towards 300 strains of non-


O157:H7 E. coli










Qualicon





Culture





Collection





Strain #
Strain Name
Serovar
BAX ® Result





DD655

Escherichia
coli

O101:K−:K99
Negative


DD656

Escherichia
coli

O101:K30:K99
Negative


DD683

Escherichia
coli

Serovar unknown
Negative


DD743

Escherichia
coli

Serovar unknown
Negative


DD1715

Escherichia
coli

O136:HNM
Negative


DD1716

Escherichia
coli

O158:H23
Negative


DD1718

Escherichia
coli

O128:H2
Negative


DD1719

Escherichia
coli

O28:HNM
Negative


DD1720

Escherichia
coli

O26:HNM
Negative


DD1721

Escherichia
coli

O114:H32
Negative


DD1722

Escherichia
coli

O127:HNM
Negative


DD1725

Escherichia
coli

O125:H19
Negative


DD1726

Escherichia
coli

O126:H2
Negative


DD1727

Escherichia
coli

O44:H18
Negative


DD1728

Escherichia
coli

O55:HNM
Negative


DD1729

Escherichia
coli

O111:HNM
Negative


DD1730

Escherichia
coli

O86:H25
Negative


DD1731

Escherichia
coli

O167:H5
Negative


DD1732

Escherichia
coli

O143:HNM
Negative


DD1733

Escherichia
coli

O142:H6
Negative


DD1734

Escherichia
coli

O124:H30
Negative


DD1735

Escherichia
coli

O144:HNM
Negative


DD1756

Escherichia
coli

O25:H12
Negative


DD1757

Escherichia
coli

O152:HNM
Negative


DD1758

Escherichia
coli

O63:HNM
Negative


DD1759

Escherichia
coli

O15:H4
Negative


DD1760

Escherichia
coli

O6:H1
Negative


DD1761

Escherichia
coli

O27:HNM
Negative


DD1761

Escherichia
coli

O27:HNM
Negative


DD1762

Escherichia
coli

O164:HNM
Negative


DD1762

Escherichia
coli

O164:HNM
Negative


DD1764

Escherichia
coli

O8:H4
Negative


DD1766

Escherichia
coli

O80:H26
Negative


DD1767

Escherichia
coli

O85:H1
Negative


DD1768

Escherichia
coli

O153:H7
Negative


DD1769

Escherichia
coli

O139:H1
Negative


DD1770

Escherichia
coli

O115:H18
Negative


DD1771

Escherichia
coli

O148:H28
Negative


DD1772

Escherichia
coli

O159:H20
Negative


DD1795

Escherichia
coli

O26:HNM
Negative


DD1796

Escherichia
coli

O86:HNM
Negative


DD1797

Escherichia
coli

O111:HNM
Negative


DD1798

Escherichia
coli

O28:HSM
Negative


DD1799

Escherichia
coli

O142:H(−)
Negative


DD1800

Escherichia
coli

O128:HNM
Negative


DD1801

Escherichia
coli

O142:HNM
Negative


DD1802

Escherichia
coli

O6:HNM
Negative


DD1803

Escherichia
coli

O25:H(−)
Negative


DD1804

Escherichia
coli

O124:H(−)
Negative


DD1805

Escherichia
coli

O143:HNM
Negative


DD1807

Escherichia
coli

O26:H(−)
Negative


DD1808

Escherichia
coli

O111:HNM
Negative


DD1809

Escherichia
coli

O111:HNM
Negative


DD1810

Escherichia
coli

O28:H16
Negative


DD1811

Escherichia
coli

O127:H40
Negative


DD1812

Escherichia
coli

O127:H10
Negative


DD1814

Escherichia
coli

O6:H(−)
Negative


DD1817

Escherichia
coli

O29:H(−)
Negative


DD1818

Escherichia
coli

O136:H8
Negative


DD1819

Escherichia
coli

O18:HNM
Negative


DD1820

Escherichia
coli

O86:H8
Negative


DD1821

Escherichia
coli

O55:H(−)
Negative


DD1822

Escherichia
coli

O28:H8,43
Negative


DD1824

Escherichia
coli

O125:HNM
Negative


DD1825

Escherichia
coli

O25:H8
Negative


DD1827

Escherichia
coli

O20:HNM
Negative


DD1828

Escherichia
coli

O143:HNM
Negative


DD1831

Escherichia
coli

O26:H11
Negative


DD1832

Escherichia
coli

O86:HNM
Negative


DD1833

Escherichia
coli

O55:H9
Negative


DD1834

Escherichia
coli

O29:H51
Negative


DD1835

Escherichia
coli

O127:H(−)
Negative


DD1836

Escherichia
coli

O125:H(−)
Negative


DD1839

Escherichia
coli

O15:H(−)
Negative


DD1840

Escherichia
coli

O143:HNM
Negative


DD1841

Escherichia
coli

O124
Negative


DD1842

Escherichia
coli

O78:HNM
Negative


DD1843

Escherichia
coli

O26:HNM
Negative


DD1844

Escherichia
coli

O119:HSM
Negative


DD1845

Escherichia
coli

O55:HNM
Negative


DD1847

Escherichia
coli

O128:H2,3,6
Negative


DD1848

Escherichia
coli

O126:H27
Negative


DD1849

Escherichia
coli

O27:HNT
Negative


DD1851

Escherichia
coli

O20:HNM
Negative


DD1852

Escherichia
coli

O152:H2,3,7
Negative


DD1853

Escherichia
coli

O124:H8
Negative


DD1854

Escherichia
coli

O44:HNT
Negative


DD1855

Escherichia
coli

O119:H(−)
Negative


DD1856

Escherichia
coli

O144:H25
Negative


DD1857

Escherichia
coli

O144:H42
Negative


DD1858

Escherichia
coli

O111:HNM
Negative


DD1859

Escherichia
coli

O125:H(−)
Negative


DD1860

Escherichia
coli

O126:H27,6
Negative


DD1861

Escherichia
coli

O126:H(−)
Negative


DD1862

Escherichia
coli

O27:HNM
Negative


DD1864

Escherichia
coli

O15:HNM
Negative


DD1865

Escherichia
coli

O144:HNM
Negative


DD1866

Escherichia
coli

O18:H(−)
Negative


DD1869

Escherichia
coli

O55:H(−)
Negative


DD1870

Escherichia
coli

O125:H19
Negative


DD1871

Escherichia
coli

O119:H26
Negative


DD1872

Escherichia
coli

O126:H10
Negative


DD1873

Escherichia
coli

O27:H12
Negative


DD1874

Escherichia
coli

O8:HNM
Negative


DD1875

Escherichia
coli

O15:H6
Negative


DD1876

Escherichia
coli

O124:H8,6,2
Negative


DD1878

Escherichia
coli

O152:H6,8,12
Negative


DD1880

Escherichia
coli

O55:H7
Negative


DD1882

Escherichia
coli

O114:H10
Negative


DD1883

Escherichia
coli

O125:HNM
Negative


DD1884

Escherichia
coli

O158:H7
Negative


DD1886

Escherichia
coli

O164:HNM
Negative


DD1889

Escherichia
coli

O152:H10
Negative


DD1890

Escherichia
coli

O143:HNM
Negative


DD1891

Escherichia
coli

O164:HNM
Negative


DD1893

Escherichia
coli

O114:H8,10
Negative


DD1894

Escherichia
coli

O114:H(−)
Negative


DD1906

Escherichia
coli

O127:H11
Negative


DD1907

Escherichia
coli

O164:HNM
Negative


DD1908

Escherichia
coli

O25:H7
Negative


DD1909

Escherichia
coli

O15:HNM
Negative


DD1910

Escherichia
coli

O164:HNM
Negative


DD1911

Escherichia
coli

O136:H(−)
Negative


DD1913

Escherichia
coli

O26:H11
Negative


DD1915

Escherichia
coli

O28:H(−)
Negative


DD1918

Escherichia
coli

O164:HNM
Negative


DD1919

Escherichia
coli

O8:HNM
Negative


DD1922

Escherichia
coli

O63:H6
Negative


DD1924

Escherichia
coli

O136:H16
Negative


DD1925

Escherichia
coli

O152:HNM
Negative


DD1927

Escherichia
coli

O111:HNM
Negative


DD1930

Escherichia
coli

O158:HNM
Negative


DD1931

Escherichia
coli

O127:H(−)
Negative


DD1932

Escherichia
coli

O78:HNM
Negative


DD1936

Escherichia
coli

O29:H10
Negative


DD1996

Escherichia
coli

O6:H10
Negative


DD1997

Escherichia
coli

O20:HNM
Negative


DD1998

Escherichia
coli

O63:HNM
Negative


DD1999

Escherichia
coli

O29:HNM
Negative


DD2000

Escherichia
coli

O29:H25
Negative


DD2001

Escherichia
coli

O143:HNM
Negative


DD2002

Escherichia
coli

O115:HNM
Negative


DD2019

Escherichia
coli

O148:H(−)
Negative


DD2020

Escherichia
coli

O139
Negative


DD2026

Escherichia
coli

O153:H(−)
Negative


DD2028

Escherichia
coli

O139
Negative


DD2030

Escherichia
coli

O159:H27
Negative


DD2034

Escherichia
coli

O115:HNM
Negative


DD2036

Escherichia
coli

O115:HNM
Negative


DD2037

Escherichia
coli

O115:HNM
Negative


DD2039

Escherichia
coli

O80:HNM
Negative


DD2040

Escherichia
coli

O80:HSM
Negative


DD2041

Escherichia
coli

O153:HNM
Negative


DD2047

Escherichia
coli

O80:HNM
Negative


DD2119

Escherichia
coli

Serovar unknown
Negative


DD2130

Escherichia
coli

Serovar unknown
Negative


DD2132

Escherichia
coli

Serovar unknown
Negative


DD2133

Escherichia
coli

Serovar unknown
Negative


DD2152

Escherichia
coli

Serovar unknown
Negative


DD2414

Escherichia
coli

Serovar unknown
Negative


DD2415

Escherichia
coli

Serovar unknown
Negative


DD2429

Escherichia
coli

O112:H18
Negative


DD2430

Escherichia
coli

O11:H10
Negative


DD2431

Escherichia
coli

O167:H5
Negative


DD2432

Escherichia
coli

O165:H(−)
Negative


DD2433

Escherichia
coli

O163:H19
Negative


DD2434

Escherichia
coli

O1:H7
Negative


DD2435

Escherichia
coli

O166:H4
Negative


DD2436

Escherichia
coli

O91:HNM
Negative


DD2437

Escherichia
coli

O50:H4
Negative


DD2438

Escherichia
coli

O118:HNM
Negative


DD2439

Escherichia
coli

O145:HNM
Negative


DD2440

Escherichia
coli

O121:H10
Negative


DD2441

Escherichia
coli

O117:H4
Negative


DD2442

Escherichia
coli

O82:HNM
Negative


DD2443

Escherichia
coli

O157:H19
Negative


DD2444

Escherichia
coli

O84:H21
Negative


DD2445

Escherichia
coli

O113:H21
Negative


DD2446

Escherichia
coli

O39:HNM
Negative


DD2447

Escherichia
coli

O38:H26
Negative


DD2448

Escherichia
coli

O5:H4
Negative


DD2449

Escherichia
coli

O103:H8
Negative


DD2450

Escherichia
coli

O45:H10
Negative


DD2451

Escherichia
coli

O135:HNM
Negative


DD2452

Escherichia
coli

O4:H5
Negative


DD2453

Escherichia
coli

O2:H4
Negative


DD2457

Escherichia
coli

O146:H21
Negative


DD2458

Escherichia
coli

O121:HNM
Negative


DD2459

Escherichia
coli

O113:H21
Negative


DD2461

Escherichia
coli

O153:H25
Negative


DD2462

Escherichia
coli

O153:H25
Negative


DD2463

Escherichia
coli

O146:H21
Negative


DD2472

Escherichia
coli

O45:H2
Negative


DD2473

Escherichia
coli

O45:H2
Negative


DD2474

Escherichia
coli

O2:H32
Negative


DD2477

Escherichia
coli

O55:H7
Negative


DD2480

Escherichia
coli

O4:HNM
Negative


DD2483

Escherichia
coli

O145:HNM
Negative


DD2484

Escherichia
coli

O25:HNM
Negative


DD2485

Escherichia
coli

O157:H19
Negative


DD2487

Escherichia
coli

O136:HNM
Negative


DD2490

Escherichia
coli

O5:HNM
Negative


DD2491

Escherichia
coli

O2:H7
Negative


DD2500

Escherichia
coli

O124:HNM
Negative


DD2501

Escherichia
coli

O124:HNM
Negative


DD2502

Escherichia
coli

O112:HNM
Negative


DD2503

Escherichia
coli

O135:HNM
Negative


DD2505

Escherichia
coli

O28:HNM
Negative


DD2508

Escherichia
coli

O127:HNM
Negative


DD2511

Escherichia
coli

O127:HNM
Negative


DD2512

Escherichia
coli

O4:HNM
Negative


DD2514

Escherichia
coli

O2:H5
Negative


DD2515

Escherichia
coli

O5:HNM
Negative


DD2517

Escherichia
coli

O2:H5
Negative


DD2518

Escherichia
coli

O2:H7
Negative


DD2519

Escherichia
coli

O91:H21
Negative


DD2520

Escherichia
coli

O113:H7
Negative


DD2521

Escherichia
coli

O103:H2
Negative


DD2522

Escherichia
coli

O91:HNM
Negative


DD2523

Escherichia
coli

O91:HNM
Negative


DD2525

Escherichia
coli

O91:H21
Negative


DD2526

Escherichia
coli

O145:HNM
Negative


DD2530

Escherichia
coli

O103:H2
Negative


DD2533

Escherichia
coli

O113:H21
Negative


DD3124

Escherichia
coli

O2
Negative


DD3127

Escherichia
coli

O7
Negative


DD3130

Escherichia
coli

O8
Negative


DD3132

Escherichia
coli

O2
Negative


DD3166

Escherichia
coli

O2
Negative


DD3197

Escherichia
coli

O75
Negative


DD3199

Escherichia
coli

O2
Negative


DD3204

Escherichia
coli

O2
Negative


DD3208

Escherichia
coli

O103
Negative


DD3210

Escherichia
coli

O103
Negative


DD3785

Escherichia
coli

O111:K58(64):H−
Negative


DD3790

Escherichia
coli

O111:K58(64):H−
Negative


DD4088

Escherichia
coli

Serovar unknown
Negative


DD5883

Escherichia
coli

O55:H10
Negative


DD5884

Escherichia
coli

O91:H−
Negative


DD5887

Escherichia
coli

O111:H−
Negative


DD5901

Escherichia
coli

O5:H−
Negative


DD5902

Escherichia
coli

O26:H11
Negative


DD5903

Escherichia
coli

O26:H11
Negative


DD5904

Escherichia
coli

O26:H11
Negative


DD5905

Escherichia
coli

O26:H11
Negative


DD5906

Escherichia
coli

O55:H7
Negative


DD9703

Escherichia
coli

O26:H11
Negative


DD9704

Escherichia
coli

O26:H11
Negative


DD9705

Escherichia
coli

O26:H11
Negative


DD9706

Escherichia
coli

O26:H11
Negative


DD9707

Escherichia
coli

O26:H11
Negative


DD10922

Escherichia
coli

Serovar unknown
Negative


DD12804

Escherichia
coli

Serovar unknown
Negative


DD12849

Escherichia
coli

Serovar unknown
Negative


DD12851

Escherichia
coli

O142:H+
Negative


DD12859

Escherichia
coli

O−:H−
Negative


DD12884

Escherichia
coli

Serovar unknown
Negative


DD12887

Escherichia
coli

O157:H2
Negative


DD12888

Escherichia
coli

O157:H2
Negative


DD12889

Escherichia
coli

O157:H4
Negative


DD12890

Escherichia
coli

O157:H11
Negative


DD12891

Escherichia
coli

O157:H12
Negative


DD12892

Escherichia
coli

O157:H29
Negative


DD12893

Escherichia
coli

O157:H32
Negative


DD12894

Escherichia
coli

O157:H43
Negative


DD12895

Escherichia
coli

O157:H43
Negative


DD12896

Escherichia
coli

O157:H44
Negative


DD12897

Escherichia
coli

O157:H54
Negative


DD12900

Escherichia
coli

Serovar unknown
Negative


DD12901

Escherichia
coli

O−:H−
Negative


DD12977

Escherichia
coli

Serovar unknown
Negative


DD13041

Escherichia
coli

Serovar unknown
Negative


DD13073

Escherichia
coli

Serovar unknown
Negative


DD13074

Escherichia
coli

Serovar unknown
Negative


DD13076

Escherichia
coli

Serovar unknown
Negative


DD13086

Escherichia
coli

Serovar unknown
Negative


DD13143

Escherichia
coli

Serovar unknown
Negative


DD13196

Escherichia
coli

Serovar unknown
Negative


DD13198

Escherichia
coli

Serovar unknown
Negative


DD13263

Escherichia
coli

Serovar unknown
Negative


DD13292

Escherichia
coli

Serovar unknown
Negative


DD13317

Escherichia
coli

Serovar unknown
Negative


DD13318

Escherichia
coli

Serovar unknown
Negative


DD13319

Escherichia
coli

Serovar unknown
Negative


DD13320

Escherichia
coli

Serovar unknown
Negative


DD13347

Escherichia
coli

Serovar unknown
Negative


DD13348

Escherichia
coli

Serovar unknown
Negative


DD13349

Escherichia
coli

O45:H2
Negative


DD13350

Escherichia
coli

O45:H2
Negative


DD13351

Escherichia
coli

O45:H2
Negative


DD13352

Escherichia
coli

O45:H2
Negative


DD13353

Escherichia
coli

O45:H2
Negative


DD13354

Escherichia
coli

O45:
Negative


DD13355

Escherichia
coli

O45:
Negative


DD13479

Escherichia
coli

Serovar unknown
Negative


DD13481

Escherichia
coli

Serovar unknown
Negative


DD13493

Escherichia
coli

O104:H4
Negative


DD13832

Escherichia
coli

Serovar unknown
Negative


DD13833

Escherichia
coli

Serovar unknown
Negative


DD13834

Escherichia
coli

Serovar unknown
Negative
















TABLE 5







Exclusivity demonstrated towards 185 non-E. coli strains









Qualicon




Culture




Collection

BAX ®


Strain #
Strain Name
Result





DD227

Citrobacter
koseri

Negative


DD373

Klebsiella
pneumoniae

Negative


DD375

Enterobacter
cloacae

Negative


DD375

Enterobacter
cloacae

Negative


DD376

Enterobacter
aerogenes

Negative


DD383

Citrobacter
freundii

Negative


DD383

Citrobacter
freundii

Negative


DD569

Pseudomonas
fluorescens

Negative


DD572

Aeromonas
hydrophila

Negative


DD576

Pseudomonas
mendocina

Negative


DD577

Pseudomonas
stutzeri

Negative


DD584

Salmonella
typhi

Negative


DD585

Salmonella
typhi

Negative


DD586

Salmonella
typhimurium

Negative


DD592

Yersinia
enterocolitica

Negative


DD610

Staphylococcus
aureus

Negative


DD657

Klebsiella
ozaenae

Negative


DD659

Lactococcus
lactis

Negative


DD700

Shigella
sonnei

Negative


DD707

Salmonella
newport

Negative


DD715

Bacillus
cereus

Negative


DD739

Salmonella
stanley

Negative


DD741

Salmonella
gallinarum

Negative


DD919

Salmonella
paratyphi

Negative


DD966

Salmonella
napoli

Negative


DD1074

Enterobacter
cloacae

Negative


DD1081

Shigella
boydii

Negative


DD1082

Shigella
dysenteriae

Negative


DD1085

Salmonella
binza

Negative


DD1248

Salmonella
panama

Negative


DD1251

Salmonella
Kedougou

Negative


DD1329

Salmonella
braenderup

Negative


DD1332

Salmonella
anatum

Negative


DD1336

Salmonella
thompson

Negative


DD1352

Salmonella
agona

Negative


DD1356

Salmonella
bredeney

Negative


DD1429

Salmonella
anfo

Negative


DD1435

Salmonella
brandenburg

Negative


DD1469

Salmonella
ealing

Negative


DD1482

Salmonella
pullorum

Negative


DD1509

Salmonella
bovismorbificans

Negative


DD1510

Salmonella
bareilly

Negative


DD1521

Salmonella
abaetetuba

Negative


DD1523

Salmonella
berkeley

Negative


DD1525

Salmonella
betioky

Negative


DD1526

Salmonella
austin

Negative


DD1530

Salmonella
altendorf

Negative


DD1535

Salmonella
brookfield

Negative


DD1543

Salmonella
adelaide

Negative


DD1547

Salmonella
aberdeen

Negative


DD1548

Salmonella
abony

Negative


DD1552

Salmonella
alabama

Negative


DD2357

Proteus
mirabilis

Negative


DD2389

Hafnia
alvei

Negative


DD2399

Yersinia
aldovae

Negative


DD2552

Enterococcus
faecium

Negative


DD2554

Enterococcus
faecalis

Negative


DD2558

Citrobacter
freundii

Negative


DD2558

Citrobacter
freundii

Negative


DD2559

Citrobacter
amalonaticus

Negative


DD2559

Citrobacter
amalonaticus

Negative


DD2560

Citrobacter
koseri

Negative


DD2560

Citrobacter
koseri

Negative


DD2561

Citrobacter
koseri

Negative


DD2584

Enterobacter
hormaechei

Negative


DD2584

Enterobacter
hormaechei

Negative


DD2586

Klebsiella
planticola

Negative


DD2599

Pantoea
agglomerans

Negative


DD2600

Enterobacter
gergoviae

Negative


DD2601

Enterobacter
cancerogenus

Negative


DD2604

Enterobacter
amnigenus

Negative


DD2604

Enterobacter
amnigenus

Negative


DD2631

Vibrio
fluvialis

Negative


DD2632

Vibrio
vulnificus

Negative


DD3097

Citrobacter
freundii

Negative


DD3114

Pantoea
agglomerans

Negative


DD3785

Escherichia
coli

Negative


DD3863

Salmonella
oranienburg

Negative


DD3882

Salmonella
broughton

Negative


DD3898

Salmonella
thompson

Negative


DD3915

Salmonella
haardt

Negative


DD3982

Pseudomonas aeruginosa

Negative


DD3984

Salmonella
java

Negative


DD4022

Salmonella
enteritidis

Negative


DD4036

Salmonella
livingstone

Negative


DD4102

Salmonella
saintpaul

Negative


DD4656

Citrobacter
freundii

Negative


DD4659

Citrobacter
freundii

Negative


DD5533

Salmonella
infantis

Negative


DD5588

Hafnia
alvei

Negative


DD6121

Prot.
Mirabilis

Negative


DD6177

Salmonella
arkansas

Negative


DD6250

Salmonella
santiago

Negative


DD6523

Klebsiella
oxytoca

Negative


DD6719

Escherichia
hermanni

Negative


DD6735

Salmonella
albany

Negative


DD6832

Shigella
sonnei

Negative


DD7083

Serratia
marcesens

Negative


DD7111

Salmonella
infantis

Negative


DD10001

Cronobacter
sakazakii

Negative


DD10005

Cronobacter
sakazakii

Negative


DD10006

Cronobacter
sakazakii

Negative


DD10006

Enterobacter
sakazakii

Negative


DD10009

Cronobacter
sakazakii

Negative


DD10011

Cronobacter
sakazakii

Negative


DD10014

Cronobacter
sakazakii

Negative


DD10016

Cronobacter
sakazakii

Negative


DD10017

Cronobacter
sakazakii

Negative


DD10019

Cronobacter
sakazakii

Negative


DD10023

Cronobacter
sakazakii

Negative


DD11232

Vibrio
mimicus

Negative


DD11330

Cronobacter
sakazakii

Negative


DD11334

Cronobacter
sakazakii

Negative


DD11335

Cronobacter
sakazakii

Negative


DD11336

Cronobacter
sakazakii

Negative


DD11338

Cronobacter
sakazakii

Negative


DD11339

Cronobacter
sakazakii

Negative


DD11340

Cronobacter
sakazakii

Negative


DD12720

Enterobacter
sakazakii

Negative


DD12720

Cronobacter
sakazakii

Negative


DD12721

Cronobacter
sakazakii

Negative


DD12722

Cronobacter
sakazakii

Negative


DD12723

Cronobacter
sakazakii

Negative


DD12725

Cronobacter
sakazakii

Negative


DD12726

Cronobacter
sakazakii

Negative


DD12727

Cronobacter
sakazakii

Negative


DD12728

Cronobacter
sakazakii

Negative


DD12729

Cronobacter
sakazakii

Negative


DD12731

Cronobacter
sakazakii

Negative


DD12732

Cronobacter
sakazakii

Negative


DD12733

Cronobacter
sakazakii

Negative


DD12760

Enterobacter
cloace

Negative


DD12760

Enterobacter
cloace

Negative


DD12761

Cronobacter
muytjensii

Negative


DD12763

Cronobacter
sakazakii

Negative


DD12764

Cronobacter
sakazakii

Negative


DD12769

Enterobacter
cloace

Negative


DD12777

Pantoea
species

Negative


DD12907

Salmonella
heidelberg

Negative


DD12914

Salmonella
kentucky

Negative


DD12960

Salmonella
senftenberg

Negative


DD12968

Salmonella
haardt

Negative


DD13005

Salmonella
typhimurium

Negative


DD13014

Salmonella
heidelberg

Negative


DD13035

Salmonella
Choleraesuis I
Negative


DD13056

Salmonella
senftenberg

Negative


DD13064

Salmonella
tennessee

Negative


DD13066

Salmonella
tennessee

Negative


DD13067

Salmonella
havana

Negative


DD13068

Salmonella
lexington

Negative


DD13069

Salmonella
mbandaka

Negative


DD13071

Salmonella
montevideo

Negative


DD13075

Salmonella
cubana

Negative


DD13079

Salmonella
newport

Negative


DD13081

Salmonella
virchow

Negative


DD13135

Enterobacter
cloacae

Negative


DD13136

Enterobacter
aerogenes

Negative


DD13144

Citrobacter
koseri

Negative


DD13145

Pantoea
agglomerans

Negative


DD13157

Citrobacter
freundii

Negative


DD13159

Enterobacter
cloacae

Negative


DD13160

Enterobacter
homaechei

Negative


DD13161

Enterobacter
asburiae

Negative


DD13162

Enterobacter
homaechei

Negative


DD13163

Enterobacter
turicensis

Negative


DD13164

Enterobacter
helveticus

Negative


DD13165

Enterobacter novel species
Negative


DD13166

Enterobacter
helveticus

Negative


DD13185

Enterobacter
cloacae

Negative


DD13186

Enterobacter
amnigenus

Negative


DD13187

Enterobacter
amnigenus

Negative


DD13344

salmonella
berta

Negative


DD13477

Citrobacter
brakii

Negative


DD13499

Enterobacter
turicensis

Negative


DD13500

Enterobacter
pulveris

Negative


DD13524

Enterobacter
helveticus

Negative


DD13525

Enterobacter
pulveris

Negative


DD13526

Enterobacter
turicensis

Negative


DD13531

Citrobacter
gillenii

Negative


DD13532

Citrobacter
murliniae

Negative


DD13533

Citrobacter
rodentium

Negative


DD13534

Citrobacter
werkmanii

Negative


DD13535

Citrobacter
farmeri

Negative


DD13630

Salmonella
hadar

Negative


DD13901

Citrobacter
+ G1:H186 freundii
Negative
















Sequence Listing


Number of SEQ ID NOS: 13





SEQ ID NO 1


LENGTH: 20


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Primer





SEQUENCE: 1


ccgcgtgtta cgtccgggct





SEQ ID NO 2


LENGTH: 20


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Primer





SEQUENCE: 2


ttgcataggc gtcaccctga





SEQ ID NO 3


LENGTH: 20


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Primer





SEQUENCE: 3


tgaaacccgc gtgttacgtc





SEQ ID NO 4


LENGTH: 20


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Primer





SEQUENCE: 4


aggcgtcacc ctgaccgtta





SEQ ID NO 5


LENGTH: 20


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Primer





SEQUENCE: 5


gcgtgttacg tccgggctac





SEQ ID NO 6


LENGTH: 20


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Primer





SEQUENCE: 6


gggtttgcat aggcgtcacc





SEQ ID NO 7


LENGTH: 20


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Primer





SEQUENCE: 7


gggctacgtc aagccgaaac





SEQ ID NO 8


LENGTH: 20


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Primer





SEQUENCE: 8


gcataggcgt caccctgacc





SEQ ID NO 9


LENGTH: 21


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Probe





SEQUENCE: 9


tcaagccgaa acacgaattc c





SEQ ID NO 10


LENGTH: 22


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Probe





SEQUENCE: 10


aattcccctg gagccggtaa aa





SEQ ID NO 11


LENGTH: 21


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Probe





SEQUENCE: 11


gaaacacgaa ttcccctgga g





SEQ ID NO 12


LENGTH: 21


TYPE: DNA


ORGANISM: Artificial Sequence


FEATURE: 


OTHER INFORMATION: Probe





SEQUENCE: 12


gccggtaaaa ggagccggta c





SEQ ID NO 13


LENGTH: 122


TYPE: DNA


ORGANISM: Escherichia coli


FEATURE: 





SEQUENCE: 13


TGAAACCCGCGTGTTACGTCCGGGCTACGTCAAGCCGAAACACGAATTCC


CCTGGAGCCGGTAAAAGGAGCCGGTACCACCCTGTGGGTTTATAACGGTC


AGGGTGACGCCTATGCAAACCC





Claims
  • 1. A method for detecting the presence of E. coli O157:H7 in a sample, said sample comprising nucleic acids, said method comprising: (a) providing a reaction mixture comprising a primer pair selected from group consisting of primer pair SEQ ID NO:1 and SEQ ID NO:2, primer pair SEQ ID NO:3 and SEQ ID NO:4, primer pair SEQ ID NO:5 and SEQ ID NO:6, primer pair SEQ ID NO:7 and SEQ ID NO:8, and a combination thereof;(b) performing PCR amplification of said nucleic acids of said sample using the reaction mixture of step (a); and(c) detecting the amplification of step (b), whereby a positive detection of amplification indicates the presence of E. coli O157:H7 in the sample.
  • 2. The method of claim 1, wherein said reaction mixture further comprises a nucleic acid probe.
  • 3. The method of claim 2, wherein said nucleic acid probe comprises SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a combination thereof.
  • 4. The method of claim 2, wherein said probe further comprises a detectable label and quenching molecule.
  • 5. The method of claim 1, wherein the sample comprises a food sample or a water sample.
  • 6. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, wherein the isolated polynucleotide is linked to a detectable label.
  • 7. An isolated polynucleotide comprising a primer region having a nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 and a probe region having a nucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, wherein said polynucleotide further comprises an 18-carbon non-amplifiable linker joining said primer region to said probe region, and wherein said polynucleotide further comprises a detectable label.
  • 8. A replication composition for use in performance of PCR, comprising: (a) a primer pair selected from group consisting of primer pair SEQ ID NO:1 and SEQ ID NO:2, primer pair SEQ ID NO:3 and SEQ ID NO:4, primer pair SEQ ID NO:5 and SEQ ID NO:6, primer pair SEQ ID NO:7 and SEQ ID NO:8, and a combination thereof; and(b) nucleic acid probe comprises SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a combination thereof; and(c) thermostable DNA polymerase.
  • 9. A kit for detection of E. coli O157:H7 in a sample, comprising the replication composition of claim 8.
  • 10. A tablet comprising the replication composition of claim 8.
  • 11. A method for detecting the presence of E. coli O157:H7 in a sample, said sample comprising nucleic acids, said method comprising: (a) providing a reaction mixture comprising a suitable primer pair for amplification of a polynucleotide sequence comprising SEQ ID NO:13;(b) performing PCR amplification of said nucleic acids of said sample using the reaction mixture of step (a); and(c) detecting the amplification of step (b), whereby a positive detection of amplification indicates the presence of E. coli O157:H7 in the sample.
  • 12. The method of claim 11, wherein the polynucleotide sequence of (a) consists of SEQ ID NO:13.
  • 13. The method of claim 11, wherein said primer pair for amplification of SEQ ID NO:13 is selected from group consisting of primer pair SEQ ID NO:1 and SEQ ID NO:2, primer pair SEQ ID NO:3 and SEQ ID NO:4, primer pair SEQ ID NO:5 and SEQ ID NO:6, primer pair SEQ ID NO:7 and SEQ ID NO:8, and a combination thereof.
  • 14. The method of claim 11, wherein said reaction mixture further comprises a nucleic acid probe.
  • 15. The method of claim 14, wherein said nucleic acid probe comprises SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a combination thereof.
  • 16. The method of claim 14, wherein said probe further comprises a detectable label and quenching molecule.
  • 17. The method of claim 11, wherein the sample comprises a food sample or a water sample.
PCT Information
Filing Document Filing Date Country Kind
PCT/US2019/049699 9/5/2019 WO 00
Provisional Applications (1)
Number Date Country
62727755 Sep 2018 US