WO2015003171A2 - Compositions, kits, and related methods for detecting and/or monitoring shiga toxin producing escherichia coli - Google Patents
Compositions, kits, and related methods for detecting and/or monitoring shiga toxin producing escherichia coli Download PDFInfo
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- WO2015003171A2 WO2015003171A2 PCT/US2014/045485 US2014045485W WO2015003171A2 WO 2015003171 A2 WO2015003171 A2 WO 2015003171A2 US 2014045485 W US2014045485 W US 2014045485W WO 2015003171 A2 WO2015003171 A2 WO 2015003171A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Definitions
- This invention relates to detection or monitoring or both of Shiga toxin producing E. coli ("STEC").
- Shiga toxin (stx)-producing Escherichia coli (“STEC”) serotypes There are more than 200 Shiga toxin (stx)-producing Escherichia coli (“STEC”) serotypes, but many have not been implicated in causing illness. STEC may cause devastating illnesses, particularly in children, of varying severity, from diarrhea (often bloody), hemorrhagic colitis, and abdominal cramps to kidney disorders. Outbreaks of illnesses caused by STEC have been epidemiologically related to contact with animals and consumption of meat and fresh produce. Shiga toxin will bind to tissues in the kidneys and cause hemolytic uremic syndrome ("H US”), leading to kidney failure and death. STEC also may cause asymptomatic infections and extraintestinal infections. Enterohemorrhagic E.
- H US hemolytic uremic syndrome
- EHEC EH coli
- STEC STEC coli
- EH EC infections like STEC infections, result in hemorrhagic colitis, which may progress into life-threatening HUS.
- E. coli 0157:H7 is the most notorious STEC/EHEC strain most often associated with the most severe forms of disease. 0157: H7 is a known food-borne pathogen increasingly causing illness worldwide.
- non-0157 STEC isolates have also been linked to illnesses and outbreaks of disease.
- Six O groups have been described by the U.S. Center for Disease Control (“CDC") to be the cause of the majority of non-0157 STEC disease. These serotypes have been identified as 026, 045, O103, Olll, 0121, and 0145, and are commonly referred to as the "big six" non-0157 STEC. It is estimated that non-0157 STEC may cause diarrhea at frequencies similar to other enteric bacterial pathogens, such as Salmonella and Shigella. Non-0157 STEC also causes infections resulting in HUS.
- coli 0157:H7 and non-0157 shiga toxin-producing E.coli (STEC) strains are associated with severe illnesses such as hemorrhagic colitis (HC) and as mentioned above HUS, and have become an increasing concern to the beef industry, regulatory agencies, and the public (Bosilevac et al. 2011. Appl Environ Microbiol 77:2103-2112.).
- the U.S. Department of Agriculture Food Safety and Inspection Service (USDA FSIS) has determined, in addition to E. coli 0157:H7, six most frequent STEC serogroups are adulterants in raw, non-intact beef products or components of such products. These six most frequent O serogroups were identified by the U.S.
- the invention features a method for testing a sample for the presence of a virulent Escherichia coli, the method including detecting the presence of (i) ecf (e.g., the ec/operon, ecfl, ecf2, ecf3, and ec/4) and (ii) wzx and/or stx (e.g., stxl or stx2 or any stx described herein) in the sample, wherein detection of (i) ec/ and (ii) wzx and/or stx in the sample is taken as an indication that the sample includes the virulent E. coli strain.
- ecf e.g., the ec/operon, ecfl, ecf2, ecf3, and ec/4
- wzx and/or stx e.g., stxl or stx2 or any st
- the detection of (i) and/or (ii) can be detection of a nucleic acid encoding (i) ecf and/or (ii) wzx and/or stx (e.g., stxl or stx2 or any stx described herein).
- detection of (i) or (ii) can include detection of an (i) ecf (e.g., ecfl, ecf2, ecf3, and ecf4) polypeptide and/or (ii) a wzx polypeptide and/or stx (e.g., stxl or stx2) polypeptide.
- Exemplary samples include virtually any material possibly contaminated with an E. coli pathogen.
- Samples include any food, water, biological, environmental or pharmaceutical sample as disclosed herein. Virtually any sample suspected of being contaminated with a virulent E. coli is tested using the methods and compositions described in this application.
- Exemplary samples include pharmaceutical, environmental (e.g., air, soil, lakes, rivers, or other water samples including sewage) or agricultural samples (e.g., those collected from agricultural watersheds as well as those collected from field and farm environments), samples obtained from cattle or other livestock including chickens and turkeys (such as during live animal production or during animal harvest), finished food products (e.g., for human or animal consumption), food ingredients and raw food materials, food samples (e.g., drinks and beverages (unpasteurized fresh-pressed juices such as apple cider), dairy products, yogurt, and cheese made from raw milk as well as raw, frozen, or processed foods), meat samples (e.g., raw ground beef, high fat ground beef, raw ground beef components (e.g., beef and veal bulk packed manufacturing trimmings and other beef and veal components such as primal cuts, sub primal cuts, head meat, cheek meat, esophagus meat, heart, and advanced meat recovery product intended for grinding)), produce such as fruits (e.g., grapes, apples, peaches, or strawberries),
- Samples may also be collected to investigate foodborne outbreaks such as those originating in a restaurant or a food processing plant. Other samples are collected to facilitate checking the safety of a foodstuff suspected of being contaminated by a pathogen. Such a foodstuff may be for human or animal consumption, and may be in the form of a food or a beverage. Samples may be enriched as desired according to standard methods. The methods provide for testing to determine the presence or absence of the markers described herein according to standard techniques well known in the art.
- the detection of ec/ and wzx is taken as an indication of the presence of E. coli 0157:1-17. In these or other embodiments, detection of ec/ and the absence of wzx is taken as an indication of the presence of non-0157:H7 shiga toxin (six)-containing E. coli (STEC). In these or yet other embodiments, detection of ec/ and stx is taken as an indication of the presence of enterohemorrhagic Escherichia coli (EHEC).
- the sample is obtained following enrichment of the sample, such as high fat ground beef, beef trim, or produce (for example, fruits such as grapes, apples, peaches, or strawberries and/or vegetables such as lettuce, spinach, radishes and alfalfa sprouts).
- high fat ground beef, beef trim, or produce for example, fruits such as grapes, apples, peaches, or strawberries and/or vegetables such as lettuce, spinach, radishes and alfalfa sprouts.
- the detecting can include, e.g., contacting the sample with an oligonucleotide (e.g., an oligonucleotide with a detectable label) that hybridizes to a portion of a nucleic acid encoding the ecf operon, a nucleic acid encoding wzx, a nucleic acid encoding stxl, or a nucleic acid encoding stx2.
- an oligonucleotide e.g., an oligonucleotide with a detectable label
- These detection methods may include a hybridization assay selected from the group including of a Transcription Mediated Amplification (TMA) reaction, a Nucleic Acid Sequence-Based Amplification (NASBA) reaction, a Polymerase Chain Reaction (PCR) reaction, a hybridization protection assay, or a non-amplified hybridization reaction.
- TMA Transcription Mediated Amplification
- NASBA Nucleic Acid Sequence-Based Amplification
- PCR Polymerase Chain Reaction
- hybridization protection assay a non-amplified hybridization reaction.
- the method may include a polypeptide detection assay, e.g., an immunoassay.
- the polypeptide detection methods generally include, e.g., contacting the sample with a molecule (e.g., a molecule with a detectable label) that specifically binds to a polypeptide selected from the group including of ecfl, ecf2, ecf3, ecf4, wzx, stxl, and stx2. Examples of such molecules include an antibody or fragment thereof.
- the invention features a method for producing a packaged foodstuff free of a virulent Escherichia coli adulterant, the method including the steps of a) providing a sample obtained from a foodstuff; b) testing the foodstuff for the presence of (i) ecf (e.g., the ecf operon, ecfl, ecf2, ecf3, and ec/4) and (ii) wzx and/or stx (e.g., stxl or stx2) in the sample, wherein absence of (i) ec/ and (ii) wzx and/or stx in the sample is taken as an indication that the sample is free of pathogenic E. coli adulterant; and c) packaging the foodstuff identified as free of the pathogenic E. coli adulterant (e.g., packaging the foodstuff in a carton, container, plastic wrap, or a foodstuff tray wrapped with plastic).
- the invention features a method for producing a packaged lot of meat free of a virulent Escherichia coli adulterant, the method including the steps of a) providing a sample obtained from a lot of meat (e.g., where the sample is obtained following enrichment of a meat sample);
- ecf e.g., the ecf operon, ecfl, ecf2, ecf3, and ec/4
- wzx and/or stx e.g., stxl or stx2
- absence of (i) ec/ and (ii) wzx and/or stx in the sample is taken as an indication that the sample is free of pathogenic E. coli adulterant
- packaging meat identified as free of the pathogenic E. coli adulterant e.g., packaging the meat in a carton, container, plastic wrap, or a meat tray wrapped with plastic.
- the detection of (i) and/or (ii) can be detection of a nucleic acid encoding (i) ecf (e.g., the ecf operon, ecfl, ecf2, ecf3, and ec/4) and/or (ii) wzx and/or stx (e.g., stxl or stx2).
- ecf e.g., the ecf operon, ecfl, ecf2, ecf3, and ec/4
- wzx and/or stx e.g., stxl or stx2
- detection of (i) or (ii) can include detection of (i) an ecf (e.g., ecfl, ecf2, ecf3, and ec/4) polypeptide and/or (ii) a wzx polypeptide and/or stx (e.g., stxl or stx2) polypeptide.
- an ecf e.g., ecfl, ecf2, ecf3, and ec/4
- a wzx polypeptide and/or stx e.g., stxl or stx2
- the detection of ec/ and wzx is taken as an indication of the presence of E. coli 0157:1-17. In these or other embodiments, detection ec/ and the absence of wzx is taken as an indication of the presence of non-0157:H7 shiga toxin (stx)-containing E. coli (STEC). In these or yet other embodiments detection of ec/ and stx is taken as an indication of the presence of enterohemorrhagic Escherichia coli (EHEC).
- the detecting can include, e.g., contacting the sample with an oligonucleotide (e.g., an oligonucleotide with a detectable label) that hybridizes to a portion of a nucleic acid encoding the ec/ operon, a nucleic acid encoding wzx, a nucleic acid encoding stxl, or a nucleic acid encoding stx2.
- an oligonucleotide e.g., an oligonucleotide with a detectable label
- These detection methods may include a hybridization assay selected from the group including of a Transcription Mediated Amplification (TMA) reaction, a Nucleic Acid Sequence-Based Amplification (NASBA) reaction, a Polymerase Chain Reaction (PCR) reaction, a hybridization protection assay, or a non-amplified hybridization reaction.
- TMA Transcription Mediated Amplification
- NASBA Nucleic Acid Sequence-Based Amplification
- PCR Polymerase Chain Reaction
- hybridization protection assay a non-amplified hybridization reaction.
- the method may include a polypeptide detection assay, e.g., an immunoassay.
- the polypeptide detection methods can also include, e.g., contacting the sample with a molecule (e.g., a molecule with a detectable label) that specifically binds to a polypeptide selected from the group including of ecfl, ecf2, ecf3, ecf4, wzx, stxl, and stx2 or any stx described herein. Examples of such molecules include an antibody or fragment thereof.
- the foregoing methods can, e.g., further include shipping the packaged meat.
- the lot of meat can include, e.g., raw ground beef, high fat ground beef, raw ground beef components (e.g., beef and veal bulk packed manufacturing trimmings and other beef and veal components such as primal cuts, sub primal cuts, head meat, cheek meat, esophagus meat, heart, and advanced meat recovery product intended for grinding).
- sample provided for enrichment is, e.g., about 200 g to about 500 g (e.g., about 325 g to about 375 g).
- the invention features a method for producing a lot of produce free of a pathogenic Escherichia coli adulterant, the method including the steps of a) providing a sample obtained from a lot of produce (e.g., where the sample is obtained following enrichment of a produce sample);
- the detection of (i) and/or (ii) can be detection of a nucleic acid encoding (i) ecf (e.g., the ecf operon, ecfl, ecf2, ecf3, and ec/4) and/or (ii) wzx and/or stx (e.g., stxl or stx2).
- ecf e.g., the ecf operon, ecfl, ecf2, ecf3, and ec/4
- wzx and/or stx e.g., stxl or stx2
- detection of (i) or (ii) can include detection of (i) an ecf (e.g., ecfl, ecf2, ecf3, and ecf4) polypeptide and/or (ii) a wzx polypeptide and/or stx (e.g., stxl or stx2) polypeptide.
- an ecf e.g., ecfl, ecf2, ecf3, and ecf4
- a wzx polypeptide and/or stx e.g., stxl or stx2
- the detection of ec/ and wzx is taken as an indication of the presence of E. coli 0157:1-17. In these or other embodiments, detection ec/ and the absence of wzx is taken as an indication of the presence of non-0157:H7 shiga toxin (six)-containing E. coli (STEC). In these or yet other embodiments detection of ec/ and stx is taken as an indication of the presence of enterohemorrhagic Escherichia coli (EHEC).
- the detecting can include, e.g., contacting the sample with an oligonucleotide (e.g., an oligonucleotide with a detectable label) that hybridizes to a portion of a nucleic acid encoding the ec/ operon, a nucleic acid encoding wzx, a nucleic acid encoding stxl, or a nucleic acid encoding stx2.
- an oligonucleotide e.g., an oligonucleotide with a detectable label
- These detection methods may include a hybridization assay selected from the group including of a Transcription Mediated Amplification (TMA) reaction, a Nucleic Acid Sequence-Based Amplification (NASBA) reaction, a Polymerase Chain Reaction (PCR) reaction, a hybridization protection assay, or a non-amplified hybridization reaction.
- TMA Transcription Mediated Amplification
- NASBA Nucleic Acid Sequence-Based Amplification
- PCR Polymerase Chain Reaction
- hybridization protection assay a non-amplified hybridization reaction.
- the method may include a polypeptide detection assay, e.g., an immunoassay.
- the polypeptide detection methods can also include, e.g., contacting the sample with a molecule (e.g., a molecule with a detectable label) that specifically binds to a polypeptide selected from the group including of ecfl, ecf2, ecf3, ecf4, wzx, stxl, and stx2. Examples of such molecules include an antibody or fragment thereof.
- the foregoing methods can, e.g., further include shipping the packaged produce.
- the lot of produce can include, e.g., fruit or vegetables (such as lettuce, spinach, cabbage, celery, cilantro, coriander, cress sprouts, radishes, or alfalfa sprouts).
- sample provided for enrichment is, e.g., about 200 g to about 500 g (e.g., about 325 g to about 375 g).
- the detecting of (i) and detecting of (ii) can be performed in a single or multiple reaction mixtures.
- the invention features a composition including (i) a first oligonucleotide that specifically hybridizes to a nucleic acid encoding the ec/operon, or portion thereof (e.g., ecfl, ecf 2, ecf 3, or ec/4), and (ii) a second oligonucleotide that specifically hybridizes to a nucleic acid encoding wzx, stxl, or stx2.
- the first and or second oligonucleotide can be, e.g., detectably labeled.
- compositions can, e.g., further including primers for performing a Transcription Mediated Amplification (TMA) reaction, a Nucleic Acid Sequence-Based Amplification (NASBA) reaction and/or a Polymerase Chain Reaction (PCR) reaction.
- TMA Transcription Mediated Amplification
- NASBA Nucleic Acid Sequence-Based Amplification
- PCR Polymerase Chain Reaction
- the invention features a composition including (i) a first amplicon produced by a method of amplifying a nucleic acid encoding the ec/ operon (e.g., ecfl, ecf2, ecf3, or ec/4) and (ii) a second amplicon produced by a method of amplifying a nucleic acid encoding wzx, stxl, or stx2.
- the method of amplifying the nucleic acid is selected from Transcription Mediated Amplification (TMA) reaction, a Nucleic Acid Sequence-Based Amplification (NASBA) reaction and a Polymerase Chain Reaction (PCR) reaction.
- TMA Transcription Mediated Amplification
- NASBA Nucleic Acid Sequence-Based Amplification
- PCR Polymerase Chain Reaction
- the first and/or second amplicon can be, e.g., detectably labeled.
- the invention also relates to the use of ECF such as the ecf operon/gene cluster (e.g., ECF2-1 and ECF2-2 described herein) to detect virulent STECs including virulent non-0157:H7 STEC and virulent non- 0157:1-17 EHEC.
- ECF such as the ecf operon/gene cluster (e.g., ECF2-1 and ECF2-2 described herein) to detect virulent STECs including virulent non-0157:H7 STEC and virulent non- 0157:1-17 EHEC.
- ECF such as the ecf operon/gene cluster (e.g., ECF2-1 and ECF2-2 described herein) to detect virulent STECs including virulent non-0157:H7 STEC and virulent non- 0157:1-17 EHEC.
- Use of this nucleic acid target, in combination, with other targets such as Z5866, rfb 0 i5 7 , WZX0157
- the invention accordingly relates to compositions, kits, and methods used for the detection of E. coli STEC.
- the invention is based at least in part on the discovery that certain E. coli sequences are surprisingly efficacious for the detection of 0157:1-17 and virulent non-0157 STECs such as the big six: 026, 045, O103, 0111, 0121, and 0145.
- particular regions of 0157:1-17 STEC have been identified as useful targets for nucleic acid amplification and, which when used in combination, provide improvements in relation to specificity, sensitivity, or speed of detection as well as other advantages.
- virulent non-0157:H7 STEC is meant any E.
- coli bacterium containing an fc/gene cluster other than 0157:H7.
- Exemplary virulent non-0157:H7 STEC include E. coli such as 026, 045, O103, 0111, 0121, and 0145.
- Other exemplary non-0157:H7 STEC are those containing stxl or stx2 in combination with eae and the large EHEC plasmid.
- the invention accordingly further features a first method for assigning whether a sample includes Shiga- toxin producing E.
- the method includes the steps of: (a) providing nucleic acids from a sample; (b) detecting an 0157-specific fragment and an ECF-specific fragment; (c) assigning to the sample one of the following outcomes: 1) if the 0157-specific fragment and the ECF-specific fragment are absent then the sample is negative for virulent 0157 STEC and a virulent non-0157:H7 STEC; 2) if the 0157-specific fragment is present and the ECF-specific fragment is absent then the sample is negative for a virulent non-0157:H7 STEC; 3) if the 0157-specific fragment and ECF-specific fragment are present then the sample includes virulent 0157 STEC; or 4) if the 0157-specific fragment is absent and the ECF-specific fragment is present then the sample includes a virulent non-0157:H7 STEC.
- This method typically includes an 0157-specific fragment which is rfb, wzx, or wzy as is disclosed herein.
- Exemplary virulent 0157 STEC include 0157:H7, 0157:NM, 0157:H-, 0157:H8, or 0157:H21.
- exemplary virulent, non-0157:H7 STEC includes 026, 045, O103, 0111, 0121, or 0145.
- the method also involves detection of at least two 0157-specific fragments (e.g., rfb and wzk, rfb and wzy, and wzk and wzy, or rfb, wzk, and wzy).
- Other exemplary 0157-specific fragments include katP junction and Z5866.
- the invention features a second method for assigning whether a sample includes STEC, the method includes the steps of: (a) providing nucleic acids from a sample; (b) detecting an 0157:H7-specific fragment and a ECF-specific fragment; (c) assigning to the sample one of the following outcomes: 1) if the 0157:H7-specific fragment and the ECF-specific fragment are absent then the sample is negative for 0157:1-17 STEC and a virulent non-0157:H7 STEC is present; 2) if the 0157:1-17- specific fragment is present and the ECF-specific fragment is absent then the sample is negative for a virulent non-0157:H7 STEC; 3) if the 0157:H7-specific fragment and the ECF-specific fragment are both present then the sample includes an 0157:H7 STEC; or 4) if the 0157:H7-specific fragment is absent and the ECF-specific fragment is present then the sample includes a virulent non-0157:H7 STEC.
- Exemplary 0157:H7-specific fragments include katP junction or Z5866 as is described herein.
- Exemplary virulent, non-0157:H7 STEC includes 026, 045, O103, 0111, 0121, or 0145.
- the method also involves, in certain embodiments, detection of at least two 0157:H7-specific fragments.
- the invention features a third method of assigning whether a sample includes STEC, the method includes the steps of: (a) providing nucleic acids from a sample; (b) detecting a first fragment that detects 0157 STEC and STEC lacking an ECF gene, and a second fragment that detects an ECF gene;(c) assigning to the sample one of the following outcomes: 1) if the first and second fragments are absent then the sample is negative for virulent 0157 STEC and a virulent non-0157:H7 STEC; 2) if the first fragment is present and the second fragment is absent then the sample is negative for a virulent non-0157:H7 STEC; 3) if the first fragment and second fragment are present then the sample includes virulent 0157 STEC; or 4) if the first fragment is absent and the second fragment is present then the sample includes a virulent non-0157:H7 STEC.
- Exemplary first fragments include Sil or Z0372, as is described herein.
- Exemplary virulent 0157 STEC includes 0157:1-17, 0157:NM, 0157:H-, 0157:1-18, or 0157:H21.
- exemplary virulent, non-0157:H7 STEC includes 026, 045, O103, 0111, 0121, or 0145.
- the method also involves detection of at least two first fragments (e.g., Sil and Z0372).
- the invention features a fourth method of assigning whether a sample includes STEC, the method includes the steps of: (a) providing nucleic acids from a sample; (b) detecting a first fragment that detects 0157:H7 STEC and STEC lacking an ECF gene, and a second fragment that detects the ECF gene; (c) assigning to the sample one of the following outcomes: 1) if the first and second fragments are absent then the sample is negative for 0157:H7 STEC and a virulent non-0157:H7 STEC; 2) if the first fragment is present and the second fragment is absent then the sample is negative for virulent non-0157:H7 STEC; 3) if the first fragment and second fragment are present then the sample includes an 0157:H7 STEC; or 4) if the first fragment is absent and the second fragment is present then the sample includes a virulent non-0157:H7 STEC.
- Exemplary virulent, non-0157:H7 STEC includes 026, 045, O103, 0111, 0121,
- the invention features still a method for detecting STEC in a sample, the method including the steps of: a) providing a sample including nucleic acid molecules; b) contacting the nucleic acid molecules with a virulent 0157 STEC-specific probe and an ECF-specific probe under hybridization conditions, wherein i) the virulent 0157 STEC-specific probe specifically hybridizes to a virulent 0157 STEC-specific fragment of the nucleic acid molecules; and ii) the ECF-specific probe specifically hybridizes to an ECF-specific fragment of the nucleic acid molecules; and c) detecting hybridization of the virulent 0157 STEC-specific probe and the ECF-specific probe to identify the presence or absence of the virulent 0157 STEC-specific fragment or the ECF-specific fragment as an indication of the presence of absence of STEC in the sample.
- the absence of the virulent 0157 STEC-specific fragment and absence of the ECF-specific fragment is taken as an indication that the sample is negative for virulent 0157 STEC and a virulent non-0157:H7 STEC; the presence of the virulent 0157-specific fragment and the absence of the ECF-specific fragment is taken as an indication that the sample is negative for a virulent non- 0157:H7 STEC; the presence of the virulent 0157-specific fragment and the presence of the ECF-specific fragment is taken as an indication that the sample is positive for virulent 0157 STEC; or the absence of the virulent 0157 STEC-specific fragment and the presence of the ECF-specific fragment is taken as an indication that the sample is positive for a virulent non-0157:H7 STEC.
- Exemplary virulent 0157 STEC- specific fragments include rfb, wzx, or wzy.
- Exemplary virulent 0157 STEC includes 0157:H7, 0157:NM, 0157:1-1-, 0157:1-18, or 0157:H21.
- exemplary virulent, non-0157:H7 STEC includes 026, 045, O103, 0111, 0121, or 0145.
- the method also involves detection of at least two virulent 0157 STEC-specific fragments (e.g., rfb and wzk, rfb and wzy, and wzk and wzy, or rfb, wzk, and wzy).
- Exemplary methods for detecting hybridization involve amplification or cDNA synthesis. Nucleic acid molecules, if desired, are typically purified from an environmental or a biological sample (e.g., a food sample such as meat).
- the invention features a method for detecting STEC in a sample, the method includes the steps of: a) providing a sample including nucleic acid molecules; b) contacting the nucleic acid molecules with an 0157:H7-specific probe and an ECF-specific probe under hybridization conditions, wherein i) the 0157:H7-specific probe specifically hybridizes to an 0157:H7-specific fragment of the nucleic acid molecules; and ii) the ECF-specific probe specifically hybridizes to an ECF-specific fragment of the nucleic acid molecules; and c) detecting hybridization of the 0157:H7-specific probe and the ECF- specific probe to identify the presence or absence of the 0157:H7-specific fragment or the ECF-specific fragment as an indication of the presence of absence of STEC in the sample.
- the absence of the 0157:H7-specific fragment and absence of the ECF-specific fragment is taken as an indication that the sample is negative for 0157:H7 STEC and a virulent non-0157:H7 STEC; the presence of the 0157:H7-specific fragment and the absence of the ECF-specific fragment is taken as an indication that the sample is negative for a virulent non-0157:H7 STEC; the presence of the 0157:H7-specific fragment and the presence of the ECF-specific fragment is taken as an indication that the sample is positive for an 0157:H7 STEC; or the absence of the 0157:H7-specific fragment and the absence of the ECF-specific fragment is taken as an indication that the sample is positive for a virulent non-0157:H7 STEC.
- Exemplary 0157:H7-specific fragments include katP junction or Z5866 as is described herein.
- Exemplary virulent, non-0157:H7 STEC include 026, 045, O103, 0111, 0121, or 0145.
- the method also involves detection of at least two 0157:H7-specific fragments (e.g, katP and Z5866).
- Standard methods for detecting hybridization involve amplification or cDNA synthesis.
- Nucleic acid molecules, if desired, are typically purified from an environmental or a biological sample (e.g., a food sample such as meat).
- the invention features a method for detecting STEC in a sample, the method includes the steps of: a) providing a sample including nucleic acid molecules; b) contacting the nucleic acid molecules with a first probe and a second probe under hybridization conditions, wherein i) the first probe specifically hybridizes with nucleic acid molecules of (1) a virulent 0157 STEC and (2) STEC lacking an ECF gene; and ii) the second probe specifically hybridizes to an ECF-specific fragment of the nucleic acid molecules; and c) detecting hybridization of the first probe and the second probe, wherein the presence or absence of hybridization to the first probe and the second probe is taken as indication of the presence or absence of STEC in the sample.
- the absence of hybridization to the first probe and absence of hybridization to the second probe is taken as an indication that the sample is negative for virulent 0157 STEC and a virulent non-0157:H7 STEC; the presence of hybridization to the first probe and the absence of hybridization to the second probe is taken as an indication that the sample is negative for a virulent non-0157:H7 STEC; the presence of hybridization to the first probe and the presence of hybridization to the second probe is taken as an indication that the sample is positive for virulent 0157 STEC; or the absence of hybridization to the first probe and the presence of hybridization to the second probe is taken as an indication that the sample is positive for a virulent non-0157:H7 STEC.
- Exemplary first fragments include Sil or Z0372 as is described herein.
- Exemplary virulent 0157 STEC includes 0157:H7, 0157:NM, 0157:1-1-, 0157:1-18, or 0157:1-121.
- Exemplary virulent, non-0157:H7 STEC includes 026, 045, O103, 0111, 0121, or 0145.
- the method also involves detection of at least two first fragments (e.g., Sil and Z0372). Standard methods for detecting hybridization involve amplification or cDNA synthesis. Nucleic acid molecules, if desired, are typically purified from an environmental or a biological sample (e.g. a food sample such as meat).
- the invention features an method for detecting STEC in a sample, the method including the steps of: a) providing a sample including nucleic acid molecules; b) contacting the nucleic acid molecules with a first probe and a second probe under hybridization conditions, wherein i) the first probe specifically hybridizes with nucleic acid molecules of (1) an 0157:H7 STEC and (2) STEC lacking an ECF gene; and ii) the second probe specifically hybridizes to an ECF-specific fragment of the nucleic acid molecules; and c) detecting hybridization of the first probe and the second probe, wherein the presence or absence of hybridization to the first probe and the second probe is taken as indication of the presence or absence of STEC in the sample.
- the absence of hybridization to the first probe and absence of hybridization to the second probe is taken as an indication that the sample is negative for 0157 STEC and a virulent non-0157:H7 STEC; the presence of hybridization to the first probe and the absence of hybridization to the second probe is taken as an indication that the sample is negative for a virulent non-0157:H7 STEC; the presence of hybridization to the first probe and the presence of hybridization to the second probe is taken as an indication that the sample is positive for an 0157:H7 STEC; or the absence of hybridization to the first probe and the presence of hybridization to the second probe is taken as an indication that the sample is positive for a virulent non-0157:H7 STEC.
- Standard methods for detecting hybridization involve amplification or cDNA synthesis. Nucleic acid molecules, if desired, are typically purified from an environmental or a biological sample (e.g., a food sample such as meat).
- the invention features a method for assessing the presence or absence of virulent non-0157:H7 STEC in a sample, the method includes the steps of: a) contacting nucleic acid molecules from the sample with an ECF-specific probe under hybridization conditions, wherein the ECF -specific probe specifically hybridizes to an ECF-specific region; and b) detecting hybridization of the ECF-specific probe and the nucleic acid molecules, wherein presence or absence of hybridization of the ECF-specific probe with the nucleic acid molecules indicates the presence or absence of virulent non-0157:H7 STEC in the sample.
- the nucleic acid molecules are contacted with a virulent 0157 STEC-specific probe that specifically hybridizes to a virulent 0157 STEC-specific fragment of the nucleic acid molecules, and wherein (i) absence of hybridization of the 0157 STEC-specific probe and absence of hybridization of the ECF-specific probe is taken as an indication that the sample is negative for virulent 0157 STEC and a virulent non-0157:H7 STEC; (ii) the presence of hybridization of the virulent 0157- specific fragment and the absence of hybridization of the ECF-specific fragment is taken as an indication that the sample is negative for a virulent non-0157:H7 STEC; (iii) the presence of hybridization of the virulent 0157-specific fragment and the presence of hybridization of the ECF-specific fragment is taken as an indication that the sample is positive for virulent 0157 STEC; or (iv) the absence of hybridization of the virulent 0157 STEC-specific fragment and the presence of hybridization of the ECF-
- the nucleic acid molecules may also be contacted with a 0157:H7-specific probe that specifically hybridizes to an 0157:H7-specific fragment of the nucleic acid molecules, and (i) the absence of hybridization of the 0157:H7-specific fragment and absence of hybridization of the ECF-specific fragment is taken as an indication that the sample is negative for 0157:H7 STEC and a virulent non-0157:H7 STEC; (ii) the presence of hybridization of the 0157:H7-specific fragment and the absence of hybridization of the ECF- specific fragment is taken as an indication that the sample is negative for a virulent non-0157:H7 STEC;
- the nucleic acid molecules may be contacted with a probe (a') that specifically hybridizes with nucleic acid molecules of (1) a virulent 0157 STEC and (2) STEC lacking an ECF gene; and wherein (i) the absence of hybridization to the probe (a') and absence of hybridization to the ECF-specific fragment is taken as an indication that the sample is negative for virulent 0157 STEC and a virulent non-0157:H7 STEC, (ii) the presence of hybridization to the probe (a') and the absence of hybridization to the ECF- specific fragment is taken as an indication that the sample is negative for a virulent non-0157:H7 STEC; (iii) the presence of hybridization to the probe (a') and the presence of hybridization to the ECF-specific fragment is taken as an indication that the sample is positive for virulent 0157 STEC, (iv) the absence of hybridization to the probe (a') and the presence of hybridization to the ECF-specific fragment is taken as an indication that the
- the nucleic acid molecules may be contacted with a probe (b') that specifically hybridizes with nucleic acid molecules of (1) an 0157:H7 STEC and (2) STEC lacking an ECF gene, and wherein (i) the absence of hybridization to probe (b') and absence of hybridization to the ECF-specific fragment is taken as an indication that the sample is negative for 0157 STEC and a virulent non-0157:H7 STEC; (ii) the presence of hybridization to the probe (b') and the absence of hybridization to the ECF-specific fragment is taken as an indication that the sample is negative for a virulent non-0157:H7 STEC, (iii) the presence of hybridization to the probe (b') and the presence of hybridization to the ECF-specific fragment is taken as an indication that the sample is positive for an 0157:H7 STEC, and (iv) the absence of hybridization to the probe (b') and the presence of hybridization to the ECF-specific fragment is taken as an indication that the
- the invention features targets for identifying a STEC as well as oligonucleotides or primers, alone or in combination, which are useful for identifying or amplifying such targets.
- targets for identifying a STEC as well as oligonucleotides or primers, alone or in combination, which are useful for identifying or amplifying such targets.
- Exemplary target sequences and oligonucleotides are described herein (see, for example, Figures 1-9 and Table 2 as well as other sequences described herein, respectively).
- the invention features a nucleic acid consisting of a nucleic acid sequence wherein the nucleic acid sequence is a 1318 bp Z5886 shown in Fig. 1 or a fragment thereof or sequence complementary thereto.
- the invention features a composition including a nucleic acid consisting of a nucleic acid sequence wherein the nucleic acid sequence is a fragment of the Ecf gene cluster shown in Fig. 2 or a fragment thereof or sequence complementary thereto, wherein the fragment is 1-2404 bp or 3584 - 5612 bp as shown in Fig. 2.
- Exemplary nucleic acid sequences are the 949 bp Ecf2-1 fragment or the 1050 bp Ecf2-2 fragment, each disclosed herein.
- an isolated nucleic acid sequence selected from the group consisting of: 5'-CCC TTA TGA AGA GCC AGT ACT GAA G-3' (SEQ ID NO: 1) and 5' ATT ACG CAT AGG GCG TAT CAG CAC-3' (SEQ ID NO: 2).
- Ecf primers include the following or combinations thereof:
- the invention features a composition including a nucleic acid consisting of a nucleic acid sequence wherein the nucleic acid sequence is a 1269 bp Rfb 0 i5 7 shown in Fig. 3 or a fragment thereof or sequence complementary thereto.
- the invention features a composition including a nucleic acid consisting of a nucleic acid sequence wherein the nucleic acid sequence is a 1392 bp ⁇ Nzx 01S7 shown in Fig. 4 or a fragment thereof or sequence complementary thereto.
- the invention features a composition including a nucleic acid consisting of a nucleic acid sequence wherein the nucleic acid sequence is a 1185 bp Wzy 0 i5 7 shown in Fig. 5 or a fragment thereof or sequence complementary thereto.
- the invention features a composition including a nucleic acid consisting of a nucleic acid sequence wherein the nucleic acid sequence is a 2634 bp SI L0 157 shown in Fig. 6 or a fragment thereof or sequence complementary thereto.
- the invention features a composition including a nucleic acid consisting of a nucleic acid sequence wherein the nucleic acid sequence is a 279 bp Z0344 shown in Fig. 7 or a fragment thereof or sequence complementary thereto.
- the invention features a composition including a nucleic acid consisting of a nucleic acid sequence wherein the nucleic acid sequence is a 357 bp Z0372 shown in Fig. 8 or a fragment thereof or sequence complementary thereto.
- the invention also features oligonucleotides that bind to any of the aforementioned targets as well as combinations of any of these oligonucleoetides.
- the invention further features a composition, including: a first oligonucleotide that has a target-complementary base sequence to Ecf2-1 or Ecf2-2, optionally including a 5' sequence that is not complementary to the specific target sequence.
- the invention features a composition, including: a first oligonucleotide that has a target- complementary base sequence to Ecf gene cluster, optionally including a 5' sequence that is not complementary to the specific target sequence and a second oligonucleotide.
- exemplary second oligonuclotides include, without limitation, an oligonucleotide selected from the group consisting of: a. ) an oligonucleotide that has a target-complementary base sequence to Z5886, optionally including a 5' sequence that is not complementary to the specific target sequence; b. ) an oligonucleotide that has a target-complementary base sequence to hylA, optionally
- an oligonucleotide that has a target-complementary base sequence to SIL0 157 optionally including a 5' sequence that is not complementary to the specific target sequence.
- an oligonucleotide that has a target-complementary base sequence to Z0344 optionally including a 5' sequence that is not complementary to the specific target sequence;
- an oligonucleotide that has a target-complementary base sequence to Z0372 optionally including a 5' sequence that is not complementary to the specific target sequence;
- an oligonucleotide that has a target-complementary base sequence to katP junction optionally including a 5' sequence that is not complementary to the specific target sequence.
- compositions are prepared, if desired, so that only one of the first and second oligonucleotides has a 3' end that can be extended by a template-dependent DNA polymerase.
- an oligonucleotide may include a detectably labeled hybridization probe.
- the invention provides long awaited advantages over a wide variety of standard screening methods used for distinguishing and evaluating STEC.
- the invention disclosed herein reduces not only the number of false positives typically obtained when compared to current methods but also reduces the number of tests and steps performed on a sample.
- the invention accordingly obviates many issues encountered when analyzing a sample in which many microorganism co-infections result in a high false positive rate.
- the methods of the invention provide a facile means to identify and distinguish STEC.
- the methods of the invention provide a route for analyzing virtually any number of samples for presence of STEC with high-volume throughput and high sensitivity.
- the methods are also relatively inexpensive to perform and enable the analysis of small quantities of samples found in either purified or crude extract form.
- the invention disclosed herein advantageously demonstrates specificity for distinguishing highly virulent non-0157:H7 STEC, including the big six non-0157:H7 STECs, from 0157:H7.
- Fig. 1 shows a 1318 bp sequence of Z5886. Forward and reverse primers used to generate an 80 bp amplicon are also shown.
- Fig. 2 shows a 5612 bp sequence of the ECF gene cluster as well as Ecf2-1 and Ecf2-2 fragments respectively 949 bp and 1050 bp. Forward and reverse primers used to generate a 114 bp amplicon are also shown in connection with the ECF gene cluster and Ecf2-1 gene fragment.
- Fig. 3 shows a 1269 bp sequence of Rfb 0 i5 7 . Forward and reverse primers used to generate a 141 bp amplicon are also shown.
- Fig. 4 shows a 1392 bp sequence of VJZX 01S7 . Forward and reverse primers used to generate a 122 bp amplicon are also shown. Forward and reverse primers used to generate a 167 bp amplicon are shown as well.
- Fig. 5 shows a 1185 bp sequence of wzy. Forward and reverse primers used to generate a 191 amplicon are also shown.
- Fig. 6 shows a 2634 bp sequence Forward and reverse primers used to generate a 152 amplicon are shown.
- Fig. 7 shows a 279 bp sequence of Z0344. Forward and reverse primers used to generate a 125 bp amplicon are shown.
- Fig. 8 shows a 357 bp sequence of Z0372. Forward and reverse primers used to generate a 177 bp amplicon are shown.
- Fig. 9 shows a 1489 bp sequence of katP junction. Forward and reverse primers used to generate a 101 bp amplicon are shown.
- Fig. 10 shows polymerase chain reaction (PC ) screening results testing 214 E. coli strains for identifying virulent 0157:H7 and non-0157 STEC.
- Figure 11 shows ecf-1, ecf-2, ecf-3, and ecf-4 nucleotide and polypeptide sequences.
- Figure 12 shows WZX O-antigen nucleotide and polypeptide sequences.
- Figure 13 shows Shiga Toxin nucleotide and polypeptide sequences.
- the invention relates to compositions, methods and kits for the identification, detection, and/or quantitation of E. coli STEC, which may be present either alone or as a component, large or small, of a homogeneous or heterogeneous mixture of nucleic acids in a sample taken for testing, e.g., for diagnostic testing, for screening of blood products, for microbiological detection in bioprocesses, food such as meat or dairy products, water, animals such as reservoirs of 0157:H7 and non-0157:H7 STEC such as ruminants and other animals, industrial or environmental samples, and for other purposes.
- E. coli STEC such as 0157:H7 and non-0157:H7 STEC.
- assays disclosed herein identify ecf sequences common to E. coli 0157:H7 and non-0157:H7 STEC, and differentiates E. coli STECs including virulent non-0157 STECs such as 026, 045, O103, 0111, 0121, and 0145 from other non-virulent strains and, for example, from 0157:H7.
- a preferred useful region for such differentiation is the ECF gene cluster, for example Ecf2-1 and Ecf2-2.
- E. coli 0157:H7 As a result of extensive analyses of amplification oligonucleotides specific for E. coli 0157:H7, the particular region of E. coli 0157:H7, corresponding to the region of E. coli Ecf2-1 sequence, has been identified as a target for amplification-based detection of E. coli 0157:H7 and non-0157:H7 STEC. In addition, after extensive analysis a particular region of E. coli 0157:H7 (Z5886)(hereinafter referred to as the "Z5886 region”) has been identified as still another useful target for amplification-based detection of E. coli 0157:H7.
- Z5886 region Z5886 region
- the invention relates to methods of detection of E. coli 0157:H7 and non-0157:H7 STEC in a sample of interest, amplification oligonucleotides, compositions, reaction mixtures, and kits.
- the assays described herein detect sequences specific for STEC from other non-virulent strains.
- the assays also provide for the detection of the big six virulent, non-0157:H7 STEC. It may utilize virtually any known nucleic amplification protocol such as real-time polymerase chain reaction (PCR) or real-time transcription mediated amplification (TMA), where the target-specific sequence is amplified and a fluorescent molecular torch is used to detect the amplified products as they are produced.
- PCR real-time polymerase chain reaction
- TMA real-time transcription mediated amplification
- Target detection is performed simultaneously with the amplification and detection of an internal control in order to confirm reliability of the result.
- the result of the assay consists of the classification of the sample as positive or negative for the presence or absence of STEC.
- the sample is a blood sample or a contaminated meat product where STEC is a known or suspected contaminant.
- STEC is a known or suspected contaminant.
- the presence of STEC in one or more contaminated samples may be monitored in a rapid and sensitive fashion.
- Target nucleic acids may be isolated from any number of sources based on the purpose of the amplification assay being carried out.
- the present invention provides a method for detecting and distinguishing between E. coli (e.g., 0157 STEC and virulent non-0157 strains) using a hybridization assay that may also include a nucleic amplification step that precedes a hybridization step.
- Preparation of samples for amplification of E. coli sequences may include separating and/or concentrating organisms contained in a sample from other sample components according to standard techniques, e.g., filtration of particulate matter from air, water, or other types of samples.
- the target nucleic acid may be obtained from any medium of interest, such as those described above and, in particular, contaminated food.
- Sample preparation may also include chemical, mechanical, and/or enzymatic disruption of cells to release intracellular contents, including E. coli NA or DNA.
- Preferred samples are food and environmental samples.
- Methods to prepare target nucleic acids from various sources for amplification are well known to those of ordinary skill in the art.
- Target nucleic acids may be purified to some degree prior to the amplification reactions described herein, but in other cases, the sample is added to the amplification reaction without any further manipulations.
- Sample preparation may include a step of target capture to specifically or non-specifically separate the target nucleic acids from other sample components.
- Nonspecific target preparation methods may selectively precipitate nucleic acids from a substantially aqueous mixture, adhere nucleic acids to a support that is washed to remove other sample components, or use other means to physically separate nucleic acids, including STEC nucleic acid, from a mixture that contains other components.
- Other nonspecific target preparation methods may selectively separate RNA from DNA in a sample.
- a target sequence may be of any practical length.
- An optimal length of a target sequence depends on a number of considerations, for example, the amount of secondary structure, or self-hybridizing regions in the sequence. Typically, target sequences range from about 30 nucleotides in length to about 300 nucleotides in length or greater. Target sequences accordingly may range from 3-100, 50-150, 75-200, 100-500, or even 500-800 or 900-1,100 nucleotides in length.
- the optimal or preferred length may vary under different conditions which can be determined according to the methods described herein and the sequences of the targets described herein.
- a nucleic acid comprises a contiguous base region that is at least 70%; or 75%; or 80%, or 85% or 90%, or 95%, or even 96%, 97%, 98%, 99% or even 100% identical to a contiguous base region of a reference nucleic acid.
- the degree of identity between a base region of a query nucleic acid and a base region of a reference nucleic acid can be determined by manual alignment or using any standard alignment tool known in the art such as "BLAST.” "Identity' is simply determined by comparing just the nucleic acid sequences.
- the query:reference base sequence alignment may be DNA:DNA, NA: NA, DNA:RNA, RNA:DNA, or any combinations or analogs thereof. Equivalent RNA and DNA base sequences can be compared by converting U's (in RNA) to T's (in DNA).
- Oligonucleotides An oligonucleotide can be virtually any length, limited only by its specific function in the amplification reaction or in detecting an amplification product of the amplification reaction. However, in certain embodiments, preferred oligonucleotides will contain at least about 5, 6, 7, 8, 9, or 10; or 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; or 22; or 24; or 26; or 28; or 30; or 32; or 34; or 36; or 38; or 40; or 42; or 44; or 46; or 48; or 50; or 52; or 54; or 56 contiguous bases that are complementary to a region of the target nucleic acid sequence or its complementary strand.
- the contiguous bases are preferably at least about 80%, more preferably at least about 90%, and most preferably completely complementary to the target sequence to which the oligonucleotide binds.
- Certain preferred oligonucleotides are of lengths generally between about 5-20, 5-25, 10-100; or 12-75; or 14-50; or 15-40 bases long and optionally can include modified nucleotides. Exemplary oligonucleotides are described herein. Oligonucleotides may be modified in any way, as long as a given modification is compatible with the desired function of a given oligonucleotide. One of ordinary skill in the art can easily determine whether a given modification is suitable or desired for any given oligonucleotide. Modifications include base modifications, sugar modifications or backbone modifications.
- Primers are a type of oligonucleotide used in amplification reactions. Primers have a 3' hydroxyl group which is involved in the amplification reaction.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- SDA strand displacement amplification
- NASBA nucleic acid sequence based amplification
- TMA transcription- mediated amplification
- Suitable amplification conditions can be readily determined by a skilled artisan in view of the present disclosure.
- Amplification conditions refer to conditions which permit nucleic acid amplification.
- Amplification conditions may, in some embodiments, be less stringent than "stringent hybridization conditions" as described herein.
- stringent hybridization conditions is meant hybridization assay conditions wherein a specific detection probe is able to hybridize with target nucleic acids over other nucleic acids present in the test sample. It will be appreciated that these conditions may vary depending upon factors including the GC content and length of the probe, the hybridization temperature, the composition of the hybridization reagent or solution, and the degree of hybridization specificity sought. Specific stringent hybridization conditions are disclosed herein.
- Oligonucleotides used in the amplification reactions as disclosed herein may be specific for and hybridize to their intended targets under amplification conditions, but in certain embodiments may or may not hybridize under more stringent hybridization conditions. On the other hand, detection probes generally hybridize under stringent hybridization conditions. While the Examples section infra provides preferred amplification conditions for amplifying target nucleic acid sequences, other acceptable conditions to carry out nucleic acid amplifications could be easily ascertained by someone having ordinary skill in the art depending on the particular method of amplification employed.
- the target nucleic acid of a STEC can also be amplified by a transcription- based amplification technique.
- a transcription-based amplification system is transcription-mediated amplification (TMA), which employs an RNA polymerase to produce multiple RNA transcripts of a target region.
- TMA transcription-mediated amplification
- Exemplary TMA amplification methods are described in, e.g., U.S. Pat. Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491; 5,437,990; 5,480,784; 5,554,516; and 7,374,885; and PCT Pub. Nos. WO 88/01302; WO 88/10315 and WO 95/03430.
- the methods of the present invention may include a TMA reaction that involves the use of a single primer TMA reaction, as is described in U.S. Pat. No. 7,374,885.
- the single-primer TMA methods use a primer oligomer (e.g., a NT7 primer), a modified promoter-based oligomer (or "promoter-provider oligomer"; e.g., a T7 provider) that is modified to prevent the initiation of DNA synthesis from its 3' end (e.g., by including a 3'-blocking moiety) and, optionally, a blocker oligomer (e.g., a blocker) to terminate elongation of a cDNA from the target strand.
- a primer oligomer e.g., a NT7 primer
- promoter-based oligomer or "promoter-provider oligomer”; e.g., a T7 provider
- Promoter-based oligomers provide an oligonucleotide sequence that is recognized by an RNA polymerase.
- This single primer TMA method synthesizes multiple copies of a target sequence and includes the steps of treating a target RNA that contains a target sequence with a priming oligomer and a binding molecule, where the primer hybridizes to the 3' end of the target strand.
- RT initiates primer extension from the 3' end of the primer to produce a cDNA which is in a duplex with the target strand (e.g., RNAxDNA).
- a blocker oligomer is used in the reaction, it binds to the target nucleic acid adjacent near the user designated 5' end of the target sequence.
- the 3' end of the cDNA is determined by the position of the blocker oligomer because polymerization stops when the primer extension product reaches the binding molecule bound to the target strand.
- the 3' end of the cDNA is complementary to the 5' end of the target sequence.
- the RNA:cDNA duplex is separated when RNase (e.g., RNase H of RT) degrades the RNA strand, although those skilled in the art will appreciate that any form of strand separation may be used.
- RNase e.g., RNase H of RT
- the promoter-provider oligomer includes a 5' promoter sequence for an RNA polymerase and a 3' target hybridizing region complementary to a sequence in the 3' region of the cDNA.
- the promoter-provider oligomer also has a modified 3' end that includes a blocking moiety that prevents initiation of DNA synthesis from the 3' end of the promoter-provider oligomer.
- the 3'-end of the cDNA is extended by DNA polymerase activity of RT using the promoter oligomer as a template to add a promoter sequence to the cDNA and create a functional double-stranded promoter.
- RNA polymerase specific for the promoter sequence then binds to the functional promoter and transcribes multiple RNA transcripts complementary to the cDNA and substantially identical to the target region sequence that was amplified from the initial target strand.
- the resulting amplified RNA can then cycle through the process again by binding the primer and serving as a template for further cDNA production, ultimately producing many amplicons from the initial target nucleic acid present in the sample.
- Some embodiments of the single-primer transcription-associated amplification method do not include the blocking oligomer and, therefore, the cDNA product made from the primer has an indeterminate 3' end, but the amplification steps proceed substantially as described above for all other steps.
- RTMA reverse transcription-mediated amplification
- RNA polymerase RNA polymerase
- reverse transcriptase RNA polymerase
- RTMA isothermal; the entire reaction is performed at the same temperature in a water bath or heat block. This is in contrast to other amplification reactions such as PCR that require a thermal cycler instrument to rapidly change the temperature to drive reaction.
- RTMA can amplify either DNA or RNA, and can produce either DNA or RNA amplicons, in contrast to most other nucleic acid amplification methods that only produce DNA. RTMA has very rapid kinetics, resulting in a billion-fold amplification within 15-60 minutes.
- RTMA can be combined with a Hybridization Protection Assay (HPA), which uses a specific oligonucleotide probe labeled with an acridinium ester detector molecule that emits a chemiluminescent signal, for endpoint detection or with molecular torches for real-time detection. There are no wash steps, and no amplicon is ever transferred out of the tube, which simplifies the procedure and reduces the potential for contamination.
- HPA Hybridization Protection Assay
- the RTMA reaction is initiated by treating an RNA target sequence in a nucleic acid sample with both a tagged amplification oligomer and, optionally a blocking oligomer.
- the tagged amplification oligomer includes a target hybridizing region that hybridizes to a 3'-end of the target sequence and a tag region situated 5' to the target hybridizing region.
- the blocking oligomer hybridizes to a target nucleic acid containing the target sequence in the vicinity of the 5'-end of the target sequence.
- the target nucleic acid forms a stable complex with the tagged amplification oligomer at the 3'-end of the target sequence and the terminating oligonucleotide located adjacent to or near the determined 5'-end of the target sequence prior to initiating a primer extension reaction.
- Unhybridized tagged amplification oligomers are then made unavailable for hybridization to a target sequence prior to initiating a primer extension reaction with the tagged priming oligonucleotide, preferably by inactivating and/or removing the unhybridized tagged priming oligonucleotide from the nucleic acid sample.
- Unhybridized tagged amplification oligomer that has been inactivated or removed from the system is then unavailable for unwanted hybridization to contaminating nucleic acids.
- the tagged amplification oligomer is hybridized to the target nucleic acid, and the tagged amplification oligomentarget nucleic acid complex is removed from the unhybridized tagged amplification oligomer using a wash step.
- the tagged amplification oligomentarget nucleic acid complex may be further complexed to a target capture oligomer and a solid support.
- the tagged amplification oligomers further comprise a target-closing region.
- the target hybridizing region of the tagged amplification oligomer hybridizes to target nucleic acid under a first set of conditions (e.g., stringency).
- a first set of conditions e.g., stringency
- the unhybridized tagged amplification oligomer is inactivated under a second set of the conditions, thereby hybridizing the target closing region to the target hybridizing region of the unhybridized tagged amplification oligomer.
- the inactivated tagged amplification oligomer is then unavailable for hybridizing to contaminating nucleic acids.
- a wash step may also be included to remove the inactivated tagged amplification oligomers from the assay.
- An extension reaction is then initiated from the 3'-end of the tagged amplification oligomer with a DNA polymerase, e.g., reverse transcriptase, to produce an initial amplification product that includes the tag sequence.
- the initial amplification product is then separated from the target sequence using an enzyme that selectively degrades the target sequence (e.g., RNAse H activity).
- the initial amplification product is treated with a promoter-based oligomer having a target hybridizing region and an RNA polymerase promoter region situated 5' to the target hybridizing region, thereby forming a promoter- based oligomeninitial amplification product hybrid.
- the promoter-based oligomer may be modified to prevent the initiation of DNA synthesis, preferably by situating a blocking moiety at the 3'-end of the promoter-based oligomer (e.g., nucleotide sequence having a 3'-to-5' orientation).
- the 3'-end of the initial amplification product is then extended to add a sequence complementary to the promoter, resulting in the formation of a double-stranded promoter sequence.
- Multiple copies of a RNA product complementary to at least a portion of the initial amplification product are then transcribed using an RNA polymerase, which recognizes the double-stranded promoter and initiates transcription therefrom.
- the nucleotide sequence of the RNA product is substantially identical to the nucleotide sequence of the target nucleic acid and to the complement of the nucleotide sequence of the tag sequence.
- RNA products may then be treated with a tag-targeting oligomer, which hybridizes to the complement of the tag sequence to form a tag-targeting oligomer: RNA product hybrid, and the 3'-end of the tag-targeting oligomer is extended with the DNA polymerase to produce an amplification product complementary to the RNA product.
- the DNA strand of this amplification product is then separated from the RNA strand of this amplification product using an enzyme that selectively degrades the first RNA product (e.g., RNAse H activity).
- the DNA strand of the amplification product is treated with the promoter-based oligomer, which hybridizes to the 3'-end of the second DNA primer extension product to form a promoter-based oligomenamplification product hybrid.
- the promoter-based oligomenamplification product hybrid then re-enters the amplification cycle, where transcription is initiated from the double-stranded promoter and the cycle continues, thereby providing amplification product of the target sequence.
- Amplification product can then be used in a subsequent assay.
- One subsequent assay includes nucleic acid detection, preferably nucleic acid probe-based nucleic acid detection.
- the detection step may be performed using any of a variety of known ways to detect a signal specifically associated with the amplified target sequence, such as by hybridizing the amplification product with a labeled probe and detecting a signal resulting from the labeled probe.
- the detection step may also provide additional information on the amplified sequence, such as all or a portion of its nucleic acid base sequence. Detection may be performed after the amplification reaction is completed, or may be performed simultaneous with amplifying the target region, e.g., in real time.
- the detection step allows detection of the hybridized probe without removal of unhybridized probe from the mixture (see, e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174).
- the amplification methods as disclosed herein also preferably employ the use of one or more other types of oligonucleotides that are effective for improving the sensitivity, selectivity, efficiency, etc., of the amplification reaction.
- Target capture in general, refers to capturing a target polynucleotide onto a solid support, such as magnetically attractable particles, wherein the solid support retains the target polynucleotide during one or more washing steps of the target polynucleotide purification procedure. In this way, the target polynucleotide is substantially purified prior to a subsequent nucleic acid amplification step.
- Many target capture methods are known in the art and suitable for use in conjunction with the methods described herein.
- any support may be used, e.g., matrices or particles free in solution, which may be made of any of a variety of materials, e.g., nylon, nitrocellulose, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene, or metal.
- Illustrative examples use a support that is magnetically attractable particles, e.g., monodisperse paramagnetic beads to which an immobilized probe is joined directly (e.g., via covalent linkage, chelation, or ionic interaction) or indirectly (e.g., via a linker), where the joining is stable during nucleic acid hybridization conditions.
- any technique available to the skilled artisan may be used provided it is effective for purifying a target nucleic acid sequence of interest prior to amplification.
- Any labeling or detection system or both used to monitor nucleic acid hybridization can be used to detect STEC amplicons. Such systems are well known in the art.
- Detection systems typically employ a detection oligonucleotide of one type or another in order to facilitate detection of the target nucleic acid of interest. Detection may either be direct (i.e., probe hybridized directly to the target) or indirect (i.e., a probe hybridized to an intermediate structure that links the probe to the target).
- a probe's target sequence generally refers to the specific sequence within a larger sequence which the probe hybridizes specifically.
- a detection probe may include target-specific sequences and other sequences or structures that contribute to the probe's three-dimensional structure, depending on whether the target sequence is present Essentially any of a number of well known labeling and detection system that can be used for monitoring specific nucleic acid hybridization can be used in conjunction with the present invention.
- fluorescent moieties include fluorescent moieties (either alone or in combination with "quencher” moieties), chemiluminescent molecules, and redox-active moieties that are amenable to electronic detection methods.
- preferred fluorescent labels include non- covalently binding labels (e.g., intercalating dyes) such as ethidium bromide, propidium bromide, chromomycin, acridine orange, and the like.
- probes exhibiting at least some degree of self-complementarity are desirable to facilitate detection of probe:target duplexes in a test sample without first requiring the removal of unhybridized probe prior to detection.
- structures referred to as “molecular torches” and “molecular beacons” are designed to include distinct regions of self-complementarity and regions of target-complementarity. Molecular torches are fully described in U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and molecular beacons are fully described in U.S. Pat. Nos. 5,118,801, 5,312,728, and 5,925,517.
- Methods and compositions are provided herein for the immunological detection of E. coli adulterants in a sample using ecf and wzx and/or stx.
- Such methods include enzyme-linked immunoabsorbent assays (ELISA) which is a widely used for the detection of E. coli.
- ELISA enzyme-linked immunoabsorbent assays
- antibodies e.g., monoclonal or polyclonal or fragments thereof
- stx stxl and stx2
- Test devices for immunological assays include conventional microtitre plates, dipsticks, immunofiltration, and capillary migration assays. Such systems are also useful as visual tests.
- Immunological detection systems utilizing antibodies having specificity to an ecf, wzx, or stx polypeptide are useful for simple, fast, and high- voliume screening methods, with the identification of negative and positive samples in a short time period. According to the methods, detection of ecf and wzx is taken as an indication of the presence of E. coli 0157:H7; detection of ecf and the absence of wzx is taken as an indication of the presence of non- 0157:H7 shiga toxin (stx)-containing E. coli (STEC); and detection of ecf and stx is taken as an indication of the presence of enterohemorrhagic Escherichia coli (EHEC).
- the p0157 ecf (E. coli attaching and effacing [eae] gene-positive conserved fragments) operon is especially useful in the disclosed methods.
- This operon encodes four genes as one operon: ecfl, ecf2, ecf3, and ec/4. These ecf genes are involved in bacterial cell wall synthesis encoding bacterial surface structure-associated proteins. Both ecfl and ec/2 respectively encode a polysaccharide deacetylase and a lipopolysaccharide (LPS) a-l,7-/V-acetylglucosamine transferase (also designated WabB).
- LPS lipopolysaccharide
- Ecf3 encodes an outer membrane protein associated with bacterial invasion.
- ec/4 encodes a second LPS - lipid A myristoyl transferase.
- Exemplary Ecf polypeptides (Ecfl, Ecf2, Ecf3, and Ecf 4) are described in Figure 11 as well as in Table 5 (in connection with Genbank accession number).
- Other Ecf polypeptides useful in the invention are those having identity with those described in Figure 11. Such sequence identity is typically 90%, 92% or 95% or greater between an Ecf polypeptide described herein and a polypeptide used for comparative purposes. To determine the percent identity of two polypeptides standard methods well known in the art are employed. Fragments of Ecf polypeptides are also useful in the invention.
- Wzx is an E. coli translocase.
- Exemplary wzx polypeptides are described in Figure 12 as well as in Table 5 (in connection with Genbank accession number).
- Other wzx polypeptides useful in the invention are those having identity with those described in Figure 12. Such sequence identity is typically 90%, 92% or 95% or greater between a wzx polypeptide described herein and a polypeptide used for comparative purposes. To determine the percent identity of two polypeptides standard methods well known in the art are employed. Fragments of wzx polypeptides are also useful in the invention.
- stx £. coli shiga-like toxins, e.g., stxl and stx2
- Exemplary stx polypeptides are described in Figure 13 as well as in Table 5 (in connection with Genbank accession number).
- Other stx polypeptides useful in the invention are those having identity with those described in Figure 13. Such sequence identity is typically 90%, 92% or 95% or greater between a stx polypeptide described herein and a polypeptide used for comparative purposes. To determine the percent identity of two polypeptides standard methods well known in the art are employed. Fragments of stx polypeptides are also useful in the invention.
- samples of lots of meat product e.g., a lot of meat such as raw ground beef, beef trim, high fat ground beef, and raw ground beef components for example, beef and veal bulk packed manufacturing trimmings and other beef and veal components such as primal cuts, sub primal cuts, head meat, cheek meat, esophagus meat, heart, and advanced meat recovery product intended for grinding
- meat product e.g., a lot of meat such as raw ground beef, beef trim, high fat ground beef, and raw ground beef components for example, beef and veal bulk packed manufacturing trimmings and other beef and veal components such as primal cuts, sub primal cuts, head meat, cheek meat, esophagus meat, heart, and advanced meat recovery product intended for grinding
- produce e.g. fruits or vegetables such as leafy green vegetables including lettuce and spinach
- processing may include a step for enriching for the presence of an E.
- coli adulterant from the lot of meat or produce.
- Analysis of the sample includes one or more of the nucleic acid or polypeptide detection assays described herein. If desired, multiple samples may be tested.
- the sample is then subject to a hybridization assay or to an immunological assay or both as described herein to test for the presence of (i) ec/and (ii) wzx and/or stx.
- results indicative of the absence of these markers is taken as an indication that the lot of meat or produce is free of an E. coli adulterant and may be packaged as a product.
- Methods for packaging meat and produce are well known and typically include the use of cartons, containers, plastic wrap, or trays wrapped with plastic. Packaged meat and produce products free of pathogenic E.
- coli may be subsequently shipped to a destination for sale or consumption. Shipping typically involves transport of the product from a processor to a distribution center or directly to a grocery store or restaurant or other consumer of the product. These methods and compositions are also useful for producing other products free of E. coli contamination. Examples include unpasteurized fresh-pressed juices such as apple cider, yogurt, and cheese made from raw milk. KJts
- the invention also features a kit for carrying out the described methods according to the present invention described herein.
- the kit includes nucleic acid probes or primers that may be labeled, reagents and containers for carrying out the hybridization assay, positive and negative control reagents, and instructions for performing the assay.
- Oligonucleotides, probes, and primers are readily designed nucleic acids known in the art for the ec/operon, wzx, and stx (stxl and stx2). Exemplary sequences are shown in Figures 11-13 as well as in Table 5.
- Kits may also include antibodies specific for any of the polypeptides or fragments thereof disclosed herein and appropriate reagents for an immunological-based assay for detecting an ecf, wzx, and stx polypeptide. Some kits contain at least one target capture oligomer for hybridizing to a target nucleic acid. Some kits for detecting the presence or abundance of two or more target nucleic acids contain two or more target capture oligomers each configured to selectively hybridize to each of their respective target nucleic acids.
- kits contain at least one first amplification oligomer for hybridizing to a target nucleic acid.
- Some kits for detecting the presence or abundance of two or more target nucleic acids contain two or more first amplification oligomers, each configured to selectively hybridize to their respective target nucleic acids.
- kits contain chemical compounds used in performing the methods herein, such as enzymes, substrates, acids or bases to adjust pH of a mixture, salts, buffers, chelating agents, denaturants, sample preparation agents, sample storage or transport medium, cellular lysing agents, total NA isolation components and reagents, partial generalized RNA isolation components and reagents, solid supports, and other inorganic or organic compounds.
- Kits may include any combination of the herein mentioned components and other components not mentioned herein. Components of the kits can be packaged in combination with each other, either as a mixture or in individual containers. It will be clear to skilled artisans that the invention includes many different kit configurations.
- the kits of the invention may further include additional optional components useful for practicing the methods disclosed herein. Exemplary additional components include chemical-resistant disposal bags, tubes, diluent, gloves, scissors, marking pens, and eye protection.
- Useful targets identified for such assays include those found in Figures 1-9.
- Useful oligonucleotides for amplifying such targets are found in Figures 1-9 as well.
- E. coli strains shown in Fig. 10 were cultured according to standard methods. DNA was extracted from an overnight culture and purified using a PureLink Genomic DNA Kit (Invitrogen) according to kit instructions.
- amplified DNA products were generated using a Clontech PCR kit consisting of the following master mix/reaction:
- Amplification conditions were as follows:l min at 95°C, 30 cycles of 30 seconds at 95°C denature / 90 seconds at 68°C extension, followed by 90 seconds at 68°C.
- Amplified DNA was sequenced using oligos Z5866 F-1/Z5866 R-2 to detect target region Z5886 (0157:1-17) and oligos ecf2-l F/ ecf2-l R and ecf2-2 F/ ecf 2-2 R) to detect target regions ecf2-l and ecf2-2 (STEC). Sequences of these primers are shown below in Table 1.
- Real Time PCR analysis was performed as follows. A real time master mix using the following ratio of components was prepared: lOul Power ABI SYBR Green Mixture / 7.8ul RNase-free H20 / 0.2ul Fwd/Rev primer. Primers are shown in Table 2. In a 96-well PCR plate, 2ul of DNA template was added to 18ul of real time master mix, sealed, and run on a Stratagene real time instrument using the following cycler conditions: denaturing for 10 minutes at 95°C, 40 cycles of 15 seconds at 95°C denature / 1 minute at 60°C extension.
- E. coli STECs including 0157:1-17 and virulent non-0157 STECs such as 026, 045, O103, 0111, 0121, and 0145 as well as non-virulent E. coli strains were tested.
- the data obtained from these PCR assays is summarized in Figure 10.
- Figure 10 shows PCR screening results testing 214 E. coli strains for specificity of 0157:1-17 (Z5886, rfboi 57 , wzx 0 i5 7 , Z0344, Z0372) and STEC (ecf) specific targets.
- coli strains which have a combination of 3 virulence factors: stxl or stx2 or stxl/stx2 in combination with eae S TEc and hlyA (ehx), and therefore is specific for highly virulent STEC/EHEC strains including the big six non-0157 serogroups - 026, 045, O103, 0111, 0121, and 0145. Further, we obtained 104 non-0157:H7 STEC isolates from the USDA (Bosilevac and Koohmaraie, Appl. Environ. Microbiol. 77(6):2103-2112, 2011).
- This sample is a highly virulent EHEC/STEC isolate which contains three virulent markers, stx, eae and hlyA, and therefore is correctly detected by the ec/assay herein.
- Table 3 Specificity of 0157 and STEC target regions tested by real time PCR (104 non-0157 STEC samples were tested).
- E. coli 0157:H7 detection assay combines two unique target genes, the chromosomal wzx 0157 gene and the ecfl gene which is located in a conserved ec/ operon on a large virulence plasmid.
- the large virulence plasmid is found in highly virulent EHEC strains.
- the ec/ operon encodes 4 proteins involved in cell wall synthesis which enhances colonization of E. coli in cattle.
- the sensitivity of the assay was determined by using serial 10-fold dilutions of five different E. coli 0157:H7 strains. The sensitivity or limit of detection (LOD) was defined using a 95% confidence interval.
- LOD limit of detection
- coli isolates including 130 of the FSIS regulated big six STEC strains. All isolates were tested at a concentration of le8 CFU/ml. Serotypes and presence of virulence genes such as shiga toxins 1 and 2 (stx 1 and stx 2 ), intimin (eae) and enterohemolysin (ehxA) for all E. coli isolates included in this study were tested by PCR.
- shiga toxins 1 and 2 stx 1 and stx 2
- intimin eae
- ehxA enterohemolysin
- the LOD of the E.coli 0157:H7 detection assay was determined to be le3 CFU/mL. All 117 0157H7/NM strains containing stx genes and the eae gene were successfully detected by the assay. Seven 0157:NM strains which lacked shiga toxin genes were not detected. Of the 356 non-0157:H7 E. coli isolates included in this study, none were detected by the E. coli 0157:H7 detection assay. Significance: The results of these studies show that the use of the ecfl gene in conjunction with the wzx 0157 gene accurately detects stx/eae containing pathogenic 0157:H7/NM strains.
- E. coli 0157:H7 and six serovars (026, O103, 0121, 0111, 0145, 045) are frequently implicated in severe clinical illness worldwide.
- Standard testing methods using stx, eae and O-serogroup-specific gene sequences for detecting the top six serogroups bear the disadvantage that these genes may reside, independently, in different non-pathogenic organisms leading to false positive results.
- the ecf operon has previously been identified in the large enterohemolysin-containing plasmid of eae-positive STEC.
- top six non-0157 STEC were detected in 4.0% of samples by an ecfl -detect ion assay and in 5.0% of samples by the stx/eae-based method.
- top six non-0157 STEC were detected at 1.1% by both methods.
- Estimation of false positive rates among the top six non-0157 STEC revealed a lower rate using the ecfl detection method (0.5%) compared to the eae/stx screening method (1.1%).
- the ecfl -detection assay detected STEC strains associated with severe illness not included in the FSIS regulatory definition of adulterant STEC.
- E. coli strains included in this study were acquired from Silliker Laboratories, United States Department of Agriculture (USDA) Agricultural Research Service, E. coli Reference Center Pennsylvania State University, STEC Center Michigan State University, and American Type Culture Collection (ATCC). Serotypes and presence of ecfl and virulence genes stx lt stx 2 , eae, and ehxA of all E. coli isolates are provided in detail in Tables 4a and 4b. Approximately 30% of the E. coli isolates included in this study were from food sources.
- Bacterial isolates were stored frozen at -70°C in brain heart infusion (BHI) media (Becton, Dickinson and Company, Franklin Lakes, NJ) containing 30% glycerol and were subcultured on MacConkey agar plates (Hardy Diagnostics, Santa Maria, CA) prior to testing.
- BHI brain heart infusion
- Ground Beef and Beef Trim Samples A total of 2162 pre-enriched beef samples were examined.
- the supplier prepared randomized samples of different sizes (25 g, 50 g, 75 g or 100 g) which were diluted 1:10 (225 mL, 450 mL, 675 mL, or 900 mL) in tryptic soy broth (Becton, Dickinson and Company, Franklin Lakes, NJ) and then enriched for 14 - 20 hrs at 42°C.
- Template DNA from pure bacterial cultures was prepared using PureLinkTM Genomic DNA Kits (Invitrogen, Carlsbad CA). A single colony from a MacConkey agar plate was diluted in 5 mL BHI broth and grown overnight at 35°C. One mL was then pelleted by centrifugation and used in the PureLinkTM Genomic DNA extraction kit according to the manufacturer's specified protocol. Aliquots of 2 to 5 ⁇ of the final DNA preparation were then directly transferred to the PCR reactions or stored at -20°C until further analysis.
- Template DNA from the 1065 enriched ground beef samples received from the commercial ground beef producer was prepared according to the PrepMan 3 ⁇ 4 Ultra Sample Preparation Reagent Protocol (Applied Biosystems, Foster City, CA). One mL of enrichment broth was centrifuged for 3 min. The supernatant was discarded and 100 ⁇ PrepMan 3 ⁇ 4 Ultra Sample Preparation Reagent was added. After heating at 100°C for 10 minutes the extract was centrifuged and 50 ⁇ was diluted into 450 ⁇ of nuclease-free water. Aliquots of 2 to 5 ⁇ of this DNA preparation were then directly transferred to the PCR reactions or stored at -20°C until further analysis.
- Nucleic acid was extracted from the second set of 1097 enriched beef samples (Study II) using the KingFisher * 96 magnetic particle processor (Thermo Fisher Scientific, Waltham, MA) followed by PCR analysis. An aliquot of 400 ⁇ from each sample was combined with 125 ⁇ of Roka target capture reagent containing magnetic beads that bind nucleic acids. The solution was heated to 95°C for 10 minutes using an EchoThermTM SC20 Orbital Mixing Chilling/Heating Dry Bath (Torrey Pines Scientific, Carlsbad CA). The samples were placed on the KingFisher * 96 magnetic particle processor, magnetic beads were collected and transferred into 200 ⁇ of Roka wash buffer containing detergent. The samples were mixed, collected and washed a second time.
- the final elution of the nucleic acid bound to magnetic beads was captured in a volume of 50 ⁇ consisting of 25 ⁇ TaqMan * Environmental Master Mix 2.0, 21 ⁇ RNase-free H 2 0, and 4.0 ⁇ probe (375 nM), forward and reverse primers (2.5 uM each).
- PCR Assays to Determine Presence of ecf, Virulence Genes and O-Serogroups The presence of the ecfl gene, virulence factors stx lt stx 2 , eae, ehxA and presence of O-serogroups 026, 045, O103, Olll, 0121, 0145 was determined in 501 E. coli isolates and 2162 enriched beef samples using real time PCR. The presence of ec/3 and ec/4 was also determined in 253 out of the 501 E. coli isolates. The presence of virulence factors was determined using stx lt stx 2 specific oligonucleotides (Paton et al., 1998.
- PCR amplification reactions using the Power SYBR * Green PCR Master Mix were performed in a final volume of 20 ⁇ consisting of 10 ⁇ Power SYBR * Green PCR Master Mix, 7.8 ⁇ RNase-free H 2 0, and 0.2 ⁇ forward and reverse primers (1.5 ⁇ each).
- PCR amplification reactions using the TaqMan * Environmental Master Mix 2.0 were performed in a final volume of 25 ⁇ consisting of 12.5 ⁇ TaqMan * Environmental Master Mix 2.0, 8.5 ⁇ RNase-free H 2 0, and 2.0 ⁇ probe (150 nM), forward and reverse primers (1.0 ⁇ each), with the exception of beef samples from the second study that used a final volume of 50 ⁇ as described in the previous section.
- Template DNA for the beef samples from Study II utilized nucleic acid extracted using the KingFisher * 96 magnetic particle processor as described above. Samples were amplified with an initial denaturation step at 95°C for 10 min. Then the following thermocycling conditions for the individual amplification reactions were 40 cycles (SYBR * Green) or 45 cycles (TaqMan * ) of denaturation at 95°C for 15 sec, annealing and extension at 60°C for 1 min (SYBR * Green) or 59°C for 1 min (TaqMan * ), followed by 15 sec at 95°C, 15 sec at 60°C, and 15 sec at 95°C.
- TGT TCC AGG TGG TAG GAT TCG 171 2690613-2690593
- Non-0157 E. coli isolates which were positive for the ecfl and eae and ehxA genes but negative for st ⁇ and stx 2 genes were tested by SYBFf Green real time PCR for the presence j of the bfpA gene found only in typical enteropathogenic E. coli (EPEC).
- chromosomal gene markers associated with eae-positive STEC such as nleB, espK, Z2098, and Z2099 were tested.
- E. coli isolates from various sources for the presence of ecf and other virulent genes including stxl, stx2, eae, and ehxA.
- 100 of 100 0157:H7 isolates were correctly identified including one rough strain not expressing the O antigen were positive for ecfl gene. All of these isolates contained ecf, stxl or stx2, eae and ehxA.
- 0157:NM strains containing stx are also considered adulterants in beef by the FSIS, we examined 24 0157:NM strains. Only 17 0157:NM strains containing stx and eae and ehxA were also positive for the ecfl gene while the remaining 7 E. coli 0157:NM strains, which were negative for stx and eae and exhA genes were also negative for ecfl gene (Table 4a).
- E. coli isolates negative for either stx or eae genes including 43 E. coli strains with different serotypes, 23 EPEC strains and 152 STEC isolates, were negative for the ecfl gene.
- STEC strains positive for stx and eae genes but negative for the six most frequent O serogroups were also negative for both ecfl and ehxA genes (Table 6, Table 4b). All four isolates (three E. coli 055:1-17 strains and one E. coli 0128 strain) were also negative for the eae-positive STEC markers Z2098 and Z2099, except for one E. coli 055:1-17 isolate which was positive for the Z2099 marker.
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