rfbE Gene Research Articles

Quantification of viable Escherichia coli O157:H7 in meat products by duplex real-time PCR assays

Rapid and specific detection of viable Escherichia coli O157:H7 cells in ready-to-eat (RTE) meat products, by duplex quantitative PCR (qPCR) procedures with mRNA and SYBR Green and TaqMan methodologies were developed. Specific primers and probes were designed based on the serotype of E. coli O157:H7, fliCh7 and rfbE genes. No cross-reactivity with other microorganisms was observed. The detection limit of the assays was 101 or 102CFU/g for artificially contaminated meat products, and after a 4h enrichment period at 37°C, the detection limit decreased to about 1CFU/g. Time-to completion of the assay was approximately 8h. Thus, these qPCR methods offer a useful, rapid and efficient tool for screening viable E. coli O157:H7 in RTE meat products. This tool could also be proposed for monitoring these foodborne pathogens in HACCP programs.

Rapid Detection of Viable Escherichia coli O157 by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

Conventional molecular detection methods cannot distinguish between viable and dead Escherichia coli O157 cells. In this study, the loop-mediated isothermal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect viable E. coli O157 cells. Four primers, including outer primers and inner primers, were specially designed for the recognition of six distinct sequences on the serogroups (O157) of the specific rfbE gene of the E. coli O157 genome. PMA selectively penetrated through the compromised cell membranes and intercalated into DNA. Amplification of DNA from dead cells was completely inhibited by 3.0 μg/ml PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. Exhibiting high sensitivity and specificity, PMA-LAMP is a suitable method for evaluating the inactivation efficacy of slightly acidic electrolyzed water in broth. PMA-LAMP can selectively detect viable E. coli O157 cells. This study offers a novel molecular detection method to distinguish between viable and dead E. coli O157 cells.

Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR

The objective of this study was to develop a rapid multiplex PCR (m-PCR) method using pure bacterial cultures and pork that would allow the simultaneous detection of five major foodborne pathogens likely to be found in pork (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella and Yersinia enterocolitica). Five pairs of primers were designed to identify invA gene, hlyA gene, rfbE gene, nuc gene and ail gene for Salmonella, L. monocytogenes, E. coli O157:H7, S. aureus and Y. enterocolitica, respectively. On the basis of determining specificity of the m-PCR, sensitivity was performed with an overnight enrichment step in culture media and pork. The 16S rRNA gene was targeted as an internal control gene of the presence of bacterial DNA. The results suggested that the m-PCR was an effective procedure having high specificity for the simultaneous detection of the five target pathogens. After overnight culture, the detection limit was 103 cfu/mL for the simultaneous detection of the five target pathogens and less than 10 cfu/mL for detection of a single pathogen. The m-PCR protocol successfully detected all five organisms inoculated overnight together on pork. Salmonella, S. aureus, L. monocytogenes, Y. enterocolitca and E. coli O157:H7 were detected at levels of 142, 51, 9, 33 and 670 cfu/mL, respectively, in the pork. The m-PCR assay developed in this study could provide an effective and informative supplement for routine monitoring for pork safety.

Primers with 5′ Flaps Improve the Efficiency and Sensitivity of Multiplex PCR Assays for the Detection of Salmonella and Escherichia coli O157:H7

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

A duplex SYBR Green I real-time quantitative PCR assay for detecting Escherichia coli O157:H7

PCR and hybridization assays are widely used for the detection and identification of Escherichia coli serogroups and serotypes. We used these techniques for the detection of E. coli O157:H7, a dominant serogroup among E. coli strains that are considered major public health problems worldwide. We developed a quantitative PCR assay using SYBR Green I, based on the published sequences of the rfbE and fliC genes from E. coli O157:H7. This method detected the E. coli O157:H7 O somatic antigen gene and the flagellar antigen gene simultaneously, with good specificity, sensitivity, and repeatability. The sensitivity of the assay was 2.95 x 10 copies/µL, which is 10(3) times more sensitive than obtained with a conventional PCR. The intra-assay and inter-assay coefficients of variation were less than 2%. We concluded that this duplex quantitative PCR assay is adequate for the identification and quantitative analysis of E. coli O157:H7. This provides a new identification method for clinical diagnosis of E. coli O157:H7 and for food safety analysis, as well as for molecular epidemiological studies of foodborne diseases.

Lipopolysaccharide O-antigen of enterohemorrhagic Escherichia coli O157:H7 is required for killing both insects and mammals

Studies of enterohemorrhagic Escherichia coli (EHEC) infection mechanisms using mammals require large numbers of animals and are both costly and associated with ethical problems. Here, we evaluated the pathogenic mechanisms of EHEC in the silkworm model. Injection of a clinically isolated EHEC O157:H7 Sakai into either the silkworm hemolymph or intraperitoneal fluid of mice killed the host animals. EHEC O157:H7 Sakai deletion mutants of the rfbE gene, which encodes perosamine synthetase, a monosaccharide component synthetase of the O-antigen, or deletion mutants of the waaL gene, which encodes O-antigen ligase against the lipid A-core region of lipopolysaccharide (LPS), had attenuated killing ability in both silkworms and mice. Introduction of the rfbE gene or the waaL gene into the respective mutants restored the killing ability in silkworms. Growth of both mutants was inhibited by a major antimicrobial peptide in the silkworm hemolymph, moricin. The viability of both mutants was decreased in swine serum. The bactericidal effect of swine serum against both mutants was inactivated by heat treatment. These findings suggest that the LPS O-antigen of EHEC O157:H7 plays an important defensive role against antimicrobial factors in the host body fluid and is thus essential to the lethal effects of EHEC in animals.

Assessment of a real-time PCR for the detection and characterization of verocytotoxigenic Escherichia coli

The European Union Reference Laboratory (EU-RL) has produced guidelines for a real-time PCR for the detection of verocytotoxigenic Escherichia coli (VTEC). In this study, we validated the EU-RL assay on 545 strains of VTEC and evaluated the utility of the assay for the detection of VTEC from stool specimens. The validation study on cultures showed that the EU-RL VTEC PCR was 99.3% sensitive for the detection of vtx genes; only strains harbouring vtx2f genes were not detected. The assay was 100% sensitive and 100% specific for the detection of both the eae and O157 rfbE genes. In a prospective study involving 500 stool samples, the EU-RL VTEC PCR detected vtx genes in 12.4% of specimens, compared to 3.8% specimens found to be culture-positive for E. coli O157 using the Health Protection Agency national standard culture method. This study showed that the EU-RL VTEC assay was reliable and robust, and an effective rapid screening method for the detection of VTEC from stool specimens.

Evaluation of a Multiplex Real-Time Polymerase Chain Reaction for the Quantification of Escherichia coli O157 in Cattle Feces

Cattle are asymptomatic reservoirs for Escherichia coli O157, a major foodborne pathogen. The organism generally colonizes the hindgut of cattle and is shed in the feces at low concentrations. The objective of this research was to evaluate a multiplex, real-time polymerase chain reaction (mqPCR) assay for quantification of E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets. Primer efficiency and analytical sensitivity of the assay were evaluated with a single or pooled (five strain) culture of E. coli O157. In pure culture, the minimum detection limit of the assay was 1.4×10(3) CFU/mL and 3.6×10(3) CFU/mL for the single and five-strain mixture of E. coli O157, respectively. Diagnostic sensitivity was analyzed using DNA extracted from cattle feces spiked with E. coli O157. In feces spiked with the pooled mixture of five E. coli O157 strains, the minimum detection limit was 3.6×10(4) CFU/g. We also evaluated the assay with feces from cattle experimentally inoculated with E. coli O157 by comparing the results to a culture-based method. For the majority of samples tested, the concentration of E. coli O157 detected by the real-time and culture methods was within one log difference. However, the assay could only be evaluated for cattle shedding high concentrations of E. coli O157. In conclusion, the mqPCR quantifying E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets may have use in detecting and quantifying super shedders, but is not applicable for quantification in animals shedding low concentrations (10(2) to 10(3) CFU/g feces).

Rapid Detection Viable Escherichia Coli O157 in Raw Milk Using Loop-Mediated Isothermal Amplification with Aid of Ethidium Monoazide

The distinction between viable and dead cells was a major issue in detection of pathogenic microbe in foods especially when foods had been subjected to thermal processing. The performance of a loop-mediated isothermal amplification (LAMP) assay with aid of ethidium monoazide (EMA) for detecting viable Escherichia coli O157 in raw milk was presented in this paper. Three pairs of primers were specially designed for recognizing eight distinct sequences of rfbE gene. LAMP can only amplified DNA of viable Escherichia coli O157 because EMA selectively penetrated dead cells and covalently bound to DNA, detection limit level for artificially contaminated raw milk samples by the EMA-LAMP assay was 440 cfu/mL corresponding to 3–5 cells per reaction tube, while the detection level by EMA-PCR was 4.4×104 cfu/mL. In conclusion, EMA-LAMP had offered a novel assay for distinction between viable and dead cells with promising application in food safety detection.

Effect of modified atmosphere packaging on the persistence and expression of virulence factors of Escherichia coli O157:H7 on shredded iceberg lettuce.

Fresh-cut leafy greens contaminated with Escherichia coli O157:H7 have caused foodborne outbreaks. Packaging conditions, coupled with abusive storage temperatures of contaminated lettuce, were evaluated for their effect on the potential virulence of E. coli O157:H7. Shredded lettuce was inoculated with 5.58 and 3.98 log CFU E. coli O157:H7 per g and stored at 4 and 15°C, respectively, for up to 10 days. Lettuce was packaged under treatment A (modified atmosphere packaging conditions used for commercial fresh-cut produce, in gas-permeable film with N(2)), treatment B (near-ambient air atmospheric conditions in a gas-permeable film with microperforations), and treatment C (high-CO(2) and low-O(2) conditions in a gas-impermeable film). E. coli O157:H7 populations from each treatment were determined by enumeration of numbers on MacConkey agar containing nalidixic acid. RNA was extracted from packaged lettuce for analysis of expression of virulence factor genes stx(2), eae, ehxA, iha, and rfbE. E. coli O157:H7 populations on lettuce at 4°C under all treatments decreased, but most considerably so under treatment B over 10 days. At 15°C, E. coli O157:H7 populations increased by at least 2.76 log CFU/g under all treatments. At 15°C, expression of eae and iha was significantly greater under treatment B than it was under treatments A and C on day 3. Similarly, treatment B promoted significantly higher expression of stx(2), eae, ehxA, and rfbE genes on day 10, compared with treatments A and C at 15°C. Results indicate that storage under near-ambient air atmospheric conditions can promote higher expression levels of O157 virulence factors on lettuce, and could affect the severity of E. coli O157:H7 infections associated with leafy greens.

Improved Detection of Viable Escherichia coli O157:H7 in Milk by Using Reverse Transcriptase-PCR

A sensitive reverse transcriptase-PCR (RT-PCR) method to detect viable Escherichia coli O157:H7 in milk was established. The primer sets were designed based on the nucleotide sequences of the rfbE (per) and wbdN genes in the O157 antigen gene cluster of E. coli O157:H7. RT-PCR using five different primer sets yielded DNA with sizes of 655, 518, 450, and 149-bp, respectively. All five of the E. coli O157:H7 strains were detected by RT-PCR, but 11 other bacterial species were not. The sensitivity of RT-PCR was improved by adding yeast tRNA as a carrier to the crude RNA extract. The RT-PCR amplifying the 149-bp DNA fragment was the most sensitive for detecting E. coli O157:H7 and the most refractory to the bactericidal treatments. Heat treatment at <TEX>$65^{\circ}C$</TEX> for 30 min was the least inhibitory of all bactericidal treatments. Treatment with RNase A strongly inhibited the RT-PCR of heated milk but not unheated milk. This study described RT-PCR methods that are specific and sensitive with a detection limit of 10 E. coli O157:H7 cells, and showed that pre-treating milk samples with RNase A improved the specificity to detect viable bacteria by RT-PCR.

A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products

A 7-plex PCR assay was developed to achieve an effective detection and identification of serotype Enteritidis of Salmonella spp. and shiga toxin-producing type of Escherichia coli O157 in meat products. Six DNA sequences in the invA and sdfI genes of Salmonella Enteritidis as well as rfbE, eae, stx1, and stx2 genes of E. coli O157:H7 were employed to design primers. The multiplex PCR assay could specifically and sensitively detect and identify target pathogens. Applying the assay to meat samples, the multiplex PCR assay was able to simultaneously detect and identify the two pathogens at a sensitivity of three CFU/10 g raw meats after simple 16 h enrichment in buffered peptone water. Further applying in 21 retail meat samples revealed that two samples were positive for non-shiga toxin producing E. coli O157, one sample was positive for Stx2 producing E. coli O157 and five samples were positive for Salmonella enterica Enteritidis. Taken together, the 7-plex PCR assay is a rapid and reliable method for effectively screening single or multiple pathogens occurrences in various meat products, and could also identify the Salmonella enterica Enteritidis from all Salmonella spp. and shiga toxin producing type from all E. coli strains. Considering as a non expensive screening tool, the multiplex PCR assay has a great potential in complement for food stuff analysis by conventional microbiological tests.

Development of PCR assays for detection of Escherichia coli O157:H7 in meat products

A multiplex polymerase chain reaction (PCR) procedure based on fliC h7 and rfbE genes was developed for the detection of Escherichia coli O157:H7 in raw pork meat and ready-to-eat (RTE) meat products. Two different DNA extraction procedures were evaluated for application on meat products. MasterPure™ DNA Purification kit in combination with immunomagnetic separation was found to be the best method in a meat system. The optimized PCR included an enrichment step in brilliant green bile 2% broth at 37 °C. This method was applied to artificially inoculated meat and RTE meat products with different concentrations of E. coli O157:H7. The results indicate that the PCR assay developed could sensitively and specifically detect E. coli O157:H7 in raw pork meat and RTE meat products in approximately 10 h, including a 6 h enrichment step. Thus, this method could be proposed for screening E. coli O157:H7 in raw pork and RTE meat products.

A simple PCR-based macroarray system for detection of multiple gene markers in the identification of priority enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) strains bearing the O antigenic determinants O157, O26, O111, O103, and O145 have a high rate of association with foodborne illness worldwide. To expand Canadian food inspection capability, a cloth-based hybridization array system (CHAS) was developed for the identification and characterization of priority EHEC. This method targets key virulence genes (eae, hlyA, vt1, and vt2) plus the rfbE gene specifying the O157 antigenic determinant, and the wzx genes specifying the O26, O111, O103, and O145 determinants. Multiplex PCR products incorporating a digoxigenin label were detected by hybridization with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. This method identified the relevant markers in 85 different strains bearing various combinations of the target genes (virulence and priority O-antigen markers). None of the target genes was detected in 26 different strains of other E. coli and non-E. coli bacteria. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the various markers among different bacterial strains. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the markers among various target and nontarget bacteria. The entire procedure could be completed in less than 5 h, and is useful for the identification of priority EHEC colonies isolated from foods by using enrichment culture techniques.

A Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Detection ofSalmonellaspp.,Escherichia coliO157, andListeria monocytogenesin Meat Products

To achieve an effective detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products, a multiplex real-time polymerase chain reaction (PCR) coupled with a multipathogen enrichment strategy was developed in this study. Pathogen-specific DNA sequences in the invA, rfbE, and hlyA genes were employed to design primers and TaqMan probes for identifying Salmonella spp., E. coli O157, and L. monocytogenes, respectively. An internal amplification control (IAC) utilizing a novel DNA sequence from human adenovirus was incorporated into the multiplex PCR assay to indicate false-negative results. Concurrent amplifications of multiple targets and IAC were thoroughly evaluated and optimized to minimize PCR competitions. Combined with a multipathogen enrichment in a selective enrichment broth for Salmonella, Escherichia, and Listeria (SEL), the multiplex real-time PCR assay was able to simultaneously detect all of the three organisms in artificially contaminated ground beef at a detection sensitivity of <18 CFU/10 g ground beef. Applying the assay to 26 retail meat samples including beef, chicken, turkey, and pork revealed that 12 samples were positive for one of the organisms and 3 samples were positive for two of the organisms after a 20-h enrichment in SEL. The remaining meat samples tested negative for all of the organisms by only showing amplification of the IAC. These results were confirmed by traditional culture methods testing for each individual species. Taken together, the multiplex real-time PCR assay combined with multipathogen enrichment is a rapid and reliable method for effectively screening single or multiple pathogen occurrences in various meat products.

Prevalence of Escherichia coli O157 and verocytotoxin producing E. coli (VTEC) on Danish beef carcasses

The prevalence of verocytotoxin producing Escherichia coli (VTEC), E. coli O157, and VTEC O157 in 474 swab samples from Danish beef carcasses was determined. The presence of E. coli O157 was determined by a culture method that included immunomagnetic separation (IMS) followed by real time PCR testing of isolates for verocytotoxin ( vtx) genes. E. coli O157 was recovered from 4.2% of the carcass samples and VTEC O157 from 3.4% of the samples. All VTEC O157 contaminated carcasses were from bull calves and the VTEC O157 prevalence on bull calf carcasses was 7.3%. The VTEC O157 contaminated beef carcasses were sampled again after one week of cold storage, and 15 of the 16 carcasses were then VTEC O157 negative. The presence of VTEC was determined by a duplex real time PCR assay for vtx1 and vtx2 in DNA from enrichment cultures of swabs. In total 45.4% of the samples were VTEC positive. VTEC were isolated from 21% of 77 vtx-positive samples that were identified by replication of colonies on hydrophobic grid membrane filters followed by hybridisation with vtx specific DNA probes. Fourteen of the 16 VTEC isolates were non-O157 and these strains were negative for the virulence gene eae. A real time PCR assay for the E. coli O157 specific rfbE gene was developed. In total 22.4% of the enriched samples were positive for the O157 rfbE gene. The combined results of the vtx and rfbE real time PCR screening showed that 17.5% of the carcasses potentially were contaminated with VTEC O157. Screening of carcass swabs was expanded by real time PCR testing for eae in a subset of the samples. Of 244 samples, 25.4% were positive for both vtx and eae. The eae gene was detected in 81% of the vtx-positive samples and in 46% of 67 vtx-negative samples, indicating that bacteria harbouring eae are widespread on bovine carcasses. The present study shows that real time PCR screening of carcass samples for genes encoding virulence or other genetic markers is a reliable method for rapid identification of carcasses that potentially are contaminated with VTEC.

Induction of Escherichia coli O157:H7 into the viable but non‐culturable state by chloraminated water and river water, and subsequent resuscitation

Induction of culturable Escherichia coli O157:H7 cells into a viable but non-culturable (VBNC) state by chloraminated tap water was carefully investigated; as many as 90% of initial cells entered into a VBNC state within 15 min, compared with 14% in river water within 14 weeks. To understand what specific stresses may induce E. coli O157:H7 into a VBNC state, chloraminated tap water, autoclaved river water, and media with known ingredients (PBS buffer and deionized water) at 4°C or 25°C were used to examine induction efficiency. Chloramination alone, or the combination of starvation with either low temperature or osmotic pressure, induced E. coli O157:H7 into a VBNC state, while starvation alone did not induce the bacteria into a VBNC state within 1.5 years. The mRNA of the rfbE and fliC genes was detected in the 10-month-old VBNC cells induced by river water, confirming the viability of E. coli O157:H7 VBNC cells. The VBNC cells induced by chloraminated water and the 10-month-old VBNC cells induced by river water were first resuscitated using autoinducers produced by E. coli O157:H7 itself in a serum-based medium; the VBNC cells of bovine isolates recovered more efficiently compared with those of clinical isolates. These results demonstrate a potential health risk of VBNC E. coli O157:H7 in environmental water and the utility of monitoring viable E. coli O157:H7 including VBNC cells based on the mRNA of the rfbE and fliC genes.

Attomole DNA Electrochemical Sensor for the Detection of Escherichia coli O157

Enterohemorrhagic Escherichia coli O157, a verocytotoxin (VT1/2)-producing pathogen, can be deadly because it can induce acute or chronic renal failure. To speed up the clinical diagnosis of related syndromes caused by E. coli O157, there is an urgent need for rapid, simple, and reliable analytical tools for its quantitation. In this study, we developed a novel electrochemical competitive genosensor, featuring gold-electrodeposited screen-printed electrodes (nanoAu/SPE) modified with a self-assembled monolayer of thiol-capped single-stranded DNA (capture probe), for the detection of the rfbE gene, which is specific to E. coli O157. This assay functions based on competition between the target gene (complementary to the capture probe DNA) and reporter DNA-tagged, hexaammineruthenium(III) chloride-encapsulated liposomes. The current signal of the released liposomal Ru(NH(3))(6)(3+) was measured using square wave voltammetry, yielding a sigmoidally shaped dose-response curve whose linear portion was over the range from 1 to 10(6) fmol. This liposomal competitive assay provides an amplification route for the detection of the rfbE gene at ultratrace levels; indeed, we could detect as little as 0.75 amol of the target rfbE DNA (equivalent to the amount present in 5 microL of a 0.15 pM solution).

DEVELOPMENT AND EVALUATION OF A LOOP‐MEDIATED ISOTHERMAL AMPLIFICATION METHOD FOR DETECTING ESCHERICHIA COLI O157 IN RAW MILK

ABSTRACT In the present study, we report the performance of a loop‐mediated isothermal amplification (LAMP) assay detecting foodborne pathogen Escherichia coli O157. Three pairs of primers were specially designed for recognizing eight distinct sequences of rfbE gene. Time and temperature conditions for amplification of E. coli O157 were optimized to be 40 min at 65C. The LAMP assay gave artificially contaminated raw milk sample detection limit level of 410 cfu/mL which corresponds to three to five cells per reaction tube, while the detection level of conventional polymerase chain reaction was 4.1 × 104 cfu/mL. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with the ISO 16654: 2001 reference method.PRACTICAL APPLICATIONSThe loop‐mediated isothermal amplification method reported here provides a powerful tool for the detection of Escherichia coli O157 in raw milk samples because of its specificity, sensitivity and rapidity.

An improved method for RNA extraction from carcass samples for detection of viableEscherichia coliO157:H7 by reverse-transcriptase polymerase chain reaction

To develop a rapid RNA extraction procedure for maximizing bacterial RNA yield from carcass samples with low abundance of Escherichia coli O157:H7 without pre-enrichment. Nontarget bacterial cells were added to the sample prior to RNA extraction, facilitating the co-precipitation of target RNA along with nontarget RNA and thus enhancing the recovery. This method was developed using a serial dilution of log phase target cells (E. coli O157:H7), combined with a high number of nontarget cells (E. coli K12). Cells were lysed by a bead beating method followed by RNA purification using a commercial kit. A reverse-transcriptase PCR assay for the detection of rfbE gene in E. coli O157:H7 was used to demonstrate that the procedure increased the recovery of amplifiable RNA target with a detection limit of approximately 63 CFU ml(-1) in cultures and 27.5 CFU ml(-1) in carcass liquor. An RNA extraction procedure was developed to detect low numbers (<30 viable cells ml(-1)) of E. coli O157:H7 in carcass liquor without pre-enrichment. This method could be applied for the detection of E. coli O157:H7 in low abundance on carcasses where rapid detection and early intervention is essential for safety in the livestock industry.