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Detection of Viable Enterohemorrhagic Escherichia Coli using PCR and RT-PCR


<OL> <LI> Develop QC-PCR assays for detection of viable enterohemorrhagic Escherichia coli. <LI> Develop an RT-PCR assay for detection of viable enterohemorrhagic Escherichia coli <LI> Determine the sensitivity of the QC-PCR and RT-PCR assays. <LI> Test the developed assays in artificially contaminated ground beef.

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NON-TECHNICAL SUMMARY: Development of strategies to ensure the safety of food is critical for sustaining U.S. agriculture. Recent detection methods based on DNA amplification, termed polymerase chain reaction (PCR), offer a high degree of speed, sensitivity and specificity for pathogen screening in foods. Positive results, however, must still be confirmed by time-consuming conventional methods as PCR does not differentiate viable from nonviable cells. Methodologies will be developed to rapidly detect viable enterohemorrhagic Escherichia coli (EHEC). A new DNA-based detection method, quantitative competitive PCR (QC-PCR), and an RNA-based detection method, RT-PCR of mRNA, will be developed. The slt toxin genes will be used as targets. For QC-PCR, a segment of competing DNA similar to the target DNA will be created. A constant amount of competing DNA is then added to dilutions of extracted target DNA followed by PCR amplification. The amount of target DNA is quantified by a generated standard curve. QC-PCR at two points during enrichment will be used to determine viability. For RT-PCR, mRNA , which is a known indicator of cell viability, will be used. RT-PCR amplification of slt mRNA will be used after a brief enrichment to determine viability. The sensitivity of both methods will be evaluated. The methods will be applied to artificially contaminated ground beef samples. The development of nucleic acid based methodologies to detect viable EHEC will be applicable to other foodborne pathogens and will provide a more rapid option for monitoring food safety.


APPROACH: Both assays will use primers from sltI and/or sltII of enterohemorrhagic Escherichia coli. Different amplicons from these targets will be evaluated for sensitivity. DNA or RNA will be extracted using developed methodologies. For the QC-PCR assay, extractions and subsequent PCR will be conducted at two timepoints during enrichment to determine pathogen viability. For RT-PCR, relative expression of sltI/sltII mRNA will be evaluated during enrichment using RT-PCR. Sensitivity of both methods will be determined by the use of serial dilutions of bacteria. QC-PCR and RT-PCR will also be tested for sensitivity with regard to acid and cold stressed bacteria and selective versus nonselective enrichment. Ground beef will be inoculated with serial dilutions of bacteria for application of the assays to food.


PROGRESS: 2001/07 TO 2004/01<BR>
The purpose of this project was to develop and compare nucleic acid amplification-based methods for the rapid detection of viable enterohemorrhagic Escherichia coli, including E. coli O157:H7. A quantitative competitive PCR (QC-PCR) assay was developed. Estimated CFU from QC-PCR were compared to viable plate counts. E. coli O157:H7 (10E2 to 10E8 CFU/mL) were detected and accurately quantified in broth and in milk. QC-PCR was applied using multiple timepoint quantitative competitive polymerase chain reaction (QC-PCR) to detect viable E. coli O157:H7 in ground beef enrichments. E. coli O157:H7 inoculated at 0.20 CFU/g cooked ground beef (25 g ground beef +225 ml mEC + novobiocin) was detected and confirmed as viable in less than 15 h. Application of immunomagnetic separation processes enhanced PCR detection limits 10 to 100 fold. An RT-PCR assay was developed using the stxII (Shiga toxin) mRNA as a target. Enrichment conditions were examined for their effect on stx mRNA expression of E. coli O157:H7 as measured by RT-PCR. Toxin expression was constitutive and was unaffected by media. Message RNA of stx toxin was not amplified from dead cells. E. coli O157:H7 was inoculated into trypticase soy broth or cooked ground beef enrichments. Detection by PCR and RT-PCR from both broth and meat enrichments was identical for both inocula at 1 or 10 CFU/gram. Southern blot hybridization detected amplification products at all time points in broth enrichments and at 8, 12 and 24 h for meat enrichments. The effect of storage time and growth in broth culture and in a food medium on DNA extraction efficiency and PCR detection sensitivity of E. coli O157:H7 were investigated. Detection limits were evaluated using dilution series PCR targeting the stx-II gene. The relationship between cell density and DNA yield was generally log-linear for pure cultures. When the bacteria were suspended in skim milk at a density of 10E6 CFU/ml, held at 4C and sampled at 24 h intervals, cell density, total DNA yield, and PCR detection limits remained stable throughout the entire 96 h storage period. However, when E. coli O157:H7 was grown in skim milk to a final cell density of 10E6 CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E. coli O157:H7 growth was static over 96 h of storage at 4C. This is most likely due to poor DNA purity, which consistently occurred when extracting from food matrices in which the pathogen was grown rather than stored. Future studies should address these issues. The impact of sublethal stress on expression and detection of E. coli O157:H7 stx, hly, and eae were investigated. Virulence factor expression and/or production was impacted (usually negatively) by stress conditions encountered in food indicating that an enrichment process would be necessary for application of these targets in an RT-PCR or ELISA assay. Enhanced virulence factor production was not observed under moderately acidic conditions or in apple juice.
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IMPACT: 2001/07 TO 2004/01<BR>
Ensuring food safety is an important goal for the sustainability of U.S. agriculture. The development and validation of rapid molecular-based methods offers quicker alternatives to conventional pathogen detection methods. Understanding factors that influence nucleic acid extraction and amplification from foods is critical to optimization of these rapid methods. The further development and refinement of these emerging technologies will enhance the safety of the food supply.

Drake, MaryAnne
North Carolina State University
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