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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Publications at this Location » Publication #271428

Title: The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

item Wang, Rong
item Schmidt, John
item Arthur, Terrance
item Bosilevac, Joseph - Mick

Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/20/2012
Publication Date: 1/1/2013
Citation: Wang, R., Schmidt, J.W., Arthur, T.M., Bosilevac, J.M. 2013. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests. Food Microbiology. 33: 205-212.

Interpretive Summary: There are DNA-based commercial assays to detect pathogens such as E. coli O157:H7 in foods. These assays are intended to determine whether the food is contaminated with the pathogen. Some users would like to use the assays to indicate, not only whether the pathogen is present, but if so, at what level. However, these assays have not been validated to indicate levels of the pathogen. To help answer the questions from users whether these assays should be used quantitatively to identify E. coli O157:H7 contamination levels in meat products, we designed the present study to determine the effects of physiological status of the bacteria on the accuracy of these systems for measuring E. coli O157:H7 contamination. Our data indicated that test results from two commercial assays should not be used as a direct measurement for estimating contamination levels of E. coli O157:H7 because physiological status of the bacteria plays a critical role and affects the testing results. Our study provided scientific advice regarding how to use these commercial assays properly and accurately for E. coli O157:H7 detection in meat products.

Technical Abstract: Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety and quality assurance in the beef industry. The Biocontrol GDS and the DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on real-time PCR amplification and potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. Because bacterial cell injury and recovery might create a potential problem for detecting E. coli O157:H7 by the molecular detection systems, the present study investigated the effects of bacterial physiologic status on the ability of these systems to accurately measure contamination levels by E. coli O157:H7 in meat products. While the PCR threshold cycle (CT) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, a growth comparison study indicated that despite the delayed log growth phase of frozen-injured bacteria compared to healthy cells, post-enrichment cell densities of injured or healthy bacteria did not correlate with cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by CT values from either assay. In addition, nonviable E. coli O157:H7 cells at high densities were able to persist in meat products and could be detected by both assays in pre-enriched and post-enriched beef samples. We concluded that the CT values from both systems performed on enriched samples are not an appropriate indication of pre-enrichment contamination levels of E. coli O157:H7 because bacterial physiologic status plays a critical role during sample enrichment period and affects the assay test results.