title: The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests.
abstract: Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS(®) and DuPont Qualicon BAX(®)-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (C(T)) 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, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by C(T) values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 10(6) cells/sample or higher, which are levels not typically seen in ground beef.
authors.affiliation: US Department of Agriculture, Agricultural Research Service, Roman L. Hruska US Meat Animal Research Center, Clay Center, NE 68933-0166, USA. rong.wang@ars.usda.gov
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
authors.forename: John W
authors.initials: JW
authors.lastname: Schmidt
authors.forename: Terrance M
authors.initials: TM
authors.lastname: Arthur
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Bosilevac
mesh_terms: D000818:Animals; D002417:Cattle; D019453:Escherichia coli O157 / Q000254:growth & development / Q000302:isolation & purification* / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics; D005506:Food Contamination / Q000032:analysis* / Q000191:economics; D008460:Meat / Q000191:economics / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D029968:Escherichia coli Proteins