1. To establish an EMA-real time PCR assay for quantifying viable E. coli O157:H7. <P>
2. To determine detection sensitivity and linear range of the EMA-real time PCR system for
quantitating VBNC E. coli O157:H7. <P>
3. To evaluate the performance of the EMA-real time PCR system for quantitating E. coli
O157:H7 in beef.
Findings: This project involved the development of a new technique to detect and estimate numbers of viable cells of E. coli O157:H7. Bacterial cells that are subjected to harsh processing conditions, such as heating, cooling, cleaning and sanitizing, can become undetectable via traditionally employed cultural detection methods. This state of the microbial cell is termed viable-but-non-culturable (VBNC), and while the cells may be undetectable, they still retain important metabolic and virulence genes that will allow them to cause diseases upon recovery. By combining the stain, ethidium monoazide (EMA), that can only penetrate dead cells, with real-time polymerase chain reaction (PCR), we were able to distinguish and detect only viable, including VBNC cells of E. coli O157:H7 in pure cultures and in ground beef. Various concentrations of EMA, times of exposure to halogen light for EMA denaturation and the addition of ice cooling steps were tested before conducting real-time PCR. The optimized technique had a detection sensitivity of 10 CFU/g E. coli O157:H7 in ground beef following a 24-h enrichment step. This method is demonstrated to be potentially useful at avoiding false positive results that can come from using conventional PCR, while completely preventing false-negative results that can be generated by traditional cultural methods.