How Digital PCR Will Decrease the Number of False Positive Data in STEC Detection (15 minutes presentation and open exchanges)

The last decade has seen many advances in the area of molecular diagnostics. Amplification methods such as PCR are now widely applied for detection of food pathogens. Yet, designing specific assays requires identification of unique molecular signatures, an often challenging task due to genetic relatedness. Recent advances in sequencing technology and genome comparison methods alleviated this problem as exemplified by the possibility to single out Salmonella serovars. However, for routine testing, molecular methods remain complicated when pathogen identification involves detecting several markers. This is the case for pathogenic STEC that are still defined by the concomitant presence of stx and eae virulence genes. Although significant efforts were devoted to the search of a STEC unique signature, reference methods rely on co-detection of these genes. Positive PCR results for both genes obtained in one sample do not allow concluding that one bacterium displaying both markers is present rather than two nonpathogenic bacteria bearing one marker each. Such a result is, therefore, deemed presumptive positive; and, when both genes are present, the subsequent confirmation leads to a significant rate of positives. Development in digital PCR (dPCR) may solve this problem. With this method, a single genome can be probed and determinations as to whether markers are collinear or not can be made. Digital PCR may, therefore, be a straightforward method that detects, in one PCR run, the presence of a multi-marker pathogen and alleviates the need for hazardous confirmatory steps; thus, warranting its implementation in routine testing.