Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis

Abstract

The propidium monoazide (PMA) coupled with PCR (viability PCR) is used in foodborne pathogen detection in order to detect only viable bacteria. Originally presented to fully remove the signal of dead bacteria, the limits of the viability PCR rapidly came out in the literature. In this study, the use of PMA in a viability-qPCR (v-qPCR) was assessed on viable and dead cells of Salmonella enterica subsp. enterica serovar Enteritidis. The PMA treatment protocol was modified (dark incubation duration, concentration of PMA) to evaluate if a complete negative signal of dead Salmonella was possible. However, none of these modifications was found to improve the removal of the remaining qPCR signal observed in the presence of dead bacteria. The present research also underlines that PMA may unexpectedly decrease the qPCR signal observed on living S. Enteritidis at low concentration. Finally, the use of S. Enteritidis cells killed by processes altering or not the cell-wall/membrane gives us a clue to answering the question about the non-total extinction of the signal of dead cells sample in the v-qPCR assay. Indeed, the data strongly indicate that the remaining qPCR signal observed in non-culturable cells does not only depend on the cell-wall/membrane integrity of the bacteria. According to these results, the authors suggest that for a rapid and reliable foodborne bacteria detection system, an enrichment followed by a qPCR analysis should be preferred to a v-qPCR.

Keywords: Detection; Propidium monoazide; Real-time PCR; SYBR®Green; Salmonella.