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Updated: Apr 28, 2026
Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR
Published on: February 1, 2014
Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis
Elodie Barbau-Piednoir1, Jacques Mahillon2, Julie Pillyser3
1Laboratory of Food and Environmental Microbiology, Earth and Life Institute, Université catholique de Louvain, Louvain-la-Neuve, Belgium; Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health, Brussels, Belgium; Scientific Service Foodborne Pathogens, Scientific Institute of Public Health, Brussels, Belgium.
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.
Insights
Propidium monoazide (PMA) viability quantitative PCR (v-qPCR) struggles to reliably detect only viable foodborne pathogens. Modifications to PMA treatment did not eliminate signals from dead Salmonella Enteritidis, suggesting enrichment followed by qPCR is a more dependable detection method.
Area of Science:
- Microbiology
- Molecular Biology
- Food Safety
Background:
- Propidium monoazide (PMA) coupled with PCR (viability PCR) is a method for detecting viable bacteria in food.
- Previous studies have highlighted limitations in viability PCR's ability to completely eliminate signals from dead bacteria.
Purpose of the Study:
- To assess the effectiveness of PMA in viability-qPCR (v-qPCR) for detecting viable Salmonella Enteritidis.
- To optimize PMA treatment protocols to achieve a complete negative signal from dead bacteria.
- To investigate the factors contributing to the residual qPCR signal in non-culturable cells.
Main Methods:
- Treatment of viable and dead Salmonella Enteritidis cells with PMA.
- Modification of PMA treatment parameters (dark incubation duration, PMA concentration).
- Analysis of qPCR signals from treated and untreated bacterial cells.
- Comparison of v-qPCR with traditional enrichment followed by qPCR.
Main Results:
- Modifications to the PMA treatment protocol did not improve the removal of qPCR signal from dead Salmonella.
- PMA, at low concentrations, can unexpectedly decrease the qPCR signal from viable S. Enteritidis.
- The residual qPCR signal in non-culturable cells is not solely dependent on cell-wall/membrane integrity.
Conclusions:
- Viability-qPCR using PMA has limitations in accurately detecting only viable bacteria, as demonstrated with Salmonella Enteritidis.
- The inability to completely eliminate signals from dead cells is a significant drawback.
- For reliable and rapid foodborne bacteria detection, enrichment followed by qPCR is recommended over v-qPCR.
