Viable pathogens detection in fresh vegetables by quadruplex PCR

- PMA and IAC was selected to eliminate false positive and false negative results.
- The assay could detect 101 CFU/g viable bacteria after 12 h enrichment.
- The dead cells and other bacteria did not interfere with the assay results.

Salmonella Enteritidis, Eschericha coli O157:H7 and Listeria monocytogenes are important pathogens contaminating fresh vegetables. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in fresh vegetables. To detect viable cells, propidium monoazide (PMA) was applied to eliminate the false-positive results. In addition, an internal amplification control (IAC) was added to the quadruplex PCR as indicator of false negative results that are mainly caused by the PCR inhibitors. The limit of detection (LOD) for the viable cells with or without PMA treatment was 102 CFU/mL for S. Enteritidis, 103 CFU/mL for E. coli O157:H7 and 104 CFU/mL for L. monocytogenes, showing that the PMA treatment did not influence the sensitivity. After 12 h enrichment, the PMA-mPCR-IAC combination assay could detect as low as 101 CFU/g for viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in spiked vegetables. The PMA-mPCR-IAC combination assay holds promise for the simultaneous detection of viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in fresh vegetables.