Simultaneous quantitative detection of viable Salmonella spp., Shiga toxin–producing Escherichia coli, Bacillus cereus, and Listeria monocytogenes in milk through multiplex real-time polymerase chain reaction

In our study, a multiplex real-time PCR (mRT-PCR)-based method was established to detect viable Salmonella spp. (SS), Shiga toxin-producing Escherichia coli (STEC), Bacillus cereus (BC), and Listeria monocytogenes (LM) simultaneously in milk samples. The oligonucleotide sequences of invA from SS, stx1 from STEC, entFM from BC, and mpl from LM (the target genes) were used for primer and probe design. To eliminate false-positive results, the bacterial samples, containing live and partially dead bacterial cells, were treated with 300 μM SDS, followed by 20 μM propidium monoazide (PMA). The optimized SDS-PMA-mRT-PCR (SPMP) method exhibited a 10² cfu/mL detection limit for the 4 target bacteria, similar to the accuracy of the plate count method. Our findings suggest that the developed SPMP method can rapidly detect viable SS, STEC, BC, and LM in milk samples within 7 h.