Simultaneous detection of viable Salmonella spp., Escherichia coli, and Staphylococcus aureus in bird's nest, donkey‐hide gelatin, and wolfberry using PMA with multiplex real‐time quantitative PCR

Salmonella spp., Escherichia coli, and Staphylococcus aureus are common microbial contaminants within the homology of medicine and food that can cause serious food poisoning. This study describes a highly efficient, sensitive, specific, and simple multiplex real‐time quantitative PCR (mRT‐qPCR) method for the simultaneous detection of viable Salmonella spp., E. coli, and S. aureus. Primers and probes were designed for the amplification of the target genes invA, uidA, and nuc. Dead bacterial genetic material was excluded by propidium monoazide (PMA) treatment, facilitating the detection of only viable bacteria. This method was capable of detecting Salmonella spp., E. coli, and S. aureus at 102, 102, and 101 CFU/ml, respectively, in pure culture. PMA combined with mRT‐qPCR can reliably distinguish between dead and viable bacteria with recovery rates from 95.7% to 105.6%. This PMA‐mRT‐qPCR technique is a highly sensitive and specific method for the simultaneous detection of three pathogens within the homology of medicine and food. This study established an efficient, sensitive, specific, and simple multiplex real‐time quantitative PCR (mRT‐qPCR) method for the simultaneous detection of viable Salmonella spp., Escherichia coli, and Staphylococcus aureus in bird's nest, donkey‐hide gelatin, and wolfberry. This method was capable of detecting Salmonella spp., E. coli, and S. aureus at 102, 102, and 101 CFU/ml, respectively, in pure culture. And, the PMA combined with mRT‐qPCR can reliably distinguish between dead and viable bacteria with recovery rates from 95.7% to 105.6%. This study describes a highly efficient, sensitive, specific, and simple multiplex real‐time quantitative PCR (mRT‐qPCR) method for the simultaneous detection of viable Salmonella spp., E. coli, and S. aureus.