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 10 2 , 10 2 , and 10 1  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.