Dual phage amplification-mediated multiplex detection strategies for the simultaneous detection of Salmonella enterica and Staphylococcus aureus

Multiplex bacteria detection is essential to monitor various pathogens situations where multiple pathogen species are a concern. Herein, we explored novel dual phage amplification-based strategies (PAA) combined with a multiplex detection technique for simultaneous bacteria detection in one single tube. This assay was normalized on two common foodborne pathogens: Salmonella enterica and Staphylococcus aureus . In the presence of the target bacteria, phages could specifically recognize the viable cells and replicate themselves within the cells to produce more progeny phages. After thermal lysis within 10 min, the products with DNA of the progeny phages were analyzed with the multiplex detection strategies. The specificity of the multiplex detection strategies integrated with the high sensitivity of the PAA assay allowed this assay to be rapid, specific, and sensitive for simultaneous quantification of Salmonella and Staphylococcus aureus . Optimized parameters of PAA, single and multiplex qPCR were defined, and the analytical sensitivities, specificities, and stabilities of single and multiplex reactions were validated under defined experimental conditions and in real samples. The results demonstrated wide linear ranges (10–10 8 CFU/mL), with detection limits of 10 CFU/mL for all bacteria tested. Moreover, the total detection time could be reduced to no more than 4 h. This assay was successfully applied to real food samples consistent with the results comparable to traditional plate counting methods. Due to the rapid and simple operation, low sensitivity and cost, high specificity, and the ability to distinguish live bacteria, the proposed PAA-based multiplex qPCR assay provides an effective and promising strategy for the simultaneous detection of viable bacteria. • PAA relied on novel phages allows signal amplification to increase sensitivity. • PAA combined with single detection assay can achieve sensitive quantification for either Salmonella or Staphylococcus. • Dual PAA-mediated multiplex detection assay was applied for simultaneous detection. • This assay presented wide linear ranges of 10–10 8 CFU/mL, with the LODs of 10 CFU/mL for all bacteria tested.