Multiplex polymerase spiral reaction for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus.

ABSTRACT

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are common food-borne pahogens that cause food poisoning in humans. In this study, we developed a method for the simultaneous determination of S. typhimurium and S. aureus based on multiplex polymerase spiral reaction (m-PSR) and melting curve analysis. Two pairs of primers were designed specifically to target the conserved invA gene of S. typhimurium and nuc gene of S. aureus, and the nucleic acid amplification reaction was achieved under isothermal conditions in the same reaction tube for 40 min at 61 °C, melting curve analysis of the amplification product was carried out. The distinct mean melting temperature allowed simultaneous differentiation of the two target bacteria in the m-PSR assay. The limit of detection of S. typhimurium and S. aureus that could be detected simultaneously was 4.1 × 10-4 ng genomic DNA and 2 × 101 CFU/mL pure bacterial culture. Based on this method, analysis of artificially contaminated samples showed excellent sensitivity and specificity consistent with those of pure bacterial cultures. This method is rapid, simultaneous and promises to be a useful tool for the detection of food-borne pathogens in the food industry.