Multiplex polymerase spiral reaction combined with melting curve analysis for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus

Abstract

Abstract Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are common food-borne pathogens that cause food poisoning and acute gastroenteritis in humans. In this study, we developed a method for rapid, sensitive and specific 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 sequence of S. typhimurium and nuc gene sequence of S. aureus, and the nucleic acid amplification reaction was achieved using Bst DNA polymerase under isothermal conditions in the same reaction tube. After amplification 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 20 CFU/mL pure bacterial culture for each reaction. m-PSR had similar detection limits to multiplex polymerase chain reaction. Based on this method, analysis of contaminated food samples showed excellent sensitivity and specificity consistent with those of pure bacterial cultures. In conclusion, our method is rapid, simultaneous and specific, and promises to be a useful tool for the detection of food-borne pathogens in the food industry.