Mining of novel target genes through Pan-genome analysis for Conventional PCR and Real-Time PCR detection of Staphylococcus argenteus in food

Abstract

Staphylococcus argenteus is a novel species of coagulase-positive staphylococci which was separated from Staphylococcus aureus in 2015. It can threaten human health like S. aureus but cannot identify with conventional biochemical or other convenient methods. In order to develop a more convenient, specific and sensitive detection method for S. argenteus, this study screen several candidate target genes for detecting S. argenteus specifically and conveniently using Pan-genome analysis. As a result, 20 specific target sequences of S. argenteus were found and five novel target genes of S. argenteus (S. arg_16901, S. arg_21105, S. arg_22757, S. arg_22771 and S. arg_24206) were performed well after validated assays. Based on the novel target S. arg_16901, conventional PCR and real-time fluorescence quantitative PCR methods were established to detect and identify S. argenteus strains. The specificity of these methods was robustly verified by S. argenteus and non-S. argenteus species. With extracting DNA of the dilution, the sensitivity of the PCR method was 9.8 × 106 CFU/mL, and it could differentiate and detect S. argenteus, in the 9.8 × 103 CFU/mL in the 10-dilution liquid sample without extracting DNA. After that, a novel multiplex PCR based on novel target genes S. arg_16901 and S. aureus-specific gene (nuc) was designed. This method could simultaneously differentiate S. argenteus and S. aureus strains. The results of sensitivity and specificity assays indicate that the specific target fragment S. arg_16901 not only exhibits excellent performance in detecting and differentiating S. argenteus from S. aureus in food samples but also demonstrates higher accuracy than MALDI-TOF MS. Moreover, this is the first report on the detection of S. argenteus using a species-specific gene without relying on other phenotype-based methods. Our research demonstrated that these specific target genes can be mined efficiently through a pan-genome analysis tool, and the novel PCR assays developed in this study offer a promising technique for rapid and accurate detection of S. argenteus from S. aureus and other Staphylococcus.