Novel ST-Specific Molecular Target-Based Method for Simultaneous and Quantitative Detection of Staphylococcus aureus ST7, ST188 and ST398

<i>Staphylococcus aureus</i> is a globally crucial foodborne pathogen that can cause diarrhea, vomiting, and bloodstream infection in immunocompromised individuals. <i>S. aureus</i> has three predominant sequence types (STs) (ST7, ST188 and ST398) that are prevalent clones in both food and clinical cases. This study aimed to screen ST-specific targets for <i>S. aureus</i> ST7, ST188 and ST398, and then developed a novel rapid and accurate assay for the detection of these three predominant <i>S. aureus</i> STs in food. A total of 505 <i>Staphylococcus</i> strain genome sequences including 371 sequences of 58 different STs and 134 other non-target <i>S. aureus</i> ST genome sequences were subjected to pan-genome analysis; we successfully screened five novel ST-specific targets (group_10498 and group_10499 target for <i>S. aureus</i> ST7, group_9415 and group_9419 target for <i>S. aureus</i> ST188, group_9911 target for <i>S. aureus</i> ST398). The excellent specificity and sensitivity of all the targets were confirmed by PCR assays. Based on these molecular targets, mPCR and qPCR methods were developed for specifically identifying <i>S. aureus</i>' three predominant STs without non-target bacterial interference. The limits of detection (LODs) for the mPCR assay in artificially contaminated milk were determined to be 10⁴ CFU/mL for ST7, 10⁵ CFU/mL for ST188, and 10⁴ CFU/mL for ST398, while the LODs achieved by the qPCR method were 8.6 × 10² CFU/mL, 1.2 × 10² CFU/mL, and 6.4 × 10³ CFU/mL, respectively. The testing results for actual food samples suggested that the developed mPCR or qPCR assays could be used as an alternative to standard MLST analysis, for the rapid and reliable identification of <i>S. aureus</i> STs. The novel molecular detection technology established in this study provides an efficient and reliable detection method for the prevention and control of predominant <i>S. aureus</i> ST contamination in food and has important application potential and promotion prospects.