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

## TL;DR

This paper introduces a new method to detect specific strains of Staphylococcus aureus in food, improving detection speed and accuracy.

## Contribution

A novel molecular target-based method for simultaneous detection of three S. aureus sequence types (ST7, ST188, ST398) is developed.

## Key findings

- Five novel ST-specific targets were identified for S. aureus ST7, ST188, and ST398.
- mPCR and qPCR methods achieved high specificity and sensitivity for detecting these STs in food samples.
- qPCR detection limits were significantly lower than mPCR, enabling more sensitive identification.

## Abstract

Staphylococcus aureus is a globally crucial foodborne pathogen that can cause diarrhea, vomiting, and bloodstream infection in immunocompromised individuals. S. aureus 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 S. aureus ST7, ST188 and ST398, and then developed a novel rapid and accurate assay for the detection of these three predominant S. aureus STs in food. A total of 505 Staphylococcus strain genome sequences including 371 sequences of 58 different STs and 134 other non-target S. aureus ST genome sequences were subjected to pan-genome analysis; we successfully screened five novel ST-specific targets (group_10498 and group_10499 target for S. aureus ST7, group_9415 and group_9419 target for S. aureus ST188, group_9911 target for S. aureus 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 S. aureus’ 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 S. aureus STs. The novel molecular detection technology established in this study provides an efficient and reliable detection method for the prevention and control of predominant S. aureus ST contamination in food and has important application potential and promotion prospects.