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Updated: Nov 9, 2025
Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
Published on: May 8, 2013
Pathogenic and Virulence Factor Detection on Viable but Non-culturable Methicillin-Resistant Staphylococcus aureus
Hua Jiang1, Kan Wang2, Muxia Yan1
1Department of Haematology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China.
Abstract:
Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called "standards," "guidelines," or "gold standards" are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/μl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.
Insights
A new method, PMA-CPA, can detect toxins from methicillin-resistant Staphylococcus aureus (MRSA) in viable but non-culturable (VBNC) states. This advancement improves food safety by identifying pathogens missed by standard detection methods.
Area of Science:
- Food Safety and Microbiology
- Molecular Diagnostics
- Public Health
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging foodborne pathogen, posing significant public health risks.
- Conventional methods for MRSA detection often fail to identify viable but non-culturable (VBNC) cells, limiting accurate food safety assessments.
- Existing diagnostic standards struggle to detect MRSA in its VBNC state, necessitating advanced detection techniques.
Purpose of the Study:
- To develop a sensitive Cross-Priming Amplification (CPA) method for detecting key MRSA virulence genes (sea, seb, pvl).
- To establish a rapid and accurate method for detecting MRSA in the VBNC state using Propidium Monoazide (PMA) combined with CPA (PMA-CPA).
- To validate the efficacy of the PMA-CPA assay in real-world food samples.
Main Methods:
- Development of CPA assays targeting staphylococcal enterotoxin A (sea), staphylococcal enterotoxin B (seb), and Panton-Valentine leukocidin (pvl) genes.
- Establishment of a Propidium Monoazide-Cross-Priming Amplification (PMA-CPA) assay for detecting viable MRSA cells, including those in the VBNC state.
- Comparative analysis of PMA-CPA against conventional PCR and VBNC confirmation methods using analytical sensitivity tests and real samples.
Main Results:
- CPA assays demonstrated high analytical sensitivity with limits of detection (LOD) of 75 ng/μl for sea, 107.5 ng/μl for seb, and 85 ng/μl for pvl.
- The PMA-CPA method exhibited superior rapidity, sensitivity, specificity, and accuracy compared to standard PCR assays for VBNC cell detection.
- PMA-CPA showed 100% accordance with conventional VBNC confirmation methods, successfully detecting MRSA toxins in the VBNC state in real samples.
Conclusions:
- Three CPA assays were successfully developed for detecting critical MRSA virulence genes.
- The PMA-CPA assay is a highly effective tool for detecting MRSA virulence gene expression in the VBNC state.
- This study presents the first demonstration of detecting MRSA toxins in VBNC cells, offering a significant advancement for food safety diagnostics.
