Rapid Detection of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus by Multiplex Real-Time Fluorescent PCR

Summary

This protocol uses multiplex real-time PCR with primers/probes targeting SA-specific nuc and MRSA mecA genes. It enables rapid (<1 h), qualitative detection of SA/MRSA nucleic acids in human sputum samples, overcoming limitations of culture-based methods.

Abstract

Staphylococcus aureus (S.aureus) is a common Gram-positive human pathogen that causes community-acquired and nosocomial infections, with diverse clinical manifestations ranging from local superficial lesions and food poisoning to fatal systemic infections. The discovery of antibiotics significantly reduced the mortality rate, but the problem of drug resistance has since become increasingly prominent. Since the first identification of MRSA in 1960, this strain has emerged as a global public health threat. MRSA is a major pathogen of nosocomial infections, capable of causing various severe diseases such as endocarditis, chronic osteomyelitis, pneumonia, pyogenic arthritis, and bacteremia. Therefore, rapid and accurate detection of S. aureus and its drug resistance is crucial for guiding clinical treatment.

Current routine detection methods for S.aureus and MRSA have significant limitations. The traditional bacterial culture method, serving as the "gold standard" for decades, can provide definitive species identification and drug sensitivity results, but the process is time-consuming, taking 48 to 72 h. Furthermore, this method is susceptible to contamination and relies on specialized laboratory facilities and skilled technicians. Serological testing achieves non-invasive diagnosis by detecting S.aureus antibodies in patient serum, but it cannot distinguish between active infections and past infections, nor can it identify drug-resistant strains (such as MRSA).

This study focuses on developing a novel multiplex real-time fluorescence PCR detection method to overcome the above-mentioned limitations. This method designs specific primers and TaqMan fluorescent probes targeting the species-specific nuc gene of S.aureus and the mecA gene mediating methicillin resistance, enabling simultaneous amplification and detection of S.aureus and MRSA in a single reaction system. This technology greatly reduces the detection time, providing a rapid, accurate, and cost-effective solution for S.aureus and MRSA detection. This innovative approach greatly improves clinical diagnostic efficiency and facilitates the early implementation of targeted antibiotic therapy, making important contributions to controlling drug-resistant bacterial infections.

Introduction

S. aureus is a widespread Gram-positive conditional pathogen that colonizes the nasal mucosa of healthy individuals^1,2. When host immunity is compromised, it can cause local skin infections, deep tissue invasion, or systemic diseases (e.g., pneumonia, endocarditis, osteomyelitis, and bacteremia)³. Methicillin, a semisynthetic penicillin, was clinically applied for S. aureus infections in 1959, and two years later, MRSA emerged in the UK, rapidly spreading to European countries including Denmark, France, and Switzerland⁴. MRSA resistance to β-la....

Protocol

This study was approved by the Ethics Committee of Guangdong Provincial People's Hospital (Ethics No.: KY2025-510-01). Detailed information regarding the materials used in this research (reagents, chemicals, equipment, and software) can be found in the Table of Materials.

1. Specimen collection and preservation protocol

1. Inclusion criteria: With the exception of quality control materials, identify all collected samples as positive specimens using the gold standard method (culture-based assay).
2. Human sputum collection
   1. Collect 1-3 mL of deep-coughed sputum from the lower r....

Results

Detection of S. aureus and MRSA using qPCR
Based on the species-specific nuc gene of S. aureus and the mecA drug resistance gene of MRSA, we designed primers and probes labeled with distinct fluorophores for qPCR detection. All quality control results throughout qPCR experiments consistently fell within recommended thresholds, confirming the reliability of detection outcomes (Figure 1).

In th.......

Discussion

Accurate and early detection of methicillin resistance is critical for the prognosis of infections caused by S. aureus. Early identification of S. aureus and MRSA can reduce the misuse of broad-spectrum antibiotics and decrease the selective pressure leading to the emergence and spread of bacterial resistance.

Traditional phenotypic detection methods, such as cefoxitin disk diffusion, require an 18-24 h cultivation period and exhibit high false-negative rates for heteroresist.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated nucleic acid extractor	DAAN	Smart 32	For DNA extraction
BSC-1500IIA2-X	BIOBASE	SEDA 20143222263	Biosafety cabinet
E-Centrifuge	WEALTEC		Centrifuge the residual liquid off the wall of the tube
Methicillin-resistant Staphylococcus aureus drug resistance gene detection kit	BOJIE	ZC-HX-38-2	Detection of methicillin-resistant Staphylococcus aureus resistance genes
Nucleic acid extraction kit	BOJIE	TQ-BG-001-96B	Extract nucleic acid
SLAN Fully automatic medical PCR analysis system	HONGSHI		Data Analysis
SLAN-96S Real-Time PCR machine	HONGSHI	SLAN-96S	Fluorescent quantitative PCR amplification
Thermo Scientific NanoDrop	Thermo Scientific	NanoDrop One	Quantitative analysis of DNA, RNA, and proteins
Ultra-low temperature freezers (DW-YL450)	MELING	SEDA 20172220091	-20 °C for storing reagents
Vortex-5	Kylin-bell		For mixing reagent