Duplex real-time fluorescent recombinase polymerase amplification for the rapid and sensitive detection of two resistance genes in drug-resistant Staphylococcus aureus

• Published in: Journal of microbiological methods 2022-11, Vol.202, p.106590-106590, Article 106590
• Main Authors: Shi, Zhonglin, Li, Yanan, Hu, Anzhong, Cui, Junsheng, Shao, Min, Zhu, Ling, Yang, Ke, Liu, Yong, Deng, Guoqing, Zhu, Cancan
• Format: Article
• Language: English
• Subjects: Acinetobacter baumannii; Anti-Bacterial Agents; Drug-resistant Staphylococcus aureus; ermA; Humans; mecA; Methicillin-Resistant Staphylococcus aureus - genetics; Microbial Sensitivity Tests; Recombinase polymerase amplification; Recombinases - genetics; Resistance genes; Staphylococcal Infections - diagnosis; Staphylococcal Infections - microbiology; Staphylococcus aureus - genetics
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2022.106590

In the clinic, drug-resistant Staphylococcus aureus (S. aureus) is the most common suppurative infection pathogen in humans. It can cause local infections in humans and animals, such as pneumonia, mastitis, and other systemic illnesses. At present, the detection of drug-resistant S. aureus includes traditional isolation by culture and antimicrobial susceptibility tests. However, these methods are complicated in experimental design, specialized in operation and time consuming. Therefore, a rapid and accurate drug-resistant S. aureus detection technology is urgently needed. In this study, we combined duplex pairs of fluorescent probes with recombinase polymerase amplification (RPA) to realize the simultaneous detection of two resistance genes in drug-resistant S. aureus. The method shows low detection limit, detecting 20 copies within 10 min. The analytical specificity of this method was evaluated with several related drug-resistant bacterial strains (Non-resistant S. aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae), and the positive signal was only observed with drug-resistant S. aureus. In addition, the clinical suitability of this method was verified by 30 clinical isolates. Compared with qPCR, the coincidence rate of drug resistance genes were 100% (mecA) and 96.7% (ermA), respectively. These results show that the duplex real-time fluorescent RPA assay is a rapid, low detection limit and specific detection of mecA and ermA genes in isolates of drug-resistant S. aureus. •Duplex RPA can detect mecA and ermA resistance genes simultaneously.•Duplex RPA was sufficiently sensitive for detecting 20 copies within 10 min.•Available clinical isolates were used to assess the suitability of the duplex RPA.•Duplex RPA is a rapid, high sensitivity and specificity detection method.