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.
Keywords: Drug-resistant Staphylococcus aureus; Recombinase polymerase amplification; Resistance genes; ermA; mecA.
Copyright © 2022 Elsevier B.V. All rights reserved.