New Delhi metallo-β-lactamase (NDM) is a series of enzyme conferring resistance to β-lactam antibiotics including the carbapenems. The blaNDM gene has been reported in a variety of Gram-negative bacilli, especially in the Enterobacteriaceae and Acinetobacter spp., which is deeply disconcerting for public health worldwide. In this study, recombinase polymerase amplification assays using a basic detection (Basic-RPA) and a real-time fluorescent detection (Exo-RPA) were established for detecting blaNDM gene. The RPA reactions were performed at 39 °C and finished within 20 min. Using different copy numbers of pMD18T-NDM plasmid DNA as templates, we identified the detection limit of Basic-RPA assay (1.85 × 103 copies/μL), conventional PCR assay (1.85 × 104 copies/μL), Exo-RPA assay (1.85 × 102 copies/μL) and real-time PCR assay (1.85 × 102 copies/μL). Both Basic-RPA and Exo-RPA assays were highly specific for detecting blaNDM, as there were no cross-reactions with the strains without blaNDM gene. Examination of 62 clinical samples by RPA assays and PCR assays showed the same results, suggesting that RPA assays are reliable in clinical diagnosis. The amplification time of RPA is much shorter than that of other molecular techniques, it is easy to implement and has the potential for clinical application.
Keywords: Basic-RPA; Exo-RPA; New Delhi metallo-β-lactamase (NDM); Recombinase polymerase amplification assay.
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