Design of Rapid Detection System for Five Major Carbapenemase Families (bla(KPC), bla(NDM), bla(VIM), bla(IMP) and bla(OXA-48-Like)) by Colorimetric Loop-Mediated Isothermal Amplification

PURPOSE: Carbapenemase-producing Enterobacteriaceae (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt and effective therapy. In order to quickly and comprehensively detect the five major families of carbapenemases (bla(KPC), bla(NDM), bla(VIM), bla(IMP), and bla(OXA-48-like)), colorimetric loop-mediated isothermal amplification (LAMP) was employed. MATERIALS AND METHODS: Five sets of LAMP primers were designed, each of which can, respectively, amplify all the carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-“standard strains”, including 1 bla(NDM-1), 1 bla(NDM-5), 1 bla(NDM-6), 1 bla(NDM-7), 2 bla(IMP-4), 1 bla(IMP-8), 2 bla(KPC-2), 1 bla(KPC-3), 1 bla(KPC-4), 1 bla(KPC-5), 1 bla(KPC-6), 1 bla(KPC-7), 1 bla(OXA-48) and 1 bla(OXA-181) carrier, and 1 bla(VIM) and bla(OXA-244), 1 bla(KPC-2) and bla(IMP-4), 1 bla(KPC-2) and bla(VIM-1) and 1 bla(KPC-2) and bla(NDM-1)-co-carriers, were used to establish a 25-microliter visual LAMP reaction system (kept at 65°C for 30 minutes in water bath). Color change from bright pink to yellow indicated positive amplification. In addition, 126 pre-verified clinical carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 65 CPE (23 bla(NDM), 2 bla(OXA-48-like), 1 bla(KPC) and bla(VIM), 2 bla(IMP), and 37 bla(KPC) carriers) and 61 non-CPE, were also detected. RESULTS: With the lowest detection limit of 10 colony forming units (CFU) per reaction for LAMP and 10(3) CFU per reaction for PCR, the LAMP system demonstrated dramatically higher sensitivity while retaining the same specificity. Furthermore, we demonstrated concordant results between the two methods for the 126 clinical isolates. CONCLUSION: Therefore, LAMP could be used for rapid identification of the five major carbapenemase gene families in routine clinical laboratories.