Design of Rapid Detection System for Five Major Carbapenemase Families (blaKPC, blaNDM, blaVIM, blaIMP and blaOXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.

摘要: Purpose Carbapenemase-producing Enterobacteriaceae (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt effective therapy. In order to quickly comprehensively detect the five major families carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48-like), colorimetric loop-mediated isothermal amplification (LAMP) was employed. Materials Methods Five sets LAMP primers were designed, each which can, respectively, amplify all carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-"standard strains", including 1 blaNDM-1, blaNDM-5, blaNDM-6, blaNDM-7, 2 blaIMP-4, blaIMP-8, blaKPC-2, blaKPC-3, blaKPC-4, blaKPC-5, blaKPC-6, blaKPC-7, blaOXA-48 blaOXA-181 carrier, blaVIM blaOXA-244, blaKPC-2 blaVIM-1 blaNDM-1-co-carriers, used establish 25-microliter visual reaction system (kept at 65°C 30 minutes water bath). Color change from bright pink yellow indicated positive amplification. addition, 126 pre-verified clinical carbapenem-resistant (CRE) isolates, 65 CPE (23 blaOXA-48-like, blaKPC 37 carriers) 61 non-CPE, also detected. Results With lowest detection limit 10 colony forming units (CFU) per 103 CFU PCR, demonstrated dramatically higher sensitivity while retaining same specificity. Furthermore, we concordant results between two methods isolates. Conclusion Therefore, could be rapid identification gene routine laboratories.