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

Wenjuan Feng,* Siqiang Niu,* Yanbin Chang, Xiaojiong Jia, Shifeng Huang, Ping Yang Department of Clinical Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shifeng Huang; Ping YangDepartment of Clinical Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, No. 1 Friendship Road, Yuzhong District, Chongqing, People’s Republic of ChinaTel +86-18623027077; +86-13637993035Fax +86-23-89012513; +86-23-89012513Email sfhuang@hospital.cqmu.edu.cn; 201903@cqmu.edu.cnPurpose: 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 (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-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 blaNDM-1, 1 blaNDM-5, 1 blaNDM-6, 1 blaNDM-7, 2 blaIMP-4, 1 blaIMP-8, 2 blaKPC-2, 1 blaKPC-3, 1 blaKPC-4, 1 blaKPC-5, 1 blaKPC-6, 1 blaKPC-7, 1 blaOXA-48 and 1 blaOXA-181 carrier, and 1 blaVIM and blaOXA-244, 1 blaKPC-2 and blaIMP-4, 1 blaKPC-2 and blaVIM-1 and 1 blaKPC-2 and blaNDM-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 blaNDM, 2 blaOXA-48-like, 1 blaKPC and blaVIM, 2 blaIMP, and 37 blaKPC 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 103 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.Keywords: carbapenemase-producing Enterobacteriaceae, loop-mediated isothermal amplification, blaNDM, blaKPC, blaVIM, blaIMP, blaOXA-48-like