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

Wenjuan Feng; Siqiang Niu; Yanbin Chang; Xiaojiong Jia; Shifeng Huang; Ping Yang

doi:10.2147/IDR.S301757

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

Infect Drug Resist. 2021 May 24:14:1865-1874. doi: 10.2147/IDR.S301757. eCollection 2021.

Authors

Wenjuan Feng^#¹, Siqiang Niu^#¹, Yanbin Chang¹, Xiaojiong Jia¹, Shifeng Huang¹, Ping Yang¹

Affiliation

¹ Department of Clinical Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People's Republic of China.

^# Contributed equally.

Abstract

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 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: blaIMP; blaKPC; blaNDM; blaOXA-48-like; blaVIM; carbapenemase-producing Enterobacteriaceae; loop-mediated isothermal amplification.