Rapid Detection of New Delhi Metallo-β-Lactamase Gene and Variants coding Carbapenemase with Different Activity by a PCR-based in vitro protein expression method.

Li Huang，Xiumei Hu，Man Zhou, Yinmei Yang, Jinjuan Qiao, Dianbing Wang, Junping Yu, Zongqiang Cui, Zhiping Zhang, Xian-En Zhang and Hongping Wei.　

JCM. 03363-13. March 2014, doi: 10.1128/JCM.03363-13

Abstract

　　NDM (New Delhi metallo-β-lactamase) producing bacteria are considered as potential global health threats. It is necessary to monitor NDM-1 and its variants in clinical isolates in order to understand the NDM-1 epidemic and the impact of its variants on the β-lactam resistance. To overcome the lengthy time needed for cloning and expression of NDM-1 variants, a novel PCR-based in vitro protein expression (PCR-P) method was used to detect bla_NDM-1 and its variants coding carbapenemases with different activity (functional variants). The PCR-P method combined a long fragment real-time PCR (LF-qPCR) with an in vitro cell free expression to convert the bla_NDM-1 amplicons into NDM for carbapenemase assay. The method could screen bla_NDM-1 within 3 h with a detection limit of 5 copies and identify functional variants within 1 day. Using the PCR-P to analyze 5 recent bla_NDM-1 variants, 2 functional variants bla_NDM-4 and bla_NDM-5 were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing bla_NDM-1. This novel method provides the first integrated approach for rapidly detecting the full length bla_NDM-1 and revealing its functional variants in clinical isolates.

http://jcm.asm.org/content/early/2014/03/20/JCM.03363-13.abstract