title: Rapid detection of New Delhi metallo-β-lactamase gene and variants coding for carbapenemases with different activities by use of a PCR-based in vitro protein expression method.
abstract: New Delhi metallo-β-lactamase (NDM)-producing bacteria are considered 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 β-lactam resistance. To reduce 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 blaNDM-1 and its variants coding for carbapenemases with different activities (functional variants). The PCR-P method combined a long-fragment real-time quantitative PCR (LF-qPCR) with in vitro cell-free expression to convert the blaNDM-1 amplicons into NDM for carbapenemase assay. The method could screen for blaNDM-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 blaNDM-1 variants, 2 functional variants, blaNDM-4 and blaNDM-5, were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing blaNDM-1. This novel method provides the first integrated approach for rapidly detecting the full-length blaNDM-1 and revealing its functional variants in clinical isolates.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Li
authors.initials: L
authors.lastname: Huang
authors.affiliation: Clinical Laboratory Department, Guangzhou First Municipal People's Hospital, Affiliated Hospital of Guangzhou Medical College, Guangzhou, China Southern Medical University, Guangzhou, China.
authors.forename: Xiumei
authors.initials: X
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Man
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Clinical Laboratory Department, Guangzhou First Municipal People's Hospital, Affiliated Hospital of Guangzhou Medical College, Guangzhou, China.
authors.forename: Yinmei
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Jinjuan
authors.initials: J
authors.lastname: Qiao
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Dianbing
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Junping
authors.initials: J
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Zongqiang
authors.initials: Z
authors.lastname: Cui
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China hpwei@wh.iov.cn.
authors.forename: Hongping
authors.initials: H
authors.lastname: Wei
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000302:isolation & purification; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001618:beta-Lactamases; C552864:beta-lactamase NDM-1