Rapid Detection of blaNDM, blaKPC, blaIMP, and blaVIM Carbapenemase Genes in Bacteria by Loop-Mediated Isothermal Amplification
Rapid Detection of blaNDM, blaKPC, blaIMP, and blaVIM Carbapenemase Genes in Bacteria by Loop-Mediated Isothermal Amplification
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DOI：
10.1089/mdr.2014.0040
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发表时间：
2014-12-01
影响因子：
2.6
通讯作者：
Rui, Yongyu
中科院分区：
文献类型：
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作者：
Cheng, Cancan;Zheng, Fen;Rui, Yongyu
开发并评价了环介导等温扩增（LAMP）试验，用于快速检测bla（KPC）、bla（NDM）、bla（IMP）和bla（Vim）碳青霉烯酶基因。针对每个靶基因中的8个不同区域设计了6个寡核苷酸，包括外引物、内环引物和环引物。使用两个定性标准来评估LAMP反应：颜色变化的视觉检查和荧光变化的实时检测。对于每个基因，实时检测的检测下限为10个菌落形成单位（CFU）/反应，目视检查的检测下限为100 CFU/反应。222株碳青霉烯类耐药临床分离株（包括100株铜绿假单胞菌，100株不动杆菌属，和22种肠杆菌科细菌）。同时，这些分离物通过常规的聚合酶链反应（PCR）和测序分析进行了确认。在这些临床分离株中，11株bla（NDM）、11株bla（KPC）、11株bla（Vim）和2株bla（IMP）的LAMP检测结果与常规PCR结果一致。本文建立的LAMP方法可用于快速检测细菌中bla（NDM）、bla（KPC）、bla（IMP）和bla（Vim）碳青霉烯酶基因。
A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for rapid detection of bla(KPC), bla(NDM), bla(IMP), and bla(VIM) carbapenemase genes. Six oligonucleotides, including outer, inner, and loop primers, were designed for eight distinct regions in each target gene. Two qualitative criteria were used to evaluate LAMP reactions: visual inspection of color change and real-time detection of fluorescence change. The lower detection limit was 10 colony forming units (CFU) per reaction for real-time detection and 100 CFU per reaction for visual inspection for each gene. Two hundred twenty-two carbapenem-resistant clinical isolates (including 100 Pseudomonas aeruginosa, 100 Acinetobacter sp., and 22 Enterobacteriaceae) were tested by LAMP assay. At the same time, these isolates were confirmed by conventional polymerase chain reaction (PCR) and sequencing analysis. In these clinical isolates, the results of 11 strains with bla(NDM), 11 strains with bla(KPC), 11 strains with bla(VIM), and 2 strains with bla(IMP) obtained using LAMP assays were concordant with conventional PCR. The LAMP method reported here may be a useful and powerful tool for rapid detection of bla(NDM), bla(KPC), bla(IMP), and bla(VIM) carbapenemase genes in bacteria.