Primer Evaluation for PCR and its Application for Detection of Carbapenemases in Enterobacteriaceae.
Primer Evaluation for PCR and its Application for Detection of Carbapenemases in Enterobacteriaceae.
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DOI：
10.5812/jjm.29314
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发表时间：
2016-01
影响因子：
0.6
通讯作者：
Kolar M
中科院分区：
文献类型：
--
作者：
Mlynarcik P;Roderova M;Kolar M
在过去的十年中，人类患者中耐碳青霉烯类肠杆菌科细菌的患病率有所增加。产生碳青霉烯酶的细菌通常具有多重耐药性。因此，早期识别碳青霉烯酶生产者对于防止其传播至关重要。本研究的目的是开发用于单一和/或多重 PCR 扩增测定的引物，以同时鉴定肠杆菌科细菌中的 A 类、B 类和 D 类碳青霉烯水解 β-内酰胺酶，然后评估其效率。所有编码碳青霉烯酶基因的参考序列均从GenBank下载。引物设计用于扩增以下 11 个基因：blaKPC、blaOXA、blaVIM、blaNDM、blaIMP、blaSME、blaIMI、blaGES、blaGIM、blaDIM 和 blaCMY。测试 PCR 条件以扩增不同大小的片段。创建了两个多重 PCR 组用于检测临床上重要的碳青霉烯酶。第三组引物用于检测肠杆菌科中所有已知的碳青霉烯酶。使用六种参考菌株和九种临床分离株对它们进行了评估。使用优化条件，所有碳青霉烯酶阳性对照均产生了预测的扩增子大小，并证实了单次和多重 PCR 中引物的特异性。我们在此报告了一种可靠的方法，由单一和多重 PCR 测定组成，用于筛选所有临床已知的碳青霉烯酶。在计算机和体外测试的引物可以区分碳青霉烯类耐药肠杆菌科细菌，并有助于对抗碳青霉烯类抗生素耐药性在肠杆菌科细菌中的传播。
During the last decade, the prevalence of carbapenem-resistant Enterobacteriaceae in human patients has increased. Carbapenemase-producing bacteria are usually multidrug resistant. Therefore, early recognition of carbapenemase producers is critical to prevent their spread. The objective of this study was to develop the primers for single and/or multiplex PCR amplification assays for simultaneous identification of class A, class B, and class D carbapenem hydrolyzing β-lactamases in Enterobacteriaceae and then to evaluate their efficiency. The reference sequences of all genes encoding carbapenemases were downloaded from GenBank. Primers were designed to amplify the following 11 genes: blaKPC, blaOXA, blaVIM, blaNDM, blaIMP, blaSME, blaIMI, blaGES, blaGIM, blaDIM and blaCMY. PCR conditions were tested to amplify fragments of different sizes. Two multiplex PCR sets were created for the detection of clinically important carbapenemases. The third set of primers was included for detection of all known carbapenemases in Enterobacteriaceae. They were evaluated using six reference strains and nine clinical isolates. Using optimized conditions, all carbapenemase-positive controls yielded predicted amplicon sizes and confirmed the specificity of the primers in single and multiplex PCR. We have reported here a reliable method, composed of single and multiplex PCR assays, for screening all clinically known carbapenemases. Primers tested in silico and in vitro may distinguish carbapenem-resistant Enterobacteriaceae and could assist in combating the spread of carbapenem resistance in Enterobacteriaceae.