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首页> 外文期刊>Journal of Clinical Microbiology >Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci.
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Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci.

机译：多重PCR检测肠球菌中的vanA，vanB，vanC-1和vanC-2 / 3基因。

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Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates. Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls. To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels. Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes. PCR products were detected in 63 of the 100 clinical isolates. The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all > and = 64 micrograms/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 micrograms/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 micrograms/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to > 256 micrograms/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates. DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely related to the published sequence for the vanB gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes. Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp. in the clinical laboratory. In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.
机译：从临床标本中越来越多地分离出耐万古霉素的肠球菌（VRE）。一百株肠球菌临床分离株（卡氏大肠杆菌/黄萎肠杆菌[n = 10]，粪肠球菌[n = 34]，粪肠球菌[n = 43]，鸟肠杆菌[n = 1]，鸡肠球菌） [n = 11]和牛油杆菌[n = 1]）通过直接用血琼脂平板上的菌落进行的单次多重PCR检测了vanA，vanB，vanC-1和vanC-2 / 3基因的存在。携带vanA，vanB，vanC-1或vanC-2基因的六个先前表征的VRE菌株用作对照。为了区分van基因，PCR扩增子用MspI消化，并在琼脂糖凝胶上电泳。由于vanC-2和vanC-3基因之间存在明显的序列同源性，因此该测定法无法将这些基因彼此区分开。因此，这些被称为vanC-2 / 3基因。在100个临床分离株中有63个检测到PCR产物。限制性片段长度模式与10个菌株的vanA，30个菌株的vanB，12个菌株的vanC-1、6个菌株的vanC-2以及1个菌株的vanA和vanC-1一致。具有与vanA和vanB一致的限制性片段长度模式的分离株的万古霉素MIC均≥64微克/ ml。具有与vanC-1或vanC-2一致的限制性片段长度模式的分离株的万古霉素MIC为4至8微克/ ml。没有产生PCR扩增子的分离物的万古霉素MIC为2-4微克/ ml。在四个分离株（万古霉素MIC，4至> 256微克/ ml）中产生了PCR产物，其限制性片段长度模式不同于对照vanA，vanB，vanC-1和vanC-2分离株。这些扩增子的DNA测序表明，四个分离物中的两个具有与vanB基因的公开序列密切相关的核酸序列，而两个具有与vanC-2和vanC- 3个基因。多重PCR-限制性片段长度多态性似乎是快速检测和区分耐万古霉素肠球菌基因型的有用和方便的方法。在临床实验室。在PCR扩增子出现异常限制性片段模式的情况下，可以进行DNA测序以区分van基因型。

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