A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in Enterobacteriaceae
A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in Enterobacteriaceae
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DOI：
10.1186/1471-2180-13-58
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发表时间：
2013-03-14
期刊：
影响因子：
4.2
通讯作者：
Ma, Xuejun
中科院分区：
文献类型：
--
作者：
Hu, Xiumei;Xu, Banglao;Ma, Xuejun
背景资料：编码氨基糖苷类修饰酶和16 S rRNA甲基转移酶的耐药基因是导致氨基糖苷类耐药增加的主要因素。抗菌素耐药基因谱的表征和分布提供了关于细菌治疗的潜在困难的重要信息。已经开发了几种分子方法来调查氨基糖苷类耐药基因的流行。现有的这些方法在流行病学调查中费时、费力、昂贵或灵敏度有限。因此，有必要建立一种快速、低成本、高通量、高灵敏度的检测方法来研究临床分离菌耐药基因的分布。本研究建立了一种基于GeXP分析仪的多重PCR检测方法，可同时检测aac（3）-II、aac（6 '）-Ib、aac（6'）-II、ant（3 ''）-I、aph（3 '）-VI、阿尔马和rmtB基因，并分析这些基因在肠杆菌科临床分离株中的分布。在优化的条件下，该测定实现了低至10个拷贝的7个基因中的每一个的检测限。应用该方法对56株临床分离的肠杆菌科细菌进行氨基糖苷类耐药基因的分布分析，并与传统的单一PCR方法进行比较。两种检测方法的Kappa值分别为0.831、0.846、0.810、0.909、0.887、0.810和0.825。结论：GeXP法是一种快速、经济、高通量、灵敏度高、特异性强的同时检测7种常见氨基糖苷类抗生素耐药基因的方法。
Background: The aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates.Results: In this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6')-Ib, aac(6')-II, ant(3 '')-I, aph(3')-VI, armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively.Conclusion: This GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.