Sabet Negar Shafiei, Subramaniam Geetha, Navaratnam Parasakthi, Sekaran Shamala Devi
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
J Microbiol Methods. 2007 Jan;68(1):157-62. doi: 10.1016/j.mimet.2006.07.008. Epub 2006 Aug 28.
In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.
在本研究中，我们描述了一种三重实时荧光定量PCR检测方法，该方法能够利用实时荧光定量PCR技术在一次检测中同时鉴定金黄色葡萄球菌并检测两个重要的抗生素耐药基因。在这种三重实时荧光定量PCR检测方法中，使用双标记Taqman探针在一次检测中同时检测mecA（耐甲氧西林）、femA（金黄色葡萄球菌特异性基因）和aacA-aphD（耐氨基糖苷类）基因。该检测方法可同时提供金黄色葡萄球菌鉴定以及葡萄球菌分离株中耐甲氧西林和耐氨基糖苷类情况的信息。152株临床分离株接受了这种三重实时荧光定量PCR检测。三重实时荧光定量PCR检测结果与表型抗生素敏感性试验结果相关。三重实时荧光定量PCR检测获得的结果表明，引物和探针组对金黄色葡萄球菌的鉴定具有特异性，并且能够检测耐甲氧西林和耐氨基糖苷类基因。整个检测可在3小时内完成，这是一种非常快速的方法，能够同时提供金黄色葡萄球菌鉴定以及葡萄球菌分离株抗生素耐药模式的信息。这种快速方法在微生物实验室中的应用将成为快速鉴定金黄色葡萄球菌分离株并确定其对甲氧西林和氨基糖苷类抗生素耐药模式的宝贵工具。
