引物标记技术在耐甲氧西林金黄色葡萄球菌检测中的应用  

Application of primer labeling technique in detection of MRSA

作　　者：梁有才 聂署萍[2] 吴润香[2] 范菲楠[2] 陆学东[2] 

机构地区：[1]广东医科大学研究生学院,广东湛江524000 [2]中山大学附属第八医院检验医学部,广东深圳518033

出　　处：《检验医学与临床》2017年第5期624-626,629,共4页Laboratory Medicine and Clinic

摘　　要：目的在GeXP系统中应用CY5标记的特异引物代替通用引物,建立一种检测耐甲氧西林金黄色葡萄球菌(MRSA)的方法。方法设计mecA与nuc基因引物,经聚合酶链反应(PCR)扩增琼脂糖电泳验证引物特异性并优化PCR反应条件。用荧光染料CY5标记单条引物(单独标记上游或下游引物)。以提取的MRSA、甲氧西林耐药凝固酶阴性葡萄球菌(MRCNS)、甲氧西林敏感金黄色葡萄球菌(MSSA)核酸为模板液,采用已标记引物进行单重及多重PCR,产物经琼脂糖电泳与GeXP毛细管电泳分析,验证应用效果。再通过采用混合标记引物的GeXP系统检测100株MRSA,分析其应用效果。结果单标记下游引物的GeXP-PCR的杂质扩增峰较少。经GeXP系统检测分析的MRSA、MRCNS、MSSA均出现目的特征峰,能明确辨别其含有大小不同的基因片段,结果与琼脂糖电泳一致;经GeXP系统分析的100株MRSA均含有nuc与mecA片段,与VITEKⅡCompact检测结果相符。结论单独标记下游引物应用于GeXP检测MRSA的方法可行。Objective To establish a method for detecting MRSA by using CY5-labelled specific primer to replace universal primer in GeXP system.Methods The mecA and nuc gene primers were designed.The primer specificity was verified by PCR amplification and agarose gel electrophoresis.The PCR reaction condition was optimized.Single primers were marked by fluorescent dye CY5（labeling the forward primer or reverse primer alone）.The extracted MRSA,MRCNS and MSSA nucleic acid served as the template solution.The labeled primers were adopted for conducting the single PCR and multiple PCR.The PCR products were analyzed by the agarose gel electrophoresis and GeXP capillary electrophoresis for verifying the application effect.Then 100 strains of MRSA were detected by the GeXP system using the labelled primers and their application effects were analyzed.Results The GeXP-PCR impurity amplification peaks in single labeled reverse primers were less.The target specific amplification peaks appeared in MRSA,MRCNS and MSSA detected and analyzed by the GeXP system,which containing different sizes of gene fragments was clearly distinguished,the results were consistent with the agarose gel electrophoresis;100 strains of MRSA contained the nuc and mec A fragments by the GeXP system analysis,which conformed to the results of VITEKⅡCompact detection.Conclusion Applying singly labeling reverse primer in GeXP system for detecting MRSA is feasible.

关 键 词：标记 特异引物 GeXP系统 耐甲氧西林金黄色葡萄球菌 

分 类 号：R378.11[医药卫生—病原生物学]