登革病毒TaqMan MGB实时荧光定量PCR检测方法的建立及初步应用  

Development of TaqMan MGB probe-based real-time fluorescence quantitative reverse transcription PCR for dengue virus and its application

作　　者：罗雅艳[1] 冯俊杰[1] 方丹云[1] 江丽芳[1] 

机构地区：[1]中山大学中山医学院微生物教研室,广东广州510080

出　　处：《分子诊断与治疗杂志》2012年第3期158-162,共5页Journal of Molecular Diagnostics and Therapy

基　　金：国家科技重大专项(2008ZX10004-007)

摘　　要：目的建立登革病毒TaqMan实时荧光定量PCR快速检测方法及应用于临床。方法根据1～4型登革病毒3'端非编码区的一段高度保守序列,设计一套型通用的引物和TaqMan MGB探针,以4个血清型登革病毒标准株为标准,以日本乙脑病毒和丙肝病毒作阴性对照,以包含登革病毒2型标准株(DENV-2NGC株)3'非编码区349bp片段的质粒DNA作标准品,对引物和TaqMan MGB探针的特异性、灵敏度进行分析,从而建立登革病毒实时荧光定量PCR检测方法。用该法对登革病毒野毒株和10份登革病毒患者血清进行检测。结果 TaqMan实时荧光定量PCR检测的1～4型登革病毒标准株及野毒株均为阳性,日本乙脑病毒和丙肝病毒均为阴性;检测灵敏度可达到每反应2个基因拷贝;检测的10份登革患者血清样本中,8份检测结果为阳性。结论 TaqMan MGB实时荧光定量PCR方法是一个快速、特异性强、敏感性高的检测登革病毒的方法,适用于登革病毒的临床早期诊断。Objective To establish a qRT-PCR TaqMan MGB probe-based real-time fluorescence quantitative reverse transcription PCR（TaqMan MGB qRT-PCR） detection method of dengue virus to facilitate the clinical diagnosis. Methods A pair of universal primers and TaqMan MGB probe were designed according to the 3＇noncoding region of four dengue virus serotypes. The sensitivity and specificity of this method were evaluated by using four standard dengue strains, Japanese encephalitis virus strains（JEV） and Hepatitis Virus C（HCV）（negative control） and the plasmid DNA containing the gene fragment of 349 bp in 3＇ noncoding region of DV 11 NGC（positive control）. Then, wild dengue strains and ten serum samples obtained from dengue virus-infected patients were detected by using this method. Results Four standard dengue strains and two wild dengue strains all showed positive results, with a corresponding negative results for JEV and HCV. It was found that the sensitivity of the developed assay was about 2 genomic copies per reaction. Using this method to detect ten clinical serum samples, eight serum samples were positive. Conclusion It is evident that TaqMan MGB qRT-PCR is a fast, specific, sensitive method for detecting dengue virus and it could be applied to the early clinical diagnosis of dengue virus infectioin.

关 键 词：登革病毒 TAQMAN MGB 实时定量RT-PCR 检测 

分 类 号：R450[医药卫生—治疗学]