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马尔堡、埃博拉病毒双重荧光定量PCR检测方法的建立
引用本文:杨宇,白琳,胡孔新,杨志红,胡健萍,王静.马尔堡、埃博拉病毒双重荧光定量PCR检测方法的建立[J].中华实验和临床病毒学杂志,2012,26(4):313-315.
作者姓名:杨宇  白琳  胡孔新  杨志红  胡健萍  王静
作者单位:中国检验检疫科学研究院,北京,100123
基金项目:国家自然科学基金(30900053);国家质检总局科技计划项目(20101K211);质检公益项目(2007GYJ023);十二五重大传染病专项(2011ZX10004-001)
摘    要:目的 建立一种快速、敏感、特异的双重实时荧光定量PCR方法,可同时检测马尔堡病毒和埃博拉病毒.方法 通过序列比对挑选出两种病毒基因组中高度保守的序列,分别设计引物及Taqman探针,两条探针分别标记FAM和Texas Red荧光报告基因,建立双重实时荧光定量PCR反应体系.结果 双重荧光定量PCR方法检测两种病毒阳性标准品的灵敏度分别为30.5拷贝/μl和28.6拷贝/μl,通过检测日本脑炎病毒、黄热病毒、登革热病毒无交叉反应,有较好的灵敏度和特异性.结论 建立了马尔堡、埃博拉病毒双重荧光定量PCR检测方法,实现了两种病毒同时实时定量检测,在传染病防控领域有较好的应用前景.

关 键 词:聚合酶链反应  马尔堡样病毒  埃博拉样病毒

Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus
YANG Yu , BAI Lin , HU Kong-xin , YANG Zhi-hong , HU Jian-ping , WANG Jing.Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus[J].Chinese Journal of Experimental and Clinical Virology,2012,26(4):313-315.
Authors:YANG Yu  BAI Lin  HU Kong-xin  YANG Zhi-hong  HU Jian-ping  WANG Jing
Institution:. Chinese Academy of Inspection and Quarantine, Beijing 100123, China
Abstract:Objective Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Methods Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. Results We have developed a real-time PCR method with the sensitivity of 30.5 copies/μl for Marburg virus positive plasmid and 28.6 copies/μl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. Conclusions The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.
Keywords:Polymerase chain reaction  Marburg-like virus  Ebola-like virus
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