文章摘要
谢荣辉,徐芳,朱函坪,程胤凯,傅桂明,姚苹苹,翁景清,卢亦愚,杨章女,朱智勇.一步法流行性乙型脑炎病毒TaqMan荧光定量反转录聚合酶链反应方法的建立及应用[J].中华流行病学杂志,2009,30(3):277-280
一步法流行性乙型脑炎病毒TaqMan荧光定量反转录聚合酶链反应方法的建立及应用
Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus
投稿时间:2008-08-31  
DOI:10.3760/cma.j.issn.0254-6450.2009.03.019
中文关键词: 流行性乙型脑炎病毒;荧光定量反转录.聚合酶链反应;检测
英文关键词: Japanese encephalitis virus;Real-time reverse transcription-polymerase chain reaction;Detection
基金项目:浙江省医药卫生科学研究基金资助项目(2008839)
作者单位
谢荣辉 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
徐芳 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
朱函坪 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
程胤凯 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
傅桂明 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
姚苹苹 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
翁景清 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
卢亦愚 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
杨章女 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
朱智勇 浙江省疾病预防控制中心出血热重点实验室, 杭州 310051 
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中文摘要:
      目的 建立一种特异、灵敏、快速检测流行性乙型脑炎(乙脑)病毒的荧光定鼍RT-PCR方法. 方法 根据GenBank登录的乙脑病毒毒株序列, 应用生物软件在乙脑病毒基因的保守区设计与筛选引物和探针, 对荧光定量RT-PCR反应体系与条件进行优化, 验证方法的特异性、敏感性和重复性. 并通过对蚊虫样本的检测, 以评价该方法的实际应用价值. 结果 优化结果表明荧光定量RT-PCR的最佳反应体系为0. 1 μmol/L探针浓度, 0. 2μmol/L引物浓度, 5 mmol/L Mg2+浓度. 结果表明该方法对乙脑病毒的检测有高度的特异性、通用性, 对1~4型登革热病毒、狂犬病毒、汉坦病毒(SEO型与HTN型)均无交叉反应, 检测的灵敏度达0. 1 TCID50, 重复性分析表明同一样品Gt值的重复检测4次, 其标准差均在合理范围内, 分别为0. 033、0. 079、0. 149和0. 411. 对110批蚊虫标本进行荧光定量RT-PCR检测和病毒分离榆测, 结果荧光定量RT-PCR检测出7份阳性, 而病毒分离则只检测出3份阳性, 表明建立的荧光定量RT-PCR方法更敏感, 可直接从蚊虫样本中检测乙脑病毒核酸, 从病毒核酸提取至完成检测仅需3 h左右, 且操作简便、敏感性高. 结论 研究建立的TaqMan荧光定量RT-PCR的方法具有特异、敏感、快速的特点, 适用于乙脑病原学的快速检测.
英文摘要:
      Objective To establish a TaqMan based real-time reverse transeription-polymerase chain reaction(RT-PCR)assay for the detection of Japanese encephalitis virus. Methods The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. Results Mg2+, primer and probe were optimized at 5 mmol/L, 0. 2 μmol/L and 0. 1 μmol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0. 1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. Conclusion This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.
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