登革-2型病毒一步法TaqMan荧光定量RT-PCR检测方法建立

发布时间：2019-04-23 23:00

【摘要】：目的建立一种针对登革2型病毒(DENV-2)的快速、灵敏、特异的检测方法。方法从Gen Bank下载105株DENV-2病毒毒株全基因序列,应用生物软件Bioedit进行比对分析筛选出保守序列,在保守区域设计特异引物和探针,建立检测DENV-2的TaqMan荧光定量逆转录聚合酶链反应(RT-PCR)方法。结果该方法检测灵敏度达到102copies/μL;特异性验证中除登革2型病毒有明显扩增外,与墨累谷脑炎病毒、西尼罗病毒、蜱传脑炎病毒、登革病毒1、3、4型、黄热病毒和科萨努尔森林病毒、乙型脑炎病毒均无交叉反应。重复性实验结果表明该方法的组间和组内的变异系数(CV)均2%。对人工感染DENV-2的87只蚊虫标本进行比对实验,结果荧光定量RT-PCR检测出56份阳性,传统RT-PCR只检测出16份阳性,差异有统计学意义(χ~2=37.908,P=0.000)。结论 TaqMan荧光定量RT-PCR方法检测DENV-2,特异性强、敏感性高。
[Abstract]:Objective to establish a rapid, sensitive and specific method for the detection of dengue 2 virus (DENV-2). Methods the whole gene sequence of DENV-2 virus strain was downloaded from Gen Bank, and the conserved sequence was screened by biological software Bioedit. The specific primers and probes were designed in the conserved region. To establish a TaqMan quantitative reverse transcription polymerase chain reaction (RT-PCR) method for the detection of DENV-2. Results the sensitivity of this method was up to 102copies/ 渭 L; In addition to the marked amplification of dengue 2 virus, it is associated with Muriya encephalitis virus, West Nile virus, tick-borne encephalitis virus, dengue virus 1, 3,4, yellow fever virus and Cosanur forest virus, There was no cross reaction between Japanese encephalitis virus and Japanese encephalitis virus. The repeatability test results showed that the coefficient of variation (CV) of the method was 2% among groups and within groups. A comparative experiment was conducted on 87 mosquito specimens infected with DENV-2. The results showed that 56 samples were positive by fluorescent quantitative RT-PCR and 16 positive by traditional RT-PCR (蠂 ~ 2 ~ 2 ~ 3. 908, P < 0. 000). The results showed that there was no significant difference between the two groups (蠂 ~ 2 ~ 2 ~ 2, P < 0. 008). Conclusion TaqMan fluorescence quantitative RT-PCR method has high specificity and sensitivity in detecting DENV-2,.
【作者单位】：贵州医科大学基础医学院现代病原生物学实验室;贵州医科大学公共卫生学院劳动卫生与环境卫生学教研室;贵州医科大学基础医学院免疫学教研室;
【基金】：贵州省科技厅基础平台建设项目(黔科平台[2012(4006)]) 贵州省社会发展科技公关项目(黔科合SY字[2009]3056)
【分类号】：R3416;R373.33

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