汉坦病毒通用型实时荧光定量RT-PCR方法的建立及应用

发布时间：2018-04-15 14:08

本文选题：汉坦病毒 + 基因型　；参考：《现代预防医学》2016年02期

【摘要】：目的建立一种适应汉坦病毒不同基因型别的通用实时荧光定量RT-PCR检测方法。方法利用Beacon Designer7.5软件设计引物和探针,以人工合成汉坦病毒5种具有代表性基因型别的L片段作为模板,进行实时荧光定量RT-PCR方法的建立,确定所建立方法的检测限及灵敏度并对采集的112份鼠肺进行快速检测。结果 5种模板RNA的Ct值与其稀释浓度的对数存在良好的线性关系,以HTNV为例建立的标准曲线为:y=-3.40x+46.8,R2=0.99,其最低检出限为5.32 copies/μl,对于不同基因型别的汉坦病毒,其检测最低限:HTNV为12.40 copies/μl、SEOV为5.32 copies/μl、PUUV为15.02 copies/μl、DOBV为22.14 copies/μl及SNV为24.26 copies/μl。对112份鼠肺样本的病毒检出率为17.86%,较巢式RT-PCR的检出率(8.04%)高。结论建立的通用型实时荧光定量RT-PCR方法灵敏度高,检测的广谱性好,适合啮齿类动物中携带的汉坦病毒的快速检测。
[Abstract]:Objective to establish a universal real-time quantitative RT-PCR method for the detection of Hantavirus with different genotypes.Methods the primers and probes were designed by Beacon Designer7.5 software, and the L fragments of five representative genotypes of Hantavirus were used as template to establish a real-time fluorescence quantitative RT-PCR method.The detection limit and sensitivity of the established method were determined and rapid detection of 112 rat lungs was carried out.Results there was a good linear relationship between the Ct value of RNA and the logarithm of dilution concentration of the five templates. The standard curve of HTNV was established as follows: yyyang-3.40x 46.8R2O0.99.The lowest detection limit was 5.32 copies/ 渭 l, and the lowest detection limit was 5.32 copies/ 渭 l for different Hantavirus genotypes.The lowest detection limit was 12.40 copies/ / 渭 L SEOV, 5.32 copies/ / 渭 L PUUV, 15.02 copies/ / 渭 l-1 DOBV, 22.14 copies/ 渭 l and 24.26 copies/ 渭 l, respectively.The detection rate of virus in 112 lung samples of mice was 17.86, which was higher than that of nested RT-PCR.Conclusion the universal real-time fluorescent quantitative RT-PCR method has high sensitivity and good broad-spectrum detection. It is suitable for rapid detection of Hantavirus in rodents.
【作者单位】：淮安市疾病预防控制中心;中国检验检疫科学研究院卫检所;
【基金】：国家质检总局科技计划项目(2011IK150) 淮安市预防医学会科研基金(hayf201516)
【分类号】：R440;R511

【相似文献】