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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。
Objective To establish a universal real-time fluorescence quantitative RT-PCR assay to adapt to different genotypes of Hantavirus. Methods The primers and probes were designed by Beacon Designer7.5 software. Five L-fragments with representative genotypes of Hantavirus were synthesized and used as templates to establish real-time fluorescence quantitative RT-PCR method to determine the detection of the established method Limit and sensitivity and rapid detection of 112 rat lungs collected. Results There was a good linear relationship between the Ct values of the five template RNAs and the logarithm of their dilutions. The standard curve established by HTNV was y = -3.40x + 46.8 and R2 = 0.99 with a minimum detectable limit of 5.32 copies / μl. For hantavirus of different genotypes, the detection limit was as follows: HTNV 12.40 copies / μl, SEOV 5.32 copies / μl, PUUV 15.02 copies / μl, DOBV 22.14 copies / μl and SNV 24.26 copies / μl. The detection rate of virus in 112 rat lung samples was 17.86%, higher than that of nested RT-PCR (8.04%). Conclusion The established universal real-time fluorescent quantitative RT-PCR method has high sensitivity and good broad spectrum detection and is suitable for the rapid detection of hantavirus carried in rodents.