汉坦病毒实时荧光定量PCR检测方法的建立

Development and use of a TaqMan real-time PCR assay to detect Hantavirus

目的通过建立汉坦病毒实时荧光定量PCR检测方法,快速准确地检测汉坦病毒。方法根据汉滩型和汉城型汉坦病S片段基因的序列分别设计特异性探针引物,使用含有目的基因片段的重组质粒标准品绘制标准曲线,建立检测两型汉坦病毒的实时荧光定量PCR方法。结果建立的检测方法特异性良好,与其他常见病原不发生交叉反应;最低检测限为101copies/μl,灵敏度高;不同梯度定量模板的拷贝数的对数值与Ct值之间线性关系表达式分别为y=-3.4607x+40.988(HTN),y=-3.5307x+39.356(SEO),扩增效率分别为92.2%(HTN)和92.6%(SEO),相关系数R2分别为0.9968(HTN)和0.9997(SEO),呈良好的线性关系,且重复性好。结论建立的实时荧光定量PCR灵敏度高,重复性好,可用于汉坦病毒的快速检测,并可初步辨别汉滩型和汉城型汉坦病毒,对肾综合征出血热的病原早期诊断和流行病学调查有较好的应用价值。

Objective To establish a new TaqMan real-time PCR assay to rapidly and accurately detect Hantavirus. Methods Two assays were developed to specifically identify the Hantaan（HTN）virus and Seoul（SEO）virus.The assays are based on a TaqMan real-time polymerase chain reaction（RT-PCR）with a small segment used as the target sequence. Results The assay specifically detected Hantavirus,and the assay did not cross-react with other pathogens.The assay had a sensitivity level of 101copies/μl.Assay results were linearly correlated with the DNA concentration from102~108 copies/μl as indicated by the equations y=-3.4607x＋40.988（HTN）and y=-3.5307x＋39.356（SEO）.The correlation coefficient of the standard curve was 0.9968（HTN）and 0.9997（SEO）with good repeatability.This assay differentiated between HTN and SEO viral infections. Conclusion TaqMan real-time PCR is useful at rapidly detecting Hantavirus and at epidemiological study of hemorrhagic fever with renal syndrome.