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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 detect Hantavirus rapidly and accurately by establishing real-time fluorescent quantitative PCR assay of Hantavirus. Methods Specific probe primers were designed according to the sequences of Hantaan and Han-type Hantavirus S-segment genes, and the standard curve was constructed by using the recombinant plasmid standard containing the target gene fragment to establish a real-time fluorescence quantitative PCR method. Results The detection method was established to be specific and did not cross-react with other common pathogens. The minimum detection limit was 101 copies / μl, and the sensitivity was high. The linear relationship between the logarithm of the copy number and the Ct value was (HTN), y = -3.4607x + 40.988 (HTN), y = -3.5307x + 39.356 (SEO) respectively. The amplification efficiencies were 92.2% (HTN) and 92.6% (SEO) respectively. The correlation coefficients R2 were 0.9968 (HTN) and 0.9997 (SEO), has a good linear relationship, and good repeatability. Conclusion The established real-time PCR method has high sensitivity and repeatability and can be used for the rapid detection of Hantavirus. Hantaan and Seoul-type Hantavirus can be identified initially, and the pathogens of hemorrhagic fever with renal syndrome are diagnosed early and epidemics Study has a better application value.