汉坦病毒实时荧光定量PCR检测方法的建立

发布时间：2018-03-04 17:28

本文选题：汉坦病毒　切入点：荧光定量PCR　出处：《中国病原生物学杂志》2014年04期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to establish a real-time fluorescent quantitative PCR assay for the detection of Hantavirus. Methods specific probe primers were designed according to the sequence of Hantaan type and Seoul type Hantavirus S fragment gene. The standard curve of recombinant plasmid containing target gene fragment was used to establish a real-time fluorescent quantitative PCR method for the detection of Hantavirus. The results showed that the method was specific and did not cross with other common pathogens. The lowest detection limit was 101copies/ 渭 l, the sensitivity was high, the linear relationship between logarithmic values and Ct values of different gradient quantitative templates was YYPE-3.4607x 40.988 HTNNCE-3.5307x 39.356SSEOO, the amplification efficiency was 92.2HTN) and 92.6SECON, the correlation coefficient (R2) was 0.9968m HTN) and the correlation coefficient (R _ 2) was 0.9968m (HTNs) and 0.9997m (SEOO), respectively, showing a good linear relationship, and the detection efficiency was 92.2 HTNs and 92.6SECON, respectively, and the correlation coefficient (R ~ 2 was 0.9968) and 0.9997%, respectively. Conclusion the established real-time fluorescent quantitative PCR has high sensitivity and reproducibility, and can be used for rapid detection of Hantavirus and preliminary identification of Hantaan and Seoul Hantavirus. It is valuable for the early diagnosis and epidemiological investigation of hemorrhagic fever with renal syndrome.
【作者单位】：南京军区军事医学研究所;浙江省疾病预防控制中心;
【基金】：国家重大传染病防治专项(No.2013ZX10004103,2013ZX10004-104,2013ZX10004-203,2013ZX10004-218,2012ZX10004801-004) 国家自然科学基金项目(No.81171609) 全军后勤科研“十二五”重大项目(No.AWS11C001,AWS11C009) 南京军区医学科技课题(No.12MA119,10Z039,11Z040) 江苏省科技支撑计划(社会发展)项目(No.BE2013603,BE2012609)
【分类号】：R373.9

【参考文献】