肠道病毒CODEHOP巢式RT-PCR检测及基因分型方法的建立

发布时间：2018-07-05 11:47

本文选题：肠道病毒 + 检测　；参考：《现代预防医学》2017年14期

【摘要】：目的建立一种可用于肠道病毒的快速检测和基因分型的一致简并引物巢式RT-PCR方法。方法根据genbank发表的不同肠道属病毒多聚蛋白氨基酸序列,用CODEHOP在线软件设计1对肠病毒属简并引物,并在该引物的扩增区域内用Primer 3.0软件设计了EV71、COXA16和COXA10 3个病毒的特异引物,建立了巢式RT-PCR方法,对其检测灵敏度、特异性进行了分析,并对27份手足口病患者粪便样品进行了检测。结果该方法第1轮简并引物扩增的灵敏度是17 ng;第2步分型引物扩增的灵敏度是第1步反应的100倍。27份手足口病患者粪便样品RT-PCR检测结果为阳性,其中15份样品经检测为EV71病毒阳性,10份样品经COXA16病毒阳性,2份样品经检测为COXA10病毒阳性。结论建立的一致简并引物巢式RT-PCR检测方法具有特异强、灵敏高的优势,可用于肠道病毒的检测和基因分型。
[Abstract]:Objective to establish a uniform degenerate primer nested RT-PCR method for the rapid detection and genotyping of enterovirus. Method based on the sequence of different enterovirus polyprotein amino acids published by GenBank, 1 pairs of degenerate primers were designed by CODEHOP online software, and the Primer 3 software was set up in the amplification area of the primer. With the specific primers of 3 viruses of EV71, COXA16 and COXA10, the nested RT-PCR method was established. The sensitivity and specificity of the detection were analyzed, and the fecal samples of 27 hand foot and mouth disease patients were detected. The results showed that the sensitivity of the first wheeled primer amplification was 17 ng, and the sensitivity of the second step primer amplification was first step reaction. The results of RT-PCR detection in the stool samples of 100 times.27 HFMD patients were positive, of which 15 samples were detected as EV71 virus positive, 10 samples were COXA16 virus positive and 2 samples were detected as COXA10 virus positive. Conclusion the consistent degenerate primer nested RT-PCR detection method has a strong and sensitive advantage, which can be used in the intestines. Detection and genotyping of the virus.
【作者单位】：大榭出入境检验检疫局;
【基金】：国家质检总局科技项目(2016IK179)
【分类号】：R440;R512.5

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