猪流行性腹泻病毒实时荧光定量RT-PCR检测方法的建立

发布时间：2018-01-06 16:03

本文关键词：猪流行性腹泻病毒实时荧光定量RT-PCR检测方法的建立　出处：《中国动物检疫》2016年01期 　论文类型：期刊论文

【摘要】：根据Gen Bank报道的N基因高度保守核苷酸序列,设计并合成一对引物。上下游引物与Gen Bank中登录的153株猪流行性腹泻病毒(PEDV)N基因全长序列匹配度分别是100%和97%。以本实验室分离流行毒株为模板,利用SYBR Green I荧光染料法进行RT-PCR扩增,获得扩增产物构建重组质粒作为阳性对照,建立检测猪流行性腹泻病毒核酸的方法。同一样品进行3次重复试验,变异系数0.9%。通过对临床样品进行检测和测序验证,核酸检测结果中的阳性样品准确率为100%。本研究所建立的荧光定量PCR检测方法具有快速、灵敏、准确等优点,可用于临床PEDV的检测及分子流行病学调查。
[Abstract]:According to the highly conserved N gene nucleotide sequence of Gen reported by Bank, a pair of primers were designed and synthesized. The primers of Gen and Bank on the 153 strains of porcine epidemic diarrhea virus (PEDV) sequence of N gene, respectively is 100% and 97%. is separated by our laboratory strains as template, amplified by RT-PCR using SYBR Green I fluorescence dye method, obtained PCR products to construct recombinant plasmid as a positive control, set up a method for the detection of porcine epidemic diarrhea virus nucleic acid. The same samples were repeated 3 times, the coefficient of variation was detected by 0.9%. and sequencing of clinical samples, the positive samples of nucleic acid detection results in the accurate rate of fluorescence quantitative PCR detection the establishment of the Institute of 100%. method is fast, sensitive, accurate and can be used for detection and molecular epidemiology of clinical PEDV.

【作者单位】：浙江农林大学动物科技学院;中国动物卫生与流行病学中心;金华职业技术学院农业与生物工程学院;
【基金】：浙江省科技厅项目(2014C32061,2014C02003) 金华市农业类重点研究项目(2014-2-003)
【分类号】：S852.65
【正文快照】： 猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(PEDV)引起猪的一种高度接触性肠道传染病,其特征为呕吐、腹泻、脱水[1]。本病主要发生在当年12月至P次年2月,夏季也可发生,可以感染各年龄P阶段的猪,其中哺乳仔猪最易感,发病率可达100%,平均死亡率可达50%。

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