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牛病毒性腹泻病毒SYBR,Green,Ⅰ实时荧光定量PCR检测方法的建立及应用

材料写作网    时间: 2020-10-15 04:06:07     阅读:次     手机站


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摘要:本研究旨在建立一种灵敏度高、特异性强、重复性好,能快速检测牛病毒性腹泻病毒(BVDV)的方法。根据BVDV的E2基因保守序列,设计合成一对特异性引物,建立了检测BVDV的SYBR Green Ⅰ实时荧光定量PCR方法。将标准阳性样品10倍梯度稀释检测灵敏度,用能感染奶牛的其它病毒做对照检测特异性,用临床样本检测重复性。结果表明,该方法与能感染奶牛的其它幾种病毒均无交叉反应,检出敏感度达4.87 × 101 copies/μL,比常规PCR检测方法高10倍。同时检测了5个规模化奶牛场送检的67份奶牛腹泻样本,其BVDV检出率为46.27%(31/67)。本研究成功建立了BVDV SYBR Green Ⅰ实时荧光定量PCR检测方法,为BVDV的快速诊断和定量分析提供了技术支撑,具有很好的应用前景。

关键词:牛病毒性腹泻病毒(BVDV);荧光定量PCR;E2基因;检测

中图分类号:S858.23 文献标识号:A 文章编号:1001-4942(2019)12-0106-05

Abstract The experiment was conducted to establish a method to rapidly detect bovine viral diarrhea virus with high sensitivity, high specificity and good reproducibility. In the study, a pair of specific primers were designed and synthesized according to the conservative sequence of E2 gene in BVDV, and the SYBR Green Ⅰquantitative real-time PCR detection method was established. The standard positive samples were diluted 10 times gradient to detect sensitivity. The specificity was tested with other viruses that could infect the cow, and the repeatability was tested with clinical samples. The results showed that this method did not have cross-react with other cow viruses, and the detection sensitivity was up to 4.87×101 copies/μL, which was 10 times higher than that of the conventional PCR method. At the same time, 67 diarrhea samples from 5 large-scale dairy farms were tested, ...



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