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参考GenBank上登录的犬冠状病毒(CCoV)S基因的保守序列设计并合成1对特异性引物,建立了一种快速、灵敏和特异的SYBR GreenⅠ荧光定量RT-PCR方法,用于CCoV的检测。特异性试验结果表明,该方法特异性强,与犬瘟热病毒、犬腺病毒2型、副流感病毒和犬细小病毒等无交叉反应;敏感性试验结果表明,它的最低检测限为4×10~1~5×10~1 copies/L,高于常规RT-PCR方法 10倍左右;批内和批间重复试验的变异系数均小于1.20%。利用该方法和普通RT-PCR方法分别对76份临床样品进行检测;结果显示,普通RT-PCR的阳性率为69.7%,实时荧光定量RT-PCR的阳性率为78.9%。对于60份阳性病料,随机选择2份病料进行病毒分离,用阳性血清中和其他外源病毒后都出现了CPE,进行RT-PCR扩增后测序,结果证实为CCoV。以上结果表明,本研究建立的方法可以为犬冠状病毒病的快速诊断及流行病学调查提供技术手段。
A pair of specific primers was designed and synthesized according to the conserved sequence of the canine coronavirus (CCoV) S gene registered in GenBank. A rapid, sensitive and specific SYBR GreenⅠ fluorescence quantitative RT-PCR method was established for the detection of CCoV. Specificity test results show that this method is specific and does not cross-react with canine distemper virus, canine adenovirus type 2, parainfluenza virus and canine parvovirus. The sensitivity test results show that its minimum detection limit is 4 × 10 ~ 1 ~ 5 × 10 ~ 1 copies / L, higher than the conventional RT-PCR method about 10 times; intra-assay and inter-assay repetitive test coefficient of variation were less than 1.20%. 76 clinical samples were detected by this method and ordinary RT-PCR respectively. The results showed that the positive rate of common RT-PCR was 69.7% and the positive rate of real-time fluorescence quantitative RT-PCR was 78.9%. For 60 positive disease materials, 2 disease materials were randomly selected for virus isolation. CPE was found after neutralization of other exogenous viruses with positive serum. RT-PCR was used to amplify CPE, and the result was confirmed as CCoV. The above results show that the method established in this study can provide technical means for the rapid diagnosis and epidemiological investigation of canine coronavirus disease.