犬冠状病毒SYBR GreenⅠ荧光定量RT-PCR检测方法的建立

Establishment of SYBR GreenⅠreal-time RT-PCR for the detection of canine coronavirus

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。

According to the conserved sequence of S gene of the canine coronavirus（CCoV） in GenBank,a pair of specific primers were designed for amplifying the specific fragment from CCoV.ASYBR Green Ⅰreal-time reverse transcriptase polymerase chain reaction assay（real-time RT-PCR）was established for detecting CCoV,which was rapid,sensitive and specific.The results showed that the method was highly sensitive to detect as little as 4×10^1—5×10^1 copies/L,and it was 10 times more sensitive than the conventional RT-PCR.There was no amplification from canine distemper virus,canine adenovirus type 2,canine parainfluenza virus and canine parvovirus.The intra-and inter-coefficients of variation were less than 1.20%.Seventy-six clinical samples were detected by the real-time RT-PCR and the conventional RT-PCR,and the positive rate was 78.9% and 69.7%for CCoV.Two out of 60 positive faeces samples were inoculated into CRFK cells after neutralizing other exogenous viruses with positive serum andcaused typical cytopathic effects.The partical M gene of the isolated viruses were cloned and se quenced.The result showed a high similarity with other CCoVs.In conclusion,the method can provide an effective way for the rapid diagnosis and epidemiological investigation of CCoV.