致牛腹泻主要病毒多重实时荧光定量PCR检测方法的建立与应用

Establishment and Application of Multiplex Real-time Fluorescence PCR Method for Detecting Major Viruses Causing Bovine Diarrhea

为鉴别检测引起牛病毒性腹泻的4种主要病原牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)、牛冠状病毒(Bovine coronavirus,BCV)、牛轮状病毒(Bovine rotavirus,BRV)和牛细小病毒(Bovine parvovirus,BPV),分别以BCV Nsp10基因、BVDV 5′UTR基因、BRV VP6基因和BPV VP2基因为靶基因设计引物,建立了检测上述4种病毒的实时荧光定量PCR方法。结果显示,方法的特异性强,仅检测靶标病毒的结果为阳性;方法的灵敏度高,对4种病毒BCV、BVDV、BRV和BPV相应质粒的最低检出限分别为7.77×10^(2)、7.11×10^(1)、1.74×10^(2)和2.53×10^(2)copies/μL,且重复性良好,批内和批间变异系数均小于1.0%。由于上述病毒存在交叉感染的情况较多,因此建立多重荧光定量PCR,并利用建立的多重实时荧光定量PCR方法对采集的230份牛腹泻临床样本进行检测,BVDV、BCV、BRV和BPV的阳性率分别为66.96%、3.48%、18.26%和9.57%;且存在多病毒混合感染情况,其中BVDV和BRV混合感染率为10.43%,BVDV和BPV混合感染率为3.48%,BVDV、BCV和BRV混合感染率为1.74%,BVDV、BCV和BPV混合感染率为0.87%,BVDV、BRV和BPV混合感染率为0.87%。研究建立的多重实时荧光定量PCR方法具有良好的特异性、灵敏性,可为病毒性牛腹泻的快速诊断和流行病学调查提供的技术支持。

In order to identify the four main viral pathogens causing bovine diarrhea of bovine coronavirus(BCV),bovine viral diarrhea virus(BVDV),bovine rotavirus(BRV)and bovine parvovirus(BPV),BCV Nsp10 Gene,BVDV 5'UTR gene,BRV VP6 gene and BPV VP2 gene were used as target genes to design primers,and real-time fluorescent PCR(RT-PCR)methods were established and optimized.The results showed that RT-PCR methods were all with highly specificity and only the target virus was positive,RT-PCR methods also had highly sensitivity with the minimum detection limits for BCV,BVDV,BRV and BPV of 7.11×10^(2)copies/μL,7.7×10^(1)copies/μL,1.74×10^(2)copies/μL and 2.53×10^(2)copies/μL,respectively.In addition,the stabilities of these RT-PCR methods were meet the detection acquirement with coefficient variation<1.0%.As the cross-infection of the above-mentioned viruses,multiplex fluorescent quantitative PCR was established,and the multiplex RT-PCR method was used to detect 230 clinical samples of bovine diarrhea.The results showed that the positive detection rates of BVDV,BCV,BRV and BPV were 66.96%,3.48%,18.26%and 9.57%.Moreover,mixed infections were found in these positive samples,of which 10.43%for BVDV,3.48%for BRV,BVDV and BPV,1.74%for BVDV,BCV and BRV,0.87%for BVDV,BCV and BPV,and 0.87%for BVDV,BRV and BPV.All result indicated that the established RT-PCR methods have good specificity,sensitivity and accuracy,which would be a powerful tool for simultaneously detecting the four viruses and an epidemiological investigation of viral bovine diarrhea.