牛病毒性腹泻病毒通用型及分型RT-PCR检测方法的建立及应用

Establishment and application of RT-PCR detection method for universal and differential types of Bovine viral diarrhea virus

为了建立快速检测牛病毒性腹泻病毒(Bovine viral diarrhea virus, BVDV)通用型及1型(BVDV-1)、2型(BVDV-2)的检测方法,并了解聊城地区BVDV的流行规律,试验根据BVDV-1、BVDV-2 5′端非编码区(5′UTR)基因序列分别设计BVDV通用型检测引物及BVDV-1、BVDV-2特异性检测引物,通过优化RT-PCR扩增条件分别建立BVDV通用型RT-PCR、BVDV-1分型RT-PCR、BVDV-2分型RT-PCR检测方法,并分析3种检测方法的特异性、重复性、符合性,最后采用3种检测方法对聊城地区采集的4 685份临床样品进行病原学检测分析。结果表明:3种检测方法检测牛传染性鼻气管炎病毒(IBRV)、牛副流感病毒3型(BPIV-3)、牛呼吸道合胞体病毒(BRSV)、牛轮状病毒均为阴性,检测灵敏度分别为6.84,1.08,3.03 ng;BVDV通用型RT-PCR检测方法与BVDV-1分型RT-PCR、BVDV-2分型RT-PCR检测方法的符合率均为100%;聊城地区BVDV阳性率为6.51%,其中BVDV-1和BVDV-2阳性率分别为4.50%和2.01%,BVDV-1和BVDV-2混合感染率为0.13%。说明试验建立的BVDV通用型RT-PCR、BVDV-1分型RT-PCR、BVDV-2分型RT-PCR检测方法均具有良好的特异性、敏感性、准确性和临床适用性,聊城地区普遍存在BVDV的流行,其流行优势基因型为BVDV-1。

In order to establish a rapid detection method for Bovine viral diarrhea virus(BVDV)universal type,type 1(BVDV-1)and type 2(BVDV-2),and understand the epidemic law of BVDV in Liaocheng region,in this experiment according to the BVDV-1 and BVDV-25′UTR gene sequences,BVDV-1 general detection primers,BVDV-1 specific detection primers,and BVDV-2 specific detection primers were designed respectively.Via optimizing the amplification conditions of RT-PCR,BVDV universal RT-PCR,BVDV-1 typing RT-PCR method and BVDV-2 typing RT-PCR method were established respectively;and the specificity,sensitivity and consistency of the three detection methods were analyzed,and three detection methods were used for etiological detection and analysis on 4685 clinical samples collected in Liaocheng region.The results showed that the three detection methods were negative for Bovine infectious rhinotracheitis virus(IBRV),Bovine parainfluenza virus type 3(BPIV-3),Bovine respiratory syncytial virus(BRSV),Bovine rotavirus(BRV),and the detection sensitivity were 6􀆰84 ng,1􀆰08 ng and 3􀆰03 ng respectively;the coincidence rate of universal RT-PCR detection method with BVDV-1 typing RT-PCR detection method and BVDV-2 typing RT-PCR detection method were 100%.The positive rate of BVDV in Liaocheng region was 6􀆰51%,among which the positive rates of BVDV-1 and BVDV-2 were 4􀆰50%and 2􀆰01%respectively;the mixed infection rate of BVDV-1 and BVDV-2 was 0􀆰13%.The results suggested that the established BVDV universal RT-PCR detection method,BVDV-1 typing RT-PCR detection method and BVDV-2 typing RT-PCR detection method had good specificity,sensitivity,accuracy and clinical applicability.BVDV was prevalent in Liaocheng region,and its prevalent dominant genotype was BVDV-1.