牛病毒性腹泻病毒实时荧光定量RT-PCR检测方法的建立  

Establishment Real-time Fluorescence Quantitative RT-PCR for Detection of Bovine Viral Diarrhea Virus

作　　者：黄杰 岳丰雄 徐宏军 

机构地区：[1]成都天邦生物制品有限公司,四川成都610100

出　　处：《动物医学进展》2017年第5期59-63,共5页Progress In Veterinary Medicine

摘　　要：为了建立一种快速、特异、灵敏的检测牛病毒性腹泻病毒(BVDV)Taq Man实时荧光定量RT-PCR的方法,根据NCBI GenBank上已公布的BVDV、猪瘟病毒(CSFV)核酸序列进行比对,利用Oligo 6.71软件设计一对引物及一条探针,建立了检测BVDV的Taq Man实时荧光定量RT-PCR方法。通过对该方法的特异性、重复性及其敏感性进行相关试验,结果表明该方法检测出BVDV标准毒株Oregon C_(24)V为阳性,猪圆环病毒2型、猪伪狂犬病病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪繁殖与呼吸综合征病毒和CSFV的检测均为阴性。对BVDV标准毒株最低检测量达到10^(-2.5) TCID_(50)。该方法检测同一样品重复进行8次检测,结果均一致,表明方法的重复性较好。应用该方法对6批猪瘟疫苗专用血清进行BVDV检测,阳性率为16.7%;猪瘟弱毒苗中未检出BVDV。建立的BVDV Taq Man实时荧光定量RT-PCR方法为生产无BVDV污染的猪瘟苗提供了有力的保障。The aim of this study was to establish a rapid, specific and sensitive Taq Man real-time fluorescence quantitative RT-PCR method for detection of bovine viral diarrhea virus （BVDV）.A pair of primers and a probe were designed by using the Oligo 6.71 software to detect BVDV according to the nucleic acid sequence of BVDV and classical swine fever virus （CSFV） on NCBI GenBank so as to establish the method.The specificity, repeatability and sensitivity of the method were tested, the results ,showed that BVDV Oregon C24 V strain was positive, while PCV2,PRV, TGEV, PEDV, PRRSV and CSFV test results were negative. To detect BVDV standard strains, the minimum detectable amount could reach 10-2.5 TCID50 .The method has good repeatability by testing the same sam- ple repeated eight times.BVDV positive rate of the special serum of classical swine fever vaccine was 16.7 %0. However,BVDV positive rate of classical swine fever vaccine was not detected.Therefore, this new method provided a useful tool for free from contamination with BVDV of classical swine fever vaccine.

关 键 词：牛病毒性腹泻病毒 实时荧光定量RT-PCR 猪瘟病毒 疫苗 

分 类 号：S852.653[农业科学—基础兽医学]