牛病毒性腹泻-粘膜病病毒TaqMan荧光定量RT-PCR检测方法的建立及初步应用  

Establishment and Initial Application of Fluorescent Quantitative RT-PCR for Detection of Bovine Viral Diarrhea Virus

作　　者：陈其兵 薛霜 漆世华 朱薇 张萍 谢红玲 温文生 吴玉石 

机构地区：[1]武汉中博生物股份有限公司,武汉430070

出　　处：《中国兽药杂志》2012年第4期4-6,47,共4页Chinese Journal of Veterinary Drug

摘　　要：为建立一种牛病毒性腹泻-粘膜病病毒(BVDV)的快速检测方法,根据GenBank中登录的BVDV基因序列,针对5’UTR的保守序列,设计合成了一对特异性引物和一条TaqMan荧光探针。通过对反应条件和反应体系进行优化,建立了一种能快速定量检测BVDV的荧光定量RT-PCR检测方法。通过对20份胎牛血清样品和猪瘟细胞苗半成品进行检测,对该方法的特异性、敏感性和重复性进行试验。结果显示,建立的方法能检测到BVDV,而对CSFV、PRRSV和MDBK细胞的扩增结果均为阴性,具有高度的特异性。在所检测的20份样品中,BVDV阳性6份(30%)。对所构建的标准品进行检测,在102～108拷贝/μL的范围内可以得到良好的动力学曲线,能检测低至103～104拷贝/mL的病毒量。表明所建立的检测方法具有快速、特异、灵敏、重复性好等特点,可用于临床及科研中对BVDV的快速定量检测和对牛血清及猪瘟兔化弱毒疫苗等生物制品中是否污染BVDV进行监测。According to the genomic sequences within 5 ＇ UTR of BVDV which published in Genbank, a pair of primers and a Taqman probe were designed. Then a rapid fluorescent quantitative RT - PCR method was developed by optimization of the reaction conditions and system. This method was established by specificity, sensibility, reproducibility. Twenty samples of fetal bovine serum and swine fever vaccine were detected by this methed. The experiment showed that six samples were positive and the ratio was 30%, and the detection result of CSFV, PRRSV and MDBK cells were all negative. A standard curve was achieved and the result showed that the sensitivity of this method was 10^3 - 10^4copies/mL and the linear relation was excellent. The results show that this method is fast, specific, sensitive, repeatable and can be used to monitor the pollution of BVDV in biological products and quantitative detection of BVDV.

关 键 词：牛病毒性腹泻-粘膜病病毒 荧光定量RT-PCR 检测 

分 类 号：S852.65[农业科学—基础兽医学]