牛副流感病毒3型3种基因型多重RT-PCR检测方法的建立

Establishment of a Multiplex RT-PCR Detection Method for Three Genotypes of Bovine Parainfluenza Virus Type 3

为建立检测牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV3)3种基因型的多重RT-PCR方法,根据GenBank上发表的BPIV3 3种基因型病毒株的HN基因序列设计特异性引物,优化反应体系建立多重RT-PCR方法。结果显示,方法可同时扩增出BPIV3A型150bp、B型253bp和C型342bp的特异性片段,与牛传染性鼻气管炎病毒(IBRV)、牛呼吸道合胞体病毒(BRSV)、牛病毒性腹泻病毒(BVDV)、小反刍兽疫病毒(PPRV)、牛支原体、牛布鲁氏菌、羊布鲁氏菌、牛源多杀性巴氏杆菌A型和B型均无交叉反应,A、B、C基因型BPIV3最低阳性质粒检测量分别为0.89×104、0.92×104和1.53×104拷贝/μL。本试验建立的多重RT-PCR检测方法操作方便、特异性强,应用于临床样本的检测,可快速检测BPIV3 3种基因型。

In order to develop a multiplex RT-PCR method for detecting three genotypes of bovine parainfluenza virus type 3（BPIV3）,according to the hemagglutinin-neuraminidase protein genomic sequences of three BPIV3 genotypes obtained from GenBank,three pairs of specific PCR primers were designed.A multiplex RT-PCR detecting three genotypes of BPIV3 was established and optimized.The results showed no cross-reactivity with IBRV,BRSV,BVDV,M.bovis,PPRV,B.melitensis,B.abortus,bovine P.multocidaserotype A and bovine P.multocidaserotype B.In this study,the amplification produced a series of specific fragments with lengths of 150 bp（BPIV3 a）,253 bp（BPIV3 b）and 342 bp（BPIV3 c）,respectively.The limit detection of three recombinant plasmids were 0.89×104,0.92×104 and 1.53×104 copies/μL.This multiplex RTPCR method was high specificity and simplicity of operator.It was applied to detect clinical samples and could rapidly detect three genotypes of BPIV3.