鉴别猪瘟病毒强毒和疫苗弱毒的RT-PCR检测方法的建立

A Reverse Transcription Polymerase Chain Reaction for Detection and Differentiation of Wild-type and Vaccine Strains of Classical Swine Fever Virus

根据Genbank上发表的猪瘟病毒(CSFV)全基因组序列,选择猪瘟强毒株和兔化弱毒疫苗株序列存在的差异区域,分别设计合成两条特异性的上游引物,一条只针对强毒毒株,一条只针对兔化弱毒疫苗株;同时选择高保守区域序列,设计合成了一条强毒和兔化弱毒共用的下游引物,成功建立了一种能够区分猪瘟病毒强毒株和兔化弱毒疫苗株的RT-PCR检测方法。应用该方法从猪瘟强毒株和兔化弱毒株中扩增出了大小分别为680bp和785bp的特异性片段。该方法对PRRSV、BVDV、PRV、PCV等病毒基因组扩增,结果均为阴性;该方法可以检测出最低为0.5pg的猪瘟病毒RNA,具有高度的特异性和敏感性。本方法的建立为猪瘟的实验室检测和流行病学调查奠定了坚实的基础。

A reverse transcription polymerase chain reaction （RT-PCR） was developed for the differentiation of wild- type classical swine fever virus from the C-strain vaccine with primers specific respectively for CSFV C-strain vaccine and highly virulent Shimen strain as the forward primer and a common downstream primer designed according to the highly conserved regions of CSFV genome in GenBank. By this method, a fragment of 785bp was amplified from genomic RNA of C-strain and a fragment of 680bp was amplified from genomic RNA of Shimen strain. No amplification was achieved for genomic RNAs of PRRSV, BVDV, PRV, PCV etc. The method could detect 0.5pg of CSFV RNA. The study laid the foundation for the establishment of the laboratory tests and epidemiological investigation of CSFV.