猪瘟病毒强毒株和兔化弱毒疫苗株RT-PCR快速鉴别检测方法的建立

Development of RT-PCR for detection and differentiation of wild-type and vaccine viruses of classical swine fever virus

【目的】建立猪瘟病毒强毒株和兔化弱毒疫苗株的RT-PCR快速鉴别检测方法。【方法】根据Gen-Bank中36株猪瘟病毒(CSFV)强毒株和兔化弱毒疫苗株的基因序列,分别设计了针对CSFV强毒和兔化弱毒疫苗株的特异性引物,建立了一种能区分CSFV强毒和兔化弱毒疫苗株的RT-PCR检测方法。【结果】建立的RT-PCR检测方法可以从CSFV强毒株和兔化弱毒疫苗株中分别扩增出大小为187和492 bp的特异性片段,对猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)和猪传染性胃肠炎病毒(TGEV)等猪源性病毒的检测结果均为阴性,说明该方法具有较好的特异性。对10份疑似猪瘟临床样品进行检测后发现,利用该方法可以从中分别检测出CSFV强毒和疫苗毒。【结论】建立了可鉴别检测CSFV强毒株和弱毒疫苗株的RT-PCR方法,为猪瘟的早期诊断及疫苗免疫猪和野毒感染猪的鉴别提供了技术参考。

【Objective】 The study was done to develop RT-PCR for detection and differentiation of wild-type and vaccine viruses of classical swine fever virus.【Method】 Based on the alignment of genomic sequences of 36 strains of classical swine fever virus（CSFV） including wild-type and vaccine viruses available in GenBank,2 pairs of primers specific to CSFV highly virulent Shimen strain and CSFV C-strain vaccine were designed respectively.A reverse transcription polymerase chain reaction（RT-PCR） was developed for the differentiation of wild-type CSFV from the C-strain vaccine.【Result】 The results showed that a fragment of 187 or 492 bp was amplified from genomic RNA of CSFV wild-type or C-strain vaccine respectively.No amplification was achieved to porcine reproductive and respiratory syndrome virus（PRRSV）,porcine circovirus type 2（PCV2） and transmissible gastroenteritis virus（TGEV） using designed primers respectively.It showed the specificity of developed RT-PCR was high to CSFV.CSFV highly virulent strain and C-strain vaccine in 10 collected clinical samples were determined by developed RT-PCR,the wild-type and vaccine viruses of CSFV could be detected and differentiated using this method.【Conclusion】 The developed RT-PCR can be used to detect and differentiate wild-type CSFV from pig vaccinated with the C-strain vaccine.