SYBR Green Ⅰ实时荧光定量RT-PCR检测猪瘟疫苗病毒含量

Development of SYBR green Ⅰ fluorogenetic quantitative RT-PCR assay for rapid detection of hog cholera lapinized virus

为建立一种能够快速定量检测猪瘟兔化弱毒疫苗病毒含量的实时荧光定量RT-PCR方法。对GenBank登录的25株猪瘟病毒强毒株和兔化弱毒疫苗株基因组全序列进行比较分析,在其高度保守的5′端非编码区设计1对针对猪瘟兔化弱毒疫苗的特异性引物,扩增片段为245 bp,且不与牛病毒性腹泻病毒以及其他猪源病毒发生非特异性反应。应用实时荧光定量RT-PCR法对16份猪瘟脾淋苗和细胞苗进行定量检测,结果表明,102拷贝/μL的总RNA即能得到特异性扩增,在107~102拷贝/μL线性范围内有良好的扩增曲线,并与兔体定型热反应有良好的相关性。该法具有敏感性、特异性、重复性好等优点,可望取代传统的兔热法用于猪瘟疫苗生产过程中的效价测定及指导疫苗的配制,也为猪瘟病毒分子生物学研究提供一种新的有效工具。

In order to develop a real-time RT-PCR assay for quantitative detection of hog cholera lapinized virus（HCLV）.25 strains classical swine fever virus（CSFV）genomic sequences including wild-type and vaccine virus pubulished in GenBank were aligned,and a pair of primers corresponding to the highly conserved regions of CSFV genome was designed,amplified fragment length 245 bp,which has no specific reaction with bovine viral diarrhea virus and other virus of pigs.16 samples were detected,the sensitivity of the assay was 102 copies per reaction,and the assay has good linear relationship in a wide range of 107-102 copies/μL.The method developed in this paper has the advantages of high sensibility,specificity and good stability,which can detect HCLV rapidly and accurately and provides a simple and effective tool for the development of HCLV vaccine and the molecular biological studies on classical swine fever virus.