猪瘟强毒株与兔化弱毒疫苗株的LAMP鉴别检测

为了鉴别猪瘟病毒（CSFV）野毒感染和疫苗接种,系统比较分析CSFV标准强毒株、疫苗株、不同基因亚型的野毒株的全基因组序列差异,设计一套分别针对猪瘟强毒与弱毒NS5B基因的环介导等温扩增（LAMP）引物,建立特异性的猪瘟强毒株与疫苗株的LAMP检测方法.LAMP扩增产物用罗丹明B指示剂或者琼脂糖凝胶电泳进行检测.该方法特异性好,比RT-PCR灵敏度高出1 000倍,且重复性稳定性良好,为快速准确地鉴别CSFV野毒感染和疫苗接种提供了有效的方法.

Loop-mediated Isothermal Amplification Assay for Differentiation and Detection of Virulent and Vaccine Strains of Classical Swine Fever Virus

To differentiate virulent and vaccine strains of classical swine fever virus（ CSFV）, a loop-mediated isothermal amplification assay（LAMP） was established with a set of primers based on the NS5B gene of CSFV by analyzing genomic sequences of CSFV standard virulent strains, CSFV vaccine strains, and different genetic subtypes of CSFV. The results showed that the LAMP products could be completed at a constant temperature of 65℃ , then a ladder-like pattern of products were visible on agarose gel electrophoresis, and products could be visualized with rhodamine B dye. No cross-reactivity was observed in the samples with other related viruses. Detecting limit of the LAMP assay was 1 000 fold higher than that of RT-PCR. The assay was therefore a sensitive, simple, and rapid method for differentiation and detection of virulent and vaccine strains of classical swine fever virus.