IBDV超强毒株、强毒株及疫苗株的快速鉴别诊断

Research on rapid identification for vvIBDV,cvIBDV and vaccine strains

【目的】利用限制性酶切位点的特异性,鉴别IBDV超强毒株、强毒株及疫苗株。【方法】根据IBDVVP2基因的共保守序列设计1对引物,分别以IBDV超强毒株GX8/99、陕西分离株、强毒株(XZ-1、ZZ-1、JL、GX-2、JS-2、SD-1、SD-3、HeN-4、ZJ-1、JX-2、JS-30)和疫苗株(B87、NF8)的VP2保守序列为模板,采用RT-PCR方法扩增VP2基因的共保守序列,并构建IBDV不同毒株的重组质粒。用AhaⅠ/StuⅠ和BamHⅠ/NspⅠ2组限制性内切酶分别对超强毒株、强毒株及疫苗株的重组质粒进行酶切鉴定。【结果】扩增出了IBDVVP2基因中的共保守序列,其长度为802 bp。超强毒株能被AhaⅠ/StuⅠ切出327 bp的片段,而强毒株及疫苗株则能被BamHⅠ/NspⅠ切出270 bp的片段。【结论】此种RT-PCR结合限制性内切酶酶切的鉴别方法能够很好的区分IBDV超强毒株与强毒株和疫苗株。

【Objective】 In order to distinguish very-virulent strain,virulent strain and vaccine strain,the conserved reign of conservative sequence in IBDV VP2 gene was digested by different pairs of restriction enzymes.【Method】 One pair of primers was designed by the conserved reign of conservative sequence in IBDV VP2 gene.Nucleic acid extracts from vv IBDV GX8/99,isolation strain of Shaanxi,V IBDV strain XZ-1,ZZ-1,JL,GX-2,JS-2、SD-1、SD-3、HeN-4、ZJ-1、JX-2、JS-30 and vaccine strain B87,NF8 were used as templates for RT-PCR.The productions of 802bp were cloned into plasmid.And the recombined plasmids were digested by two groups of restriction enzyme,AhaⅠ/StuⅠ and BamHⅠ/NspⅠ.【Result】 The results showed that a 327 bp fragment was cutout in veryvirulent strain,whereas a 270 bp fragment could be cutout in both virulent strain and vaccine strain,which were consistent with the putative.【Conclusion】 This method can help to detect veryvirulent IBDV in pathogenic avian accurately and quickly and has provided a new way for IBD diagnosis.