鸡传染性支气管炎病毒地高辛标记探针检测方法的的建立和应用

Establishment and application of digoxigenin-labeled nucleic acid probe for detection of infectious bronchitis virus

根据GenBank中已经发表的IBV N基因的保守序列,设计合成一对引物,利用RT-PCR技术扩增IBVN基因的582 bp的核酸片段,并制备出地高辛标记的IBV核酸探针。特异性检测结果表明,该探针能与不同毒株的IBV核酸发生特异性杂交,而与对照的NDV、鹅副粘病毒的核酸杂交反应为阴性;敏感性检测结果表明,该探针对IBV的最低检出量为10 pg,显示所制备的核酸探针用于IBV的检测是可行的。

According to the genomic sequences of N gene of infectious bronchitis virus （IBV） published in Genbank, a pair of primers was designed for amplifying the 582 bp fragment in RT - PCR experiments. The PCR product was labeled with digoxigenin as DNA probe for detection of IBV. The hybridization assay of specificity showed all RNA of IBV strains were positive, but other nucleotide extracted from NDV andPMV negative. The sensitivity test showed that as low as 10 pg of IBV＇s RNA could be detected by DIG-labeled probe. Therefore, it was concluded that the DIG-labeled probe could be used for IBV detection.