杨凌某羊场羊口疮病毒的分离鉴定

Isolation and Identification of Orf Virus in a Goat Farm of Yangling

为获得杨凌地区羊口疮病毒野毒株,在某羊场采集具有典型羊口疮症状的病羊口唇部结痂并研磨,无菌过滤后取500μL接种于犊牛睾丸原代细胞。盲传4代,测定细胞病变的第5代病毒的滴度,用羊口疮病毒B2L和F1L基因的特异性引物进行PCR检测。结果显示,第5代病毒能够致犊牛睾丸原代细胞出现细胞病变(CPE),病毒滴度为107.66 TCID50/mL;扩增出了羊口疮病毒B2L和F1L基因的片段,其大小分别为540bp和437bp,与用于设计引物的参考毒株序列的相似度分别为96.57%和96.43%,可确定为羊口疮病毒的相应基因片段,获得了羊口疮病毒分离株。

In order to obtain a wild strain of orf virus （ORFV ） in Yangling,the lip scab of a lamb with typi-cal orf symptoms from a goat farm in Yangling was collected.The scab was grinded and centrifuged suffi-ciently after dipped in PBS overnight at 4 ℃,and then the supernatant was sterilized with 0.22 μm filter and inoculated in the calf testis primary cells with 500μL.After 4 generations of blind passages,the virus titer of the 5th generation was determined and a PCR assay based on B2L and F1L genes was used to detect the ORFV.The results suggested that the 5th generation of ORFV can cause apparent CPE in calf testis primary cells and the virus titer of the 5th generation was 10^7.66 TCID50/mL.The fragments of B2L and F1L amplified by PCR were about 540 bp and 437 bp respectively,and the similarities with its reference se-quences are 96.57% and 96.43%.In conclusion,a wild strain of ORFV in Yangling was isolated in this study.