【作者】 赵晓军；

【导师】 申之义； 关平原； 张七斤；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 本试验对CEV内蒙古分离株OV/nm-05株进行传代培养,待出现典型的CPE后收毒并进行DNA的提取,根据已发表的CEV B2L基因序列,设计引物,进行B2L基因的特异性扩增,并将其克隆到pMD18-T载体后进行测序。结果表明:OV/nm-05株的B2L基因长为1133bp。核苷酸序列比较、分析结果表明:OV/nm-05株与Strain NZ2同源性最高,达97.9%,与N86.20a最低,为84.2%,说明OV/nm-05株B2L基因与参考毒株之间差异不大。本试验根据已发表的CEV NZ2株的序列,设计引物,对OV/nm-05株、CPV和新生犊牛睾丸原代细胞的核酸进行PCR扩增,结果表明:只有OV/nm-05株扩增出约440bp左右的特异性片段,其它均为阴性,说明该PCR扩增体系具有良好的特异性。对OV/nm-05株进行敏感性试验,结果表明:该PCR扩增体系可检测出1.1ng模板,说明该PCR扩增体系具有较高的敏感性。更多还原

【Abstract】 The OV/nm-05 virus was propagated and the total DNA of virus was extracted. According to the published B2L gene sequence of CEV strain in GenBank, one pair of primer was designed. The B2L gene of virus was amplificated with the primer by PCR and then cloned into pMD18-T plasmids and then sequenced and analysed in homology. The acquired sequences contained 1133bp, The homology analysis revealed that the homology of the strain NZ2 and the OV/nm-05 was 97.9%, the N86.20a and the OV/nm-05 was 84.2%. There were a little variation between OV/nm-05 strain and the referenced virus strains.According to the published sequence of strain NZ2 in GenBank, another pair of primer was designed. The virus DNA was amplificated with the primer by PCR, then PCR diagnostic method was established, and this method’s specificity and sensitivity was detected. The results indicated that the PCR method could detect the nucleic acid of OV/nm-05 virus only and a 440bp DNA fragment was amplificated. The minimum CEV DNA’s detection level was 1.1ng. This illustrated that this method was specific and sensitive for the detection of CEV.更多还原