山羊痘病毒和羊口疮病毒双重PCR检测方法的建立与初步应用  

Establishment and Rudiment Application of Duplex PCR for Goatpox Virus and Orf Virus

作　　者：向智龙[1] 程振涛[1] 卓建华 周碧君 欧德渊 鲜思美[1] 刘芳[1] 冉光鑫[1] 

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]湖北省京山县畜牧兽医局,湖北京山431800 [3]贵州省动物疫病研究所,贵州贵阳550025

出　　处：《中国畜牧兽医》2011年第1期88-91,共4页China Animal Husbandry & Veterinary Medicine

基　　金：贵州省科学技术基金项目“贵州省羊口疮的病原学与检测技术研究”(黔科合J字[2010]2260);贵州大学引进人才科研项目“贵州省羊口疮的诊断与防控技术研究”(贵大人基合字(2009)024号);贵州大学2010年“SRT”项目“羊传染性脓疱病毒贵州株PCR鉴定及分子特征分析”;贵州大学2010年研究生创新基金创新项目“贵州省羊口疮的分子诊断技术及B2L表达研究”;国家自然科学基金项目"基于山羊痘病毒P32基因的山羊粘膜免疫研究"(30940054);

摘　　要：根据GenBank发表的山羊痘病毒(goatpox virus,GPV)和羊口疮病毒(orf virus,ORFV)基因序列,分别合成针对GPVITR和ORFVH2L基因片段的2对引物,以GPV和ORFV的核酸混合物作为模板,经优化反应条件,成功建立了快速鉴别检测GPV、ORFV的双重PCR方法。特异性试验结果显示,该方法对GPV与ORFV核酸的扩增能获得289和507 bp的特异性目的片段,而对FMDV、FPV、BTV、健康山羊皮肤的核酸扩增均为阴性;敏感性试验结果显示,该方法对GPV核酸的最小检出量为4 pg,对ORFV核酸为0.4 pg;对临床采集的12份病料进行双重PCR扩增,GPV检出率为25%(3/12),ORFV的检出率为75%(9/12)。结果表明,建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于GPV或/和ORFV临床感染病例的联合检测与鉴别诊断。Two pairs of primers were designed according to the sequences of ITR gene of Goatpox virus（GPV） and H2L gene orf virus（ORFV） available in GenBank,respectively.Nucleic acids of GPV and ORFV were mixed as template,a duplex PCR assay was developed based on optimization of reaction conditions.Using the duplex PCR assay,the specific fragments 289 bp and 507 bp were amplified respectively from DNA samples of GPV and ORFV,but not amplified from foot-mouth disease virus,fowl pox virus,bluetongue virus and skin from normal healthy goat.The sensitivity of the duplex PCR reached 4 pg for GPV DNA and 0.4 pg for ORFV DNA,respectively.12 clinical material were amplified by the duplex PCR assay,detection rate of GPV was 25%（3/12）,detection rate of ORFV was 75%（9/12）.The results showed that this duplex PCR assay was rapid,sensitive and specific for simultaneous diagnosis or differentiation of GPV and/or from ORFV infections.

关 键 词：山羊痘病毒 羊口疮病毒 双重PCR 检测 

分 类 号：S858.26[农业科学—临床兽医学]