中蜂囊状幼虫病毒RT-PCR检测方法的建立与应用  

Establishment and application of RT-PCR for detection of Chinese sacbrood virus

作　　者：张明华 韦小平 周碧君[1,3,4] 文明 程振涛[1,3,4] 田琴 王开功 ZHANG Ming-hua;WEI Xiao-ping;ZHOU Bi-jun;WEN Ming;CHENG Zhen-tao;TIAN Qin;WANG Kai-gong(College of Animal Science,Guizhou University,Guiyang 550025,China;Guizhou Modern Agricultural Development Research Institute,Guiyang 550025,China;Institute of Animal Diseases,Guizhou University,Guiyang 550025,China;Guizhou Provincial Key Laboratory of Animal Diseases and Veterinary Public Health,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省现代农业发展研究所,贵州贵阳550025 [3]贵州大学动物疫病研究所,贵州贵阳550025 [4]贵州省动物疫病与兽医公共卫生重点实验室,贵州贵阳550025

出　　处：《中国兽医科学》2021年第3期275-280,共6页Chinese Veterinary Science

基　　金：贵州省基础研究计划项目(黔科基础2019[1453]);贵州省科技支撑计划项目(黔科合支撑2018[2283]);黔农科院青年科技基金项目(2020[08]);贵州省百层次创新型人才项目(黔科合人才[2016]4009号);贵州省科技平台及人才团队计划项目(黔科合平台人才[2018]5253号)。

摘　　要：为建立中蜂囊状幼虫病毒(CSBV)核酸的特异性检测方法,以CSBV结构蛋白VP3基因为靶基因,根据GenBank中序列合成1对特异性引物,建立了CSBV RT-PCR检测方法。以CSBV克隆质粒作为模板和蜜蜂6种不同病毒,分别进行特异性、敏感性、重复性试验。结果显示:所建立的RT-PCR检测方法能扩增出CSBV特异性片段,且不能扩增出蜜蜂以色列急性麻痹病毒、蜜蜂畸翅病毒、蜜蜂急性麻痹病毒、蜜蜂慢性麻痹病毒、黑蜂王台病毒、蜜蜂克什米尔病毒;CSBV阳性质粒最低检测质量浓度为2.82 fg/m L;对CSBV阳性质粒和双蒸水空白对照组重复检测3次,均能扩增出与预期一致的特异性片段。利用该检测方法对从贵州省不同养蜂场采集的100份中华蜜蜂样本进行检测,71份样本为阳性,通过序列比对分析,同源性高达99.16%,进一步证实为CSBV。结果表明,所建立的CSBV检测方法特异性强、敏感性高、重复性好,具有良好的实用性。In order to establish a specific detection method for Chinese sacbrood virus(CSBV)nucleic acid,CSBV structural protein VP 3 gene was used as the target gene,a pair of specific primers was synthesized according to the sequence in Gen Bank,and a CSBV RT-PCR detection method was established.The CSBV clone plasmid was used as a template to conduct specificity,sensitivity,and repeatability tests with 6 different honeybee viruses.The results showed that the established RT-PCR detection method can amplify specific fragments of CSBV,and can not amplify Israeli acute paralysis virus,deformed wing virus,acute bee paralysis virus,chronic bee paralysis virus,black queen cell virus,Kashmir bee virus;the minimum detection concentration of CSBV positive plasmid was 2.82 fg/m L.The CSBV positive plasmid and negative samples were repeatedly tested three times,and the specific fragments consistent with expectations were amplified.Total 100 Apis cerana cerana samples collected from different apiaries in Guizhou Province were tested using this test method,71 samples It was positive,and the homology was as high as 99.16%through sequence comparison analysis,which was further confirmed as CSBV.The results indicate that the established CSBV detection method has strong specificity,high sensitivity,good repeatability and good practicability.

关 键 词：中蜂囊状幼虫病毒 RT-PCR VP3基因 中华蜜蜂 

分 类 号：S895.133[农业科学—特种经济动物饲养]