幼虫芽孢杆菌和蜂房蜜蜂球菌双重TaqMan荧光定量PCR检测方法的建立  

DEVELOPMENT OF DUPLEX TAQMAN REAL-TIME PCR ASSAY FOR DETECTION OF PAENIBACILLUS LARVAE AND MELISSOCOCCUS PLUTON OF HONEY BEES

作　　者：何晓杰[1] 王科珂 叶尔保勒 阿斯喀 张婕妤 哈森 季新成[2] 王振宝[1,2,3] HE Xiao-jie;WANG Ke-ke;Yeerbaole;Asika;ZHANG Jie-yu;Hasen;JI Xin-cheng;WANG Zhen-bao(Yili Vocational and Technical College, Yining 835000, China;Xinjiang Entry-Exit Inspection and Quarantine Bureau, Urumqi 830063, China;Yili Entry-Exit Inspection and Quarantine Bureau, Yining 835000, China;Chongqing Technology and Business Universi（y, Chongqing 400067, China)

机构地区：[1]伊犁职业技术学院,伊宁835000 [2]新疆出入境检验检疫局,乌鲁木齐830063 [3]伊犁出入境检验检疫局,伊宁835000 [4]重庆工商大学,重庆400067

出　　处：《中国动物传染病学报》2018年第3期51-56,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家质检总局科技计划项目(2012IK023);国家质检总局科技计划项目(2016IK260);新疆维吾尔自治区自然科学基金项目(201318101-19);新疆维吾尔自治区自然科学基金项目(2016D01B059)

摘　　要：为快速鉴别诊断蜜蜂美洲幼虫腐臭病(American foulbrood,AFB)和欧洲幼虫腐臭病(European foulbrood,EFB),本研究根据Gen Bank中登录的幼虫芽孢杆菌(Paenibacillus larvae)和蜂房蜜蜂球菌(Melissiciccus pluton)16S r RNA基因序列分别设计特异性引物和探针,建立了双重Taq Man荧光定量PCR检测方法。结果表明,该方法与其他病原无交叉反应;对P.larvae和M.pluton的检测灵敏度均达10 copies/μL的阳性质粒;重复性试验结果显示批内、批间变异系数均小于2%。应用该方法对200份实验室模拟样品和200份临床样品进行了检测,与预期结果一致。本研究建立的方法可用于AFB与EFB的快速鉴别诊断和早期监测,以及蜂产品中P.larvae和M.pluton的定量分析。To establish the method of real-time PCR for rapid detection of American foulbrood and European foulbrood pathogen in honey bees, accordding to the genetic sequence of 16S rRNA of P.larvae and M.pluton from GenBank, the specific primers and TaqMan fluorescent probes were designed and synthesized, and the duplex TaqMan real-time PCR was established. The study showed that it was no reaction with other pathogens, and better amplification efficiency in detecting P.larvae and M.pluton with detection limit of 10 copies/~tL. The repeatability test results showed that the coefficient of variation between intro- and inter-reproducibility was less than2% respectively. 200 laboratory simulation samples and 200 clinical samples were tested by this method and the result was consistent to the prediction in the theory. These data suggested that the developed duplex TaqMan real-time PCR assay could be applied in rapid identification, early rapid detection and quantitative analysis ofP, larvae and M.pluton infections.

关 键 词：美洲幼虫腐臭病 幼虫芽孢杆菌 欧洲幼虫腐臭病 蜂房蜜蜂球菌 双重TaqMan荧光定量PCR 

分 类 号：S852.616[农业科学—基础兽医学] S852.611[农业科学—兽医学]