蜜蜂幼虫芽孢杆菌微滴式数字PCR检测方法的建立  

Establishment of droplet digital PCR assay for detection of Paenibacillus larvae

作　　者：王建峰 高峰[2] 郑剑 倪健波 黄素文 张体银[4] 陈先锋 WANG Jianfeng;GAO Feng;ZHENG Jian;NI Jianbo;HUANG Suwen;ZHANG Tiyin;CHEN Xianfeng(Ningbo Academy of Inspection and Quarantine,Ningbo 315012,China;Yancheng Entry-exit Inspection and Quarantine Bureau,Yancheng 224002,China;Hubei Academy of Inspection and Quarantine,Wuhan 430050,China;Fujian Entry-Exit Inspection and Quarantine Bureau,Fuzhou 350001,China)

机构地区：[1]宁波检验检疫科学技术研究院,浙江宁波315012 [2]盐城出入境检验检疫局,江苏盐城224002 [3]湖北检验检疫科学技术研究院,湖北武汉430050 [4]福建出入境检验检疫局,福建福州350001

出　　处：《畜牧与兽医》2019年第1期80-84,共5页Animal Husbandry & Veterinary Medicine

基　　金：质检总局科研项目(2017IK276);宁波科技创新团队(2015C110018)

摘　　要：为建立新型高灵敏度的幼虫芽孢杆菌微滴式数字PCR (droplet digital PCR,ddPCR)检测方法,针对幼虫芽孢杆菌16S rRNA基因设计特异性引物和探针。通过对反应体系优化,建立幼虫芽孢杆菌ddPCR检测方法,并对方法的灵敏度、线性关系、特异性及重复性进行评估。结果:建立的方法与其他6株非幼虫芽孢杆菌无交叉反应,具有良好的特异性;线性关系良好(R^2=0. 991 4);灵敏度为2. 9 copies/μL; ddPCR检测的相对标准偏差(RSD)在0. 93%～5. 27%,重复性好。试验表明本研究建立的ddPCR方法可以对幼虫芽孢杆菌进行绝对定量检测。This study was to establish a droplet digital PCR (dd PCR) for detection of Legionella pneumophila. Primers and probes were designed for dd PCR according to the 16 S rRNA gene. The dd PCR condition and its reaction system were optimized simultaneously. The dd PCR was evaluated for linearity,specificity,sensitivity and repeatability. The results showed that the high specificity of these primers and probes were evaluated by using 6 non-targeted bacteria. The detection limits of dd PCR were 2. 9 copies/μL. The method had a high degree of linearity (R^2= 0. 991 4). The relative standard deviation (RSD) of dd PCR (0. 93%-5. 27%) was of high repeatability. The dd PCR was of high sensitivity and specificity and might be used to quantitatively detect Paenibacillus larvae.

关 键 词：幼虫芽孢杆菌 微滴式数字PCR 检测 

分 类 号：S852.61[农业科学—基础兽医学]