炭疽芽孢杆菌微滴式数字PCR定量检测方法的建立  

Establishment of droplet digital PCR detection method for Bacillus anthracis

作　　者：张炜煜[1] 张立夫[1] 聂丹丹 王艳秋[1] 姚佳彤 赵逸 王岙[1] ZHANG Weiyu;ZHANG Lifu;NIE Dandan;WANG Yanqiu;YAO Jiatong;ZHAO Yi;WANG Ao(Jilin Provincial Center for Disease Control and Prevention(Jilin Provincial Institute of Public Health),Changchu 130062,China;Changchun Customs TechnologyCenter,Changchu 130062,China)

机构地区：[1]吉林省疾病预防控制中心(吉林省公共卫生研究院),吉林长春130062 [2]长春海关技术中心,吉林长春130062

出　　处：《中国实验诊断学》2025年第1期67-73,共7页Chinese Journal of Laboratory Diagnosis

基　　金：“十三五”国家重大专项“艾滋病和病毒性肝炎等重大传染病防治”(2018ZX10734401-010)。

摘　　要：目的建立炭疽芽孢杆菌的微滴式数字聚合酶链式反应(Droplet digital PCR,ddPCR)方法对炭疽芽孢杆菌实验室活动污染的定量评估提供技术支持。方法以炭疽芽孢杆菌pXO1质粒编码保护性抗原pagA基因为靶序列,优化微滴数字PCR方法的反应条件,建立实验室微环境中炭疽芽孢杆菌核酸定量方法;对比微滴式数字PCR方法和平板计数法的定量评估效果,分析ddPCR的灵敏性、特异性和重复性。结果建立的ddPCR方法最佳引物和探针终浓度分别为900nmol·L^(-1)和250nmol·L^(-1),最佳退火温度为60℃,最佳升降温速度为1℃/s,本方法的最低检测下限为1.12copies·μL^(-1),未发现与常见疫病存在交叉反应,重复性试验的变异系数小于5%。结论本研究中建立的炭疽芽孢杆菌的微滴数字PCR方法敏感性高、特异性强、重复性好,为疫情监测、流行病学调查和实验室污染微环境检测提供重要技术。Objective In order to establish droplet digital PCR(ddPCR)detection method for Bacillus anthracis,o provide technical support for the quantitative assessment of laboratory activity pollution caused by Bacillus anthracis.Methods Using the pXO1plasmid encoding the protective antigen pagA gene of Bacillus anthracis as the target sequence,optimize the reaction conditions of the droplet digital PCR method,and establish a quantitative method for Bacillus anthracis nucleic acid in the laboratory microenvironment;Compare the quantitative evaluation results of micro drop digital PCR method and plate counting method,and analyze the sensitivity,specificity,and repeatability of ddPCR.Results The optimal primer and probe final concentrations for the established ddPCR method are 900nmol·L^(-1) and 250nmol·L^(-1),respectively.The optimal annealing temperature is 60℃,and the optimal temperature rise and fall rate is 1℃/s.The minimum detection limit of this method is 1.12copies·μL^(-1),no cross reactivity with common diseases was found,and the coefficient of variation in the repeatability test was less than 5%.Conclusion The digital PCR method for Bacillus anthracis established in this study has high sensitivity,specificity,and repeatability,providing important technology for epidemiological investigation,and laboratory pollution microenvironment detection.

关 键 词：炭疽芽孢杆菌 微滴式数字PCR 平板计数 定量评估 核酸检测 

分 类 号：S85[农业科学—兽医学]