微滴式数字PCR定量检测伯氏考克斯氏体方法的建立及应用  

Establishment and Application of Droplet Digital PCR Method by Quantitative Determination of Coxiella Burnetii

作　　者：王亚鹏 曲瑶 张皓博 李嘉琪 焉鑫 孙淑芳[2,5] 孙翔翔 欧阳康[1] WANG Yapeng;QU Yao;ZHANG Haobo;LI Jiaqi;YAN Xin;SUN Shufang;SUN Xiangxiang;OUYANG Kang(Guangxi University,Nanning,Guangxi 530005,China;China Animal Health and Epidemiology Center,Qingdao,Shandong 266032,China;Shandong Agricultural University,Tai'an,Shandong 271000,China;Key Laboratory of Animal Bio-security Risk Early Warning,Prevention and Control(South),Ministry of Agriculture and Rural Affairs,Qingdao,Shandong 266032,China;Key Laboratory for Prevention and Control of Major Ruminant Diseases,Ministry of Agriculture and Rural Affairs,Qingdao,Shandong 266032,China)

机构地区：[1]广西大学,广西南宁530005 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]山东农业大学,山东泰安271000 [4]农业农村部动物生物安全风险预警及防控重点实验室(南方),山东青岛266032 [5]农业农村部反刍动物重大疫病防控重点实验室(东部),山东青岛266032

出　　处：《广西农学报》2024年第2期26-33,共8页Journal of Guangxi Agriculture

基　　金：国家重点研发计划项目(2022YFD1800702);现代农业(奶牛)产业技术体系建设专项(CARS-36)。

摘　　要：为建立伯氏考克斯氏体(Coxiella burnetii,Cb)微滴式数字PCR(Droplet Digital PCR, ddPCR)检测方法,研究以Cb IS1111基因为靶基因,并针对基因保守区域设计了特异性引物和探针,以使反应的条件更加完善,从而构建了ddPCR检测方法,并对该方法的特异性、敏感性及重复性作出了评估。结果显示,所采用的ddPCR方法最低可检测的浓度为0.52 copies/μL,敏感性较高;与动物布鲁氏菌、牛结核分枝杆菌、土拉杆菌、金黄色葡萄球菌和沙门氏菌无交叉反应,特异性强;组内重复和组间重复变异系数均小于10%,重复性好。使用本实验建立的方法检测了42份疑似感染的临床样品,检测结果与荧光PCR鉴定结果一致。表明建立的ddPCR方法的特异性强、敏感性高、重复性和准确性好,可用于Cb的快速检测和精准定量,对Cb感染的防控具有重要意义。In order to establish a Droplet Digital PCR(ddPCR)detection method for Coxiella burnetii(Cb),this study designed specific primers and probes for the conserved region of the Cb IS1111 gene to optimize the reaction conditions.The ddPCR detection method was constructed,and the specificity,sensitivity,and repeatability of the method were evaluated.The results showed that the ddPCR method used had a minimum detectable concentration of 0.52 copies/μL,indicating high sensitivity.It did not cross-react with animal Brucella,Mycobacterium bovis,Pseudomonas aeruginosa,Staphylococcus aureus,and Salmonella,demonstrating strong specificity.The intra-group and inter-group coefficients of variation were both less than 10%,indicating good repeatability.The method established in this experiment was used to detect 42 suspected infected clinical samples,and the detection results were consistent with those of fluorescence PCR identification.This suggests that the established ddPCR method has strong specificity,high sensitivity,good repeatability,and accuracy,making it suitable for the rapid detection and precise quantification of Cb,which is of great importance for the prevention and control of Cb infections.

关 键 词：伯氏考克斯氏体 Q热 微滴式数字PCR 临床检测 

分 类 号：S853[农业科学—临床兽医学]