基于微滴式数字聚合酶链式反应技术检测布鲁氏菌病患者血液中布鲁氏菌核酸的研究  

Study of detection of Brucella nucleic acid in blood of brucellosis patients based on droplet digital polymerase chain reaction

作　　者：刘海雯 徐玲 范玉 包桂英 陈俊杰 张天承 徐晴晴 赵鸿雁[3] 朴东日[3] 田国忠[3] 任锋[2] 姜海[3] Liu Haiwen;Xu Ling;Fan Yu;Bao Guiying;Chen Junjie;Zhang Tiancheng;Xu Qingqing;Zhao Hongyan;Piao Dongri;Tian Guozhong;Ren Feng;Jiang Hai(School of Public Health,Baotou Medical College,Inner Mongolia University of Science and Technology,Baotou 014060,Inner Mongolia,China;Beijing Youan Hospital,Capital Medical University,Beijing Institute of Hepatology,Beijing 100069,China;National Key Laboratory of Intelligent Tracing and Forecasting for Infectious Diseases,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Tongliao Institute of Mongolian Medicine,Tongliao 028000,Inner Mongolia,China)

机构地区：[1]内蒙古科技大学包头医学院公共卫生学院,内蒙古包头014060 [2]首都医科大学附属北京佑安医院,北京肝病研究所,北京100069 [3]中国疾病预防控制中心传染病预防控制所,传染病溯源预警与智能决策全国重点实验室,北京102206 [4]通辽市蒙医研究所,内蒙古通辽028000

出　　处：《疾病监测》2024年第6期751-755,共5页Disease Surveillance

基　　金：布鲁氏菌两种检测方法的验证(No.33021);北京市医管局“登峰”人才计划(No.DFL20221503);高层次公共卫生技术人才建设项目(No.学科带头人-02-13);2023年度北京市自然科学基金-昌平创新联合基金(No.L234046);内蒙古自治区卫生健康委员会科研计划项目(No.202201566)。

摘　　要：目的本研究旨在建立一种基于微滴式数字聚合酶链式反应(ddPCR)的检测方法,能够实现对布鲁氏菌病(布病)患者血液中布鲁氏菌核酸的定量与精准检测。方法以布鲁氏菌Bcsp31基因为靶基因,设计引物与探针并构建pUC57-Bcsp31重组阳性质粒,评价ddPCR方法检测布鲁氏菌的灵敏度、重复性与特异度。此外,收集疑似布病患者血液120份,采用ddPCR方法对样本中布鲁氏菌DNA载量进行定量检测。结果本研究建立的ddPCR检测布鲁氏菌的方法具有很高的灵敏性,最低检出限为100拷贝/μL,并且对布鲁氏菌的检测呈现出良好的特异性与准确性。120份血液样本中,ddPCR方法检出阳性103份,实时荧光定量聚合酶链式反应(qPCR)方法检出阳性52份。105份抗体阳性样本中,ddPCR方法与qPCR方法分别检出阳性93份和51份,阳性率分别为88.57%和49.52%。15份抗体阴性样本中,两种方法分别检测出阳性12份和1份。结论本研究建立了一种基于ddPCR方法的高灵敏度、高特异度的检测布鲁氏菌的方法,能够实现布病患者血液样品中布鲁氏菌核酸的定量与精准检测。尤其对于抗体阴性的疑似患者早期布病筛查具有重要意义。Objective To establish a detection assay based on digital droplet polymerase chain reaction(ddPCR)for the quantitative and accurate detection of Brucella nucleic acid in the blood of brucellosis patients.Methods By using Brucella Bcsp31 gene as the target gene,the primers and probe were designed and the recombinant positive plasmid pUC57-Bcsp31 was constructed.The sensitivity,repeatability and specificity of ddPCR assay for detection of Brucella were evaluated.In addition,120 blood samples were collected from suspected brucellosis patients,and ddPCR was used to quantitatively detect Brucella DNA load in the samples.Results The ddPCR assay established in this study had high sensitivity and the minimum detection limit was 100 copies/μL,and showed good specificity and accuracy for the detection of Brucella.Of the 120 blood samples tested,103 were positive by ddPCR,and 52 were positive by quantitative real-time PCR(qPCR).In 105 antibody positive samples,93 and 51 were detected to be positive by ddPCR and qPCR,and the positive rates were 88.57%and 49.52%,respectively.In the 15 antibody negative samples,12 and 1 samples were detected positive by the two assays,respectively.Conclusion This study established a detection assay with high sensitivity and high specificity for Brucella detection based on ddPCR,which can be used for the quantitative and accurate detection of Brucella nucleic acid in blood samples of brucellosis patients.It is of great significance for early screening of brucellosis in suspected patients with negative antibody test results.

关 键 词：布鲁氏菌 微滴式数字聚合酶链式反应 定量检测 

分 类 号：R211[医药卫生—中医学] R378.5