首页|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

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

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

BrucellaDroplet digital PCRQuantitative detection

刘海雯、徐玲、范玉、包桂英、陈俊杰、张天承、徐晴晴、赵鸿雁、朴东日、田国忠、任锋、姜海

展开 >

内蒙古科技大学包头医学院公共卫生学院,内蒙古包头 014060

首都医科大学附属北京佑安医院,北京肝病研究所 北京 100069

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

通辽市蒙医研究所,内蒙古通辽 028000

展开 >

布鲁氏菌 微滴式数字聚合酶链式反应 定量检测

布鲁氏菌两种检测方法的验证北京市医管局"登峰"人才计划高层次公共卫生技术人才建设项目2023年度北京市自然科学基金-昌平创新联合基金内蒙古自治区卫生健康委员会科研计划项目

33021DFL20221503学科带头人-02-13L234046202201566

2024

疾病监测
中国疾病预防控制中心

疾病监测

CSTPCD北大核心
影响因子:1.567
ISSN:1003-9961
年,卷(期):2024.39(6)