交叉引物恒温扩增技术快速检测结核分枝杆菌复合群和非结核分枝杆菌系统的建立  

Establishment of a cross primer isothermal amplification technique for rapid detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria

作　　者：黄文滨 陈丽萍 张望 肖婷 张满娥[2] 吴定昌 Huang Wenbin;Chen Liping;Zhang Wang;Xiao Ting;Zhang Mane;Wu Dingchang(Department of Clinical Laboratory,the First Longyan Hospital Affiliated to Fujian Medical University,Longyan 364000,China;Department of Molecular Biology Laboratory,the Second Hospital of Longyan,Fujian Province,Longyan 364000,China;Ustar Biotechnologies(Hangzhou)Ltd.,Hangzhou 310053,China)

机构地区：[1]福建医科大学附属龙岩第一医院检验科,龙岩364000 [2]福建省龙岩市第二医院分子生物实验室,龙岩364000 [3]杭州优思达生物技术有限公司,杭州310053

出　　处：《中国防痨杂志》2023年第8期734-743,共10页Chinese Journal of Antituberculosis

基　　金：福建省检验医学研究会与国家(福建)基因检测技术应用示范中心联合研创课题(2023LHYC029)。

摘　　要：目的:建立一种快速检测结核分枝杆菌复合群(MTBC)和非结核分枝杆菌(NTM)的分子交叉引物恒温扩增技术(CPA)检测系统。方法:针对分枝杆菌高度保守的16S rRNA基因序列设计6套CPA引物和3个探针。以含16S rRNA基因序列质粒(浓度为1000拷贝/μl)为阳性模板,筛选确立最佳引物探针组合,并对建立的最佳反应体系进行敏感度、特异度和包容性分析。收集福建省龙岩市第二医院2022年2月8日至2023年3月14日具有肺结核症状或体征的125例疑似肺结核患者的标本,采用分枝杆菌核酸检测试剂盒(PCR-荧光探针法)和建立的CPA法对收集到的标本进行检测,并对两种方法间的检测结果一致性进行统计分析,采用Kappa值评估。Kappa值≥0.75,说明一致性好。结果:筛选出MTBC/NTM-16S-6引物和MTBC/NTM-16S-LR-FAM-3为分枝杆菌检测系统最佳引物探针组合,检测体系最低检测限为100拷贝/μl,检测时间为40 min,能检出MTBC和临床常见NTM,且与常见呼吸道细菌无交叉反应。与实时荧光定量PCR(RT-PCR)检测方法相比,阳性预测值为95.65%(66/69),阴性预测值为85.71%(48/56),符合率为91.20%(114/125),Kappa值为0.82。结论:成功建立了一种基于CPA技术的快速检测MTBC和NTM的检测系统,能够为基层医院结核病疫情防控提供一种新的检测方法。Objective:To establish a molecular cross primer isothermal amplification(CPA)detection system for rapid detection of Mycobacterium tuberculosis complex(MTBC)and nontuberculous mycobacteria(NTM).Methods:Six sets of CPA primers and three probes were designed for 16S rRNA-a highly conservative gene sequence of mycobacterium.Using a plasmid containing 16S rRNA gene sequence(with a concentration of 1000 copies/μl)as a positive template,the optimal primer probe combination was selected,and the sensitivity,specificity,and inclusiveness of the established optimal reaction system were analyzed.Samples of 125 presumptive tuberculosis patients with signs or symptoms of tuberculosis from the Second Hospital of Longyan,Fujian Province from February 8,2022 to March 14,2023,were collected and then detected by the mycobacterium nucleic acid detection kit(PCR-fluorescence probe method)and the established CPA method.The consistency of the test results between the two methods was statistically analyzed,setting Kappa value≥0.75 as having good consistency.Results:MTBC/NTM-16S-6 primer and MTBC/NTM-16S-LR-FAM-3 were selected as the best primer probe combinations for the mycobacterium detection system.The minimum detection limit of the detection system was 100 copies/μl,and the detection time was 40 minutes.It could detect MTBC and clinically common NTM,and show no cross reaction with other common respiratory tract bacteria.Compared with the real-time fluorescence quantitative PCR(RT-PCR)detection method,the positive prediction value of the established CPA system was 95.65%(66/69),the negative prediction value was 85.71%(48/56),the total consistency rate was 91.20%(114/125),and the Kappa value was 0.82.Conclusion:A rapid detection system of MTBC and NTM based on CPA technology was successfully established,which could provide a new detection method for the prevention and control of tuberculosis epidemic in primary hospitals.

关 键 词：核酸扩增技术 分枝杆菌 结核 分枝杆菌 非典型 

分 类 号：R378.91[医药卫生—病原生物学]