结核分枝杆菌双靶标环介导恒温扩增检测方法的建立与应用  

Development and use of loop-mediated isothermal amplification(LAMP)based on 2target genes for rapid detection of Mycobacterium tuberculosis

作　　者：杨幸贵 黄俊飞 陈依江[2] 肖子宇 汪小娟 郑雯琳 陈旭 刘英[2] 陈玮[2] 袁薇[2] 胡勇 李世军 YANG Xing-gui;HUANG Jun-fei;CHEN Yi-jiang;XIAO Zi-yu;WANG Xiao-juan;ZHENG Wen-lin;CHEN Xu;LIU Ying;CHEN Wei;YUAN Wei;HU Yong;LI Shi-jun(Public Health School,Guizhou Medical University,Guiyang 550025,China;Guizhou Center for Disease Control and Prevention;The Second Affiliated Hospital Affiliated with Guizhou University of Traditional Chinese Medicine)

机构地区：[1]贵州医科大学公共卫生学院,贵州贵阳550025 [2]贵州省疾病预防控制中心实验中心 [3]贵州中医药大学第二附属医院中心实验室

出　　处：《中国病原生物学杂志》2021年第5期513-519,共7页Journal of Pathogen Biology

基　　金：贵州省科技支撑计划项目(黔科合支撑[2019]2822号,黔科合支撑[2018]2762号);贵州省科技计划项目(黔科合基础[2019]1186号);贵州省科技创新人才团队专项资金项目(黔科合平台人才[2018]5606);贵州省高层次创新型人才培养项目(黔科合[2016]4021)。

摘　　要：目的建立基于双靶标环介导恒温扩增(LAMP)的结核分枝杆菌(MTB)检测技术并应用于临床。方法以结核分枝杆菌复合群(MTBC)插入序列IS6110和MTB mtp40基因为检测靶标,分别设计1套LAMP引物。通过引物浓度和反应条件优化,建立基于双靶标的检测方法(mtp40-LAMP和IS6110-LAMP),比较2种方法的灵敏度和特异性。应用建立的mtp40-LAMP和IS6110-LAMP方法与PCR、萋-尼氏抗酸染色法和分枝杆菌分离培养法,分别对55例患者的痰标本进行检测与鉴定,通过对其结果的比较分析评价mtp40-LAMP和IS6110-LAMP方法的实用性。结果灵敏度试验显示,mtp40-LAMP和IS6110-LAMP方法可检出MTB基因组DNA最低浓度为125fg/μl,比常规的PCR方法高10~100倍。mtp40-LAMP和IS6110-LAMP方法的特异性为100%,能检测MTB菌株并能从MTBC中鉴别MTB。痰标本检测显示,IS6110-LAMP与PNB鉴定结果一致性检验的Kappa值为0.881(P<0.01),mtp40-LAMP与TCH鉴定结果的Kappa值为0.887(P<0.01)。与PCR方法和萋-尼氏抗酸染色法检测结果比较,mtp40-LAMP和IS6110-LAMP方法敏感性更高。反应体系中加入MG指示剂可直接通过肉眼观察颜色变化进行结果判定,实现了快速闭管检测。从样本处理(35min)、扩增反应(40min)到结果验证(1~2min),整个检测过程80min内即可完成。结论基于LAMP技术建立的mtp40-LAMP和IS6110-LAMP双靶标检测方法敏感性高、特异性强,能够快速、准确地检测和鉴别MTB,可作为潜在的MTB快速筛查或诊断工具。Objective To establish loop-mediated isothermal amplification(LAMP)based on 2target genes for detection of Mycobacterium tuberculosis(MTB)and use it in clinical practice.Methods Two sets of LAMP primers,respectively targeting the insertion sequence(IS6110)of the MTB complex(MTBC)and the mtp40gene of MTB,were designed.Methods based on the 2target genes(mtp40-LAMP and IS6110-LAMP)were established after optimizing the primer concentrations and reaction conditions,and their sensitivity and specificity were evaluated.Fifty-five sputum samples from patients were tested,and MTB was differentiated using mtp40-LAMP,IS6110-LAMP,PCR,Ziehl-Neelsen staining,and mycobacterial culture.Results were analyzed to evaluate the practicality of both mtp40-LAMP and IS6110-LAMP.Results Sensitivity tests indicated that the limit of detection(LoD)was 125fg/μl of MTB genomic DNA for mtp40-LAMP and IS6110-LAMP,which was 10-100-fold higher than that of conventional PCR.mtp40-LAMP and IS6110-LAMP have excellent specificity(100%).The two established methods were able to detect MTB strains and to differentiate MTB from MTBC.The consistency with which IS6110-LAMP and PNB detected MTB in sputum samples was examined,and the Kappa value was 0.881(P<0.01).The Kappa value between mtp40-LAMP and TCH was 0.887(P<0.01).tomtp40-LAMP and IS6110-LAMP had a higher sensitivity than PCR and Ziehl-Neelsen staining.Results could be directly determined by observing the color change with the naked eye when the MG indicator was added to the mixture.Rapid closed-tube detection of MTB was achieved.In addition,the whole procedure,including sample processing(35min),isothermal amplification(40min),and confirmation of results(1-2min),could be completed within 80min.Conclusion The established methods of mtp40-LAMP and IS6110-LAMP,as sensitive and specific techniques based on 2target genes,were able to detect MTB quickly and accurately and could be used as a potential screening and diagnostic tool for MTB.

关 键 词：环介导恒温扩增 结核分枝杆菌复合群 结核分枝杆菌 颜色判定 

分 类 号：R378.911[医药卫生—病原生物学]