MDA-PCR检测痕量结核分枝杆菌核酸方法的建立  

Establishment of MDA-PCR method for detecting trace amounts Mycobacterium tuberculosis nucleic acid

作　　者：周林甫 万群 赵钢 张红鸭 ZHOU Lin-fu;WAN Qun;ZHAO Gang;ZHANG Hong-ya(School of Medicine,Northwestern University,Xi'an 710068,Shaanxi,China;不详)

机构地区：[1]西北大学医学院,陕西西安710068 [2]空军军医大学西京医院神经内科,陕西西安710032 [3]深圳大学总医院神经内科,广东深圳518055

出　　处：《广东医学》2022年第6期702-706,共5页Guangdong Medical Journal

基　　金：国家重点研发计划项目(2016YFC0904500)。

摘　　要：目的建立基于多重置换扩增及PCR(MDA-PCR)技术检测痕量结核分枝杆菌(M.tb)核酸的方法。方法制备含有M.tb插入序列IS6110和IS1081的不同稀释浓度的质粒DNA标准品。MDA-PCR分别通过检测质粒DNA标准品和其他细菌标准株基因组DNA来验证该方法的敏感度及特异性。另外,通过对模拟结核性脑膜炎(TBM)患者脑脊液(CSF)标本检测,评价该方法检测临床标本的可行性。结果MDA-PCR方法对质粒DNA标准品IS6110、IS1081基因的最低检测下限分别为10、25拷贝,其检测敏感度是ST-PCR方法的40~100倍。另外,该方法对5种其他细菌标准株基因组DNA的检测结果均为阴性,无非特异性反应发生。MDA-PCR对模拟TBM患者CSF中M.tb IS6110及IS1081基因的最低检测下限均为1 CFU,其检测敏感度是ST-PCR的10^(4)~10^(5)倍。结论MDA-PCR方法可提高对CSF中痕量M.tb核酸的检测效率,为TBM早期诊断提供了一种新的研究策略。Objective To establish a method for detecting trace amounts of Mycobacterium tuberculosis(M.tb)nucleic acid based on multiple displacement amplification and PCR(MDA-PCR)technology.Methods The standard plasmids containing M.tb insert sequence IS6110 and IS1081 were diluted to different concentrations.The analytical sensitivity and specificity of MDA-PCR were tested using standard plasmid DNAs and genomic DNA extracts from five other bacterial strains.In addition,the feasibility of MDA-PCR for detection of simulated TBM CSF specimens was evaluated.Results When using standard plasmid DNAs,the MDA-PCR results showed that limits of detection of IS6110 and IS1081 were 10 copies and 25 copies,respectively.Compared with ST-PCR,MDA-PCR method could increase the detection sensitivity by around 40 to 100 times.Moreover,the specificity analysis showed the MDA-PCR did not detect the genomic DNA of five other bacterial strains,indicating high specificity.In addition,MDA-PCR was capable of detecting 1 CFU(Colony forming units)of M.tb from simulated TBM CSF specimens.Comparing with ST-PCR,the MDA-PCR could increase the detection sensitivity around 104~105 times for detection of M.tb from simulated CSF specimens.Conclusion The MDA-PCR method could improve the detection efficiency of trace M.tb nucleic acid in CSF,and provide a new research strategy for the early diagnosis of TBM.

关 键 词：多重置换扩增 PCR 结核性脑膜炎 脑脊液 

分 类 号：R740.2[医药卫生—神经病学与精神病学] R741.04[医药卫生—临床医学]