CAJ | 학술논문

目的利用DNA芯片检测技术直接检测痰标本中结核分枝杆菌耐利福平(RFP)、异烟肼(INH)相关的耐药基因(rpoB、katG/inhA),评价DNA芯片检测技术临床应用的可行性.方法对586份涂阳痰标本使用L-J培养并用终点法确定其耐药性,同时利用DNA芯片检测技术检测痰标觋本中结核分枝杆菌的rpoB、katG/inhA常见基因突变位点的突变情况,比对两种方法的检测结果,对不符合的菌株测定其相应DNA序列,评估上述试验的准确性.结果 (1)586份涂阳痰标本,其中3(+)163份、2(+)204份、1(+)217份,培养阳性584份.耐药结果显示,对INH、RFP敏感的菌株分别为361株和327株,耐药菌株分别为223株和247株,其中低浓度耐药、高浓度敏感菌株分别为93株和59株,低浓度、高浓度均耐药菌株分别为130株和188株.(2)耐药基因特异性片段扩增阳性标本367份(62.8%)、阴性217份(37.2%).对INH耐药相关基因(katG/inhA)突变检出率是28.4%,突发发生位点集中在katG315位密码子(89.8%);对RFP耐药相关基因(rpoB)突变检出率是55.9%(137/247),突变发生位点主要在rpoB 531和rpoB 526位密码子,发生率分别是68.6%和16.1%.(3)对L-J药敏结果与DNA芯片检测结果不符的菌株进行DNA序列分析,发现有漏检现象.结论 DNA芯片技术直接检测样本中结核分枝杆菌的相关耐药基因存在可行性,如直接应用于临床样本检测,关键要解决样本中DNA的提取效率、PCR的扩增效率和试验的质量控制.
목적 이용DNA심편검측기술직접검측담표본중결핵분지간균내리복평(RFP)、이연정(INH)상관적내약기인(rpoB、katG/inhA),평개DNA심편검측기술림상응용적가행성.방법 대586빈도양담표본사용L-J배양병용종점법학정기내약성,동시이용DNA심편검측기술검측담표격본중결핵분지간균적rpoB、katG/inhA상견기인돌변위점적돌변정황,비대량충방법적검측결과,대불부합적균주측정기상응DNA서렬,평고상술시험적준학성.결과 (1)586빈도양담표본,기중3(+)163빈、2(+)204빈、1(+)217빈,배양양성584빈.내약결과현시,대INH、RFP민감적균주분별위361주화327주,내약균주분별위223주화247주,기중저농도내약、고농도민감균주분별위93주화59주,저농도、고농도균내약균주분별위130주화188주.(2)내약기인특이성편단확증양성표본367빈(62.8%)、음성217빈(37.2%).대INH내약상관기인(katG/inhA)돌변검출솔시28.4%,돌발발생위점집중재katG315위밀마자(89.8%);대RFP내약상관기인(rpoB)돌변검출솔시55.9%(137/247),돌변발생위점주요재rpoB 531화rpoB 526위밀마자,발생솔분별시68.6%화16.1%.(3)대L-J약민결과여DNA심편검측결과불부적균주진행DNA서렬분석,발현유루검현상.결론 DNA심편기술직접검측양본중결핵분지간균적상관내약기인존재가행성,여직접응용우림상양본검측,관건요해결양본중DNA적제취효솔、PCR적확증효솔화시험적질량공제.
Objective To detect the related genes with rifampin and isoniazid in Mycobacterium tuberculosis in sputums using the DNA chip technique and evaluate the fensibility of the clinical application of the DNA chip technique.Methods 586 sputum smear specimen was detected using the L-J cultivation to determine their drug resistance.Simultaneously.DNA chip was employed to detect the mutation of the frequent mutable points rpoB,katG/inhA in mycobaeterium tuberculosis isolates.These two assays were compared and samples showing discrepancy were chosen for additional sequencing to evaluate the accuracy of the detection.Results(1)There were 584 culture positive sputum smear specimens including 3(+)163 specimens,2(+)204 specimens,and 1(+)217 specimens.The drug fast results displayed that 361 strains were sensitive to INH,223 strains tolerated INH in which 93 strains tolerated it in low concentration while sensitive to it in high concentration.and 130 strains tolerated it in both low and high concentration.While 327 strains were sensitive to RFP.247 strains tolerated RFP in which 59 strains tolemted it in low concentration while sensitive to it in high concentration,and 188 strains tolerated it in both low and high concentration.(2)There were 367 positive strains(62.8%)and 217 negative strains(37.2%)identified by PCR amplification of the specific resistance gene fragments.The detection rate of the katG/inhA was 28.4%,and the mutation sites were mainly focused on the katG315(89.8%).The detection rate of the rpoB was 55.9%(137/247),and the mutation sites were mainly focused on rpoB531(68.6%)and rpoB 526(16.1%).(3)The sequencing of sample,which showed discrepancy with L-J cultivation and the DNA chip confirm a certain omission ratio.Conclusions It is feasible to detect the related resistant genes in Mycobacterium tuberculosis isolates using the DNA chip technique.The key factor is to raise the efficiency of the DNA extraction,the effciency of the PCR and the quality control of the experiment to facilitate its clinical application.