焦磷酸测序技术检测结核分枝杆菌四种药物耐药性  

Pyrosequencing for rapid detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis

作　　者：郑瑞娟[1,2] 秦莲花[1,2] 周燕[1,2] 王洁[1,2] 乐军[1,2] 胡忠义[1,2] 

机构地区：[1]同济大学医学院附属上海市肺科医院,上海200433 [2]上海市结核病(肺)重点实验室,上海200433

出　　处：《中华检验医学杂志》2011年第2期115-120,共6页Chinese Journal of Laboratory Medicine

基　　金：国家十一五“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项资助项目（2008ZXl0003-003）;上海市科委课题资助项目（10DZ2272300）;上海市科委医学重点项目资助课题（10411955100）;上海市卫生局科研计划课题资助项目（2007Y59）

摘　　要：目的 利用焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星的耐药性，并对其临床应用进行评价。方法 收集上海市肺科医院2008--2009年临床确诊的肺结核患者的痰标本培养阳性结核分枝杆菌89株。所有菌株按《结核病诊断实验室检验规程》进行分枝杆菌培养、菌种鉴定。另外10株已知药敏结果的结核分枝杆菌来自上海市肺科医院菌株库。利用焦磷酸测序技术，以10株已知药敏结果和经直接测序已知耐药基因突变情况的结核分枝杆菌为试验菌株，探索焦磷酸测序检测结核分枝杆菌rpoB、katG、gyrA、邢耐药基因的最佳模式，并用该条件检测89株结核分枝杆菌临床分离株，检测结果与Bactec960药敏法进行比较。结果 rpoB、gyrA基因检测宜采用焦磷酸测序序列分析模式，katG、rm基因检测宜采用焦磷酸测序单核苷酸多态性模式。以Bactec960药敏法测定结果为判断标准，则焦磷酸测序法检测89株结核分枝杆菌临床分离株对利福平、异烟肼、氧氟沙星、阿米卡星耐药性的敏感度分别为98．0％、64．1％、79．5％、78．3％，特异度分别为97．5％、100．O％、90．0％、100．0％，准确性分别为97．8％、77．5％、85．4％、94．4％，该法检测利福平、氧氟沙星、阿米卡星的检测结果与Bactee960药敏检测结果一致性较好，Kappa值均≥0．7。结论 焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星耐药性具有快速、准确、高通量的优点，是一种可对结核分枝杆菌多种药物耐药性进行快速检测的方法。Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikaein by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequeneing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates, when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid, ofloxacin and amikacin is 98.0%, 64. 1%, 79. 5%, 78.3% respectively, the specificity is 97.5%, 100.0%, 90.0% ,100.0% respectively, the accuracy is 97.8%, 77.5%, 85.4%, 94.4% respectively, tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to t

关 键 词：分枝杆菌 结核 抗结核药 序列分析 DNA 微生物敏感性试验 

分 类 号：R446.5[医药卫生—诊断学]