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中国防痨杂志 ›› 2019, Vol. 41 ›› Issue (6): 609-615.doi: 10.3969/j.issn.1000-6621.2019.06.005

• 论著 • 上一篇    下一篇

新一代微测序基因芯片的设计及其耐药性检测效果的初步研究

孙照刚(),张洪静,李自慧,孙琦,吕琳娜,潘丽萍,Sandy Gilbert,张宗德,许绍发,James Xia()   

  1. 101149 首都医科大学附属北京胸科医院 北京市结核病胸部肿瘤研究所(孙照刚、张洪静、李自慧、孙琦、吕琳娜、潘丽萍、张宗德、许绍发、James Xia);GenoSensor Corporation,USA(Sandy Gilbert、James Xia)
  • 收稿日期:2019-03-20 出版日期:2019-06-10 发布日期:2019-06-04
  • 通信作者: 孙照刚,James Xia E-mail:sunzg75@163.com;james.xia@genosensorcorp.com
  • 基金资助:
    北京市医院管理局临床医学发展专项经费(XMLX201812);首都卫生发展科研专项(首发)(2018-2Z-1042);首都卫生发展科研专项(首发)(2016-2-1043);首都临床特色应用项目重点项目(Z181100001718181)

Design of a next generation microsequencing gene microarray and preliminary study of its effect on drug resistance detection

Zhao-gang SUN(),Hong-jing ZHANG,Zi-hui LI,Qi SUN,Lin-na LYU,Li-ping PAN,Gilbert Sandy,Zong-de ZHANG,Shao-fa XU,Xia James()   

  1. Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China;GenoSensor Corporation,USA(Sandy Gilbert、James Xia)
  • Received:2019-03-20 Online:2019-06-10 Published:2019-06-04
  • Contact: Zhao-gang SUN,Xia James E-mail:sunzg75@163.com;james.xia@genosensorcorp.com

摘要:

目的 设计和评价微测序基因芯片检测结核分枝杆菌(MTB)耐药性的价值。方法 于北京结核病临床数据和样本资源库选取MTB实验室标准菌株(H37Rv)和109株MTB临床分离株,采用绝对浓度法进行表型药物敏感性试验(简称“表型药敏试验”)和基因测序法检测MTB耐药相关基因突变(包括inhAkatGrpsLgyrArrseisrpoBembB基因)。以荧光标记探针为基本检测原理设计制备微测序基因芯片,检测109株MTB临床分离株耐药基因突变,分析微测序基因芯片的检测效能。结果 通过表型药敏试验及基因测序检测,明确了109株MTB临床分离株耐药相关基因(inhAkatGrpsLgyrArrseisrpoBembB基因)突变位点及突变形式。微测序耐药检测基因芯片初期设计包含了katGinhArpoBgyrAembBrpsLrrseis基因的目前已报道的所有突变位点的全部已知突变形式,共计220条探针,并确认出67条优秀探针。采用包含67条探针的微测序耐药检测基因芯片对109株MTB临床分离株进行检测。结果发现,与测序结果相比,除检测embB基因突变的2条探针外,其余65条探针检测碱基突变形式的符合率均超过95%,其中42条探针检测的符合率达到100.00%。以测序结果为参照标准,基因芯片检测对embB基因、gyrA基因、inhA基因、katG基因、rpoB基因、rpsL基因、eis基因和rrs基因突变检测的敏感度分别为64.21%(61/95)、80.00%(8/10)、95.83%(23/24)、98.55%(68/69)、92.63%(88/95)、93.85%(61/65)、75.00%(3/4)和94.44%(17/18);特异度分别为92.86%(13/14)、100.00%(99/99)、97.65%(83/85)、87.50%(35/40)、85.71%(12/14)、63.64%(28/44)、100.00%(105/105)和98.90%(90/91);Kappa值分别为0.29、0.88、0.92、0.88、0.68、0.60、0.85、0.93。结论 本研究研发设计了一套包含67条探针微测序耐药基因检测芯片,经过初步验证后证明其检测MTB耐药相关基因突变的效能较好,对embB基因的耐药检测探针还需进一步优化,使之更好的满足临床需求。

关键词: 分枝杆菌, 结核, 结核, 抗多种药物性, 微生物敏感性试验, 基因, 突变, 芯片分析技术, 技术评估, 生物医学

Abstract:

Objective To design and evaluate the value of next generation of microsequencing gene microarray on detecting the drug resistance of Mycobacterium tuberculosis (MTB).Methods The standard laboratory strains of the MTB H37Rv and 109 clinical isolates selected from the Beijing tuberculosis data and clinical sample data base were tested the phenotypic drug susceptibility using the absolute concentration method, and the drug resistance-related gene mutation of MTB (including inhA, katG, rpsL, gyrA, rrs, eis, rpoB and embB) using the gene sequencing method. The fluorescent labeled probes were set as the fundamental testing principle to design and prepare the microsequencing gene microarray, and then the 109 MTB clinical isolates were detected to analyze the detection performance of the microsequencing gene microarray.Results Based on the results of phenotypic drug susceptibility test and gene sequencing test, the mutation sites and forms of drug resistance-related genes (inhA, katG, rpsL, gyrA, rrs, eis, rpoB and embB) in 109 clinical isolates were identified. The microsequencing gene microarray contained 220 probes of the mutations that had been reported in the above genes, and 67 predominant probes were identified. The microsequencing drug-resistance gene microarray containing 67 probes was used to detect the mutations in 109 clinical isolates. The results showed that, except for the two probes in embB gene, the remaining 65 probes had more than 95% coincidence rate in the base mutation pattern detection, compared with the sequencing results, even 42 probes of them reached a coincidence rate of 100.00%. Taking sequencing results as the reference, the sensitivity of gene microarray detection method for embB gene, gyrA gene, inhA gene, katG gene, rpoB gene, rpsL gene, eis gene and rrs gene were 64.21% (61/95), 80.00% (8/10), 95.83% (23/24), 98.55% (68/69), 92.63% (88/95), 93.85% (61/65), 75.00% (3/4) and 94.44% (17/18), respectively. The specificity of the above genes were 92.86% (13/14), 100.00% (99/99), 97.65% (83/85), 87.50% (35/40), 85.71% (12/14), 63.64% (28/44), 100.00% (105/105) and 98.90% (90/91), respectively. After statistical analysis (Kappa test), the Kappa values of the above genes were 0.29, 0.88, 0.92, 0.88, 0.68, 0.60, 0.85 and 0.93, respectively.Conclusion The microsequencing gene microarray for drug-resistance detection with a set of 67 probes had been designed and developed. After preliminary verification, it showed a good performance on the detection of MTB drug-resistant gene mutation. However, for the embB gene, the detection probes needs to be further optimized, to better fulfill the clinical requirement.

Key words: Mycobacterium tuberculosis, Tuberculosis, multidrug-resistant, Microbial sensitivity tests, Genes, Mutation, Microchip analytical procedures, Technology assessment, biomedical