摘要
20世纪80年代以来，结核病(tuberculosis，TB)的疫情有重新上升的趋势，一个主要因素是结核分枝杆菌耐药性问题。随着联合药物治疗的使用，使高耐药及多耐药菌株不断出现和传播，对化疗效果及结核病的流行产生了极大影响，增加了死亡率。据世界卫生组织估计，目前全球约有20亿人感染结核分枝杆菌(Mycobacterium tuberculosis，MTB)，其中约有5000万人感染耐药结核分枝杆菌。不合理用药引起的结核分枝杆菌内一些酶的基因突变或缺失是产生致结核分枝杆菌耐药性的主要原因。耐药结核分枝杆菌，特别是多重耐药结核分枝杆菌的增加，严重影响了结核病的化疗效果，给结核病控制带来了极大的困难。传统的药敏试验是建立在生物生长代谢过程中的表型上，这种方法所需时间长，通常为1～2个月，不能满足现代短程化疗的需求。因此，结核分枝杆菌耐药性的快速准确检测，为尽快制定最佳治疗方案提供依据，将有效地降低耐药菌株在人群中的扩散。
近年来，由于分子生物学技术的发展，一线抗结核药物的耐药分子机制逐步被阐明，也相应地建立了一些用基因技术对结核分枝杆菌耐药性进行检测的方法，为快速检测结核分枝杆菌的耐药基因开辟了一条新的途径。目前较为成熟的基因诊断方法有DNA直接测
第四军医大学硕士学位论文
序法归ireet Sequencing，Ds)、杂交法、多聚酶链反应一单链构象多态
性分析(polymerase Chain Reaetion一Single一strand Conformation
polymo甲hism，pCR一ssCp)法等。而PeR一sseP又以其操作简便，
费用较低和快速敏感等优点，更适合在临床推广使用。
多耐药性结核病(Multiple一Drug Resistanee几berulbosis，
MDR一TB)是指对2种或2种以上抗结核药物耐药的结核分枝杆菌
引起的结核病，它是人类消灭结核病的一大障碍。利福平(rifampin，
RFp)、链霉素(strepto娜cin，sM)和异烟脐(isoniazid，INH)
是主要的抗痔一线药物，其耐药性的出现对感染患者的治疗带来很
大困难。建立一种敏感、特异并且能同时检测多个药物耐药性的方
法，成为临床迫切需要解决的问题，也是目前国内外研究的热点。
目前虽有PCR-SSCP检测耐药基因的报道，但均为单一基因的检测，
从而使目前标准的化疗方案治疗失败。为此，本研究的主要目的为:
建立一种同时检测孙P、SM和E，H主要耐药基因rpoB、rpsL、katG
基因突变的方法，为临床早期治疗提供快速而准确可靠的方法及依
据。
首先建立检测3个耐药基因的multi一PCR方法。已经证实，RFP
耐药性的产生主要是由于编码RNA聚合酶的p亚单位的编码基因
印oB发生突变，大约90%一95%的突变主要发生在rp0B基因中央
的81饰区域内(507一533位27个氨基酸密码子)。编码结核分枝杆
菌核糖体16srRNA的rrs基因与512蛋白质的印sL基因发生突变导
致耐链霉素。印sL基因突变是耐sM的主要分子机制，rpsL基因突
变占SM耐药株的60%一70%，其突变多见于43，88位点。刀呵H耐
药主要与过氧化氢酶一过氧化物酶的编码基因katG有关，还与即hC
启动子、ixlllA基因的突变有关。K叔G基因突变占D妊J耐药株的50
%一70%，主要发生在315、304位点。基于此，本实验在设计PCR
特异性引物时涵盖了这3个基因突变的高发区域。rpoB基因特异性
引物根据Genebank:L27989序列设计:P15’一CCC AGGACGTGG
第四军医大学硕士学位论文
AGG CGA TCAC一3’;P25’一TGC CGC ACC AAI，CGC TGC TC一3’，
扩增片段365bp。;印sL基因特异性引物根据Genebank:L08011序
列设计:P，5’一CCG CGT GII’A CAC CAC CAC TC一3’;P25’一CTA
o认Goe oee AAo eGA一3’，扩增片段Zolbp。kato基因特异性弓}
物根据Genebank:X68081序列设计:P15’一CGG CGATGAGCG
TTA CAG一3’;P:5’一CGT CCT TGG CGG TGT ATTG一3’，扩增片段
458bP。
实验中对multi一PcR反应体系中退火温度、M犷+浓度、模板DNA
用量、多重引物用量、循环次数多面进行优化，选择最佳反应条件，
成功建立了multi一PCR扩增方法。对H37Rv标准株和临床分离株分
别采用常规PCR和multi一PCR同时进行扩增，结果两种扩增方法均
能扩增出预期的目的片段，符合率达100%。
按绝对浓度间接法进行药敏试验。78株临床分离株中，敏感株
22株，耐药株56株，耐多药株51株。其中，耐利福平46株，高耐
药株33株，低耐药株13株;耐链霉素43株，高耐药株34株，低
耐药株9株;耐异烟腆45株，高耐药株35株，低耐药株10株。
用单基因PCR一SSCP法分别扩增结核分枝杆菌H37Rv和78株临
床分离株的rp0B、印sL、katG基因，并将扩增产物进行sscP电泳
分析。共检出48株突变株，其中耐药株中检出47株(83.9%)，敏
感株中检出1株(4 .5%)。印OB基因突变发生率为87%(40/46)，
另检出1株敏感株突变;rpsL基因突变发生率为74.4%(犯/43);
katG基因突变发生率为“.7%(30/45)。
将56株耐药临床株和1株PCR一SSCP法检出突变的敏感株的扩
增产物切下进行胶回收，然后进行测序分析。共检出50株突变株，
其中1株即为ssCP法检出突变的敏感株。检出rpoB基因突变发生
率89.1%(4一/46);印sL基因突变发生率74.4%(犯/43);katG基
因突变发生率68.9%(31/45)。
The epidemic of tuberculosis got a recovery from 1980s in last century, because of the drug resistance of mycobacterium tuberculosis. The epidemic situation and chemotherapy effect experienced a great change result from multidrug resistant and high resistant strains occurred or transmitted. WHO estimate 2 billion individuals or so infected with mycobacterium tuberculosis in the world and about 50 million in them caused by resistant strains now. Most resistant strains related to the gene mutation or deletion of some enzymes in mycobacterium tuberculosis owing to unreasonable chemotherapy. Those strains, especially the increase of multi-drug resistant organisms, led negative impact on chemotherapy effect and brought trouble to control tuberculosis. The traditional drug sensitive test, which based on the mechanism of microorganism metabolism cost time usually from 1 to 2 mouths and can not meet the requirement from modern short course chemotherapy, so the rapid and accurate detecting of drug resistance in mycob
acterium tuberculosis will be provide gist for establish the best chemotherapy project and effective lower the diffusion of drug-resistant strains in crowed.
Along with the development of molecular biology recently, the
mechanism of drug resistance of the basic drugs to treat TB were established step by step, and develop some detections of mycobacterium tuberculosis using of gene technique, which opened a new way for the test of resistant strains rapidly. Now some better methods includes direct sequencing, hybridization and Polymerase Chain Reaction-Single- Strand conformation polymorphism but the last one is more optional for clinic application because of convenience, low cost and sentivity.
Multi-drug resistance tuberculosis, which is resistant to more than two anti- tuberculosis drugs, threw the shadow on our determination of elimination tuberculosis. The resistant of the most commonly used anti-tuberculosis drugs, mostly includeing rifampin, streptomycin and isoniazid, brought much trouble for patient. It is urgent for clinic to establish a sensitive and sepecific protocol aimed at multi-drug resistant detection at the same time.Although there were reports about the PCR-SSCP detection of drug- resistant genes now, failing to detect the majority of the drug-resistant genes at the time. This leads an ineffective treatment with standard chemotherapy. For this, the main research objective of the theme is as follows: to develop a method in order to test RFP, SM and IHN resistant gene mutation named rpoB, rpsL and katG respectively at the same time, and provide rapid , nicety and trusty means for early of clinic theraty.
First, to develop the multi-PCR technique. It has been verified that RFP resistance result from the mutation of rpoB which encode subunit in RNA polymerase and about 90%~95% mutation occurred at the 81bp region (507~533 site 27 amino acid codons) in the center of rpoB gene. The mutation of rrs gene encode 16SrRNA and rpsL gene encode S12 protein lead resistant to streptomycin. The mutation of rpsL, 60%~
70% occupied SM-resistant strains, usually happened at the site of 43 and 88, is the major molecular mechanism for SM resistant. The katG gene of the encoded hydrogen oxide - peroxidase is the main reason that leads to the resistance of mycobacterium tuberculosis to isoniazid, and associated with the mutation of genes of the inhA and aphC promoter. KatG gene mutation 50% ~70% occupied INH-resistant strains, mostly occurred at the site of 315 and 304. For this purpose, the experiment designed the specific primers to cover as much as possible the mutation sites for the drug-resistant genes. The specific primer of the rpoB gene according to the Genbank: L27989, P1 5'-GAT CAA GAG TCA GAC GGT GTT C-3'; P2 5'-ACG GTG TTG TCC TTC TCC AG-3', the extended section was 365bp; The specific primer of the rpsL gene according to the Genbank: L08011, P, 5'-ACA CCA CCA CTC CGA AGA AG-3'; P25'-TGC GTA TCC AGC GAA CCG-3', the extended section was 201bp; The specific primer of the katG gene was self-designed
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