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目的分析临床分离的耐多药结核分枝杆菌的3种二线抗结核药物[卷曲霉素(CAP)、卡那霉素(KAN)和氧氟沙星(OFX)]耐药相关基因的分子特征,及其与不同Spoligotyping基因型的相关性。方法对临床分离的128株耐多药结核分枝杆菌进行Spoligotyping分型,并对其5个基因区域(tly A、eis、rrs、gyr A和gyr B)进行聚合酶链反应测序和分析。结果在60.0%(6/10株)的CAP耐药和0.8%(1/118株)的CAP敏感菌株中存在rrs-A1401G突变;在84.6%(11/13株)KAN耐药菌株在rrs基因或eis启动区存在突变,其中有53.8%(7/13株)存在rrs-A1401G突变,23.1%(3/13株)存在eis-C(-10)A突变,7.7%(1/13株)存在eis-G(-14)T突变,3.5%(4/115株)的KAN敏感菌株存在eis-C(-12)T突变。在83.3%(53/60株)的OFX耐药菌株中存在gry A基因突变,最常见的突变发生在第94位密码子(37/60株,61.7%),其次为第90位密码子(12/60株,20.0%)。还有5.0%(3/60株)的菌株存在gry B基因突变。共有84.4%(108/128株)的菌株被鉴定为北京基因型,没有发现菌株的基因型与耐药相关突变位点间存在关联性。使用核酸测序检测CAP、KAN和OFX耐药的敏感度和特异度分别是60.0%和99.4%、84.6%和99.4%、90.0%和100.0%。结论耐多药菌株中最常见的基因突变位点是:rrs-1401、eis-10,以及gyr A-94、-90和-91。这些结果为结核分枝杆菌相关药物耐药性的快速检测提供了良好的基础。
Objective To analyze the molecular characteristics of three second-line anti-tuberculosis drugs [CAP, kanamycin and ofloxacin (OFX)] in clinical isolates of multidrug-resistant Mycobacterium tuberculosis , And their correlation with different Spoligotyping genotypes. Methods 128 clinical isolates of Mycobacterium tuberculosis were genotyped by Spoligotyping, and their 5 regions (tly A, eis, rrs, gyr A and gyr B) were sequenced and analyzed by polymerase chain reaction. Results There were rrs-A1401G mutations in 60.0% (6/10 strains) CAP resistant strains and 0.8% (1/118 strains) CAP sensitive strains. In 84.6% (11/13 strains) of KAN-resistant strains, the rrs gene There were mutations of rrs-A1401G in 53.8% (7/13 strains), eis-C (-10) A mutation in 23.1% (3/13 strains) and 7.7% (1/13 strains) in eis- There was eis-G (-14) T mutation and 3.5% (4/115) KAN-sensitive strains had eis-C (-12) T mutation. The gry A gene mutation was found in OFX-resistant strains of 83.3% (53/60 strains). The most common mutation occurred at codon 94 (37/60 strains, 61.7%), followed by codon 90 12/60 strains, 20.0%). There are 5.0% (3/60 strains) strains gry B gene mutation. A total of 84.4% (108/128) isolates were identified as Beijing genotypes. No association was found between the genotypes of the isolates and the resistance-related mutation sites. The sensitivity and specificity of using nucleic acid sequencing to detect CAP, KAN and OFX resistance were 60.0% and 99.4%, 84.6% and 99.4%, 90.0% and 100.0%, respectively. Conclusions The most common sites of mutations in multidrug resistant strains are: rrs-1401, eis-10, and gyr A-94, -90, and -91. These results provide a good basis for rapid detection of drug resistance in M. tuberculosis.