应用不同引物检测耐氧氟沙星结核分枝杆菌gyrA基因点突变对结果判定的影响  

Effect of detecting gyrA gene point mutation of ofloxacin-resistant Mycobacterium tuberculosis on the result by using different primers

作　　者：张健源[1,2] 程君[1,2] 任卫聪[1,2] 刘毅[1,2] 赵雁林[3] 孙勇[1,2] 王伟[1,2] 李传友[1,2] 

机构地区：[1]北京市结核病胸部肿瘤研究所 [2]首都医科大学附属北京胸科医院细菌免疫学研究室,101149 [3]中国疾病预防控制中心结核病预防控制中心国家结核病参比实验室

出　　处：《中华临床医师杂志（电子版）》2013年第15期116-120,共5页Chinese Journal of Clinicians(Electronic Edition)

基　　金："十二.五"国家科技重大专项(结核快速诊断和鉴别诊断试剂的研制)(2013ZX10003002-004)

摘　　要：目的探讨以PCR-SSCP技术为基础,应用不同引物检测耐氧氟沙星结核分枝杆菌gyrA基因点突变以及固定/终止液的组成对判定结果可能会产生的影响。方法采用2对引物,经PCR-SSCP检测,利用银染显色的方法显现耐氧氟沙星结核分枝杆菌gyrA基因点突变的DNA单链位移。结果对比标准株H37Rv及H37Ra gyrA基因的单链带位置,在被测试的89株菌株中发生位置变化的;对氧氟沙星的耐药程度>10μg/ml的分别有4株与14株,约占89株检测总数的4.49%和87.5%,对氧氟沙星的耐药程度介于≥2且≤10μg/ml之间的26株中,有与标准株gyrA基因的单链带位置发生改变的分别有20株与7株,约占89株检测总数的22.47%和7.87%;对氧氟沙星的耐药程度<2μg/ml的47株中分别有2株与11株,约占89株检测总数的2.25%和12.36%。用含5.0%乙醇和4.5‰冰醋酸配比的固定/终止液可为利用银染法显现核酸凝胶分离DNA提供温和的反应环境,操作过程方便,观测效果较为理想。结论应用不同引物检测耐氧氟沙星结核分枝杆菌gyrA基因点突变会对结果的判定产生影响,采用含5.0%乙醇、4.5‰冰醋酸的固定/终止液可为应用非变性聚丙烯酰胺凝胶分离核酸检测的银染着色法提供温和的反应环境,并产生清晰可辨的被检测标本的DNA单链条带。Objective To explore possible impact to the determination results, on the basis of PCR-SSCP technique, using different primers detecting Mycobacterium tuberculosis resistant to ofloxacin gyrA gene mutation, and of the fixed/stop solution composition. Methods Using two different pairs of primers detected by PCR-SSCP testing, using silver staining method revealed DNA single strand displacement of Mycobacterium tuberculosis resistant to ofloxacin point mutation in gyrA gene. Results To be compared to the position with single stranded gyrA gene of the standard strains H37Rv and H37Ra, position changed among the tested 89 strains, resistant degree ~ 10 I.tg/ml of ofloxacin were 4 strains and 14 strains, accounting for about 4.49% and 87.5% of 89 strains; degree of resistance between ≥2 - 10 μg/ml to ofloxacin of 26 tested strains in single and standard strain gyrA gene with pos.ition change were 20 strains and 7 strains, respectively accounting for about 6.747% and 21.35% to 89 of the total detection, and the other 2 strains and 11 strains resistant to ofloxacin〈2 μg/ml of 47 strains, accounting for about 2.25% and 12.36% of the total number of 89 detecti. Conclusions The application of different primers to detect Mycobacterium tuberculosis resistant to ofloxacin gyrA gene point mutation will affect the determination results, the use of fixed stop solution containing ethanol 5.0% and glacial acetic acid 4.5‰ for the application of non-denaturing polyacrylamide coagulation gum isolated nucleic acid detection method provides a mild reaction environment for silver-colored dying and produce a legible single chain DNA samples to be detected with.

关 键 词：分枝杆菌 结核 氧氟沙星 银染色法 GYRA基因 

分 类 号：R440[医药卫生—诊断学]