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目的以氧氟沙星为例,探讨阈值的划分对实验室早期检测出耐氧氟沙星结核分枝杆菌的影响。结合DNA序列分析探讨gyrA基因点突变与结核分枝杆菌对氧氟沙星耐受程度的相关性。方法 (1)在含氧氟沙星改良罗氏培养基上检测101株结核分枝杆菌临床分离株对氧氟沙星的敏感性。(2)测序以检测耐氧氟沙星结核分枝杆菌gyrA基因点突变的特征。结果 (1)52株耐氧氟沙星5.0μg/ml的临床分离株中,17株能在含氧氟沙星10.0μg/ml的改良罗氏培养基上生长(约占32.69%),49株对氧氟沙星5.0μg/ml敏感的临床分离株中,有2株能在含氧氟沙星2.0μg/ml的改良罗氏培养基上生长(约占4.08%)。(2)基因扩增产物测序发现;耐氧氟沙星结核分枝杆菌gyrA基因点突变主要发生第90、91和94位点上,有10株在不同的gyrA基因位点上发生了碱基缺失或插入的变异,101株被检测的结核分枝杆菌临床分离株中,发生在第95位点的碱基突变率为100%。结论 (1)判定耐药结核分枝杆菌上、下限的阈值对耐药结核菌的早期检出具有重要的作用。(2)发生在耐氧氟沙星结核分枝杆菌gyrA基因上的点突变是多变的,但与结核分枝杆菌对氧氟沙星的耐受程度没有明显的相关性。
Objective To use ofloxacin as an example to study the influence of thresholds on the early detection of Mycobacterium tuberculosis resistant to ofloxacin in laboratory. DNA sequence analysis of gyrA gene point mutation and the relationship between M. tuberculosis tolerance of ofloxacin. Methods (1) To detect the susceptibility of 101 clinical isolates of Mycobacterium tuberculosis to ofloxacin on the modified Roche medium containing ofloxacin. (2) sequencing to detect the characteristics of the point mutation in the gyrA gene of the Mycobacterium tuberculosis resistant to ofloxacin. Results (1) Among 52 clinical isolates of 5.0 ofloxacin 5.0μg / ml, 17 strains were able to grow on modified Roche medium containing about 10.0μg / ml ofloxacin (about 32.69%), and 49 Of the clinical isolates susceptible to ofloxacin 5.0 μg / ml, 2 were able to grow on modified Roche medium containing about 2.0 μg / ml ofloxacin (about 4.08%). (2) Sequencing of gene amplification products found that the point mutations of gyrA gene of Mycobacterium tuberculosis were mainly occurred on the 90th, 91th and 94th spots, and 10 of them had bases at different gyrA loci In the 101 strains of Mycobacterium tuberculosis clinical isolates that were deleted or inserted, the mutation rate at the 95th base was 100%. Conclusion (1) It is important to determine the upper and lower thresholds of drug-resistant Mycobacterium tuberculosis to early detection of drug-resistant Mycobacterium tuberculosis. (2) Point mutations in the gyrA gene of Mycobacterium tuberculosis resistant to ofloxacin are variable, but have no significant correlation with the tolerability of ofloxacin in Mycobacterium tuberculosis.