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目的 建立一种灵敏、特异、快速检测结核分支杆菌耐药相关基因突变的方法 ,用于临床对利福平耐药的快速诊断。方法 将 1 4条特异性探针固定在尼龙膜上 ,通过在下游引物标记生物素的方法得到生物素标记的结核分支杆菌DNAPCR扩增产物 ,与固定在尼龙膜上的特异性探针杂交 ,杂交物通过链酶亲和素标记辣根过氧化物酶及底物 (四甲基联苯胺 )显色判定结果。将杂交结果与基因测序结果及药敏试验结果进行对比分析。结果 采用逆向点杂交法共检测 2 3株耐利福平及1 1株敏感型菌株 ,与药敏试验结果、测序结果符合率分别为 2 8/ 34和 30 / 34。所发现突变类型依次为 :第 531位突变 1 1株 ,第 52 6位突变 7株 ,第 533位突变 3株。未检测到第 51 6位、第 51 3位碱基突变。结论 该方法可用于临床结核分支杆菌对利福平耐药性的快速检测
Objective To establish a sensitive, specific and rapid detection of Mycobacterium tuberculosis resistance-related gene mutations for rapid clinical diagnosis of rifampicin resistance. Methods A total of 14 specific probes were immobilized on nylon membrane. The biotin-labeled Mycobacterium tuberculosis DNA PCR products were obtained by labeling the biotin in the downstream primer and hybridized with the specific probe immobilized on the nylon membrane. Hybrids were detected by streptavidin horseradish peroxidase and substrate (tetramethylbenzidine) color determination results. The results of hybridization and gene sequencing and susceptibility test results were compared. Results Twenty-three strains of rifampin-resistant and 11 strains of susceptible strains were detected by reverse dot blot hybridization. The coincidence rates with susceptibility test results and sequencing results were 28/34 and 30/34, respectively. The types of mutations found were: the first 531 mutations 11, the 526th mutation 7, the 533th mutation 3. No 51st and 6th base mutations were detected. Conclusion This method can be used for rapid detection of rifampicin resistance in clinical Mycobacterium tuberculosis