摘要
目的 建立简单的杂交方法特异地检测聚合酶链反应 (PCR)扩增的结核杆菌 DNA。方法 用玻璃粉提纯结核分枝杆菌 DNA,d UTP-脲 DNA糖基化酶 (UDG)防污染 ,用 5′端标记生物素的引物进行 PCR扩增 ,5′端带标记的扩增产物与固定在微孔板上特异探针杂交 ,然后用酶标链霉亲和素进行酶联免疫法检测。结果 对 10 0份来自结核病人高度怀疑有结核分枝杆菌的痰标本检测 ,扩增杂交法检出 40份阳性 ,比抗酸染色法 (15 / 10 0 ) ,培养法 (2 8/ 10 0 )和 PCR电泳法 (35 / 10 0 )检出率高 ,而 5 0份来自无结核感染者的痰标本 ,几种方法检查均为阴性。结论 该方法可灵敏、特异、客观地检测临床标本中的结核分枝杆菌 ,比传统 PCR—电泳法优越。
Objective To develop a simple hybridization assay for objective detection of mycobacterium tuberculosis from clinical specimens.Methods DNA template purified by using glass-powder absorption method was amplified by the specific primers of which one was labeled with biotin at 5′ end.Then,PCR products were detected by hybridization with capture probes precoated onto maxisorp microwellplate and the hybrids were detected in manner similar to ELISA.Results The method developed by us could detect target bacterium with higher sensitivity and specificity comparing with the conventional PCR electrophoresis method.100 sputum specimens which may be contain mycobacterium tuberculosis were used to evaluate the method.The results demonstrated that 40 of 100 sputum specimens were positive by PCR ELISA method while only 15/100 and 28/100 by fast staining and culture respectively,and 35/100 by PCR electrophoresis.Negative result was found in 50 sputum specimens without mycobacterium tuberculosis by the methods.Conclusion Through the optical densities,the method developed by us can objectively detect mycobacterium tuberculosis clinical specimens with high specificty and sensitivity.
出处
《四川医学》
CAS
2000年第4期299-301,共3页
Sichuan Medical Journal
关键词
结核分枝杆菌
DNA
微孔杂交
比色检测
Mycobacterium tuberculosis Amplification Coating Hybridization
