聚合酶链反应和DNA探针联合检测临床标本中结核杆菌的研究

Detection of Mycobacterium tuberculosis in clinical samples by PCR and DNA probe

目的为提高聚合酶链反应(PCR)检测未培养临床标本中结核杆菌的敏感性和特异性。方法首先通过琼脂糖凝胶电泳检测PCR扩增产物,然后用Southern法转移至膜上,与地高辛标记的人型结核杆菌DNA探针杂交。结果 PCR电泳检测的灵敏度为1pg,而DNA探针检测将灵敏度提高到100fg。24种受试菌株中,只有结核分支杆菌复合体和蟾分支杆菌有245hp扩增带,其中牛型结核分支杆菌与188bp探针不杂交。对225例临床标本进行涂片、PCR电泳和探针检测,51例非结核性标本三者均阴性;1例非结核分支杆菌感染者的痰标本涂片和培养均阳性,PCR电泳和探针检测均阴性。菌种鉴定为快生长分支杆菌;173例结核性标本三者阳性率分别为16.2%、37.6%和50.3%。此外,将PCR电泳图谱分为四型,并探讨了其影响因素。结论结果表明PCR是结核病早期诊断和鉴别诊断的一种有价值的检测手段,而与探针联合检测可提高阳性检出率,避免漏判和误判。

Objective To improve the sensitivity and specificity of polymerase chain reaction (PCR) for detecting M. tuberculosis in uncultural clinical samples. Methods PCR amplification products were identified with agarose gel electrophoresis (PCR-electrophoresis), then Southern blotted onto a membrane and hybridized with a digoxigenin-labeled M. tuberculosis DNA probe (PCR-probe). Resalts The sensitivity of the purefied DNA for PCR-electrophoresis was 1 picogram, that for PCRprobe was increased to 100 femtogram, tn 24 species tested, only M. tuberculosis complex and M. xenopi DNA produced 245-bp amplification bands, hut the band from M. boris did not hybridize with 188-bp digoxigenin-labeled probe. 225 clinical samples were examined by smear, PCR-electrophoresis and PCR-probe, 51 nontuberculous clinical samples were all negative, the positive rates of 173 tuberculous samples were 16.2%, 37.6% and 50.3%, respectively. The smear and culture of 1 sputum sample from a patient infectecd by nontuberculous mycobacteria were all positive, but its PCR-electrophoresis and PCR-probe were all negative. Identification of species showed that it was a quickly growing mycobacterium. Otherwise, the figures of electrophoresis were classified as four types, and influential factors were also discussed. Conclusions The results showed that PCR was a valuable detective tool for early diagnosis and differential diagnosis of tuberculosis, and PCR-probe could increase positive rate of the detection and avoid misjudgements of results.