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

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).

RESULTS

The sensitivity of the purefied DNA for PCR-electrophoresis was 1 picogram, that for PCR-probe was increased to 100 femtogram. In 24 species tested, only M. tuberculosis complex and M. xenopi DNA produced 245-bp amplification bands, but the band from M. bovis 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 infected 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.