[Study on detection of the Mycobacteria DNA in formalin-fixed, paraffin-embedded tissue samples by triplex polymerase...

To supply an additional differential diagnostic method for pathological diagnosis of Mycobacterium tuberculosis complex and nontuberculous mycobacteria infections in formalin-fixed, paraffin-embedded tissue samples by triplex-PCR. Three pairs of oligonucleotide primer were used in triplex-PCR. A 383 bp DNA fragment encoding part of the 65 kD mycobacterial surface antigen, a 123 bp fragment corresponding to a specific Mycobacterium tuberculosis complex sequence which was the insertion sequence 6110 (IS6110) and a 268 bp fragment for human beta-globin were amplified by triplex-PCR respectively. The sensitivity of the triplex-PCR-electrophoresis for the mycobacteria DNA was 0.6 picogram. The specific bands of 383 bp and 123 bp among the amplified DNA from Mycobacterium tuberculosis, M. bovis, M. bovis BCG and M. simiae were present in the agarose gel. By contrast, only a band of 383 bp was found among the nontuberculosis mycobacteria which contained M. avium, M. chelonae, M. scrofulaceum, M. xenopi, M. kansasii, M. intracellulare and M. smegmatis. Compared with the standard strains, there was an additional 268 bp band in simulated clinic samples infected by mycobacteria, 209 formalin-fixed, paraffin-embedded tissue samples of the patients diagnosed as scrofula by clinic doctor at first visit were examined by triplex polymerase chain reaction. Among them, 193 tissue samples of the patients pathologically diagnosed as scrofula, tuberculous granulomatous tissue or tuberculous granulomatous inflammation were positive: the specific hands of 383 bp, 123 bp and 268 bp were present in the agarose gel and this tallied with Mycobacterium tuberculosis complex infection. Of 16 tissue samples of the patients pathologically diagnosed as suspicious scrofula, 15 samples were same positive results and this tallied with Mycobacterium tuberculosis complex infection, too; 1 sample could find the specific bands of 383 bp and 268 bp which were present in the agarose gel and this tallied with nontuberculous mycobacteria infection. The results showed that the triplex-PCR could detect and identify the DNA of Mycobacterium tuberculosis complex and nontuberculous mycobacteria except M. simiae. It is a valuable detecting method which has high sensitivity and specificity.