Comparison of DNA targets for amplification by polymerase chain reaction for detection of Mycobacterium tuberculosis in sputum.

PCR assays for detection of Mycobacterium tuberculosis were compared using three different primer pairs to amplify fragments of IS 6110, pab and mtp 40 genes. The amplified products were detected by gel electrophoresis. The detection limit of PCR assay for amplification of IS 6110, pab and mtp 40 genes was 10,100 and 100 femtograms of purified M. tuberculosis DNA, respectively. Fifty-six smear-positive sputum samples were tested for the sensitivity of detection. Two sputum specimens contained inhibitors that were not removed after treatment. Of 54 specimens, 53 was positive for M. tuberculosis and one was positive for mycobacteria other than Mycobacterium tuberculosis (MOTT) by culture method. The one that grew MOTT was PCR-negative. Of 53 culture-positive found to contain M. tuberculosis, amplified products were detected in 47 (90%), 28 (53%) and 17 (30%) samples in which the target for amplification was IS 6110, pab and mtp 40 genes, respectively. This study confirms the potential of IS 6110 amplification by polymerase chain reaction for rapid detection of M. tuberculosis in clinical specimens.

The studies of HLA class II (HLA-DR) system by PCR-SSO typing and the relationship with serological method.

By using two methods together, namely, the conventional serological typing and the new method which is oligonucleotide typing, in order to study the HLA system, it was concluded that the new method was better. Some subtypes were detectable from the new method, whereas, the serological typing yielded negative results such as DRB1 subtype "0103". This method, therefore, provided more information in order to study the HLA system. However, the new method was twice as expensive as the conventional serological method.

Multiplex PCR to detect the dystrophin gene deletion in Thai patients.

We have demonstrated the usefulness of the multiplex PCR to directly detect the dystrophin gene mutation. Prenatal diagnosis and confirmation of clinical diagnosis of DMD/BMD via non invasive technique are now possible. Nine DMD and one BMD patients were tested. Five DMD patients demonstrated deletion. Thus, this multiplex PCR could detect deletion in approximately 50 per cent of DMD/BMD Thai patients. Eighty per cent of the deletions were in the distal part, whereas, 20 per cent were in the proximal part. We are planning to establish other molecular techniques such as linkage analysis, cDNA hybridization and immunostaining of dystrophin protein to improve a mode of diagnosis and management of DMD/BMD patients in the Thai community.