Purpose: To evaluate the reliability of the gyrB PCR-RFLP technique in differentiating clinical
Mycobacterium tuberculosis
complex isolates.
Materials and Methods: A primer pair MTUB-f and MTUB-r for
M.
tuberculosis complex (MTBC) was used to differentiate 79 mycobacterial isolates by specific amplification of the 1,020-bp fragment of the
gyrB gene (
gyrB-PCR1). The MTBC isolates were further differentiated using a set of specific primers MTUB-756-Gf and MTUB-1450-Cr that allowed selective amplification of the
gyrB fragment specific for
M.
tuberculosis (
gyrB-PCR2). The DNA polymorphisms in the 1,020-bp
gyrB fragment for 7
M.
tuberculosis strains confirmed by PCR as well as 2 reference strains;
M.
tuberculosis H37Rv and
M.
bovis BCG were analyzed with the restriction enzyme
Rsa1.
Results: Seventy-seven (97.5%) isolates were positive for
gyrB-PCR1 and thus identified as members of
M.
tuberculosis complex (MTBC) and two (2.6%) isolates were negative and identified as Mycobacteria other than tuberculosis (MOTT). All the
M.
tuberculosis isolates showed the typical
M.
tuberculosis specific
Rsa1 RFLP patterns (100, 360, 560-bp) while 360 and 480-bp fragments were generated from
M.
bovis BCG.
Conclusion: The gyrB PCR-RFLP using the endonuclease
Rsa1 can be used to differentiate
M.
tuberculosis from
M.
bovis in clinical isolates.
