Abstract:

In this study, the use of the rpoB gene to identify mycobacterial species was evaluated. The
rpoB gene encodes the highly conserved beta subunit of RNA polymerase. It is known to be
more discriminatory in bacterial species identification compared to the widely used I6S
rRNA gene sequence. This attribute can be exploited for correct identification of
mycobacterial strains identified according to the 16S rRNA but requiring further
identification to (sub-) species level.
A gene fragment of approximately 760 bp was targeted using primer pair My9 - MycoR. The
study involved evaluating the primer set on 30 reference mycobacterial strains from the
public culture collection (BCCM/ITM). The tests were performed using an optimised inhouse
PCR system followed by gel electrophoresis and sequencing of the amplicons. BLAST
analysis was performed to identify the species. Twenty-five (83%) of the strains were in
agreement with the sequence data available from GenBank (NCBI). For three species namely
Mycobacterium sphagni, M. komossense and M. parafortuitum, the sequence information was
not available on GenBank (NCBI). Test attributes including repeatability and reproducibility
were performed on two strains (M. ulcerans and M. abscessus) and results were consistent
with tested strains.

The rpoB was further evaluated on 71 mycobacterial strains from BCCM/ITM, which
required further identification. According to the test results, thirty one of the strains (43%)
were identified to the respective species. Using the rpoB gene sequence, it was possible to
distinguish M. marinum (1TM M000140/NCTC 2275) from M. ulcerans and M.
montmartrense (NCBI strain 028/ITM 981197) from M. peregrinum and M. septicum which
16S rRNA is not able to do.
The rpoB gene sequence has an added value in mycobacterial species identification.
However, some rpoB gene sequences are not available on Genbank. It is therefore important
to establish a complete rpoB gene sequence data bank for mycobacterial species especially
the non-tuberculous mycobacteria (NTM) to improve their identification and to aid in disease
diagnosis.