Abstract:
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OBJECTIVES: To characterise mycobacterial clinical isolates
based on amplification of the rpoB gene.
SETTING: One hundred and thirty-five mycobacterial
isolates cultured from suspected pulmonary tuberculosis
(TB) patients were identified phenotypically. Molecular
characterisation of the isolates was performed based on
amplification of the rpoB gene, using duplex polymerase
chain reaction (DPCR), PCR-restriction fragment length
polymorphism (RFLP) and nested PCR-based sequence
analysis techniques.
RESULTS: The DPCR assay identified 129 of 135 (95.5%)
clinical isolates as Mycobacterium tuberculosis complex
species. Restriction enzyme analysis of the rpoB PCR
product using Hind II identified 134 of the 135 (99.3%)
isolates as M. tuberculosis complex, while nested PCR
sequence analysis of the rpoB gene identified 133/133
examined isolates (100%) as M. tuberculosis species. No
mycobacteria other than M. tuberculosis (MOTT) were
detected among the studied isolates.
CONCLUSION: DPCR, PCR/RFLP Hind II and nested
PCR sequence analysis of the rpoB gene techniques showed
comparable efficiency in the characterisation of Mycobacterium
isolates. Nested PCR sequence analysis of the
rpoB gene was superior to PCR/RFLP for characterisation
of suspected M. tuberculosis isolates, while the DPCR
technique showed less sensitivity. As PCR-RFLP requires
less sophisticated laboratory facilities than nested PCR
sequence analysis, it would be more appropriate to be
adopted for accurate characterisation of mycobacteria
in countries with a weak infrastructure. |