Comparative assays of the rpoB gene for identification of Mycobacterium tuberculosis isolated from patients in Sudan.
OBJECTIVE
To characterise mycobacterial clinical isolates based on amplification of the rpoB gene.


METHODS
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.


CONCLUSIONS
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.
El-Eragi
A M S
AM
Central Veterinary Research Laboratories, Soba, Khartoum, Sudan. eleragi@hotmail.com
Hamid
M E
ME
Saeed
N S
NS
Ahmed
A H
AH
Lee
K
K
Kook
Y-H
YH
Mukhtar
M M
MM
D001426:Bacterial Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D011237:Predictive Value of Tests; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D013397:Sudan; D014376:Tuberculosis / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D015252:Deoxyribonucleases, Type II Site-Specific; C024400:GTYRAC-specific type II deoxyribonucleases