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Free Content Comparative assays of the rpoB gene for identification of Mycobacterium tuberculosis isolated from patients in Sudan

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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.
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Keywords: Mycobacterium tuberculosis; PCR; Sudan; rpoB

Document Type: Regular Paper

Affiliations: 1: Central Veterinary Research Laboratories, Soba, Khartoum, Sudan 2: Department of Preventive Medicine and Public Health, Faculty of Veterinary Medicine, Khartoum North, Sudan 3: National Health Laboratories, Ministry of Health, Khartoum, Sudan 4: Department of Medicine, Faculty of Medicine, University of Khartoum, Khartoum, Sudan 5: Department of Microbiology, College of Medicine, Seoul National University, South Korea 6: Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan

Publication date: June 1, 2007

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  • The International Journal of Tuberculosis and Lung Disease publishes articles on all aspects of lung health, including public health-related issues such as training programmes, cost-benefit analysis, legislation, epidemiology, intervention studies and health systems research. The IJTLD is dedicated to the continuing education of physicians and health personnel and the dissemination of information on tuberculosis and lung health world-wide.

    Certain IJTLD articles are selected for translation into French, Spanish, Chinese or Russian. They are available on the Union website

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