Mycobacterium tuberculosis Detection based on mpt64 amplification by Nested-PCR in Sputum samples

Introduction: Tuberculosis (TB) is an old problem that is currently considered as a great challenge, mostly in developing countries. It may be a lethal disease. Thus, rapid diagnosis of Mycobacterium tuberculosis (MTB) infection plays a critical role in controlling the spread of TB, whereas conventional methods may take up to several weeks or longer to diagnose the infection. Hence, nested polymerase chain reaction (NCR) assay was applied for direct identification of the MTB DNA presence in sputum samples. The aim of the study was the development of a direct NCR method using mpt64 specific primers for rapid diagnosis of MTB infection. Materials and Methods: To development of study, eight positive and negative sputum specimens obtained from Masih Daneshvari hospital pulmonary TB center, were studied. After smear preparation genomic DNA was extracted and mpt64 was amplified using NCR method. While doing work we paying attention to PCR standardization and precautions to avoid sample contamination. Results: After evaluation gained appropriate results from purified genomic DNA by AGE and biophotometer, the standardized NCR products were evaluated by Agarose Gel Electrophoresis. Five of 7 positive samples were positive, and one of the negative samples was negative using our NCR assay. Conclusion: Based on the results of this study, we could be successful in the NCR technique’s optimization to our system for disese detection, while it can be apply as a more rapid, accurate, inexpensive, and specific diagnostic assay for direct detection of MTB DNA.