The Number Of Points Digestion Joint Sscp In The Detection Of Mycobacterium Tuberculosis Inha Full Gene Mutation

Background: The incidence of tuberculosis keeps on rising and it remains one of the top serious contagious disease all over the world in recent twenty years. Meanwhile, the drug-resistant M. tuberculosis isolates are daily on the increase, which seriously threaten the control and treatment of tuberculosis. Isoniazid is the most important first line antitubercular agents. Five genes have been implicated in INH resistance: katG, inhA, aphC, kasA and ndh, in which katG and inhA are the most important. InhA gene is 807bp, which codes an NADH-dependent enoyl-acyl carrier protein (ACP) reductase. About 10-35% of the clinical isolates of isoniazid-resistant M. Tuberculosis contains mutations of inhA gene. PCR-SSCP digesting with restriction endonuclease Hae II technique was adopted in detection of inhA genetic mutations in MTB isolates, therefore to get full mutation information. Methods: A total of 48 isolates of M. Tuberculosis, including 23 drug-resistant MTB isolates were collected. Their genome DNA were extracted, the target gene were gained by PCR, the products were purified and digested with restriction endonuclease Hae II. Single-stranded conformational polymorphism method was used to detect mutations in these isolates. DNA analysis were used to identify mutant sequences when conformation change was found. Results: No inhA mutation has been identified in isoniazid-sensitive isolates while conformation changes were found in five isoniazid-resistant isolates. The latter were confirmed as mutations by DNA analysis. The mutation rate was 21.8% in isoniazid-resistant isolates. New missense mutation included A26E, R27K, V28A, Q32A, L54V and S72T. Multiple mutations were found in one isolate. Most of these mutations were near the NADH binding region of InhA. Conclusion: PCR-SSCP digesting with restriction endonuclease Hae II technique is available for detecting inhA genetic mutations in MTB isolates. This new method will help the molecular epidemiological research of M. tuberculosis. It also proves that mutations of inhA gene are diversiform.