Mutation Detection Of Drug-Resistant Mycobacterium Tuberculosis On DNA Chip

Tuberculosis (TB), one of the most deadly and common infectious diseases, claims 3,000,000 lives a year worldwide. The global spread of the disease is further complicated by the ubiquitous appearance of drug-resistant, especially multidrug-resistant strains that, by definition, are resistant to at least rifampin (RIF) and isoniazid. However, most laboratories incubate solid medium cultures for 4 to 6 weeks to achieve a better sensitivity. Therefore, there is an urgent need to develop simple, fast, and cost-effective methods to identify drug-resistance in mycobacteria. Molecular characterization of drug-resistance of Mycobacterium tuberculosis has been widely studied, and it is possible to utilize analytical biotechniques, for example DNA chip, to detect drug-resistance of Mycobacterium tuberculosis fast and accurately.Herein, a new method for mutation detection, termed as hybridization-ligation based on DNA chip, was developed and used to identify drug-resistance of Mycobacterium tuberculosis. In the research, rpoB gene in Mycobacterium tuberculosis was cloned. Point-mutation library of drug-resistance gene was constructed. Solid-phase proofreading PCR and silver staining of colloidal gold were applied to detect the point mutation. Meanwhile, solid phase hybridization ligation based on DNA chips was developed to detect mutations. In 55 rifampin-resistant clinical samples, DNA fragments of rpoB gene in the length of 443 bp including 81 bp hotspot region were sequenced. We detected these rifampin-resistant strains of Mycobacterium tuberculosis by solid-phase hybridization ligation.The results showed that rifampin-resistance of Mycobacterium tuberculosis was detected rapidly bysolid-phase hybridization ligation.