Construction and evaluation of mycobacteriophage D29 expressing green fluorecent protein to detect Mycobacterium tuberculosis in sputum

Abstract

Tuberculosis is a major challenge to public health system. The inadequate use of medicine and the high number of patients who abandon the treatment have been selecting multidrug resistant Mycobacterium tuberculosis strains. This has introduced difficulties to treat patients hosting these bacteria. In addition, methods currently being used for diagnosis by most public laboratories are time consuming and present a very low sensibility. It is very important to develop a faster, highly sensible, and low cost method to treat tuberculosis disease. The objective of this study is to set up a very quickly method using a recombinant D29 phage carrying GFP gene with an acetamidase promoter to used as a tool for tuberculosis diagnosis. This gene will be inserted in a site direct cloning using 2 restriction enzyme site. After a routine sample treatment, the purified product will be incubated with the recombinant phage, the best time contact to produce an amount of green protein for quickly detection in a Fluorescent ELISA system will be evaluated. By using this approach, the time for Mycobacterium tuberculosis detection will be shortened to only 48 hours and less expensive if compared to PCR techniques. (AU)