Tuberculosis remains a devastating disease of mankind resulting in 3 million deaths per year. There are 10 million new cases of tuberculosis worldwide, and for the first time after a 32 year decline, the number of new cases of tuberculosis in the United States has increased for the last 3 years. In New York city alone, there was a 36% rise in the number of tuberculosis cases, predominantly in the black and hispanic populations. This rise of tuberculosis is most likely associated with increase in AIDS patients and so is still likely to worsen. M. avium infections are a primary cause of fatality of AIDS patients. There are effective chemotherapeutic regimens for effective treatment of tuberculosis, but rapid diagnosis of M. tuberculosis infection is essential in the effective treatment. Accurate diagnostic methods of M. tuberculosis take a minimum of 9 days to 6 weeks. The goal of this proposal is to develop a novel diagnostic test, using newly developed shuttle phasmids and a cloned luciferase gene. By developing the first efficient transfection system and a novel E. coli-mycobacteria shuttle vector, we have introduced recombinant DNA molecules into both M. smegmatis and BCG vaccine strains for the first time. This novel vector, termed a shuttle phasmid, replicates in myco- bacteria as a phage and in E. coli as a plasmid. This vector has been successfully used to introduce and stably express the first selectable marker gene for mycobacteria genetic research. We plan to construct shuttle phasmids using phages that are specific for M. tuberculosis. By cloning in a promoterless luciferase gene, we will construct promoter probe vectors for analysis of mycobacterial promoters. We will clone a promoter that is efficiently expressed in M. tuberculosis to express the cloned luciferase genes in M. tuberculosis and thus, develop a shuttle phasmid which can be used for the diagnosis of M. tuberculosis infection. We plan to use this exquisitely diagnostic shuttle phasmid as a basis for detecting viable M. tuberculosis cells in human sputum, blood, or cerebral spinal fluid samples, by mixing phage particles with the samples in an appropriate media and measuring the photons emitted. This novel approach should produce an extremely specific diagnostic test that is rapid, extremely sensitive and non-radioactive for diagnosis of mycobacterial disease.
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