DIAGNOSIS OF TUBERCULOSIS BY RECOMBINANT SHUTTLE PHASMID

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date8/1/897/31/92

Funding

  • National Institutes of Health

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)
  • Applied Microbiology and Biotechnology
  • Microbiology (medical)
  • Medical Laboratory Technology
  • Infectious Diseases
  • Pulmonary and Respiratory Medicine

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