eprintid: 10593 rev_number: 29 userid: 363 dir: disk0/00/01/05/93 datestamp: 2012-01-30 18:58:57 lastmod: 2019-03-08 22:23:43 status_changed: 2012-01-30 18:58:57 type: thesis_degree metadata_visibility: show contact_email: alexabhatti@gmail.com item_issues_count: 0 eprint_status: archive creators_name: Bhatti, Alexandra creators_email: Alexabhatti@gmail.com title: Optimization of rTDMH as a Reagent Toward Improving the Sensitivity of the RT-PCR Based Diagnosis for Mycobacterium Tuberculosis ispublished: unpub divisions: sch_gsph_infectiousdiseasesmicrobiology full_text_status: public keywords: NA abstract: Current diagnostic tools being used for tuberculosis lack the speed and sensitivity necessary to successfully combat the current tuberculosis epidemic. Real-time Polymerase Chain Reaction, RT-PCR, can provide the rapid and specific diagnosis that is currently in demand in the global community. Its disadvantage is that due to the waxy and robust nature of the M. tuberculosis membrane, not enough genomic DNA is present to provide for amplification in a RT-PCR. It was previously found in our laboratory that hydrolysis of one of abundant glycolipid of mycobacterial envelope, Trehalose, 6,6’-dimycolate, by a recombinant TDM-specific hydrolase caused rapid lysis of cell (Yong et.al. manuscript submitted). In this study, we tested if rapid lysis by TDM-specific hydrolase (rTDMH) can be exploited in conjunction with the RT-PCR to develop a sensitive diagnosis of tuberculosis. Results demonstrated that by incubation of both attenuated M. tuberculosis, and virulent M. tuberculosis with rTDMH for lysis and subsequent usage of this lysate in a RT-PCR assay, yields sensitive amplification of mycobacterial DNA. rTDMH-mediated lsyis could facilitate amplification of even 10 bacilli, the rTDMH treated cells show amplification while lack of treatment failed to detect these bacilli These results were consistent in in-vitro liquid culture and in complex sputum samples spiked with the mycobacteria, showing that incubation with rTDMH can improve the sensitivity of the RT-PCR. Statement of Public Health relevance: Using rTDMH with RT-PCR as an improved diagnostic tool for tuberculosis due to the rapid, accurate and sensitive nature of the assay could provide the global community with a much better method of diagnosing a disease that has plagued the world for thousands of years. Tuberculosis infects 9 million people and kills 3 million people every year and presently one-third of the world’s population is infected with it. A better diagnostic tool could result in reducing the spread of disease; reducing the mortality associated with disease, especially in HIV infected individuals; and on a broader scale, could reduce the economic burden associated with the disease date: 2012-01-30 date_type: published pages: 69 institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Ojha, Anil etdcommittee_name: Martinson, Jeremy etdcommittee_name: Terry, Martha etdcommittee_name: Beatty, Rodger etdcommittee_email: ano7@pitt.edu etdcommittee_email: jmartins@pitt.edu etdcommittee_email: MATERRY@pitt.edu etdcommittee_email: rbear3@pitt.edu etdcommittee_id: ANO7 etdcommittee_id: JMARTINS etdcommittee_id: MATERRY etdcommittee_id: RBEAR3 etd_defense_date: 2011-12-01 etd_approval_date: 2012-01-30 etd_submission_date: 2011-11-30 etd_release_date: 2012-01-30 etd_access_restriction: 1_year etd_patent_pending: FALSE assigned_doi: doi:10.5195/pitt.etd.2011.10593 thesis_type: thesis degree: MPH citation: Bhatti, Alexandra (2012) Optimization of rTDMH as a Reagent Toward Improving the Sensitivity of the RT-PCR Based Diagnosis for Mycobacterium Tuberculosis. Master's Thesis, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/10593/1/Bhatti_aab_etd2011.pdf