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Optimization of rTDMH as a Reagent Toward Improving the Sensitivity of the RT-PCR Based Diagnosis for Mycobacterium Tuberculosis

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 (Unpublished)

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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 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


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairOjha, Anilano7@pitt.eduANO7
Committee MemberMartinson, Jeremy JMARTINS
Committee MemberTerry, MarthaMATERRY@pitt.eduMATERRY
Committee MemberBeatty, RBEAR3
Date: 30 January 2012
Date Type: Publication
Defense Date: 1 December 2011
Approval Date: 30 January 2012
Submission Date: 30 November 2011
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 69
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Infectious Diseases and Microbiology
Degree: MPH - Master of Public Health
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: NA
Date Deposited: 30 Jan 2012 18:58
Last Modified: 08 Mar 2019 22:23


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