Ultra-Sensitive Diagnosis of AIDS-Related Tuberculosis

DESCRIPTION provided by applicant This grant will develop a novel strategy to enhance the analytical sensitivity of polymerase chain reaction PCR based detection of bacteria in samples We will commercialize the strategy in the form of a pre PCR andquot turbo chargerandquot to be marketed in conjunction with PCR based molecular diagnostic systems The current STTR project will apply this strategy to a task for which an ultra sensitive molecular method is needed namely the same day diagnosis of smear negative and extra pulmonary tuberculosis TB in HIV coinfected patients Current molecular TB tests are not sensitive enough for this task Under ideal conditions PCR can detect a single nucleic acid molecule However when applied to real samples its sensitivity is blunted by background nucleic acid and inhibitory substances To address this limitation we developed a molecular viability testing MVT method that is fold to andgt fold more sensitive than standard PCR when used to detect bacteria in complex samples The overall Phase Phase STTR project will develop a semi automated MVT system for ultra sensitive detection of Mycobacterium tuberculosis cells in sputum and in cerebrospinal fluid CSF Its intended use is the same day diagnosis of smear negative pulmonary TB and extrapulmonary TB which are more commonly seen in HIV coinfected people than in the general population Specific aims of this Phase STTR grant are to demonstrate a manual MVT assay for M tuberculosis in sputum and CSF with an analytical limit of detection to fold better than existing PCR tests and to validate an inexpensive single use andquot blenderandquot system for efficient release of target RNA from M tuberculosis cells in sputum and CSF In Phase of the project MVT will be automated to yield a single step pre PCR unit that will be commercialized in conjunction with existing qPCR system s PUBLIC HEALTH RELEVANCE The proposed technology termed MVT will be useful for diagnostic applications that require the greatest possible sensitivity in the same day detection of cellular pathogens in samples Although the current grant focuses on AIDS related tuberculosis MVT can improve the sensitivity of PCR in a variety of other infectious disease diagnostic tasks including the detection of other AIDS opportunistic infections biowarfare agents and sterile site infections It will be a significant addition to the current molecular diagnostic toolbox