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 "turbo-charger" 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 5-fold to >10-fold more sensitive than standard PCR when used to detect bacteria in complex samples. The overall Phase 1 + Phase 2 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 1 STTR grant are 1 ) to demonstrate a manual MVT assay for M. tuberculosis in sputum and CSF, with an analytical limit of detection 5- to 10-fold better than existing PCR tests; and 2 ) to validate an inexpensive single-use "blender" system for efficient release of target RNA from M. tuberculosis cells in sputum and CSF. In Phase 2 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 Statement: 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.
Project Terms: Acquired Immunodeficiency Syndrome; Address; AIDS diagnosis; AIDS-Related Opportunistic Infections; Aliquot; Animals; Bacteria; Bacterial Infections; base; Biological Assay; Biological Warfare; Cells; Central Nervous System Tuberculosis; Cerebrospinal Fluid; clinical research site; Communicable Diseases; Complex; Culture Media; Cytolysis; Data; design; Detection; Diagnosis; Diagnostic; forest; General Population; Generations; Grant; Growth; HIV; Hour; improved; Individual; Infection; Lung; Manuals; Marketing; Measures; Messenger RNA; Methods; microbial; microorganism; Molecular; Mycobacterium tuberculosis; new technology; novel strategies; Nucleic Acids; Nutrient; Nutritional; pathogen; Patients; Peru; Phase; Physiology; Polymerase Chain Reaction; Process; public health relevance; Pulmonary Tuberculosis; ratiometric; Refractory; Relative (related person); Resistance; response; Ribosomal RNA; RNA; RNA-Directed DNA Polymerase; rRNA Precursor; Sampling; Signal Transduction; Site; Small Business Technology Transfer Research; South Africa; Sputum; Sterility; System; Tail; Technology; Testing; theories; Time; Tuberculosis
