Rapid Molecular Detection of Tuberculosis without PCR amplification

DESCRIPTION provided by applicant TB is an infection caused by species of the Mycobacterium tuberculosis complex MTBC In million people died from the disease which is unacceptable given that TB is curable The lack of a technology for accurate TB detection in limited resource settings is a major obstacle in the fight against the disease However available detection methods have poor performance and are not suited for diagnosis in limited resource settings The most commonly used method to detect TB is smear microscopy a procedure developed more than years ago that only detects of the cases Here we propose to develop a next generation molecular diagnostic test that can dramatically improve TB diagnosis The new platform will be based on a novel technology capable of detecting single molecules using simple equipment In the single molecule technique probe hybridization to a target nucleic acid is directly detected without PCR amplification The technique is capable of detecting CFU ml of Mycobacterium tuberculosis cells which is below the limit of detection of the leader PCR based TB detection assay In addition the technique applies a disruptive force to the probe target duplexes which makes detection highly specific even at room temperature Thanks to its non enzymatic detection approach reagents and equipment are simple and robust ideal for settings with limited resources Our overall goal is to develop a molecular platform for TB testing that will be easy to use without training accurate rapid inexpensive portable and battery operated In this proposal we will demonstrate all the properties of the final product by developing a non automated TB assay and testing it using sputum samples spiked with known quantities of the bacillus The focus of Aim is to develop an efficient method to capture MTBC RNA targets from sputum samples containing MTBC cells Aim focuses on developing a multiplex SM Scanning test and determining the limit of detection cross reactivity sensitivity and specificity of the test If successful the new platfom could support other tests that are urgently needed in settings with limited resources such as TB drug susceptibility Ebola and sexually transmitted diseases PUBLIC HEALTH RELEVANCE We propose the development of a rapid sensitive and affordable molecular diagnostic test for the detection of active tuberculosis in limited resource settings