DIRECT DETECTION OF DRUG RESISTANT MTB WITHOUT PCR

DESCRIPTION (provided by applicant): Drug resistance is a significant problem in the fight against the bacterial pathogen Mycobacterium tuberculosis. Resistance to rifampin, a first-line antituberculosis drug, is almost always the result of mutations in the rpoB gene of the pathogen. In order to speed the diagnosis of drug resistant tuberculosis, it is possible to detect these mutations by a variety of available methods. However, all such methods require the PCR reaction, which is too expensive for routine use in most countries. There is a critical need for new diagnostic methods that detect genetic markers of rifampin resistance at a fraction of the cost of current methods. Our Tessera Array Technology detects single-copy chromosomal genes of M. tuberculosis without the need for PCR amplification. The method involves high-affinity labeling of pathogen DNA in crude or partially purified lysates, hybridization of the labeled DNA to sequence-specific probes, and detection of the labeled, hybridized DNA. A test for detecting M. tuberculosis has been developed by AGTI and is currently in pre-clinical trials. We propose to expand the test for simultaneous taxonomic detection of M. tuberculosis and genotypic detection of mutations that cause rifampin resistance. The specific aims of Phase I of the project are to adapt the Tessera platform to accommodate probes for rpoB; to enhance test sensitivity by optimizing cell lysis methods; and to conduct a non-clinical trial on known rifampin-sensitive and rifampin-resistant clinical isolates of M. tuberculosis.