Multi-drug resistant TB (MDR-TB) poses a significant threat to treatment and is a Category C bioterror agent. The current identification of MDR-TB still relies on culture and takes 2-6 weeks because of the slow growth of M. tuberculosis. Despite recent progress in understanding the molecular basis of drug resistance in M. tuberculosis, a rapid and reliable molecular method to identify drug resistance is still lacking. Molecular identification of mutations in the genes associated with drug resistance offers an opportunity to rapidly detect drug resistant TB and eliminates the need for the time-consuming phenotype-based susceptibility testing. However, the current molecular methods such as PCR-SSCP, heteroduplex formation etc. are tedious and do not demonstrate required sensitivity or high-throughput sample screening capability. Although DNA sequencing is the most accurate and reliable method for mutation detection, it is expensive, ill-suited for high throughput screening of multiple genes involved in resistance or for resistance mutations not clustered in a sizable target gene. In a preliminary study, we have developed a microarray methodology using overlapping short oligoprobes that can reliably and rapidly detect pncA mutations in pyrazinamide-resistant strains. In this project, we would like to extend this work to incorporate the use of microarray in detection of mutations in multiple genes involved in resistance to several other TB drugs. This technique allows for analysis of all important drug resistance genes in a single hybridization. The goal of this Phase I application is to develop a simple and high-throughput microarray test for rapid detection of MDR-TB.
The specific aims of this Phase I SBIR application are: (1) To design microarray oligoprobes based on multiple genes involved in M. tuberculosis drug resistance; (2) To fabricate microarray chip; (3) To evaluate the chip using random mutant libraries; (4) To test the feasibility of microarray hybridization profiles for detection of MDR-TB. Such a rapid microarray test will provide timely information for the treatment and containment of MDR-TB. ? ?
| Volokhov, Dmitriy V; Chizhikov, Vladimir E; Denkin, Steven et al. (2009) Molecular detection of drug-resistant Mycobacterium tuberculosis with a scanning-frame oligonucleotide microarray. Methods Mol Biol 465:395-417 |
