Mycobacterium tuberculosis drug resistance detection by pyrosequencing method

Having an accurate and timely diagnostics tool for infectious diseases is important for clinician to make decisions on appropriate treatments and patient management. The conventional way of detecting Mycobacterium tuberculosis and its resistance is by culture followed by phenotypic drug susceptibility testing. This laborious process usually takes 4 to 12 weeks due to the slow growing nature of the organism. To improve the speed of diagnosis, we have explored the pyrosequencing platform to detect mutations in the rpoB gene, which is associated with rifampicin drug resistance in M. tuberculosis. The commonly known mutations on the 81-bp core region of rpoB were sequenced in two reactions to obtain quality reads. Validation with wild type M. tuberculosis genomic DNA and synthetic mutant sequences showed pyrograms with low background and was highly specific. Linearity analysis demonstrated high correlation with the amount of mutant variants in a mixed population, thus able to predict as low as 5% of mutant. Pyrosequencing has proven to be valuable tool for detection of single nucleotide polymorphism mutations in M. tuberculosis rpoB 81-bp core region.​Master of Science (Biomedical Engineering