Mycobacterium tuberculosis drug resistance detection by pyrosequencing method

Abstract

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.