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 sus...
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| Format: | Theses and Dissertations |
| Language: | English |
| Published: |
2014
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| Online Access: | http://hdl.handle.net/10356/61081 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | 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. |
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