alexa Rapid detection of ethambutol-resistant Mycobacterium tuberculosis directly from sputum samples by radioisotope (32P)-based PCR dot blot hybridization and sequencing methods.
Microbiology

Microbiology

Journal of Microbial & Biochemical Technology

Author(s): Rosilawati ML, Yasmon A

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Abstract AIM: to develop an assay system of a radioisotope (32P)-based PCR dot-blot hybridization technique and evaluation of the assay directly for TB sputum samples to detect mutation at codon 306 of embB gene of Mycobacterium tuberculosis related with ethambutol (EMB) resistance. METHODS: one hundred and sixteen of sputum samples were used in this study. Bacterial genome in sputum samples was extracted and tested for mutation at codon 306 of embB gene by the developed PCR dot blot assay using a radioisotope (32P)-labeled oligonucleotide. The positive results were confirmed by DNA sequencing. RESULTS: all 116 sputum samples were PCR positive for M. tuberculosis. Of 116 samples, three (2.59\%) were EMB resistant-M. tuberculosis (MTB) and showed a substitution mutation (ATG/Met'-->GTG/Val) at codon 306 of embB gene. None of mutation was detected at codon 299 of embB gene. CONCLUSION: we successfully developed a radioisotope (32P)-based PCR dot blot hybridization technique for detection of mutation at codon 306 of embB gene related with EMB resistant M. tuberculosis. The assay can detect a large number of samples that is suitable for monitoring, surveillance, and epidemiology studies.
This article was published in Acta Med Indones and referenced in Journal of Microbial & Biochemical Technology

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