title: Rapid detection of ethambutol-resistant Mycobacterium tuberculosis directly from sputum samples by radioisotope (32P)-based PCR dot blot hybridization and sequencing methods.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Centre for Application of Isotopes and Radiation Technology, National Nuclear Energy Agency, Jl. Lebak Bulus Raya No. 49, Jakarta, Indonesia. marialinarosilawati@yahoo.com
authors.forename: Maria L
authors.initials: ML
authors.lastname: Rosilawati
authors.forename: Andi
authors.initials: A
authors.lastname: Yasmon
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004977:Ethambutol / Q000494:pharmacology; D005240:Feasibility Studies; D005838:Genotype; D006801:Humans; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization / Q000379:methods; D010430:Pentosyltransferases / Q000235:genetics; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013183:Sputum / Q000737:chemistry / Q000382:microbiology*; D013997:Time Factors; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D003062:Codon; D010761:Phosphorus Radioisotopes; D004977:Ethambutol; C540269:EmbB protein, Mycobacterium tuberculosis; D010430:Pentosyltransferases