title: [Detection of KatG 315 gene mutation associated with isoniazid resistance in Mycobacterium tuberculosis by template-directed dye-terminator incorporation with fluorescence polarization detection technology].
abstract: OBJECTIVE
  To study the relationship between KatG 315 mutation and isoniazid (INH) resistance in M. tuberculosis by template-directed dye-terminator incorporation with fluorescence polarization detection (TDI-FP), and to develop a new method that can detect INH-resistant M. tuberculosis precisely and quickly.
  
  
  METHODS
  Isolates of M. tuberculosis resistant to INH from 82 tuberculosis patients were cultured on culture medium with different INH concentrations. DNA of all M. tuberculosis samples was extracted and amplification of 271 bp KatG gene fragment was carried out by polymerase chain reaction (PCR). After digestion of the excess primers and dNTPs in PCR products by clean-up reagents, template-directed dye-terminator incorporation reaction was performed, and the Acyclo-terminators labeled Rhodamine 110 (R110) or 6-carboxytetramethylrhodamine (TAMRA) were incorporated into the detection primers specifically. Then fluorescence polarization value was measured using Victor2 multi-label counter and the genotypes of the mutation site in 315 condon of KatG gene of all samples were investigated.
  
  
  RESULTS
  There were 15 out of the 29 isolates of M. tuberculosis with high INH-resistance showed G-->C mutation in 315 condon of KatG gene, including 4 isolates with both mutated and non-mutated trains, the mutation frequency being 52%. In 32 isolates of M. tuberculosis with low INH-resistance, 15 were mixed infection, the mutation frequency being 47%. No mutation in 315 condon of KatG gene was found in 21 susceptible isolates of M. tuberculosis.
  
  
  CONCLUSIONS
  The G-->C mutation in 315 condon of KatG gene was associated with INH-resistance of M. tuberculosis. TDI-FP technology was a reliable, easy to use and high-through method to detect mutation of KatG gene. It may be a useful technique for the diagnosis of INH-resistance in the future.
authors.affiliation: Institute of Gentice Diagnosis, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Wen-hong
authors.initials: WH
authors.lastname: Zhang
authors.forename: Jin-rong
authors.initials: JR
authors.lastname: Zhao
authors.forename: Ming
authors.initials: M
authors.lastname: Luo
authors.forename: Zeng-xian
authors.initials: ZX
authors.lastname: Zhang
authors.forename: Yu-jie
authors.initials: YJ
authors.lastname: Bai
authors.forename: Xiao-jun
authors.initials: XJ
authors.lastname: Yan
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D002374:Catalase; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D005798:Genes, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D017354:Point Mutation; D014397:Tuberculosis, Pulmonary / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D007538:Isoniazid