[Detection of KatG 315 gene mutation associated with isoniazid resistance in Mycobacterium tuberculosis by template-directed dye-terminator incorporation with fluorescence polarization detection technology].
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.
Wang
Yan
Y
Institute of Gentice Diagnosis, Fourth Military Medical University, Xi'an 710032, China.
Zhang
Wen-hong
WH
Zhao
Jin-rong
JR
Luo
Ming
M
Zhang
Zeng-xian
ZX
Bai
Yu-jie
YJ
Yan
Xiao-jun
XJ
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
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D007538:Isoniazid