[Establishment of a method with micro-channel electrophoresis for detecting the mutations of isoniazid-resistant genes in Mycobacterium tuberculosis].
OBJECTIVE
To establish a method for the detection of isoniazid resistance-associated mutations in katG gene and inhA gene in Mycobacterium tuberculosis with single-stranded conformation polymorphism (SSCP) analysis by the micro-channel electrophoresis chip system.


METHODS
The polymer solutions of acrylamide and its derivatives were used for sieving matrices with small amount of acridine orange as the fluorescent tag. A novel pair of primers was designed to link an extra segment that could be base paired with the mutation region to the PCR products of wild type inhA gene, which made the single strands of wild type inhA fragments present a unique conformation.


RESULTS
The wild type fragments of katG gene and the fragments with mutation at codon 315 can be distinguished, and the inhA fragments with wild type and mutated regulatory sequence can be distinguished by this method. Twenty-two out of the 23 resistant strains were detected from 30 clinical isolates, the efficiency being 95%.


CONCLUSIONS
It is demonstrated the high speed and sensitivity in detecting the mutations of isoniazid-resistant genes in Mycobacterium tuberculosis by micro-channel electrophoresis, and this method may be applicable in clinical detection of isoniazid-resistant strains.
Wang
Ji
J
Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China.
Cui
Zhen-ling
ZL
Jing
Feng-xiang
FX
Jin
Qing-hui
QH
Zhao
Jian-long
JL
Hu
Zhong-yi
ZY
Xu
Yuan-sen
YS
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004586:Electrophoresis / Q000379:methods*; D007538:Isoniazid / Q000494:pharmacology*; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D010544:Peroxidases / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010088:Oxidoreductases; D010544:Peroxidases; C076136:catalase HPI; C085257:InhA protein, Mycobacterium; D007538:Isoniazid