Rapid Diagnosis of Isoniazid Resistance by Detection of Mutations in katG and inhA of Mycobacterium tuberculosis from Korea

ABSTRACT

29 isoniazid (INH) resistant isolated strains and INH sensitive reference strain (H37Rv) of Mycobacterium tuberculosis were analysed by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and NciI restriction mapping for the detection of mutations in katG gene and inhA gene. The katG gene was divided into 3 parts (Akat, Bkat, Ckat; each part is about 800 bp) and amplified, inhA gene was amplified as a whole. Each of the amplified 800 bp DNA was digested into small fragments of less than 400 bp with restriction enzymes for the direct PCR-SSCP analysis. Firstly, 10 strains were analysed. All the 10 isolates showed clearly distinct SSCP patterns in Bkat from that of the reference strain, but only two isolates showed distinct SSCP patterns in Akat, and no isolated strain showed any distinct SSCP patterns in Ckat. 10 isolates also showed distinct SSCP patterns in inhA. NciI restriction mapping of Bkat showed mutation in codon 463 in 7 strains among 10 isolated strains. With these results an early detection strategy for the INH resistant M. tuberculosis was applied to the rest of 19 isolated INH resistant strains. Firstly, isolates were screened by Ncsl mapping in Bkat, and 13 strains showed mutations in codon 463. Secondly, the rest of 6 INH resistant isolates were analysed by PCR-SSCP with restriction enzyme digestion (PCR-SSCP-RE) in Bkat, and all the strains showed distinct SSCP patterns from that of the INH sensitive reference strain. This proved our strategy as effective and economic and time saving method in early detection of INH resistant M. tuberculosis.