Two types of techniques for detection of single nucleotide polymorphism in 315 codon of katG gene of MTB are developed. Isoniazid resistance of MTB is associated with point mutations in the mentioned codon. Two primer sets with additional competitive blocking primer containing 3’-terminal phosphate group (for elimination of unspecific amplification) allow detecting the most frequent point mutations AGC→ACC and AGC→AGA in 315 codon of katG gene. PCR with primer set of two primers one of which contains five LNA-monomers allows to determine an occurrence of any type from six known mutations in 315 codon of katG gene, i.e. to differentiate wild type and isoniazid-resistant MTB. Purity and structure of 17 bp long primers with LNA-modified nucleotides were characterized by time-of-flight MALDI-mass spectrometry. Duplex of 17 bp length formed by two complementary oligonucleotides with LNA-monomers was studied using melting.
Keywords: point mutation, polymerase chain reaction, mycobacteria tuberculosis, isoniazid, LNA-monomer
