Molecular technologies used in detecting of sensitive and isoniazid-resistant Mycobacterium tuberculosis

Abstract

Two variants for the detection of single nucleotide polymorphisms in codon 315 of the katG gene of Mycobacterium tuberculosis (MTB) (mutations in this gene are associated with resistance to isoniazid, which is an antituberculosis drug of the first line) have been developed. Two sets of primers, either of which included an additional competitive blocking primer with a 3′-terminal phosphate group (in order to prevent nonspecific amplification), permitted the identification of the most frequent AGC → ACC and AGC → AGA point mutations in codon 315 of the katG gene. Conduction of PCR with a set of two primers, one of which contained five LNA monomers, permitted the detection of any of the six known mutations in codon 315 of the katG gene and, thereby, for the discrimination between isoniazid-sensitive and isoniazid-resistant MTB. The purity and structure of the 17 bp long primers containing LNA-modified nucleotides were characterized by time-of-flight MALDI mass spectrometry, and the 17 bp duplex formed by two LNA-containing complementary oligonucleotides was analyzed by thermal denaturation. The molecular genetic test systems created for differentiating between the wild-type MTB isolates and isoniazid-resistant MTB (an antituberculosis drug of the first line) can be used in clinical laboratories equipped with standard PCR devices; such systems permit the shortening of the time required for the detection of isoniazid resistance of MTB: from 1–3 months by the standard bacteriological methods to 1–3 days by PCR.