Identification of wild-type Mycobacterium tuberculosis isolates and point mutations associated with isoniazid resistance

• Published in: Molecular biology (New York) 2010-08, Vol.44 (4), p.559-567
• Main Authors: Limanskaya, O. Yu, Mukhina, T. N, Stepanshina, V. N, Shemyakin, I. G, Wu, X, Zhang, J, Fesenko, T. V, Pokrovskiy, V. A, Limanskii, A. P
• Format: Article
• Language: English
• Subjects: Bacteria; Biochemistry; Biomedical and Life Sciences; Drug resistance; Genomics and Transcriptomics; Human Genetics; Life Sciences; Mass spectrometry; Molecular biology; Mutation; Mycobacterium tuberculosis; Polymerase chain reaction; Tuberculosis
• ISSN: 0026-8933; 1608-3245
• DOI: 10.1134/S0026893310040096

Isoniazid resistance in Mycobacterium tuberculosis (MBT) is associated with point mutations in codon 315 of the katG gene. Two PCR technique were developed for detection of point mutations in codon 315. Most frequent point mutations (AGC → ACC and AGC → AGA) were identified in codon 315 by using 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. PCR with a set of two primers, one of which contained five locked nucleic acid monomers (LNA), permits one to detect any of six known mutations in codon 315 of katG and, thereby, discriminate between isoniazid-sensitive and resistant MBT isolates. The structure and purity of the 17-nt long LNA-containing oligonucleotides were characterized by MALDI-TOF mass spectrometry; and the 17 bp duplex formed by two LNA-containing complementary oligonucleotides was analyzed by thermal denaturation.