Use of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis to detect a point mutation in the catalase-peroxidase gene (katG) of Mycobacterium tuberculosis

Zelalem Temesgen, Koji Satoh, James R. Uhl, Bruce C. Kline, Franklin R. Cockerill

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

We have previously reported that a significant percentage (44%) of isoniazid-resistant Mycobacterium tuberculosis strains carry an arginine to leucine mutation in codon 463 (R463L) in the catalase-peroxidase gene (katG). For the current study, we compared the utility of one mutation screening method, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, with a reference method, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), to detect this mutation. The PCR-SSCP method detects mutations by electrophoretic mobility shifts of single-stranded DNA in nondenaturing polyacrylamide gels. The RFLP method detects a loss in an MspI restriction site which occurs when the R463L is present. Eighty one M. tuberculosis strains, including the wild type strain H37Rv, with isoniazid susceptibility in the range < 0.12 to > 32 μg ml-1 were evaluated. The results for the PCR-SSCP method were in complete agreement with the PCR-MspI RFLP reference method. Of 81 M. tuberculosis strains analysed, 13 showed mobility shifts by the PCR-SSCP method and all of those strains carried the R463L as detected by the PCR-MspI RFLP method. All of the remaining 54 strains had PCR-SSCP and PCR-MspI RFLP results identical to the wild type (R463) M. tuberculosis strain, H37Rv. It is concluded that the described PCR-SSCP is a reliable method for screening M. tuberculosis strains for the katG R463L mutation.

Original languageEnglish (US)
Pages (from-to)59-63
Number of pages5
JournalMolecular and Cellular Probes
Volume11
Issue number1
DOIs
StatePublished - Feb 1997

Keywords

  • Catalase-peroxidase gene
  • Isoniazid
  • Mycobacterium tuberculosis
  • PCR-SSCP

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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