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

Abstract

We have previously reported that a significant percentage (44%) of isoniazid-resistantMycobacterium tuberculosisstrains 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 anMspI restriction site which occurs when the R463L is present. Eighty oneM. tuberculosisstrains, including the wild type strain H37Rv, with isoniazid susceptibility in the range <0·12 to >32 μg ml−1were evaluated. The results for the PCR-SSCP method were in complete agreement with the PCR-MspI RFLP reference method. Of 81M. tuberculosisstrains 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. tuberculosisstrain, H37Rv. It is concluded that the described PCR-SSCP is a reliable method for screeningM. tuberculosisstrains for thekatGR463L mutation.