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.
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 Mspl 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 micrograms ml-1 were evaluated. The results for the PCR-SSCP method were in complete agreement with the PCR-Mspl 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-Mspl RFLP method. All of the remaining 54 strains had PCR-SSCP and PCR-Mspl 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.
Temesgen
Z
Z
Department of Internal Medicine, Mayo Clinic, Rochester MN 55905, USA.
Satoh
K
K
Uhl
J R
JR
Kline
B C
BC
Cockerill
F R
FR
D001426:Bacterial Proteins; D004269:DNA, Bacterial / Q000032:analysis*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D010544:Peroxidases / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010544:Peroxidases; C076136:catalase HPI; D007538:Isoniazid