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

被引:23
作者
Temesgen, Z
Satoh, K
Uhl, JR
Kline, BC
Cockerill, FR
机构
[1] MAYO CLIN & MAYO FDN,DIV CLIN MICROBIOL,DEPT PATHOL & LAB MED,ROCHESTER,MN 55905
[2] MAYO CLIN & MAYO FDN,DEPT INTERNAL MED,DIV INFECT DIS,ROCHESTER,MN 55905
[3] MAYO CLIN & MAYO FDN,DEPT BIOCHEM & MOL BIOL,ROCHESTER,MN 55905
[4] TOKYO NATL CHEST HOSP,TOKYO 204,JAPAN
关键词
Mycobacterium tuberculosis; isoniazid; catalase-peroxidase gene; PCR-SSCP;
D O I
10.1006/mcpr.1996.0077
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 mu g 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. Ail of the remaining 54 strains had PCR-SSCP and PCR-Mspl RFLP results identical to the wild type (R463) M. tuberculosis strain, H37Rv. II is concluded that the described PCR-SSCP is a reliable method for screening M. tuberculosis strains for the katG R463L mutation. (C) 1997 Academic Press Limited.
引用
收藏
页码:59 / 63
页数:5
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