Rapid detection of gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae by denaturing high-performance liquid chromatography Katsumi Shigemura a , Toshiro Shirakawa b , Hiroshi Okada c , Kazushi Tanaka a , Tohru Udaka d , Sadao Kamidono a , Soichi Arakawa a , Akinobu Gotoh b, * a Division of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Japan b International Center for Medical Research, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan c Division of Urology, Teikyo University School of Medicine, Japan d Research Laboratory, Transgenomic Inc., Tokyo, Japan Received 5 June 2004; received in revised form 16 August 2004; accepted 19 August 2004 Available online 21 September 2004 Abstract The detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that is used to detect mutations in human DNA. This is the first report that this technique is used as a tool to detect mutations in genes encoding fluoroquinolone resistance in Neisseria gonorrhoeae . Eighty-one strains of N. gonorrhoeae were used in this study. Genomic DNA from each strain was subjected to PCR amplification of 225 bp in gyrA and 166 bp in parC spanning the fluoroquinolone-resistance determining regions (QRDRs). After we performed DNA sequencing of these amplicons and identification of mutations in the QRDRs, DHPLC was undertaken to investigate whether its results correlate the distinctive chromatogram with their DNA mutations pattern. The profilings detected by DHPLC completely corresponded to the results of the DNA sequencing in mutation patters in gyrA and parC genes. They resulted in the following amino acid substitutions: Ser-91Phe, Asp-95Gly, and Asp-95Asn in gyrA; and Gly-85Asp, Asp-86Asn, Ser-87Arg, and Ser-88Pro in parC, respectively. These mutations existed alone or as combinations, and we identified five mutations patterns in gyrA and six in parC including wild-type. These mutations and their patterns could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This novel detection system facilitates the detection of resistance 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.08.004 * Corresponding author. Tel.: +81 78 382 5694; fax: +81 78 382 5693. E-mail address: gotoh@med.kobe-u.ac.jp (A. Gotoh). Journal of Microbiological Methods 59 (2004) 415 – 421 www.elsevier.com/locate/jmicmeth