Detection of fluoroquinolone resistance SNPs in gyrA and parC genes of Neisseria gonorrhoeae using MALDI-TOF mass-spectrometry

Central Research Institute of Dermatology and Venereology, Moscow 107076, Russia). For many known mechanisms of the drug resistance in microorganisms are described genetic markers (specific changes in the genome of microorganism, in the majority of the cases representing single nucleotide polymorphism). The search for the new methods, which make possible to identify single nucleotide changes with the greater effectiveness and at smaller prime is actual for the solution of the problem of the identification of the resistant strains. In this work a new approach of the determination of single nucleotide polymorphisms is proposed. It is based on the reactions of mini-sequencing and/or sequencing with the subsequent Matrix-Assisted Laser Desorption/Ionisation Time Of Flight Mass-Spectrometry (MALDI-TOF MS) of the reaction products. The approach was tested on a clinical group of Neisseria gonorrhoeae strains to investigate specific single nucleotide polymorphisms in genes gyrA and parC (the genetic markers of the bacterium fluoroquinolone resistance). The results of the nucleotide polymorphism determination was completely agreed with the data, obtained earlier with the use of a "gold standard" (sequencing with the classical gel-electrophoresis separation of the reaction products). There is specific interest in the method of sequencing of the short DNA sequences using MALDI-TOF MS. The new high-throughput approach of the single nucleotide polymorphisms determination in bacterial genes considerably increases the effectiveness of the methods of microorganism's identification, genotyping and determining the genetic markers of the drug resistance.