Establishment of an assay for identifying mutation of clarithromycin -resistance gene of Helicobacter pylori by real time PCR with a novel fluorescence quencher / 预防医学

ABSTRACT

Objective To establish a real time PCR assay with a novel fluorescence quencher for identification of mutation of clarithromycin -resistance gene of Helicobacter pylori.Methods Two mutations of 23S rDNA gene in Helicobacter pylori,No.2142 and 2143,were chosen as targets for detection,and then the primers and the probe with a novel fluorescence quencher were designed.The genome DNA of Helicobacter pylori was extracted,and then detected by real time PCR reported here.Meanwhile,the specificity,reproducibility and sensitivity of the assay were evaluated.Finally,the real time PCR described here,the real time PCR based on TaqMan,and a sequencing assay were applied to detect 55 Helicobacter pylori strains isolated from clinical specimens,respectively.The results from three assays were compared with each other in order to further evaluate the applicability of this assay in clinic.Results It indicated that the mutation points related to clarithromycin -resistance,A2142G and A2143G,were identified by real time PCR with a novel fluorescence quencher rapidly and accurately.Moreover the coefficient of variation was less than 5%.The limit of detection was 100 copies/reaction.While this assay was applied directly to detect 55 Helicobacter pylori strains,the results were in accordance with those obtained from a TaqMan real time PCR and a sequencing assay,respectively.Conclusion The real time PCR described here was a simple,reliable and accurate approach and substituted for the TaqMan real time PCR for identification of two mutation points of clarithromycin -resistance,A2142G and A2143G in Helicobacter pylori.Thus,a novel tool for diagnosis of gene mutation was provided and the results might be regarded as a substantial evidence for clinical individual therapy.