Introduction:
It is estimated that more than half of the world's population is infected with Helicobacter pylori (HP). Increasing HP resistance to clarithromycin also increases the risk of treatment failure. The aim of this study was to develop a Taqman probe based asymmetric real-time PCR assay for simultaneous detection of HP infection and identification of point mutations in the 23S rRNA gene responsible for clarithromycin resistance.
Materials and methods
Primers for the HP 23S rRNA gene were selected from a region close to commonly reported mutations, while the Taqman probe was selected from a region across the mutations. During PCR amplification, annealing of the Taqman probe on the HP target sequence (if present) is indicated by increasing fluorescence. During the high-resolution (HRM) melt stage, the melting of Taqman probe occurs at different temperature for the normal (clarithromycin susceptible) and the mutant (clarithromycin non-susceptible) targets. For identification of HP, the PCR was evaluated for analytical sensitivity & specificity, limit-of-detection (LOD), linearity and precision against known HP and other bacterial isolates. For identification of clarithromycin resistance, the PCR was tested against HP isolates with known clarithromycin susceptibilities, as previously determined by Etest.
Results:
This real-time PCR gave a performance of 100% sensitivity (78/78) and 100% specificity (25/25). For mutation detection, 62 phenotypically sensitive isolates showed an average Tm of 75.8°C (SD 0.3°C), compared to a Tm ranging from 71.5°C to 73.8°C (depending on mutation type) for 16 clarithromycin-resistant isolates. PCR reaction efficiency was 93% over range of 4.7E+07 to 4.7E+04 bacteria per reaction, while the LOD was 15 bacteria per reaction. For the inter-assay & intra-assay reproducibility, one ATCC sensitive strain and two clarithromycin resistant strains (A2142G & A2143G) were tested in 5 individual PCR runs. Both Ct value and Tm were consistent.
Conclusion:
This PCR assay demonstrates the capability to both detect HP, and to differentiate wild-type and clarithromycin resistant mutations in the 23s rRNA gene.