Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori

Abstract

We developed a single-vessel multiplex real-time PCR assay that detects Helicobacter pylori infection and identified the four existing alleles of the 23S rRNA genes of H. pylori-the wild-type sequence and the three mutations conferring clarithromycin resistance-using allele-specific Scorpion primers directly on biopsy specimens. The Scorpion primers combine a primer and a probe in a single molecule and are able to distinguish single-nucleotide polymorphism. Fluorescent signals, produced when the probes are annealed, are read in four channels by a SmartCycler thermocycler. The assay was first applied successfully on 4 reference and 61 clinical strains. MICs of clarithromycin were determined by the Etest method. A perfect concordance was obtained between Etest and Scorpion PCR. Mixed populations were better detected by Scorpion PCR. We examined 259 biopsies from 229 patients by culture, PCR-restriction fragment length polymorphism (RFLP), and Scorpion PCR. One biopsy, positive for culture, exhibited inhibitors for both PCR-RFLP and Scorpion PCR. Twelve biopsies were positive for PCR-RFLP and Scorpion PCR but negative for culture with concordant determination of mutations in the 23S rRNA genes by the two PCR assays. Three biopsies were positive for Scorpion PCR only. Compared to culture, the sensitivity of Scorpion PCR was 98.3% and the specificity was 92.5%. The Scorpion PCR assay provides a highly accurate, rapid, and precise method for the detection and determination of mutations conferring clarithromycin resistance to H. pylori.

Evaluation of a New Test, GenoType HelicoDR, for Molecular Detection of Antibiotic Resistance inHelicobacter pylori

“…Other genotyping methods have been developed for the detection of antibiotic resistance in H. pylori based on the LiPA technology (32,33), real-time PCR (2,18,19,27), fluorescence in situ hybridization (11), microarray (5, 35), or others (34). Most of them are in-house tests that are not commercially available, and they detect only clarithromycin resistance.…”

Chemokines and Antimicrobial Peptides Have a cag -Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

“…pylori detection and determination of cag PAI status. H. pylori detection in human gastric biopsy specimens was performed by bacterial culture and Scorpion PCR as previously described (17). H. pylori strains from human biopsy specimens were cultured, and 24-h cultures were used for DNA extraction using a MagNA Pure compact system and DNA isolation kit (Roche) according to the manufacturer's instructions.…”

Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

“…Alternatively, clinical laboratories use the Etest method as it is considered less technically demanding (21,22). However, the Etest and the agar dilution method are more tedious and time-consuming to perform than molecular methods such as PCR or fluorescence in situ hybridization (FISH) (23)(24)(25). PCR-based methods have been used as a suitable alternative (18,(25)(26)(27) but can be affected by DNA contamination or degradation (12,28).…”

“…However, the Etest and the agar dilution method are more tedious and time-consuming to perform than molecular methods such as PCR or fluorescence in situ hybridization (FISH) (23)(24)(25). PCR-based methods have been used as a suitable alternative (18,(25)(26)(27) but can be affected by DNA contamination or degradation (12,28). FISH is typically based on fluorescently labeled DNA probes that hybridize with specific rRNA sequences of microorganisms (23,29).…”