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.

FIG. 1.

Example of a sensitivity assay performed on the SmartCycler thermocycler with 10-fold serial dilution of the genomic DNA purified from strain J99. (A) The fluorescence signal was from FAM dye of the 23SScWT-specific Scorpion primer. (B) Second derivative curves of fluorescence for FAM dye. (C) Standard curve plotting log concentrations versus C_Ts, calculated linear fit equation, and correlation coefficient. (D) Fluorescence curves for Texas red. (E) Fluorescence curves for Cy3 dye. (F) Fluorescence curves for Cy5 dye.

FIG. 2.

Distribution of C_T values determined by Scorpion PCR according to culture and PCR-RFLP results among the 119 biopsy specimens displaying a C_T value during the 50 cycles of the assay (16 biopsy specimens displayed two C_T values for two different genotypes). White bars, biopsy specimens positive for culture; black bars, biopsy specimens positive for PCR-RFLP but negative for culture; gray bars, biopsy specimens negative for culture and PCR-RFLP.