Quadriplex Real-Time PCR Assay Using allele specific Scorpion Primers for detection of mutations conferring Clarithromycin resistance in Helicobacter pylori

Université de Poitiers, EA 3807, Poitiers, 86021, France; CHU Poitiers, Laboratoire de Bactériologie, Poitiers, 86021, France; Université de Poitiers, EA 3806, Poitiers, 86021, France; CHU de Poitiers, Service d'Hépato-gastroentérologie, Poitiers, 86021, France

^* To whom correspondence should be addressed. Email: c.burucoa{at}chu-poitiers.fr.

	Abstract

We developed a single-vessel multiplex real-time PCR assay that detect H. pylori infection and identify 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-RFLP and Scorpion-PCR. One biopsy, positive for culture, exhibits 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, sensitivity of Scorpion-PCR was 98.3%, specificity 92.5%. The Scorpion-PCR assay provides a highly accurate, rapid and precise method for the detection and determination of mutations conferring clarithromycin resistance in H. pylori.