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Journal of Clinical Microbiology, July 2008, p. 2320-2326, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.02352-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Université de Poitiers, EA 3807, Poitiers 86021, France,¹ CHU Poitiers, Laboratoire de Bactériologie, Poitiers 86021, France,² Université de Poitiers, EA 3806, Poitiers 86021, France,³ CHU de Poitiers, Service d'Hépato-gastroentérologie, Poitiers 86021, France⁴

Received 7 December 2007/ Returned for modification 22 January 2008/ Accepted 29 April 2008

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.

Published ahead of print on 7 May 2008.

Supplemental material for this article may be found at http://jcm.asm.org/.