Detection of clarithromycin-resistant helicobacter pylori strains by a preferential homoduplex formation assay

Abstract

It has been shown that resistance to clarithromycin, a major cause of failure in Helicobacter pylori eradication therapy, is associated with point mutations in the 23S rRNA gene. We sought to apply the preferential homoduplex formation assay (PHFA), a novel technique for the efficient detection of point mutations, to detection of the mutations. PHFA was performed on streptavidin-coated microtiter plates with biotin- and dinitrophenyl-labeled amplicons to detect the wild-type gene or each mutant gene. DNA samples were extracted from gastric juice specimens of 412 patients with H. pylori infection and were applied to the assay. The detection threshold of PHFA was as few as 10 gene copies. The sensitivity of PHFA for the detection of H. pylori infection was higher than those of culture and the rapid urease test. A total of 337 (81.8%) samples had the wild-type gene, 38 (9.2%) had the A2144G mutation, and 37 (9.0%) contained both the wild type and a mutation (A2144G in 30 samples, A2143G in 5 samples, and A2143G plus A2144G in 2 samples). About half the strains isolated from patients with mixed infection were susceptible by the agar dilution method (MIC, <0.1 mg/liter). Therefore, PHFA can detect clarithromycin-resistant H. pylori strains, even in patients with mixed infections with the wild type, that are not detectable by the agar dilution method.

FIG. 1

PCR-PHFA. Double-labeled PCR amplicons were prepared with biotin- and DNP-labeled primers (probes). Unlabeled amplicons were prepared with unlabeled primers (samples). Labeled and unlabeled amplicons were mixed and were denatured at 98°C for 10 min and were then cooled slowly to 70°C at a rate of 1°C/10 min. (A) When labeled and unlabeled amplicons were identical, the population of double-labeled double-stranded DNA decreased due to dilution with the unlabeled molecule. (B) When two amplicons differed by even a single base, preferential homoduplex formation occurred. The population of double-labeled double-stranded DNA did not decrease. Double-labeled double-stranded DNA was captured on a streptavidin-coated microtiter plate via biotin-streptavidin affinity and was quantified with alkaline phosphatase (ALP)-conjugated anti-DNP antibody and p-nitrophenyl phosphate (p-NPP) as the substrate.