Rapid colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA in gastric biopsy specimens

Abstract

A very simple, practical, sensitive, and specific colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA is described. This assay, which combines a sensitive sandwich DNA hybridization reaction and a colorimetric protocol similar to those used in conventional enzyme immunoassays, was shown to be suitable for detecting H. pylori-infected gastric biopsy specimens and for monitoring the eradication of the pathogen after treatment. The specificity and sensitivity of the colorimetric hybridization assay were tested by assaying 27 H. pylori strains (4 reference and 23 clinical isolates), 9 strains of other Helicobacter spp. or Campylobacter spp., and 11 clinical isolates of other urease-positive bacteria. The likelihood of H. pylori detection in gastric biopsy specimens by the colorimetric hybridization assay was evaluated with 23 H. pylori-positive and 41 H. pylori-negative biopsy specimens on the basis of positive and negative results, respectively, of culture, rapid urease test, histological examination, and PCR. Biopsy specimens from 33 treated patients, endoscopied 4 to 8 weeks after the end of treatment, were also tested. All H. pylori strains showed positive results in the colorimetric hybridization assay, presenting optical densities at 450 nm (OD 450 s) of >3.0. None of the other Helicobacter spp., Campylobacter spp., or the clinical isolates of other urease-positive bacteria showed OD 450 s equal to or greater than the cutoff (mean OD 450 cutoff, 0.208). The colorimetric hybridization assay detected all 23 H. pylori-positive biopsy specimens (mean OD 450 , 2.910 ؎ 0.295), while none of the H. pylori-negative biopsy specimens was shown to be positive in the assay (mean OD 450 , 0.108 ؎ 0.025). H. pylori was considered to be not eradicated from three of the posttreatment biopsy specimens by culture, rapid urease test, histological examination, and PCR. They were all positive by the colorimetric hybridization assay, and their OD 450 s were >3.0. The colorimetric hybridization assay also detected two other H. pyloripositive patients. Specimens from these two patients had negative culture, rapid urease test, and histology results, and a specimen from one of them also tested negative by PCR. These results indicate that the colorimetric hybridization assay is a suitable method both for the diagnosis of H. pylori in biopsy specimens and for the follow-up of patients after the end of treatment.

Detection of Point Mutations Associated with Resistance of Helicobacter pylori to Clarithromycin by Hybridization in Liquid Phase

“…The sensitivity and the specificity of the method were determined. We detected a 10-fold increase in amplicons with the hybridization reaction compared to the electrophoresis gel, as was reported in previous studies (11,15). The limit of detection of this assay was 50 pg of specific DNA.…”

Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori

“…Sensitivity and species specificity of PCR. The lower limit of detection by the single-step PCR with ureA or ssp primers was 50 to 500 fg of genomic DNA, theoretically corresponding to about 3 ϫ 10 1 to 3 ϫ 10 2 helical bacteria containing 1.8 fg of DNA/cell (20). Specific PCR products were not obtained when DNAs from 36 other species of bacteria, including Helicobacter fennelliae and Helicobacter cinaedi, were tested as described by Kawamata et al (18).…”

“…The PCR mixture contained 5 l of 10ϫ PCR buffer (N808-0160; Perkin-Elmer Cetus), 4 l of 2.5 mM deoxynucleoside triphosphate, 2 l of 10 M oligonucleotide primers, 1 U of AmpliTaq polymerase (Perkin-Elmer Cetus), 37.8 l of molecular-biology-grade distilled water, and 1 l of sample DNA. Prior to the addition of sample DNA, each reaction mixture was overlaid with a drop of mineral oil (Sigma) (20). The mixtures were incubated for 5 min at 94°C for initial denaturation of the target DNA and then subjected to 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C (ureA) or 53°C (ssp) for 1 min, and extension at 72°C for 1 min (18).…”

“…H. pylori colonizes the mucosa of the human stomach and causes nonspecific chronic gastritis as well as, probably, gastroduodenal ulcers and carcinoma (20,21). In developed countries, H. pylori seropositivity is found in 40 to 50% of the population aged 20 to 60 years (21), but the mode of spread remains unclear (3).…”

Comparative Evaluation of Colorimetric Microtiter Plate Systems for Detection of Herpes Simplex Virus in Cerebrospinal Fluid

“…This step, however, takes an additional 12 to 24 h to complete, delaying the use of test results for clinical intervention. The ideal postamplification detection system would combine the increased sensitivity and specificity of Southern blotting with a rapid turnaround time (2,14,19,20,32). For this purpose, enzyme-linked adsorbent microtiter plate (MTP) systems have been adapted for amplicon detection (40).…”