PCR-Denaturing Gradient Gel Electrophoresis and Two Feces Antigen Tests for Detection of Helicobacter pylori in Mice

Abstract

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57Bl/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.

Significance of Transiently Positive Enzyme-Linked Immunosorbent Assay Results in Detection of Helicobacter pylori in Stool Samples from Children

“…Thus, it remains possible that these Helicobacter species were coincidental, rather than causal, for initial antigen positivity. In a rodent model, however, Sjunnesson and colleagues found significant cross-reactivity in HpSA between the H. pylori and other Helicobacters, including H. bilis and H. canis (H. winghamensis and 99-5507 were not tested) (22). Thus, we believe between 6.8% and 50% of stool Helicobacters in children represent non-pylori species and that these may create false-positive antigen tests.…”

Analysis of allelic variants of rdxA associated with metronidazole resistance in Helicobacter pylori: detection of common genotypes in rdxA by multiplex allele-specific polymerase chain reaction

“…Molecular characterization of sensitive and resistant strains has yielded information that could be useful in assessing the genotypic association with Mtz resistance, phylogenetic evolution, and epidemiological study. Some of the molecular techniques used to study genotypic variations include sequencing, rapid amplified polymorphic DNA (RAPD), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), real-time-PCR, denaturing gel gradient electrophoresis, ribotyping, and pulse field gel electrophoresis; however, these methods are sophisticated (requiring high-end instrumentation), time-consuming, and expensive (Kansau et al, 1996;Tee, 1997;Sjunnesson et al, 2003;Rimbara et al, 2013).…”

Prevalence of Helicobacter pylori vacA and cagA Genotypes in Ethiopian Dyspeptic Patients

“…Estimation of size of the PCR products was done by using Gene ruler 100-bp DNA ladder (Fermentas, Vilnius, Lithuania). The products of each PCR assay were visualized by electrophoresis in a 1.5% agarose gel containing ethidium bromide (15).…”

“…Denaturing gradient gel electrophoresis (DGGE) analysis of the PCR products was performed in a DCode system (Bio-Rad, Hercules, Calif.) as recently described (1 vacA and cagA genotyping. Detection of H. pylori vacA and cagA genes was performed on gastric biopsy specimens and isolates positive for H. pylori by PCR-DGGE as previously described (15,19). As a positive control, H. pylori (CCUG 17874) DNA (ϳ0.1 ng) was added to the reaction mixture, while 5 l of sterile deionized Millipore-filtered water was added to the reaction mixture as a negative control.…”