Fluorogenic PCR-based quantitative detection of a murine pathogen, Helicobacter hepaticus

Helicobacter hepaticus infection in mice is being used as an animal model f or elucidating the pathogenesis of gastrointestinal and biliary diseases in humans. H, hepaticus, which forms a spreading film on selective agar, is n ot amenable to routine quantitative counts of organisms in tissues using a CFU method. In this study, a fluorogenic PCR-based assay was developed to q uantitatively detect H. hepaticus in mouse ceca and feces using the ABI Pri sm 7700 sequence detection system. A pair of primers and a probe for this a ssay were generated from the H. heparicus cdtB gene (encoding subunit B of the H, hepaticus cytolethal distending toxin), Using this assay, the sensit ivity for detection of H. hepaticus chromosomal DNA prepared from pure cult ure was 20 fg. which is equivalent to approximately 14 copies of the H, hep aticus genome based on an estimated genome size of approximate to1.3 Mb det ermined by pulsed-field gel electrophoresis, H. hepaticus present in feces and cecal samples from H, hepaticus-infected mice was readily quantified. T he selected PCR primers and probe did not generate fluorescent signals from eight other helicobacters (H, canis, H, cineadi, H.felis, Ii. mustelae, H, nemestrinae, H, pullorum, H. pylori, and H, rodentium). A fluorescent sign al was detected from 20 ng of H. bilis DNA but with much lower sensitivity (10(6)-fold) than from H, hepaticus DNA, Therefore, this assay can be used with high sensitivity and specificity to quantify H. hepaticus in experimen tally infected mouse models as well as in naturally infected mice.