Fluorogenic PCR-based quantitative detection of a murine pathogen, Helicobacter hepaticus.
Helicobacter hepaticus infection in mice is being used as an animal model for elucidating the pathogenesis of gastrointestinal and biliary diseases in humans. H. hepaticus, which forms a spreading film on selective agar, is not amenable to routine quantitative counts of organisms in tissues using a CFU method. In this study, a fluorogenic PCR-based assay was developed to quantitatively detect H. hepaticus in mouse ceca and feces using the ABI Prism 7700 sequence detection system. A pair of primers and a probe for this assay were generated from the H. hepaticus cdtB gene (encoding subunit B of the H. hepaticus cytolethal distending toxin). Using this assay, the sensitivity for detection of H. hepaticus chromosomal DNA prepared from pure culture was 20 fg, which is equivalent to approximately 14 copies of the H. hepaticus genome based on an estimated genome size of approximately 1.3 Mb determined by pulsed-field gel electrophoresis. H. hepaticus present in feces and cecal samples from H. hepaticus-infected mice was readily quantified. The selected PCR primers and probe did not generate fluorescent signals from eight other helicobacters (H. canis, H. cineadi, H. felis, H. mustelae, H. nemestrinae, H. pullorum, H. pylori, and H. rodentium). A fluorescent signal 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 experimentally infected mouse models as well as in naturally infected mice.
Ge
Z
Z
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. zge@mit.edu
White
D A
DA
Whary
M T
MT
Fox
J G
JG
D000818:Animals; D002432:Cecum / Q000382:microbiology; D017931:DNA Primers; D004195:Disease Models, Animal; D005243:Feces / Q000382:microbiology; D005260:Female; D005456:Fluorescent Dyes; D016998:Helicobacter / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D016481:Helicobacter Infections / Q000382:microbiology*; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D005456:Fluorescent Dyes