A PCR-RFLP method for the detection of Helicobacter hepaticus in frozen or fixed liver from B6C3F1 mice.
Establishing the diagnosis of Helicobacter hepaticus infection in mouse liver has recently become important for the interpretation of rodent carcinogenicity bioassays. A seminested primer polymerase chain reaction (PCR) amplification of the bacterial 16S ribosomal RNA gene in combination with a restriction fragment length polymorphism (RFLP) assay was designed to identify and distinguish H. hepaticus from H. muridarum and H. bilis in mouse liver. The PCR-RFLP assay was applied to formalin-fixed, paraffin-embedded and, when available, corresponding frozen liver tissues from male and female B6C3F1 mice with or without histologic evidence of infection from various National Toxicology Program 2-yr bioassay studies. PCR products consistent with H. hepaticus were detected in 10-80% of livers from mice in studies with other evidence of infection that were frozen or fixed for less than 24 hr but not in liver fixed for several weeks. The sensitivity of the PCR-RFLP assay for H. hepaticus on formalin-fixed, paraffin-embedded mouse liver varied between studies from markedly decreased when compared to the results from frozen liver or histologic evaluation to nearly equivalent or more sensitive than histologic evaluation. The PCR-RFLP results appeared dependent on the duration of fixation and bacterial load but not on the presence of hepatitis, sampling from neoplastic or nonneoplastic liver, or sex of the mouse.
Malarkey
D E
DE
Department of Microbiology, Pathology, and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh 27606, USA. David_Malarkey@ncsu.edu
Ton
T V
TV
Hailey
J R
JR
Devereux
T R
TR
D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis*; D005260:Female; D005557:Formaldehyde; D005629:Frozen Sections; D016998:Helicobacter / Q000235:genetics*; D016481:Helicobacter Infections / Q000473:pathology; D008099:Liver / Q000382:microbiology* / Q000473:pathology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D016707:Tissue Fixation
D004269:DNA, Bacterial; D005557:Formaldehyde