A PCR-RFLP METHOD FOR THE DETECTION OF HELICOBACTER-HEPATICUS IN FROZEN OR FIXED LIVER FROM B6C3F(1) MICE

Establishing the diagnosis of Helicobacter hepaticus infection in mous e liver has recently become important for the interpretation of rodent carcinogenicity bioassays. A seminested primer polymerase chain react ion (PCR) amplification of the bacterial 16S ribosomal RNA gene in com bination with a restriction fragment length polymorphism (RFLP) assay was designed to identify and distinguish H. hepaticus from H. muridaru m and H. bilis in mouse liver. The PCR-RFLP assay was applied to forma lin-fixed, paraffin-embedded and, when available, corresponding frozen liver tissues from male and female B6C3F(1) mice with or without hist ologic 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 bur nor in liver fixed for several weeks. The sensitivity of the PCR-RFLP assay f or H. hepaticus on formalin-fixed, paraffin-embedded mouse liver varie d between studies from markedly decreased when compared to the results from frozen liver or histologic evaluation to nearly equivalent or mo re sensitive than histologic evaluation. The PCR-RFLP results appeared dependent on the duration of fixation and bacterial load but nor on t he presence of hepatitis, sampling from neoplastic or nonneoplastic li ver, or sex of the mouse.