Fachbereich Veterinärmedizin



    Effect of formalin fixation and long-term storage on the detectability of bovine viral-diarrhoea-virus (BVDV) RNA in archival brain tissue using polymerase chain reaction (1994)

    Zeitschriftenartikel / wissenschaftlicher Beitrag
    Gruber, A D (WE 12)
    Moennig, V
    Hewicker-Trautwein, M
    Trautwein, G
    Zentralblatt für Veterinärmedizin. Reihe B. Journal of veterinary medicine. Series B; 41(10) — S. 654–661
    ISSN: 0514-7166
    Pubmed: 7597858
    Institut für Tierpathologie

    Robert-von-Ostertag-Str. 15
    Gebäude 12
    14163 Berlin
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    Abstract / Zusammenfassung

    Detection of DNA or RNA in formalin-fixed, paraffin-embedded tissues using polymerase chain reaction (PCR) may be hindered by degradation of nucleic acids during tissue collection, preparation and archivation. This study describes investigations on the effect of formalin fixation and prolonged storage of paraffin-embedded tissues on bovine viral-diarrhoea (BVD)-virus RNA as a model system. Brain tissues from eight persistently BVDV-infected calves containing high amounts of the virus were fixed in 5% neutral-buffered formalin or 10% non-buffered formalin for different fixation times, respectively, and paraffin embedded. Subsequent detection of an 803 bp fragment from single tissue sections using nested PCR after reverse transcription (nested RT-PCR) demonstrated a loss of detectability of viral RNA after more than 10 days (10% non-buffered formalin) and 3 months (5% neutral-buffered formalin) of fixation. Additional studies with 280 initially BVDV-positive brain tissues from 25 persistently BVDV-infected calves after storage of up to 10 years revealed a loss of detectable RNA after more than 1 year of storage. For estimation of the higher sensitivity of nested RT-PCR compared to single step RT-PCR, serially diluted BVD virus suspensions were examined using both methods. Nested RT-PCR was found to be about 100-fold more sensitive than single-step RT-PCR, and is therefore recommended as the appropriate technique for archival studies.