Detection of bovine viral diarrhea virus RNA in formalin-fixed, paraffin-embedded brain tissue by nested polymerase chain reaction.
Isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissues is delicate due to its fragility and ubiquitous ribonucleases. For retrospective studies, however, a convenient procedure for the detection of RNA in archived material is of great value. Bovine viral diarrhea (BVD) virus is a member of the pestivirus genus in the family Flaviviridae. Different protocols for the isolation of BVD virus RNA from fresh and autolytic as well as from routinely formalin-fixed and paraffin-embedded brain tissue of BVDV-infected calves were compared. The polymerase chain reaction (PCR) after reverse transcription (RT-PCR) was carried out subsequently for the detection of viral RNA. Using proteinase K digestion of deparaffinized tissue sections without additional ribonuclease inhibitors and subsequent nested PCR, a 803 bp fragment of the gene coding for the nonstructural protein p125 of BVD virus could be consistently detected. In addition, BVD virus RNA was detected by RT-PCR from non-fixed brain tissue after 10 days of autolysis.
Gruber
A D
AD
Institute of Pathology, School of Veterinary Medicine, Hannover, Germany.
Greiser-Wilke
I M
IM
Haas
L
L
Hewicker-Trautwein
M
M
Moennig
V
V
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000382:microbiology*; D001921:Brain / Q000382:microbiology*; D002417:Cattle; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification*; D005557:Formaldehyde; D008969:Molecular Sequence Data; D010232:Paraffin; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D016707:Tissue Fixation
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D005557:Formaldehyde; D010232:Paraffin