title: Nested reverse transcriptase-polymerase chain reaction (RT-PCR) for typing ruminant pestiviruses: bovine viral diarrhea viruses and border disease virus.
abstract: A nested reverse transcription (RT) polymerase chain reaction (PCR) assay was evaluated for differentiating reference bovine viral diarrhea virus (BVDV) strains, BVDV from diagnostic accessions, modified-live virus (MLV) BVDV strains in bovine viral vaccines, and a reference border disease virus (BDV). The detection level of this assay was compared to viral infection in cell culture. The PCR assay was used to distinguish 3 ruminant pestiviruses, types 1 and 2 BVDV, and type 3 BDV. The consensus (first) PCR assay detected all 3 ruminant pestiviruses, a result of the shared sequence homology. The consensus PCR product was subjected to a second (nested) PCR which used type-specific primers. The nested PCR was able to differentiate the 3 ruminant pestiviruses. Viral stocks of BVDV were diluted 10-fold and processed for the 2-step PCR assay. The sensitivity of this 2-step PCR assay was compared to viral infectivity in cell culture based on identical volumes of the system tested (cell culture assay and processing for RNA). The RT-PCR type-specific assay differentiated BVDV laboratory reference strains (12), diagnostic laboratory isolates (15), 2 MLV BVDV vaccine strains, and a BDV strain. The 30 ruminant pestiviruses typed included: (1) 27 reference strains and diagnostic laboratory isolates; 18 cytopathic (CP) type 1 strains, 3 CP type 2 strains, 3 noncytopathic (NCP) type 1 strains, and 3 NCP type 2 strains; (2) 2 MLV strains, type 1; and (3) 1 CP BDV type 3. The PCR assay had a detection limit of 10 TCID50/0.025 mL of virus when 3 separate BVDV were tested. This 2 step RT-PCR assay would be useful for the typing of ruminant pestiviruses, particularly BVDV isolates from the diagnostic laboratory.
authors.affiliation: Department of Infectious Diseases and Physiology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078, USA. rfulton@okway.okstate.edu
authors.forename: R W
authors.initials: RW
authors.lastname: Fulton
authors.forename: J M
authors.initials: JM
authors.lastname: d'Offay
authors.forename: J T
authors.initials: JT
authors.lastname: Saliki
authors.forename: L J
authors.initials: LJ
authors.lastname: Burge
authors.forename: R G
authors.initials: RG
authors.lastname: Helman
authors.forename: A W
authors.initials: AW
authors.lastname: Confer
authors.forename: S R
authors.initials: SR
authors.lastname: Bolin
authors.forename: J F
authors.initials: JF
authors.lastname: Ridpath
mesh_terms: D000818:Animals; D001681:Biological Assay; D018083:Border disease virus / Q000145:classification* / Q000235:genetics; D002417:Cattle; D002418:Cattle Diseases / Q000145:classification / Q000175:diagnosis / Q000821:virology*; D003937:Diagnosis, Differential; D001908:Diarrhea Viruses, Bovine Viral / Q000145:classification* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction