title: A universal 'one-tube' RT-PCR protocol for amplifying isolates of bovine viral diarrhoea virus.
abstract: The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5'-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, ERNs, E1 and E2 genes: (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10(6) infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.
authors.affiliation: Institute for Medical Microbiology, Infectious and Epidemic Diseases, Veterinary Faculty, Ludwig-Maximilians University, Munich, Germany. Martin.Pfeffer@micro.vetmed.uni-muenchen.de
authors.forename: M
authors.initials: M
authors.lastname: Pfeffer
authors.forename: M V
authors.initials: MV
authors.lastname: Freyburg
authors.forename: O R
authors.initials: OR
authors.lastname: Kaaden
authors.forename: M
authors.initials: M
authors.lastname: Beer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis*; D002417:Cattle; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D001908:Diarrhea Viruses, Bovine Viral / Q000737:chemistry / Q000145:classification* / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D019085:Fluorescent Antibody Technique, Direct / Q000662:veterinary; D014644:Genetic Variation / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D017422:Sequence Analysis, DNA; D012845:Sindbis Virus / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral