title: Direct RT-PCR from serum enables fast and cost-effective phylogenetic analysis of bovine viral diarrhoea virus.
abstract: Studies of the molecular epidemiology of viral diseases are dependent on the analysis of large numbers of samples from infected individuals, and the assembly of relevant sequence databases are a prerequisite to investigate chains of infection. As part of research in support of the Scottish BVDV eradication campaign, we have established a direct RT-PCR method for the high throughput amplification and analysis of the informative 5'-untranslated region of the BVDV genome. Heat-treatment followed by a one-step RT-PCR, performed in 96-well plates, produced sufficient material for sequence analysis from 0.5 μl of serum or plasma. Of 93 samples assayed, only five failed to give full sequence data for the region amplified and these were subsequently successfully analysed in single tube format reactions. This approach improved the speed of analysis, reduced costs, operator time and the potential for contamination, and may allow analysis of samples for which volumes are too low for conventional RNA isolation. It also has the potential for wider application in both human and animal disease research in which high throughput and low cost would increase the size of datasets that can be obtained.
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Penicuik, Midlothian EH26 0PZ, UK. claudia.bachofen@moredun.ac.uk
authors.forename: Claudia
authors.initials: C
authors.lastname: Bachofen
authors.forename: Kim
authors.initials: K
authors.lastname: Willoughby
authors.forename: Ruth
authors.initials: R
authors.lastname: Zadoks
authors.forename: Paul
authors.initials: P
authors.lastname: Burr
authors.forename: Dominic
authors.initials: D
authors.lastname: Mellor
authors.forename: George C
authors.initials: GC
authors.lastname: Russell
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis* / Q000821:virology; D002417:Cattle; D030241:Diarrhea Virus 1, Bovine Viral / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D017720:Molecular Epidemiology / Q000379:methods; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D017422:Sequence Analysis, DNA; D044967:Serum / Q000821:virology*; D014730:Veterinary Medicine / Q000191:economics / Q000379:methods*
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral