title: Cross-priming amplification for detection of bovine viral diarrhoea virus species 1 and 2.
abstract: OBJECTIVE
  The aim of the study was the development of cross-priming amplification for ubiquitous detection of bovine viral diarrhoea virus (BVDV) species 1 and 2.
  
  
  RESULTS
  Three and five specific primers, respectively, for the detection of BVDV-1 and BVDV-2, were designed on the basis of the sequences of the 5'UTR region. Incubation temperature and reaction time were determined. The optimal incubation conditions using water bath were 63°C for 75 min. Reverse transcription step (RT) was not required. The results were visualized under UV-light as a bright yellow fluorescence in positive samples. Additional method for results interpretation was agarose gel electrophoresis. Positive samples showed the presence of ladder-like banding patterns, formed by harpin-like cross-priming amplification (CPA) products. Sensitivity of CPA was compared with conventional RT-PCR and real-time RT-PCR. The CPA detection limit was 3500 copies for BVDV-1 and 80000 copies for BVDV-2 per reaction. For RT-PCR it was 350 and 80 copies for BVDV-1 and BVDV-2, respectively, and for real-time RT-PCR it was 35 copies for BVDV-1 and 80 copies for BVDV-2. The sensitivity of the developed method is sufficient to detect persistently infected (PI) animals. Positive results were found in 24 of 25 BVDV isolates belonging to species 1 and 2. Additionally, one false-negative result for BVDV-2 was detected. There were no false-positive results in negative samples and in the negative control. Both sets of primers used for the detection of BVDV-1 and BVDV-2 were not able to detect atypical pestiviruses. CPA positive results were confirmed by RT-PCR and real-time RT-PCR.
  
  
  CONCLUSIONS
  CPA is a rapid method for the detection of BVDV-1 and BVDV-2 in field samples from PI animals.
  
  
  CONCLUSIONS
  This is the first report on the application of the CPA method for the detection of BVDV.
authors.affiliation: Department of Virology, National Veterinary Research Institute, Puławy, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Kuta
authors.affiliation: Department of Swine Diseases, National Veterinary Research Institute, Puławy, Poland.
authors.forename: G
authors.initials: G
authors.lastname: Woźniakowski
authors.affiliation: Department of Virology, National Veterinary Research Institute, Puławy, Poland.
authors.forename: M P
authors.initials: MP
authors.lastname: Polak
mesh_terms: D000818:Animals; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis / Q000821:virology*; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D030241:Diarrhea Virus 1, Bovine Viral / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D030242:Diarrhea Virus 2, Bovine Viral / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D030243:Hemorrhagic Syndrome, Bovine / Q000175:diagnosis / Q000821:virology*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010802:Phylogeny
chemical_list: D017931:DNA Primers
keywords: BVDV-1 and BVDV-2; bovine viral diarrhoea virus; cross-priming 3 amplification; detection; isothermal amplification