Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry

E. Kosinova, I. Psikal, B. Robesova and K. Kovarcik
Additional contact information
E. Kosinova: Veterinary Research Institute, Brno, Czech Republic
I. Psikal: Veterinary Research Institute, Brno, Czech Republic
B. Robesova: Veterinary Research Institute, Brno, Czech Republic
K. Kovarcik: Veterinary Research Institute, Brno, Czech Republic

Veterinární medicína, 2007, vol. 52, issue 6, 253-261

Abstract: Quantitative real-time RT-PCR (qRT-PCR) assay was developed for the detection and quantification of bovine viral diarrhea virus (BVDV) in clinical samples from persistently infected cattle. qRT-PCR was optimized to quantify the number of BVD virus copies using Light Cycler® detection system and intercalation fluorogenic dye SYBR Green I. A universal set of primers was selected from a highly conserved 5' untranslated region (5'UTR) to detect BVDV type I and II simultaneously. Quantification of BVDV cDNA was accomplished using a calibration curve generated from 10-fold serial dilutions of standard plasmid DNA in the range 1-108 copies/μl. Analysis of 290 bp amplicons enabled monitoring of the viral RNA/BVDV level in a total of five BVDV strains (BVD-NADL, A03/3004, DB03/2943, KA04/3124, KV05/3412) and sixteen bulk milk samples, and in bovine sera of persistent carriers originating from Czech farms, as well as in a batch of calf serum for cell culture. Melting temperatures of amplicons (Tm) of BVDV strains of the same genotype group I as the NADL reference strain showed variability of the thermal points, however significant differences were observed in Tm values between the representatives of genotype group I and II. Low concentrations of BVD virus in bulk milk samples were also qualitatively identified by conventional RT-PCR. Highly reproducible data were obtained as the coefficients of variation of threshold cycles values in intra-assay and inter-assay were less than 0.85% and 2.76%, respectively. The results give enough evidence of suitability of qRT-PCR assay for quantitative analysis of BVDV in clinical samples.

Keywords: bovine viral diarrhea virus; RNA; real-time RT-PCR; SYBR Green I (search for similar items in EconPapers)
Date: 2007
References: View complete reference list from CitEc
Citations:

Downloads: (external link)
http://vetmed.agriculturejournals.cz/doi/10.17221/1882-VETMED.html (text/html)
http://vetmed.agriculturejournals.cz/doi/10.17221/1882-VETMED.pdf (application/pdf)
free of charge

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:caa:jnlvet:v:52:y:2007:i:6:id:1882-vetmed

DOI: 10.17221/1882-VETMED

Access Statistics for this article

Veterinární medicína is currently edited by Ing. Helena Smolová Ph.D.

More articles in Veterinární medicína from Czech Academy of Agricultural Sciences
Bibliographic data for series maintained by Ivo Andrle ().