Vet Med - Czech, 2007, 52(6):253-261 | DOI: 10.17221/1882-VETMED

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

E. Kosinova, I. Psikal, B. Robesova, K. Kovarcik

Veterinary Research Institute, Brno, Czech Republic

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

Published: June 30, 2007  Show citation