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

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 (R) 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 - 10(8) copies/mu l. 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.