Medycyna Wet. 62 (2), 165-169, 2006
Stadejek T., Podgórska K., Pejsak Z.
Applying Real Time PCR for detecting pestiviruses
Pestiviruses cause serious diseases in domestic and wild ruminants and swine. The aim of the study was to apply Real Time PCR with SYBR Green I intercalating dye for detecting pestiviruses. The study was performed with RNA extracted from BVDV-1 cell culture and CSFV, DNA templates with known copy numbers synthesized from CSFV, BVDV-1 and BDV and on cDNA samples representing different pestivirus species and strains. Two PCR kits were used in the study: AmpliTaq Gold (Applied Biosystems) with added fluorescent dyes, and QuantiTect SYBR Green PCR kits (Qiagen). PCR primers were selected from 5’UTR. All pestivirus species, or species-related samples were detected using the applied method and the results were observed as amplification curves. The specificity of amplification was confirmed by estimating the melting temperature of the PCR products. It was demonstrated that the melting temperature of amplicons obtained in reaction with QuantiTect kit was 86-87°C while those obtained with AmpliTaq Gold was 90-92°C. A higher assay sensitivity of 40 copies of CSFV, 400 copies of BVDV and 40 000 copies of BDV templates was obtained using the QuantiTect SYBR Green PCR kit. It should be stressed that the above method does not facilitate pestivirus species identification and may only be a preliminary method for detecting pestivirus infections in swine and must be supplemented with CSFV specific assay.
Keywords: pestivirus, Real Time PCR