Pathogenic aspects of the secondary structure at the 5'-untranstlated regions specific to pastivirus

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Pestivirus strains isolated from diseased cattle and cell cultures were subjected to (1)nucleotide sequencing following the nested RT-PCR to amplify the 5'-untranstlated region of pestivirus genomes, (2)viral titration by using real-time PCR. Viral RNA samples were isolated by the single-step guanidinium isothiocyanate-phenol-chloroform method. The RNA solution was subsequently subjected to complementary deoxyribonucleic acids synthesis by reverse transcription. The oligonucleotide primers amplified the expected targets in real-time RT-PCR analysis using the SmartCycler. The universal primers amplified a 254 bp fragment which was evident on agarose gel electrophoresis. The conventional RT-PCR used for the specific detection of pestivirus species requires visualization step by agarose gel electrophoresis. While we describe here a rapid method to detect pestiviral RNA by real-time RT-PCR using SYBR Green I. Therefore, a simple and rapid detection and identification procedure for pestivirus strains was developed in this study.