Application of single-strand conformation polymorphism to the study of bovine viral diarrhea virus isolates.
Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products is a genetic screening technique for rapid detection of nucleotide substitutions in PCR-amplified genomic DNA or cDNA. It is based on the observation that partially formamide-denatured double-stranded DNA migrates as 2 single-stranded DNA molecules when electrophoresed in nondenaturing polyacrylamide gels. The mobility depends on the 3-dimensional conformation of the strand under the conditions used. It is possible to discriminate between DNA strands differing in only 1 nucleotide. The method was applied to the analysis of Bovine Viral Diarrhea Virus (BVDV) isolates. Reference and Argentinian strains were assessed for variations in their 5' untranslated region (5'-UTR). The PCR products of the 5'-UTR ends were formamide denatured and compared by SSCP analysis in nondenaturing 15% polyacrylamide and 15% polyacrilamide-5% glycerol gels. The reference strains SD-1, Singer, and Oregon C24V had differences in electrophoretic patterns. Despite the high conservation among the 5'-UTR of pestiviruses, the method allowed discrimination among all 9 Argentinian isolates. The 5'-UTR of a fetal kidney-derived isolate (1R93) was PCR amplified and cloned in a plasmid vector; the SSCP analysis of 30 PCR products obtained by direct amplification over randomly selected clones produced 5 different banding patterns, indicating the existence of viral quasispecies. The results show that SSCP may be used to identify and differentiate among BVDV isolates.
Instituto de Virologia, Centro de Investigación en Ciencias Veterinarias y Agronómicas, INTA-Castelar, Morón, Buenos Aires, Argentina.
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LR
Jones
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EL
Weber
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis* / Q000235:genetics; D002417:Cattle; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics*; D007668:Kidney / Q000473:pathology / Q000821:virology; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000662:veterinary; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D020121:5' Untranslated Regions