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.
Jones
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LR
Instituto de Virologia, Centro de Investigación en Ciencias Veterinarias y Agronómicas, INTA-Castelar, Morón, Buenos Aires, Argentina.
Weber
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EL
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