The application of single-strand conformation polymorphism (SSCP) technique for the analysis of molecular heterogeneity of grapevine virus A

Abstract

The results of the analysis of grapevine virus A (GVA) isolates by single-strand conformation polymorphism (SSCP) confirm that this technique is very helpful in rapid and relatively low cost preliminary analysis of molecular heterogeneity of viruses. The results clearly show that the reliability of SSCP analysis of GVA depends on oligonucleotide primers for successful RT-PCR amplification of the highest possible number of molecular variants of the virus. Among 7 pairs of GVA-specific primers designed in different laboratories only two, those from Canada (117038 and C7273) and Switzerland (MP and CPdt), allowed positive RT-PCR amplification of all our isolates of the virus mechanically transmitted from various grapevines to Nicotiana benthamiana. With SSCP analysis of 238 bp DNA fragments complementary to part of ORF5 of GVA, produced by RT-PCR using the first pair of primers, we were able to detect 1-35 nt differences between GVA isolates. The DNA fragments, about 986 bp, complementary to part of ORF3 and ORF4, ORF5 and 3'UTR of GVA, produced by RT-PCR using the second pair of primers, were useful for SSCP analysis only after their digestion with the restriction enzyme DdeI. The results strongly suggest that SSCP analysis of 238 nt fragment of ORF5 of GVA along with DdeI/SSCP analysis of about 986 nt 3'terminal fragment of the virus allow rapid and reliable determination of the number of dominant nt sequence variants of GVA present in a single N. benthamiana or grapevine plant.