38th Annual Meeting of the Society for Invertebrate Pathology

Classical single-strand conformational polymorphism (SSCP) analysis is based on the observation that single-stranded DNA fragments attain a number of conformational forms which may be separated by electrophoresis in native polyacrylamide gels giving a characteristic pattern of electrophoretic bands. Even minute sequence changes (e.g. point mutations) may have significant effect on electrophoretic pattern of single-stranded DNA. Changes of gel temperature during electrophoresis increase the sensitivity of mutation detection in PCR products; this technique was named MSSCP (where M stands for "multitemperature"). We have applied this method modified in our laboratories for characterization of baculovirus DNA fragments. A series of degenerate primers were synthesized after the comparison of the polyhedrin gene sequences of over 20 baculoviruses. Two sets of oligonucleotides were used as universal primers in PCR reactions containing genomic DNA from an array of nucleopolyhedrosis viruses including these of Autographa californica, Anticarsia gemmatalis, Spodoptera frugiperda, Lymantria dispar, Lymantria monacha and many others. PCR products were denatured and subjected to MSSCP electrophoresis where, after silver staining, they gave ssDNA band patterns characteristic for each baculovirus species. This technique can be potentially applied to detect baculoviruses in insects collected in the field, as well as to plant tissues and the excrements or bodies of predators without need for sequencing the PCR products. Often MSSCP can be used not only for species determination but also as an indication of genomic variability which can be related to infectivity