SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS OF PCR-AMPLIFIED RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACERS TO DIFFERENTIATE SPECIES OF ASPERGILLUS SECTION FLAVI

A novel genetic approach for classifying the species of Aspergillus Se ction Flavi is described here. This approach consists of PCR amplifica tion of the 5.8S ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II) with universal primers and of analysis of the PCR product by the principle of single-strand conformation polymo rphism (SSCP), The approximately 570- to 590-bp PCR products were dena tured and subjected to electrophoresis on a polyacrylamide gel supplem ented with 20% formamide, The SSCP patterns of these species became mo re distinct by the addition of formamide to the gel and by visualizati on with ethidium bromide staining, A little interspecific length polym orphism among amplified ribosomal DNAs was enhanced to be detected by PCR-SSCP analysis, This analysis was capable of classifying 67 of the 68 Aspergillus Section Flavi strains tested into the following four gr oups, regardless of origin: A, flavus/A. oryzae, A. parasiticus/A. soj ae, A. tamarii, and A. nomius. The results of restriction fragment len gth polymorphism analysis with PCR products of the ITS regions were co nsistent with those of PCR-SSCP analysis, except for A. nomius, which was not clearly differentiated from A, parasiticus/A. sojae. Nonradiol abelled PCR-SSCP analysis is inexpensive and practical to perform with out special apparatus or skill and should assist in fungal morphologic al identification.