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 Section Flavi is described here. This approach consists of PCR amplification 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 polymorphism (SSCP). The approximately 570- to 590-bp PCR products were denatured and subjected to electrophoresis on a polyacrylamide gel supplemented with 20% formamide. The SSCP patterns of these species became more distinct by the addition of formamide to the gel and by visualization with ethidium bromide staining. A little interspecific length polymorphism 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 groups, regardless of origin: A. flavus/A. oryzae, A. parasiticus/A. sojae, A. tamarii, and A. nomius. The results of restriction fragment length polymorphism analysis with PCR products of the ITS regions were consistent with those of PCR-SSCP analysis, except for A. nomius, which was not clearly differentiated from A. parasiticus/A. sojae. Nonradiolabelled PCR-SSCP analysis is inexpensive and practical to perform without special apparatus or skill and should assist in fungal morphological identification.
Kumeda
Y
Y
Osaka Prefectural Institute of Public Health, Japan.
Asao
T
T
D001230:Aspergillus / Q000145:classification*; D001483:Base Sequence; D004271:DNA, Fungal / Q000032:analysis*; D004275:DNA, Ribosomal / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational
D004271:DNA, Fungal; D004275:DNA, Ribosomal