Design of a primer for ribosomal DNA internal transcribed spacer with enhanced specificity for ascomycetes

A primer able to amplify the internal transcribed spacers (ITS) of the ribosomal DNA (rDNA), having enhanced specificity for ascomycetes, was identified by reviewing fungal ribosomal DNA sequences deposited in GenBank. The specificity of the primer, named ITS4A, was tested with DNA extracted from several species of ascomycetes, basidiomycetes, zygomycetes, mastigomycetes and mitosporic fungi (formerly deuteromycetes) and also from plants. The PCR annealing temperature most specific for ascomycetes was found to be 62 degrees C and 64 degrees C for the primer pairs ITS5 + ITS4A and ITS1F + ITS4A, respectively. At these annealing temperatures, all ascomycetous DNA samples were amplified efficiently with the ITS4A primer. The sensitivity limit was in the range 10(-14) g of DNA. This primer could also provide useful tools in suggesting the affinities of many mitosporic fungi with their perfect states.