title: Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts.
abstract: BACKGROUND
  The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set.
  
  
  RESULTS
  Numerous sequences for PCR primers were tested to develop PCR assays with a wide range of fungal compatibility and high discrimination from plant DNA. A nested set of 4 primers was developed that reflected these criteria and performed well amplifying ITS regions of fungal rDNA. Primers in the 5.8S sequence were also developed that would permit separate amplifications of ITS1 and ITS2. A range of basidiomycete fruiting bodies and ascomycete cultures were analyzed with the nested set of primers and Restriction Fragment Length Polymorphism (RFLP) fingerprinting to demonstrate the specificity of the assay. Single ectomycorrhizal root tips were similarly analyzed. These primers have also been successfully applied to Quantitative PCR (QPCR), Length Heterogeneity PCR (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses of fungi. A set of wide-range plant-specific primers were developed at positions corresponding to one pair of the fungal primers. These were used to verify that the host plant DNA was not being amplified with the fungal primers.
  
  
  CONCLUSIONS
  These plant primers have been successfully applied to PCR-RFLP analyses of forest plant tissues from above- and below-ground samples and work well at distinguishing a selection of plants to the species level. The complete set of primers was developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of fungi where samples also contain high levels of background plant DNA, such as verifying ectomycorrhizal morphotypes or characterizing phylosphere communities.
authors.affiliation: Dynamac Corporation, National Health and Environmental Effects Research Laboratory, Corvallis, OR, USA. kendall@lifetime.oregonstate.edu
authors.forename: Kendall J
authors.initials: KJ
authors.lastname: Martin
authors.forename: Paul T
authors.initials: PT
authors.lastname: Rygiewicz
mesh_terms: D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics*; D021901:DNA, Intergenic; D005658:Fungi / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021901:DNA, Intergenic