Detection and discrimination of four Aspergillus section Nigri species by PCR

Species of Aspergillus section Nigri are not easily distinguished by traditional morphological techniques, and typically are identified by DNA sequencing methods. We developed four PCR primers to distinguish between Aspergillus niger, Aspergillus welwitschiae, Aspergillus carbonarius and Aspergillus tubingensis, based on species-conserved differences in the calmodulin gene sequence. PCR amplification from total DNA using these primers was species specific; no amplification occurred from nontarget species DNA for each primer pair. Species-specific PCR could distinguish between species in mixed DNA templates, indicating a utility in determining culture uniformity of isolated Aspergillus strains. In addition, with these primer sets, each species could be detected in soil following mixed-species inoculation with Aspergillus spores. This indicates that PCR with these species-specific primers may be useful in determining the distribution of Aspergillus species in environmental samples without the need for species identification from isolated strains, as well as detecting species that may be infrequently isolated by culture-based methods. Significance and Impact of the StudyAspergillus section Nigri includes several species that produce ochratoxin A and fumonisin, two mycotoxins impacting food safety and human health. However, identification of these species is difficult without DNA sequencing. Using species-specific calmodulin gene sequences, we designed PCR primers to distinguish among Aspergillusniger, Aspergillus welwitschiae, Aspergillus carbonarius and Aspergillus tubingensis as accurately and more quickly than by sequence analysis. We demonstrated that these PCR methods can be used to detect and identify each species within mixed populations without fungal isolation. This will facilitate assessing at-risk foods for potential mycotoxin contamination by these species.