Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR

Authors: Palumbo, Jeffrey - Jeff; Sarreal, Siov; Kim, Jong Heon

Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2023
Publication Date: 12/18/2023
Citation: Palumbo, J.D., Sarreal, S.L., Kim, J. 2023. Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR. Letters in Applied Microbiology. 76(12). Article ovad142. https://doi.org/10.1093/lambio/ovad142.
DOI: https://doi.org/10.1093/lambio/ovad142

Interpretive Summary: Aspergillus ochraceus, A. westerdijkiae and A. steynii are species belonging to section Circumdati that produce ochratoxin. These species, and aflatoxin-producing species Aspergillus flavus and A. parasiticus, are the major sources of potential mycotoxin contamination of tree nuts and other crops. We developed a digital PCR assay to identify each of these species in tree nut orchard soil. We demonstrated that this assay is specific to the target species we aim to detect, and can be used to measure relative population sizes of each species in a mixed sample, by measuring relative amounts of each species DNA. This quantitative digital PCR assay provides a way to measure populations of ochratoxin- and aflatoxin-producing species in orchard environments without the need for isolating individual fungi and identifying them through microbiological techniques.

Technical Abstract: Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR assay to detect and quantify Aspergillus ochraceus, A. westerdijkiae, A. steynii, A. flavus, and A. parasiticus in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with relative DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi, and Nigri were generally not detectable using this dPCR method. Detectable non-target species (A. fresenii, A. melleus, A. sclerotiorum, and A. subramanianii) were discernable from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity.