Location: Foodborne Toxin Detection and Prevention ResearchTitle: Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil
|Hua, Sui Sheng|
|Palumbo, Jeffrey - Jeff|
|PARFITT, DAN - University Of California, Davis|
|O Keeffe, Teresa|
Submitted to: Mycotoxin Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/13/2018
Publication Date: 3/26/2018
Publication URL: http://handle.nal.usda.gov/10113/6033230
Citation: Hua, S.T., Palumbo, J.D., Parfitt, D., Sarreal, S.L., O'Keeffe, T.L. 2018. Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil. Mycotoxin Research. 34(3):187-194. https://doi.org/10.1007/s12550-018-0313-6.
Interpretive Summary: The main objectives of this study were to develop a ddPCR assay and design primer/TaqMan probe sets specifically used for differentiating and quantifying both atoxigenic and aflatoxigenic strains in soil system. The traditional microbilogical methods are laborious and impractical for soil samples as well as large numbers of samples. This method eliminates the need for isolation and identification of individual fungal isolates by molecular markers. ddPCR assays allows for rapid and accurate determination population sizes of each atoxigenic and aflatoxigenic strains in soil environment. The ddPCR assay can be used in multiples ways in biocontrol research for elimination of aflatoxin in crops and food chain.
Technical Abstract: Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus strains has emerged as one of the practical strategy for reducing aflatoxins contamination in food. Droplet digital PCR (ddPCR) is a new DNA quantification platform without an external DNA calibrator. For ddPCR, a single PCR mixture is divided into water-in-oil droplets, in which PCR is carried out. Each droplet is assigned as positive or negative by a threshold fluorescence signal intensity, and the absolute quantity of target DNA molecules is determined using Poisson statistics. Four of the atoxigenic A. flavus strains used in this study, were shown to have deletion in afalatoxin biosynthetic gene cluster and missing norA gene but omtA is present. TaqMan probe, omt-FAM and norA-HEX were designed for developing a ddPCR assay to analyze soil population of mixtures of A. flavus. Paired atoxigenic and aflatoxigenic mixtures were inoculated into a soil system and incubated at 28oC for two and four weeks. DNA was extracted from each soil sample and used for ddPCR assays. The results indicated that individual A. flavus strain has different growth rate. Higher ratio of atoxigenic to aflatoxigenic strains significantly increased soil population size of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR as a means to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains.