Food allergies affect millions of Americans.  Peanut is one of eight major allergens cited in the Food Allergen Labeling and Consumer Protection Act (FALCPA) and is one of the most prominent in terms of both the number and severity of reactions it causes.  Allergic individuals rely almost exclusively on avoidance of offending foods to prevent reactions, so sensitive and accurate detection methods are necessary to protect public health.  PCR-based methods have become increasingly important for allergen detection and are especially useful for confirmation of results from traditional ELISA and other antibody-based detection methods.  Previous work has shown real-time PCR to be highly sensitive and specific, as well as robust in a wide variety of complex and difficult food matrices.  This work describes the extension of a previously developed triplex real-time PCR assay for peanut to a droplet digital PCR (ddPCR) format.  The droplet digital PCR assay uses the same primer and probe sequences as the real time PCR assay and was adapted using a probe-mixing triplex format.  Extension to ddPCR was carried out via thorough optimizations of primer and probe concentrations as well as various aspects of the thermal profile, including annealing/extension temperature, annealing/extension time, and cycle number.  Performance of the real time PCR assay and the ddPCR assay for peanut will be compared.    <br/>

Droplet digital PCR for detection of food allergens: Development and evaluation of a method for peanut

ACS Fall 2020 Virtual Meeting

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