Optimised multiplex droplet digital PCR is more precise, but not more sensitive, than real-time PCR for the detection of allergenic peanut

Abstract

The United States requires labelling of food products containing major allergens, such as peanut, through the Food Allergen Labeling and Consumer Protection Act. Accurate labelling requires sensitive, specific and robust detection methods, and PCR-based techniques have proven highly effective. This article describes the transition of a previously developed multiplex real-time PCR assay for allergenic peanut to a droplet digital PCR format. The triplex droplet digital PCR assay was developed in a probe mixing format and directly compared to the established real-time PCR assay. Data are provided for thorough optimisation in the digital format, including the effects of primer and probe concentration, cycle number and annealing/extension time. Optimisation parameters influenced relative location and separation of droplet clusters but not final copy number. The droplet digital PCR assay was linear over five orders of magnitude; its lower limit of detection was 0.05 pg DNA per reaction, more sensitive than published digital PCR allergen assays. It was more precise, but not more sensitive, than the previously established real-time PCR assay.

Keywords:

• Allergen
• peanut
• real-time PCR
• digital PCR limit of detection
• limit of quantification

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was funded by the United States Food and Drug Administration.