Development and Evaluation of a Real-Time PCR Multiplex Assay for the Detection of Allergenic Peanut Using Chloroplast DNA Markers

Peanut is one of the most commonly consumed allergy-causing foods in the United States. Prevention of accidental consumption by allergic individuals is assisted by methods that effectively identify the presence of peanut in food, even at trace levels. This study presents a multiplex real-time polymerase chain reaction (PCR) assay that uses chloroplast markers ( matK, rpl16, and trnH-psbA) to specifically detect peanut in three types of foods: baked goods, chocolate, and tomato sauces. Food matrices were spiked with raw peanut at concentrations ranging from 0.1 to 10⁵ ppm. The assay was evaluated with respect to linear range and reaction efficiency. High reaction efficiencies were generally obtained across 6-7 orders of magnitude. Limits of detection were between 0.1 and 1 ppm, and reaction efficiencies were mostly within the preferred range of 100 ± 10%. Our results indicate that real-time PCR assays using chloroplast markers can be a valuable tool for peanut detection.