LAUSR

Abstract Walnut is one of the most widely consumed and highly allergenic tree nuts in North America. The two species most frequently consumed in the United States are English walnut (Juglans regia) and black walnut (Juglans nigra). Ensuring accurate labeling to protect allergic individuals from accidental consumption demands methods that accurately identify the presence of walnut in food. We developed a novel real-time PCR assay that targets two chloroplast regions (rbcL-accD and psbM-trnD) and implements LNA probes to specifically detect walnut in three types of foods: baked goods, salad greens, and creamy dips. This is the first assay for walnut detection that uses LNA oligos to increase specificity and prevent significant cross-reactivity with pecan and other tree nuts by blocking their amplification signal. Food matrices were spiked separately with English and black walnut at concentrations that ranged from 0.1 to 105 mg/kg (parts per million, or ppm). Reaction efficiencies within the optimal range of 100 ± 10% were obtained across 6 to 7 orders of magnitude with a limit of detection of 0.1 ppm. Our results show that real-time PCR assays using chloroplast markers and LNA probes are an effective and accurate tool for allergenic walnut detection.