Multiplex, quantitative, ligation-dependent probe amplification for determination of allergens in food

Legislation requires labeling of foods containing allergenic ingredients. Here, we present a robust 10-plex quantitative and sensitive ligation-dependent probe amplification method, the allergen-multiplex ligation-dependent probe amplification (MLPA) method, for specific detection of eight allergens: sesame, soy, hazelnut, peanut, lupine, gluten, mustard, and celery. Ligated probes were amplified by polymerase chain reaction (PCR), and amplicons were detected using capillary electrophoresis. Quantitative results were obtained by comparing signals with an internal positive control. The limit of detection varied from approximately 5 to 400 gene copies, depending on the allergen. The method was tested using different foods spiked with mustard, celery, soy, or lupine flour in the 1-0.001% range. Depending on the allergen, sensitivities were similar or better than those obtained with qPCR. The allergen-MLPA method is modular and can be adapted by adding probe pairs for other allergens. The DNA-based allergen-MLPA method will constitute a complementary method to the traditional protein-based methods.