Detection of celery (Apium graveolens) allergen in foods of animal and plant origin by droplet digital PCR assay

Celery is included among the allergenic foods that, under the EU 1169/2011 regulation, must be declared in the ingredient list. However, disposition covers only allergens that are voluntary used as ingredients and not the accidental presence of allergens in a food as consequence of cross contamination. To guarantee compliance with food allergen regulations and protect health of food-allergic consumers are needed specific and sensitive methods to detect the presence of allergens in foods. Detection of allergens relies of protein- and DNA-based methods. Real-time PCR (RT-PCR) targeting sequences from the mannitol dehydrogenase (Mtd) gene is currently the method of choice for detection and quantification of celery in foods. However, quantification by RT-PCR methods needs standard calibration curves of the target DNA. To overcome this limitation in the present study the use of a droplet digital PCR (dd-PCR) assay has been proposed for the quantification of celery in foods. A preliminarily optimization of the dd-PCR protocol was conducted using serial DNA dilution extracted from celery powder. Ideal primer probe concentrations were 0.9 μM of both forward and reverse primers and 0.250 μM of probe. The optimal annealing temperature was at 60 °C. The limit of detection (LOD) was 0.20 ± 0.12 Cp/μL while the limit of quantification (LOQ) was 0.83 ± 0.20 Cp/μL. The dd-PCR assay showed no cross-reactivity with other vegetal species, indicating a good specificity. No effect of food matrix was observed on the dd-PCR performance. The method was able to quantify the presence of celery in commercial foods of animal and plant origin.