A novel single-tube nested real-time PCR method to quantify pistachio nut as an allergenic food: Influence of food matrix

In this work, five nuclear genes (Pis v 1, Pis v 2, Pis v 3, Pis v 4 and Pis v 5) were investigated for their potential application as specific markers for pistachio nut. Pis v 2 gene was selected as the best marker for pistachio identification and targeted by a novel single-tube nested real-time PCR method. The approach allowed amplifying 1 pg of pistachio nut DNA (1.6 copies), as well as 100 and 5 mg/kg of pistachio nut in wheat material (pasta) or ice-cream, respectively, being successfully validated regarding precision/trueness. Wheat material (pasta) matrix affected sensitivity, still maintaining acceptable performance parameters, including precision and trueness. Ice-cream matrix presented the lowest Cq values, thus enabling a more efficient amplification. Herein, a highly sensitive, specific and cost-effective single-tube nested real-time PCR targeting the Pis v 2 gene is proposed, for the first time, as a novel quantitative and validated approach for pistachio nut analysis as an allergenic food.