Development and validation of two triplex real-time PCR systems for the simultaneous detection of six cereal species in processed meat products

Abstract

Meat protein can be substituted by plant protein in meat products, and cereals are particularly well-suited for this purpose as they are high in protein content and have good bioavailability. The aim of the present work was to develop real-time PCR assays for the detection and quantification of the six most commonly used cereals barley, oat, rye, maize, rice, and wheat in processed meat products. Emulsified sausages were produced with cereal flours (0.0005–0.1%). Additionally, the effect of different post-processing methods (grilling or storage), packaging materials (cans and artificial sausage casings), and cooking temperatures (75 °C, 117 °C, and 121 °C) were investigated. A negative influence on the detectability of DNA was observed with increasing cooking temperature. All other production parameters showed no significant effect. The limit of quantification was as low as 5 ppm of plant protein for all production conditions, which corresponded to 36–58 mg flour per kg of sausage. The validation of the production of reference material was more accurate for the sausages produced at low than at medium temperatures.Overall, the two triplex real-time PCR systems can be applied for specific, sensitive, and simultaneous detection of these six cereal species in meat products. However, the reference material for quantification has to be carefully chosen due to the negative influence of heat treatment on the detectability of DNA.