Development and validation of a SYBR-Green i real-time PCR protocol to detect hazelnut (Corylus avellana L) in foods through calibration via plasmid reference standard

Many tree nuts are considered to be a serious problem in food safety, because of the presence of causative factors in IgE-mediated food allergies. Among these, hazelnut (Corylus avellana L.) seeds are largely used in a range of confectionery products and contain many well-characterized allergens. DNA-based methods and ELISA tests may prove to be useful to assess the presence of hidden ingredients in foods. The aim of this work was the development and validation of a species-specific SYBR Green I real-time PCR protocol for the detection of hazelnut in foods. A novel efficient primer pair on the Cor a 8 genomic coding region was designed by preparing a plasmid vector-based Internal reference standard to calibrate the PCR. A good sensitivity, down to 20 (genomic) and 15 (plasmid) DNA copies, was established. All of the commercial samples considered in our study (containing hazelnut as ingredient or as a potential trace cross-contamination) were effectively amplified by PCR, showing a perfect correspondence with an ELISA commercial test, employed as a reference standard method.