Detection by real time PCR of walnut allergen coding sequences in processed foods

• Published in: Food chemistry Vol. 202; pp. 334 - 340
• Main Authors: Linacero, Rosario, Ballesteros, Isabel, Sanchiz, Africa, Prieto, Nuria, Iniesto, Elisa, Martinez, Yolanda, Pedrosa, Mercedes M., Muzquiz, Mercedes, Cabanillas, Beatriz, Rovira, Mercè, Burbano, Carmen, Cuadrado, Carmen
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2016
• Subjects: Allergens - analysis; Allergens - immunology; Antigens, Plant - analysis; Antigens, Plant - immunology; Enzyme-Linked Immunosorbent Assay; Food Analysis - methods; Juglans; Juglans - genetics; Juglans - immunology; Juglans regia; Pressure processing; Processed foods; Real-time PCR; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Thermal processing; Walnut allergen detection; Pressure processing; Ct; HHP; RT; Processed foods; Real-time PCR; Thermal processing; Walnut allergen detection; LTP; NTC; CTAB; Juglans regia
• ISSN: 0308-8146; 1873-7072
• DOI: 10.1016/j.foodchem.2016.01.132

•A qRT-PCR method using walnut allergen coding-sequences was developed.•The method is specific, reliable, and sensitive. LOD, 2.5pg of walnut DNA.•HHP and autoclave treatments can affect DNA quality required for PCR amplification.•The method was accurate and specific for detection of hidden walnut in commercial foods. A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB–phenol–chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays.