Relative quantification of walnuts and hazelnuts in bakery products using real-time polymerase chain reaction

Two versions of real-time polymerase chain reaction (PCR) for relative quantification of walnuts and hazelnuts in bakery products were developed. The first method is a duplex real-time PCR with 5'-nuclease (TaqMan) probes labelled with FAM and JOE for walnuts and hazelnuts, respectively. The second method uses two real-time PCR assays for walnuts and hazelnuts with TaqMan probes labelled with FAM, carried out in separate microtubes, with normalisation of the results achieved by adding wheat as an internal standard. The internal standard is quantified in a duplex format using the TaqMan probe labelled with JOE. Both methods produced linear calibration curves (r(2) = 0.9813 for the former method, r(2) = 0.9992 for the latter) in the range 10-90% (w/w) for walnut-hazelnut mixtures. Almost identical calibration curves were obtained also for walnut-hazelnut mixtures diluted with inert matrix (oat flakes; the inert matrix accounting for 80 and 96% (w/w), respectively). Practical applicability of the developed methods was demonstrated on bakery products from the market. The developed methods will be useful for food control laboratories to facilitate authentication of bakery products with nut filling.