Semi-quantitative estimation of the walnut content in fillings of bakery products using real-time polymerase chain reaction with internal standard material

A method for absolute quantification of walnuts in fillings of bakery products was developed. Macadamia nuts were used as an internal standard material. A duplex real-time polymerase chain reaction (PCR) with 5′-nuclease (TaqMan) probes labelled with FAM and JOE for walnuts and the internal standard, respectively, was used. Difference between threshold cycle values (Δc T) for the analyte and the internal standard, plotted against logarithm of contents, was used to construct the calibration line. A level of 5 % (w/w) of the internal standard material was found to be suitable for quantification of walnuts in nut fillings, the calibration line being linear. The developed method was applied to bakery products from the market, and crucial factors for its routine applicability have been identified in sampling and sample preparation. The present study demonstrates that quantification of walnuts in the fillings of bakery products should be achievable by real-time PCR with an internal standard material when the reference filling, which is used for calibration, is comparable to the fillings of the samples, a calibration line of low variability is obtained, and the sample material is properly homogenized before weighing the analytical sample.