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Matrix-normalised quantification of species by threshold-calibrated competitive real-time PCR: Allergenic peanut in food as one example

Author:
Holzhauser, Thomas, Kleiner, Kornelia, Janise, Annabella, Röder, Martin
Source:
Food chemistry 2014 v.163 pp. 68-76
ISSN:
0308-8146
Subject:
DNA, allergenicity, cookies, dough, enzyme-linked immunosorbent assay, food matrix, ice cream, milk chocolate, nucleotide sequences, peanut milk, peanuts, quantitative polymerase chain reaction
Abstract:
A novel method to quantify species or DNA on the basis of a competitive quantitative real-time polymerase chain reaction (cqPCR) was developed. Potentially allergenic peanut in food served as one example. Based on an internal competitive DNA sequence for normalisation of DNA extraction and amplification, the cqPCR was threshold-calibrated against 100mg/kg incurred peanut in milk chocolate. No external standards were necessary. The competitive molecule successfully served as calibrator for quantification, matrix normalisation, and inhibition control. Although designed for verification of a virtual threshold of 100mg/kg, the method allowed quantification of 10–1000mg/kg peanut incurred in various food matrices and without further matrix adaption: On the basis of four PCR replicates per sample, mean recovery of 10–1000mg/kg peanut in chocolate, vanilla ice cream, cookie dough, cookie, and muesli was 87% (range: 39–147%) in comparison to 199% (range: 114–237%) by three commercial ELISA kits.
Agid:
5422623