International Collaborative Trial of a Real-time PCR Approach for the Relative Quantitation of Horse DNA

Following the UK/EU Horse-meat issue of 2013, a real-time PCR approach for the relative quantitation of horse DNA was published. An international collaborative trial was subsequently organised to evaluate the repeatability and reproducibility of the method in line with IUPAC guidance. Thirteen international laboratories participated in the trial, representing laboratories from EU member states and the United States of America. Participants analysed five blindly labelled test samples, representing DNA extracted from 0.1, 0.5, 1, 5, and 20% (w/w) horse meat in a beef meat background. Each test sample was represented by four units in the experimental design, and each unit represented by triplicate PCR technical replicates. Values for the PCR efficiency and r-squared associated with the calibration curves from the collaborative trial showed acceptance with EC published acceptance criteria. A mixed effects model, based on maximum likelihood, was used to analyse the data. The relative repeatability standard deviation (RSDr ) was less than 15% and the relative reproducibility standard deviation (RSDR) was less than 26% across all samples analysed, inclusive of the 0.1% (w/w) gravimetric sample. These estimates fulfil the acceptance criteria for the precision associated with a method subject to a collaborative trial as outlined in published IUPAC guidance. A small but consistent positive bias (less than 15%) was observed, thought to be mainly contributed by the sample preparation approach. The method is now being considered for international standardisation.