A validated real-time PCR test for simultaneous detection of Gallus gallus and Meleagris gallopavo in feed instead of an impossible poultry test

Abstract

Description of the subject. Use of all processed animal proteins (PAPs) in animal feeds was banned in the EU due to the outbreak of bovine spongiform encephalopathy (BSE). This total feed ban was progressively lifted for non-ruminant PAPs from 2013 onwards. Use of ruminant PAPs in feed remains totally prohibited. In order to extend the reintroduction of poultry PAPs in pig feed, and of pig PAPs in poultry feed, as recently decided, poultry species and pig detection tests were required to check that there is no intra-specific recycling. Objectives. In this study, we describe the official EURL-AP real-time PCR method for the simultaneous detection of chicken (Gallus gallus L.) and turkey (Meleagris gallopavo L.), the two most widely used poultry species in PAPs. Method. Classic quality criteria for the validation of a PCR method are considered. A cut-off is required for the interpretation of results. Transferability was tested through an interlaboratory study. Results. The developed PCR assay amplifies an 84 bp fragment encompassing the mitochondrial DNA on 12S ribosomal RNA, tRNA-Val and 16S ribosomal RNA sequences. The qualitative method was successfully assessed for several performance criteria: specificity, sensitivity, efficiency and robustness. The applicability of the test was verified on poultry PAPs and on compound feed containing 0.1% in mass fraction of poultry PAPs. An interlaboratory validation study demonstrated that the proposed real-time PCR method is transferable. Conclusions. The combined chicken-turkey real-time PCR assay is fit for the purpose of detecting poultry PAPs.