Development and evaluation of real-time PCR targets for the detection of insect in feed

Insects are rich in proteins and could be an alternative source of proteins to feed animals. Numerous companies started the production of insects at small or larger scale as feed for chicken and fish. Most of the business models for feed production are based on the black soldier fly (Hermetia illucens) or the mealworm (Tenebrio molitor). In Europe, these novel feed are not yet authorized and products are commercialized outside Europe or eventually used as pet food (e.g. wild birds). For further authorization in Europe, many questions must be clarified concerning the presence of antinutritional compounds, the risk associated to pathogens, to residues (pesticides, antibiotics, heavy metals) and to allergens.
To authorize such products on the market, methods to detect if a product really contains insects and to authenticate insect products are also mandatory. European Commission Regulation No 51/2013 named the Polymerase Chain Reaction as a reference method to determine the constituents of animal origin in feed.
Targets focused on insects (target common to all insects) and targets specific to particular insect species are required. PCR methods are developed at CRA-W in this way. Among the methods developed, three of them already gave interesting results. The first one (81 bp) is specific to all insects excepting individuals from the Diptera order. The two others (94 bp and 114 bp) are specific to Tenebrio molitor. The specificity of the targets was tested against 45 insect species and on 10 commercial insect-based feed products (real-processed feed material samples). The sensitivity of the method was assessed through the AFNOR XP V03-020-2 standard approach using the LOD6 method. The three methods reached the recommended performance criteria (LOD≤ 20 copies).