Insects are rich in proteins and could be an alternative source of macronutrients to feed animals and humans. Over the past few years, numerous companies have started producing insects for feed purposes. In Europe, the processed animal proteins obtained from seven insect species have been authorised for aquaculture by Commission Regulation (EU) 2017/893 since 1 July 2017. Methods of authentication are required to check the conformity of the products. In this study, we propose a real-time PCR method for the specific detection of the black soldier fly (Hermetia illucens L.), one of the most widely used insects for feed production. The developed PCR assays amplify a 67 bp fragment based on the mitochondrial COX3 gene coding for subunit 3 of the cytochrome c oxidase. The qualitative method was tested according to several performance criteria. The specificity was tested against 51 insect species. The specificity was also checked against plant species and other animal species such as crustaceans, mammals and birds. The sensitivity, efficiency and robustness of the PCR test were successfully tested. The applicability of the test was proven through the analysis of real-life processed samples (industrial meals) of H. illucens.

Use of edible insects as an alternative source of proteins in food and feed is increasing. These last years, numerous companies in Europe have started producing insects for food and feed purposes. In the European Union, the use of edible insects for human consumption falls within Regulation (EU) No. 2015/2283 on novel foods. For feed, Commission Regulation (EU) 2017/893 authorizes seven insect species as processed animal proteins for aquaculture. Methods of authentication are required to check the conformity of the products. In this study, we propose a real-time polymerase chain reaction (PCR) method for the specific detection of the lesser mealworm (Alphitobius diaperinus), one of the species included in the shortlist of authorized insects. The selected target is the cadherin gene with a single-copy (per haploid genome) illustrated by our experimental evidence. The PCR test amplified a 134-bp fragment of the cadherin gene. The qualitative method was assessed toward several performance criteria. Specificity was checked against 54 insect species next to other animal and plant species. The sensitivity, efficiency, robustness, and transferability of the PCR assay were also successfully tested. Finally, the applicability of the test was assessed on real-life processed samples (industrial meals) of A. diaperinus. The study also showed that there seems to be a huge confusion on the correct labeling of the marketed mealworms. We did not succeed to get Alphitobius laevigatus samples. They all appeared to belong to the A. diaperinus taxon.

The consumption and farming of insects are gaining global attention as sustainable alternatives to conventional protein sources. Industrial processing of insects into powders or pastes complicates species identification, raising concerns about product authenticity, food safety, and potential fraud. In Western countries, particularly in Europe, the sector is expanding under a stringent regulatory framework, especially regarding rearing substrates, which hinders economic development. This study aimed to assess the species authenticity of commercial insect-based food and feed products and detect the presence of animal-derived DNA from unauthorized substrates. A total of 119 samples (pure insect meals and processed products) were collected from various origins. Species-specific real-time PCR assays targeted Tenebrio molitor, Hermetia illucens, Alphitobius diaperinus, Acheta domesticus, Bombyx mori, and Gryllodes sigillatus, alongside assays for ruminant, porcine, and poultry DNA. High-throughput sequencing (HTS) using metabarcoding confirmed and broadened species detection. Most samples contained the declared species; however, cases of mislabeling, substitution, and cross-contamination were observed. A few insect meals contained animal DNA which could suggest potential use of prohibited substrates. These findings highlight the urgent need for standardized authentication methods and improved transparency to ensure regulatory compliance, consumer trust, and sustainable development of the insect-based sector.

The silkworm, Bombyx mori, is reared on a large scale, mainly for silk production. The waste from this silk production, like pupae, is underused. As an edible insect, B. mori is a good source of protein in human food and animal feed. In recent years, European legislation on the use of insects has evolved and a multitude of European companies have initiated the rearing of insects specifically for food and feed applications. Regarding animal feed, Commission Regulations (EU) 2021/1372 and 2021/1925 authorize eight insect species, including silkworm, as processed animal proteins for use in fish, pig, and poultry feed. The incorporation of edible insects into the human diet falls within Regulation (EU) No. 2015/2283 concerning novel foods. Implementation of authentication methods is imperative to ensure the conformity of the products. In the present study, we propose a specific real-time PCR method for the detection of silkworm (B. mori). The developed PCR test amplifies a 98 bp fragment of the cadherin gene. This gene is present in a single-copy per haploid genome, as demonstrated by experimental evidence. The qualitative method was successfully evaluated on the performance criteria of specificity, sensitivity, efficiency, robustness, and transferability. The applicability of the test was assessed on samples of B. mori from industry. Light microscopy and DNA metabarcoding approaches were used as a complement to genomic analysis as a means of providing authentication of the samples.

Description. Since 2001, the number of bovine spongiform encephalopathy cases has drastically declined. A gradual lifting of the feed ban for aquaculture was possible in 2013 but this partial relaxation makes its control much more complex. Objectives. The objective was to have a practical overview of the current analytical situation and to highlight current and foreseen analytical gaps in the context of a progressive lifting of the ban. Method. Feed samples, collected before the partial lifting as regards aquafeed in 2013, were firstly analyzed by light microscopy. All samples were additionally analyzed by PCR in order to study the difficulties which would be faced with feeds produced after the relaxation. ELISA for the detection of milk proteins was also performed on samples in which ruminant DNA was detected. Results. When keeping the interpretation to the sole light microscopy results, all samples were in accordance to the legal requirements. Adding the PCR results made the interpretation more difficult. DNA of terrestrial animals was detected in nearly 65% of the samples. Apart from aquafeeds, the presence of ruminant DNA could be explained by the use of dairy products confirmed in most cases by ELISA. In aquafeeds, 23% (13/57) of the samples tested positive for ruminant DNA but only three of them also tested positive for milk proteins. Conclusions. The study underlined the crucial need for complementary analytical solutions in order to identify the source of the detected DNA.

Un test PCR en temps réel validé pour la détection simultanée de Gallus gallus et Meleagris gallopavo dans les aliments pour bétail au lieu d’un test volaille impossible Description du sujet. L’utilisation de toutes protéines animales transformées (PATs) dans les aliments pour animaux a été interdite dans l’UE en raison de l’épidémie d’encéphalopathie spongiforme bovine (ESB). Cette interdiction totale a été progressivement levée à partir de 2013 pour les PAT de non-ruminant. L’utilisation de PAT de ruminant dans l’alimentation reste totalement interdite. Afin d’étendre la réintroduction des PATs de volaille dans les aliments pour porc et de PATs de porc dans les aliments pour volaille comme décidé récemment, des tests de détection visant les espèces de volaille et le porc étaient nécessaires pour vérifier qu’il n’y a pas de recyclage intra-spécifique. Objectifs. Dans cette étude, nous décrivons la méthode de PCR en temps réel officielle de l’EURL-AP pour la détection simultanée du poulet (Gallus gallus L.) et de la dinde (Meleagris gallopavo L.), les deux espèces de volaille les plus utilisées. Méthode. Les critères de qualité classiques pour la validation d’une méthode PCR sont considérés. Un seuil (valeur de cut-off) est requis pour l’interprétation des résultats. La transférabilité a été testée par une étude interlaboratoire. Résultats. La méthode PCR développée amplifie un fragment de 84 pb situé sur les séquences de l'ARN ribosomal 12S, d'ARNt-Val et d'ARN ribosomal 16S de l’ADN mitochondrial. La méthode qualitative a été évaluée avec succès sur plusieurs critères de performance : spécificité, sensibilité, efficience et robustesse. L’applicabilité du test a été vérifiée sur des PATs de volaille et sur des aliments composés contenant 0,1 % en fraction massique de PAT de volaille. Une validation interlaboratoire a également démontré que la méthode de PCR en temps réel proposée est transférable. Conclusions. La méthode PCR en temps réel combinant la détection du poulet et de la dinde est adaptée à la détection des PATs de volaille. 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.