The utilization of insects as a source of essential nutrients holds considerable promise, with the potential to serve as both feed and food. Consequently, there is a necessity to develop control systems, as the undeclared addition of insects to food products and/or non-compliance with labelling regulations may pose health risks and result in financial losses for consumers. This review describes methods for identifying and detecting insect species by targeting biomolecules such as DNA, proteins, saccharides, and metabolites, with a particular focus on DNA-based approaches. This review provides a detailed overview of the application of polymerase chain reaction (PCR) and DNA sequencing methods that are suitable for the analysis of edible and forage insects. The main focus is on identifying species that are approved for use as novel foods or insect feeds within the European Union (e.g., house cricket (Acheta domesticus), common mealworm (Tenebrio molitor), migratory locust (Locusta migratoria), lesser mealworm (Alphitobius diaperinus), black soldier fly (Hermetia illucens), banded cricket (Gryllodes sigillatus), field cricket (Gryllus assimilis), silkworm (Bombyx mori)). However, insect species of global relevance are also discussed. The suitability of DNA analysis methods for accurate species identification, detection of (un)labeled contaminants, and monitoring of genetic diversity has been demonstrated.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Department of Food Science Czech Agrifood Research Center Drnovská 507 73 161 00 Prague Czech Republic

Department of Genetics and Breeding Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamýcká 129 165 00 Prague Czech Republic

Zobrazit více v PubMed

Fuso A., Leni G., Prandi B., Lolli V., Caligiani A. Novel foods/feeds and novel frauds: The case of edible insects. Trends Food Sci. Technol. 2024;147:104457. doi: 10.1016/j.tifs.2024.104457. DOI

Poma G., Cuykx M., Da Silva K.M., Iturrospe E., van Nuijs A.L., van Huis A., Covaci A. Edible insects in the metabolomics era. First steps towards the implementation of entometabolomics in food systems. Trends Food Sci. Technol. 2022;119:371–377. doi: 10.1016/j.tifs.2021.12.018. DOI

Spatola G., Giusti A., Mancini S., Tinacci L., Nuvoloni R., Fratini F., Di Iacovo F., Armani A. Assessment of the information to consumers on insects-based products (Novel Food) sold by e-commerce in the light of the EU legislation: When labelling compliance becomes a matter of accuracy. Food Control. 2024;162:110440. doi: 10.1016/j.foodcont.2024.110440. DOI

Baiano A. Edible insects: An overview on nutritional characteristics, safety, farming, production technologies, regulatory framework, and socio-economic and ethical implications. Trends Food Sci. Technol. 2020;100:35–50. doi: 10.1016/j.tifs.2020.03.040. DOI

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) Turck D., Bresson J.L., Burlingame B., Dean T., Fairweather-Tait S., Heinonen M., Hirsch-Ernst K.I., Mangelsdorf I., McArdle H., et al. Guidance on the preparation and presentation of an application for authorisation of a novel food in the context of Regulation (EU) 2015/2283. Efsa J. 2016;14:e04594.

Zurek L., Gorham J.R. Wiley Handbook of Science and Technology for Homeland Security. Wiley; Hoboken, NJ, USA: 2008. Insects as vectors of foodborne pathogens; p. 1.

Graczyk T.K., Knight R., Tamang L. Mechanical transmission of human protozoan parasites by insects. Clin. Microbiol. Rev. 2005;18:128–132. doi: 10.1128/CMR.18.1.128-132.2005. PubMed DOI PMC

Gałęcki R., Bakuła T., Gołaszewski J. Foodborne diseases in the edible Insect industry in Europe—New challenges and old problems. Foods. 2023;12:770. doi: 10.3390/foods12040770. PubMed DOI PMC

Garofalo C., Osimani A., Milanović V., Taccari M., Cardinali F., Aquilanti L., Riolo P., Ruschioni S., Isidoro N., Clementi F. The microbiota of marketed processed edible insects as revealed by high-throughput sequencing. Food Microbiol. 2017;62:15–22. doi: 10.1016/j.fm.2016.09.012. PubMed DOI

Chiang Y.-C., Tsen H.-Y., Chen H.-Y., Chang Y.-H., Lin C.-K., Chen C.-Y., Pai W.-Y. Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples. J. Microbiol. Methods. 2012;88:110–116. doi: 10.1016/j.mimet.2011.10.021. PubMed DOI

Grabowski N.T., Klein G. Microbiology of processed edible insect products—Results of a preliminary survey. Int. J. Food Microbiol. 2017;243:103–107. doi: 10.1016/j.ijfoodmicro.2016.11.005. PubMed DOI

Vale-Hagan W., Singhal S., Grigoletto I., Totaro-Fila C., Theodoridou K., Koidis A. Edible insects in mixed-sourced protein meals for animal feed and food: An EU focus. Food Humanit. 2023;1:1180–1187. doi: 10.1016/j.foohum.2023.09.011. DOI

Liu Z., Xia L., Wu Y., Xia Q., Chen J., Roux K.H. Identification and characterization of an arginine kinase as a major allergen from silkworm (Bombyx mori) larvae. Int. Arch. Allergy Immunol. 2009;150:8–14. doi: 10.1159/000210375. PubMed DOI

Binder M., Mahler V., Hayek B., Sperr W.R., Schöller M., Prozell S., Wiedermann G., Valent P., Valenta R., Duchêne M. Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen. J. Immunol. 2001;167:5470–5477. doi: 10.4049/jimmunol.167.9.5470. PubMed DOI

Veys P., Baeten V. Protocol for the isolation of processed animal proteins from insects in feed and their identification by microscopy. Food Control. 2018;92:496–504. doi: 10.1016/j.foodcont.2018.05.028. DOI

Pečová M., Javůrková Z., Bartlová M., Pospiech M. Detection of edible insect as a component of snack bars using histochemical method. J. Food Compos. Anal. 2024;132:106312. doi: 10.1016/j.jfca.2024.106312. DOI

Belghit I., Lock E.-J., Fumière O., Lecrenier M.-C., Renard P., Dieu M., Berntssen M.H., Palmblad M., Rasinger J.D. Species-specific discrimination of insect meals for aquafeeds by direct comparison of tandem mass spectra. Animals. 2019;9:222. doi: 10.3390/ani9050222. PubMed DOI PMC

Francis F., Mazzucchelli G., Baiwir D., Debode F., Berben G., Caparros Megido R. Proteomics based approach for edible insect fingerprinting in novel food: Differential efficiency according to selected model species. Food Control. 2020;112:107135. doi: 10.1016/j.foodcont.2020.107135. DOI

Ulrich S., Kühn U., Biermaier B., Piacenza N., Schwaiger K., Gottschalk C., Gareis M. Direct identification of edible insects by MALDI-TOF mass spectrometry. Food Control. 2017;76:96–101. doi: 10.1016/j.foodcont.2017.01.010. DOI

Karnaneedi S., Johnston E.B., Bose U., Juhász A., Broadbent J.A., Ruethers T., Jerry E.M., Kamath S.D., Limviphuvadh V., Stockwell S., et al. The Allergen Profile of Two Edible Insect Species—Acheta domesticus and Hermetia illucens. Mol. Nutr. Food Res. 2024;68:2300811. doi: 10.1002/mnfr.202300811. PubMed DOI

Jeong K.Y., Son M., Lee J.Y., Park K.H., Lee J.-H., Park J.-W. Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori. J. Korean Med. Sci. 2015;31:18–24. doi: 10.3346/jkms.2016.31.1.18. PubMed DOI PMC

Pospiech M., Pečová M., Bartlová M., Javůrková Z., Kopecká A., Šebelová K., Pospíšil O., Kulma M., Folke J., Tremlová B., et al. Development of Indirect Sandwich ELLA for Detection of Insects in Food. Appl. Sci. 2024;14:10794. doi: 10.3390/app142310794. DOI

Son Y.-J., Hwang I.-K., Nho C.W., Kim S.M., Kim S.H. Determination of carbohydrate composition in mealworm (Tenebrio molitor L.) larvae and characterization of mealworm chitin and chitosan. Foods. 2021;10:640. doi: 10.3390/foods10030640. PubMed DOI PMC

Tata A., Massaro A., Marzoli F., Miano B., Bragolusi M., Piro R., Belluco S. Authentication of edible insects’ powders by the combination of DART-HRMS signatures: The first application of ambient mass spectrometry to screening of novel food. Foods. 2022;11:2264. doi: 10.3390/foods11152264. PubMed DOI PMC

Tramuta C., Gallina S., Bellio A., Bianchi D.M., Chiesa F., Rubiola S., Romano A., Decastelli L. A set of multiplex polymerase chain reactions for genomic detection of nine edible insect species in foods. J. Insect Sci. 2018;18:3. doi: 10.1093/jisesa/iey087. PubMed DOI PMC

Köppel R., Schum R., Habermacher M., Sester C., Piller L.E., Meissner S., Pietsch K. Multiplex real-time PCR for the detection of insect DNA and determination of contents of Tenebrio molitor, Locusta migratoria and Achaeta domestica in food. Eur. Food Res. Technol. 2019;245:559–567. doi: 10.1007/s00217-018-03225-5. DOI

Jilkova D., Marien A., Hulin J., Zdenkova K., Fumiere O., Cermakova E., Berben G., Debode F. Detection of Acheta domesticus by real-time PCR in food and feed. J. Insects Food Feed. 2024;1:1–16. doi: 10.1163/23524588-00001067. DOI

Garino C., Winter R., Broll H., Winkel M., Braeuning A., Reich F., Zagon J. Development and validation of a novel real-time PCR protocol for the detection of buffalo worm (Alphitobius diaperinus) in food. Food Control. 2022;140:109138. doi: 10.1016/j.foodcont.2022.109138. DOI

Kim M.-J., Kim S.-Y., Jung S.-K., Kim M.-Y., Kim H.-Y. Development and validation of ultrafast PCR assays to detect six species of edible insects. Food Control. 2019;103:21–26. doi: 10.1016/j.foodcont.2019.03.039. DOI

Mohamadzade Namin S., Yeasmin F., Choi H.W., Jung C. DNA-based method for traceability and authentication of Apis cerana and A. dorsata honey (Hymenoptera: Apidae), using the NADH dehydrogenase 2 gene. Foods. 2022;11:928. doi: 10.3390/foods11070928. PubMed DOI PMC

Marien A., Sedefoglu H., Dubois B., Maljean J., Francis F., Berben G., Guillet S., Morin J.F., Fumière O., Debode F. Detection of Alphitobius diaperinus by Real-Time Polymerase Chain Reaction With a Single-Copy Gene Target. Front. Vet. Sci. 2022;9:718806. doi: 10.3389/fvets.2022.718806. PubMed DOI PMC

Zagon J., di Rienzo V., Potkura J., Lampen A., Braeuning A. A real-time PCR method for the detection of black soldier fly (Hermetia illucens) in feedstuff. Food Control. 2018;91:440–448. doi: 10.1016/j.foodcont.2018.04.032. DOI

Watanabe S., Masamura N., Satoh S.-y., Hirao T. Technique for the identification of insect species in processed foods based on three short DNA sequences. Food Control. 2023;153:109908. doi: 10.1016/j.foodcont.2023.109908. DOI

Giusti A., Spatola G., Mancini S., Nuvoloni R., Armani A. Novel foods, old issues: Metabarcoding revealed mislabeling in insect-based products sold by e-commerce on the EU market. Food Res. Int. 2024;184:114268. doi: 10.1016/j.foodres.2024.114268. PubMed DOI

Hillinger S., Saeckler J., Domig K.J., Dobrovolny S., Hochegger R. Development of a DNA Metabarcoding Method for the Identification of Insects in Food. Foods. 2023;12:1086. doi: 10.3390/foods12051086. PubMed DOI PMC

Siozios S., Massa A., Parr C.L., Verspoor R.L., Hurst G.D. DNA barcoding reveals incorrect labelling of insects sold as food in the UK. PeerJ. 2020;8:e8496. doi: 10.7717/peerj.8496. PubMed DOI PMC

Garrido-Sanz L., Senar M.À., Piñol J. Estimation of the relative abundance of species in artificial mixtures of insects using low-coverage shotgun metagenomics. Metabarcoding Metagenom. 2020;4:e48281. doi: 10.3897/mbmg.4.48281. DOI

Hrbek V., Zdenkova K., Jilkova D., Cermakova E., Jiru M., Demnerova K., Pulkrabova J., Hajslova J. Authentication of Meat and Meat Products Using Triacylglycerols Profiling and by DNA Analysis. Foods. 2020;9:1269. doi: 10.3390/foods9091269. PubMed DOI PMC

Cermakova E., Lencova S., Mukherjee S., Horka P., Vobruba S., Demnerova K., Zdenkova K. Identification of Fish Species and Targeted Genetic Modifications Based on DNA Analysis: State of the Art. Foods. 2023;12:228. doi: 10.3390/foods12010228. PubMed DOI PMC

Šmarda J., Doškař J., Pantůček R., Růžičková V., Koptíková J. Metody Molekulární Biologie. Masarykova Univerzita; Brno, Czech Republic: 2005.

Dingle T.C., Sedlak R.H., Cook L., Jerome K.R. Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clin. Chem. 2013;59:1670–1672. doi: 10.1373/clinchem.2013.211045. PubMed DOI PMC

Huggett J.F., O’Grady J., Bustin S. qPCR, dPCR, NGS—A journey. Biomol. Detect. Quantif. 2015;3:A1. doi: 10.1016/j.bdq.2015.01.001. PubMed DOI PMC

Debode F., Marien A., Gérard A., Francis F., Fumière O., Berben G. Development of real-time PCR tests for the detection of Tenebrio molitor in food and feed. Food Addit. Contam. Part A. 2017;34:1421–1426. doi: 10.1080/19440049.2017.1320811. PubMed DOI

Marien A., Dubois B., Anselmo A., Veys P., Berben G., Kohl C., Maljean J., Guillet S., Morin J.F., Debode F. Detection of Bombyx mori as a Protein Source in Feedingstuffs by Real-Time PCR with a Single-Copy Gene Target. Agriculture. 2024;14:1996. doi: 10.3390/agriculture14111996. DOI

Robin E.D., Wong R. Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells. J. Cell. Physiol. 1988;136:507–513. doi: 10.1002/jcp.1041360316. PubMed DOI

Paracchini V., Petrillo M., Lievens A., Kagkli D.-M., Angers-Loustau A. Nuclear DNA barcodes for cod identification in mildly-treated and processed food products. Food Addit. Contam. Part A. 2019;36:1–14. doi: 10.1080/19440049.2018.1556402. PubMed DOI

Ballin N.Z., Vogensen F.K., Karlsson A.H. Species determination—Can we detect and quantify meat adulteration? Meat Sci. 2009;83:165–174. doi: 10.1016/j.meatsci.2009.06.003. PubMed DOI

Meiklejohn K.A., Damaso N., Robertson J.M. Assessment of BOLD and GenBank—Their accuracy and reliability for the identification of biological materials. PLoS ONE. 2019;14:e0217084. doi: 10.1371/journal.pone.0217084. PubMed DOI PMC

Ratnasingham S., Hebert P.D. BOLD: The Barcode of Life Data System (http://www.barcodinglife.org) Mol. Ecol. Notes. 2007;7:355–364. doi: 10.1111/j.1471-8286.2007.01678.x. PubMed DOI PMC

Kjærandsen J. Current state of DNA barcoding of Sciaroidea (Diptera)—Highlighting the need to build the reference library. Insects. 2022;13:147. doi: 10.3390/insects13020147. PubMed DOI PMC

Yu D.W., Ji Y., Emerson B.C., Wang X., Ye C., Yang C., Ding Z. Biodiversity soup: Metabarcoding of arthropods for rapid biodiversity assessment and biomonitoring. Methods Ecol. Evol. 2012;3:613–623. doi: 10.1111/j.2041-210X.2012.00198.x. DOI

Hebert P.D., Ratnasingham S., De Waard J.R. Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. Ser. B Biol. Sci. 2003;270((Suppl. 1)):S96–S99. doi: 10.1098/rsbl.2003.0025. PubMed DOI PMC

Zdenkova K., Akhatova D., Fialova E., Krupa O., Kubica L., Lencova S., Demnerova K. Detection of meat adulteration: Use of efficient and routine-suited multiplex polymerase chain reaction-based methods for species authentication and quantification in meat products. J. Food Nutr. Res. 2018;57:351.

Barido F.H., Desti D., Pramono A., Abdurrahman Z.H., Volkandari S.D., Cahyadi M. Validating duplex-PCR targeting ND2 for bovine and porcine detection in meat products. Food Chem. Mol. Sci. 2023;7:100181. doi: 10.1016/j.fochms.2023.100181. PubMed DOI PMC

Zhou X., Li Y., Liu S., Yang Q., Su X., Zhou L., Tang M., Fu R., Li J., Huang Q. Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification. GigaScience. 2013;2:2047-217X-2-4. doi: 10.1186/2047-217X-2-4. PubMed DOI PMC

Deiner K., Bik H.M., Mächler E., Seymour M., Lacoursière-Roussel A., Altermatt F., Creer S., Bista I., Lodge D.M., de Vere N., et al. Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Mol. Ecol. 2017;26:5872–5895. doi: 10.1111/mec.14350. PubMed DOI

Taberlet P., Coissac E., Pompanon F., Brochmann C., Willerslev E. Towards next-generation biodiversity assessment using DNA metabarcoding. Mol. Ecol. 2012;21:2045–2050. doi: 10.1111/j.1365-294X.2012.05470.x. PubMed DOI

Elbrecht V., Leese F. Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol. PLoS ONE. 2015;10:e0130324. doi: 10.1371/journal.pone.0130324. PubMed DOI PMC

SVS Inspektoři SVS Odhalili Při Kontrole Skladu Potravin v Praze Nelegální Potravinu z Hmyzu. 2022. [(accessed on 27 May 2025)]. Available online: https://bezpecnostpotravin.cz/inspektori-svs-odhalili-pri-kontrole-skladu-potravin-v-praze-nelegalni-potravinu-z-hmyzu/

Weissman D.B., Gray D.A., Pham H.T., Tijssen P. Billions and billions sold: Pet-feeder crickets (Orthoptera: Gryllidae), commercial cricket farms, an epizootic densovirus, and government regulations make for a potential disaster. Zootaxa. 2012;3504:67–88. doi: 10.11646/zootaxa.3504.1.3. DOI