insect, , black soldier fly, detection,; real-time PCR; Hermetia illucens
Abstract :
[en] 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.
Disciplines :
Entomology & pest control
Author, co-author :
Marien, A; Walloon Agricultural Research Center (CRA-W), > Unit Traceability and Authentication
Debode, F; Walloon Agricultural Research Center (CRA-W), > Unit Traceability and Authentication
Aerts, C; Walloon Agricultural Research Center (CRA-W), > Unit Traceability and Authentication
Ancien, C; Walloon Agricultural Research Center (CRA-W), > Unit Traceability and Authentication
Francis, Frédéric ; Université de Liège - ULiège > Agronomie, Bio-ingénierie et Chimie (AgroBioChem) > Gestion durable des bio-agresseurs
Berben, G; Walloon Agricultural Research Center (CRA-W), > Unit Traceability and Authentication,
Association française de Normalisation (AFNOR), 2008. Détection et quantification des organismes végétaux génétiquement modifiés et produits dérivés. Partie 2: méthodes basées sur la réaction de polymérisation en chaîne. AFNOR Standard XP-V-03-020-2. AFNOR, Saint-Denis La Plaine, France
Barroso, F.G., De Haro, C., Sánchez-Muros, E.V., Martinez-Sánchez, A. and Pérez-Banón, C., 2014. The potential of various insect species for use as food for fish. Aquaculture 422-423: 193-201
Broeders, S., Huber, I., Grohmann, L., Berben, G., Taverniers, I., Mazzara, M., Roosens, N. and Morisset, D., 2014. Guidelines for validation of qualitative real-time PCR methods. Trends in Food Science & Technology 37: 115-126
Cameron, S.L., 2014. Insect mitochondrial genomics: implications for evolution and phylogeny. Annual Review of Entomology 59: 95-117
Cavelier, L., Johannisson, A. and Gyllensten, U., 2000. Analysis of mtDNA copy number and composition of single mitochondrial particles using flow cytometry and PCR. Experimental Cell Research 259: 79-85
Codex Committee on Methods of Analysis and Sampling (CCMAS), 2010. Guidelines on performance criteria and validation of methods for detection, identification and quantification for specific DNA sequences and specific proteins in foods. Report No. CAC/GL 74-2010. CCMAS, Rome, Italy. Available at: https://tinyurl.com/ ydx8uak6
Cook, L.G., Gullan, P.J. and Trueman, H.E., 2002. A preliminary phylogeny of the scale insects (Hemiptera: Sternorrhyncha: Coccoidea) based on nuclear small-subunit ribosomal DNA. Molecular Phylogenetics and Evolution 25: 43-52
Dawnay, N., Ogden, R., McEwing, R., Carvalho, G.R. and Thorpe, R.S., 2007. Validation of the barcoding gene COI for use in forensic genetic species identification. Forensic Science International 173: 1-6
Debode, F., 2017. Développement de méthodologies pour la détection des plantes génétiquement modifiées. PhD thesis, Université Catholique de Louvain, Belgium, 391 pp. Available at: http://hdl. handle.net/2078.1/186329
Debode, F., Janssen, E. and Berben, G., 2007. Physical degradation of genomic DNA of soybean flours does not impair relative quantification of its transgenic content. European Food Research and Technology 226: 273-280
Debode, F., Janssen, E., Bragard, C. and Berben, G., 2017c. Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in GM constructions. Food Additives and Contaminants: part A 34: 1398-1409
Debode, F., Janssen, E., Marien, A. and Berben, G., 2012. DNA detection by conventional and real-time PCR after extraction from vegetable oils. Journal of the American Oil Chemists' Society 89: 1249-1257
Debode, F., Janssen, E., Marien, A., Devlin, R.H., Lieske, K., Mankertz, J. and Berben, G., in press. Detection of transgenic Atlantic and Coho salmon by real-time PCR. Food Analytical Methods, https:// doi.org/10.1007/s12161-018-1214-1
Debode, F., Marien, A., Gérard, A., Francis, F., Fumière, O. and Berben, G., 2017a. Development of real-time PCR targets for the detection of Tenebrio molitor in food and feed. Food Additives & Contaminants: part A 34: 1421-1426
Debode, F., Marien, A., Janssen, E. and Berben, G., 2010. Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects. Analytical and Bioanalytical Chemistry 396: 2151-2164
Debode, F., Marien, A., Janssen, E., Bragard, C. and Berben, G., 2017b. The influence of amplicon length on real-time PCR results. Biotechnologie, Agronomie, Société et Environnement 21: 3-11
Diener, S., Zurbrügg, C. and Tockner, K., 2009. Conversion of organic material by black soldier fly larvae: establishing optimal feeding rates. Waste Management & Research 27: 603-610
European Union Reference Laboratory for Animal Proteins in feedingstuff (EURL-AP), 2013. Detection of horse DNA using real-time PCR. EURL-AP, Gembloux, Belgium. Available at: https:// tinyurl.com/y7o4dtse
European Union Reference Laboratory for Animal Proteins in feedingstuff (EURL-AP), 2014a. Detection of ruminant DNA in feed using real-time PCR. EURL-AP, Gembloux, Belgium. Available at: https://tinyurl.com/y9nycxvb
European Union Reference Laboratory for Animal Proteins in feedingstuff (EURL-AP), 2014b. DNA extraction using the 'Wizard Magnetic DNA purification system for food' kit. EURLAP, Gembloux, Belgium. Available at: https://tinyurl.com/ycjxqlgp
European Commission (EC), 2017. Commission regulation (EU) 2017/893 of 24 May 2017 amending Annexes I and IV to Regulation (EC) No 999/2001 of the European Parliament and of the Council and Annexes X, XIV and XV to Commission Regulation (EU) No 42/2011 as regards the provisions on processed animal protein. EC, Brussels, Belgium. Available at: https://tinyurl.com/ycu9yrkk
Garikipati, D.K., Gahr, S.A. and Rodgers, B.D., 2006. Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes. Journal of Endocrinology 190: 879-888
Hebert, P.D., Ratnasingham, S. and De Waard, J.R., 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B: Biological Sciences 270: S96-S99
Hoogendoorn, M. and Heimpel, G.E., 2001. PCR-based gut content analysis of insect predators: using ribosomal ITS-1 fragments from prey to estimate predation frequency. Molecular Ecology 10: 2059-2067
International Organisation for Standardization (ISO), 2005. ISO 21571:2005. Foodstuffs-methods of analysis for the detection of genetically modified organisms and derived products-nucleic acid extraction. ISO, Geneva, Switzerland
Malewski, T., Draber-Monko, A., Pomorski, J., Los, M. and Bogdanowicz, W., 2010. Identification of forensically important blowfly species (Diptera: Calliphoridae) by high-resolution melting PCR analysis. International Journal of Legal Medicine 124: 277-285
Mandal, S.D., Chhakchhuak, L., Gurusubramanian, G. and Kumar, N.S., 2014. Mitochondrial markers for identification and phylogenetic studies in insects-a review. DNA Barcodes 2: 1-9
Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiu, A., Elliott, M., Farrell, A.P., Forster, I., Gatlin, D.M., Goldburg, R.J., Hua, K. and Nichols, P.D., 2009. Feeding aquaculture in an era of finite resources. Proceedings of the National Academy of Sciences of the USA 106: 15103-15110
Pons, J., 2006. DNA-based identification of preys from non-destructive, total DNA extractions of predators using arthropod universal primers. Molecular Ecology Notes 6: 623-626
Pons, J., Barraclough, T.G., Gomez-Zurita, J., Cardoso, A., Duran, D.P., Hazell, S., Kamoun, S., Sumlin, W.D. and Vogler, A.P., 2006. Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology 55: 595-609
Riddick, E., 2014. Insect protein as a partial replacement for fishmeal in the diets of juvenile fish and crustaceans. In: Morales-Ramos, J.M., Rojas, M.G. and Shapiro-Ilan, D. (eds.) Mass production of beneficial organisms. Elsevier, New York, NY, USA, pp. 565-579
Rodriguez, A., Rodriguez, M., Córdoba, J.J. and Andrade, M.J., 2015. Design of primers and probes for quantitative real-time PCR methods. Methods in Molecular Biology 1275: 31-56
Sambrook, J., Fritsch, E.F. and Maniatis, T., 1989. Molecular cloning. A laboratory manual (2nd Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA
Sheppard, S.K., Bell, J., Sunderland, K.D., Fenlon, J., Skervin, D. and Symondson, W.O.C., 2005. Detection of secondary predation by PCR analyses of the gut contents of invertebrate generalist predators. Molecular Ecology 14: 4461-4468
Van Huis, A., 2012. Potential of insects as food and feed in assuring food security. Annual Review of Entomology 58: 563-583
Wells, J.D. and Škaro, V., 2014. Application of DNA-based methods in forensic entomology. In: Primorac, D. and Schanfield, M. (eds.) Forensic DNA applications: an interdisciplinary perspective. CRC Press, Boca Raton, FL, USA
