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Setting up of a Next Generation Sequencing (NGS)-based method for the identification of insect species in feeds
Project
Project code: BFR-LMS-08-1322-785
Contract period: 01.01.2021
- 31.12.2021
Purpose of research: Experimental development
The recent EU Regulation 2017/893, entered into force the 1st of July 2017, allowed a shortlist of seven insect species (Hermetia illucens, Musca domestica, Tenebrio molitor, Alphitobius diaperinus, Acheta domesticus, Gryllodes sigillatus and Gryllus assimilis) to be included in the formulation of feeds for aquaculture. Previously, the addition of any insect to any feed for farmed animals was not allowed, due to the risk of prion-derived diseases. The introduction of this new Regulation raises the issue to switch from a classical detection method based on microscopy to a more sophisticated and species-specific method. A species-specific method to detect the presence of insects in these feeds and to discriminate between allowed and not allowed species needs to be developed, in order to comply with the new Regulation. Given that thousands of insect species exist but only seven are allowed for inclusion in feeds, the method needs the power to distinguish between genetically distant but also very close species (for example between two species of Gryllus, one allowed and one not allowed). Most importantly, the method needs to be open, because in the future more insects might be added to the current list. This shortlist was in fact drawn up by the EC based on the existing scientific evidences on the insects currently reared in EU, and it is easy to predict that more species will be introduced in the near future. Molecular investigations using DNA guarantee enough specificity for species identification, but classical endpoint or real-time PCR have the limitation to be closed protocols, which need to be re-established and re-validated every time a new species is added to the existing list. Next Generation Sequencing (NGS) technology has been established already since several years, and applications in food and feed authenticity testing are increasing. The main advantage of this technique is that it can provide genetic information on whatever organism is present within the analysed sample, without the need of a predefined target (untargeted analysis), making it theoretically able to detect any unexpected contamination. However, PCR-free procedures like whole genome sequencing (WGS) require a large amount of high qualityng material, which is difficult to obtain from processed foods or feeds in most cases. Conversely, DNA metabarcoding performed exploiting the high-throughput capacity of NGS platforms is a powerful tool for characterizing the biodiversity, and it relies on the preparation of libraries of amplicons targeting specific conserved regions, in which DNA or RNA fragments are coupled to adapters to allow PCR amplification and sequencing. The identification of the obtained sequence relies on the existence of a large updated database, through which sequences can be compared. The protocol proposed in this project will be based on the NGS of a small region included in the COI gene, a mitochondrial gene well known to be able to discriminate animals at species level, that has been already tested successfully on insects. Within this project, a NGS method for insect species differentiation in feed will be established.
Section overview
Subjects
- Process engineering
- Toxicology
- Recirculation systems
- Mariculture
