Abstract (eng)
PCR-HRM analysis is a powerful tool for food adulteration screening due to its ability to rapidly detect DNA sequence variations without the need for subsequent processing. This makes it highly suitable for differentiating species in complex food matrices, securing food authenticity and quality. In this master´s thesis, the aim was to develop a PCR-HRM method to distinguish between seven insect species legally permitted in EU feed: black soldier fly, silkworm, house cricket, tropical house cricket, housefly, lesser mealworm and mealworm. DNA from ten insect species and three meat species was extracted using two different kits, with the DNeasy® mericon® Food Kit providing superior and more consistent results. After performing multiple primer designs and optimizations, a five-primer set was developed that allowed for the successful differentiation of melt curves for the insect species. Initially, melt curves appeared in three clusters: black soldier fly and silkworm; mealworm, crazy red cricket and housefly; tropical house cricket and house cricket. The key challenge was to resolve these melt curve clusters, particularly between species with similar DNA sequences, such as black soldier fly and silkworm, and mealworm and housefly. The method was further applied by analyzing commercially available feed samples, where insect DNA was successfully detected in twelve samples where the presence of insect content was stated. However, the feed sample with mixed insect DNA from black soldier fly and mealworm displayed challenges in identifying the species present, suggesting that the method requires further refinement for mixed samples analysis. The applicability of the PCR-HRM method was also demonstrated using DNA extracts obtained with a CTAB extraction method, and the results confirmed that this approach could provide reproducible and distinct melt curves for various insect species. Nevertheless, additional work is needed to improve its robustness in complex, highly processed products and to improve the repeatability for species such as house cricket, whose melt profiles exhibited more variability in food samples. To conclude, PCR-HRM analysis shows great potential as a rapid, simple, and cost-effective tool for feed authenticity testing. With further optimization, it could effectively replace more complex DNA-based techniques, providing dependable detection and differentiation of legally permitted insect species in feed products.