Molecular based identification of insect ingredients in animal feeds

Since January 1st 2018 seven different insects species have been allowed to be used in aquafeed (Regulation (EU) 2017/893) in the European Union. Tenebrio molitor and Hermetia illucens are the most attractive species that could suitably be used in aquafeed for their availability and market price. Taking into consideration that no official methods for insect detection in feed are available yet and the microscopy analysis have not a good accuracy, therefore, sensitive detection methods are urgently required to monitor compliance with the law since no threshold is in place. A nucleic acid-based approach like PCR is still considered straightforward method for target detection; for this reason it was decided to design suitable primers against a conservative mitochondrial gene for the detection of the two insect species and consequently the primers efficiency for the target and their cross reactivity were evaluated using qPCR. DNA samples used in the qPCR were extracted from different untreated insects, insect meals and aquafeed with different percentages of target insect meal inclusion and were used to evaluate the accuracy and sensitivity of the analysis. Results show that the designed pairs of primers do not cross react with the no target samples. Hermetia DNA at 10ng/µl in purity gave a reproducible signal about Ct=23 and the Tenebrio DNA at 10ng/µl gave a signal at Ct=20. The target species were detected in aquafeed with different insect inclusion and the qPCR analysis output gives different values of Ct do to the different level of inclusion, lower with high inclusion, higher with low inclusion level for both the target species. The qPCR method has confirmed its accuracy and suitability for the detection of these ingredients in animal feeds within a range of 0.03 to 0.5 g/g (r= 0.98, n=10). Moreover, these primers that revealed a good efficiency and affinity, are going to be used to implement a portable biosensor for field analysis based on a fluorescence detection technique as a faster and easier system of detection that could be useful at industrial level.