METHODS FOR THE DETECTION AND QUANTIFICATION OF NEMATODE PARASITES IN FISH AND FISH PRODUCTS

摘要

A molecular method based on real time PCR for the detection of the presence of Anisakis spp. and Pseudoterranova spp. parasites in fish fillets and fish- derived food products, such as babyfood, surimi, fish slices, fish sticks and the like, as well as for performing a relative quantification of nematode larvae content, comprises the steps of: - preparing a first amplicon from the ITS-I region specifically able to identify all species belonging to Anisakis and Pseudoterranova species, and a second amplicon able to amplify DNA from any host DNA, such as fish, and from any organic component or foodstuff, said amplicons being located on redundant genomic regions providing more power to detect a PCR product in degraded samples; - testing the primer pairs in the same real time PCR conditions on reference samples made from various mixtures of Anisakid nematodes and fish; - for fish fillets (not products): quantifying the larval mass in the fish sample by calculation versus a known reference; - for fish fillets and products: calculating the amount of Anisakid DNA versus the amount of total DNA in each sample. A mo lecular method based on real time PCR f or discriminating dif f erent Anis akid species in order to test the geographic provenance of Anisakid-containing f ish, comprises : - aligning the ITS l sequences of dif f erent Anisakid species typical of dif ferent predetermined seas, - designing a real time PCR assay based on detection of sequence variations, - determining whether a fish (and product containing fish) coming from a predetermined sea is contaminated by a given Anisakid species.