National Reference Centre for Anisakiasis (C.Re.N.A.)
Assessment of nematodes parasites belonging to Anisakidae family by two steps:
- Identification of larvae (stored in 70% alcohol) by fish inspection and morphology characterization by optical microscopy.
The identification of parasites is carried out by phenotypic test. The former consists in microscopic observation and comparison with morphological identification keys, for genus identification of nematodes of the Anisakidae family.
- Molecular characterization PCR-RFLP or multiplex PCR of ITS-DNA region, mtDNA cox2 and/or ITS DNA region sequence analyses. These methods are able to identify species of Anisakidae parasites such as Anisakis type I (A. simplex s.s., A. pegreffii, A. typica, A. ziphidarum, A. nascetti, A. berlandi) and Anisakis type II (A. physeteris, A. brevispiculata, A. paggiae), Pseudoterranova (P. decipiens s.s., P. krabbei, P. bulbosa, P. azarasi, P. cattani), Contracaecum (C. osculatum, C. rudolphii) and Hysterothylacium (H. aduncum, H. fabri) genera. Real Time-PCRs are carried out for the identification of Anisakids DNA trace such as Anisakis spp and Pseudoterranova spp in different processed fish products. Sequencing is carried out to confirm diagnosis and for species/strain typing.
Details of the exams are reported in the tables below.
The Standard Operative Procedures (POS/PdP) are certified according to UNI CEI EN ISO/IEC 17025:2005.
Molecular and Optical-microscopy diagnostic
| Pathogen | Matrix | Method | POS/PdP |
| Anisakidae larvae
|
Viscera, coelomic cavity and muscle tissue of fish | Optical-microscopy observation | POS-CRENA 03 |
| Anisakis spp | Anisakis larvae | PCR-RFLP | POS CRENA 02 |
| Anisakis spp, Pseudoterranova spp | Processed fish products | Real Time PCR | POS CRENA 05 |
| Pseudoterranova spp | Pseudoterranova larvae | mtDNA cox2 sequence analyses | POS CRENA 06 |
| Nematodes of the Anisakidae family (Anisakis, Pseudoterranova, Hysterothylacium, Contracaecum osculatum, Contracaecum rudolphii) | Anisakidae larvae | Multiplex PCR | PdP CRENA 07 |
| Nematodes of the Anisakidae family | Purified PCR products | PCR Sequencing | – |
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