CAMPIONI DI RIFERIMENTO CONSERVATI IN ETANOLO.

Il tasso di amplificabilità dei 48 campioni di tessuto conservati in etanolo (Tabella 3) è risultato del 63,3%. I tassi di amplificabilità per specie sono riportati in Tabella 7. L’amplificabilità è stata superiore al 50% dei campioni per 12 specie su 16 (75%). In particolare, è stato possibile amplificare il 100% dei campioni di Sgombro, Trota, Ricciola, Sardina, Razza e Rana pescatrice. Bassi valori di amplificabilità sono invece stati osservati per Orata (44,4%), Acciuga, Salmone (33,3%) e Molo (25%).

Per quanto riguarda la concentrazione, il valore medio totale è risultato10,4 ng/µl. Assumendo 10 ng/µl come valore soglia per l’invio degli ampliconi al sequenziamento, 8 specie su 16 presentavano un valore medio di concentrazione superiore a questa soglia minima. Tuttavia, anche tra le specie con un valore medio di concentrazione inferiore a 10 ng/µl la maggior parte (87,5%) presentava almeno un amplicone sequenziabile. Infatti il 50% degli ampliconi di Scorfano, Triglia, Merluzzo e Pesce spada era sequenziabile, così come il 43% di quelli di Acciuga e Salmone. Solo per i campioni di Orata e Molo non è stato possibile ottenere delle concentrazioni idonee al sequenziamento.

75

CAPITOLO 7

CONCLUSIONI

In questa tesi è stato valutato per la prima volta l’utilizzo delle FTA®_{cards per l’estrazione e}

l’amplificazione di DNA da tessuto muscolare, sia fresco che conservato in etanolo, di specie di uccelli, pesci e mammiferi. Il protocollo messo a punto, che prevede un pretrattamento in etanolo e una riequilibratura con buffer del tessuto fresco, prima delle fasi previste dal protocollo FTA®cards, ha permesso di amplificare ottenendo una concentrazione di amplificato sufficienti per il sequenziamento più dell’80% dei campioni freschi e più del 60% dei campioni conservati in etanolo.

Le FTA®cards presentano diversi vantaggi rispetto alle metodiche di estrazione classica, soprattutto in relazione alla facilità dello stoccaggio del DNA. Infatti possono essere tenute a temperatura ambiente e possono pertanto essere trasportate e stoccate con estrema facilità e costi ridotti. Considerando che negli studi d’identificazione molecolare di specie si rende spesso necessaria l’acquisizione di campioni di DNA da luoghi geograficamente distanti per la creazione di proprie banche dati di DNA di riferimento, tali caratteristiche possono rappresentare un’importante vantaggio sia pratico che economico. Inoltre, la possibilità di amplificare direttamente le FTA®cards consente di ottimizzare le tempistiche di esecuzione dell’analisi.

In conclusione, questo lavoro ha dimostrato la validità dell’impiego delle FTA®

cards nei protocolli d’identificazione di specie e la loro potenzialità come strumento utile per l’ispezione molecolare. Questo approccio si rivela sempre più importante ai fini della necessità di garantire la tracciabilità dei prodotti di origine animale, per combattere le frodi e tutelare la salute e gli interessi economici dei consumatori.

76

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Nel documento Sviluppo di un protocollo basato sull'impiego di FTA®cards per l'identificazione molecolare di specie edibili (pesci, uccelli e mammiferi) ai fini del controllo ispettivo (pagine 74-92)