Methodology and application of PCR-RFLP for species identification in tuna sashimi.

Lin Yao,Meng Qu,Yingying Guo,Fengling Li,Lianzhu Wang,Yuxiu Zhai,Yanhua Jiang,Jianping Lu

doi:10.1002/fsn3.1552

Abstract

The Thunnini, or tuna, comprise many species with very different commercial values. The principal raw tuna product on the market is sashimi, for which the species used is difficult to identify through conventional morphological analysis. The present study amplified the cytochrome b gene (Cytb) of 4 major tuna species used for preparing sashimi—yellowfin tuna (Thunnus albacares), southern bluefin tuna (Thunnus maccoyii), bigeye tuna (Thunnus obesus), and Atlantic bluefin tuna (Thunnus thynnus)—and 4 species commonly mislabeled as components of tuna sashimi—albacore tuna (Thunnus alalunga), skipjack tuna (Katsuwonus pelamis), striped marlin (Tetrapturus audax), and swordfish (Xiphias gladius). Polymerase chain reaction (PCR) amplicons were digested with 5 restriction enzymes—Eco147 I, Hinf I, Mbo I, Xag I, and Hind II—to obtain characteristic restriction maps of the above‐mentioned raw tuna species and the commonly mislabeled species. An identification method using PCR restriction fragment length polymorphism (PCR‐RFLP) was established and validated using 39 commercial tuna sashimi samples, which verified that this method provides results consistent with those obtained by classical sequencing. PCR‐RFLP has several advantages over classical sequencing, such as simplicity, speed and accuracy. This technique could support species identification for raw tuna and sashimi.

Highlights

Of the 10 samples labeled as Atlantic bluefin tuna, the Polymerase chain reaction (PCR)-RFLP results identified 8 samples as Atlantic bluefin tuna and the other 2 samples as albacore tuna, which is consistent with the sequence alignment results and phylogenetic tree analysis
Out of the 6 samples labeled as southern bluefin tuna, the PCR-RFLP results identified all samples as southern bluefin tuna, which was consistent with the results of the sequence alignment and phylogenetic tree analysis
Of the 13 samples labeled as yellowfin tuna, the PCR-RFLP results identified 10 samples as yellowfin tuna and the other 3 samples as bigeye tuna which was consistent with the results obtained using sequence alignment and phylogenetic tree analysis

Summary

Introduction

Five species-specific restriction enzymes were selected and used to digest the PCR amplicons of the Cytb gene: Eco147 I, Hinf I, Mbo I, Xag I, and Hind II (for identification of samples dd and jy only). There were no restriction sites for Mbo I and Xag I in the Cytb PCR amplicon of albacore tuna; a single band of 357-bp length was obtained upon digestion by these enzymes.

Results

Conclusion