Keijiro Sezaki

Rapid identification of eels Anguilla japonica and Anguilla anguilla by polymerase chain reaction with single nucleotide polymorphism-based specific probes

Standard molecular techniques, such as sequencing and restriction fragment length polymorphism analysis after polymerase chain reaction (PCR) amplification are relatively complicated, and species identification can take a long time when using such techniques. We established a quick method, using PCR with species-specific TaqMan Minor Groove Binder (MGB) probes based on single nucleotide polymorphism (SNP) to distinguish the two eel species Anguilla japonica and Anguilla anguilla. This method can be used in processed products. Partial sequences of the mitochondrial 16S rRNA gene were compared between A. japonica and A. anguilla to design a primer pair common to both A. japonica and A. anguilla and probes specific to A. japonica and A. anguilla. Different fluorescence intensities were produced in two PCR microtubes each containing A. japonica and A. anguilla-specific probes for one target sample. We observed the fluorescence intensity of PCR products in microtubes under ultraviolet transillumination, with similar results to those obtained by real-time PCR. Therefore, SNP-based PCR is a powerful tool for identifying materials of processed foods from either A. japonica or A. anguilla.

A simple method to distinguish two commercially valuable eel species in Japan Anguilla japonica and A. anguilla using polymerase chain reaction strategy with a species‐specific primer

In order to distinguish the two eel species Anguilla japonica and A. anguilla irrespective of unprocessed and processed samples, a quick, convenient method using polymerase chain reaction (PCR) with a species-specific primer was established. The comparison of partial sequences of the mitochondrial 16S ribosomal RNA gene between A. japonica and A. anguilla facilitated designing of a primer pair common to both A. japonica and A. anguilla and a primer specific to A. japonica. PCR was carried out with the three primers for total 110 specimens of A. japonica and A. anguilla. PCR products with the species-specific primer showed two bands for A. japonica and one band for A. anguilla in an agarose gel electrophoresis. This analytical procedure required only a short period and is more convenient than PCR-restriction fragment length polymorphism, one of the standard methods employed for fish species identification.

Identification of alfonsino, Beryx mollis and B. splendens collected in Japan, based on the mitochondrial cytochrome b gene, and their comparison with those collected in New Caledonia

The sequences spanning 307 bp of the mitochondrial cytochrome b gene were determined for 45 sepcimens of Beryx splendens and seven specimens of B. mollis collected in Japan, resulting in identification of 11 and three haplotypes in the two species, respectively. The parsimony tree was constructed from the determined sequences and those registered into the GenBank data-base as species A and W of B. splendens collected in New Caledonia, featuring with two clades. The first clade comprised species W from New Caledonia and B. mollis in the present study, whereas the second one contained species A from New Caledonia and B. splendens in the present study. These results demonstrate a large geographic distribution for both B. splendens and B. mollis. Some of the haplotypes found in Japan were identical to those of New Caledonia for both B. mollis and B. splendens, suggesting levels of gene flow at the trans-oceanic scale.