Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: A practical application to eggs and larvae

To develop a practical method for identifying Japanese eel Anguilla japonica eggs and larvae to species by a sequence detection system using a real-time polymerase chain reaction (PCR), we examined (i) the sensitivity of the system using samples at various developmental stages, and (ii) influences of intra- and interspecific DNA sequence variations in the PCR target region. PCR amplifications with extracted DNA solution at 7.0 ng l^-1 or lower were efficient at distinguishing A. japonica from other anguillids. A single egg at the gastrula or later developmental stages could also be identified. Two sequence variations in the PCR target region were observed in 2 out of 35 A. japonica collected from three localities, and from four year classes at a single locality. These mutations, however, did not affect the result of species identification achieved by A. japonica-specific PCR primers and probe. The accuracy of this PCR-based method of species identification will help in field surveys of the species.