マルチプレックスPCR法を用いた日本産マンボウ属2種のミトコンドリアDNAの簡易識別法

抄録

Morphological identification of two ocean sunfish species (genus Mola) is not an easy task due to the giant body size and poor understanding of their intraspecific variation. A rapid, reliable method of identifying mitochondrial DNA from two Mola species in Japanese waters was designed for PCR-based genotyping of the mitochondrial control region. Two allele-specific primers were developed for the mitochondrial control region of each species: CRMolaAL and CRMolaBL for Mola spp. A and B, respectively. Using the same reverse primer (H884-12S), specific primers for Mola spp. A and B were designed to amplify ca. 400- and 500-bp fragments, respectively. A pair of fish universal primers (L1969- 16S and H2582-16S) amplifying ca. 700 bp was used as an internal control. Multiplex PCR reactions including these five primers produced a species-specific fragment for either Mola sp. A or B with an internal control. This approach has major advantages over other molecular species-identification methods in speed and costefficiency, and greatly helps in determining Mola species.