A diagnostic multiplex polymerase chain reaction method to identify Japanese internal apple-feeding Lepidopteran pests: Grapholita molesta, Grapholita dimorpha (Lepidoptera: Tortricidae), and Carposina sasakii (Lepidoptera: Carposinidae)

We present a multiplex polymerase chain reaction method to differentiate between three Japanese internal apple-feeding pests: the oriental fruit moth, Grapholita molesta (Busck), Grapholita dimorpha Komai, and the peach fruit moth, Carposina sasakii Matsumura. A 1,342-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in each species. The three species showed consistent and diagnostic differences in the region of the COI gene, from which three species-specific forward primers were designed. The forward primers along with one universal reverse primer were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. This method enabled easy, immediate, and accurate identification of internal feeding Lepidoptera in apples and other fruits.