Simple and on-site DNA purification for LAMP reaction applicable to non-adult tephritid fruit fly (Diptera: Tephiritidae)

Tephritid fruit flies are important pests of fruits and vegetables worldwide, but few studies have examined their ecology and life history in their native range because non-adult individuals of closely related species are morphologically indistinguishable. To identify non-adult individuals collected from host plants, flies examined in earlier studies were painstakingly reared to adulthood. Using Zeugodacus scutellatus Hendel as a model species, this study established a simple DNA extraction and purification method based on loop-mediated isothermal amplification (LAMP) reaction for on-site molecular identification of non-adult fruit flies. The non-adult individual body fluids were sampled using a sterilized toothpick and then diluted in TE buffer and heated for a short time. Alternatively, eggs were crushed in TE buffer using a sterilized toothpick and then heated. After heated buffer was added to the LAMP reaction mixture as template DNA and the mixture was heated for incubation of polymerase, the LAMP reaction detected the amplified DNA of Z. scutellatus. This purification method, characterized by heating after dilution with TE buffer, is simple and rapid. Our purification method, which obviates large centrifuges, thermal cyclers, and other apparatus, provides lower-cost and better on-site molecular identification of non-adult fruit flies than methods described in earlier reports. The LAMP-based molecular identification technique and our purification method are particularly useful for studying quarantined pest organisms that cannot be removed from their native areas.