Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for quick identification of the Japanese salamander Hynobius tokyoensis

Abstract

Species identification using molecular techniques has recently become com-mon for various taxa. Loop-mediated isothermal amplification (LAMP) is one of the easiest and least expensive molecular identification methods. Although few studies have developed LAMP assays for amphibians, we believe that LAMP is also useful for identifying endangered amphibians. Hynobius tokyoensis and H. lichenatus occur in Honshu, Japan, and have parapatric distributions. They are similar morphologically, especially at early developmental stages, including eggs and larvae. Hynobius tokyoensis has been listed as a national endangered species in Japan since 2020, and unambiguous identification of these species is therefore important for their conservation and management. In this study, we developed a LAMP primer set for the mitochondrial cytochrome b region to detect H. tokyoensis, and we evaluated the LAMP assay using total genomic DNA from four H. tokyoensis and three H. lichenatus individuals from across most of their ranges. Our LAMP primer set could distinguish these two species. This study should help to establish LAMP assays for other endangered species and morpho-logically similar species.