An application of PCR-RFLP species identification assay for environmental DNA detection

PeerJ

article

Takeshi Igawa¹  Teruhiko Takahara²  Quintin Lau⁴  Shohei Komaki⁵ 

[1] Amphibian Research Center, Hiroshima University;Faculty of Life and Environmental Science, Shimane University;Estuary Research Center, Shimane University;Department of Evolutionary Studies of Biosystems, Sokendai ,(The Graduate University for Advanced Studies);Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank Organization, Disaster Reconstruction Center, Iwate Medical University

关键词: Amphibian;    Environmental DNA;    Distribution range;    Species identification;    Monitoring survey;    Water sample;    Frog species;    Tadpole;   

DOI  :  10.7717/peerj.7597

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Recent advancement of environmental DNA (eDNA) methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by applying quantitative PCR (qPCR) or next generation sequencing to eDNA. However, the cost of eDNA detection using these approaches can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers in starting eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (Rana japonica, Rana ornativentris, and Rana tagoi tagoi) in various spatial scales including an area close to the Fukushima nuclear power plant where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.

【 授权许可】

CC BY