Real-time multiplex PCR for simultaneous detection of multiple species from environmental DNA: an application on two Japanese medaka species

Abstract

Information about species distribution is crucial to ecological studies. Environmental DNA (eDNA) analysis has recently been used to estimate the distribution of aquatic organisms. Several analytical methods including metabarcoding and species-specific PCR are being used for eDNA analysis. However, when only a few species are targeted, metabarcoding is not cost-effective because of the wasted consumption of read due to amplification of non-target species DNA. On the other hand, species-specific PCR requires tests to be repeated multiple times resulting in consuming more DNA templates, and experimental consumables. Here we propose a methodological framework for simultaneously detecting a few species using real-time multiplex PCR. We developed the species-specific primer-probe sets for two species of Japanese medaka (Oryzias latipes and o. sakaizumii), and we used them in the real-time multiplex PCR. In aquarium experiment, even when the species abundances were biased, both species were simultaneously detected in all samples. In a field survey, eDNA analysis and capture survey produced consistent results in all sampling sites, including sites with low fish densities. eDNA analysis using real-time multiplex PCR can be easily applied to other aquatic organisms, enabling a more cost-effective distribution survey of multiple target organisms.

Figure 1

Distribution of the two medaka species determined by the environmental DNA survey with real-time multiplex polymerase chain reaction (PCR) and the capture survey. Results of the capture survey are consolidated data of PCR-restriction fragment length polymorphism analysis in our study and Kume and Hosoya. Marks on each site indicate inhabiting species determined by capture survey (circle; Oryzias latipes, triangle; O. sakaizumii, diamond; both species). Closed boxes of red (O. latipes) and blue (O. sakaizumii) indicate the positive results of real-time multiplex PCR with three replications. All photographs were taken by ST. This map was created using QGIS version 2.8 (http://www.qgis.org/en/site/) based on the Administrative Zones Data (http://nlftp.mlit.go.jp/ksj/gml/datalist/KsjTmplt-N03-v2_3.html) and the Rivers Data (http://nlftp.mlit.go.jp/ksj/gml/datalist/KsjTmplt-W05.html) which were obtained from free download service of the National Land Numerical Information (http://nlftp.mlit.go.jp/ksj/index.html, edited by ST). There was no need of obtaining permissions for editing and publishing of map data.