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

Satsuki Tsuji,Hiroki Yamanaka,Tadao Kitagawa,Iori Teramura,Naoki Shibata,Yuka Iguchi

doi:10.1038/s41598-018-27434-w

Satsuki Tsuji, Hiroki Yamanaka + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-27434-w

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Journal: Scientific Reports	Publication Date: Jun 14, 2018
Citations: 31	License type: open-access

Affiliation: Ryukoku University, Kindai University

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.

Highlights

To overcome these obstacles, a technique called “environmental DNA analysis” has recently been used[6,7]
Real-time multiplex PCR, as opposed to real-time single PCR, shortens the processing times and reduces the use of reagents[19,20]. This methodological framework was applied for detecting the Oryzias species complex in Japan, generally called ‘medaka’ fish, which includes O. latipes and O. sakaizumii
No DNA was detected in any experimental control or PCR negative controls (PCR-NCs), confirming the absence of cross-contamination during sample processing

Summary

Introduction

A technique called “environmental DNA (eDNA) analysis” has recently been used[6,7]. When multiple species are targeted, PCR is required for each individual species[17,18] so that the cost in time or money and the amount of DNA samples would increase depending on the number of target species To address these PCR issues, a new methodological framework is suggested for simultaneous detection of a limited number of species by analyzing eDNA using real-time multiplex PCR. Real-time multiplex PCR, as opposed to real-time single PCR, shortens the processing times and reduces the use of reagents[19,20] In this study, this methodological framework was applied for detecting the Oryzias species complex in Japan, generally called ‘medaka’ fish, which includes O. latipes and O. sakaizumii (hereafter, latipes and sakaizumii, respectively). The developed primer probe sets were tested with respect to whether they could detect only respective target species using real-time multiplex PCR with a mixture of latipes and sakaizumii genomic DNA. The effectiveness of these primer probe sets was tested by determining the distributions of the two medaka species in their known natural habitats

Methods

Results

Conclusion