Information on the species distribution in an environment is fundamental for the conservation of rare species and eradication of invasive species. However surveillance of species distribution generally requires much time and effort. Recently, environmental DNA (eDNA) analysis has been developed as a new method for estimating the distribution of fish, amphibians, and mammals by detecting their genetic markers from water and soil. Basically, a PCR test should be repeated for each of the target species when single PCR (basic PCR) is adopted where a set of primers is used for eDNA analysis of each species. While, the basic PCR test requires much time, consumables, and eDNA samples. In the present study, the multiplex PCR, which can detect multiple markers using multiple sets of primers at once, was applied to the eDNA analysis. In laboratory experiments, we determined optimal combination of concentrations of primers and probes for multiplex PCR based on the detection limit of each of three target species: ayu (Plecoglossus altivelis altivelis), temperate seabass (Lateolabrax japonicas) and flathead grey mullet (Mugil cephalus cephalus). Probes for each target were labeled with TAMRA, JOE, and FAM, respectively. As an application of the multiplex PCR for field study, water samples were obtained from 15 rivers flowing into Osaka Bay, Japan, and analyzed with multiplex PCR and basic PCR targeting the three species to compare the detection capabilities of the two methods.
Results/Conclusions
Using artificially synthesized DNA of each target species as PCR templates, optimal combination of concentrations of primers and probes for multiplex PCR was determined as 900 nM of each primer and 375 nM, 66 nM, 45 nM of TAMRA, JOE, and FAM probes, respectively. Detection sensitivity was reduced when the copy number of minor species was <1/1000 compared to the most abundant species. In the analyses of field samples, the detection results determined by multiplex PCR and basic PCR were highly consistent with a 91% match. Although there were some exceptional cases where a species was detected only in one of the two methods, undetected cases were not biased against one method. Simultaneous multi-species detection using multiplex PCR would provide an efficient way of eDNA analysis in terms of time, cost, and the amount of DNA sample compared with basic PCR.