Use of droplet digital PCR for estimation of fish distribution, abundance and biomass in environmental DNA surveys
Environmental DNA (eDNA) analysis has been recently developed to estimate the distribution of aquatic animals and the abundance/biomass by quantifying the number of target DNA copies with quantitative real-time PCR. A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. Here, we tested ddPCR merits for the eDNA study by two experiments; Exp. 1) We evaluated the quantification accuracy of quantitative real-time PCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp,Cyprinus carpio, and Exp. 2) We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in 25 ponds.
Exp. 1) We found that ddPCR quantified the concentration of the carp eDNA along with their abundance and biomass more accurately than real-time PCR analysis, especially at low eDNA concentrations. Thus, ddPCR is better suited to measure eDNA concentration in the water, and it provides more accurate results for the abundance and biomass of the target species than real-time PCR. In addition, the analytical errors were smaller in ddPCR than in real-time PCR. Also, we found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by both ddPCR and real-time PCR.
Exp. 2) Using pond water samples, we found that ddPCR had higher detection rates of bluegill eDNA than real-time PCR with either of the PCR reagents [Gene Expression Master Mix (GEMM), and Environmental Master Mix 2.0 (EMM), Life Technologies], especially at low DNA concentrations. Limits of DNA detection analysis, which were tested by spiking the bluegill DNA to the DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Thus, we found that ddPCR had higher detection rate than real-time PCR. In eDNA studies, PCR amplification is often inhibited by the presence of organic and inorganic matter. In ddPCR, the sample is partitioned into thousands of nano-liter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. The results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR.
Results were published by Doi et al. 2015 in PLOS ONE 10: e0122763 (for Exp.1), and in Environmental Science & Technology (DOI: 10.1021/acs.est.5b00253, for Exp. 2)