Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). 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. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.

近年来，研究人员开发了环境DNA（environmental DNA, eDNA）分析方法，通过实时荧光定量聚合酶链式反应（quantitative real-time PCR, qPCR）对目标DNA的拷贝数进行定量，以此估算水生动物的分布范围；微滴式数字聚合酶链式反应（droplet digital PCR, ddPCR）作为一种新型定量PCR技术，可将PCR反应体系划分为数千个微滴并检测每个微滴内的扩增情况，从而实现对目标DNA的直接定量。本研究通过设置不同数量普通鲤鱼的中宇宙实验（mesocosm experiments），利用eDNA评估了qPCR与ddPCR在估算物种丰度和生物量时的定量准确性，结果显示相较于qPCR，ddPCR能够更精准地定量鲤鱼eDNA浓度，且与鲤鱼丰度和生物量的相关性更强，尤其在低eDNA浓度条件下表现更为突出，此外ddPCR的分析误差也显著低于qPCR，综上ddPCR更适用于水体中eDNA浓度的检测，相较于qPCR能够为目标物种的丰度和生物量估算提供更为准确的结果；同时无论是采用ddPCR还是qPCR进行分析，鲤鱼丰度与eDNA浓度的相关性均强于生物量与eDNA浓度的相关性，这表明对于体重变异较小的物种，通过eDNA分析能够更准确地估算其种群丰度。