Rapid environmental DNA detection with isothermal amplification methods: a comparison with the real-time quantitative PCR in the natterjack toad (Epidalea calamita).

Abstract

Species detection relying on environmental DNA (eDNA) traditionally relies on Polymerase Chain Reaction (PCR). This amplification technique takes in general a minimum of one hour and requires in most cases access to specialized laboratories and equipment. Isothermal amplification techniques, originating from clinical research, are emerging as rapid and field-deployable alternative methods. Yet, whether their sensitivity, specificity, and cost/time-efficiency are comparable to the traditional real-time quantitative PCR (qPCR) remains to be assessed. In this work, we compare the performance of loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) with qPCR to detect the natterjack toad (Epidalea calamita) from aquatic filtrations. This objective was achieved in three steps. First, we developed LAMP and RPA primers specific to the target species based on its mitochondrial genome. Second, we amplified DNA extracts from E. calamita and from tissues of closely related species using real-time fluorescence detection with LAMP, RPA, and qPCR commercially available protocols to evaluate and compare the sensitivity and specificity of the three methods. Finally, we validated all protocols on eDNA extracts recovered from water samples from ponds surveyed by field scientists. We show that RPA had a sensitivity equivalent to that of the qPCR, while LAMP sensitivity was ten times lower. All our assays amplified the species DNA in eDNA samples where the species was observed by field scientists. Through this work, we thus propose a robust methodology for implementing isothermal amplification assays for eDNA applications and show that RPA is a good alternative to qPCR for detecting conservation-priority species (i.e. protected or invasive species) when time and lab facilities are constrained.