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

Environmental DNA (eDNA) detection methods have traditionally relied on Polymerase Chain Reaction (PCR), which typically takes at least an hour to complete and requires specialized labs and equipment. Isothermal amplification techniques, pioneered in clinical research, offer a faster and more accessible alternative. However, their sensitivity, specificity, and cost/time efficiency compared to real-time quantitative PCR (qPCR) has yet to be determined.

This study compares the performance of loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) with qPCR in detecting the natterjack toad (Epidalea calamita) from aquatic filtrations. The researchers developed LAMP and RPA primers based on the target species' mitochondrial genome and then amplified DNA extracts from E. calamita and related species using real-time fluorescence detection with LAMP, RPA, and qPCR.

Sensitivity, specificity, and validation of all protocols on eDNA extracts recovered from surveyed water samples were evaluated.

The results show that RPA had a sensitivity equivalent to qPCR, while LAMP's sensitivity was ten times lower. All assays amplified the species DNA in eDNA samples where the natterjack toad was observed by field scientists. This work proposes a robust methodology for implementing isothermal amplification assays in eDNA applications and demonstrates that RPA serves as a viable alternative to qPCR when time and lab facilities are limited.