The use of multiple markers and internal positive controls significantly improves species eDNA detection rates and data reliability

In recent years, environmental DNA analyses became increasingly integrated to detect and monitor the presence abundance of rare organisms, especially in inaccessible aquatic habitats. Although it is generally proven that detection probabilities eDNA surveys exceed those obtained via conventional techniques, these molecular approaches are, however, also subjected limitations levels uncertainty. Besides improvements can be made terms sampling design, volumes filtered water, effective quantity finally analysed, sensitivity inherently determined by number target copies suspended water column. Here we show multiplexing different primer/probe assays for same species, but targeting amplicons situated at loci, a surprisingly overlooked aspect substantially contribute reduce increase single-species detections. By empirically testing large natural samples ddPCR, reveal use multiple markers significantly lower LOD LOQ elusive such as invasive American bullfrog endangered European weather loach variety bodies, ponds, lakes, streams, canals, etc. Especially very low concentrations both our results showed analysing mulitple loci increased lowered stochasticity effects, thus ultimately reduces PCR costs when analysed multiplex. The validation more than one assay taregtting single may further confidence positive Finally, illustrate implementation internal controls (IPC's), an absolute must accurate workflows reliable interpretation generated data. IPC’s not only help track down degraded inhibited samples, avoid false-negative detections, offers insights into extraction efficiency, indispensable quantification population densities. Overall, findings provide strong support on combination with ensures rates generates robust