Influence of accuracy, repeatability and detection probability in the reliability of species-specific eDNA based approaches.

Environmental DNA (eDNA) barcoding has a high potential to increase the cost-efficiency of species detection and monitoring in aquatic habitats. However, despite vast developments in the field, many published assays often lack detailed validation and there is little to no commonly (agreed upon) standardization of protocols. In this study, we evaluated the reliability of eDNA detection and quantification using published primers and assays targeting the Freshwater Pearl Mussel as a model organism. We first assessed limits of detection for two different target genes (COI and 16S) following the MIQE guidelines, and then tested the reliability of quantification in a double-blind mesocosm experiment. Our results reveal that different methodological indicators, namely accuracy, repeatability and detection probability affected the reliability of eDNA measurement at the different levels tested. The selection of the optimal analytical method was mainly determined by detection probability. Both the COI and 16S assays were highly specific for the targeted organism and showed similar accuracy and repeatability, whilst the limit of detection was clearly lower for the COI based approach. In contrast, the reliability of eDNA quantification hinged on repeatability, reflected by the scattering (r2 = 0.87) around the relationship between eDNA and mussel density in mesocosms. A bootstrapping approach, which allowed for the assignment of measures associated with repeatability of samples, revealed that variability between natural replicates (i.e. accuracy) strongly influenced the number of replicates required for a reliable species detection and quantification in the field.

Development of a micro-fluidic manifold for copper monitoring utilising chemiluminescence detection.

The progressive development of a micro-fluidic manifold for the chemiluminescent detection of copper in water samples, based on the measurement of light emitted from the Cu(ii) catalysed oxidation of 1,10-phenanthroline by hydrogen peroxide, is reported. Micro-fluidic manifolds were designed and manufactured from polymethylmethacrylate (PMMA) using three micro-fabrication techniques, namely hot embossing, laser ablation and direct micro-milling. The final laser ablated design incorporated a reagent mixing channel of dimensions 7.3 cm in length and 250 x 250 microm in width and depth (triangular cross section), and a detection channel of 2.1 cm in length and 250 x 250 microm in width and depth (total approx. volume of between 16 to 22 microL). Optimised reagents conditions were found to be 0.07 mM 1,10-phenanthroline, containing 0.10 M cetyltrimethylammonium bromide and 0.075 M sodium hydroxide (reagent 1 delivered at 0.025 mL min(-1)) and 5% hydrogen peroxide (reagent 2 delivered at 0.025 mL min(-1)). The sample stream was mixed with reagent 1 in the mixing channel and subsequently mixed with reagent 2 at the start of the detection channel. The laser ablated manifold was found to give a linear response (R(2) = 0.998) over the concentration ranges 0-150 microg L(-1) and be reproducible (% RSD = 3.4 for five repeat injections of a 75 microg L(-1) std). Detection limits for Cu(ii) were found to be 20 microg L(-1). Selectivity was investigated using a copper selective mini-chelating column, which showed common cations found in drinking waters did not cause interference with the detection of Cu(ii). Finally the optimised system was successfully used for trace Cu(ii) determinations in a standard reference freshwater sample (SRM 1640).