Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions

Abstract

The use of sensitive methods is key for the detection of target taxa, from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex PCRs, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analysing dilution series of DNA extracts and field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl DNA extract. celPCR was suitable for quantifying target DNA and direct inference of DNA concentrations from RFU was possible after accounting for primer effects. Furthermore, multiplex celPCRs and dPCRs were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.

Lateral and longitudinal fish environmental DNA distribution in dynamic riverine habitats

“…Though celPCR assay optimization (Sint et al., 2012; Thalinger et al., 2016) cannot fully account for differences in amplification efficiency between primer pairs (Thalinger et al., 2020), primer bias is an unlikely cause for the strong P. phoxinus signals, because detection patterns and signal strength did not differ between the salmonids. Nevertheless, celPCR produces a semiquantitative measure of target copy numbers and without direct comparison to a fully quantitative approach (i.e., digital PCR); it is not possible to infer absolute eDNA concentrations from RFU (Thalinger et al., 2020). Most likely, the strong eDNA signals of P. phoxinus can be attributed to a combination of the aforementioned physiological and morphological differences.…”

“…Target DNA signal strength was determined via capillary electrophoresis on the QIAxcel (QIAGEN) with the associated software QIAxcel Screengel version 1.6.0.10 using the method AM320‐30s. The fluorescent signal measured in Relative Fluorescence Units (RFU) was used as a semiquantitative measure of target DNA (Thalinger et al., 2019, 2020), and signals ≥0.08 RFU were deemed positive. To verify the applicability of this approach, 40 samples positive for S. trutta were retested with digital PCR (Appendix S1: a).…”

Lateral and longitudinal fish eDNA distribution in dynamic riverine habitats

“…However, the difference in eDNA signal strength was principally found in November at ∼60 L/s, when all but one individual of P. phoxinus survived. Though celPCR assay optimization (Sint et al, 2012; Thalinger et al, 2016) can not fully account for differences in amplification efficiency between primer pairs (Thalinger et al, 2020), primer bias is an unlikely cause for the strong P. phoxinus signals, because detection patterns and signal strength did not differ between the salmonids. Nevertheless, celPCR produces a semi-quantitative measure of target copy numbers and without direct comparison to a fully quantitative approach (i.e.…”

“…Nevertheless, celPCR produces a semi-quantitative measure of target copy numbers and without direct comparison to a fully quantitative approach (i.e. digital PCR), it is not possible to infer absolute eDNA concentrations from RFU (Thalinger et al, 2020). Most likely, the strong eDNA signals of P. phoxinus can be attributed to a combination of the aforementioned physiological and morphological differences.…”

“…Target DNA signal strength was determined via capillary electrophoresis on the QIAxcel (QIAGEN) with the associated software QIAxcel Screengel version 1.6.0.10 using the method AM320-30s. The fluorescent signal measured in Relative Fluorescence Units (RFU) was used as a semi-quantitative measure of target DNA (Thalinger, Pütz, & Traugott, 2020; Thalinger et al, 2019) and signals ≥0.08 RFU were deemed positive. To verify the applicability of this approach, 40 samples positive for S. trutta were re-tested with digital PCR (Supporting Information (SI) 1a).…”