Investigation of Limit of Detection and Quantification for digital PCR assays: the case study of Azadinium spinosum (Dinophyceae) in Irish waters

Azaspiracids (AZAs) are a group of lipophilic toxins associated with Azaspiracid Poisoning (AZP) in humans following consumption of contaminated shellfish. AZA toxins are produced by some species of marine nano dinoflagellates from the cosmopolitan family Amphidomataceae. Ireland is one of the countries most affected globally by AZA events, where blooms of toxigenic Azadinium spinosum have been leading to prolonged closures of shellfish farms. DNA-based techniques such as quantitative PCR (qPCR) and digital PCR (dPCR) are extremely sensitive methods that have shown great potential for routine screening of environmental samples, with the former being successfully applied in the detection and relative quantification of some species of Amphidomataceae in Irish waters. In contrast, dPCR has been used to a lesser extent despite its ability to enable absolute quantification of low-copy targets and potential to mitigate enzymatic inhibitory effects. Thus, the present study tested the accuracy of two dPCR systems (Standard BioTools’ BiomarkHD and QIAGEN’s QIAcuity Four) using a TaqMan probe-based assay targeting the LSU-rDNA gene in samples of known target concentration. Limit of Detection (LOD) and Quantification (LOQ) thresholds were defined to infer transferability, repeatability, and reproducibility of the assay across the different systems. Both dPCR methods showed a positive correlation between and within platforms, and high sensitivity and accuracy in detecting and quantify low-copy targets. These are promising results especially in view of implementing these platforms into National Monitoring Programmes for monitoring harmful phytoplankton.