ABSTRACT
Biomonitoring of marine life has been enhanced in recent years by the
integration of innovative DNA-based approaches, which offer advantages
over more laborious conventional techniques (e.g. direct capture) and
greater taxonomic resolution especially in complex life cycles and early
life stages. However, tradeoffs between throughput, sensitivity and
quantitative measurements must be made when choosing between the
prevailing molecular methodologies (i.e. metabarcoding or qPCR/dPCR).
Thus, the aim of the present study was to demonstrate the utility of a
microfluidic-enabled High Throughput quantitative PCR platform (HT-qPCR)
for the rapid and cost-effective development and validation of a
DNA-based multi-species biomonitoring toolkit, using larvae of 24
commercially targeted bivalve and crustacean species as a case study.
The workflow was divided into three main phases: definition of target
taxa and establishment of reference databases (PHASE 1); in
silico selection/development and in vitro assessment of
molecular assays (PHASE 2); and protocol optimization and field
validation (PHASE 3). Of a total of 85 assays in silico , 42 were
eventually chosen and validated in vitro . Genetic signal showed
good correlation with direct visual counts by microscopy, but also
showed the ability to provide quantitative data at the highest taxonomic
resolution (species level) in a time- and cost-effective fashion.
This study developed a
biomonitoring toolkit, demonstrating the considerable advantages of this
state-of-the-art technology in boosting the development and application
of panels of molecular assays for the monitoring and management of
natural resources that can be applied to a range of monitoring
programmes.
Keywords : DNA, High Throughput, qPCR, biomonitoring, shellfish