“The Dilemmas of Digital PCR: Technology Transfer for Fish Health Diagnostics (7024)”

The Dilemmas of Digital PCR: Technology Transfer for Fish Health Diagnostics

Introduction:

Viral myopathies of farmed Atlantic salmon threaten the aquaculture industry, resulting in economic losses and poor fish welfare. Rapid, sensitive and cost-effective detection of myocarditis-related viruses in farmed salmon is crucial for early interventions and appropriate mitigation for disease management.

Droplet digital polymerase chain reaction (ddPCR) is a relatively new evolution of PCR with the advantage of absolute nucleic acid quantification, unlike Real Time-PCR (qPCR), which provides relative quantification information. ddPCR is also reportedly better suited to multiplex assays due to reduced competitive effects.

This project aims to develop a multiplex ddPCR assay for three RNA myocarditis-related viruses. Three viruses significantly affecting Irish farmed salmon include Salmonid Alphavirus (SAV) initiating pancreas disease (PD), Piscine Orthoreovirus (PRV) causing Heart Skeletal Muscle Inflammation (HSMI) and Piscine Myocarditis Virus (PMCV), triggering Cardiomyopathy Syndrome (CMS).

Methodology:

This project planned to optimise qPCR molecular detection assays in singleplex, transfer these singleplex assays to ddPCR and subsequently develop a ddPCR multiplex assay.

Existing Taqman® probe-based qPCR assays for SAV, PRV and PMCV were optimised using the Applied Biosystem 7500 qPCR machine, then tested in singleplex using the Stilla Naica Prism 3 ddPCR machine. Two probe-based detection methods were tested using ddPCR: Hydrolysis probes (TaqMan®) and Universal Nucleic Acid Detection probes (Rainbow Technology – Pxlence).

Results:

Transfer of the specific qPCR assays to the Stilla Naica Prism 3 ddPCR system has been challenging. Technical issues include the availability of probe dyes compatible with both systems, the probe quencher types available with certain dyes and limitations multiplexing more than two minor groove binder (MGB) probes. In preparation for multiplex development, a combination of MGB and QSY (non-fluorescent quencher technology by Thermo Fisher Scientific) probes were tested. QSY probes had low fluorescence signal compared to MGB probes on ddPCR. To overcome these issues, Universal Nucleic Acid Detection probe technology (Rainbow) was trialed in singleplex.

Conclusions:

This project, while in early stages, has already shown that transferring molecular detection assays from a qPCR platform to a digital one may not be as straightforward as expected. Careful consideration of the digital platform and probe-based assay type is necessary, as probe dye and quencher compatibility are key factors for successfully multiplexed ddPCR assays. In this instance, extensive optimisation and redesign of key assay components was required to move viral RNA detection qPCR assays onto a ddPCR platform. Optimisation experiments remain ongoing.

1. LINEHAN, STEPHANIE, MARINE INSTITUTE, Presenter