B-026 Transforming PCR protocols in pandemic response with automated non-contact liquid dispensing
Authors: Henry L , Pitt F , Mulla S , Redgwell T , Grovewood D , Buckley-Taylor R , Lee S , Fedele G , Hill G , Harper P , Fraser D 
Published: October 2024

Abstract

Background
During the COVID-19 pandemic, effective Polymerase Chain Reaction (PCR) protocols were crucial for detecting SARS-CoV-2 in clinical samples, playing a key role in rapid infection control and easing the strain on healthcare services. However, with the critical need for fast and accurate pathogen surveillance, the manual execution of PCR workflows presented numerous challenges, including limited throughput, risk of human error and variability, and risk to human health. Several automated liquid handling systems from a range of vendors were considered to prepare extraction plates for this process. Ultimately, RFL decided on dragonfly® discovery, a non-contact positive displacement dispenser.

Methods
The analytical sensitivity for the UltraDx SARS-CoV-2 N1/N2/RP assay was evaluated by the RFL development team using RNA extracted from a panel of virus inputs from a Qnostics Analytical Q Panel, Product Number: SCV2AQP. Samples were extracted using a modified RNA extraction procedure for Thermo MagMax reagents and consumables with dragonfly discovery. dragonfly discovery was utilized to distribute a combination of inactivation buffer and beads into a 96-well kingfisher flex plate. This process was accomplished using a peristaltic pump and a 6-way Auto Feed Reservoir (AFR) to ensure a constant buffer flow. Simultaneously, the AFR ensured that the beads were kept in constant suspension, resulting in precise dispensing across all wells. The EPCR assay, UltraDx SARS-CoV-2 N1/N2/RP assay (LGC, Biosearch Technologies), utilized two CDC-characterized amplicon primer sets alongside two additional probes using fluorescence-based (FAM dye) detection. The input RNA extracted from clinical samples was combined with RT-PCR reaction mixes, before the reaction arrays were amplified. The reactions were then measured by a single scan on a multi-channel fluorescence reader.

Results
The UltraDx SARS-CoV-2 N1/N2/RP assay (when performed using the manufacturer method and in the absence of dragonfly discovery) has an LoD of 80 copies per reaction for the N1 assay, and 20 copies per reaction for the N2 assay, across 20 replicates for each copy number tested. Using the same/equivalent reagents and control materials, inclusion of dragonfly discovery showed an LoD of 70 copies/mL (0.9 copies/reaction) sensitivity when compared to the LoD suggested by the manufacture.

Conclusions
Integrating dragonfly discovery into the UltraDx SARS-CoV-2 N1/N2/RP assay led to an 80-fold reduction in the limit of detection for the N1 assay compared to the reagent manufacturer's recommendation. Some of this improvement can be attributed to dragonfly discovery's non-contact positive displacement dispensing, coupled with the AFR pump mechanism that ensures a continuous suspension of magnetic beads, enhancing accuracy and precision. dragonfly discovery's versatility supports a robust business-as-usual (BAU) process and is easily scalable for pandemic-level testing. Its user-friendly software interface requires zero computer programming knowledge, providing end-users with the flexibility to run multiple protocols on the same instrument, making it ideal for BAU assay processing. The instrument played a pivotal role in the UltraDx SARS-CoV-2 N1/N2/RP assay conducted by UKHSA during the pandemic, and SPT Labtech will continue to support UKHSA in their mission to protect communities from the impact of health threats.