Abstract
We present INSIGHT (Isothermal NASBA-Sequencing-based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a barcoded isothermal NASBA reaction that combines point-of-care diagnosis with next generation sequencing, aiming to achieve population-scale COVID-19 testing. INSIGHT combines the advantages of near-patient with centralised testing. Stage 1 allows a quick decentralised readout for early isolation of pre-symptomatic or asymptomatic patients. The same reaction products can then be used in a highly multiplexed sequencing-based assay in Stage 2, confirming the near-patient testing results and facilitating centralised data collection. Based on experiments using commercially acquired human saliva with spiked-in viral RNA as input, the INSIGHT platform gives Stage 1 results within one to two hours, using either fluorescence detection or a lateral flow (dipstick) readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. INSIGHT Stage 2 can be performed by directly pooling and sequencing all post-amplification barcoded Stage 1 products from hundreds of thousands of samples with minimal sample preparation steps. The 95% limit of detection (LoD-95) for INSIGHT is estimated to be below 50 copies of viral RNA per 20 μl of reaction. Our two-stage testing strategy is suitable for further development into a rapid home-based and point-of-care assay, and is potentially scalable to the population level.
Competing Interest Statement
Q.W., C.S., S.A.T. and A.R.B. are inventors on a patent application (GB2007724.4) submitted by Genome Research Limited, with the aim of making this technology freely available for research and deployment. In the past three years, S.A.T. has consulted for Genentech and Roche, and sits on Scientific Advisory Boards for Foresite Labs, Biogen and GlaxoSmithKline. T.B. is a consultant at ATDBio.
Footnotes
*** IMPORTANT: This protocol has not been validated on patient samples and should not be used for clinical diagnosis without further validation and certification. ***
*** This is ongoing research to develop a testing strategy for the SARS-CoV-2 virus. The Wellcome Sanger Institute is not in a position to develop this into a commercial product, but would be open to discussions with third parties about how they could develop this further to achieve the societal benefits of this work. ***
Methods and Results sections updated details on dipstick readout and sequencing
2 Molecular beacons were synthesised in the ATDBio laboratory using a K&A H-8 SE DNA Synthesiser, and purified by reversed-phase HPLC.
3 Molecular beacon is reconstituted with annealing buffer (10 mM Tris pH 8 with 10 μM MgCl2) to the final concentration of 10 μM. Beacon is then annealed by incubation at 85 °C for 5 min, then gradual cooling to 4 °C by 0.1 °C/s before the NASBA reaction.
