INSIGHT: a population scale COVID-19 testing strategy combining point-of-care diagnosis with centralised high-throughput sequencing

Abstract

We present here INSIGHT (Isothermal NASBA-Sequencing based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a combination of an isothermal NASBA reaction and next generation sequencing. From commercially acquired human saliva with spiked-in viral RNA as input, the first stage employs isothermal amplification of viral RNA to give a rapid result in one to two hours, using either fluorescence detection or a dipstick readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. In the first stage, fluorescent viral RNA detection can be consistently achieved at 10-100 copies per 20 µl reaction. The second stage pools post-amplification barcoded products from multiple samples for scalable sequencing that could be centralised, to further improve the accuracy of the test in a massively parallel way. Our two-stage testing strategy is suitable for further development into a home-based or point-of-care assay, and is potentially scalable to population level. Affiliations:* Molecular beacons were synthesised in the ATDBio laboratory using a K&A H-8 SE DNA Synthesiser, and purified by reversed-phase HPLC.

Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay

“…Previous reports have also highlighted the importance of primer design and optimization to achieve efficient NASBA. 22,42,43 We observed that primers with fairly similar basic criteria showed significantly different NASBA efficiencies. Therefore, after an initial round of screening for sensors, and zeroing in on an efficient sensor, we again screened for the most efficient NASBA primers for the selected sensor.…”

ApharSeq: An Extraction-free Early-Pooling Protocol for Massively Multiplexed SARS-CoV-2 Detection

“…We believe that our approach, and future improvements and modifications thereof, might form the basis for such an improvement of several orders of magnitude relative to current testing strategies. Specifically, the ability to pool hundreds or thousands of samples as early as possible, without losing the sample-specific information, is a crucial improvement on all currently published NGS-based tests 6,23,24,25,26,27 . The early pooling approach proposed here, which might be applicable to other protocols, elicits proportional reductions in costs and labor that directly translate to higher throughput in testing.…”

Science’s Response to COVID-19

“…151 It is noteworthy that other isothermal amplification techniques (some of which will be discussed below) are also in existence and have been used for viral detection, for instance: recombinase polymerase amplification (RPA); 152 rolling cycle amplification (RCA); 153 and nucleic acid sequence-based amplification (NASBA). 154 We will discuss LAMP as an exemplar. One key benefit of this method over traditional PCR is that it is an isothermal reaction, and hence can be run in simple setups without the need for a thermocycler.…”