Primers and probes

Primers and probes were ordered from IDT or Biolegio and can be found in the Supplementary Information in Supplementary Tables 3, 4, and 5.

VLP preparation

The nucleic acid sequence of the N-gene of SARS-CoV-2 (accession number: NC_045512) was ordered from GeneArt (Thermo Fisher Scientific). The N-gene was cloned into a MS2 VLP expression plasmid backbone (Addgene #128233) using Type IIs assembly. The sequence-verified (Eurofins Genomics) plasmid (Addgene #155039) was then transformed into Rosetta 2 (DE3) pLysS cells (Merck Millipore). An overnight culture was used to inoculated 200 mL of Terrific Broth (Merck) supplemented with 35 µg/mL of Chloramphenicol (Merck) and 50 µg/mL of Kanamycin (Merck), and grown at 37 °C, 200 r.p.m. until an OD of 0.8. The culture was induced by supplementing with 0.5 mM IPTG (Merck) and grown at 30 °C for a further 16 h. Cells were collected at 3220 × g at 4 °C and stored at −20 °C for later purification.

All protein purification steps were performed at 4 °C. The cell pellet was resuspended in 4 mL Sonication Buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 5 mM CaCl2, and 100 mM NaCl) with 700 U RNase A (Qiagen), 2500 U BaseMuncher (Expedeon), and 200 U Turbo DNase (Thermo Fisher Scientific). The cells were sonicated for a total of 2 min (50% amplitude, 30 s on, 30 s off) on wet ice. The lysate was then incubated for 3 h at 37 °C. The lysate was centrifuged at 10,000 × g for 10 min at room temperature in a microcentrifuge. The supernatant was then filtered with a 5 µm cellulose acetate (CA) filter before being mixed 1 : 1 with 2× Binding Buffer (100 mM monosodium phosphate monohydrate pH 8.0, 30 mM Imidazole, 600 mM NaCl).

Supernatant was applied to a 5 mL HiTrap® TALON® Crude column (Cytiva) with a HiTrap® Heparin HP column (Cytiva) in series on an ÄKTA pure (Cytiva) primed with Binding Buffer (50 mM monosodium phosphate monohydrate pH 8.0, 15 mM Imidazole, 300 mM NaCl). The protein was eluted with a linear gradient of elution buffer (50 mM monosodium phosphate monohydrate pH 8.0, 200 mM Imidazole, 300 mM NaCl) and then desalted and buffer exchanged into STE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl) using an Amicon Ultra-15 10 K Centrifuge Filter (Merck). The protein concentration was measured using the Qubit Protein Assay Kit and Qubit 3 Fluorometer (Thermo Fisher Scientific). The protein was then diluted in STE buffer, aliquoted, and stored at −80 °C.

Reverse-transcriptase droplet digital PCR

Droplet digital PCR was performed using the Bio-Rad QX200 Droplet Digital PCR system. Reactions were set up using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad) with primer and probe concentrations of 500 nM and 125 nM, respectively. Data were exported in CSV format and analysed using a custom Python implementation (https://github.com/mcrone/plotlydefinerain) of an online tool (http://definetherain.org.uk). The online tool uses a positive control to define positive and negative droplets using K-means clustering, with rain being determined as anything outside three standard deviations from the mean of the positive and negative clusters. It then calculates final concentration based on Eq. 1.

$$c = – {it{ln}}frac{{N_{mathrm{{neg}}}}}{N}/V_{mathrm{{droplet}}}$$

(1)

c = calculated concentration (copies/µL)

Nneg = number of negative droplets

N = total number of droplets

Vdroplet = average volume of each droplet (0.91 × 10−3 µL).

Dynamic light scattering

DLS was performed using a Zetasizer Nano (Malvern Panalytical) according to the manufacturer’s instructions.

Quantitative PCR

qPCR experiments were designed using the combination of SAS JMP and Riffyn. Primers, probes, and their relative concentrations were those recommended by the CDC and were ordered from IDT. TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific), TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific), or Luna Universal Probe One-Step RT-qPCR (NEB) were used as the relevant master mixes. qPCR reactions were otherwise set up according to the manufacturer’s instructions and thermocycling settings (according to the CDC protocol). Liquid transfers were performed using an Echo 525 (Labcyte). Plates were sealed with MicroAmp Optical Adhesive Films (Thermo Fisher Scientific) and spun at 500 × g in a centrifuge. An Analytik Jena qTower3 auto was used for thermocycling and measurements were taken in the FAM channel.

LwCas13a purification

A plasmid expressing LwCas13 [pC013-Twinstrep-SUMO-huLwCas13a was a gift from Feng Zhang (Addgene plasmid # 90097)] was transformed into Rosetta 2 (DE3) pLysS cells (Merck Millipore). An overnight culture was inoculated into 1 L of Terrific Broth (Merck) supplemented with 35 µg/mL of Chloramphenicol (Merck) and 50 µg/mL of Kanamycin (Merck), and was grown at 37 °C, 160 r.p.m. to an OD of 0.6. The culture was then induced with 0.5 mM IPTG (Merck), cooled to 18 °C, and grown for a further 16 h. Cells were collected at 3220 × g at 4 °C and stored at −20 °C for later purification.

All protein purification steps were performed at 4 °C. The cell pellet was resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM dithiothreitol (DTT)) supplemented with protease inhibitors (cOmplete Ultra EDTA-free tablets, Merck) and BaseMuncher (Expedeon), and sonicated for a total of 90 s (amplitude 100% for 1 s on, 2 s off). Lysate was cleared by centrifugation for 45 min at 38,758 × g at 4 °C and the supernatant was filtered through a 5 µm CA filter.

Supernatant was applied to a 5 mL StrepTrap™ HP column (Cytiva) on an ÄKTA pure (Cytiva). The buffer of the system was changed to SUMO digest buffer (30 mM Tris-HCL pH 8, 500 mM NaCl, 1 mM DTT, 0.15% Igepal CA-630). SUMO digest buffer (5 mL) supplemented with SUMO enzyme (prepared in-house) was then loaded directly onto the column and left to incubate overnight. The cleaved protein was then eluted with 5 mL of SUMO digest buffer. The elution fraction was diluted 1 : 1 with Ion Exchange low salt buffer (20 mM HEPES pH 7, 1 mM DTT, 5% Glycerol), applied to a Hitrap® SP HP column (Cytiva), and eluted using a gradient of the ion exchange high-salt buffer (20 mM HEPES pH 7, 2000 mM NaCl, 1 mM DTT, 5% Glycerol). The eluted protein was then pooled, concentrated, and buffer exchanged into Storage buffer (50 mM Tris-HCl pH 7.5, 600 mM NaCl, 2 mM DTT, 5% Glycerol) using an Amicon Ultra-15 30 K Centrifuge Filter (Merck). The protein concentration was measured using the Qubit Protein Assay Kit and Qubit 3 Fluorometer (Thermo Fisher Scientific). The protein was then diluted, aliquoted, and stored at −80 °C.

crRNA transcription and quantification

DNA was ordered as ssDNA oligonucleotides from IDT and resuspended at 100 µM in Nuclease Free Duplex Buffer (IDT). Oligos contained a full-length reverse strand and a partial forward strand that contained only the T7 promoter sequence. Oligos were annealed by combining forward and reverse strands in equimolar concentrations of 50 µM and heating to 94 °C for 5 min and slow cooling (0.1 °C/s) to 25 °C in a thermocycler.

RNA was then in vitro transcribed using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions with a DNA template of 100 nM. Reactions were incubated for 16 h at 37 °C. DNAse I was then added and incubated for 15 min at 37 °C.

Automated purification was performed using the CyBio FeliX liquid-handling robot (Analytik Jena) using RNAClean XP beads (Beckman Coulter) according to the manufacturer’s instructions.

For automated quantification, samples were loaded into a 384 PP Echo plate (Labcyte). Qubit RNA BR Dye and Qubit RNA BR Buffer (Thermo Fisher Scientific) were premixed at a ratio of 1 : 200 and loaded into a 6-well reservoir (Labcyte). Experimental design was performed using a custom Python script and Riffyn with each sample having four technical replicates that were randomly distributed in a Greiner 384 PS Plate (Greiner Bio-One). A standard curve of 9 concentrations (0, 5, 10, 15, 20, 40, 60, 80, 100 ng/µL) was prepared using the standards provided with the Qubit RNA BR Kit (Thermo Fisher Scientific).

A volume of 9.95 µL of the mix of Qubit Dye and Qubit buffer was added to each well using an Echo 525 (Labcyte). A volume of 0.05 µL of sample was then added to each well using the Echo 525 (Labcyte) and the plate was sealed with a Polystyrene Foil Heat Seal (4titude) using a PlateLoc Thermal Microplate Sealer (Agilent). Plates were centrifuged at 500 × g for 1 min before being kept in the dark for 3 min.

Plates were read using a CLARIOstar Plus (BMG Labtech) plate reader, using the following settings: excitation wavelength of 625–15 nm, dichroic of 645 nm, and emission of 665–15 nm and the Enhanced Dynamic Range (EDR) function. RNA molar concentration values were calculated, and the concentration was then normalized, RNA aliquoted and subsequently stored at −80 °C.

CRISPR-Cas13a assays with PCR amplification

Experiments were designed and randomized using SAS JMP and Riffyn. Targets were pre-amplified using the Luna Universal One-Step RT-qPCR kit (NEB) with a primer concentration of 500 nM for 45 cycles. All concentrations are final CRISPR reaction concentrations and the final CRISPR reaction volumes were 5 µL. An Echo 525 (Labcyte) was used to transfer CRISPR Master Mix (50 nM LwCas13a, 1 U/mL murine RNAse inhibitor (NEB), 4 mM Ribonucleotide Solution Mix (NEB), 1.5 U/µl T7 RNA Polymerase (Thermo Fisher Scientific) and 1.25 ng/µL HEK293F background RNA) in Nuclease Reaction Buffer (20 mM HEPES pH 6.8, 60 mM NaCl, 9 mM MgCl2) to a 384-well Small Volume LoBase Microplate (Greiner Bio-One). crRNA (25 nM) and 200 nM poly-U fluorescent probe (5′-/56-FAM/rUrUrUrUrU/3IABkFQ/-3′) were then added separately. An Echo 550 (Labcyte) was used to transfer pre-amplified products from a 384 LDV Plus Echo plate (Labcyte) to initiate the reaction, the plate was sealed, spun at 500 × g for 1 min and read using a CLARIOstar Plus (BMG Labtech) plate reader with an excitation wavelength of 483-14 nm, emission of 530-30 nm, dichroic filter of 502.5 nm, and EDR enabled. Double orbital shaking of 600 r.p.m. for 30 s was performed before the first cycle. The reactions were incubated at 37 °C with readings taken every 2 min. Each reaction was normalized between a water input (background fluorescence) as 0 and an RNase I (Thermo Fisher Scientific) input (0.25 U) as 1 (RNase I cleaves all of the fluorescent probe and thus serves as a positive relative control).

Colorimetric LAMP reactions with VLPs

Experiments were designed and randomized using SAS JMP and Riffyn. Colorimetric LAMP reactions (NEB WarmStart® Colorimetric LAMP 2× Master Mix) were performed with a lower final reaction volume of 5 µL. Master Mix, primers, and template were transferred to a 384-well small volume LoBase plate (Greiner Bio-One) using an Echo 525 (Labcyte). The plate was then sealed with a MicroAmp Optical Adhesive Film (Thermo Fisher Scientific) and centrifuged for 1 min at 500 g. The plate was incubated at 65 °C in a CLARIOstar Plus (BMG Labtech) plate reader and absorbance measurements were taken at 415 nm every minute for 60 min. Double orbital shaking of 600 r.p.m. for 30 s was performed before the first, sixth, and eleventh cycles.

RNA extraction

RNA extraction was performed using a custom Analytik Jena CyBio FeliX script (available on reasonable request) for the Analytik Jena InnuPREP Virus DNA/RNA Kit-FX or the Promega Maxwell HT Viral TNA Kit. Samples of 200 µL were run and eluted in 50 µL of RNase Free Water.

qPCR patient validation

Clinical material (viral transport medium from throat/nose swabs), provided for validation by NWLP, included samples left over after clinical diagnosis as per standard practice for the validation of new assays and platforms. Patient samples were stored at room temperature for no more than 48 h after the initial analysis by NWLP before they were purified and analysed on our platform. Results (Ct values) were compared directly with those obtained by NWLP. As NWLP uses a nested PCR method, Ct values were reported as being the summation of the first and second PCR steps.

qPCR reactions were set up using the TaqPath 1-Step RT-qPCR Master Mix, CG kit, and the CDC N1 Primers according to the manufacturer’s instructions and thermocycling settings (according to the CDC protocol). Final reaction volumes were 10 µL with 5 µL of extracted RNA template. Liquid transfer of the qPCR master mix was performed using an Echo 525 (Labcyte) from a 6-well reservoir (Labcyte). Extracted RNA templates were transferred using a multichannel pipette. Plates were sealed with MicroAmp Optical Adhesive Films (Thermo Fisher Scientific) and spun at 500 × g in a centrifuge. An Analytik Jena qTower3 auto was used for thermocyling and measurements were taken in the FAM channel.

CRISPR-Cas13a assays with RT-RPA amplification

Experiments were designed and randomized using SAS JMP and Riffyn. Targets were pre-amplified using the TwistAmp Liquid Basic Kit (TwistDx) supplemented with 0.5 U/µL Murine RNase Inhibitor (NEB) and 0.08 U/µL Omniscript (Qiagen). Final reactions had a final volume of 14 µL and were set up in Echo 384 LDV Plus plates (final primer concentration of 0.45 µM and 2 µL of purified patient RNA template) and incubated at 42 °C for 30 min in a CLARIOstar Plus (BMG Labtech) plate reader with double orbital shaking of 600 r.p.m. for 30 s at 5 min. All concentrations are final CRISPR reaction concentrations and the final CRISPR reaction volumes were 5 µL. An Echo 525 (Labcyte) was used to transfer CRISPR Master Mix (50 µM LwCas13a, 1 U/µL Murine RNase inhibitor (NEB), 4 mM Ribonucleotide Solution Mix (NEB), 1.5 U/µL T7 RNA Polymerase (Thermo Fisher Scientific), and 1.25 ng/µL HEK293F background RNA) in Nuclease Reaction Buffer (20 mM HEPES pH 6.8, 60 mM NaCl, 9 mM MgCl2) to a 384-well Small Volume LoBase Microplate (Greiner Bio-One). crRNA (25 nM) and 200 nM poly-U fluorescent probe (5′-/56-FAM/rUrUrUrUrU/3IABkFQ/-3′) were then added separately. An Echo 550 (Labcyte) was used to transfer pre-amplified products (0.25 µL) from the 384 LDV Plus Echo plate (Labcyte) to initiate the reaction, the plate was sealed, centrifuged at 500 × g for 1 min and read using a CLARIOstar Plus (BMG Labtech) plate reader with an excitation wavelength of 483-14 nm, emission of 530-30 nm, dichroic filter of 502.5 nm and EDR enabled. Double orbital shaking of 600 r.p.m. for 30 s was performed before the first cycle. The reactions were incubated at 37 °C with readings taken every 2 min. Each reaction was normalized between a water input as 0 (background fluorescence) and an RNase I (Thermo Fisher Scientific) input (0.25 U) as 1 (RNase I cleaves all of the fluorescent probe and thus serves as a positive relative control).

Colorimetric LAMP reactions with patient samples

Experiments were designed and randomized using SAS JMP and Riffyn. Colorimetric LAMP reactions (NEB WarmStart® Colorimetric LAMP 2× Master Mix) were performed as previously described11 but with a lower final reaction volume of 5 µL and template of 2 µL. Master Mix, primers, and template were transferred to a 384-well Small Volume LoBase plate (Greiner Bio-One) using an Echo 525 and Echo 550 (Labcyte). The plate was then sealed with a MicroAmp Optical Adhesive Film (Thermo Fisher Scientific) and centrifuged for 1 min at 500 x g. The plate was incubated at 65 °C in a CLARIOstar Plus (BMG Labtech) plate reader and absorbance measurements were taken at 415 nm every minute for 60 min. Double orbital shaking of 600 rpm for 30 seconds was performed before the 1st, 6th, and 11th cycles.

Ethics statement

Surplus clinical material was used to validate the assay as per normal practice and does not require ethical review.

Reporting summary

Further information on research design is available in the Nature Research  Reporting Summary linked to this article.



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