Rapid detection of SARS-CoV-2 RNA in saliva via Cas13

Abstract

Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore. The device consists of a single-use gravity-driven microfluidic cartridge inserted into a compact instrument for automated running of the assay and readout of fluorescence within 60 min. DISCoVER can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a sensitivity of 40 copies μl^-1, and was 94% sensitive and 100% specific when validated (against quantitative PCR) using total RNA extracted from 63 nasal-swab samples (33 SARS-CoV-2-positive, with cycle-threshold values of 13-35). The device correctly identified all tested clinical saliva samples (10 SARS-CoV-2-positive out of 13, with cycle-threshold values of 23-31). Rapid point-of-care nucleic acid testing may broaden the use of molecular diagnostics.

Extended Data Fig. 1 |. rLAMP with T7-Floop primer mechanism.

FIP and BIP primers, assisted by F3 and B3 primers to XX, bind to the target DNA at F2c and B2c, adding complementary regions of DNA to the amplicon (F1c and B1c). Their complementarity to F1 and B1 result in loop structures, which facilitate further FIP and BIP binding at the loops (F2c and B2c) and extension, amplifying the target via formation of long concatemers. Floop and Bloop primers can additionally bind to the loops and extend, further increasing amplification. Addition of the T7 promoter at the 5’ end of the loop primer provides a substrate for T7 polymerase to transcribe, resulting in an RNA product containing inverted repeats of the target amplicon.

Extended Data Fig. 2 |. Validation of the microfluidic system.

(a) Screen of process control primer sets on commercial saliva samples. (b) Screen of T7 promoter locations on ACTB primer set on RNA extract and commercial saliva samples. (c) Schematic of cartridge design. The reaction can be separated in three steps: 1. amplification reaction; 2. post-amplification metering + Cas13 mix; 3. Cas13 reaction in detection chambers. Reagents stored on the cartridge are separated via a proprietary hydrophobic solution to avoid premature initiation of the reactions. After the LAMP reaction, the sample is split into two reactions: The left part of the cartridge will expose the sample to N gene crRNA (step 2a) while the right side of the cartridge will act as internal control with only ACTB crRNA (step 2b). (d) Results of on-board DISCoVER on 25 individual saliva samples (20 samples with SARS-CoV-2 genomic RNA at 500 cp/μL, 5 expected negative samples). A 2-fold fluorescence increase over blank cartridges at 10 minutes was the experimentally determined criteria for positive results. Samples with value below this threshold at 10 minutes were declared negative.

Fig. 1 |. DISCoVER microfluidic system for rapid and automated molecular diagnostics.

Patient samples, such as saliva, are collected and heat-inactivated in direct lysis buffer, followed by loading onto a single-use, gravity-driven microfluidic cartridge. The inactivation step ranges from 5 to 20 min depending on institutional regulations. The cartridge is then inserted into a companion instrument that automatically runs the DISCoVER assay in a closed system to minimize reaction contamination. In step 1, an initial rLAMP reaction employs two mechanisms for amplification of target nucleic acids. RFU, relative fluorescence units. Cas13 enzymes are programmed with a guide RNA to specifically recognize the desired RNA molecules over non-specifically amplified products. Subsequent activation of Cas13 ribonuclease activity, in step 2, results in cleavage of reporter molecules for saturated signals within 5 min of CRISPR detection. Including actuation time of the device, time to readout in a finalized system is 48 min. In the left half of the cartridge, guide RNAs targeting SARS-CoV-2 enable rapid and selective detection of attomolar concentrations of virus. The mirrored half of the cartridge is used for an internal process control, enabling a negative test result by ensuring the presence of adequate patient samples. By exploiting template switching and CRISPR programmability, the point-of-care DISCoVER system can contribute to increased surveillance of diverse pathogens.

Fig. 2 |. Direct nucleic acid detection with CRISPR–Cas enzymes and LAMP.

a, Cas13 and Cas12 detection kinetics at varying activator concentrations. Line and shaded regions denote mean ± standard deviation (s.d.) with n = 3 technical replicates. b, Cas13 and Cas12 time to half-maximum fluorescence. †, time to half-maximum fluorescence was too rapid for reliable detection. ††, time to half-maximum fluorescence could not be determined within the 120 min assay runtime. Values are mean ± s.d. with n = 2 technical replicates. c, Schematic of SARS-CoV-2 genome sequence, with LAMP primer set locations indicated. d, Representative fluorescence plots of LAMP amplification of 100 copies μl⁻¹ of synthetic SARS-CoV-2 RNA or NTC. Shaded regions denote mean ± s.d. with n = 3 technical replicates. e, Time to threshold of nine screened LAMP primer sets, targeting synthetic SARS-CoV-2 RNA or NTC. Replicates that did not amplify are represented at 0 min. Error bars represent mean ± s.d. of amplified technical replicates, n = 4. f, LOD assay of LAMP using N set 1 primer set. Replicates that did not amplify are represented at 0 min. Error bars represent mean ± s.d. of amplified technical replicates, n = 4.

Fig. 3 |. rLAMP for two layers of nucleic acid amplification.

a, Schematic of rLAMP mechanism for exponential DNA amplification using F3/B3 and FIP/BIP primers, resulting in higher-order inverted repeat structures. Red arrows indicate location of T7 promoter sequence, inserted in the mBIP primer. Upon T7 transcription, the resulting RNA contains one or more copies of the Cas13 target sequence (orange). b, Schematic of the location of different T7 promoter locations on the rLAMP dumbbell structure and loop primers. c, rLAMP time to threshold of six distinct T7 promoter insertions. Replicates that did not amplify are represented at 0 min. Error bars represent mean ± s.d. of amplified technical replicates, n = 4. d, Denaturing PAGE gels of mBIP rLAMP products to verify T7-mediated transcription. AfeI cleaves in the crRNA target region of templated products, which is expected to result in a single major transcribed species. e, Kinetics of T7 transcription and Cas13 detection on mBIP rLAMP products. Values are mean ± s.d. with n = 3 technical replicates. f, Cas13 detection of eight technical replicates of mBIP rLAMP amplification on genomic RNA, depicted as fold change over NTC at different reaction endpoints.

Fig. 4 |. DISCoVER for extraction-free detection of saliva.

a, Direct saliva lysis conditions were tested for compatibility with the DISCoVER workflow. Replicates that did not amplify are represented at 0 min. Error bars represent mean ± s.d. of amplified technical replicates, n = 4. IA, inactivation reagent; QE, QuickExtract. b, Schematic of contrived saliva sample generation, quantification via RT–qPCR, and detection via DISCoVER to determine analytical sensitivity. c, Fold change in DISCoVER signal relative to NTC on SARS-CoV-2-positive saliva samples at 5 min of Cas13 detection. Error bars show mean ± s.d. with n = 20 biological replicates. d, Fold change in DISCoVER signal relative to NTC on 30 negative saliva samples collected before November 2019, at 5 min of Cas13 detection.

Fig. 5 |. Validation of DISCoVER on patient samples.

a, Schematic of rLAMP multiplexing with SARS-CoV-2 (N gene) and human internal control (RNase P) primer sets. b, DISCoVER signal of SARS-CoV-2-positive saliva samples after multiplexed rLAMP. Values are mean ± s.d. with n = 3 technical replicates. c, Fold change in DISCoVER signal relative to NTC on 30 negative clinical nasal samples at 5 min of Cas13 detection. d, Fold change in DISCoVER signal relative to NTC on 33 positive clinical nasal samples at 5 min of Cas13 detection.

Fig. 6 |. Design of an automated microfluidic-driven diagnostic system.

a, Left: illustration of the final instrument in which the cartridge is inserted. Inset shows picture of the entire microfluidic gravity-driven cartridge. Right: images of the sample metering and rLAMP reaction chamber (1), rLAMP metering and Cas13 mixing chamber (2) and the detection chamber (3). b, Front (left) and rear (right) illustration of the instrument components, including detection system, heaters, air-driven valves and mechanical valves. c, Schematic of the cartridge function. The reaction can be separated in four steps: saliva metering (1), amplification reaction (2), post-amplification metering + Cas13 mix (3), and Cas13 reaction in detection chambers (4). Reagents stored on the cartridge are separated via a proprietary hydrophobic solution to avoid premature initiation of the reactions. After the LAMP reaction, the sample can be split into two reactions: the left part of the cartridge will expose the sample to N gene crRNA (step 3a), while the right side of the cartridge will act as internal control with only RNase P crRNA (step 3b). Saliva samples went through only step 3a, while clinical samples went through steps 3a and 3b.

Fig. 7 |. Experimental validation of the microfluidic-driven diagnostic system.

a, Heat map depicting the fold change in DISCoVER signal on negative and positive saliva samples relative to the NTC. b, Heat map depicting the fold change in SARS-CoV-2 RNA positive and negative clinical samples from nasal swabs, relative to NTC. Ct values for N gene target by qPCR detection is listed on the heat map axis. c, Graph depicting raw fluorescence over time for both detection chambers (blue for RNase P and red for N gene) in NTC (left), negative (middle) and positive (right, Ct 16) samples. Inset within each plot is fluorescent images of detection chamber for NTC cartridge (no viral sample), negative and positive clinical sample (with RT–qPCR Ct ranging from 16 to 21). The left detection chamber is specific for N gene detection, whereas the right chamber is specific for RNase P. d, Results of on-board DISCoVER on ten clinical saliva samples and three negative clinical saliva samples. Fold increase is calculated over a blank cartridge. A two-fold fluorescence increase over blank cartridges at 50 min was the experimentally determined criterion for positive results. Samples with value below this threshold at 5 min were declared negative.