WO2022047127A1 - Compositions and methods for detection of coronavirus - Google Patents

Abstract

The disclosure provides for primers that can detect SARS-CoV-2 nucleic acids in a highly specific and efficient manner using loop-mediated isothermal amplification.

Description

COMPOSITIONS AND METHODS FOR DETECTION OF CORONAVIRUS

CROSS REFERENCE TO RELATED APPLICATIONS

[ 0001 ] This application claims priority to U.S. Provisional Application Serial No. 63/072,044, filed August 28, 2020, the disclosures of which are incorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

[ 0002 ] This invention was made with Government support under Grant No. R33AI129077, awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

[ 0003 ] The disclosure provides for primers that can detect SARS-CoV-2 nucleic acids in a highly specific and efficient manner using loop-mediated isothermal amplification.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[ 0004 ] Accompanying this filing is a Sequence Listing entitled, “Sequence- Listing_ST25” created on August 27, 2021 and having 50,457 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

[ 0005] The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus, is a major global health threat. A still unknown proportion of people, especially the elderly and those with preexisting conditions, are at high risk of a severe course of COVID-19, leading to a high burden on health care systems worldwide. Further, because of limited testing capacity, only people with symptoms are usually tested for SARS-CoV-2 infection, although studies have confirmed that many individuals infected with SARS- CoV-2 are asymptomatic carriers of the virus. This suggests that infection control strategies focusing on symptomatic patients are not sufficient to prevent virus spread. [ 0006] Therefore, large-scale diagnostic methods are needed to determine the spread of the virus in populations quickly, comprehensively, and sensitively. This would allow for the rapid isolation of infected persons during an existing wave of infection. In addition, continuous and repeated testing of large groups within a population may be required as a long-term strategy to contain new outbreaks while keeping societies and economies functional until effective vaccines become available.

BRIEF DESCRIPTION OF THE DRAWINGS

[ 0007 ] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the disclosure and, together with the detailed description, serve to explain the principles and implementations of the invention.

[ 0008 ] Figure 1A-B presents diagrams of the loop-mediated isothermal amplification (LAMP) process. (A) Presents the location of the various LAMP primers. FIP (forward inner primer), BIP (backward inner primer), F3 (forward primer) and B3 (backward primer), FL (forward loop primer) and BL (backward loop primer). (B) Presents the various amplification products generated by using LAMP during each round of amplification, until the double hairpin product is formed and the exponentially amplified. [ 0009] Figure 2A-D presents selected SARS-CoV-2 genes and embodiments of exemplary methods of the disclosure. (A) The organization of select SARS-CoV-2 genes and locations of possible primer sites. (B) An exemplary method using RT-LAMP with Casl2 mediated detection, whereby the products are visualized using a lateral flow assay. (C) An exemplary method using RT-LAMP with Cast 2 mediated detection, whereby the fluorescent products are measured using a fluorescence plate reader device. (D) A workflow for using an exemplary one-pot high-throughput exemplary method of the disclosure that is compatible with automated handling.

[ 0010 ] Figure 3 shows that adding L-Serine (Final -100 mM) to the LAMP assay reaction buffer significantly improved the results from the assay. RNA template was spiked into saliva; 5 pL was used as input into 25 pL total reaction. Primers were the 1051_2 set of LAMP primers.

[ 0011 ] Figure 4 presents the design and results of an exemplary RT-LAMP assay of the disclosure that uses quenching of unincorporated amplification signal reporters (QUASR) Primer detection. QUASR provides for endpoint detection of RT-LAMP reactions, based upon quenching of unincorporated amplification signal reporters. One of the loop primers (LF or LB) or inner primers (FIP or BIP) is labeled with a fluorescent dye. The reaction mixture also contains a short probe, labeled with a dark quencher at the 3' end, and complementary to 7-13 bases at the 5' end of the dye labeled primer. The quench probe is present at slight excess relative to the labeled primer and has Tm > 10 °C below the temperature of the LAMP reaction, such that it remains dissociated during the amplification. After incubation, the reaction is cooled to ambient temperature, resulting in dark quenching of fluorescent primers (negative reactions) or highly fluorescent amplicons (positive reactions).

[ 0012 ] Figure 5 presents the design and results of an exemplary RT-LAMP assay of the disclosure that uses fluorescence of loop primer upon self-dequenching (FLOS). FLOS primers provide for real-time monitoring yielding Ct values. FLOS-RT-LAMP is a direct detection approach, whereby a labelled loop probe is self-quenched in its unbound state and fluoresces only when bound to its target (amplicon) generated by the RT-LAMP reaction.

[ 0013 ] Figure 6 presents the results of a RT-LAMP assay for SARS-CoV-2 whereby a series of primers that bind to the N gene portion of the SARS-CoV-2 gene were screened. Primer 1051_2 provided the best result of the tested primers. NTC = No template control that monitors contamination and primer-dimer formation that could produce false positive results.

[ 0014 ] Figure 7 presents the results of a RT-LAMP assay for SARS-CoV-2 whereby another series of primers that bind to the N gene portion of the SARS-CoV-2 gene and RdRP were screened. Primer 1051_2 provided the best result, followed by xp-3. NTC = No template control that monitors contamination and primer-dimer formation that could produce false positive results. NTC = No template control that monitors contamination and primerdimer formation that could produce false positive results.

[ 0015 ] Figure 8 presents the results of a RT-LAMP assay for SARS-CoV-2 whereby a series of spike primers were screened. NTC = No template control that monitors contamination and primer-dimer formation that could produce false positive results. Primer 1051 2 provided the best result, followed by SPI1-1, SPI2-2 and SPI2-3.

[ 0016] Figure 9 presents the results of a RT-LAMP assay for SARS-CoV-2 whereby the effects of increasing the primer concentration up to 5X for the best performing primers were evaluated. NTC = No template control that monitors contamination and primer-dimer formation that could produce false positive results.

[ 0017 ] Figure 10 presents the results of a multiplex RT-LAMP assay for SARS-CoV- 2 using non-FLOS primers with FLOS primers. NTC = No template control that monitors contamination and primer-dimer formation that could produce false positive results.

[ 0018 ] Figure 11 presents the results of a multiplex RT-LAMP assay for SARS-CoV- 2 using various permutations of the best performing primers. NTC = No template control that monitors contamination and primer-dimer formation that could produce false positive results. [ 0019] Figure 12 presents a comparison of the results for SARS-CoV-2 using qRT- PCR assay with the N1 primer v. RT-LAMP assay with the 1051 primer. The limit of detection (LoD) for the qRT-PCT assay was found to be 3 copies/reaction, while the LoD of the RT-LAMP assay was found to be 15 copies/reaction. The RT-LAMP assay could also detect 3-4 copies/reaction, but not all replicates can succeed.

[ 0020 ] Figure 13 presents the results of a multiplex RT-LAMP assay for SARS-CoV- 2. * = all replicates can amplify 3 copies of RNA. NTC = No template control that monitors contamination and primer-dimer formation that could produce false positive results.

[ 0021 ] Figure 14 presents the results of a RT-LAMP assay using patient samples. The patient sample was mixed at 1 : 1 ratio with BioRad (BR) lysis buffer, QE buffer, UTM, and H2O, and then 5ul of the mixture was added directly to the LAMP mastermix for the LAMP reaction. Extracted pure RNA was mixed with the same buffers at 1 : 1 ratio, and directly added to the LAMP mastermix for the LAMP reaction as positive controls. H2O was mixed with the same buffers at 1 : 1 ratio and directly added to the LAMP mastermix for the LAMP reaction as negative controls. Two primers were tested: 1051 and Mth-N. An independent 1051 -Flos primer was also tested using pure RNA as template. The LAMP reaction was terminated after 45 min at 65 °C. RNase inhibitor and carrier RNA included in the reaction. It was found that the patient sample + H2O or directly added to the reaction worked the best. BR, QE and UTM (no heating step) all inhibited the reaction. The 1051 primer worked better than Mth-N, and the 1051 -FLOS primer worked well and can be used for one-pot reactions. NTC = No template control that monitors contamination and primerdimer formation that could produce false positive results. The circles indicate false positive by checking with the amplification curves, but the software produced some very small Ct values for those.

[ 0022 ] Figure 15 presents the results of a sample direct RT-LAMP assay using 4 different samples from patients. 5 pl of samples were directly added to the LAMP reaction. 1051 and Mth-N Primers were used with no RNase inhibitor and carrier RNA.

PS1=M15711; PS2=F20432; PS3=H38600; and PS4=S41782. It was found that the 1051 primer worked better than Mth-N; and PS4 which has the highest viral loads can be detected by direct LAMP.

[ 0023 ] Figure 16 presents the results of a sample direct multiplex RT-LAMP assay using the 1051 primer with other primers. The reaction volume was increased to 100 pL and the sample input was 10 pL.

[ 0024 ] Figure 17 presents the results of an additional primer screen of various LAMP sets of primers. Extracted SARS-CoV-2 RNA was used as the template. The LAMP primers are all directed to the N-gene of SARS-CoV-2. The 1051 primer set was run with the LF- 1051_2 primer or without. NTC = No template control. It was found that the MAI and MA89 LAMP primer set provided better results than the 1051_2 LAMP primer set.

[ 0025 ] Figure 18 presents the results of additional tests with the best performing primer sets. The template for the LAMP reaction was patient swab samples in universal transport media (UTC). The primer sets were either run singly e.g., 1051_2 primer set alone, or with other primers sets (multiplex), e.g, 1051_2 primer set being run with the MA89 primer set in the same LAMP reaction. NTC = No template control. It was found that the LAMP reaction that contained the 1051_2 primer set that was multiplexed with the MA89 primer set or with the MAI primer set, provided similar results to the 1051_2 primer set being run with Mam. The multiplex runs provided better then results than 1051 2 primer set being run alone, except for the multiplex sample that contained three different primer sets. The 1051_2 and MA89 primer sets were selected for the additional LAMP reaction experiments.

[ 0026] Figure 19A-C looks at whether the combined LAMP primer set (1051_2 + MA89) could generate results from unprocessed patient samples (swab or saliva). The patient samples were directly added into the LAMP reaction without any sample processing steps. (A) Results of LoD tests for swab samples in universal transport media and saline. (B) Experiment with 20 replicates to confirm LoD. (C) Results of a LAMP assay with saliva samples. 10 pL of input saliva per 100 pL reaction volume, and 20 pL of input saliva per 100 pL reaction volume.

[ 0027 ] Figure 20 shows that patient samples collected in a reduced media volume will increase the detection sensitivity. A patient sample collected in 0.3 mL of universal transport media gave better results than the patient sample collected in 3 mL of universal transport media. 5 pL of raw sample in universal transport media was used as input into a 25 pL total reaction volume.

[ 0028 ] Figure 21A-B provides the results of the use of both SYTO9 and QUASR LAMP Primers in a one-pot LAMP assay. QUASR primers are Cy5 labeled, which use a different fluorescence detection channel from the SYTO9 dye. (A) SYTO9 provides real time results, thus allowing for a Tt value for quantification. (B) QUASR provides end-point results, and increased assay sensitivity.

[ 0029] Figure 22 shows that heating the sample at 95 °C for 5 min improved the LAMP kinetics but not the detection limit. Diluting the original universal transport media (UTM) sample by half using H2O reduced the inhibition by inhibitors, and improved LAMP kinetics. The raw sample to reaction volume ratio should be <=1:10. The assay LoD is 8720 copies/mL sample in UTM using 5 pL input diluted half by water in 25 pL total reaction. [ 0030 ] Figure 23 demonstrates that using larger input and reaction volumes increased the detection limit. The assay LoD is 3490 copies/mL sample in UTM using 5 pL input in 50 pL total reaction. The assay LoD is 349 copies/mL sample in UTM using 10 pL input in 100 pL total reaction, which is similar to CDC assay.

[ 0031 ] Figure 24A-B provides that the LAMP assay has a detection limit similar to CDC qRT-PCR assay, when using extracted RNA samples. Serial dilutions of RNA samples are used as templates for both (A) qRT-PCR and (B) LAMP. The LoD are equivalent, both ~ 4 copies/reaction.

[ 0032 ] Figure 25A-C shows that using FAM labeled FIP LAMP primer and biotin labeled BIP LAMP primer allowed for lateral flow detection for at-home tests. (A) Diagram of the mechanism for detection on the lateral flow detection strips. (B) Presents an image of two lateral flow strips, the left strip indicating a negative result for SARS-CoV-2, the right strip indicating a positive result for SARS-CoV-2 using 50 copies/rxn. (C) Shows the results of lateral flow strips which were inserted into sample tubes to read the LAMP reaction results. Going from left to right, l^rst strip, 4 copies of SARS-CoV-2 per reaction; 2^nd strip, 50 copies of SARS-CoV-2 per reaction; 3^rd and 4^th strips, BIP primer is not labelled with Biotin; 5^th strip, 600 copies of SARS-CoV-2 per reaction; 6^th strip, non-template control; and 7^th strip, water only strip.

SUMMARY

[ 0033 ] The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS- CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop- mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. The primers used in an RT- LAMP assay need to have high specificity and efficiency for SARS-CoV-2 to determine whether SARS-CoV-2 is present in a sample even at low copy number (e.g., < 100) and with minimal sample handling.

[ 0034 ] In a particular embodiment, the disclosure provides for a set of loop-mediated isothermal amplification (LAMP) primers comprising a Forward Outer (F3) primer, a Backward Outer (B3) primer, a Forward Inner Primer (FIP) primer, a Backward Inner Primer (BIP) primer, optionally a Forward Loop (LF) primer, and optionally a Backward Loop (LB) primer, wherein the set of primers comprise one or more of primers having the sequence(s) of:

(i) F3 : TGAATAAGCATATTGACGCATAC (SEQ ID NO: 1) or a sequence that is at least 87% identical to SEQ ID NO: 1, or a sequence that is at least 24-30 nucleotides in length and contains the sequence of SEQ ID NO: 1 ;

(ii) B3: TGAGTTGAGTCAGCACTG (SEQ ID NO:2) or a sequence that is at least 83.3% identical to SEQ ID NO:2, or a sequence that is at least 19-25 nucleotides in length and contains the sequence of SEQ ID NO:2;

(iii) FIP: TAAGGCTTGAGTTTCATCAGCCCATTCCCACCAACAGAGC (SEQ ID NO:3) or a sequence that is at least 87.5% identical to SEQ ID NO:3, or a sequence that is at least 41-46 nucleotides in length and contains the sequence of SEQ ID NO:1;

(iv) BIP: CGCAGAGACAGAAGAAACAGCATTGTTGCAATTGTTTGGAGAA (SEQ ID NO:4) or a sequence that is at least 86% identical to SEQ ID NO:4, or a sequence that is at least 44-50 nucleotides in length and contains the sequence of SEQ ID NO:4;

(v) LF: TTCTTCTTTTTGTCCTTTTTAG (SEQ ID NO:5) or a sequence that is at least 87% identical to SEQ ID NO:5, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO:5; and/or

(vi) LB: AAACTGTGACTCTTCTTCCTGC (SEQ ID NO:6) or a sequence that is at least 87% identical to SEQ ID NO:6, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO:6; wherein the one or more primers may further comprise one or more fluorophores or capture moieties located internally and/or at the end(s) of the primers; and wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples. In a further embodiment, the set of primers comprise the primers having the sequences of: F3: TGAATAAGCATATT GACGCATAC (SEQ ID NO:1); B3: TGAGTTGAGTCAGCACTG (SEQ ID NO:2); FIP: TAAGGCTTGAGTTTCATCAGCCCATTCCCACCAACAGAGC (SEQ ID NO:3); BIP: CGCAGAGACAGAAGAAACAGCATTGTTGCAATTGTTTGG AGAA (SEQ ID NO: 4); LF: TTCTTCTTTTTGTCCTTTTTAG (SEQ ID NO:5); and LB: AAACTGTGACTCTTCT TCCTGC (SEQ ID NO:6); wherein the primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primer. In another embodiment, the set of primers comprise the primers having the sequences of: F3: TGAATAAGCATATT GACGCATAC (SEQ ID NO:1); B3: TGAGTTGAGTCAGCACTG (SEQ ID NO:2); FIP: 6FAM/TAAGGCTTGAGTTTCATCAGCCCATTCCCACCAACAG AGC (SEQ ID NO:3), wherein 6FAM is 6-Carboxyfluorescein; BIP: Biotin/CGCAGAGACAGAAGAAACAGC ATTGTTGCAATTGTTTGGAGAA (SEQ ID NO:4); LF: TTCTTCTTTTTGTCCTTT TTAG (SEQ ID NO:5); and LB: AAACTGTGACTCTTCTT CCTGC (SEQ ID NO:6), wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples. In yet a further embodiment, the set of primers comprise the primers having the sequences of: F3: TGAATAAGCATATTGACGCATAC (SEQ ID NO:1); B3: TGAGT TGAGTCAGCACTG (SEQ ID NO:2); FIP: *-TAAGGCTTGAGTTTCATCAGCCC ATTCCCACCAACAGAGC (SEQ ID NO:3), wherein * is a fluorescent dye; BIP: Biotin/CGCAGAGACAGAAGAAACAGCATTGTTGCAATTGTTTGGAGAA (SEQ ID NO:4); LF: TTCTTCTTTTTGTCCTTTTTAG (SEQ ID NO:5); and LB: AAACTGTGAC TCTTCTTCCTGC (SEQ ID NO:6), wherein the set of primers is used to detect SARS-CoV- 2 nucleic acid in one or more samples. In yet another embodiment, * is a fluorescent dye selected from 6-Carboxyfluorescein (6FAM), TAMRA fluorophore (6TAMN), and cy5. [ 0035] In a certain embodiment, the disclosure provides for a set of loop-mediated isothermal amplification (LAMP) primers comprising a Forward Outer (F3) primer, a Backward Outer (B3) primer, a Forward Inner Primer (FIP) primer, a Backward Inner Primer (BIP) primer, optionally a Forward Loop (LF) primer, and optionally a Backward Loop (LB) primer, wherein the set of primers comprise one or more of primers having the sequence(s) of:

(a) F3: CTGCCACTAAAGCATACAATGT (SEQ ID NO: 7) or a sequence that is at least 87% identical to SEQ ID NO:7, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO:7;

(b) B3 : TTGATGGCACCTGTGTAGG (SEQ ID NO: 8) or a sequence that is at least 84.2% identical to SEQ ID NO:8, or a sequence that is at least 20-25 nucleotides in length and contains the sequence of SEQ ID NO: 8;

(c) FIP: GTGCAATTTGCGGCCAATGTTTGTTTTTCAAGGAAATTTTGGGGA CCAG (SEQ ID NO:9) or a sequence that is at least 90% identical to SEQ ID NO:9, or a sequence that is at least 51-55 nucleotides in length and contains the sequence of SEQ ID NO:9;

(d) BIP: CCAGCGCTTCAGCGTTCTTCTTTTTCAACCACGTTCCCGAAGG (SEQ ID NO:10) or a sequence that is at least 86% identical to SEQ ID NO:10, or a sequence that is at least 44-50 nucleotides in length and contains the sequence of SEQ ID NO: 10; (e) LF: CAGTTCCTTGTCTGATTAGTTC (SEQ ID NO: 11) or a sequence that is at least 87% identical to SEQ ID NO: 11, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO: 11; and/or

(f) LB: AATGTCGCGCATTGGCATGG (SEQ ID N:O12) or a sequence that is at least 75% identical to SEQ ID NO: 12, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO: 12; wherein the one or more primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primers; and wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples. In a further embodiment, the set of primers comprise the primers having the sequences of: F3: CTGCCACTAAAGCATACA ATGT (SEQ ID NO:7); B3: TTGATGGCACCTGTGTAGG (SEQ ID NO:8); FIP: GTGC AATTTGCGGCCAATGTTTGTTTTTCAAGG AAATTTTGGGGACCAG (SEQ ID NO:9); BIP: CCAGCGCTT CAGCGTTCTTCTTTTTCAACCACG TTCCCGAAGG (SEQ ID NO: 10); LF: CAGTTCCTT GTCTGATTAGTTC (SEQ ID NO: 11); and LB: AATGTCG CGCATTGGCATGG (SEQ ID NO: 12); wherein the one or more primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primers; and wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples. In yet a further embodiment, the set of primers comprise the primers having the sequences of: F3: CTGCCACTAAAGCATACAATGT (SEQ ID NO:7); B3: TTGATGGCA CCTGTGTAGG (SEQ ID NO: 8); FIP: FAM/GTGCAATTTGCGGCCAATGTTTGTTTTTC AAGGAAATTTTGGGGACCAG (SEQ ID NO:9); BIP: Biotin/CCAGCGCTTCAGCGTTC TTCTTTTTCAACCACGTTCCCGAAGG (SEQ ID NO: 10); LF: CAGTTCCTTGTCTGAT TAGTTC (SEQ ID NO: 11); and LB: AATGTCGCGCATTGG CATGG (SEQ ID NO: 12). In another embodiment, the set of primers comprise the primers having the sequences of: F3: CTGCCACTAAAGCATACAATGT (SEQ ID NO:7); B3: TTGATGGCACCTGTGTAGG (SEQ ID NO:8); FIP: *-GTGCAATTTGCGGCCAATGTTTGTTTTTCAAGGAAATTTT GGGGACCAG (SEQ ID NO:9), wherein * is a fluorescent dye; BIP: CCAGCGCTTCA GCGTTCTTCTTTTTCAACCACGTTCCCGAAGG (SEQ ID NOTO); LF: CAGTTCCTT GTCTGATTAGTTC (SEQ ID NO: 11); and LB: AATGTCGCGCATTGGC ATGG (SEQ ID NO: 12). In another embodiment, * is a fluorescent dye selected from 6-Carboxyfluorescein (6FAM), TAMRA fluorophore (6TAMN), and cy5.

[ 0036] In a particular embodiment, the disclosure further provides for a set of loop- mediated isothermal amplification (LAMP) primers comprising a Forward Outer (F3) primer, a Backward Outer (B3) primer, a Forward Inner Primer (FIP) primer, a Backward Inner Primer (BIP) primer, optionally a Forward Loop (LF) primer, and optionally a Backward Loop (LB) primer, wherein the set of primers comprise one or more of primers having the sequence(s) of:

(1) F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13) or a sequence that is at least 83.3% identical to SEQ ID NO: 13, or a sequence that is at least 19-24 nucleotides in length and contains the sequence of SEQ ID NO: 13;

(2) B3: TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14) or a sequence that is at least 77.3% identical to SEQ ID NO: 14, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO: 14;

(3) FIP: GCGGCCAATGTTTGTAATCAGTTCCTTTTAACCCAAGGAAATTT TGGG GAC (SEQ ID NO: 15) or a sequence that is at least 90% identical to SEQ ID NO: 15, or a sequence that is at least 52-57 nucleotides in length and contains the sequence of SEQ ID NO: 15;

(4) BIP: GTCGCGCATTGGCATGGAAGTTTTTATGGCACCTGTGTAGGTCA (SEQ ID NO: 16) or a sequence that is at least 88.6% identical to SEQ ID NO: 16, or a sequence that is at least 45-50 nucleotides in length and contains the sequence of SEQ ID NO: 16;

(5) LF: TTGTCTGATTAGTTCCTG (SEQ ID NO: 17) or a sequence that is at least 83.3% identical to SEQ ID NO: 17, or a sequence that is at least 19-24 nucleotides in length and contains the sequence of SEQ ID NO: 17; and/or

(6) LB: CACACCTTCGGGAACGTGGT (SEQ ID NO: 18) or a sequence that is at least 75% identical to SEQ ID NO:18, or a sequence that is at least 21-26 nucleotides in length and contains the sequence of SEQ ID NO: 18; wherein the one or more primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primers; and wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples. In a further embodiment, the set of primers comprise the primers having the sequences of: F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13); B3: TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14); FIP: GCGG CCAATGTTTGTAATCAGTTCCTTTTAACCCAAGGAAATTTTGGGGAC (SEQ ID NO: 15); BIP: GTCGCGCATTGGCATGGAAGT TTTT ATGGCACCTGTGTAGGTCA (SEQ ID NO: 16); LF: TTGTCTGATTAGTTCCTG (SEQ ID NO: 17); and LB: CACACC TTCGGGAACGTGGT (SEQ ID NO: 18); wherein the primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primer. In yet a further embodiment, the set of primers comprise the primers having the sequences of: F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13); B3: TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14); FIP: 6FAM/GCGGCCAATGTTTGTAATCAGTTCCTTTTAACCCAAG GAAATTTTGGGGAC (SEQ ID NO: 15); BIP: Biotin/GTCGCGCATTGGCATGGAAGTT TTTATGGCACCTGTGTAGGTCA (SEQ ID NO: 16); LF: TTGTCTGATTAGTTCCTG (SEQ ID NO: 17); and LB: CACACCTTCGGGAAC GTGGT (SEQ ID NO: 18); wherein the primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primer. In yet another embodiment, the set of primers comprise the primers having the sequences of: F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13); B3: TTTGAAATTTGG ATCTTTGTCA (SEQ ID NO: 14); FIP: *-GCGGCCAATGTTTGTAATCAGTTCCTTTTA ACCCAAGGAAATTTTGGGGAC (SEQ ID NO: 15), wherein * is a fluorescent dye; BIP: GTCGCGCATTG GCATGGAAGTTTTTATGGCACCT GTGTAGGTCA (SEQ ID NO: 16); LF: TTGTCTGA TTAGTTCCTG (SEQ ID NO: 17); and LB: CACACCTTCGG GAACGTGGT (SEQ ID NO: 18); wherein the primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primer. In a further embodiment, * is a fluorescent dye selected from 6-Carboxyfluorescein (6FAM), TAMRA fluorophore (6TAMN), and cy5.

[ 0037 ] In a particular embodiment, the disclosure provides a LAMP assay for detecting SARS-CoV-2 in a sample, comprising: a set of LAMP primers disclosed herein. In yet a further embodiment, a LAMP assay disclosed herein further comprises a set of spike protein LAMP primers having the sequence of: spike-3F3: CTCTATTGCCATACCCACA (SEQ ID NO: 19); spike-3B3: CTTGTGCAAAAACTTCTTGG (SEQ ID NO:20); spike- 3FIP: CATTCAGTTGAATCACCACAAATGTGTGTTACCACAGAAATTCTACC (SEQ ID NO:21); spike-3BIP: GTTGCAATATGGCAGTTTTTGTACATTTGTCTTGTTCAA CAGCTAT (SEQ ID NO:22); and spike-3LF: GTACAATCTACTGATGTCTTGGTCA (SEQ ID NO:23).

[ 0038 ] In a certain embodiment, the disclosure also provides for a LAMP assay for detecting SARS-CoV-2 in a sample, comprising: two sets of LAMP primers disclosed herein. In another embodiment, the disclosure provides for a LAMP assay disclosed herein, and a primer selected from SEQ ID NO:24: ACTCCAGCCTTA/3BHQ 1/ or SEQ ID NO:25: ACTCCAGCCTTA/3BHQ 2/, wherein 3BHQ 1 is Black Hole Quencher 1, and wherein 3HBQ 2 is Black Hole Quencher 2; wherein if the fluorescent dye is 6FAM then the primer is SEQ ID NO:24 (ACTCCAGCCTTA/3BHQ 1); and wherein if the fluorescent dye is 6FAM is 6TAMN or cy5, then the primer is SEQ ID NO:25 (ACTCCAGCCTTA/3BHQ 2). [ 0039] In a particular embodiment, the disclosure provides for a LAMP assay disclosed herein which comprises: one or more sets of primers disclosed herein, a Bst DNA polymerase; a reverse transcriptase; isothermal amplification buffer; dNTP mix; MgSOr: L- Serine; RNase inhibitor; Carrier RNA; and SYTO9.

[ 0040 ] In a certain embodiment, the disclosure provides a method to determine whether a sample comprises SARS-CoV-2 nucleic acids, comprising: carrying out the LAMP assay disclosed herein on a sample at 60-65 °C for 10 - 30 minutes to generate amplification products; performing a trans-cleavage assay by incubating at an elevated temperature a second reaction mixture comprising the amplification products from the previous step, a programmable nuclease that has been complexed with gRNA specific to corresponding gene sequences of SARS-CoV-2, and a single-stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is trans-cleaved by the programmable nuclease if SARS-CoV-2 gene amplification products are present in the second reaction mixture; and detecting whether SARS-CoV-2 is the sample based upon detecting transcleaved detector nucleic acid fragments. In another embodiment, the sample is an environmental sample or a sample from a subject. In yet another embodiment, the sample is an oropharyngeal (OP) specimen, nasal mid-turbinate swab, anterior nares (nasal swab) specimen, nasopharyngeal wash/aspirate, oral swab, throat swab or saliva sample obtained from a human patient. In a certain embodiment, the LAMP reaction and the trans-cleavage assay are performed as a single-tube reaction. In a further embodiment, the programmable nuclease is a Cast 2 nuclease, a Cast 3 nuclease, or a Cast 4 nuclease. In yet a further embodiment, the programmable nuclease is a Cas 12a nuclease. In another embodiment, the polypeptide sequence of the programmable nuclease has at least 85% sequence identity to SEQ ID NO:26, 27, 28 or 29. In yet another embodiment, the polypeptide sequence of the programmable nuclease has at least 95% sequence identity to SEQ ID NO:29. In a further embodiment, the gRNA is specific to the N-gene, E-gene, or human Rnase P gene of SARS- CoV-2.

[ 0041 ] In a certain embodiment, the disclosure also provides a method to determine whether a sample comprises SARS-CoV-2 nucleic acids, comprising: carrying out the LAMP assay disclosed herein on a sample at 60-65 °C for 10 - 30 minutes to generate amplification products; and detecting whether SARS-CoV-2 is the sample by using a lateral flow assay strip, wherein if amplification products are produced in the LAMP assay, then the presence of the amplification products can be determine by a band being present at biotin binding test line on the lateral flow assay strip, and wherein if amplification products are not produced, then there is no band present at biotin binding test line of the lateral flow assay strip. In a further embodiment, the sample is an environmental sample or a sample from a subject. In yet a further embodiment, the sample is an oropharyngeal (OP) specimen, nasal mid-turbinate swab, anterior nares (nasal swab) specimen, nasopharyngeal wash/aspirate, oral swab, throat swab or saliva sample obtained from a human patient. In another embodiment, the method is carried out at a laboratory, at a hospital, at a physician office/laboratory (POLs), at a clinic, at a remote site, or at home. In a particular embodiment, the method is carried out at home.

[ 0042 ] In a certain embodiment, the disclosure provides a method to determine whether a sample comprises SARS-CoV-2 nucleic acids, comprising: carrying out a LAMP assay disclosed herein on a sample at 60-65 °C for 10 - 30 minutes to generate amplification products and then cooling the LAMP assay to room temperature; and measuring the sample for fluorescence using a fluorescent detection device, wherein if the sample fluoresces more than background, indicates that the sample comprises SARS-CoV-2 nucleic acids. In a further embodiment, the detection device is a plate reader or spectrophotometer. In yet a further embodiment, the method is carried out using automation equipment comprising robotic handlers. In another embodiment, more than 90 samples can be run at a time using said method.

[ 0043 ] The disclosure also provides kits and/or articles of manufacture containing LAMP primer sets as provided herein. The kits can further comprise reagents for performing a LAMP reaction and may also contain additional reagents for performing trans-cleavage assays. The kit may further include lateral flow strips for detection of amplified products.

DETAILED DESCRIPTION

[ 0044 ] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a primer" includes a plurality of such primers and reference to "the subject" includes reference to one or more subjects, and so forth.

[ 0045 ] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[ 0046] Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

[ 0047 ] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of or "consisting of."

[ 0048 ] The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.

[ 0049] The coronavirus disease 2019 (COVID- 19) pandemic, caused by the SARS- CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus, is a major global health threat. An active SARS-CoV-2 infection can be diagnosed by detecting either the viral genome or viral antigens in appropriate human samples. Assays for detecting SARS-CoV-2 antigens are limited by the sensitivity, specificity, and production speed of diagnostic antibodies, whereas detecting viral RNA only requires specific oligonucleotides. Therefore, an assay that detects SARS-CoV-2 RNA facilitates testing of large cohorts.

[ 0050 ] The SARS-CoV-2 diagnostic pipeline that has proven to be successful and that is currently used in many test centers consists of three steps: collecting nasopharyngeal or oropharyngeal swab specimens, isolation of total RNA, and specific detection of the viral genome by RT-qPCR. The latter comprises a reverse transcriptase (RT) step, which translates the viral RNA into DNA, followed by a semiquantitative DNA polymerase chain reaction using oligonucleotides specific for the viral cDNA (qPCR). As a result, a short piece of the viral genome is strongly amplified and then is detected by a sequence-specific oligonucleotide probe labeled with a fluorescent dye. This procedure includes several steps that require sample handling; therefore, the detection process in a clinical diagnostic laboratory takes about 3 to 24 hours or more, depending on the number of samples and process optimization of the test center. In addition, in the context of the CO VID-19 pandemic, many of the reagents required are only slowly being replenished due to insufficient production capacity or lack of international transport. Therefore, increasing daily test capacities for RT-qPCR-based diagnostics for SARS-CoV-2 RNA detection is currently limited. To accelerate and optimize such diagnostics, new scalable methods for RNA isolation and the detection of viral genomes are needed.

[ 0051 ] An alternative to RT-qPCR is reverse transcription loop-mediated isothermal amplification (RT-LAMP). RT-LAMP reactions include a reverse transcriptase and a DNA polymerase with strong strand displacement activity and tolerance for elevated temperatures and up to six DNA oligonucleotides of a certain architecture. Samples with potential template molecules are added to the reaction and incubated for 10 to 60 min at a constant temperature (e.g., 65 °C). Accordingly, a specialized thermocycler is not needed, only a heat block. The oligonucleotides act as primers for the reverse transcriptase, and additional oligonucleotides for the DNA polymerase are designed so the DNA products loop back at their ends. These, in turn, serve as self-priming templates for the DNA polymerase. In the presence of a few RNA template molecules, a chain reaction is set in motion, which then runs until the added reagents (in particular, the deoxynucleotide triphosphates) are used up. LAMP exhibits increased sensitivity and specificity due to an exponential amplification feature that utilizes 6 different target sequences simultaneously identified by separate distinct primers in the same reaction. LAMP assays are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection.

[ 0052 ] The LAMP system principally employs four core primers, namely FIP (forward inner primer), BIP (backward inner primer), F3 (forward primer) and B3 (backward primer) to recognize six different regions of the target sequences (see FIG. 1). First, FIP anneals to the template, and extension occurs using a polymerase with displacement activity (such as Bst polymerase), the product obtained from FIP is then displaced by the extension reaction associated with the F3 primer. Subsequently, an extension reaction occurs using BIP on the product of FIP, and not on the template DNA due to displacement by DNA synthesis associated with the B3 primer. These reactions result in a product with a dumbbell-like structure which is essential for LAMP to establish isothermal amplification as the loop structures are always single stranded and can be annealed by FIP or BIP. This loop structure formation eliminates the denaturing step, which is otherwise essential in PCR for obtaining single-stranded DNA, and also establishes a cyclic reaction between the dumbbell-like structure and its complementary product, leading to elongated products with various copies of the target sequence produced. Two optional primers, LF (loop forward) and LB (loop backward), can be added to the amplification reaction to enhance the reaction speed. This modification, also known as “accelerated LAMP”, was a later modification of the classical, four-primer LAMP. Though the exact mechanism is unclear, the LF/LB primers presumably accelerate the four primer LAMP reaction by creating additional binding sites for the autocycling FIP/BIP primers. The auto-cycling leads to the formation of “cauliflower-like” DNA structures, which essentially are DNA concatemers with loops interspersed between alternating inverted repeats. Formation of these multimeric products of the target region represents a successful amplification of the target DNA. LAMP is an ultrasensitive nucleic acid amplification method that can detect minute quantities of DNA or RNA templates within roughly an hour, far outstripping normally utilized RT-PCR methods, particularly with the current demands for rapid and sensitive testing. As the growing number of suspected COVID- 19 cases exceeds the capacity of many hospitals, many patients remain untested impeding efforts to the control the disease. Accordingly, the rapid, point-of-care diagnostic for the COVID- 19 described herein fulfills an unmet need.

[ 0053 ] The disclosure provides for a LAMP assay that utilizes a specific set of LAMP primers, e.g, FIP-H051.2, BIP-H051.2, F3-H051.2, B3-H051.2, LF-1051.2, and LP-1051.2 (see Table 2) that exhibited, in the studies presented herein, noticeably superior properties in regards to specificity and amplification efficiency for SARS-CoV-2 nucleic acids in comparison to other known primers. These primers were developed by screening primers generated by tiling across the N gene and E gene of SARS-CoV-2. Due to the superior properties of the primers disclosed herein, they can be used to identify SARS-CoV-2 nucleic acids without needing any RNA isolation/purification step; and exhibit a lower limit of detection (LOD) for target sequences, provides for less non-specific amplification, and exhibits a shorter time to show curve than other primers. The disclosure further provides a series of spiked primers for enrichment of targeted SARS-CoV-2 sequences: spike2-3F3, spike2-3B3, spike2-3FIP, spike2-3BIP, and spike2-3LF (see Table 2). These spike2 primers can be used in a LAMP assay disclosed herein to increase the sensitivity of the LAMP assay. [ 0054 ] Typically, the measurement of LAMP products relies on end-point analysis and requires post-amplification processing, leading to possible cross-contamination or detection of non-specific LAMP amplicons. Some of these methods include: resolving amplified products on agarose gel electrophoresis turbidity analysis of positive reactions due to the accumulation of magnesium pyrophosphate (Mg2P₂O7), detection of dsDNA under UV- light in presence of an intercalating dyes like SYBR Green I or EvaGreen and addition of metal ion indicators like, calcein/Mn²⁺ and hydroxynapthol blue dye (HNB). Amongst these, the use of intercalating fluorescent dyes has been favored for clinical diagnostics as they are more sensitive and relatively tolerant towards opaque substances like proteins, which are known to affect turbidimetric signal. A major disadvantage, however, of using non-specific detection methods is the increased likelihood of detecting false positives. This is despite the fact that LAMP relies on 4-6 different primers to independently recognize 6-8 independent regions on the target sequence suggesting, at least in theory, a higher degree of specificity than a two-primer PCR. Though the mechanism of non-specific amplification remains unclear, it is assumed that cis and trans priming amongst the six primers, could be responsible for this phenomenon. Thus, indirect detection of amplification products remains one of major shortcomings of LAMP technology.

[ 0055 ] In a particular embodiment, the LAMP assay disclosed herein is a modified LAMP assay that incorporates a primer or probe which is labelled with a label (e.g, a fluorophore) in order to monitor or determine amplicon formation. Examples of such modified LAMP assays, include LAMP assays which incorporate one of the following techniques: fluorescence of loop primer upon self-dequenching (FLOS) LAMP, HyBeacon probes, Guanine quenching principle, alternately binding quenching probe competitive LAMP (ABC-LAMP), fluorophore-modified primer with ethidium bromide, universal quenching probe (QProbe), and graphene oxide (GO) based FRET. In another embodiment, the LAMP assay disclosed herein is a modified LAMP assay that incorporates multiple primers or probes that are labelled with labels (e.g, fluorophore(s), FRET pairs, and/or quencher(s)) in order to monitor or determine amplicon formation. Examples of such modified LAMP assays, include LAMP assays which incorporate one of the following techniques: detection of amplification by release of quenching (DARQ), quenching of unincorporated amplification signal reporters (QUASR), toehold-mediated strand exchange reaction, termed one-step strand displacement (OSD), molecular beacon, lightCycler, assimilating probe, and mediator displacement (MD) LAMP. The specifics for each of the following techniques can be found in Becherer et al. (Anal. Methods, 12:717-746 (2020), the disclosure of which is incorporated herein in-full.

[ 0056] Fluorophore labelled nucleic acids that specifically hybridize, in a sequence dependent manner, to a transiently generated single-stranded DNA structure, have proven to be an ideal solution to any non-specific, dye-based detection system. Examples of such include the, hydrolysis-based TaqMan™ probes specifically developed for qPCR and molecular beacons among host of others. Due to the atypical amplification chemistry of iNAAT’s and LAMP per se, seamless application of any of these probe technologies, specifically developed for qPCR have proven to be technically challenging. Attempts however have been made to develop a probe-based detection system for LAMP include: loss- of-signal guanine quenching, gain-of-signal fluorescence using labeled primers, detection of amplification by release of quenching (DARQ), assimilating probe, one-step-toe-hold (OSD) reporter and more recently, molecular beacons. The use of self-quenching fluorogenic probes as an alternative approach to detect and monitor LAMP reactions in real-time has been recently developed and is termed Fluorescence of Loop Primer Upon Self Dequenching- LAMP (FLOS-LAMP). The FLOS-LAMP utilizes a labelled loop probe quenched in its unbound state, fluoresces only when bound to its target (amplicon). For the LAMP reactions, the fluorophore is conjugated internally to the primer sequence, not on the end, and the expected fluorescent property of primary and secondary deoxy oligonucleotide structure can change (10-fold) upon hybridization. The FLOS probe can comprise different fluorophores (FAM, JOE and ROX), enhancing the versatility of the assay.

[ 0057 ] Any non-dumbbell shaped nucleic acid structure(s) that are presumably formed in a typical LAMP reaction, is unable to offer a binding site to the FLOS-probe. As a result, no spurious signal gets generated. This is a major technical advancement considering the fact that the majority of the real-time LAMP assays used in clinical diagnostics use the indirect detection approach (e.g. intercalating dyes) which cannot discriminate between a genuine amplicon and background. As a result, some form of additional, post-amplification, confirmatory step is usually implemented, for e.g. dissociation curve analysis, to confirm the veracity of the detected signal. Such post-amplification analysis can be a challenge to implement especially in a POC setting, where any unreasonable complications to interpret the data can negatively affect the turnaround time. The use of FLOS-LAMP obviates such postamplification processing, resulting in greater simplicity as well as accuracy. In the instant disclosure, it has been found that a FLOS-LAMP assay with a FLOS probe (e.g, AAACTGTGACTCTTCTTCC/i6-FAMK/GC; SEQ ID NO:30) allowed for identification of SARS-CoV-2 nucleic acids in a highly specific and efficient manner from a sample.

[ 0058 ] The disclosure also provides for end point determination of SARS-CoV-2 nucleic acids produced from a LAMP reaction disclosed herein using quenching of unincorporated amplification signal reporters (QUASR). QUASR uses a primer for LAMP, either the inner primers FIP or BIP, or the loop primers, LF and LB are suitable, and that is labeled with a fluorophore at the 5' end (e.g, the FIP-tlO51.2-QUASR primer). As amplification proceeds, the fluorophore-labeled primers are incorporated into the amplicon. Also included is a short quencher probe, typically with 7-13 bases complementary to the 5' end of the labeled primer (e.g., FIPc-tlO51.2-QUASR-IBQ primer). The quencher probe is modified at the 3' end with a dark quencher (e.g, Iowa Black quencher (IBQ) or Black Hole quencher (BHQ)). Importantly, the melting temperature of the quenching probe annealed to the labeled primer (typically 20-30 min of incubation), the reaction is stopped and cooled down by removing the reaction tubes to ambient temperature. Upon cooling, any free primer that has not been incorporated into an amplicon hybridizes with the quenching probe, resulting in close proximity between the fluorophore and the quencher. However, any labeled primer that has been incorporated into an amplicon is unavailable to hybridize with the quenching probe and thus remains bright. Excess quenching probe ensures that fluorescence is fully quenched in negative reactions.

[ 0059] For QUASR, labeled FIP or BIP primer generally provides a brighter signal than labeled LF or LB primer, since the former are used at twice the concentration of the latter in the LAMP reaction and thus incorporated to a higher degree into amplicons. Besides adding a fluorophore to the chosen primer and including an excess of complementary quenching probe, QUASR does not require altering any LAMP reaction conditions (e.g, time of amplification, primer concentration, or temperature). QUASR at room temperature outperforms SYTO dyes at end point discrimination. A successful QUASR amplification results in a high degree of incorporation of labeled primers into an amplicon and thus a high residual fluorescence that allows even clearer discrimination between positive and negative reactions. Negative QUASR reactions look nearly identical to positive QUASR reactions at elevated temperatures, where the fluorophore-labeled primer and quench probe are dissociated in solution but become very dark as the temperature drops below the annealing temperature of the quench probe. In contrast, positive QUASR reactions typically become brighter as they cool due to the temperature dependence of fluorescence quantum yield. The combined effect is greatly increased signal discrimination as the reaction cools. By comparison, discrimination between positive and negative reactions with intercalating SYTO dyes is optimal at higher temperatures (below the melting temperature of the amplicon). By combining multiple QUASR primer sets specific for different targets, spectrally multiplexed detection can be achieved. QUASR based LAMP assays are favorable in low resource settings where real-time monitoring is impractical. For such applications, discriminating a positive SARS-CoV-2 test from a negative SARS-CoV-2 test at a defined end point (e.g, 30 min of amplification) is instrumentally simpler than real-time quantitative detection and is simpler for a non-specialist to interpret. By combining a LAMP assay disclosed herein with a QUASR reaction allows for non-inhibitory, bright, single-step, closed-tube, and multiplexed detection of SARS-CoV-2.

[ 0060 ] The disclosure further provides that the LAMP assay can be combined with a programmable nuclease (e g, CRISPR-Casl2) based assay to detect the presence or absence of SARS-CoV-2 nucleic acids in a sample. The programmable nuclease-based assay functions to amplify positive SARS-CoV-2 samples from the LAMP assay via trans cleavage of a single-stranded detector nucleic acid by the programmable nuclease, such that attomolar concentration of amplicons can generate a detectable signal.

[ 0061 ] In a particular embodiment, a programmable nuclease complexed with guide nucleic acid sequences to detect the presence or absence of, and/or quantify the amount of, nucleic acids from SARS-CoV-2 generated by a LAMP assay disclosed herein. The programmable nuclease detection assays disclose herein feature low coast, portability, and accurate detection of SARS-CoV-2 and may be performed using commercially available reagents and devices.

[ 0062 ] In a particular embodiment, a programmable nuclease can be used for detection of a target nucleic acid from SARS-CoV-2 in a sample (e.g, a subject’s sample or an environmental sample). For example, a programmable nuclease can be complexed with a guide nucleic acid that hybridizes to a target sequence of a target nucleic acid from SARS- CoV-2. The complex can be contacted to a sample from a subject. The subject may or may not be infected with SARS-CoV-2. The target nucleic acid in the sample can be reversed transcribed and isothermally amplified using a LAMP assay disclosed herein. If the subject is infected with SARS-CoV-2, the guide nucleic acid hybridizes to the target nucleic acid leading to activation of programmable nuclease. Upon activation, the programmable nuclease can cleave a detector nucleic acid, wherein the detector nucleic acid comprises a detectable label attached to a polynucleotide (e.g, polydeoxyribonucleotide or polyribonucleotide). In some embodiments of the assay, upon cleavage of the polynucleotide, the detectable label emits a detectable signal, which is then captured and quantified (e.g, the detectable label is a fluorophore and the detectable signal is fluorescence). Upon detection of the detectable label, it can be determined that the sample from the subject contained target nucleic acids from SARS-Cov-2. In further embodiments, the target nucleic acid comprises the N gene or the E gene of SARS-CoV-2 and can be assayed by using the compositions and methods of the disclosure.

[ 0063] The compositions and method of use thereof disclosed herein include using a programmable nuclease such as a Casl2 protein, a Casl3 protein, or a Casl4 protein to assay for, detect and/or quantify a nucleic acid from SARS-CoV-2. In a particular embodiment, a Cast 2 protein, a Cast 3 protein, or a Cast 4 protein is used for detection of a target nucleic acid from SARS-CoV-2 in a sample from a subject. For example, a Casl2 protein, a Casl3 protein, or a Cast 4 protein is complexed with a guide nucleic acid that hybridizes to a target sequence of a target nucleic acid from SARS-CoV-2. The complex can be contacted to a sample from a subject or an environmental sample. The sample may or may not comprise

SARS-CoV-2. For use in an assay with a Cast 2 protein, a Cast 3 protein or a Cast 4 protein, a target nucleic acid in the sample can be reverse transcribed back into RNA. If the subject is infected with SARS-CoV-2, the guide nucleic acid hybridizes to the target nucleic acid or amplicon thereof leading to activation of the Casl2 protein, the Casl3 protein, or the Casl4 protein. Upon activation, the Casl2 protein, the Casl3 protein, or the Casl4 protein can cleave a detector nucleic acid, wherein the detector nucleic acid comprises a detectable label attached to the nucleic acid for cleavage by the Casl2 protein, the Casl3 protein, or the Cast 4 protein. In another embodiment, upon cleavage of the detector nucleic acid, the detectable label emits a detectable signal, which can then be captured and quantified (e.g, the detectable label is a fluorophore and the detectable signal is fluorescence). Upon detection of a detectable label, it can be determined that the sample from the subject comprise the target nucleic acids from SARS-CoV-2. In yet another embodiment, the target nucleic acid comprises the N gene or the E gene of SARS-CoV-2 and can be assayed by using the compositions and methods disclosed herein.

[ 0064 ] In some embodiments, a programmable nuclease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:26, 27, 28 or 29, can be used for detection of a target nucleic acid from SARS-CoV-2. For example, a programmable nuclease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity sequence identity to SEQ ID NOs: SEQ ID NOs:26, 27, 28 or 29 can be complexed with a guide nucleic acid that hybridizes to a target sequence of a target nucleic acid from SARS-CoV-2. The complex can be contacted to a subject’s sample or an environmental sample. The target nucleic acid of the sample can be reverse transcribed and amplified by a LAMP assay disclosed herein. If the subject is infected with SARS-CoV-2, the guide nucleic acid hybridizes to the target nucleic acid leading to activation of programmable nuclease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity sequence identity to SEQ ID NOs:26, 27, 28 or 29 can cleave a detector nucleic acid, wherein the detector nucleic acid comprises a detectable label attached to a nucleic acid. In some embodiments of the assay, upon cleavage the detector nucleic acid, the detectable label emits a detectable signal, which can then be captured and quantified (e.g, the detectable label is a fluorophore and the detectable signal is fluorescence). Upon detection of a detectable label, it can be determined that the sample from the subject contained target nucleic acids from SARS-CoV- 2. In some embodiment, the target nucleic acid comprises the N gene or the E gene of SARS- CoV-2 and can be assayed for using the compositions and methods disclosed herein.

[ 0065] The compositions and methods disclosed herein can be used as a companion diagnostic with medicaments used to treat SARS-CoV-2, or can be used in reagent kits, point-of-care diagnostics, over-the-counter diagnostics, or at-home testing. The compositions and methods disclosed herein can be used for diagnosis of COVID-19 or for screening of asymptomatic populations for public health surveillance purposes or for determination of status with regards to return to work or return to school. The methods of the disclosure may be used as a point-of-care diagnostic or as a lab test for detection of a target nucleic acid from SARS-CoV-2, and thereby, detection of a COVID-19 in a subject from which the sample was taken. The compositions and methods of the disclosure may be used in various sites or locations, such as in laboratories, in hospitals, in physician offices/laboratories (POLs), in clinics, at remote sites, or at home.

[ 0066] Also described herein are methods, reagents, and devices for detecting the presence of a target nucleic acid from SARS-CoV-2 in a sample. The methods, reagents, and devices for detecting the presence of a target nucleic acid from SARS-CoV-2 in a sample can be used as a rapid lab test for the detecting a target nucleic acid of interest. In particular, provided herein are methods, reagents, and devices, wherein the rapid lab tests can be performed in a single system. The target nucleic acid may be a portion of a nucleic acid from SARS-CoV-2. The target nucleic acid may be a portion of an RNA or DNA or an amplicon thereof from SARS-CoV-2.

[ 0067 ] In some embodiments, programmable nucleases disclosed herein are activated by RNA or DNA to initiate trans cleavage activity of a detector nucleic acid. A programmable nuclease as disclosed herein is, in some cases, binds to a target RNA to initiate trans cleavage of a detector nucleic acid, and this programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, a programmable nuclease as disclose herein binds to a target DNA to initiate trans cleavage of a detector nucleic acid, and this programmable nuclease can be referred to as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein is capable of being activated by a target RNA or a target DNA. For example, a Casl3 protein, such as Casl3a, disclosed herein is activated by a target RNA nucleic acid or a target DNA nucleic acid to transcollaterally cleave RNA detector nucleic acids. In another embodiment, the Cast 3 binds to a target ssDNA which initiates trans cleavage of RNA detector nucleic acid.

[ 0068 ] The detection of the target nucleic acid in the sample may indicate the presence of SARS-CoV-2 in the sample and may provide information for taking action to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual. The detection of the target nucleic acid from SARS- CoV-2 is facilitated by a programmable nuclease. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid from SARS-CoV-2, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity, which can also be referred to as “collateral” or “transcollateral” cleavage.

[ 0069] Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety is released from the detector nucleic acid and generates a detectable signal that is immobilized on a support medium. Often the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid. Sometimes the detection moiety binds to a capture molecule on the support medium to assess the presence or level of the target nucleic acid associated with an ailment, such as a disease. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid. These assays, which leverage the transcollateral cleavage properties of CRISPR-Cas enzymes. The assays of the disclosure can be performed in a microfluidic device or genechip.

[ 0070 ] In a particular embodiment, the disclosure further provides for Cas 12 detection of a target nucleic acid from SARS-CoV-2. In this case, nucleic acids (RNA) from a sample are reverse transcribed and amplified into cDNA. Any Cas 12 protein disclosed herein is complexed with a guide nucleic acid designed to hybridize to a nucleic acid sequence of the reverse transcribed and amplified DNA. In the presence of reverse transcribed and amplified DNA, Cas 12 is activated to transcollaterally cleave a detector nucleic acid, emitting a detectable signal (e.g, fluorescence).

[ 0071 ] Also described herein is a kit for detecting a target nucleic acid from SARS- CoV-2. The kit may comprise any one or more of the following: SARS-Cov-2 spike primers; a support medium; a plurality of LAMP primers; guide nucleic acid sequences targeted to a target nucleic acid sequence from SARS-CoV-2; a programmable nuclease capable of being activated when complexed with a guide nucleic acid and a target nucleic acid from SARS- CoV-2; and a single-stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal.

[ 0072 ] Some methods are described herein utilize an editing technique, such as a technique using an editing enzyme or a programmable nuclease and guide nucleic acid, to detect a target nucleic acid from SARS-CoV-2. An editing enzyme or a programmable nuclease in the editing technique can be activated by one or more target nucleic acids, after which the activated editing technique can be activated by one or more target nucleic acids, after which the activated editing enzyme or activated programmable nuclease can cleave nearby single-stranded nucleic acids, such as detector nucleic acids having a detection moiety. A target nucleic acid from SARS-CoV-2 can be amplified by a LAMP assay disclosed herein and then an editing technique can be used to detect the marker. In some instances, the editing technique can comprise an editing enzyme or programmable nuclease that, when activated, cleaves nearby RNA or DNA as the readout of the detection. The methods as described herein in some instances comprise obtaining a cell-free RNA sample, reverse transcribing the RNA sample to cDNA, amplifying the cDNA, and using an editing technique to cleave detector nucleic acids, and reading the output of the editing technique. In other instance, the method comprises obtaining a fluid sample from a patient, and without amplifying a nucleic acid of the fluid sample, using an editing technique to cleave detector nucleic acids, and detecting the nucleic acid. The method can also comprise using singlestranded detector DNA, cleaving the single-stranded detector DNA using an activated editing enzyme, wherein the editing enzyme cleaves at least 50% of a population of single-stranded detector DNA as measured by a change in color. A number of samples, guide nucleic acids, programmable nucleases or editing enzymes, support mediums, target nucleic acids, singlestranded detector nucleic acids, and reagents that are used with said devices, systems, fluidic devices, kits and methods disclosed herein.

[ 0073] Also disclosed herein are detector nucleic acids and methods detecting a target nucleic acid using the detector nucleic acids. Often, the detector nucleic acid is a protein- nucleic acid. For example, a method of assaying for a target nucleic acid from SARS-CoV-2 in a sample comprises performing LAMP on the sample followed by contacting the sample with a plurality of complexes comprising a guide nucleic acid, each guide nucleic acid sequence comprising a segment that is reverse complementary to a segment of a target nucleic acid sequence from SARS-CoV-2 with a target nucleic acid population and programmable nucleases that exhibits sequence independent cleavage upon forming complexes comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein- nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of one or more of the target nucleic acid populations in the sample and wherein absence of the signal indicates an absence of the target nucleic acid population in the sample. Often, the protein-nucleic acid is an enzyme-nucleic acid or an enzyme substrate-nucleic acid. The nucleic acid can be DNA, RNA, or a DNA/RNA hybrid. The methods described herein use a programmable nuclease, such as the CRISPR/Cas system, to detect a target nucleic acid (e.g, SARS-CoV-2 RNA.

[ 0074 ] A number of samples are consistent with the methods, reagents, and devices disclosed herein. These samples can comprise a target nucleic acid from SARS-CoV-2 for detection. Generally, a sample from an individual or an animal, or an environmental sample can be obtained to test for presence of SARS-CoV-2. A biological sample from the individual may be a sample selected from blood, serum, plasma, saliva, urine, mucosal, peritoneal, cerebrospinal, gastric secretions, nasal secretions, sputum, pharyngeal exudates, urethral or vaginal secretions, an exudate, an effusion, or tissue. A tissue sample may be dissociated or liquefied prior to use in the methods of the disclosure. A sample from an environment may be from soil, air or water. In some instances, the environmental sample is collected by using a swab. In a particular embodiment, the sample is unprocessed and used directly in the methods of the disclosure. In another embodiment, the sample is diluted with a buffer or a fluid or concentrated prior to application to the detection system or be applied neat to the detection system. Sometimes, less than 20 pL of the sample is used in the methods of the disclosure. The sample in some embodiments is in a volume of 1 pL, 2 pL, 3 pL, 4 pL, 5 pL, 6 pL, 7 pL, 8 pL, 9 pL, 10 pL, 11 pL, 12 pL, 13 pL, 14 pL, 15 pL, 16 pL, 17 pL, 18 pL, 19 pL, 20 pL, 25 pL, 30 pL, 35 pL, 40 pL, 45 pL, 50 pL, 55 pL, 60 pL, 65 pL, 70 pL, 75 pL, 80 pL, 85 pL, 90 pL, 95 pL, 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, or a range that includes or is between any two of the foregoing values.

[ 0075] A number of target nucleic acids from S ARS-CoV -2 can be used in the methods disclosed herein. The methods of the disclosure can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid. In some cases, the sample has at least 2 target nucleic acids. In some cases, the sample has 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 target nucleic acids. In some cases, the method detects target nucleic acids present at rate of one copy per 10¹ non-target nucleic acids, 10² non-target nucleic acids, 10³ non-target nucleic acids, 10⁴ non-target nucleic acids, 10⁵ non-target nucleic acids, 10⁶ non-target nucleic acids, 10⁷ non-target nucleic acids, 10⁸ non-target nucleic acids, 10⁹ non-target nucleic acids, IO¹⁰ non-target nucleic acids, or a range that includes or is between any two of the foregoing values.

[ 0076] A number of target nucleic acids from S ARS-CoV -2 are consistent with the methods or compositions disclosed herein. In a particular embodiment, the methods of the disclosure detect two or more target nucleic acid sequences present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 target nucleic acid sequences from SARS-CoV-2. In other cases, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 target nucleic acid sequences from SARS-CoV-2, or a range that includes or is between any two of the foregoing values.

[ 0077 ] Any of the above disclosed samples are consistent with systems, assays, and programmable nucleases disclosed herein and can be used as a companion diagnostic with SARS-CoV-2, or can be used in reagent kits, point-of-care diagnostics, over-the-counter diagnostics, or at-home self-test.

[ 0078 ] A number of reagents are compatible with the methods, compositions and devices disclosed herein. The reagents described herein for detecting SARS-CoV-2 comprise a plurality of LAMP primers and optionally multiple guide nucleic acids, each guide nucleic acid being specific to a target nucleic acid segment indicative of SARS-CoV-2. Each guide nucleic acid binds to the target SARS-CoV-2 nucleic acid comprising a segment of a nucleic acid sequence from SARS-CoV-2 as described herein. Each guide nucleic acid is complementary to a target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid.

[ 0079] Disclosed herein are methods of assaying for a plurality of target S ARS-CoV - 2 nucleic acids as described herein. For example, a method of assaying a plurality of target SARS-CoV-2 nucleic acids in a sample comprises contacting the sample with LAMP primers comprising any of the F3, B3, FIP, BIP and optionally the FL and BL primers described herein under conditions such that a SARS-CoV-2 present in the sample is amplified. In an optional step, the sample comprising the LAMP-amplified nucleic acids is contacted to a complex comprising a plurality of guide nucleic acid sequences, each guide nucleic acid comprising a segment that is reverse complementary to a segment of the target SARS-CoV-2 nucleic acid, and programmable nucleases that exhibit sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target SARS-CoV-2 nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of one or more target SARS-CoV-2 nucleic acids of the plurality of target SARS-CoV-2 nucleic acids in the sample and wherein absence of the signal indicates an absence of the target SARS-CoV-2 nucleic acids in the sample.

[ 0080 ] A programmable nuclease is a nuclease that is capable of being activated when complexed with a guide nucleic acid and target SARS-CoV-2 nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity. Trans cleavage activity can be nonspecific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and generate a signal. A signal can be a calorimetric, a potentiometric, an amperometric, an optical (e.g, fluorescent, colorimetric, etc.), or a piezo-electric signal. Often, the signal is present prior to detector nucleic acid cleavage and changes upon detector nucleic acid cleavage. Sometimes, the signal is absent prior to detector nucleic acid cleavage and is present upon detector nucleic acid cleavage. The detectable signal can be immobilized on a support medium for detection. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target SARS-CoV-2 nucleic acid. The CRISPR-Cas nucleoprotein complex can comprise a Cas protein (also referred to as a Cas nuclease) complexed with a guide nucleic acid, which can also be referred to as CRISPR enzyme. A guide nucleic acid can be a CRISPR RNA (crRNA). Sometimes, a guide nucleic acid comprises a crRNA and a trans-activating crRNA (tracrRNA).

[ 0081 ] Several programmable nucleases are consistent with the methods and devices of the disclosure. For example, CRISPR/Cas enzymes are programmable nucleases used in the methods and systems disclosed herein. CRISPR/Cas enzymes can include any of the known Classes and Types of CRISPR/Cas enzymes. Programmable nucleases disclosed herein include Class 1 CRISPR/Cas enzymes, such as the Type I, Type III, or Type IV CRISPR/Cas enzymes. Programmable nucleases disclosed herein also include the Class 2 CRISPR/Cas enzymes, such as the Type II, Type V, and Type VI CRISPR/Cas enzymes. Preferable programmable nucleases included in the several assays disclosed herein (e.g, for assaying for coronavirus in a device, such as a microfluidic device or a lateral flow assay) and methods of use thereof include a Type V or Type VI CRISPR/Cas enzyme.

[ 0082 ] In some embodiments, the Type V CRISPR/Cas enzyme is a programmable Casl2 nuclease. Type V CRISPR/Cas enzymes (e.g, Casl2 or Casl4) lack an HNH domain. A Casl2 nuclease disclosed herein cleaves nucleic acids via a single catalytic RuvC domain. The RuvC domain is within a nuclease, or “NUC” lobe of the protein, and the Casl2 nucleases further comprise a recognition, or “REC” lobe. The REC and NUC lobes are connected by a bridge helix and the Casl2 proteins additionally include two domains for PAM recognition termed the PAM interacting (PI) domain and the wedge (WED) domain. A programmable Casl2 nuclease can be a Casl2a (also referred to as Cpfl) protein, a Casl2b protein, Casl2c protein, Casl2d protein, or a Casl2e protein. In some cases, a suitable Casl2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to any one of SEQ ID NO: l-4, or to any one of accession number: ATB19153.1, WP_021736722.1, 5B43 A, 5KK5 A, 5XH7_A, 5XH6_A, WPJ20110811.1, WP_093729503.1, WP 075579848.1, NLM08782.1, WP_120110807.1, WP_037975888.1, WP_081839471.1, WP_073043853.1, WP_031492824.1, WP_078933213.1, WP_048112740.1, WP_119227726.1, HAW84277.1, KFO67989.1, WP_044110123.1, KIE18657.1, OFY19591.1, HHV41458.1, HHU53715.1, WP_167604087.1, WPJ34744521.1, WP_167554110.1, WP 134711643.1, NDP22346.1, PKP47250.1, WP_003040289.1, ATB19155.1, WP 014550095.1, WP_003034647.1, SCH45297.1, 5NG6_A, WP_104928540.1, 6I1L_A, WP_117448340.1, 6GTC_A, 5MGA A, WP_071304624.1, OHB41002.1, WP 137013028.1, 5NFV_A, WP_039871282.1, EFI70750.1, HHU98002.1, WP 004339290.1, WP_118080156.1, KAF3979590.1, WP_104505765.1, WP_077541740.1, OQB16057.1, SelWP_089081092.1, NLA83350.1, PIN76207.1, WP_016301126.1, WP_078273923.1, WP_112742561.1, EFI15981.1, WP_118231964.1, WP_157236615.1, WP_115369192.1, WP 014085038.1, WP_117970347.1, WP_112132723.1, WP_065256572.1, HAG50355.1, HGP76849.1, WP_118025820.1, WP_097554884.1, WP 068647445.1, WPJ22892441.1, WP_015504779.1, WP_115099143.1, WP_118379198.1, WP_112744621.1, WP_045971446.1, WP_055225123.1, WP 118371518.1, WP 020988726.1, WP_117689699.1, SWP_117996653.1,

WP_117685196.1, SOLA16049.1, WP_087408205.1, SWP_055272206.1, SeHAQ63770.1, WP 154266439.1, WP_118190996.1, WP_055237260.1, HCF15406.1, SWP_117714171.1, GFI53642.1, WP_117918179.1, WP_118163031.1, OLA30477.1, and WP_148871677. Table 1. Casl2 Polypeptide sequences

[ 0083 ] Alternatively, the type V CRISPR/Cas enzyme is a programmable Cast 4 nuclease. A Casl4 protein of the present disclosure include 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the Casl4 protein, but form a RuvC domain once the protein is produced and folded. A naturally occurring Cast 4 protein functions as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid. A programmable Casl4 nuclease can be a Casl4a protein, a Casl4b protein, a Casl4c protein, a Casl4d protein, a Casl4e protein, a Casl4f protein, a Casl4g protein, a Casl4h protein, or a Casl4u protein. In some cases, a suitable Casl4 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to any one of Accession numbers or WP_048402777.1, WP_077210027.1,

WP 083314917.1, WP_018297204.1, WP_014302663.1, WP_088264504.1, CAB0532088.1, CAB0624945.1, CAB0539033.1, CAB1026072.1, WP_014309766.1, CAB0670887.1, CAB0991295.1, WP_014306233.1, WP_082258207.1, CAB0668382.1, WP_014307728.1, WP_071570672.1, CAB0674275.1, CAB0772394.1, WP_016830429.1, CAB0715588.1, CAB0625127.1, WP_014309003.1, CAB1027305.1, WP_088267460.1, WP_072588167.1, CAB0529362.1, WP_018024637.1, WP_080754080.1, WP_052251263.1, WP_082345415.1, VH000656.1, WP_082346565.1, WP_126416583.1, SUO87902.1, WP_115324474.1, WP 051952360.1, WP_083290301.1, WP_041729458.1, WP_025296977.1,

WP_114949752.1, WP_164978155.1, OQD32504.1, WP_070565815.1, WP_101678534.1, WP_024963699.1, WPJ58381790.1, WP_126714927.1, WP_143335301.1, WP_167616985.1, WP_099981414.1, WP_070361639.1, WP_108252736.1, WP 070840746.1, WP_101733443.1, WP_071565490.1, WP_083330183.1, SDE63285.1, WP_070562523.1, WP_049620073.1, KMY22947.1, WP_070529210.1, WP_080975047.1, WP 158396338.1, WP_042531761.1, WP_165002380.1, WPJ51549778.1, WP_082121333.1, WP_083317802.1, WP_157034441.1, WP_144779956.1,

WP_058237269.1, WP_075664776.1, WP_115685609.1, WPJ50386218.1, WP_015382092.1, WP_103759126.1, WP_070791257.1, WP_087117126.1, WP 034986778.1, WP_070551100.1, WP_035011874.1, NNO11674.1, WP_092150518.1, SeWP_071056232.1, WP_070761730.1, WP_012361195.1, WP_095537844.1,

WP 034966247.1, WP_111725437.1, WP_034665223.1, WP_048378674.1, WP 095555610.1, WP_051483496.1, WPJ22086339.1, WP_035007949.1, WP 126843983.1, WP_034980529.1, and VDG62294.1.

[ 0084 ] In some embodiments, the Type VI CRISPR/Cas enzyme is a programmable Casl3 nuclease. The general architecture of a Cast 3 protein includes an N-terminal domain and two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains separated by two helical domains. The HEPN domains each comprise aR-X4-H motif. Shared features across Cast 3 proteins include that upon binding of the crRNA of the guide nucleic acid to a target nucleic acid, the protein undergoes a conformational change to bring together the HEPN domains and form a catalytically active RNase. Thus, two activatable HEPN domains are characteristic of a programmable Cast 3 nuclease of the present disclosure. However, programmable Cast 3 nucleases also consistent with the present disclosure include Cast 3 nucleases comprising mutations in the HEPN domain that enhance the CAS 13 proteins cleavage efficiency or mutations that catalytically inactivate the HEPN domains.

[ 0085] A programmable Cas 13 nuclease can be a Cas 13a protein (also referred to as “c2c2”), a Casl3b protein, a Casl3c protein, a Casl3d protein, or a Casl3e protein. In some cases, a subject C2c2 protein includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to an amino acid sequence as set forth in any one of accession number: WP_012985477.1, WP_099225408.1, WP_118907415.1, WP_036091002.1, WP_036059185.1, WP_013443710.1 , WP_034560163.1 , WP_034563842.1 , WP_071146234.1 ,

WP 149023847.1, WP_092118911.1, HCK24590.1, WP_149023846.1, WP_021747205.1, WP_015770004.1, WP_071125398.1, WP_021746774.1, WP_146998208.1,

WP 146959607.1, WP_149023848.1, QID24124.1, 5XWY_A, ERL25782.1, 5XWP_A, WP 021746003.1, WP_071124126.1, BBM48975.1, NLU52303.1, NLU32351.1, OQX30025.1, HHT89324.1, WP_092122836.1, NMA74573.1, WP_132694182.1, PJI41863.1, SUS05702.1, WP_076398593.1, WP_163266467.1, WPJ50214158.1, KAA6404670.1, WP_114086813.1, PIT01667.1, WP_100176879.1, WP_073955355.1, WP 133357912.1, KAA6204339.1, WP_103203632.1, WP_036059184.1,

WP 013067728.1, WP_023911507.1, WP_146746344.1, RAP39618.1, WP_149023845.1, WP_150215210.1, WPJ37134457.1, WP_080615427.1, WP_108028905.1, WP 031473346.1, KAA6204514.1, and WP_079495749.1.

[ 0086] In a certain embodiment, the programmable nuclease is Cas 13. In a further embodiment, the Casl3 is selected from Casl3a, Casl3b, Casel3c, Casl3d, and Casl3e. In another embodiment, the programmable nuclease can be Mad7 or Mad2. In yet another embodiment, the programmable nuclease is Casl2. In yet a further embodiment, the Casl2 is selected from Casl2a, Casl2b, Casel2c, Casl2d, and Casl2e. In a certain embodiment, the programmable nuclease is selected from Csml, Cas9, C2c4, C2c8, C2c5, C2cl0, C2c9, or CasZ. In another embodiment, the Csml can be smCmsl, miCmsl, obCmsl, or suCmsl. In yet another embodiment, Casl3a is C2c2. In a further embodiment, CasZ is Casl4a, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, Casl4i, Casl4j, or Casl4k. In yet another embodiment, the programmable nuclease is a type V CRISPR-Cas system. In a further embodiment, the programmable nuclease is a type VI CRISPR-Cas system. In another embodiment, the programmable nuclease is a type III CRISPR-Cas system. In yet another embodiment, the programmable nuclease originated from Leptotrichia shahii, Listeria selligeri, Leptotrichia buccalis, Leptotrichia waden, Rhodobacter capsulatus, Herbinix hemicellulosilytica, Paludibacter propionicigenes, Lachnospiraceae bacterium, Eubacterium rectale, Listeria new yorkensis, Clostridium aminophilum, Prevotella sp., Capnocytophaga canimorsus, Lachnospiraceae bacterium, Bergeyella zoohelcum, Prevotella intermedia, Prevotella buccae, Alistipes sp., Riemerella anatipestifer , Prevotella aurantiaca, Prevotella saccharolytica, Prevotella intermedia, Capnocytophaga canimorsus, Porphyromonas gulae, Prevotella sp., Porphyromonas gingivalis, Prevotella intermedia, Enterococcus italicus, Lactobacillus salivarius, or Thermus thermophilus. In a further embodiment, the Cas 13 is selected from LbuCasl3a, LwaCasl3a, LbaCasl3a, HheCasl3a, PprCasl3a, EreCasl3a, CamCasl3a, and LshCasl3a. The trans cleavage activity of the CRISPR enzyme can be activated when the crRNA is complexed with the target nucleic acid. The trans cleavage activity of the CRISPR enzyme can be activated when the guide nucleic acid comprising a tracrRNA and crRNA are complexed with the target SARS-CoV-2 nucleic acid.

[ 0087 ] Described herein are reagents comprising a detection moiety, wherein the detector nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a first detectable signal. As used herein, a detector nucleic acid is used interchangeably with report or reporter molecules. In some embodiments, the detector nucleic acid is a singlestranded nucleic acid comprising deoxyribonucleotides. In other cases, the detector nucleic acid is a single-stranded nucleic acid comprising ribonucleotides. The detector nucleic acid can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In other embodiments, the detector nucleic acid is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some embodiments, the detector nucleic acid comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some embodiments, the detector nucleic acid has only ribonucleotide residues. In other embodiments, the detector nucleic acid has only deoxyribonucleotide residues. In some embodiments, the detector nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or is range that includes or is between any two of the foregoing lengths.

[ 0088 ] The single-stranded detector nucleic acid comprises a detection moiety capable of generating a first detectable signal. In a certain embodiment, the detector nucleic acid comprises a protein capable of generating a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g, fluorescent, luminescent, etc.), or piezo-electric. In some embodiments, a detection moiety is located on one side of the detector nucleic acid cleavage site. In further embodiments, a quenching moiety is on the other side of the cleavage site. In some embodiments, the quenching moiety is 5’ to the cleavage site and the detection moiety is 3’ to the cleavage site. In other embodiments, the detection moiety is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. In further embodiments, the quenching moiety is at the 5’ terminus of the detector nucleic acid. In alternate embodiments, the quenching moiety is at the 3’ terminus of the detector nucleic acid. In further embodiments, the detection moiety is at the 5’ terminus of the detector nucleic acid. In alternate embodiments, the detection moiety is at the 3’ terminus of the detector nucleic acid.

[ 0089] A detection moiety can be an infrared fluorophore. A detection moiety can be a fluorophore that emits fluorescence at 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, or a range that includes or is between any two of the foregoing wavelengths. A detection moiety can be a fluorophore that emits a fluorescence in the same range as fluorescein, 6-fluoresceine, IRDYE 700, TYE 665, Alexa Fluor, or ATTO TM633. A detection moiety can be fluorescein, 6-fluoresceine, IRDYE 700, TYE 665, Alexa Fluor, or ATTO TM633.

[ 0090 ] A quenching moiety can be chosen based on its ability to quench the detection moiety. A quenching moiety can quench the fluorescence emitted by a fluorophore at 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, or a range that includes or is between any two of the foregoing wavelengths. A quenching moiety can quench fluorescence emitted by fluorescein, 6-fluoresceine, IRDYE 700, TYE 665, Alexa Fluor, or ATTO TM633. A detection moiety can be fluorescein, 6-fluoresceine, IRDYE 700, TYE 665, Alexa Fluor, or ATTO TM633. A quenching moiety can be Iowa Black RQ, Iowa Black FQ, and Black Hole quencher.

[ 0091 ] The generation of the detectable signal from the release of the detection moiety indicates that cleavage by the programmable nuclease has occurred and that the sample contains the target nucleic acid. In some embodiments, the detection moiety comprises a fluorescent dye. In a certain embodiment, the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some embodiments, the detection moiety comprises an infrared (IR) dye. In some embodiments, the detection moiety comprises and ultraviolet (UV) dye.

[ 0092 ] A number of detection devices and methods are consistent with the methods disclosed herein. For example, any device that can measure or detect a calorimetric, potentiometric, amperometric, optical (e.g, fluorescent, luminescent, etc.), or piezo-electric signal. For optical signals, the signals can be visualized by eye (e.g, a lateral flow assay), or by use of detection device (e.g, a microplate reader, camera, etc.). In a particular embodiment, a lateral flow assay is used to detection a target nucleic acid from SARS-CoV-2 (e.g., see FIG. 2B).

[ 0093] Lateral flow assay (LFA) based devices are among very rapidly growing strategies for qualitative and quantitative analysis. Lateral flow assays are performed over a strip, different parts of which are assembled on a plastic backing. These parts include a sample application pad, a conjugate pad, a nitrocellulose membrane and an adsorption pad. The nitrocellulose membrane is further divided into test and control lines. Pre-immobilized reagents at different parts of the strip become active upon flow of liquid sample. Lateral flow assays combine unique advantages of biorecognition probes and chromatography. Lateral flow assays basically combine a number of variants such as formats, biorecognition molecules, labels, detection systems and application. LFA strips give qualitative or semi- quantitative results which can be observed by naked eyes. Conventional LFAs are normally qualitative and give answers as a ‘yes’ or ‘no’ result. A good LFA biosensor should have the following properties: biocompatibility, high specificity, high sensitivity, rapidity of analysis, reproducibility/precision of results, wide working range of analysis, accuracy of analysis, high through-put, compactness, low cost, simplicity of operation, portability, flexibility in configuration, possibility of miniaturization, potential of mass production and on-site detection.

[ 0094 ] For use in applications described herein, kits and articles of manufacture are also described herein. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

[ 0095] For example, the container(s) can comprise one or more SARS-CoV-2 assay components (e.g, LAMP primers) described herein, optionally in a composition or in combination with another agent (e.g, QUASR primers or FLOS probe) as disclosed herein. The container(s) optionally have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprise a compound with an identifying description or label or instructions relating to its use in the methods described herein.

[ 0096] A kit will typically comprise one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint for use of a compound described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[ 0097 ] A label can be on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g, as a package insert. A label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein. These other therapeutic agents may be used, for example, in the amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

[ 0098 ] After packaging the formed product and wrapping or boxing to maintain a sterile barrier, the product may be terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, the product may be prepared and packaged by aseptic processing.

EXAMPLES

[ 0099] The following examples are illustrative and non-limiting to the scope of the devices, methods, reagents, systems, and kits described herein.

[ 00100] Construction of SARS-CoV-2 Template Genome. SARS-CoV-2 target sequences are designed from a SARS-CoV-2 template that is generated by aligning all available SARS-CoV-2 genomes from GISAID using Clustal Omega.

[ 00101] Generation of SARS-CoV-2 Target RNAs for screening assays. Target RNAs are generated from synthetic gene fragments of the viral genes of interest. First a PCR step was performed on the synthetic gene fragment with a forward primer that contained a T7 promoter. Next, the PCR product is used as a template for an in vitro transcription (IVT) reaction at 37 °C for 2 h. The IVT reaction is then treated with TURBO DNase (Thermo) for 30 minutes at 37 °C, followed by a heat-denaturation step at 75 °C for 15 minutes. RNA is purified using RNA Clean and Concentrator 5 columns (Zymo Research). RNA is quantified by Nanodrop and Qubit and diluted in nuclease-free water to working concentrations.

[ 00102] Primers for LAMP Assay. The following considerations were taken into account for the LAMP primers: generation of a product that is long enough to be targeted by the LAMP primers, but short enough to be rapidly amplified; and use of primers to the N gene region of SARS-CoV-2 that takes into account of partial degradation by RNases during processing. By tiling across the N gene and E gene a series of primers were generated using PrimerExplorer v5 (primerexplorer.jp/e/). These primers were then screened for time-to- threshold (Tt) for targeted SARS-CoV-2 product formation using different copy numbers, with spike in primers, and in multiplex fashion (see FIGs. 5-10, 17, and 18). Of the primers that were screened, three sets of LAMP primers (1051.2, MA80, MAI) were identified as having the best specificity and efficiency in RT-LAMP assays (e.g, see FIGs. 5-7, 17 and 18). A set of spiked primers were also generated in order to enrich the SARS-CoV-2 nucleic acids directly from clinical samples. Additional primers were generated for direct identification of amplified products using a FLOS-RT-LAMP assay and a QUASR-RT- LAMP assay. All of the foregoing primers are presented in Table 2.

[00103] Table 2. Primers for LAMP assay and primers using FLOS and QUASR techniques.

Lamp Assay Primers

Name Sequence t!051.2-F3 TGAATAAGCATATTGACGCATAC (SEQ ID NO: 1) t!051.2-B3 TGAGTTGAGTCAGCACTG (SEQ ID NO:2) tl 051.2-FIP TAAGGCTTGAGTTTCATCAGCCCATTCCCACCAACAGAGC (SEQ ID NO:3) t!051.2-BIP CGCAGAGACAGAAGAAACAGCATTGTTGCAATTGTTTGGAGAA (SEQ ID NO:4) t!051.2-LF TTCTTCTTTTTGTCCTTTTTAG (SEQ ID NO:5) t!051.2-LB AAACTGTGACTCTTCTTCCTGC (SEQ ID NO:6)

MAP89-F3 CTGCCACTAAAGCATACAATGT (SEQ ID NO: 7)

MAP89-B3 TTGATGGCACCTGTGTAGG (SEQ ID NO:8)

MAP89-FIP GTGCAATTTGCGGCCAATGTTTGTTTTTCAAGGAAATTTTGGGGACCAG (SEQ ID NO:9)

MAP89-BIP CCAGCGCTTCAGCGTTCTTCTTTTTCAACCACGTTCCCGAAGG (SEQ ID NOTO)

MAP89-LF CAGTTCCTTGTCTGATTAGTTC (SEQ ID NO: 11)

MAP89-LB AATGTCGCGCATTGGCATGG (SEQ ID NO: 12)

MA-I-F3 ACAAGC T f I'CGGCAGACG (SIX? ID XO: 13)

MA-1-B3 TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14)

MA-l-FIP GCGGCCAATGTTTGTAATCAGTTCCTTTTAACCCAAGGAAATTTTGGGGAC (SEQ

ID NO: 15)

MA-l-BIP GTCGCGCATTGGCATGGAAGT TTTT ATGGCACCTGTGTAGGTCA (SEQ ID NO: 16)

MA-l-LF TTGTCTGATTAGTTCCTG (SEQ ID NO: 17)

MA-l-LB CACACCTTCGGGAACGTGGT (SEQ ID NO: 18) spike2-3F3 CTCTATTGCCATACCCACA (Si:Q ID XO: 19) spike2-3B3 CTTGTGCAAAAACTTCTTGG (SEQ ID NO:20) spike2-3FIP CATTCAGTTGAATCACCACAAATGTGTGTTACCACAGAAATTCTACC (SEQ ID

NO:21) spike2-3BIP GTTGCAATATGGCAGTTTTTGTACATTTGTCTTGTTCAACAGCTAT (SEQ ID

NO:22) spike2-3LF GTACAATCTACTGATGTCTTGGTCA (SEQ ID NO:23)

RPP-F3 TTGATGAGCTGGAGCCA (SEQ ID NO:31)

RPP-B3 CACCCTCAATGCAGAGTC (SEQ ID NO:32)

RPP-FIP GTGTGACCCTGAAGACTCGGTTTTAGCCACTGACTCGGATC (SEQ ID NO:33)

RPP-BIP CCTCCGTGATATGGCTCTTCGTTTTTTTCTTACATGGCTCTGGTC (SEQ ID NO:34) RPP-LF ATGTGGATGGCTGAGTTGTT (SEQ ID NO:35)

RPP-LB CATGCTGAGTACTGGACCTC (SEQ ID NO:36)

FLOS- LAMP primer

Name Sequence

LB-tlO51.2- AAACTGTGACTCTTCTTCC/i6-FAMK/GC (SEQ ID NO:30)

FLOS

QUASR- LAMP Primers

Name Sequence

NO:40)

*RPP-FIP- 6FAM/6TAMN/cy5-GTGTGACCCTGAAGACTCGGTTTTAGCCACTGACTCGGATC

QUASR (SEQ ID NO:41)

*RPP-FIP-IBQ AGGGTCACAC/3BHQ 1/ (SEQ ID NO:42) or AGGGTCACAC3/3BHQ 2/ (SEQ ID

NO:42)

LAMP primers- At home lateral flow assay test

Name Sequence t!051.2-FIP- 6FAM/TAAGGCTTGAGTTTCATCAGCCCATTCCCACCAACAGAGC (SEQ ID NO:3)

6FAM t!051.2-BIP- Biotin/CGCAGAGACAGAAGAAACAGCATTGTTGCAATTGTTTGGAGAA (SEQ ID

Biotin NO:4)

MAP89-FIP- 6FAM/GTGCAATTTGCGGCCAATGTTTGTTTTTCAAGGAAATTTTGGGGACCAG

6FAM (SEQ ID NO:9)

MAP89-BIP- Biotin/CCAGCGCTTCAGCGTTCTTCTTTTTCAACCACGTTCCCGAAGG (SEQ ID

Biotin NO: 10)

MA- 1 -FIP- 6FAM/GCGGCC AATGTTTGTAATC AGTTCCTTTTAACCC AAGGAAATTTTGGGGAC

6FAM (SEQ ID NO: 15)

MA- 1 -BIP- Biotin/GTCGCGCATTGGCATGGAAGTTTTTATGGCACCTGTGTAGGTCA (SEQ ID

Biotin NO: 16)

RPP = The human ribonuclease (RNase) P reference sequence (GenBank accession number

U94316.1)

*When FIP is labeled with FAM, BHQ1 is used as the quencher; when FIP is labeled with TAMN or Cy5, BHQ2 is used as the quencher.

[ 00104 ] Screening LAMP assay for SARS-CoV-2. A LAMP screening assay was performed with target RNA and a series of LAMP primers as designed and detailed above. Briefly, the LAMP assay is prepared using the following reaction components:

Final Concentration in LAMP assay

1 OX Isothermal Amplification IX Isothermal Amplification Buffer

Buffer 20 mM Tris-HCl pH 8.8@25°C

10 mM (NH4)2SO4

50 mM KC1 2 mM MgSO4 0.1% Tween® 20

100 mM MgS04 8 mM

10 mM dNTPs 1.4 mM each

Warmstart Bst 2.0 8 U

Warmstart RTx 7.5 U

250 uM SYTO9 5 uM

L-Serine 100 mM

Rnase Inhibitor (40 U/uL) 4 U carrier RNA (1 ug/uL) 100 ng

Template 5-10 uL

H2O make it up to 25 uL

[ 00105] The LAMP primers are added at a final concentration of 0.2 pM for F3 and B3, 1.6 pM for FIP and BIP, and 0.8 pM for LF and LB. Reactions are performed independently or multiplex for N-gene, E-gene and RNase P using 2 pL of input RNA at 60- 65 °C for 10 - 30 minutes. Generally, the outer primer (F3, B3): the inner primer (FIP, BIP): the molar ratio of the loop primer (LF, LB) is 1:8:4.

[ 00106] LAMP assay using samples from subjects. A sample (e.g, environmental sample, water sample, sewage sample, subject sample, etc.) that is suspected of having SARS-CoV-2 is collected. Typically, the sample collected from human subjects is a nasopharyngeal swab sample. However, other samples from human subjects may also be used, including oropharyngeal (OP) specimen, nasal mid-turbinate swab, anterior nares (nasal swab) specimen, nasopharyngeal wash/aspirate, oral swab, throat swab and saliva. Lysis buffer (Lucigen, Inc.) is added to the sample, and the sample is incubated with agitation for 5 min. An aliquot of the sample is then directly used in the LAMP assay. Alternatively, the RNA may be extracted prior to the use in the LAMP assay. However, this RNA extraction step is optional due to the high sensitivity and specificity of the 1051.2, MAP89, and MA-1 LAMP primer sets. The reaction components for LAMP assay are described above. 5 pL of the sample was used in the LAMP assay. The reactions were incubated at 60-65 °C for 10 - 30 minutes. It was surprisingly found that the LAMP assay provided much better results if L- Serine (final 100 mM) was added into the LAMP assay reaction mixture (e.g, see FIG. 3). [ 00107] FLOS-LAMP assay. The samples were processed as above for the LAMP assays, but the LAMP assay further comprised a FLOS probe (e.g, LB-tl051.2-FLOS primer). The reactions are incubated at 60-65 °C for 10 - 30 minutes and monitored using real-time fluorimeter. The instrument can be programed to acquire the fluorescence signal every 30 sec. Amplification is noted positive by analysis software when any signal attained slope value, >20 mV from the baseline (0 mV). The time-to-positivity (Tp) value (minutes) is obtained by dividing the numerical value on the x-axis scale, designated as Time (30 sec Interval) by a factor of 2.

[ 00108] QUASR-LAMP assay. The samples were processed as above for the LAMP assays, except that the LAMP assay comprised a fluorescently labeled primer (e.g, FIP- tlO51.2-QUASR) and further comprised a quencher containing primer (e.g, FlPc-tl 051.2- QUASR-IBQ). The Quencher containing probe is typically added at 1.5 x the concentration of the corresponding fluorescently labeled primer. After incubation at 60-65 °C for 10 - 30 minutes, the reaction is cooled to ambient temperature, resulting in dark quenching of fluorescent primers (negative reactions for SARS-CoV-2) or highly fluorescent amplicons (positive reactions for SARS-CoV-2) (e.g, see FIG. 3).

[ 00109] LAMP assay with CRISPR-Cas 12-based detection. The LAMP assay is performed as described above. LbCasl2atrans-cleavage assays are performed similarly to those described in Chen et al. (Science 360, 436-439 (2018)). A total of 50 nM LbCasl2a (NEB) is preincubated with 62.5 nM gRNA in 1 x NEBuffer 2.1 for 10 min at 37 °C. After formation of the RNA-protein complex, the lateral flow cleavage reporter (156- FAM/TTATTATT/3Bio/, IDT) is added to the reaction at a final concentration of 500 nM. RNA-protein complexes are used immediately or stored at 4 °C for up to 24 h before use.

[ 00110] Lateral Flow Assay for CRISPR-Casl2-based detection. A lateral flow strip (Milenia HybriDetect 1, TwistDx) is added to the reaction tube and a result was visualized after approximately 2 min. A single band, close to the sample application pad indicated a negative result, whereas a single band close to the top of the strip or two bands indicated a positive result. Lateral flow strips are interpreted visually or are quantified using ImageJ’s gel analyzer tool and signal was normalized to the maximum signal intensity.

[ 00111] Lateral Flow Assay for at-home SARS-CoV-2 tests. The LAMP reaction is run as above, except that the FIP primer is labelled with FAM and the BIP primer is labelled with biotin (e.g, see FIG. 25A). After the LAMP assay, a lateral flow strip (Milenia HybriDetect 1, TwistDx) is added to the reaction tube and a result was visualized after approximately 2 min (e.g., see FIG. 25B-C). If a LAMP amplicon is present, then the FAM residue of the amplicon is bound by anti -FAM antibody conjugated to a gold particle. The LAMP amplicon will bind to the test line (as well as biotin labelled primers). Due to the presence of the gold particle, a visible band will be formed at the test line by a LAMP amplicon. If there is no LAMP amplicon, no band should be visible at the test line.

[ 00112] A lateral flow strip (Milenia HybriDetect 1 , TwistDx) is then added to the reaction tube and a result was visualized after approximately 2 min. A single band, close to the sample application pad indicated a negative result, whereas a single band close to the top of the strip or two bands indicated a positive result. Lateral flow strips are interpreted visually or are quantified using ImageJ’s gel analyzer tool and signal was normalized to the maximum signal intensity.

[ 00113] It will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

IN THE CLAIMS:

1. A set of loop-mediated isothermal amplification (LAMP) primers comprising a

Forward Outer (F3) primer, a Backward Outer (B3) primer, a Forward Inner Primer (FIP) primer, a Backward Inner Primer (BIP) primer, optionally a Forward Loop (LF) primer, and optionally a Backward Loop (LB) primer, wherein the set of primers are selected from the group consisting of:

(A) (i) F3: TGAATAAGCATATTGACGCATAC (SEQ ID NO: 1) or a sequence that is at least 87% identical to SEQ ID NO: 1, or a sequence that is at least 24-30 nucleotides in length and contains the sequence of SEQ ID NO: 1;

(ii) B3: TGAGTTGAGTCAGCACTG (SEQ ID NO:2) or a sequence that is at least 83.3% identical to SEQ ID NO:2, or a sequence that is at least 19-25 nucleotides in length and contains the sequence of SEQ ID NO:2;

(iii)FIP : TAAGGCTTGAGTTTCATCAGCCCATTCCCACC AACAGAGC (SEQ ID NO:3) or a sequence that is at least 87.5% identical to SEQ ID NO:3, or a sequence that is at least 41-46 nucleotides in length and contains the sequence of SEQ ID NO: 1;

(iv)BIP:

CGCAGAGACAGAAGAAACAGCATTGTTGCAATTGTTTGGAGAA (SEQ ID NO:4) or a sequence that is at least 86% identical to SEQ ID NO:4, or a sequence that is at least 44-50 nucleotides in length and contains the sequence of SEQ ID NO:4;

(v) LF: TTCTTCTTTTTGTCCTTTTTAG (SEQ ID NO: 5) or a sequence that is at least 87% identical to SEQ ID NO:5, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO:5; and/or

(vi)LB: AAACTGTGACTCTTCTTCCTGC (SEQ ID NO:6) or a sequence that is at least 87% identical to SEQ ID NO:6, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO:6;

(B) (a) F3: CTGCCACTAAAGCATACAATGT (SEQ ID NO: 7) or a sequence that is at least 87% identical to SEQ ID NO:7, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO:7;

(b) B3: TTGATGGCACCTGTGTAGG (SEQ ID NO: 8) or a sequence that is at least 84.2% identical to SEQ ID NO: 8, or a sequence that is at least 20-25 nucleotides in length and contains the sequence of SEQ ID NO: 8;

43 (c) FIP: GTGCAATTTGCGGCCAATGTTTGTTTTTCAAGGAAATTTTG GGGACCAG (SEQ ID NO: 9) or a sequence that is at least 90% identical to SEQ ID NO:9, or a sequence that is at least 51-55 nucleotides in length and contains the sequence of SEQ ID NO:9;

(d) BIP: CCAGCGCTTCAGCGTTCTTCTTTTTCAACCACGTTCCCGAAGG (SEQ ID NO: 10) or a sequence that is at least 86% identical to SEQ ID NO:10, or a sequence that is at least 44-50 nucleotides in length and contains the sequence of SEQ ID NO: 10;

(e) LF: CAGTTCCTTGTCTGATTAGTTC (SEQ ID NO: 11) or a sequence that is at least 87% identical to SEQ ID NO: 11, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO: 11; and/or

(f) LB: AATGTCGCGCATTGGCATGG (SEQ ID N:O12) or a sequence that is at least 75% identical to SEQ ID NO: 12, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO: 12;

(C) (1) F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13) or a sequence that is at least 83.3% identical to SEQ ID NO: 13, or a sequence that is at least 19-24 nucleotides in length and contains the sequence of SEQ ID NO: 13;

(2) B3: TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14) or a sequence that is at least 77.3% identical to SEQ ID NO: 14, or a sequence that is at least 23-28 nucleotides in length and contains the sequence of SEQ ID NO: 14;

(3) FIP: GCGGCCAATGTTTGTAATCAGTTCCTTTTAACCCAAGGAAATTT TGGG GAC (SEQ ID NO: 15) or a sequence that is at least 90% identical to SEQ ID NO: 15, or a sequence that is at least 52-57 nucleotides in length and contains the sequence of SEQ ID NO: 15;

(4) BIP: GTCGCGCATTGGCATGGAAGTTTTTATGGCACCTGTGTAG GTCA (SEQ ID NO: 16) or a sequence that is at least 88.6% identical to SEQ ID NO: 16, or a sequence that is at least 45-50 nucleotides in length and contains the sequence of SEQ ID NO: 16;

(5) LF: TTGTCTGATTAGTTCCTG (SEQ ID NO: 17) or a sequence that is at least 83.3% identical to SEQ ID NO: 17, or a sequence that is at least 19-24 nucleotides in length and contains the sequence of SEQ ID NO: 17; and/or

(6) LB: CACACCTTCGGGAACGTGGT (SEQ ID NO: 18) or a sequence that is at least 75% identical to SEQ ID NO: 18, or a sequence that is at least 21-26 nucleotides in length and contains the sequence of SEQ ID NO: 18;

44 wherein the one or more primers may further comprise one or more fluorophores or capture moieties located internally and/or at the end(s) of the primers; and wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples.

2. The set of LAMP primers of claim 1, wherein the set of primers comprise the primers having the sequences of:

F3: TGAATAAGCATATTGACGCATAC (SEQ ID NO:1);

B3: TGAGTTGAGTCAGCACTG (SEQ ID NO:2);

FIP: TAAGGCTTGAGTTTCATCAGCCCATTCCCACCAACAGAGC

(SEQ ID NO:3);

BIP : CGC AGAGAC AGAAGAAAC AGC ATTGTTGC AATTGTTTGGA

GAA (SEQ ID NO: 4);

LF: TTCTTCTTTTTGTCCTTTTTAG (SEQ ID NO:5); and

LB: AAACTGTGACTCTTCTTCCTGC (SEQ ID NO:6); wherein the primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primer.

3. The set of LAMP primers of claim 1 or claim 2, wherein the set of primers comprise the primers having the sequences of:

F3: TGAATAAGCATATTGACGCATAC (SEQ ID NO:1);

B3: TGAGTTGAGTCAGCACTG (SEQ ID NO:2);

FIP : 6FAM/TAAGGCTTGAGTTTC ATC AGCCC ATTCCCACCAACA

GAGC (SEQ ID NO:3), wherein 6FAM is 6-Carboxyfluorescein;

BIP: Biotin/CGCAGAGACAGAAGAAACAGCATTGTTGCAATTGTTT

GGAGAA (SEQ ID NO:4);

LF: TTCTTCTTTTTGTCCTTTTTAG (SEQ ID NO:5); and

LB: AAACTGTGACTCTTCTTCCTGC (SEQ ID NO: 6), wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples.

4. The set of LAMP primers of claim 1 or claim 2, wherein the set of primers comprise the primers having the sequences of:

F3: TGAATAAGCATATTGACGCATAC (SEQ ID NO:1);

45 B3: TGAGTTGAGTCAGCACTG (SEQ ID NO:2);

FIP: *-TAAGGCTTGAGTTTCATCAGCCCATTCCCACCAACAGAGC (SEQ ID N0:3), wherein * is a fluorescent dye;

BIP: Biotin/CGCAGAGACAGAAGAAACAGCATTGTTGCAATTGTTT

GGAGAA (SEQ ID NO:4);

LF: TTCTTCTTTTTGTCCTTTTTAG (SEQ ID NO:5); and

LB: AAACTGTGACTCTTCTTCCTGC (SEQ ID NO: 6), wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples.

5. The set of LAMP primers of claim 4, wherein * is a fluorescent dye selected from 6- Carboxyfluorescein (6FAM), TAMRA fluorophore (6TAMN), and cy5.

6. The set of LAMP primers of claim 1, wherein the set of primers comprise the primers having the sequences of:

F3: CTGCCACTAAAGCATACAATGT (SEQ ID NO:7);

B3: TTGATGGCACCTGTGTAGG (SEQ ID NO: 8);

FIP : GTGCAATTTGCGGCC AATGTTTGTTTTTC AAGGAAATTTTGGGG ACCAG (SEQ ID NO: 9);

BIP: CCAGCGCTTCAGCGTTCTTCTTTTTCAACCACGTTCCCGAAGG (SEQ ID NOTO);

LF: CAGTTCCTTGTCTGATTAGTTC (SEQ ID NO: 11); and/or

LB: AATGTCGCGCATTGGCATGG (SEQ ID NO: 12); wherein the one or more primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primers; and wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples.

7. The set of LAMP primers of claim 6, wherein the set of primers comprise the primers having the sequences of:

F3: CTGCCACTAAAGCATACAATGT (SEQ ID NO:7);

B3: TTGATGGCACCTGTGTAGG (SEQ ID NO: 8);

FIP : GTGCAATTTGCGGCC AATGTTTGTTTTTC AAGGAAATTTTGGGG

ACCAG (SEQ ID NO: 9); BIP: CCAGCGCTTCAGCGTTCTTCTTTTTCAACCACGTTCCCGAAGG

(SEQ ID NO: 10);

LF: CAGTTCCTTGTCTGATTAGTTC (SEQ ID NO: 11); and

LB: AATGTCGCGCATTGGCATGG (SEQ ID NO: 12); wherein the one or more primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primers; and wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples.

8. The set of LAMP primers of claim 6 or claim 7, wherein the set of primers comprise the primers having the sequences of:

F3: CTGCCACTAAAGCATACAATGT (SEQ ID NO:7);

B3: TTGATGGCACCTGTGTAGG (SEQ ID NO: 8);

FIP : F AM/GTGC AATTTGCGGCC AATGTTTGTTTTTC AAGGAAATTTT GGGGACCAG (SEQ ID NO:9);

BIP: Biotin/CCAGCGCTTCAGCGTTCTTCTTTTTCAACCACGTTCCCGA AGG (SEQ ID NOTO);

LF: CAGTTCCTTGTCTGATTAGTTC (SEQ ID NO: 11); and

LB: AATGTCGCGCATTGGCATGG (SEQ ID NO: 12).

9. The set of LAMP primers of claim 6 or claim 7, wherein the set of primers comprise the primers having the sequences of:

F3: CTGCCACTAAAGCATACAATGT (SEQ ID NO:7);

B3: TTGATGGCACCTGTGTAGG (SEQ ID NO: 8);

FIP: *-GTGCAATTTGCGGCCAATGTTTGTTTTTCAAGGAAATTTTG GGGACCAG (SEQ ID NO:9), wherein * is a fluorescent dye;

BIP: CCAGCGCTTCAGCGTTCTTCTTTTTCAACCACGTTCCCGAAGG (SEQ ID NOTO);

LF: CAGTTCCTTGTCTGATTAGTTC (SEQ ID NO: 11); and

LB: AATGTCGCGCATTGGCATGG (SEQ ID NO: 12).

10. The set of LAMP primers of claim 9, wherein * is a fluorescent dye selected from 6- Carboxyfluorescein (6FAM), TAMRA fluorophore (6TAMN), and cy5.

11. The set of LAMP primers of claim 1, wherein the set of primers comprise the primers having the sequences of:

F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13);

B3: TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14);

FIP : GCGGCCAATGTTTGTAATCAGTTCCTTTTAACCCAAGGAA

ATTTTGGGGAC (SEQ ID NO: 15);

BIP : GTCGCGC ATTGGC ATGGAAGTTTTTATGGC ACCTGTGTAG

GTCA (SEQ ID NO: 16);

LF: TTGTCTGATTAGTTCCTG (SEQ ID NO: 17); and/or

LB: CACACCTTCGGGAACGTGGT (SEQ ID NO: 18); wherein the one or more primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primers; and wherein the set of primers is used to detect SARS-CoV-2 nucleic acid in one or more samples.

12. The set of LAMP primers of claim 11, wherein the set of primers comprise the primers having the sequences of:

F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13);

B3: TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14);

FIP : GCGGCCAATGTTTGTAATCAGTTCCTTTTAACCCAAGGAA

ATTTTGGGGAC (SEQ ID NO: 15);

BIP : GTCGCGC ATTGGC ATGGAAGTTTTTATGGC ACCTGTGTAGG

TCA (SEQ ID NO: 16);

LF: TTGTCTGATTAGTTCCTG (SEQ ID NO: 17); and

LB: CACACCTTCGGGAACGTGGT (SEQ ID NO: 18); wherein the primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primer.

13. The set of LAMP primers of claim 11 or claim 12, wherein the set of primers comprise the primers having the sequences of:

F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13);

B3: TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14);

FIP: 6FAM/GCGGCCAATGTTTGTAATCAGTTCCTTTTAACCCAAGG

AAATTTTGGGGAC (SEQ ID NO: 15);

48 BIP: Biotin/GTCGCGCATTGGCATGGAAGTTTTTATGGCACCTGTGT

AGGTCA (SEQ ID NO: 16);

LF: TTGTCTGATTAGTTCCTG (SEQ ID NO: 17); and

LB: CACACCTTCGGGAACGTGGT (SEQ ID NO: 18); wherein the primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primer.

14. The set of LAMP primers of claim 11 or claim 12, wherein the set of primers comprise the primers having the sequences of:

F3: ACAAGCTTTCGGCAGACG (SEQ ID NO: 13);

B3: TTTGAAATTTGGATCTTTGTCA (SEQ ID NO: 14);

FIP: *-GCGGCCAATGTTTGTAATCAGTTCCTTTTAACCCAAGGAA ATTTTGGGGAC (SEQ ID NO: 15), wherein X is a fluorescent dye;

BIP : GTCGCGC ATTGGC ATGGAAGTTTTTATGGC ACCTGTGTAGG

TCA (SEQ ID NO: 16);

LF: TTGTCTGATTAGTTCCTG (SEQ ID NO: 17); and

LB: CACACCTTCGGGAACGTGGT (SEQ ID NO: 18); wherein the primers may further comprise one or more fluorophores located internally and/or at the end(s) of the primer.

15. The set of LAMP primers of claim 14, wherein * is a fluorescent dye selected from 6- Carboxyfluorescein (6FAM), TAMRA fluorophore (6TAMN), and cy5.

16. A LAMP assay for detecting SARS-CoV-2 in a sample, comprising: the set of LAMP primers of claim 1.

17. The LAMP assay of claim 16, the LAMP assay further comprising: a set of spike protein LAMP primers having the sequence of: spike-3F3: CTCTATTGCCATACCCACA (SEQ ID NO: 19); spike-3B3: CTTGTGCAAAAACTTCTTGG (SEQ ID NO:20); spike-3FIP: CATTCAGTTGAATCACCACAAATGTGTGTTACCA CAGAAATTCTACC (SEQ ID NO:21); spike-3BIP : GTTGC AATATGGC AGTTTTTGTAC ATTTGTCTTGT

TCAACAGCTAT (SEQ ID NO:22); and

49 spike-3LF: GTACAATCTACTGATGTCTTGGTCA (SEQ ID NO:23).

18. A LAMP assay for detecting SARS-CoV-2 in a sample, comprising: a combination of two sets of LAMP primers presented in claim 1.

19. A LAMP assay for detecting SARS-CoV-2 in a sample, comprising: the set of LAMP primers of claim 3.

20. A LAMP assay for detecting SARS-CoV-2 in a sample, comprising: the set of LAMP primers of claim 4 or claim 5; and a primer selected from ACTCCAGCCTTA/3BHQ 1/ or ACTCCAGCCTTA/3BHQ 2/, wherein 3BHQ 1 is Black Hole Quencher 1, and wherein 3HBQ 2 is Black Hole Quencher 2; wherein if the fluorescent dye is 6FAM then the primer is ACTCCAGCCTTA/3BHQ_1; and wherein if the fluorescent dye is 6FAM is 6TAMN or cy5, then the primer is ACTCCAGCCTTA/3BHQ 2.

21. A LAMP assay for detecting SARS-CoV-2 in a sample, comprising: the set of LAMP primers of claim 8.

22. A LAMP assay for detecting SARS-CoV-2 in a sample, comprising: the set of LAMP primers of claim 9 or claim 10; and a primer selected from ACTCCAGCCTTA/3BHQ 1/ or ACTCCAGCCTTA/3BHQ 2/, wherein 3BHQ 1 is Black Hole Quencher 1, and wherein 3HBQ 2 is Black Hole Quencher 2; wherein if the fluorescent dye is 6FAM then the primer is ACTCCAGCCTTA/3BHQ_1; and wherein if the fluorescent dye is 6FAM is 6TAMN or cy5, then the primer is ACTCCAGCCTTA/3BHQ 2.

23. A LAMP assay for detecting SARS-CoV-2 in a sample, comprising: the set of LAMP primers of claim 13.

24. A LAMP assay for detecting SARS-CoV-2 in a sample, comprising: the set of LAMP primers of claim 14 or claim 15; and

50 a primer selected from ACTCCAGCCTTA/3BHQ 1/ or ACTCCAGCCTTA/3BHQ 2/, wherein 3BHQ 1 is Black Hole Quencher 1, and wherein 3HBQ 2 is Black Hole Quencher 2; wherein if the fluorescent dye is 6FAM then the primer is ACTCCAGCCTTA/3BHQ_1; and wherein if the fluorescent dye is 6FAM is 6TAMN or cy5, then the primer is ACTCCAGCCTTA/3BHQ 2.

25. The LAMP assay of any one of claims 16 to 21, wherein the LAMP assay further comprises: a Bst DNA polymerase; a reverse transcriptase; isothermal amplification buffer; dNTP mix;

MgSCL;

L-Serine;

RNase inhibitor;

Carrier RNA; and

SYTO9.

26. A method to determine whether a sample comprises SARS-CoV-2 nucleic acids, comprising: carrying out the LAMP assay of any one of claims 16 to 25 on a sample at 60-65 °C for 10 - 30 minutes to generate amplification products; performing a trans-cleavage assay by incubating at an elevated temperature a second reaction mixture comprising the amplification products from the previous step, a programmable nuclease that has been complexed with gRNA specific to corresponding gene sequences of SARS-CoV-2, and a single-stranded detector nucleic acid comprising a detection moiety, wherein the detector nucleic acid is trans-cleaved by the programmable nuclease if SARS-CoV-2 gene amplification products are present in the second reaction mixture; and detecting whether SARS-CoV-2 is the sample based upon detecting trans-cleaved detector nucleic acid fragments.

27. The method of claim 26, wherein the sample is an environmental sample or a sample

51 from a subject.

28. The method of claim 27, wherein the sample is an oropharyngeal (OP) specimen, nasal mid-turbinate swab, anterior nares (nasal swab) specimen, nasopharyngeal wash/aspirate, oral swab, throat swab or saliva sample obtained from a human patient.

29. The method of any one of claims 26 to 28, wherein the LAMP reaction and the transcleavage assay are performed as a single-tube reaction.

30. The method of any one of claims 26 to 29, wherein the programmable nuclease is a Casl2 nuclease, a Casl3 nuclease, or a Casl4 nuclease.

31. The method of claim 30, wherein the programmable nuclease is a Cast 2a nuclease.

32. The method of claim 30 or claim 31, wherein the polypeptide sequence of the programmable nuclease has at least 85% sequence identity to SEQ ID NO:26, 27, 28 or 29.

33. The method of claim 32, wherein the polypeptide sequence of the programmable nuclease has at least 95% sequence identity to SEQ ID NO:29.

34. The method of any one of claims 26 to 33, wherein the gRNA is specific to the N- gene, E-gene, or human Rnase P gene of SARS-CoV-2.

35. A method to determine whether a sample comprises SARS-CoV-2 nucleic acids, comprising: carrying out the LAMP assay of any one of claims 19, 21 and 23 on a sample at 60-65 °C for 10 - 30 minutes to generate amplification products; and detecting whether SARS-CoV-2 is the sample by using a lateral flow assay strip, wherein if amplification products are produced in the LAMP assay, then the presence of the amplification products can be determine by a band being present at biotin binding test line on the lateral flow assay strip, and wherein if amplification products are not produced, then there is no band present at biotin binding test line of the lateral flow assay strip.

36. The method of claim 35, wherein the sample is an environmental sample or a sample

52 from a subject.

37. The method of claim 36, wherein the sample is an oropharyngeal (OP) specimen, nasal mid-turbinate swab, anterior nares (nasal swab) specimen, nasopharyngeal wash/aspirate, oral swab, throat swab or saliva sample obtained from a human patient.

38. The method of any one of claims 35 to 37, wherein the method is carried out at a laboratory, at a hospital, at a physician office/laboratory (POLs), at a clinic, at a remote site, or at home.

39. The method of claim 38, wherein the method is carried out at home.

40. A method to determine whether a sample comprises SARS-CoV-2 nucleic acids, comprising: carrying out the LAMP assay of any one of claims 20, 22 and 24 on a sample at 60-65 °C for 10 - 30 minutes to generate amplification products and then cooling the LAMP assay to room temperature; and measuring the sample for fluorescence using a fluorescent detection device, wherein if the sample fluoresces more than background, indicates that the sample comprises SARS-CoV-2 nucleic acids.

41. The method of claim 40, wherein the detection device is a plate reader or spectrophotometer.

42. The method of claim 41, wherein the method is carried out using automation equipment comprising robotic handlers.

43. The method of claim 41 or claim 42, wherein more than 90 samples can be run at a time using said method.

44. A kit comprising any of the LAMP primer sets of claim 1.

45. The kit of claim 44, further comprising a programmable nuclease that has been complexed with gRNA specific to corresponding gene sequences of SARS-CoV-2, and a

53 single-stranded detector nucleic acid comprising a detection moiety.

54