WO2021231706A1

WO2021231706A1 - Methods and compositions for rapid testing for a target nucleic acid including viral nucleic acid

Info

Publication number: WO2021231706A1
Application number: PCT/US2021/032218
Authority: WO
Inventors: Samuel Zev Williams; Shan WEI
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2020-05-13
Filing date: 2021-05-13
Publication date: 2021-11-18

Abstract

Disclosed herein are compositions and methods for the detection of at least one bacteria or virus using a one-step assay. One virus that can be detected using the disclosed compositions and methods is SARS-CoV-2. In particular, the disclosure relates to a method of detection of the bacteria and viruses using specific primers and reagents and methods designed to detect bacteria and viruses using high-performance loop-mediated isothermal amplification.

Description

METHODS AND COMPOSITIONS FOR RAPID TESTING FOR A TARGET NUCLEIC ACID INCLUDING VIRAL NUCLEIC ACID

CROSS-REFERENCE TO OTHER APPLICATIONS

The present application claims priority to U.S. Patent Application Serial Nos. 63/024,388 filed May 13, 2020; 63/038,763 filed June 13, 2020; 63/079,922 filed September 17, 2020; and 63/086,552 filed October 1, 2020, all of which are hereby incorporated by reference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

The present invention was made with government support under grants HD086327 and HD100013 awarded by the National Institutes of Health. The government has certain rights in the present invention.

FIELD

This disclosure relates to compositions and methods for the detection of at least one pathogen, e.g., bacteria or virus, using a one-step assay. The viruses to be detected include at least SARS-CoV-2. In particular, the disclosure relates to a method of detection of pathogens, e.g., bacteria or viruses, using specific primers and reagents in a high-performance loop- mediated isothermal amplification method.

BACKGROUND

The Coronavirus disease 2019 (COVID-19) pandemic caused by SARS-coronavirus 2 (SARS-CoV-2) has created a global health emergency.

Widespread molecular diagnostic testing for the virus is crucial for prompt diagnosis and quarantine, treatment, and for a managed response to the ongoing SARS-Cov-2 pandemic, especially given the apparently high rate of asymptomatic viral shedding (He et al. 2020).

The primary method for testing for active disease has been various forms of nucleic acid amplification tests (NAAT), including quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). However, the majority of these assays require multiple steps including extended sample pretreatments, and/or RNA extraction and subsequent amplification and detection of specific regions of viral RNA, which can only be performed by shipping clinical samples to centralized high-complexity laboratories to perform the testing in batches (Broughton et al. 2020; Nagura-Ikeda et al. 2020; Joung et al. 2020; Lamb et al. 20202; Yan et al. 2020)

The FDA has recently approved several simple and rapid molecular diagnostic tests for SARS-CoV-2, such as Abbott ID NOW and Cepheid’s Xpert Xpress, which can be performed at the point-of-care (POC). However, these systems rely on specialized, proprietary instruments and consumables which have contributed to the limited capacity to scale testing for widespread testing both in the United States and globally (Cheng et al. 2020).

Thus, there is a critical need for a SARS-CoV-2 diagnostic test that can be performed at the point-of-care with prompt delivery of results and without the need for costly and scarce proprietary equipment and reagents.

SUMMARY

Disclosed herein is a method and assay for detection of pathogens, such as bacteria and viruses, based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), but with significant modifications made to enable detection of single-copy levels of virus in less than 30 minutes directly from clinical samples including but not limited to viral transport media and saliva, using only a single fluid transfer step and readily available reagents and equipment such as a simple heat block with a simple colorimetric readout that can be interpreted with the unaided eye. The new method and assay are designated high-performance loop-mediated isothermal amplification (HP-LAMP). While the focus herein was the detection of SARS-CoV-2, the current methods can be used to detect other pathogens including bacteria and viruses.

The current disclosure provides compositions, methods, and kits for detecting the presence of nucleic acids of certain bacteria and viruses including SARS-CoV-2. The disclosed method and assay are rapid, inexpensive, sensitive, and specific. In certain embodiments, the current disclosure allows the detection and the determination of which specific bacteria or virus or viruses are found in a single sample.

In certain aspects, the disclosure provides for methods of detecting a nucleic acid of SARS-CoV-2 virus in a one-step assay, i.e., a single sample. In some embodiments, the methods comprise amplifying the nucleic acid of the viruses using at least one LAMP primer, wherein the at least one LAMP primer comprises the nucleotide sequence of SEQ ID NOs: 1- 6. In some embodiments, the methods comprise amplifying a nucleic acid of SARS-CoV-2 virus with at least one LAMP primer, under conditions to allow for initiation of amplification of at least part of the nucleotide sequence from the oligonucleotide, and detecting the amplification products, i.e., amplified nucleic acid, thereby detecting the virus, wherein the at least one LAMP primer comprises the nucleotide sequence of SEQ ID NOs: 1-6. In some embodiments, all of the LAMP primers comprising SEQ ID NOs: 1-6 are used in the method.

In some embodiments, the method further comprises contacting the sample with a thermostable DNA polymerase to amplify the reaction.

In some embodiments, the method further comprises contacting the sample with reverse transcriptase.

In some embodiments, the method further comprises contacting the sample with additional reagents including but not limited to human genomic DNA, bacteriophage lambda DNA, DNase, RNase inhibitor, dUTP/UDG, and combinations thereof.

In some embodiments, the presence of the amplification products is detected using colorimetry, fluorescence, turbidity or precipitation.

In some embodiments, the method is performed in a volume of about 500 pL.

In some embodiments, the method is performed at a temperature of about 63°C.

In some embodiments, the method is performed for a time of about 30 minutes.

In some embodiments, the method is performed in a high throughput platform. In this embodiment, plates with multiple wells are used, and the method is performed in a volume of about 20 pL and for a time of about 20 minutes.

In some aspects, the current disclosure provides for methods of detecting a nucleic acid of bacteria or viruses in a one-step assay, i.e., a single sample. In some embodiments, the methods comprise amplifying the nucleic acid of the bacteria or viruses using at least one LAMP primer under conditions to allow for initiation of amplification of at least part of the nucleotide sequence from the oligonucleotide, and detecting the amplification products, i.e., amplified nucleic acid, thereby detecting the bacteria or virus in the sample. In some embodiments, the method further comprises contacting the sample with a thermostable DNA polymerase to amplify the reaction.

In some embodiments, the presence of the amplification products is detected using colorimetry, fluorescence, turbidity or precipitation. In some embodiments, the method is performed in a volume of about 500 μL.

In some embodiments, the method is performed at a temperature of about 63°C.

In some embodiments, the method is performed for a time of about 30 minutes.

In some embodiments, the method is performed in a high throughput platform. In this embodiment, plates with multiple wells are used, and the method is performed in a volume of about 20 μL and for a time of about 20 minutes.

In addition to the foregoing methods, the present disclosure further provides nucleic acid primers for detecting a nucleic acid of the SARS-CoV-2 virus. In some embodiments, the set of LAMP primers specifically amplifies a SARS-CoV-2 nucleic acid. An exemplary set of LAMP primers for amplification of a SARS-CoV-2 nucleic acid includes an F3 primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 1, a B3 primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 2, an FIP primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 3, a BIP primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 4, a LF primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 5, and a LB primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 6, or the reverse complement of any of SEQ ID NOs: 1-6. In one example, the set of LAMP primers for SARS-CoV-2 nucleic acid amplification includes primers comprising, consisting essentially of, or consisting of the nucleic acid sequence each of SEQ ID NOs: 1-6.

In other aspects, the disclosure provides a kit for the detection of a nucleic acid of bacteria or virus. In certain embodiments the kit includes one or more LAMP primers.

In other aspects, the disclosure provides a kit for the detection of a nucleic acid of the SARS-CoV-2 virus and/or the detection of the SARS-CoV-2 virus. In certain embodiments the kit includes one or more LAMP primers chosen from the group consisting of SEQ ID NOs: 1- 6.

In some embodiments, the kits further include additional reagents for performing the method chosen from the group consisting of: nucleic acid polymerase; reverse transcriptase; genomic DNA; lambda phage DNA; DNase; RNase inhibitor; dUTP/UDG; ssRNA; reagents for detection of the amplification products; and combinations thereof.

In yet further embodiments, the reagents for the detection of the amplification products allow colorimetric detection and include but are not limited to phenol red, neutral red, cross red, Cresol red, and m-Cresol purple. In some embodiments, the reagents for the detection of the amplification products allow fluorescent detection and include but are not limited to propidium iodide, Picogreen, SYBR green, and Syto 9.

In some embodiments, the kits further include additional equipment for performing the method chosen from the group consisting of: 1.5mL LoBind microcentrifuge tubes; ice and containers to hold ice; a 63.0°C dry bath or heat block; 95°C dry bath or heat block; a mini centrifuge; a mini vortex mixer; and combinations thereof.

In embodiments, where the method is being used in a high throughput platform, the kits can include the LAMP primers and additional reagents contained in wells of a plate used for high throughput platforms.

In certain embodiments, the kits additionally comprise instructions for detecting a nucleic acid of of bacteria or virus, including SARS-CoV-2 virus, according to the methods and using the compositions disclosed herein. In certain embodiments, the kits include controls including but not limited to positive controls for the viruses including SARS-CoV-2, and human nucleic acid, and negative controls.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, certain embodiments of the invention are depicted in drawings. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 - Primer design. A set of 6 LAMP primers targeting the middle of the Orflab gene. Sequences and primers matching to the + strand of virus genome are shown in pink, while those matching to the - strand are shown in blue. Illustration of GC % of SARS-CoV-2 genome was from UCSC genome browser (Kent et al. 2002; Fernandez et al. 2020)

Figure 2 - HP- LAMP assay for rapid and direct SARS-CoV-2 testing of clinical sample. Figure 2A is an illustrative schematic of the workflow of HP- LAMP assay. 20 μL of samples can be added to 480 μL reaction solution consisting of reaction master mix and lysis buffer, mixed and 250 μL are aliquoted with a transfer pipette and then incubated on heat block for 30 minutes. The reaction is stopped by placing the samples on ice and results can be interpreted by color due to a pH sensitive dye in the master mix (yellow = positive; red = negative). Figure 2B is an illustrative schematic of the workflow of HP-LAMP assay using saliva. Figure 2C shows an example of colorimetric results on negative and positive clinical samples.

Figure 3 - The performance of HP-LAMP testing for SARS-CoV-2 using VTM. Figure 3 A is a graph of the estimation of limit of detection using clinical samples selected to represent a wide range of Ct values. Each dot represents the Ct value of target 2 of one sample, and a sample with discordant testing result shown as a filled in dot. Error bar indicates Mean ± SD. Figure 3B is a graph of the results of testing on randomly selected positive and negative clinical samples. Each dot represents the Ct value of target N2 in Roche cobas SARS-CoV-2 Test of one sample, and a sample with discordant testing result is shown as a filled in dot. Error bars indicates mean ± SD.

Figure 4 is a schematic of the two-fold serial dilution to determine the LoD of the HP- LAMP assay.

Figure 5 - The performance of HP- LAMP testing for SARS-Cov-2 using saliva. Figure 5A shows the results of determining the limit of detection (LoD) of the HP-LAMP assay. The concentrations indicated show copies of heat-inactivated SARS-CoV-2 per mΐ of saliva. NC = negative control with no SARS-CoV-2 added. ‘ + ’ = Positive HP-LAMP result; = Negative HP-LAMP result. The color of each box is taken directly from its corresponding reaction tube. Figure 5B shows the results of the cross-reactivity of HP-LAMP assay on common pathogens. Inactivated known respiratory pathogens (n = 21) along with inactivated SARS-CoV-2 virus were tested using HP-LAMP assay. All pathogens showed negative detection results in HP- LAMP assay, expect for SARS-CoV-2 virus. Figure 5C shows representative results of HP- LAMP testing on clinical samples.

Figure 6 - The performance of HP-LAMP testing for SARS-Cov-2 using pooled samples of saliva. Figure 6 is an illustration of the HP-LAMP assay for sample pooling. A negative matrix was created by pooling 4 known negative saliva samples. A positive or negative pool of 5 samples (N = 5) was created by pooling a known positive or negative sample with the negative matrix. 20 positive (n = 20) and 20 negative (n = 20) pools were subjected to HP-LAMP assay.

DETAILED DESCRIPTION

Abbreviations

LAMP- Loop-mediated isothermal amplification

RT-LAMP- Reverse transcriptase loop-mediated isothermal amplification

HP-LAMP- High performance loop-mediated isothermal amplification

VTM- viral transport medium or media

LoD- limit of detection A Field-Deployable Rapid Testing Method for Pathogens including Viruses including SARS- CoV-2

To safely re-open economies and prevent future outbreaks, rapid, frequent, point-of- need, SARS-CoV-2 diagnostic testing is necessary. However, existing field-deployable COVID-19 testing methods require the use of uncomfortable swabs and trained providers in PPE, while saliva-based methods must be transported to high complexity laboratories for testing.

Disclosed herein is a method and assay for detection of bacteria and viruses, including SARS-CoV-2, based on reverse transcriptase loop-mediated isothermal amplification (RT- LAMP), but with significant modifications made to enable detection of single-copy levels of virus in less than 30 minutes directly from clinical samples including but not limited to viral transport media and saliva, using only a single fluid transfer step and readily available reagents and equipment such as a simple heat block with a simple colorimetric readout that can be interpreted with the unaided eye. The new method and assay are designated high-performance loop-mediated isothermal amplification (HP-LAMP). Illustrative workflows are shown in Figures 2A and 2B. While the focus herein was the detection of SARS-CoV-2, the current methods can be used to detect other pathogens including bacteria and viruses.

The disclosed method was used for the direct detection of SARS-CoV- 2 in viral transport media in 30 minutes with a LoD as low as 2.5 copies/mΐ (see, e.g., Example 2).

The disclosed method was also used for the direct detection of SARS-CoV-2 in saliva with a limit of detection of 1.4 copies of virus per mΐ of saliva and a sensitivity and specificity of greater than 96%, on par with traditional RT-PCR based methods using swabs. The disclosed method can deliver results using only a single fluid transfer step and simple heat block. Additionally, shown herein was the 100% accuracy of testing of 120 patient samples in 40 pools comprised of 5 patient samples each with either all negative or a single positive patient sample. See, e.g., Examples 3 and 4.

The disclosed method can also be used in a high-throughput platform with minor modifications. See, e.g., Example 5.

Loop-mediated isothermal amplification (LAMP) is a targeted nucleic acid amplification method that utilizes a combination of primer sets and a DNA polymerase with high strand displacement activity to specifically replicate a region of DNA (Notomi et al. 2000). At least four primers, which are specific for six regions within a target nucleic acid sequence, are typically used for LAMP. The primers include a forward outer primer (F3), a backward outer primer (B3), a forward inner primer (FIP), and a backward inner primer (BIP). A forward loop primer (LF), and a backward loop primer (LB) can also be included in some embodiments. The amplification reaction produces a stem-loop DNA with inverted repeats of the target nucleic acid sequence. Reverse transcriptase can be added to the reaction for amplification of RNA target sequences. This variation is referred to as RT-LAMP.

Compared with traditional PCR, LAMP has several distinct advantages for point-of- care testing of clinical samples for viruses, including SARS-CoV-2. While traditional PCR requires a costly and complex thermocycler, the entire LAMP amplification reaction is performed at a single temperature, and thus requires only a heat block or water bath. The polymerase used in LAMP (e.g., Bacillus stearothermophilus or Bst) is more robust than that used in traditional PCR and can therefore function in the presence of PCR inhibitors frequently found in bodily fluids such as saliva and viral transport media, without the need to first purify the RNA. Because there is no need for thermocycling, DNA amplification is faster than with PCR and the product can be visualized with the unaided eye in real time using colorimetry, precipitation, turbidity, or fluorescence. The addition of a reverse transcription (RT) step (RT- LAMP) that can occur in the same master mix at the start of the reaction allows for detection of RNA targets, such as RNA viruses such SARS-CoV-2. Finally, with a protocol that is compatible with a wide range of global suppliers for reagents, and does not require specialized equipment, the method can be rapidly adopted and scaled and is thus useful for detection of other pathogens, including bacteria and viruses, known and those not yet discovered.

While RT-LAMP has been used for SARS-CoV-2 detection by several groups (Zhang et al. 2020; Lamb et al. 2020; Yu et al 2020), these methods require a prior extraction step or lengthy sample treatment (which makes it difficult to deploy in the field), multiple fluid transfer steps, or lack the accuracy and limit of detection necessary for clinical implementation and are therefore not suitable for clinical testing outside of a laboratory. A unique feature of the disclosed method is that it does not require RNA isolation and/or cell lysis and could be applied directly to clinical samples.

The ability to test at the point-of-care and return results within 30 minutes without the need for RNA extraction/purification or specialized equipment has practical advantages for onsite screening and detection of those with a higher viral load. In particular, the F1P-LAMP test may be useful as a primary screening to provide a quick diagnostic for patients at the early stage of spreading and without significant symptoms, when the patients are normally reported to have 10⁴ to 10⁷ copies/mL virus load (Wylie et al. 2020). Thus, this method would also lend itself to widespread testing and testing in resource-poor settings. The equipment costs for performing the HP-LAMP assay are very low, performing the assay requires only a pipette, a mini centrifuge, a vortexer, and two heat blocks that retail for about 250 USD each. In contrast, the equipment costs for RT-PCR based methods is greater than 45,000 USD while the automated Roche cobas 6800 unit costs about 350,000 USD.

If purchasing the consumable reagents individually using off-the-shelf components, the costs for the HP-LAMP assay is about 80 USD per assay, and about 16 USD per assay when pooling five samples. The cost for pre-made HP-LAMP cocktails is 20-25 USD per assay (Sorrento Therapeutics). The consumable cost for RT-PCR based methods is 20-60 USD per assay.

In summary, HP-LAMP enables rapid detection of viruses, including SARS-CoV-2, directly from saliva without the need for a laboratory, using a simple, one-step protocol. HP- LAMP has an LoD of less than 2 viral copies per mΐ of saliva, and a positive and negative percentage agreement of greater than 96% and greater than 97%, respectively, comparable to the gold-standard RT-PCR based methods that must be run in a high-complexity laboratory. The simple workflow may also allow adaptation for at-home testing and pooling strategies. Thus, HP-LAMP can enable rapid and accurate results in the field using saliva, without need of a high-complexity laboratory.

In order to use HP-LAMP to specifically detect a target nucleic acid, a number of non- obvious, and in many cases surprising, changes had to be made to the existing protocol.

Primer design: RT-LAMP utilizes a set of primers (typically 4-6 primers) that are complimentary to a specific region of the target RNA or DNA. Typically, primers are designed to target GC-rich regions of the viral RNA because GC-rich regions bind more tightly to primers. Consequently, as an example, the standard approach to primer design for HP-LAMP- based detection of SARS-CoV-2 has been to design primer sets targeting the GC-rich regions of the virus. These regions are found towards the ends of the viral RNA. However, it was reasoned that if there were degradation of the viral RNA, the ends of the virus would most likely be lost first, and that would render the primers ineffective. But, if there is degradation of the virus from the ends of the viral RNA, the central region would be more likely to remain intact. Consequently, the target was the opposite direction of the standard approach and the primers were designed to target the central portion of the virus. However, in the case of SARS- CoV-2, the central region is GC-poor (AT-rich). Thus, the design of primers for this region is different from how primers are typically designed; the standard parameters for primer design would not work here. Instead, when designing these primers, the parameters were changed such that a larger-than-usual primer-mediated-loop- structures would be permitted. Because of this, extra steps were taken to ensure that the primer itself did not form stable secondary structures or self-dimerize. After using this approach to design 8 sets of primers (6 primers in each set), these resulting eight sets were tested (along with previously published primer sets) to determine which primer set was the most sensitive and specific. See, e.g., Example 1. This same theory- designing of primers to target the central portion of the target nucleic acid- would be used for the design of primers for use in the disclosed method to detect other nucleic acids, including viral nucleic acids.

The final primer set used are shown below in Table 1.

Even with the improved primer sets, the RT-LAMP reaction was still not sufficiently sensitive to detect fewer than 200 viral copies/mΐ in saliva, which is far higher than the less than 2 viral copies/mΐ limit considered necessary for testing clinical samples. In order to achieve the necessary greater than 100-fold improvement in sensitivity while maintaining a 100% specificity, the RT-LAMP reaction conditions were systemically modified to improve performance. It was found that sensitivity and specificity of the assay could be markedly improved by adding carrier DNA, carrier RNA, and RNase inhibitors, as well as by increasing the reaction volume.

Volume: Many enzymatic reactions, such as DNA ligation, perform better in small reaction volumes. Much of the standard approach to HP-LAMP has been to do the testing in the smallest possible volume (e.g., 25 μL). However, while the small-volume approach works with high sensitivity when testing isolated viral RNA, the experiments showed that it had a poor sensitivity in detecting viral RNA target directly on clinical samples in saliva or transport media. Instead, the volume of the reaction was increased 10- 20 x fold (e.g., 250-500 μL) in order to improve the sensitivity for clinical samples. This increased the total number of viral copies present in the reaction master mix and was necessary in order for us to be able to have sufficient sensitivity to detect viral RNA directly in clinical samples.

Lysis buffer: Typically, lysis buffer needs pH buffering capacity, enzymes (protease K, DNAse), and detergent. Several changes were made to the lysis buffer in order get the assay to work and to be adequately sensitive and specific:

The RT-LAMP assay usually uses a proteinase enzyme in the reaction buffer to degrade proteins and improve sensitivity of the reaction. However, using proteinase enzymes would require the additional step of a heat inactivation or adding a stop solution. This additional step: (a) makes the test difficult to perform at the point of care; and (b) increases the time, cost and complexity of performing the assay. The new buffer that did not contain the proteinase, surprisingly actually performed better. It should be noted that there was no showing of a significant benefit of adding thermolabile proteinase in the buffer when used in a method testing saliva samples and thus, when used in a method with saliva samples, the buffer should not contain proteinase. However, the buffer can contain thermolabile proteinase when being used in a method of testing other samples, e.g., VTM.

The RT-LAMP assay usually uses a DNAse treatment to degrade any DNA present at the start of the reaction. However, the DNAse typically used requires a step of a heat inactivation or adding a stop solution. This adds an extra step which: (a) makes the test difficult to perform at the point of care; and (b) increases the time, cost and complexity of performing. Instead DNAse was used that is inactivated at lower temperatures (55°C) and hence is naturally inactivated in the course of performing the reaction (which is performed at 63°C).

For testing of saliva, people are typically using much harsher lysis conditions. This approach was tested and it resulted in no improvement. dUTP/UDG: Based on experiments, it was found that because the assay is so sensitive, there was a risk that carry-over amplification product from prior samples could contaminate the new sample and lead to false -positive results (i.e., to reduce the specificity). To solve this, dUTP was added in the reaction mix which gets incorporated into the LAMP product. In the master mix, Antarctic Thermolabile uracil-DNA N-glycosylase (UDG) is added which degrades any UTP-containing product from prior reactions but is itself inactivated at temperatures above 50°C.

Genomic DNA (gDNA) spike-in: In the process of developing of the assay, a buffer with genomic DNA spiked in as a negative control was used so that there were no observed false positive results due to non-specific binding of the primers to genomic DNA. Surprisingly, not only did was there no observation of an increase in false positive results, but there was a marked decrease in false positive results. Thus, it was discovered that genomic DNA spike-in helped improve the specificity of the method.

Direct from mouth: Most methods for testing saliva have saliva samples placed into a collection container, often with a special buffer. In addition to adding cost and complexity, it also adds a step that can make point-of-care testing (and chain-of-custody) issues more complicated. In the disclosed method, there is the option of either having samples placed into a container (which does not require any buffer to be present) or, alternatively, having the saliva (1-20 mΐ) removed directly from the patient’s mouth using the disposable transfer pipette and then placed directly into the tube containing the master mix. Dilute in HBSS buffer: In some rare cases, the saliva may be very viscous, particularly if someone is dehydrated or in patients who are symptomatic from a respiratory virus, such as patients symptomatic with COVID-19. A simple buffer, such as HBSS, can be added to the saliva and the diluted saliva can be processed using the disclosed method.

Tube selection: For samples with a low amount of RNA, low retention tubes are commonly used (“regular tubes”). These “regular tubes” were tried in the disclosed method, but surprisingly, gave a high rate of false-positive results. Better results were obtained when using DNA LoBind tubes instead of the regular low retention tubes.

The present methods can be used in a high-throughput platform resulting in an even more rapid reaction time. A few modifications are made to the method for use in a high- throughput platform.

In some embodiments, the high-throughput platform is plate based. In some embodiments, a 96-well plate is used. In some embodiments, a 384-well plated is used.

Extraction: When the method is performed in a high throughput manner, RNA is extracted from the sample, using any method known in the art.

Volume: The volume of the reaction cocktail is reduced from about 500 mΐ to about 20 mΐ and the per patient volume is reduced to about <80 mΐ. It is noted that while the increased volume did have an effect on the sensitivity of the assay in a non-high throughput platform, for the high throughput platform, the reduced volume was sufficiently specific and sensitive.

Other Differences Include: Use of an internal extraction control (e.g., primers specific for RNase P or actin), bacteriophage lambda DNA replacing genomic DNA), and no use of an RNase inhibitor. Additionally, a plate reader can be used to detect the amplification products by fluorescence.

Synthetic Primers for Detection of SARS-CoV-2 Using LAMP

The current disclosure provides for isolated nucleic acid sequences such as primers from specific portions of the particular viral genomes. As discussed, these specific primers were designed in the center of the viral genome considering degradation as well as the possible cross-reactivity based upon sequence alignments and assay sensitivity. Thus, the primers disclosed herein are particularly useful in that they can be used in the HP-LAMP method disclosed herein or in other LAMP methods or other single reaction methods to detect virus, specifically SARS-CoV-2.

Additionally, the primers are non-naturally occurring compositions. SARS-CoV-2 are enveloped, single-stranded RNA viruses. As such, the primers of the current disclosure comprise cDNA that do not occur in nature and the nucleic acid sequences of the current disclosure are markedly different in structure from naturally occurring viral RNA sequences.

In one aspect, the disclosure provides for at least one primer that is useful in detecting the presence of a nucleic acid of SARS-CoV-2 and/or the SARS-CoV-2 virus itself. In certain embodiments, the primers target the center of the SARS-CoV-2 genome. In certain embodiments, primers target the ORFlab gene of SARS-CoV-2. In certain embodiments, the primers are used in a LAMP method or assay or an HP-LAMP method or assay. In certain embodiments, the primers are selected from the primers comprising the sequences of SEQ ID NOs: 1-6.

The nucleic acid primers disclosed herein can be prepared by any method known to one of skill in the art without limitation.

LAMP primers include oligonucleotides between 15 and 60 nucleotides in length. In some embodiments, the set of LAMP primers specifically amplifies a SARS-CoV-2 nucleic acid. An exemplary set of LAMP primers for amplification of a SARS-CoV-2 nucleic acid includes an F3 primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 1, a B3 primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 2, an FIP primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 3, a BIP primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 4, a LF primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 5, and a LB primer comprising a nucleic acid with at least 90% sequence identity to SEQ ID NO: 6, or the reverse complement of any of SEQ ID NOs: 1-6. In one example, the set of LAMP primers for SARS-CoV-2 nucleic acid amplification includes primers comprising, consisting essentially of, or consisting of the nucleic acid sequence each of SEQ ID NOs: 1-6.

Also provided by the present disclosure are primers that include variations to the nucleotide sequences shown in any of SEQ ID NOs: 1-6, as long as such variations permit detection of the target nucleic acid molecule. For example, a primer can have at least 90% sequence identity such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleic acid including the sequence shown in any of SEQ ID NOs: 1-6. In such examples, the number of nucleotides does not change, but the nucleic acid sequence shown in any of SEQ ID NOs: 1-6 can vary at a few nucleotides, such as changes at 1, 2, 3, 4, 5, or 6 nucleotides.

The present disclosure also provides primers that are slightly longer or shorter than the nucleotide sequences shown in any of SEQ ID NOs: 1-6, as long as such deletions or additions permit amplification and/or detection of the desired target nucleic acid molecule. For example, a primer can include a few nucleotide deletions or additions at the 5'- or 3'-end of the primers shown in any of SEQ ID NOs: 1-6, such as addition or deletion of 1, 2, 3, 4, 5, or 6 nucleotides from the 5'- or 3'-end, or combinations thereof (such as a deletion from one end and an addition to the other end). In such examples, the number of nucleotides changes.

Also provided are primers that are degenerate at one or more positions (such as 1, 2, 3, 4, 5, or more positions), for example, a primer that includes a mixture of nucleotides (such as 2, 3, or 4 nucleotides) at a specified position in the primer. In other examples, the primers disclosed herein include one or more synthetic bases or alternative bases (such as inosine). In other examples, the primers disclosed herein include one or more modified nucleotides or nucleic acid analogues, such as one or more locked nucleic acids (see, e.g., U.S. Pat. No. 6,794,499) or one or more superbases (Nanogen, Inc., Bothell, Wash.). In other examples, the primers disclosed herein include a minor groove binder conjugated to the 5' or 3' end of the oligonucleotide (see, e.g., U.S. Pat. No. 6,486,308).

Methods and Systems Utilizing HP-LAMP to Detect Pathogens including Viruses including SARS-CoV-2

The methods and systems of the present disclosure may be used to detect nucleic acids from pathogens including bacteria and viruses as well as the bacteria and viruses themselves, in research and clinical settings, from any sample. Viruses that can be detected using the disclosed methods include but are not limited to SARS-CoV-2.

A preferred sample is a biological sample. A biological sample may be obtained from a tissue of a subject or bodily fluid from a subject including but not limited to nasopharyngeal aspirate, oropharyngeal aspirate, blood, cerebrospinal fluid, saliva, serum, plasma, urine, sputum, bronchial lavage, pericardial fluid, or peritoneal fluid, or a solid such as feces. Preferred samples include but are not limited to saliva.

The subject may be any animal, particularly a vertebrate and more particularly a mammal, including, without limitation, a cow, dog, human, monkey, mouse, pig, or rat. In one embodiment, the subject is a human.

A sample may also be a research, clinical, or environmental sample. One such sample is viral transport media. Another such sample is waste water.

Samples also include isolated nucleic acids, such as DNA or RNA isolated from a tissue or bodily fluid from a subject or other source of nucleic acids. Methods for extracting nucleic acids such as RNA and/or DNA from a sample are known to one of skill in the art; such methods will depend upon, for example, the type of sample in which the nucleic acid is found. Nucleic acids can be extracted using standard methods. For instance, rapid nucleic acid preparation can be performed using a commercially available kit (such as kits and/or instruments from Qiagen, Roche Applied Science, Thermo Scientific, bioMerieux, or Epicentre. In other examples, the nucleic acids may be extracted using guanidinium isothiocyanate, such as single-step isolation by acid guanidinium isothiocyanate-phenol- chloroform extraction.

Additional applications include, without limitation, detection of the screening of blood products (e.g., screening blood products for infectious agents), biodefense, food safety, environmental contamination, forensics, and genetic-comparability studies. The present disclosure also provides methods and systems for detecting viral nucleic acids in cells, cell culture, cell culture medium and other compositions used for the development of pharmaceutical and therapeutic agents.

When the sample is saliva, the method can include a step of heat inactivation. This step can be performed from about 1 minute to about 10 minutes, or from about 2 minutes to about 8 minutes, or from about 3 minutes to about 7 minutes, or from about 4 minutes to about 6 minutes, or for about 5 minutes.

In some embodiments, the temperature of the heat inactivation is performed from about 90°C to about 100°C, or from about 95°C to about 98°C, or at about 95°C.

The disclosed methods are highly sensitive and/or specific for detection of bacterial and viral nucleic acids, including SARS-CoV-2. In some examples, the disclosed methods can detect presence of at least 10 copies of nucleic acids, including SARS-CoV-2 nucleic acids, in a sample or a particular reaction volume (such as per pi reaction). In particular, non-limiting examples, the disclosed methods have a limit of detection of about 1.3 copies per mΐ, or about 1.4 copies per mΐ, or about 1.5 copies per mΐ, or about 1.6 copies per mΐ, or about 1.7 copies per mΐ, or about 1.8 copies per mΐ, or about 1.9 copies per mΐ, or about 2.0 copies per mΐ, or about 2.1 copies per mΐ, or about 2.2 copies per mΐ, or about 2.3 copies per mΐ, or about 2.4 copies per mΐ, or about 2.5 copies per mΐ, or about 3.0 copies per mΐ, or about 3.5 copies per mΐ, or about 4.0 copies per mΐ, or about 4.5 copies per mΐ, about 5.0 copies per mΐ, or about 6.0 copies per mΐ, or about 7.0 copies per mΐ, or about 8.0 copies per mΐ, or about 9.0 copies per mΐ, or about 10 copies per mΐ, of DNA or NRA including SARS-CoV-2 RNA. However, one of ordinary skill in the art will recognize that the limit of detection of an assay depends on many factors (such as reaction conditions, amounts and quality of starting material, and so on) and the limit of detection using particular LAMP primer sets, such as those disclosed herein, may be even less with modifications to the assay conditions. In some examples, the disclosed methods can predict with a sensitivity of at least 90% and a specificity of at least 90% for presence of nucleic acid, including SARS-CoV-2 nucleic acid, such as a sensitivity of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% and a specificity of at least of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%.

The present disclosure provides a method for detecting nucleic acid from a bacteria or virus such as SARS-CoV-2, or of detection of the bacteria or virus itself, in any sample, including the steps of: contacting the sample with set of LAMP primers; subjecting the nucleic acid and primer to amplification conditions; and detecting the presence of amplification product, wherein the presence of the amplification products indicates the presence of nucleic acid of the bacteria or virus and the bacteria or virus in the sample.

In some embodiments, the method includes the use of at least one set of HP-LAMP primers. In further embodiments, the HP-LAMP primers are designed as disclosed herein using the central region of a viral genome for the target sequences. In some embodiments, the at least one set of HP-LAMP primers is specific for SARS-CoV-2. In some embodiments, the at least one set of HP-LAMP primers is specific for the ORFlab gene of SARS-CoV-2. In yet further embodiments, for use of the method to detect SARS-CoV-2, primers comprising the sequences SEQ ID NOs: 1-6 are used. In a further embodiment, the method comprises contacting sample or the nucleic acid from the sample with HP-LAMP primers comprising SEQ ID NOs: 1-6. In further embodiments, the at least one set of HP-LAMP primers includes at least one F3 primer, at least one B3 primer, at least one FIP primer, at least one BIP primer, at least one LF primer and at least one LB primer.

In other embodiments, the methods include contacting a sample (such as a sample including or suspected to include SARS-CoV-2 nucleic acids) with at least one set of HP- LAMP primers specific for SARS-CoV-2 nucleic acid (for example, a set of primers including the sequences of SEQ ID NOs: 1-6) under conditions sufficient for amplification of the SARS- CoV-2 nucleic acid and producing an amplification product.

In some embodiments, the methods further include reverse transcription of SARS-CoV- 2 RNA in the sample, for example by contacting the sample with a reverse transcriptase. Contacting the sample with reverse transcriptase may be prior to contacting the sample with the one or more sets of HP-LAMP primers or may be simultaneous with contacting the sample with the one or more sets of HP-LAMP primers (for example in the same reaction mix with the HP-LAMP primers). In a further embodiment, the present disclosure also provides a method for detecting nucleic acid from a virus such as SARS-CoV-2, or of detection of the virus itself, in any sample, including the steps of: contacting the sample with set of LAMP primers; and further contacting the sample with a thermostable DNA polymerase to amplify the reaction. Polymerases that can be used in the method include but are not limited to Bst DNA polymerase.

Exemplary DNA polymerases include: Bst DNA polymerase; Bst DNA polymerase large fragments; Bst 2.0 DNA polymerase; Bst 2.0 WarmStart™ DNA polymerase (New England Biolabs, Ipswich, Mass.); Phi29 DNA polymerase; Bsu DNA polymerase; OmniAmp™ DNA polymerase (Lucigen, Middleton, Mich.); Taq DNA polymerase; VentR® and Deep VentR®DNA polymerases (New England Biolabs); 9° Nm™DNA polymerase (New England Biolabs); Klenow fragment of DNA polymerase I; PhiPRDl DNA polymerase; phage M2 DNA polymerase; T4 DNA polymerase; and T5 DNA polymerase.

In some examples, about 1 to 20 U (such as about 1 to 15 U, about 2 to 12 U, about 10 to 20 U, about 2 to 10 U, or about 5 to 10 U) of DNA polymerase is included in the reaction.

In some examples, the polymerase has strand displacement activity and lacks 5'-3' exonuclease activity. In one embodiment, the DNA polymerase is Bst 2.0 WarmStart ™ DNA polymerase (New England Biolabs, Ipswich, Mass).

In some embodiments, the target SARS-CoV-2 nucleic acid is RNA, and a reverse transcriptase is additionally included in the HP-LAMP assay. Exemplary reverse transcriptases include MMLV reverse transcriptase, AMV reverse transcriptase, and ThermoScript™ reverse transcriptase (Life Technologies, Grand Island, N.Y.), Thermo-X™ reverse transcriptase (Life Technologies, Grand Island, N.Y.).

The method can further include the use of a lysis buffer comprising genomic DNA and DNase. In some embodiments the genomic DNA is human. In some embodiments, the DNase is inactivated at a temperature at which the method is being performed. In some embodiments, this temperature is about 63° C. In some embodiments, this temperature is lower than about 63° C. In some embodiments, this temperature is about 55° C to about 70° C.

In some embodiments, when the lysis buffer is being used in a high-throughput platform, bacteriophage lambda DNA is used rather than human genomic DNA.

In some embodiments the method can further include a reagent to prevent carry-over of amplification products from prior samples. In some embodiments, this reagent is dUTP used in conjunction with uracil-DNA N-glycosylase (UDG).

In some embodiments, the method further uses ssRNA ladder. In some embodiments, the method further uses an RNase inhibitor. In some embodiments, the RNase inhibitor is murine.

It will be understood by those of skill in the art that the sample can be contacted or incubated with any of these reagents, simultaneously or sequentially. If the sample is contacted or incubated with any of the reagents sequentially, the order of contact or incubation is not critical.

Following incubation of the reaction mixture, the amplification product is detected by any suitable method. The detection methods may be quantitative, semi-quantitative, or qualitative. Accumulation of an amplification product (for example, compared to a negative control, such as a reagent only control) indicates presence of SARS-CoV-2 nucleic acids in the sample. In some examples, accumulation of an amplification product is detected by measuring the turbidity of the reaction mixture (for example, visually or with a turbidimeter). In other examples, amplification product is detected using gel electrophoresis, for example by detecting presence or amount of amplification product with agarose gel electrophoresis. In some examples, amplification product is detected using a colorimetric assay, such as with an intercalating dye (for example, propidium iodide, Syto 9, SYBR Green or Picogreen) or a chromogenic reagent (see, e.g., Goto et al., BioTechniques 46:167-172, 2009).

In some embodiments, the amplification products are detected and/or measured via turbidity. In some embodiments, the turbidity is caused by magnesium pyrophosphate precipitate in solution as a by-product of amplification. This precipitation can be easy visualized by the naked eye or via simple photometric detection approaches for small volumes. The reaction can be followed in real-time. When turbidity is used for detection, a visual reagent can be used in the method such as Tris-EDTA visual reagent. An apparatus can also be used, such as a turbidimeter.

In some embodiments, the amplification products are detected and/or measured using fluorescence. In some embodiments, when using fluorescence, intercalating dyes are added to the reaction. Intercalating dyes include but are not limited to SYTO 9, SYBR Green, LC Green, Eva Green, BEBO, BEXTO, and other DNA binding dyes.

In some embodiments, the amplification products are detected and/or measured using colorimetry. A visible color change that can be seen with the naked eye without the need for expensive equipment, or for a response that can more accurately be measured by instrumentation. Dye molecules intercalate or directly label the DNA, and in turn can be correlated with the number of copies initially present. Hence, LAMP can also be quantitative. Dyes that can be used for colorimetric amplification detection include but are not limited to phenol red, neutral red, cross red, Cresol red, and m-Cresol purple.

In further embodiments, amplification product is detected by a fluorescent indicator dye such as calcein (see, e.g., Tomita et al., Nat. Protoc. 3:877-882, 2008). In other examples, amplification products are detected using a detectable label incorporated in one or more of the HP-LAMP primers. The detectable label may be optically detectable, for example, by eye or using a spectrophotometer or fluorimeter. In some examples, the detectable label is a fluorophore, such as those described above. In some examples, the label is detected in realtime, for example using a fluorescence scanner (such as ESEQuant, Qiagen).

One of skill in the art can select one or more detectable labels for use in the methods disclosed herein. Other methods of detection and/or measurement of amplification products known in the art or later developed can be used in the disclosed method. One of skill in the art can determine what additional reagents need to be added to the method for detection and/or measurement and can adjust the reaction volume as needed.

In some embodiments, the method uses a volume of the total reaction of about 100 μL to about 1000 μL. In some embodiments, the volume of the total reaction is about 150 μL to about 750 μL. In some embodiments, the volume of the total reaction is about 200 μL to about 600 μL. In some embodiments, the volume of the total reaction is about 250 μL to about 500 μL. In some embodiments, the volume of the total reaction is about 300 μL to about 400 μL. In some embodiments, the volume of the total reaction is about 500 μL.

The total volume of the reaction includes the reagents used for the method as well as the sample. In some embodiments, the ratio of reagents to sample is about 80:20; or 85:15; or 90:10; or 95:5; or 96:4; or 97:3; or 98:2 or 99:1.

In some embodiments, the volume of the sample is about 5 μL to about 20 μL.

In some embodiments, the method is performed in a 1.5mL microcentrifuge tube.

The method is performed at temperature and for time to allow the amplification reaction to occur.

In some embodiments, the temperature of the method is performed at about 63°C. In some embodiments, this temperature is lower than about 63° C. In some embodiments, this temperature is from about 55° C to about 70° C.

In some embodiments, the method is performed at a time of about 10 minutes to about 90 minutes. In some embodiments, the method is performed at a time of about 15 minutes to about 60 minutes. In some embodiments, the method is performed at a time of about 30 minutes. In some embodiments, the method is performed in a high-throughput manner or platform.

In these embodiments, the method uses a volume of the total reaction of about 5 μL to about 50 μL. In some embodiments, the volume of the total reaction is about 7 μL to about 40 μL. In some embodiments, the volume of the total reaction is about 10 μL to about 35 μL. In some embodiments, the volume of the total reaction is about 12 μL to about 30 μL. In some embodiments, the volume of the total reaction is about 15 μL to about 25 μL. In some embodiments, the volume of the total reaction is about 20 μL.

In these embodiments, the nucleic acid is extracted from the sample. In some embodiments, the volume of the nucleic acid from the sample is about 1 μL to about 10 μL. In some embodiments, the volume of the nucleic acid from the sample is about 5 μL

In these embodiments, the method is performed in a 96- well or a 384-well plate.

In some embodiments, all of the reagents needed to perform the method are contained in the wells of the plate, then the sample, e.g., nucleic acid, is added to the wells.

In some embodiments, the method is performed at a time of about 10 minutes to about 90 minutes. In some embodiments, the method is performed at a time of about 15 minutes to about 60 minutes. In some embodiments, the method is performed at a time of about 15 minutes to about 30 minutes. In some embodiments, the method is performed at a time of about 20 minutes.

A further embodiment of the present disclosure is a system for the detection of nucleic acid from a virus, or detection of the virus itself, such as SARS-CoV-2 in any sample. The system includes at least one subsystem, wherein the subsystem includes HP-LAMP primers. In some embodiments, the system includes at least one subsystem wherein the subsystem includes HP-LAMP primers comprising SEQ ID NOs: 1-6. The system can also include additional subsystems for the purpose of: reverse transcribing the nucleic acid from the sample; amplifying the reaction; and detection of the amplification products. In some embodiments, the subsystem for amplifying the reaction is Bstl polymerase. In some embodiments, the subsystem for detection includes but is not limited to reagents for colorimetric detection, fluorescent detection, turbidity, and precipitation. In some embodiments, the system includes additional subsystems including but not limited to: buffers; dUTP/UDG; lysis buffer comprising DNase and genomic DNA; RNase inhibitor; and ssRNA. In some embodiments, the DNase is inactivated at a temperature at which the method is being performed. In some embodiments, the genomic DNA is human.

One embodiment for performing the HP-LAMP method for detection of SARS-CoV-2 can include the following reagents.

1. LAMP primers (Table 1- CUFC-FIP (SEQ ID NO: 1), CUFC-BIP (SEQ ID NO: 2), CUFC-LF (SEQ ID NO: 3), CUFC-LB (SEQ ID NO: 4), CUFC-F3 (SEQ ID NO: 5), CUFC- B3 (SEQ ID NO:6)), approximately 50 nt x2, approximately 25nt x4. No end modification. No special purification.

2. lOOmM dUTP (e.g., Thermo Scientific, R0133),

3. Antarctic Thermolabile UDG (uracil-DNA N-glycosylase) (e.g., NEB, M0372)

4. 5mM SYTO 9 (e.g., Invitrogen, S34854)

Optional: Allows reaction to be visualized by fluorescence. 483 Excitation Max, 503 Emission Max (Can use FITC filter). Can be visualized with blue light (470 nm) like gel imager.

5. WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) (e.g., NEB, M1800L).

Alternative 1: Bst 2.0 WarmStart DNA Polymerase (e.g., NEB, M0538), WarmStart RTx Reverse Transcriptase (e.g., NEB, M0380), dNTPs, phenol red

Alternative 2: LavaLAMP™ RNA Component Kit (e.g., Lucigen, 30096-1), or LavaLAMP™ RNA Master Mix (e.g., Lucigen, 30086-1), phenol red

Alternative 3: Loopamp® RNA Amplification Kit (RT-LAMP) (e.g., EIKEN chemical, LMP246), dNTPs, phenol red

6. 1-fold TE buffer pH 8.0 (e.g., Invitrogen, AM9849)

7. Tween-20 (e.g., Sigma-Aldrich, P9416)

8. ezDNase (e.g., Invitrogen, 11766051)

9. Human genomic DNA (e.g., Coriell, NA12777)

10. ssRNA ladder (e.g., NEB, N0362S)

11. RNase inhibitor, murine (e.g., NEB, M0314)

The method also can use the following equipment. 1. 1.5mL LoBind microcentrifuge tube (e.g., Eppendorf, 022431021)

2. Ice and a container to hold the ice

3. Sterile disposal transfer pipette (e.g., Fisherbrand, 13-711-20)

4. 63.0°C dry bath or heat block (e.g., Fisherbrand, 14-955-219)

5. 95°C dry bath or heat block (e.g. Fisher brand, 14-955-219)

6. Mini centrifuge; and

7. Mini vortex mixer

One embodiment of the HP-FAMP method to detect SARS-CoV-2 can include the following steps.

Preparation of HP-FAMP+ Cocktail (for about 40 tubes):

1. Prepare a 25-fold primer mix of FAMP primers (see Table 1): Primer mix (CUFC-FIP, CUFC-BIP, CUFC-FF, CUFC-FB, CUFC-F3, CUFC-B3) can be prepared by assembling 40mM CUFC-FIP and CUFC-BIP, IOmM CUFC-FF and CUFC-FB, and 5mM CUFC-F3 and CUFC-B3 primers in nuclease-free water. Can make 200mM individual stock solutions of CUFC-FIP, and CUFC-BIP, IOOmM individual stock solutions of CUFC-FF, CUFC-FB, CUFC-F3, and CUFC-B3 using 0.1 -fold TE, store at -20°C, thaw at 4°C before use. In a sterile low retention 2mF tube (on ice rack), add 540mE nuclease-free water, 360mE 200mM CUFC-FIP and CUFC-BIP, 180mE IOOmM CUFC-FF and CUFC-FB, 90mE IOOmM CUFC-F3 and CUFC-B3. This is 1800pF 25- fold primer mix in total. Vortex mix. Store at -20°C. Thaw at 4°C before use.

2. Prepare Fysis buffer base: The lysis buffer base is 0.1-fold TE buffer pH 8.0 with 0.1% tween-20. 0.1 -fold TE is prepared by combining 5mF 1-fold TE pH 8.0 and 45mF nuclease-free water in a sterile 50mF tube. The lysis buffer base can be prepared by adding IOmE tween-20 to lOmF 0.1-fold TE. The lysis buffer base is stored at room temperature. Before use, invert 5 times.

3. Thaw 25-fold primer mix of FAMP primers, lOOmM dUTP, 5mM SYTO 9, WarmStart® Colorimetric FAMP 2X Master Mix (DNA & RNA) at 4°C. Mix thoroughly by vortexing for 3 seconds and then spinning down, repeat 3 times. Place reagents on an isofreezer microtube chiller rack or on ice (on an aluminum rack). If using an -20°C isofreezer rack, rinse it under DI water 3-5 times, dry with a paper towel and wipe using RNase away. 4. Prepare Lysis buffer: To 900μL of lysis buffer base, add 18μL ezDNase and 13.5μL of 20ng/μL (or 9μL 30ng/μL) human genomic DNA from a normal male. Vortex to mix and spin down for 4 times, and incubate at RT for 15-18 min. 802.7μL lysis buffer is needed. Lysis buffer can be saved on ice to be used within 30 min, or saved at -20°C for long term storage.

5. Make a 1/10 dilution of ssRNA ladder (NEB, N0362S) in nuclease-free water (8μL + 72μL nuclease-free water). Vortex mix and keep on isofreezer rack or ice.

6. Prepare HP-LAMP+ Cocktail: The HP-LAMP+ Cocktail can be prepared in an Eppendorf DNA LoBind 50mL tubes on ice by combining 7626μL nuclease-free water, 10 mL WarmStart® Colorimetric LAMP 2X Master Mix (625μL x 16), 28μL lOOmM dUTP, 4μL Antarctic Thermolabile UDG, 2μL 5mM SYTO 9, 603.8μL 1-fold TE pH 8.0, 802.7μL 25-fold primer, 802.7μL lysis buffer, 57.3μL 1/10 ssRNA, and 80.6 μL RNase Inhibitor (NEB, M0314). Invert 10 times. Invert 3 time and vortex for 10 sec, repeat twice. Place on ice (rack).

7. Immediately dispense 497μL HP-LAMP+ Cocktail into 1.5mL DNA LoBind microcentrifuge tubes (Eppendorf, 022431021) on ice or isofreezer chiller racks. Thoroughly mixing before aliquoting and maintaining cold temperature for the reagents are critical for even performance of cocktails.

8. Close the caps tightly, wipe the tops of the caps with RNase away. Store at -20°C until use.

9. Perform a quality control run using a negative saliva sample with heat-inactivated SARS-CoV-2 virus (ATCC® VR-1986HK) spike-in at 1-fold to 2-fold LOD.

Performing the HP-LAMP+ Assay:

1. Count the number of HP-LAMP+ Cocktail tubes (1.5 ml microcentrifuge tubes prefilled with 497 mΐ of reaction cocktail) required for the assay (2 tubes per sample) and place these tubes at 4°C for at least 30 minutes to thaw.

2. If the saliva sample is frozen (-20°C), hand-thaw it and then place it back on ice. 3. Thoroughly mix the saliva sample.

4. Heat inactivate 30-50 mΐ of the saliva 95°C for 5 minutes.

5. Place heat-inactivated sample on ice for 5 minutes then spin for 1 second on mini spin centrifuge.

6. Take 5 mΐ of this sample and add to the bottom of a 1.5 ml microcentrifuge tube prefilled with reaction cocktail, and repeat on a second HP-LAMP+ Cocktail tube.

7. Mix and place the tube back on ice.

8. Repeat the same procedure for the second tube.

9. Clean the outside of both the tubes and place them on 63°C heat block for 30 minutes (close the lid tightly).

10. After the 30 minute incubation remove the tubes from the heat block and place on ice at least for 2 minutes. Record the results (preferably take a picture with the tube’s side label facing the camera).

11. Record colorimetric results. The assay is performed in duplicate for each sample.

Red=Negative Y ellow=Positive lb: Alternative method: Add 10μL clinical sample directly into a 1.5mL LoBind microcentrifuge tube (Eppendorf, 022431021) containing the reaction mix (230μL) and lysis buffer (10μL). Repeat for a second tube. Proceed directly to step 4. lc: Alternative method 2: Add 20μL clinical sample directly into a 1.5mL LoBind microcentrifuge tube (Eppendorf, 022431021) containing the reaction mix (460μL) and lysis buffer (20μL), mix and proceed to step 5.

Kits

In another aspect, the present disclosure provides kits that can be used to detect a nucleic acid of a virus or the virus itself or the nucleic acid of bacteria or the bacteria itself. The kit can be used to detect nucleic acid from SARS-CoV-2.

In certain embodiments, the kit comprises HP-LAMP primers. In certain embodiments, the HP-LAMP primers are specific for SARS-CoV-2. In certain embodiments, the kit comprises at least one primer chosen from the group consisting of SEQ ID NOs: 1-6. In certain embodiments, the kit comprises all of the primers comprising SEQ ID NOs: 1-6.

In some embodiments, one or more primers (such as one or more sets of primers), are provided in pre-measured single use amounts in individual, typically disposable, tubes, wells, microfluidic devices, or equivalent containers. In this example, the sample to be tested for the presence of the target nucleic acids can be added to the individual tube(s) or well(s) and amplification and/or detection can be carried out directly. The kit may also include additional reagents for the detection and/or amplification of SARS-CoV-2 nucleic acids, such as buffer(s), nucleotides (such as dNTPs), enzymes (such as DNA polymerase and/or reverse transcriptase), or other suitable reagents. The additional reagents may be in separate container(s) from the one or more primers or may be included in the same container as the primer(s).

In certain embodiments, the kit further contains a thermostable DNA polymerase. In some embodiments, the thermostable DNA polymerase is BST1 polymerase. In some embodiments, the thermostable DNA polymerase is Bst 2.0 WarmStart DNA polymerase.

In certain embodiments, the kit further contains reverse transcription.

In certain embodiments, the kit further contains a lysis buffer comprising genomic DNA and DNase. In some embodiments the genomic DNA is human. In some embodiments, the DNase is inactivated at a temperature at which the method is being performed. In some embodiments, this temperature is about 63° C. In some embodiments, this temperature is lower than about 63 C. In some embodiments, this temperature is about 55° C to about 63° C.

In some embodiments the kit further contains a reagent to prevent carry-over of amplification products from prior samples. In some embodiments, this reagent is dUTP used in conjunction with uracil-DNA N-glycosylase (UDG).

In some embodiments, the kit further contains ssRNA ladder.

In some embodiments, the kit further contains RNase inhibitor.

In certain embodiments, the kit additionally comprises reagents and instructions for detecting a nucleic acid of SARS-CoV-2 according to the disclosed methods.

In certain embodiments, the reagents of the kit can be contained in a composition. For example, the compositions can comprise suitable preservatives prevent degradation of the composition, suitable buffers to modulate the pH of the composition, suitable diluents to alter the viscosity of the compositions, and the like. The reagents of the kit may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the nucleic acid(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, multi-well plates, ampoules, or bottles.

In other embodiments, the kits comprise one or more containers to hold the components of the kit.

In further embodiments, the kit can contain additional reagents including but not limited to buffers such as TE and nuclease free water. In yet further embodiments, the kit can contain equipment including but not limited to: 1.5mL LoBind microcentrifuge tubes; ice and containers to hold ice: a 63.0°C dry bath or heat block; 95°C dry bath or heat block; a mini centrifuge; and a mini vortex mixer.

In further embodiments, the kit can include primers for controls. In certain embodiments, the HP- LAMP control primers are specific for RNase P. In certain embodiments, the kit comprises at least one primer chosen from the group consisting of SEQ ID NOs: 7-12. In certain embodiments, the HP-LAMP control primers are specific for actin. In certain embodiments, the kit comprises ate least one primer chosen from the group consisting of SEQ ID NOs: 13-18.

One of ordinary skill in the art can select additional suitable positive and negative controls for the assays disclosed herein.

One embodiment of the present disclosure is a kit comprising various containers comprising various components for the detection of SARS-CoV-2 (HP-LAMP) (see, e.g., Table 13).

A further embodiment of the present disclosure is a kit for performing a high throughput method. In one embodiment the kit comprises the components for the detection of SARS- CoV-2 (HP-LAMP) for high throughput platforms (see, e.g., Table 9).

In some embodiments, a kit for performing a high throughput method for the detection of SARS-CoV-2 (HP-LAMP) comprises a 96-well or 384-well plate, wherein the wells contain various reagents for performing the disclosed method. These reagents can include the following: HP-LAMP primers for the detection of SARS-CoV-2 (i.e., primers with sequences SEQ ID NO: 1-6) or HP-LAMP primers for the detection of controls (i.e., primers with sequences SEQ ID NOs: 7-12 and/or 13-18); thermostable DNA polymerase; reverse transcriptase; dUTP/UDG; DNase; lambda phage DNA; ss RNA ladder; buffers; and reagents for detection, such as fluorescent detection.

EXAMPLES

The present invention may be better understood by reference to the following nonlimiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention.

Example 1- Primer Design

To develop HP-LAMP, novel primers for targeting the SARS-CoV-2 virus were designed. PrimerExplorer V5 and the SARS-Cov-2 reference genome NC_045512v2 were used to design the HP-LAMP primers. The primers were matched against human reference genome Hgl9 and Human Coronavirus reference genome to ensure specificity (Hatcher et al. 2017; Kent at el. 2002) (Figure 1).

Existing primers used for RT-PCR and LAMP based nucleic acid testing of SARS- CoV-2 target the GC-rich regions located at the 5' and 3' ends of the virus. However, because salivary exonucleases degrade viral RNA from the ends, the primers were designed to target the central portion of the virus that would be better protected. Eight sets of six LAMP primers targeting SARS-CoV-2 reference genome (NC_045512.2) were designed (Figure 1). The central region of SARS-CoV-2 genome is GC-poor (AT-rich), making it difficult to select primer candidates across the genome with optimal annealing temperatures when following standard parameters for primer design. Therefore, the primers were designed to permit large primer-mediated loop-structures while ensuring that the primers did not form stable secondary structures or self-dimerize. The known SARS-CoV-2 genomic sequence were also aligned with those of six other human coronaviruses (SARS-CoV, MERS-CoV, HCoVHKU- 1, HCoV- NL63, HCoV-OC43 and HCoV-229E) to ensure no cross-reactivity.

These eight primer sets along with previously published primer sets (Zhang et al. 2020) were tested using serial dilutions of 500 to 0.5 copies of SARS-CoV-2 RNA standard spiked into a 5 mΐ standard RT-LAMP reaction (see Examples 2 and 3). The in-house designed primer set designated V5 detected 100 to 10- 1 copies level viral RNA in water, representing a 10- to 100-fold improvement in sensitivity and equivalent specificity compared with previously published primer sets. In-silico inclusivity analysis of primer set V5 performed by aligning all primer sequences against all (n = 16,453) complete SARS-CoV-2 genomic sequences deposited in the NCBI Virus database on September 15, 2020 showed a 100% match for the ORFlab gene was found for 98.8% of SARS-CoV-2 strains (n = 16,264), 1 mismatch was found to 1.2% (n = 182), and 2 mismatches were found to 0.04% (n = 7) of strains deposited in the NCBI Virus database (Brister et al. 2015), respectively (Table 2).

No instances of more than two mismatches were found. In silico crossreactivity/exclusivity was performed by aligning the V5 primer sequences against sequences of 32 common viruses as well as coronaviruses related to SARS-CoV-2. Both the Forward Inner Primer (FIP) and the Backward Inner Primer (BIP) consist of 2 sections of non- continuous genomic sequences and were aligned separately to increase the sensitivity of alignment of cross reactivity. In total, 6 primers corresponding to 8 sections of virus genome were assessed in silico for potential cross-reactivity against 32 common respiratory pathogens including six other human coronaviruses (SARS-CoV, MERS-CoV, HCoV-HKU-1, HCoV-NL63, HCoV-OC43 and HCoV-229E) (Table 3). None of the pathogens tested have a match against the total sequence length of the SARS-CoV-2 primers greater than the recommended threshold of 80%, except for SARS-CoV virus. The greatest percentage match is 92.0% on part 1 (approximately 50%) of the FIP primer, and 95.8% on the Loop Backward (LB) primer against SARS-CoV virus.

The final primer set was designed to target a central portion of the SARS-CoV-2 genome and was able to detect 100 copies of viral RNA per reaction with no false positive amplification in the negative control. It was used for further development of HP-LAMP assay and is termed as HP-LAMP primer set.

The sequences for the primers used for detection of SARS-CoV-2 are shown in Table

1.

Table 1 - LAMP Primers for Detection of SARS-CoV-2

Table 2- In Silico Inclusivity Analysis

Table 3 - Cross-Reactivity In Silico Analysis

Example 2 - HP-LAMP Testing of Viral Transport Medium

Materials and Methods

HP-LAMP assay reagents preparation

A 25-fold primer mix of LAMP primers (CUFC1-FIP, CUFC1-BIP, CUFC1-LF, CUFC-LB, CUFC1-F3, CUFC1-B3; Table 1) was prepared by assembling 40 mM FIP and BIP, 10 mM CUFC1-LF and CUFC1-LB, and 5 mM CUFC1- F3 and CUFC1-B3 primers in nuclease-free water (Ambion, AM9937). A 2X colorimetric RT-LAMP master mix was prepared by adding 3.5 μL 100 mM dUTP (Thermo Scientific, R0133), 0.5 μL Antarctic Thermolabile UDG (NEB, M0372S), and 0.25 μL 5 mM SYTO 9 (Invitrogen, S34854) to 1250 μL WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB, M1800S/L).

The reaction mix for one 250 μL reaction was prepared by mixing 125 μL 2X colorimetric RT-LAMP master mix, 10 μL 25-fold LAMP primer mix, and 95 μL nuclease- free water. These values can be scaled up according to the actual number of samples.

Two 250 μL reactions were used to test one sample. Lysis buffer consisted of 0.1 -fold buffer TE pH 8.0 (Ambion, AM9848) with 0.1% TWEEN-20, 1% volume (e.g., 1 μL enzyme added to 100μL buffer) Thermolabile Proteinase K (NEB, P8111S), 2% volume ezDNase (Invitrogen, 11766051), and 0.3 ng/μL human genomic DNA from a normal male. For one reaction, 460 μL of reaction mix and 20 μL of lysis buffer were preloaded in a clean 1.5 mL DNA LoBind microcentrifuge tube (Eppendorf, 022431021) and kept on ice until use.

Initial testing of SARS-Cov-2 RNA-spiked and selected clinical samples

The limit of detection (LoD) was determined by testing serial dilutions in IX HBSS (Gibco, 14025-092) of the SARS-Cov-2 RNA standard (Exact Diagnostics, COV019) 0.5 μL spike-in with 20 μL viral transport medium (VTM) (CDC SOP#: DSR-052-02), instead of clinical samples, following the optimized protocol detailed above. To determine the LoD, serial dilution was performed using 4-10 repeats. 95% Cl was calculated using Clopper-Pearson method.

To determine the LoD with clinical samples, a set of 20 positive clinical samples was selected to represent the range of Ct values detected using a Roche cobas 6800 system for SARS-Cov-2 and 10 negative samples were subjected to the testing using the optimized LAMP protocol.

Testing of clinical samples

A second set of 20 positive clinical samples consisting of viral transport media inoculated with a nasopharyngeal swab sample obtained as part of routine clinical testing was chosen at random. From each clinical specimen, 20 μL was placed directly into a 1.5 mL DNA LoBind microcentrifuge tube (Eppendorf, 022431021) containing the reaction mix (460μL) and lysis buffer (20 μL ). The solution was mixed using a sterile disposal transfer pipette (Fisherbrand, 13-711-20) by gentle pipetting 12 times. Using the same sterile disposable transfer pipette, 250μL of the 500μL solution was placed into a new 1.5 mL DNA LoBind microcentrifuge tube. Both tubes with approximately 250 μL each were placed in a 63.0°C dry bath (Fisherbrand, 14- 955-219) and incubated for 30 minutes. The tubes were then placed on ice for 1 minute to pause the reaction and the colorimetric results were read (red = negative, yellow = positive).

This study was reviewed and approved by the Institutional Review Board of Columbia University Irving Medical Center (CUIMC IRB) (Protocol# AAAS9910). The study used residual specimens from clinical care de-identified by indirect identifier. Consent was exempted for these samples by CUIMC IRB. All methods were carried out in accordance with relevant guidelines and regulations.

Results

LAMP primers and reaction conditions were designed and optimized for high performance, direct rapid colorimetric HP-LAMP testing for SARS-CoV-2 (Figure 2). The final primer set targeted the middle of ORFlab, the largest SARS-CoV-2 gene (Figure 1) and has relatively low GC%. The optimal reaction temperature was determined experimentally and set to be 63°C. The workflow enables direct testing of clinical samples without the need for RNA isolation or cell lysis (Figure 2A) (Broughton et al. 2020; Zhang et al. 2020). The set-up requires only a pipette and tips, a transfer pipette, a mini heat block, and a box of ice; no special equipment or devices are needed. A colorimetric output for simple interpretation of results was used without the need for extra equipment. HP-LAMP amplification of the targeted DNA results in decreased pH. A pH sensitive dye added to the reaction mixture resulted in a color change from red to yellow in a positive test and remained red in negative tests (Figure 2C). For samples with low copy number of virus, a positive signal was displayed only in one of the two tubes, in which case we considered the results as positive.

Viral transport medium contains inhibitors that reduce sensitivity of amplification and it was found that it reduced the sensitivity of the HP-LAMP reaction by 30 to 100-fold compared with buffers such as HBSS (Broughton et al. 2020). Nonetheless, samples collected as part of clinical care that had been placed in viral transport media continued to be used so that: (a) the existing workflow could be kept as consistent as possible; and (b) single nasopharyngeal swab sample tested in parallel using the disclosed test and the Roche cobas system could be performed. To determine the LoD of the assay, viral RNA standards were spiked into viral transport media. Serial dilution experiments, conducted in quadruplicate, consistently showed positive results down to 2.5 copies/ μL. Results with copy number below 2.5 copies/ μL were inconsistent, and thus 2.5 copies/μL was determined to be the LoD.

To compare the Ct values of clinical samples tested on the Roche cobas system with the HP- LAMP results, positive clinical samples from routine clinical testing for COVID-19 were selected to represent the broad range of Ct value and tested using the HP-LAMP assay (Figure 3A). These samples showed Ct values ranging from 18.52 to 34.42 for RT-PCR Target 1, and 18.69 to 36.61 for RT-PCR Target 2 on the Roche cobas 6800 system.

Ten negative clinical samples were also tested. Samples with Ct value of < 30 for target 1 and < 31 for target 2, corresponding to approximately 0.168-0.252 TCID50/mL14, had stable performance and high accuracy in testing results (Figure 3A). Hence the LoD of clinical samples using the HP-LAMP assay was Ct < 30 for RT-PCR target 1 and < 31 for RT-PCR target 2. Five out of 13 samples with high Ct values tested positive, and all of the false negative samples were not necessarily the ones with highest Ct value, indicating that the presence of detectable virus was not within the linear range due to low copy numbers. All of the 10 negative samples were negative by HP-LAMP, indicating that it had a specificity of 100% (binomial 97.5% unidirectional confidence limit 69.2-100%) (Figure 3A, Table 4).

To estimate the detection power of the HP-LAMP test on actual clinical specimens, a second set of 20 clinical samples tested positive and 10 clinical samples tested negative by standard test were randomly selected and tested. Samples had a Ct value ranging from 17.46 to 35.71 for Target 1, and 17.94 to 38.12 for Target 2. Eight samples were within, while 12 samples were below, the predicted LoD of the rapid testing method (Figure 3B). Eight out of 8 samples within LoD were tested positive and were in concordance with clinical testing results. Nine out of 12 samples below LoD were tested positive.

Taken together, these results indicated that HP-LAMP has the 75% (9/12) positive percentage agreement (PPA) below LoD, 100% (8/8) PPA within LoD, and 100% (10/10) negative percentage agreement (NPA) (Table 4). Together, the rapid testing generated an 85% (17/20) PPA, 100% (10/10) NPA, and 90% (27/30) accuracy for randomly selected clinical samples. Table 4 - Summary of Direct HP-LAMP Testing Using Clinical Samples

Example 3 - HP-LAMP Testing of Saliva Materials and Methods Ethics

The study was reviewed and approved by the Columbia University Institutional Review Board (IRB) (#AAAS9893) and all methods were carried out in accordance with relevant guidelines and regulations. All study subjects signed informed consent prior to participating. Participant enrollment

Study participants were enrolled at New York Presbyterian Hospital when they underwent routine clinical testing for SARS-CoV-2 from 04/29/2020 to 06/1/2020 at the cough and fever clinic or a COVID-19 testing tent. Study participants were enrolled at Jackson Memorial Hospital (JMH) when they underwent routine clinical testing for SARS-CoV-2 upon presentation to the emergency room from 08/14/2020 to 09/10/2020.

Sample collection

Nasopharyngeal (NP) swab and saliva samples were obtained from participants following CDC-recommended protocols. Nasopharyngeal swab samples were transported in 3 ml. viral transport medium (VTM) and subjected to routine clinical testing for RT-PCR-based SARS-CoV-2 testing. Saliva samples were self-collected by each participant, by spitting approximately 1 mL of saliva into a clean 50 mL DNA LoBind Conical Tube (Eppendorf, 0,030,122,232). Saliva samples were shipped with -80°C ice packs and stored at -80°C until use. SARS-CoV-2 positive (n = 30) and SARS-CoV-2 negative (n = 35) samples were included in this study. Contrived samples for direct saliva testing

Contrived samples were prepared using SARS-CoV-2 Standard (200,000 cp/mL) (Exact Dx, COV019) spike-in or inactivated virus (ATCC, VR-1986HK). SARSCoV- 2 RNA Standard was diluted in nuclease-free water (Ambion, AM9937), and 1 to 100 copies of viral RNA were spiked into each reaction along with 5-20 μL of saliva from healthy individuals as detailed below. Inactivated virus was spiked into saliva from healthcare workers who tested negative for SARS-CoV-2, and serially diluted to the targeted concentration using additional negative saliva.

Primers

The sequences for the primers used are shown in Table 1. CDC 2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Primers were also included as a reference (CDC 2020).

Preparing one-step HP-LAMP reaction master mix (HP-LAMP)

A 25-fold primer mix of LAMP primers (SEQ ID NOs: 1-6; Table 1) was prepared by assembling 40 mM FIP and BIP, 10 mM. LF and LB, and 5 mM F3 and B3 primers in nuclease- free water (Ambion, AM9937).

A 2 x colorimetric RT-LAMP master mix was prepared by adding 3.5μL 100 mM dUTP Thermo Scientific, R0133), 0.5 μL Antarctic Thermolabile UDG (NEB, M0372S), and 0.25μL 5 mM SYTO 9 (Invitrogen, S34854) to l,250 μL WarmStart Colorimetric LAMP 2 x Master Mix (DNA & RNA) (NEB, M1800S/L). The final reaction mix for one reaction includes 250μL 2 x colorimetric RT-LAMP master mix, 20 μL 25-fold LAMP primer mix, and 190 μL nuclease-free water, 20 μL of lysis buffer ((0.1% tween-20, 2% volume (i.e., 2μL added to 100μL) ezDNase (Invitrogen, 11,766,051)), 0.3 ng/μL lysis buffer volume of carrier DNA (human genomic DNA from a normal male e.g., 6 ng carrier DNA for 20 μL lysis buffer), and approximately 9 ng/μL lysis buffer volume of carrier RNA (NEB, N0362S, ~ 250 ng/μL), 2μL RNase Inhibitor, Murine (NEB, M0314S/L), 15 μL buffer TE pH 8.0 (Ambion AM9849), and can be scaled up according to the actual number of samples.

Lysis buffer was mixed with the carrier gDNA and incubated at RT for approximately 15 minutes before use. For each reaction, 497 μL of the final reaction mix was preloaded in a clean 1.5 mL LoBind microcentrifuge tube (Eppendorf, 022,431,021), stored at -20°C, and thawed at 4°C before use. This was the final reaction mix used for the HP-LAMP assay, and each sample was tested in duplicate. HP-LAMP assay was quality controlled using negative saliva with 1-2 x LoD inactivated SARS-CoV-2 virus spike-in, or 25 copies SARS-CoV-2 virus RNA standard. SARS-CoV-2 detection on saliva samples using HP-LAMP without RNA extraction

Saliva samples were subjected to a 95°C heat inactivation for 5 minutes (Batejat et al. 2020; Formsgaard and Rosenstierne 2020), and then cooled on ice. 5μL of saliva sample was added to the one-step HP-LAMP final reaction mix, mixed by gentle pipetting using a transfer pipette (Fisherbrand, 13-711-20), and incubated at 63°C for 30 min in a portable heat block (Fisherbrand, 14-955-219). The reaction was paused by placing on ice for 1 minute, and the colorimetric results were then recorded visually and by camera.

Determining the limit of detection (LoD)-analytical sensitivity

The limit of detection (LoD) is defined as the lowest concentration at which 19/20 replicates (or approximately 95% of all true positive replicates) are positively detected.

To determine the LoD of HP-LAMP, intact SARS-CoV-2 (ATCC VR-1986HK, Batch 70,037,676) with a known virus concentration (1.77 x 10⁵ copies/mΐ) was spiked into saliva from healthcare workers who tested negative for SARS-CoV-2 using the Roche cobas system. The following two fold dilution series was tested: 88.5, 44.2, 22, 11, 5.5, 2.75, 1.38, and 0.69 copies/mΐ of saliva. The dilutions of 5.5, 2.75, 1.38, and 0.69 copies/mΐ were tested in triplicate to determine the ‘preliminary LoD’. Spiked saliva specimens were tested according to protocol for the HP-LAMP Assay. The preliminary LoD was then confirmed with 20 additional replicates (Figure 4). The LoD of the HP-LAMP Assay was determined to be 1.38 copies/mΐ of saliva. At this LoD, 19/20 (95%) individual replicates at a concentration of 1.38 copies/mΐ of saliva tested positive (Figure 4).

Determining cross-reactivity (analytical specificity)

Wet testing was performed to evaluate potential cross-reactivity/exclusivity of the assay with other organisms using ZeptoMetrix Corporation NATtrol Respiratory Verification Panel (ZeptoMetrix, NATRVP-IDI) including 19 respiratory pathogens, NATtrol Coronavirus- SARS Stock (ZeptoMetrix, NATSARS-ST), NATtrol MERSCoV Stock (ZeptoMetrix, NATMERS-ST), and NATtrol SARS-Related Coronavirus 2 (SARS-CoV-2) External Run Control (ZeptoMetrix, NATSARS(COV2)-ERC). Samples were prepared by spiking 3μL inactivated, intact viral particles or bacterial cells using the panels/organisms into negative saliva samples and were subsequently processed using HP-LAMP. Virus and bacteria were tested at concentrations similar to or greater than the SARSCoV- 2 virus External Run Control (50,000 copies/mL).

Clinical evaluation

The performance of HP-LAMP was compared to test results from paired nasopharyngeal (NP) swab samples. The study was conducted with symptomatic patients from Jackson Memorial Hospital (JMH) and Columbia University Irving Medical Center (CUIMC) who each provided a paired NP and saliva sample on the same day. NP samples were immediately processed in the clinical pathology laboratory using FDA authorized Roche cobas (Roche 2020), Cepheid (Cepheid 2020), Qiagen (Qiagen 2020), or EliTech (GendFinder) (Osang Healthcare 2020) systems for SARS-CoV-2 testing at JMH and CUIMC (depending on the available testing option at the time of testing). Saliva was collected in blinded sterile tubes (Eppendorf, 0,030,122,232) without any preservatives and sent to Columbia University Fertility Center for testing by HP-LAMP. A total of 65 samples were tested: 30 samples that were positive for SARS-CoV-2 by NP swab and 35 that were negative by NP swab. After testing, results were sent back to JMH for unblinding. Samples containing food particles or blood were not excluded.

Interfering substances

To determine whether endogenous or exogenous substances that could be found in saliva could interference with the assay, negative saliva samples were tested after addition of each of the substances in the concentrations as listed in Table 8 and then spiked with purified, intact, inactivated viral particles at 5x LoD (6.9 copies/mΐ, total of 34 viral copies per reaction tube) and run using HP-LAMP. For negative control samples, no viral particles were spiked into saliva containing each of the potentially interfering compounds.

Effect of freezing saliva

To determine whether freezing of saliva would affect test performance, FDA SARS- CoV-2 negative saliva samples were spiked with intact, inactivated SARS-CoV-2 virus at 2x LOD and 4x LOD and tested fresh and after freeze/thaw cycle with a 48hr freeze cycle. A total of 20 samples were tested, 10 of which were prepared at 2x the established LoD, and 10 samples prepared at 4x of the established LoD.

Results

The HP-LAMP reaction conditions were modified to improve performance. The sensitivity and specificity of the assay could be markedly improved by adding carrier DNA, carrier RNA, and sensitivity and specificity of the assay could be markedly improved by adding carrier DNA, carrier RNA, and RNase inhibitors, as well as by increasing the reaction volume and introducing a heat-inactivation step. Because of the risk that carry-over product from prior samples could cross contaminate a new sample and lead to false-positive results, Deoxyuridine Triphosphate (dUTP) and Antarctic thermolabile uracil-DNA N-glycosylase (UDG) was added to the reaction mixture to incorporate dUTP into the HP-LAMP product and digest the HP- LAMP carry-over. To determine the limit of detection (LoD) of HP-LAMP, twofold serial dilutions of intact virus were spiked into negative saliva in concentrations ranging from 88.5 to 0.69 copies/mΐ of saliva (Figure 5A). At the LoD of 1.38 copies/mΐ of saliva, 19/20 replicates (95%) were positively detected. At 2 x LoD (2.7 copies/ mΐ of saliva), 20/20 replicates (100%) were detected (Figures 4 and 5A).

This LoD was comparable to other U.S. Food and Drug Administration (FDA) Emergency Use Authorization (EUA) authorized swab- and saliva-based tests that must be run in centralized high complexity laboratories, including swab-based assays, such as LabCorp’s COVID-19 RT-PCR test (approximately 15.625 copies/reaction), the Centers for Disease Control and Prevention (CDC) 2019-nCoV Real-Time RT-PCR panel (approximately 100 to -0.5 copies/μL), SalivaDirect (6 copies/μL), Fluidigm Corporation’s Advanta Dx (6.25 copies/μL), as well as rapid point-of-care swab tests, such Quidel Lyra Direct (34 copies/μL), though these were tested using different reference panels and thus direct comparison was difficult.

Wet testing for cross-reactivity/exclusivity was performed to evaluate potential crossreactivity/exclusivity of the assay with 21 respiratory pathogens. All results, except for the SARS-CoV-2, of wet bench testing were negative (Table 5 and Figure 5B).

Clinical evaluation of F1P-LAMP was performed by comparing results from 65 blinded, paired, nasopharyngeal (NP) swab and saliva samples collected at the same time from symptomatic patients at Jackson Memorial Hospital (JMH) and Columbia University Irving Medical Center (CUIMC). Samples were collected throughout the day without the need for study subjects to be fasting or have previously rinsed their mouths. Samples containing food debris, thick mucus or frank blood were included in the analysis and were not excluded. A representative image of test results of some of the samples collected is shown in Figure 5C.

Out of 30 individuals with NP swab specimens that tested positive, 29 were positive by HP-LAMP, a positive percentage agreement (PPA) of 96.7% (95% CI= 82.8-99.9%). Out of 35 individuals with NP swab specimens that tested negative, 34 were negative by the Columbia University Fertility Center SARS-CoV-2 Rapid Saliva Assay, a percentage negative agreement (NPA) of 97% (95% CI= 85.1-99.9%). Of note, the single false positive had previously tested positive for SARS-CoV-2 from a NP swab collected two weeks prior. See Table 6.

The RT-PCR cycle threshold (Ct) values for SARS-CoV-2 target N2 from the NP swab from these positive samples ranged from to 14.2MT.6. See Table 7.

Testing was performed to determine if endogenous or exogenous substances found in saliva could interfere with the HP-LAMP assay. Interference was observed in the presence of mouthwash (5% v/v), toothpaste (1% w/v), nasal allergy spray (1% v/v), and zinc (2% w/v). Of note, in the clinical validation study, saliva samples were collected throughout the day without study subjects having to refrain from eating or drinking or avoiding any specific substances. See Table 8. All samples (20/20) tested positive in both the fresh and freeze/thaw treatments.

Table 5 - Cross-Reactivity Wet testing

Table 6 - Positive and negative percentage agreement of HP-LAMP for detection of SARS- CoV-2 in saliva compared with nasopharyngeal swab RT-PCR results

Table 7 - N2 Ct Values for NP Samples and Testing Platform

Table 8 - Interfering Substances

Example 4 - HP-LAMP Testing in Pooled Samples

Sample pooling allows multiple people to be tested at once in a single assay. This enables testing of more individuals in a shorter time using fewer resources and is, therefore, an important public health tool (USFDA COVID-19 Update July 18, 2020).

To evaluate the ability of HP-LAMP to be used with pooled samples, using the materials and methods of Example 3, sample pooling of 5 individual samples (N = 5) was performed. 20 known positive samples (N2 Ct < 33) and 100 known negative samples were used to generate 20 positive pools and 20 negative pools for evaluation of pool testing using HP-LAMP assay. The negative sample matrix was created by individually pooling 80 negative clinical samples into 20 pools of N = 4 before adding either a single positive or negative sample to create the final testing pool.

Twenty positive pools and 20 negative pools of five were tested by HP-LAMP. HP- LAMP accurately detected 20/20 (100%) positive pools and 20/20 (100%) negative pools (Figure 6).

Example 5 - High Throughput HP-LAMP

A high throughput assay to detect SARS-CoV-2 was performed using a 96- well plate and the primers in Table 1 (SEQ ID NOs: 1-6). The wells of the plate were preloaded with 20 ul of test cocktail (HP-LAMP cocktail with HP-LAMP primers targeting SARS-CoV-2 (SEQ ID NOs: 1-6) and a paired well preloaded with 20 ul of control cocktail (HP-LAMP cocktail with Internal control primers targeting RNAse P (SEQ ID NOs: 7-12) or targeting rActin (SEQ ID NOs: 13-18)). The cocktail was the same as for the HP-LAMP cocktail used in Examples 2 and 3, except that Lambda DNA was used instead of human genomic DNA (to enable the use of the RNAse P internal control), and RNAse inhibitor was not used.

Materials and Methods

Reagents used for the high throughput HP-LAMP assay are listed in Table 9.

Table 9 - Reagents for High Throughput LAMP Assay

Primers

The sequences for the primers used are shown in Table 1 and Table 10. Additional optional control primers are shown in Table 11. Table 10 - LAMP Primers for Internal Control RNAse P

Table 11 - LAMP Primers for Internal Control rActin

High Throughput HP-LAMP Assay Reagents

Primer Mix

A 25-fold primer mix of LAMP primers (either the CUFC-SARS-CoV-2 set (Table 1) or Internal control set (Tables 10 and 11) was prepared by assembling 40mM FIP and BIP, and IOmM LF and LB, and 5mM F3 and B3 primers in nuclease-free water (Ambion, AM9937). 200mM individual stock solutions of FIP and BIP, IOOmM individual stock solutions of LF, LB, F3, B3 can be prepared using 0.1-fold TE, store at -20°C, thaw at 4°C before use.

In a sterile low retention 2mL tube (on ice rack), 540 μL nuclease-free water, 360μL 200mM FIP and BIP, 180μL IOOmM LF and LB, 90μL IOOmM F3 and B3 were added resulting in an 1800μL 25-fold primer mix in total. The mixture was vortexed and small aliquots made to avoid repeat freeze-and-thawed.

Once thawed, all materials (including plate and plated samples) should be at 4°C unless otherwise indicated.

2X Color

A 25-fold or 25x primer mix of LAMP primers was thawed as well as lOOmM dUTP, 5mM SYTO 9, WarmStart® Colorimetric LAMP 2X Master Mix, and Human genomic DNA at 4°C. Each component was thoroughly vortexed for 3 seconds and then spun down, repeated 3 times. Then, the reagents were placed in ice. Antarctic Thermolabile UDG and ezDNase were spun down and placed in ice.

To one tube of 1250μL WarmStart® Colorimetric LAMP 2X Master Mix, 3.5μL lOOmM dUTP, 0.5μL Antartic Thermolabile UDG, and 0.25μL 5mM Syto-9 was added and mixed by vortexing and spun down and kept on ice. (Note: this can be scaled up as needed.)

Lysis buffer

200μL of 0.1% Tween 20 in 0.1-fold TE pH 8.0 was pipetted into a clean 1.5 mL DNA LoBind microcentrifuge tube. 4μL ezDNase and 3 μL of 20 ng / m L lambda DNA was added and mixed by vortexing, and spun down 3 times, incubated at room temperature for 15-18 minute and placed on ice. ssRNA

In a 1.5 mL DNA LoBind microcentrifuge tube, 1:10 dilution of ssRNA ladder was made in nuclease-free water (2μL ssRNA ladder + 18μL nuclease-free water), mixed by vortexing, spun down and placed on ice.

High Throughput HP-LAMP Testing Protocol

The Reaction Cocktail is prepared as follows in Table 12. Table 12 - Reaction Cocktail Preparation

20 μL of the Reaction Cocktail was dispensed per well of the plate. (Note: plate can be sealed and stored at -20°until future use. 200 ul of samples (VTM, PBS, or saliva) were extracted using EUA approved kit (e.g.,

Omega Biotek or Zymo) into nuclease free, molecular grade water. 5 ul of extracted RNA from each patient was loaded into the plate well containing the test cocktail and an additional 5 ul of the extracted RNA was loaded into the well containing the control cocktail. The plate was mixed by brief vortexing or flipping the plate and spun down and then kept on ice. The plate was then incubated at 63 °C in a fluorescence plate reader and fluorescence is monitored.

For example in a BioRad CFX96 (or equivalent): The thermocycler was pre-warmed to 25°C. The plate with samples was loaded, incubated at 25°C for 1 minute, followed by 63°C real-time monitor of SyBr green signal with heated lid on for 30-40 minutes. The plate reading time should also be counted. For example, on a BioRad CFX96, setting 15 sec/cycle with SyBr Green signal reading at the end of each cycle, the actual time is approximately 26sec/cycle with approximately 11 sec plate reading. The actual plate reading time on different models may be different and need to be determined in advance.

The baseline threshold was set to approximately 1000 RFU, and positive detection cutoff to 30 minutes.

Quality control was run by the testing protocol using controls. The controls consisted of VTM spiked-in with heat-inactivated SARS-CoV-2 virus (ATCC® VR-1986HK) at 5x FOD (positive control) or no spike-in (negative control). Both positive and negative samples should be carried through the entire extraction process. Nuclease-free water is used as non-template control (NTC). Determining the limit of detection using inactivated SARS-CoV-2 virus.

The limit of detection (LoD) is defined as the lowest concentration at which 19/20 replicates (or approximately 95% of all true positive replicates) are positively detected. The is established using a dilution series of heat-inactivated SARS-CoV-2 virus (ATCC VR- 1986HK), spiked into negative anterior nasal swab clinical matrix in VTM.

To determine the preliminary LoD, test ½ serial dilutions (40 copies/μL to 0.31 copies/μL) of virus was spiked into pooled clinical negative matrix (swab VTM or saliva). Each dilution was tested with 3 replicates (6 wells) (see Figure 4). Each spiked replicate should be processed through the entire assay, beginning with RNA extraction using the FDA approved kit (e.g., Zymo Quick-DNA/RNA Viral MagBead - DX on a KingFisher instrument) using the same extraction used for the clinical samples that were already extracted and that you will be testing in the clinical validation.

Example 6 - FDA Emergency Use Authorization Application Data

MEASURAND

Specific nucleic acid sequences from the genome of the SARS-CoV-2 targeting a region in the ORFlab gene.

PROPOSED INTENDED USE

The Columbia University Fertility Center SARS-CoV-2 Rapid Saliva Assay is a rapid, reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) assay intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in saliva collected without preservatives in a sterile container from individuals suspected of COVID-19 by their healthcare provider. Testing is limited to Columbia University Fertility Center which is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in saliva specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Laboratories within the United States and its territories are required to report all test results to the appropriate public health authorities. The Columbia University Fertility Center SARS-CoV-2 Rapid Saliva Assay is intended for use by qualified and trained clinical laboratory personnel specifically instructed and trained in the techniques of RT-LAMP and in vitro diagnostic procedures. The assay is only for use under the Food and Drug Administration's Emergency Use Authorization. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information. Negative results for SARS- CoV-2 RNA from saliva should be confirmed by testing of an alternative specimen type if clinically indicated.

INSTRUMENTS AND REAGENTS USED TO PERFORM THE HP-LAMP+ SARS- COV-2 DIAGNOSTIC ASSAY

Table 13 - Instruments and Reagents Used to Perform the HP-LAMP+ SARS-CoV-2 Diagnostic Assay

DEVICE DESCRIPTION AND TEST PRINCIPLE

1) Product Overview/Test Principle

The SARS-CoV-2 Rapid Saliva Assay is a rapid (<45 min), single-tube, extraction- free, RT-LAMP method for SARS-CoV-2 detection using a saliva sample collected in a sterile tube without the need for preservatives (Figure 2B). The ability to test in under 45 minutes with only a single pipetting means that testing can be done in real-time enabling prompt quarantine and simplifying contact tracing. In the future, the test can be scaled and broadly implemented at the point-of-care because: (1) it does not require specialized equipment for RNA extraction; (2) it does not require the use of a thermocycler for PCR; (3) it does not require multiple pipetting or fluid-transfer steps; and (4) it provides results in under 45 minutes. Because the test requires only the use of two heat blocks, disposable transfer pipettes, and microcentrifuge tubes pre-filled with the reaction cocktail, it avoids the supply-chain and manufacturing limitations required for specialized laboratory equipment and swabs, or specialized collection tubes and preservative. Because the test uses saliva, it is more conducive to repeated testing, and avoids the need for nasal or nasopharyngeal (NP) swabbing which is a deterrent to testing for some individuals.

Saliva is heat-inactivated and then directly placed into a microcentrifuge tube containing the reaction cocktail, incubated at 63°C for 30 minutes. Results are interpreted using a simple color indicator visible to the unaided eye (red= negative, yellow= positive). The reaction cocktail contains all the necessary components for: (1) lysis of the viral particles; (2) reverse-transcription of the viral RNA; (3) amplification of the targeted region using six primers designed to uniquely detect SARS-CoV-2 viral RNA in a region better protected from degradation by endogenous exonucleases; (4) protection of the viral RNA and DNA from degradation; (5) reducing inhibition of the reaction by components of saliva; and (5) color indication of the presence or absence of viral RNA.

2 ) Description of Test Steps Saliva (approximately 1 ml) is collected in a sterile container and heat inactivated (95 °C for 5 minutes) and then cooled on ice. 5 ul of saliva is added to a 1.5 ml microcentrifuge tube x2, pre-filled with the reaction cocktail and mixed. The microcentrifuge tubes are then placed in a 63°C heatblock for 30 minutes The reaction is the stopped by placing the tubes in ice and then the tubes are visualized to determine the results. 3) Control Materials to be Used

Two controls are run with each batch: Positive control: A positive control is used and consists of heat-inactivated S ARS-CoV-2 virus control at 5X limit of detection (LoD) spiked into a negative saliva sample (see “Negative control” below). This serves as an “extraction” and performance control and is run through the entire assay. Negative control: A no template control (NTC) is used and consists of a pool of confirmed negative saliva sample (negative swab sample run on the Roche cobas) and negative RT-PCR of extracted saliva sample. Confirmed pool is then aliquoted to ensure consistency between runs. This negative control saliva sample serves as a control for contamination of reagents and false amplification.

4) Assay results and interpretation 1) S ARS-CoV-2 RT-LAMP Test Controls

Positive Control: The positive control should yield a “detected” result in 2/2 tubes Negative Control: The negative control should yield a “not detected” result in 2/2 tubes.

2) Examination and Interpretation of Patient Specimen Results All test controls should be examined prior to interpretation of patient results. If the controls are not valid, the patient results cannot be interpreted. A colorimetric indicator is used with a positive signal considered equivalent to a Ct value of < 35. Results will be interpreted according to the following criteria: Table 14 - Results Interpretation

PERFORMANCE EVALUATION

The results for Limit of Detection (LoD) - analytical sensitivity are shown in Example 3 and Figure 4.

The results for Inclusivity (analytical sensitivity) are shown in Example 1 and Table 2. The results for Cross-reactivity (analytical specificity) are shown in Examples 1 and 3 and Tables 3 and 5.

The results for Interfering substances are shown in Example 3 and Table 8.

The results of the Effect of freezing saliva are shown in Example 3.

The results of Clinical Evaluation are shown in Example 3 and Tables 6 and 7.

LABORATORY SOP / PROTOCOL FOR PERFORMING THE SARS-COV-2 RAPID SALIVA ASSAY

Materials:

HP-LAMP+ Cocktail (Pre-made)

Saliva samples

Sani-Cloth bleach germicidal wipes 63°C Heat block (Fisher brand, 14-955-219)

95°C Heat block (Fisher brand, 14-955-219)

Transfer pipettes

Pipettes and low retention filter tips Crushed ice Mini spin Vortex

Set-up:

Thawing the HP-LAMP+ cocktail tubes.

Count the number of HP-LAMP+ cocktail tubes (1.5 ml microcentrifuge tubes pre-filled with 497 mΐ of reaction cocktail) required for the assay (2 tubes per sample) and place these tubes at 4°C for at least 30 min to thaw.

Protocol:

1. Take all the necessary precautions such as wearing PPE, double gloves, masks, safety glasses, appropriate hoods, etc. 2. Clean the hood with sani-cloth, and place cleaned heat blocks one on each side. Set the temperatures to 63 °C and 95 °C. Check with the thermometer to confirm that the desired temperature is achieved. If not, adjust the temperature on the heat block to get the desired temperature. 3. If the saliva sample is frozen (-20°C), hand-thaw it and then place it back on ice.

4. Using the 1 ml transfer pipettes, thoroughly mix the saliva sample.

5. Heat inactivate ~30 mΐ of the saliva 95°C for 5 min.

6. Place the heat-inactivated sample on ice for 5 min then spin for 1 second on mini spin centrifuge. 7. Using a plO pipette (adjusted to 5 mΐ), pipette the saliva sample up and down 5 times, take 5 mΐ of this sample and add to the bottom of 1.5 ml microcentrifuge tube prefilled with reaction cocktail, and repeat on a second HP-LAMP+ cocktail tube.

8. Using a 1 ml Transfer pipette, mix the tube sixteen times and place it back on ice.

9. Discard the transfer pipette appropriately; use a new one following the same procedure for the second tube.

10. Clean the outside of both the tubes and place them on 63°C heat block for 30 min (close the lid tightly).

11. After the 30 min incubation remove the tubes from the heat block and place on ice for at least 2 min. Clean the outside of the tube and record the results (preferably take a picture with the tube’s side label facing the camera).

12. Record colorimetric results. The assay is performed in duplicate for each sample.

13. Discard the tubes appropriately using a biohazard container containing hydrogen peroxide in it.

REFERENCES

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Brister, et al. NCBI viral genomes resource. Nucleic Acids Res 43, D571-577 (2015). Broughton, et al. CRISPR-Casl2-based detection of SARS-CoV-2. Nat. Biotechnol. 38, 870-874 (2020).

Centers for Disease Control and Prevention. Research use only 2019 — novel coronavirus (2019-nCoV) real-time RT-PCR primers and probes. (2020).

Cepheid. Xpert ® Xpress SARS-CoV-2 Instructions for Use. Coronavirus Disease 2019 ( COVID-19) Emergency Use Authorizations for Medical Devices (2020).

Cheng, et al. Diagnostic testing for severe acute respiratory syndrome-related coronavirus-2: A narrative review. Ann. Intern. Med. 2, 13 (2020).

Fernandes, et al. The UCSC SARS-CoV-2 genome browser. Nat Genet 52, 991-998

(2020).

Fomsgaard and Rosenstierne. An alternative workflow for molecular detection of SARS-CoV-2 — escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020. Eurosurveillance 25, 1-8. (2020).

He, et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat. Med. 26, 1491-1493 (2020).

Joung, et al. Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing. N. Engl. J. Med. 15, 1492-1496 (2020).

Kent, et al. The human genome browser at UCSC. Genome Res. 12, 996-1006

(2002).

Lamb, et al. Rapid detection of novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification. PLoS ONE 15, e0234682 (2020).

Nagura-Ikeda, et al. Clinical evaluation of self-collected saliva by quantitative reverse transcription-PCR (RT-qPCR), direct RT-qPCR, reverse transcription-loop-mediated isothermal amplification, and a rapid antigen test to diagnose COVID-19. J. Clin. Microbiol. 58, 9 (2020).

Notomi. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, 63e- 663 (2000). Osang Healthcare. GeneFinder ™ COVID-19 PLUS Real Amp Kit Instructions for Use. Coronavirus Disease 2019 ( COVID-19 ) Emergency Use Authorizations for Medical Devices 2020, 1-10 (2020).

QIAGEN GmbH. QIAstat-Dx Respiratory SARS-CoV-2 Panel Instructions for Use (Handbook). Coronavirus Disease 2019 ( COVID-19 ) Emergency Use Authorizations for Medical Devices, 1-120 (2020).

Roche Molecular Systems, Inc. cobas SARS-CoV-2 Instructions for Use. (Coronavirus Disease 2019 (COVID-19) Emergency Use Authorizations for Medical Devices, 2020).

U.S. Food & Drug Administration. Coronavirus ( COVID-19) Update: FDA Issues First Emergency Authorization for Sample Poolingin Diagnostic Testing. (2020, July 18).

Wyllie, et al. Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. medRxiv (2020).

Yan, et al. Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay. Clin. Microbiol. Infect. 1, 1-10 (2020).

Yang, et al. Rapid detection of SARS-CoV-2 using reverse transcription RT-LAMP method. medRxiv 395, 565 (2020).

Zhang, et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv 164, 1453 (2020).

Claims

1. A method for detecting the presence of SARS-CoV-2 nucleic acid in a sample comprising: a. contacting the sample with at least one loop-mediated isothermal amplification (LAMP) primer specific for the SARS-CoV-2 virus; b. subjecting the sample and the primers to amplification conditions; c. detecting the presence of amplification product, wherein the presence of amplification product from the primer specific to SARS-CoV-2 virus indicates the presence of nucleic acid from SARS-CoV-2 virus in the sample.

2. The method of claim 1, wherein the at least one LAMP primer comprises the nucleotide sequence chosen from the group consisting of SEQ ID NOs: 1-6.

3. The method of claim 1, wherein the at least one LAMP primer is about 90% identical to the sequences chosen from the group consisting of SEQ ID NOs: 1-6.

4. The method of claim 1 , wherein the sample is chosen from the group consisting of nasal swabs, nasopharyngeal aspirates, oropharyngeal aspirates, feces, saliva, plasma, serum, whole blood, spinal fluid, semen, amniotic fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, and tissue.

5. The method of claim 1, wherein the sample is saliva.

6. The method of claim 5, wherein the saliva is heat inactivated and contacted with the at least one LAMP primer directly.

7. The method of claim 1, wherein the sample is from a human subject.

8. The method of claim 1, further comprising contacting the sample with reverse transcriptase.

9. The method of claim 1, further comprising contacting the sample with a thermostable DNA polymerase to amplify the reaction.

10. The method of claim 1, further comprising contacting the sample with human genomic DNA.

11. The method of claim 1 , further comprising contacting the sample with a DNase, wherein the DNase is inactivated at about 63° C.

12. The method of claim 1, further comprising contacting the sample with dUTP and uracil-DNA N-glycosylase (UDG).

13. The method of claim 1, further comprising contacting the sample with an RNase inhibitor.

14. The method of claim 1, wherein the presence of the amplification products is detected using colorimetry, fluorescence, turbidity or precipitation.

15. The method of claim 1, wherein the method is performed in a volume of about

500 μL.

16. The method of claim 1, wherein the method is performed at a temperature of about 63°C.

17. The method of claim 1, wherein the method is performed for a time of about 30 minutes.

18. The method of claim 1, wherein the method is performed in a high-throughput platform comprising plates with wells, and wherein the method is performed in a volume of about 20 μL and for a time of about 20 minutes.

19. A kit for carrying out the method of any of claims 1-18 comprising at least one LAMP primer chosen from the group consisting of SEQ ID NOs: 1-6.

20. The kit of claim 19, further comprising additional reagents for performing the method chosen from the group consisting of: nucleic acid polymerase; reverse transcriptase; genomic DNA; DNase; RNase inhibitor; dUTP/UDG; ssRNA; reagents for detection of the amplification products; and combinations thereof.

21. The kit of claim 20, wherein the reagents for the detection of the amplification products allow colorimetric detection and are chosen from the group consisting of phenol red, neutral red, cross red, Cresol red, and m-Cresol purple.

22. The kit of claim 20, wherein the reagents for the detection of the amplification products allow colorimetric detection and are chosen from the group consisting of propidium iodide, Picogreen, SYBR green, and Syto 9.

23. The kit of claim 20, further comprising additional equipment for performing the method chosen from the group consisting of: 1.5mL LoBind microcentrifuge tubes; ice and containers to hold ice: a 63.0°C dry bath or heat block; 95°C dry bath or heat block; a mini centrifuge; a mini vortex mixer; and combinations thereof.

24. The kit of claim 20, further comprising control sequences and instructions for use.

25. The kit of claim 20, wherein the LAMP primers and additional reagents are contained in wells of a plate used for high throughput platforms.

26. The kit of claim 25, further comprising LAMP primers for controls chosen from the group consisting of RNase P and actin.

27. A synthetic nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6.

28. A method for detecting the presence of a nucleic acid from bacteria or virus in a sample comprising: a. contacting the sample with at least one loop-mediated isothermal amplification (LAMP) primer specific for the bacteria or virus; b. subjecting the sample and the primers to amplification conditions, wherein the amplification conditions include contacting the sample with a thermostable DNA polymerase; c. contacting the sample with additional reagents selected from the group consisting of human genomic DNA, DNase, RNase inhibitor, dUTP/UDG, and combinations thereof; and c. detecting the presence of amplification product, wherein the presence of amplification product from the primer specific to bacteria or virus indicates the presence of nucleic acid from bacteria or virus in the sample, and wherein the method is performed at a temperature of about 63 °C for about 30 minutes and at a volume of about 500 μL.

29. The method of claim 28, wherein the sample is chosen from the group consisting of nasal swabs, nasopharyngeal aspirates, oropharyngeal aspirates, feces, saliva, plasma, serum, whole blood, spinal fluid, semen, amniotic fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, and tissue.

30. The method of claim 28, wherein the sample is saliva.

31. The method of claim 30, wherein the saliva is heat inactivated and contacted with the at least one LAMP primer directly.

32. The method of claim 28, wherein the sample is from a human subject.

33. The method of claim 28, further comprising contacting the sample with reverse transcriptase.

34. The method of claim 28, wherein the presence of the amplification products is detected using colorimetry, fluorescence, turbidity or precipitation.

35. The method of claim 28, wherein the method is performed in a high- throughput platform comprising plates with wells, and wherein the method is performed in a volume of about 20 μL and for a time of about 20 minutes.

36. The method of claim 28, wherein the LAMP primers are designed to amplify the center of the genome of the bacteria or virus.

37. A kit for carrying out the method of any of claims 28-36.

38. The kit of claim 37, wherein the reagents for the detection of the amplification products allow colorimetric detection and are chosen from the group consisting of phenol red, neutral red, cross red, Cresol red, and m-Cresol purple.

39. The kit of claim 37, wherein the reagents for the detection of the amplification products allow colorimetric detection and are chosen from the group consisting of propidium iodide, Picogreen, SYBR green, and Syto 9.

40. The kit of claim 37, further comprising additional equipment for performing the method chosen from the group consisting of: 1.5mL LoBind microcentrifuge tubes; ice and containers to hold ice: a 63.0°C dry bath or heat block; 95°C dry bath or heat block; a mini centrifuge; a mini vortex mixer; and combinations thereof.

41. The kit of claim 37, further comprising control sequences and instructions for use.

42. The kit of claim 37, wherein the LAMP primers and additional reagents are contained in wells of a plate used for high throughput platforms.