WO2022036025A2 - Rt-lamp assays to detect sars-cov-2 rna using molecular beacons - Google Patents

Abstract

Provided are compositions comprising molecular beacons, a reverse transcriptase and a polymerase and methods for detecting and assessing SARS-CoV-2 virus infection in a sample from a subject. The disclosed methods comprise a loop-mediated isothermal amplification reaction using molecular beacons specific for the SARS-CoV-2 virus. In some aspect, the provided herein are methods for multiplexed detection SARS-CoV-2 virus. Also provided herein are kits comprising the disclosed compositions.

Description

RT-LAMP ASSAYS TO DETECT SARS-COV-2 RNA USING MOLECULAR BEACONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. provisional application number 63/064,734, filed August 12, 2020, and to U.S. provisional application number 63/137,953, filed January 15, 2021, the disclosures of which foregoing applications are incorporated herein by reference in their entireties for any and all purposes.

SEQUENCE LISTINGS

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 11, 2021, is named 103241_006760_SL.txt and is 34,723 bytes in size.

FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of detecting coronavirus using RT- LAMP with molecular beacons.

BACKGROUND OF THE INVENTION

[0004] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but tests are expensive and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and Loop Mediated Isothermal Amplification (RT-LAMP) provides an alternative that uses alternative and often cheaper reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to detect bulk amplification of DNA; however a common artifact is nonspecific DNA amplification, complicating detection. Methods have been proposed based on DNA sequencing or CAS enzymes. These methods are promising, but as presently designed they typically require opening of RT-LAMP tubes and secondary manipulation of reaction products, which has the potential to result in contamination of subsequent reactions with amplification products from previous assays. [0005] There is an urgent need for fast and effective methods for detecting and monitoring coronavirus infections. The present disclosure addresses these needs.

SUMMARY OF THE INVENTION

[0006] In meeting these long-felt needs, the present disclosure provides compositions comprising molecular beacons. In one aspect, the compositions comprise at least one molecular beacon that comprises at least one nucleic acid sequence of SEQ ID NOs. 1-80. In another aspect, the compositions comprise at least one molecular beacon that comprises at least one of nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77- 80 and further comprises at least one locked nucleic acid (LNA). In another aspect, the compositions comprise a DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P. In yet another aspect, the compositions comprise a reverse transcriptase comprising an amino acid sequence of SEQ ID NO. 82.

[0007] Also disclosed herein are methods for detecting a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in a sample from a subject. The method comprises, with molecular beacons, carrying out a reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on a reaction mixture comprising the sample; incubating the reaction mixture under polymerase reaction conditions to produce a reaction product comprising amplified SARS-CoV-2 nucleic acid; and, detecting the reaction product.

[0008] Methods are also provided herein for detecting multiplexed detection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in two or more samples from two or more subjects. The methods comprise, with molecular beacons, carrying out within a same reaction mixture a reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on each of the two or more samples, incubating the reaction mixture under polymerase reaction conditions to produce a reaction product comprising amplified SARS-CoV- 2 nucleic acid for each of the two or more samples; and, detecting the reaction product.

[0009] Methods are also disclosed for monitoring a response to a medication in a subject need thereof. The methods comprising obtaining a first sample from the subject at a first time point; obtaining a second sample from the subject at a second time point following administration of the medication to the subject; determining an amount of SARS-CoV-2 virus nucleic acids in the first and second samples, wherein the determining comprises performing, with molecular beacons, reverse transcription-based loop mediated isothermal amplification (RT- LAMP) (i) on a reaction mixture comprising the first sample and (ii) on a reaction mixture comprising the second sample; incubating the reaction mixtures (i) and (ii) under polymerase reaction conditions to produce a first and a second reaction product, respectively, comprising amplified SARS-CoV-2 nucleic acids for each of the first and second samples, respectively; and, comparing the amount of SARS-CoV-2 virus nucleic acids in the first and second reaction products, wherein a decrease in the amount of SARS-CoV-2 virus nucleic acids from the first reaction product relative to the second reaction product indicates a positive response to the medication.

[0010] Kits are also provided comprising the disclosed composition of molecular beacons; packaging for the compositions; and, instructions to use thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed compositions, methods, and kits there are shown in the drawings exemplary embodiments of compositions, methods and kits; however, these should not be limited to the specific embodiments disclosed.

[0013] In the drawings:

[0014] Figure 1. LAMP-BEAC: RT-LAMP assayed using molecular beacons. The molecular beacon used is shown at the top in the annealed hairpin form, which is quenched. Binding of the beacon to the target complementary sequence separates the fluorescent group and the quencher, allowing detection of fluorescence. The red loops on the beacon indicate locked nucleic acids used to increase binding affinity.

[0015] Figures 2A-2D. Reaction progression curves comparing RT-LAMP assayed using an intercalating dye and LAMP-BEAC. Fig. 2A) Conventional RT-LAMP assay detecting SARS-CoV-2 RNA. Time after reaction initiation is shown on the x-axis, relative fluorescence intensity is shown on the y-axis. The RNA used was the Twist commercial positive control; copy numbers in the reaction mixture are shown in the key at the bottom. Fig. 2B) Detection of SARS- CoV-2 RNA using LAMP-BEAC. Markings are as in Fig. 2A). Fig. 2C) and Fig. 2D)Thermal melting curves to characterize amplification products. The results shown are for reactions in A and B; the key to samples tested is at the bottom. Reaction products were cooled to room temperature, then slowly heated for the melt curve analysis. Fig. 2C) Characterization of products generated using conventional RT-LAMP and the Twist RNA template. The x-axis shows the temperature, the y-axis shows fluorescence intensity. Fig. 2D) Characterization of products generated using LAMP-BEAC. Markings are as in Fig. 2A).

[0016] Figures 3A-3H. A multiplex LAMP-BEAC method assaying four amplicons. Assays were carried out using a LAMP-BEAC amplicon to detect human STATH RNA (A) and three amplicons to detect SARS-CoV-2 (Figs. 3B-3D). For these assays, synthetic SARS-CoV-2 RNA was diluted into saliva (inactivated as described by Rabe et al., 2020); copies per microliter are shown by the color code in the lower right. For Figs. 3A-3D, the x-axis shows time after starting the assay, and the y-axis shows fluorescence intensity. Figs. 3E-3H shows melt curve analysis for samples in Figs. 3A-3D. For Figs. 3E-3H, the x-axis shows temperature, and the y- axis shows fluorescence intensity.

[0017] Figures 4A-4C. Validation of multiplexed LAMP-BEAC on 20 clinical samples using multiplexed assays. Fig. 4A) and Fig. 4B) Fluorescence was measured after an hour of LAMP amplification and the maximum endpoint fluorescence observed in any amplification for Asle (Fig. 4A) or Penn (Fig. 4B) lamp primers (y-axis) was compared to previously measured qPCR measurements of SARS-CoV-2 copy number from the same samples (x-axis). Vertical dotted line indicates 100 copies/ml corresponding to approximately 10,000 RNA copies/ml in the unpurified NP swab eluate. Horizontal dashed line indicates a potential LAMP cutoff of two times the highest fluorescence seen in uninfected saliva controls. Fig. 4C) In the same LAMP reactions, the fluorescence of human STATH gene targeted LAMP beacons was measured and the maximum endpoint value observed compared to those seen in water controls. Dashed line indicates two times the highest fluorescence seen in the water controls.

[0018] Figure 5A-X. Reaction progression curves for LAMP-BEAC reactions carried out on clinical saliva samples. Each column represents a single sample assayed. Sample names are listed in FIGs. 7A-7B. Heavy boxes indicate positive samples.

[0019] Figures. 6A-6P. Comparing laboratory-purified and commercial polymerase enzymes using a quadruplex LAMP-BEAC assay of saliva samples spiked with synthetic SARS- CoV-2 RNA. Assays were carried out using an amplicon to detect human STATH RNA (A) and three amplicons to detect SARS-CoV-2 (Figs. 6B-6D). For these assays, synthetic SARS-CoV-2 RNA was diluted into saliva (inactivated as described by Rabe et al., 2020); copies per microliter are shown by the color code in the lower right. For Figs. 6A-6D, the x-axis shows time after starting the assay, and the y-axis shows fluorescence intensity. Figs. 6E-6H shows melt curve analysis for samples in Figs. 6A-6D. For Figs. 6E-6H, the x-axis shows temperature, and the y- axis shows fluorescence intensity. (Figs. 6I-6P) Assays are exactly as in Figs. 6A-6H, but commercial reverse transcriptase and DNA polymerase (NEB Warm Start LAMP Kit master mix product number E1700L) were used instead of the laboratory purified polymerases

[0020] FIGs. 7A-7B. List of clinical samples and results of assays for SARS-CoV-2 and human RNA.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0021] The disclosed compositions, methods, kits, and devices may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed compositions, methods, kits, and devices are not limited to the specific compositions, methods, kits, and devices described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed compositions, methods and kits.

[0022] Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed compositions, methods, kits, and devices are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

[0023] Throughout this text, the descriptions refer to compositions and methods of using said compositions. Where the disclosure describes or claims a feature or embodiment associated with a composition, such a feature or embodiment is equally applicable to the methods of using said composition. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using a composition, such a feature or embodiment is equally applicable to the composition.

[0024] When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Further, reference to values stated in ranges include each and every value within that range. All ranges are inclusive and combinable. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0025] It is to be appreciated that certain features of the disclosed compositions, methods and kits which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions, methods and kits that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0026] Definitions

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, certain preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0028] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0029] As used herein, the articles “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0030] As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0031] The term “sample” or “biological sample” refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample can be of any biological tissue or fluid. Frequently the sample will be a “clinical sample” which is a sample derived from a patient. Such samples include, but are not limited to, nasal cells, buccal cells, saliva, bone marrow, cardiac tissue, sputum, blood, serum, breast milk, cerebrospinal fluid, endolymph, perilymph gastric juice, mucus, lymphatic fluid, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, bile, sebum, semen, sweat, tears, vaginal secretion, vomit, peritoneal fluid, and pleural fluid, or cells therefrom. Samples can also include sections of tissues such as frozen sections taken for histological purposes.

[0032] As used herein, “higher” refers to expression levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or more, and/or 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold higher or more, and any and all whole or partial increments therebetween, than a control reference. A disclosed herein an expression level higher than a reference value refers to an expression level (mRNA or protein) that is higher than a normal or control level from an expression (mRNA or protein) measured in a healthy subject or defined or used in the art. [0033] As used herein, “lower” refers to expression levels which are at least 10% lower or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower or more, and/or 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold lower or more, and any and all whole or partial increments in between, than a control reference. A disclosed herein an expression level lower than a reference value refers to an expression level (mRNA or protein) that is lower than a normal or control level from an expression (mRNA or protein) measured in a healthy subject or defined or used in the art.

[0034] As used herein, the terms “control,” or “reference” can be used interchangeably and refer to a value that is used as a standard of comparison.

[0035] The term “SARS-CoV-2” refers to the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which belongs to the genus of P virus belonging to the family of Coronaviridae. SARS-CoV-2 has homology of only 79.5% and 40% with SARS-Cov and MERS-Cov. SARS-CoV-2 (which causes the disorder COVID- 19) can cause severe pneumonia in human.

[0036] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0037] The term “treatment” as used within the context of the present invention is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for the disease or disorder. As used herein, the term “treatment” and associated terms such as “treat” and “treating” means the reduction of the progression, severity and/or duration of a disease condition or at least one symptom thereof. The term ‘treatment’ therefore refers to any regimen that can benefit a subject. The treatment can be in respect of an existing condition or can be prophylactic (preventative treatment). Treatment can include curative, alleviative or prophylactic effects. References herein to “therapeutic” and “prophylactic” treatments are to be considered in their broadest context. The term “therapeutic” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylactic” does not necessarily mean that the subject will not eventually contract a disease condition. Thus, for example, the term treatment includes the administration of an agent prior to or following the onset of a disease or disorder thereby preventing or removing all signs of the disease or disorder. As another example, administration of the agent after clinical manifestation of the disease to combat the symptoms of the disease comprises “treatment” of the disease.

[0038] As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. ESTs, chromosomes, cDNAs, mRNAs, and rRNAs are representative examples of molecules that can be referred to as nucleic acids.

[0039] A “subject” or “patient,” as used therein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is a human.

[0040] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0041] Description

[0042] The present invention includes devices, compositions, methods and kits suited for both laboratory use and point-of-care settings.

[0043] The compositions disclosed herein comprise molecular beacons specific for SARS-CoV-2 RNA. In one aspect, the compositions comprise at least one molecular beacon that comprises at least one nucleic acid sequence of SEQ ID NOs. 1-80. In one embodiment, the compositions comprise (1) a molecular beacon that comprises one nucleic acid sequence of SEQ ID NOs. 1-80 and (2) a molecular beacon that comprises another nucleic acid sequence of SEQ ID NOs. 1-80.

[0044] In another aspect, the compositions comprise at least one molecular beacon that comprises at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and further comprises at least one locked nucleic acid (LNA). In one embodiment, the compositions comprise (1) a molecular beacon that comprises one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and at least one LNA and (2) a molecular beacon that comprises another nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and at least one LNA.

[0045] In some embodiments, the compositions comprise at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30 or more molecular beacons. In some embodiments, at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30 or more molecular beacons comprise at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30 or more locked nucleic acid (LNA).

[0046] The molecular beacons contemplated by the present disclosure are listed on Table 1 below. The molecular beacons of the present invention include not only the sequences included in the compositions described herein but also others having between about 99% and about 60% sequence homology with the molecular beacons included in the compositions. For example, a molecular beacon that differs from those sequences provided herein by a few nucleotides but still shares about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more sequence homology would be understood by one skilled in the art to be included in the present invention as long as it is capable of specifically hybridizing to and/or detecting the SARS-CoV-2 RNA.

[0047] In some embodiments, the molecular beacons comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 or more nucleic acids. In some embodiments, the molecular beacon comprises 5' fluorophore at the 5' end with a fluorescent dye that is covalently attached and 3' quencher (non fluorescent) dye that is covalently attached to the 3' end of the molecular beacon; or also the placement of the dyes at opposite ends (fluorophore at 3’ end and quencher at 5’ end).

[0048] In some embodiments, the molecular beacons comprise one or more fluorescently labeled nucleic acids. In some embodiments, the molecular beacons comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more fluorescently labeled nucleic acids. In some embodiments, the nucleic acids are labeled by a cyan, a green, a yellow, a red or a blue fluorescent dye. Fluorescent dyes are known in the art and include but are not limited to cyanine dyes (e.g. Cy2, Cy3, or Cy5) or SYTO9™ (Invitrogen), or Alexa Fluor™ (ThermoFisher) dyes.

[0049] Table 1: Oligonucleotides used in the disclosed LAMP-BEAC assay.

Bold font in Table 1 refers to nucleic acid sequences that comprise at least one LNA and that are tested herein (i.e. SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77- 80). [0050] In one aspect, the compositions disclosed herein comprise a DNA polymerase large fragment lacking the 5 ’-exonuclease domain from strain 10 of bacterium Geobacillus stearothermophilus . In one embodiment, the DNA polymerase large fragment from strain 10 of bacterium Geobacillus stearothermophilus comprises at least one mutation in the Bst polA gene, such as, but not limited to, a D434A mutation, a R445A mutation, or a R445P mutation within SEQ ID NO. 81 (listed below). In another embodiment, the compositions disclosed herein comprise a DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81 as listed below.

[0051] BstlO_D434A_polALF amino acid sequence (SEQ ID NO. 81):

[0052] MESPSSEEEKPLAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPI VGIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGV SFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHL VRKAAAIWALERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGEE LAEQLRTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAP YHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIP IRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAM DIFQVSEDEVTPNMRRQAKAVNFGIVYGISAYGLAQNLNISRKEAAEFIERYFESFPGVK RYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKK AMIDLNARLKEERLQARLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVD YHYGSTWYDAK.

[0053] In another aspect, the compositions disclosed herein comprise a reverse transcriptase (RT) from human immunodeficiency virus 2 (HIV2). In one embodiment, the RT disclosed herein comprises substitutions relative to the wild type HIV2 RT. In one embodiment, the substitutions comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids relative to the wild type HIV2 RT. In one embodiment, the substitutions comprise at least 10, 11, 12, 13, 14, 15, 16, or more amino acids. In one embodiment, the substitutions comprise at least 13, or at least 14 or more amino acids relative to the wild type HIV2 RT (SEQ ID NO. 83, as listed below). In one embodiment, the substitutions comprise the following amino acids and positions relative to the wild type HIV2 RT (SEQ ID NO. 83): Ml IT; V167I; Ml 84V; G211N; I250V; M287L; E396D; I397T; G431K; A437T; V458I; S515N; I517L; and, I526T. In yet another embodiment, the compositions disclosed herein comprise a reverse transcriptase comprising an amino acid sequence of SEQ ID NO. 82 as listed below. In yet another embodiment, the substitutions provide the presently disclosed HIV2 RT (also referenced as HIV2 RTMOD2, SEQ ID NO. 82) with a higher thermostability as compared to the wild type.

[0054] HIV2 RTMOD2 amino acid sequence (SEQ ID NO. 82):

[0055] AVAKVEPIKITLKPGKDGPKLRQWPLTKEKIEALKEICEKMEKEGQLEE APPTNPYNTPTFAIKKKDKNKWRMLIDFRELNKVTQDFTEIQLGIPHPAGLAKKRRITVL DVGDAYFSIPLHEDFRPYTAFTLPSVNNAEPGKRYIYKVLPQGWKGSPAIFQHTMRQILE PFRKANKDVIIIQYVDDILIASDRTDLEHDRVILQLKELLNNLGFSTPDEKFQKDPPYHW MGYELWPTKWKLQKIQLPQKEVWTVNDIQKLVGVLNWAAQLYPGIKTKHLCRLIRGK LTLTEEVQWTELAEAELEENRIILSQEQEGHYYQEEKELEATVQKDQDNQWTYKIHQED KILKVGKYAKVKNTHTNGIRLLAQVVQKIGKEALVIWGRIPKFHLPVERDTWEQWWDN YWQVTWIPDWDFVSTPPLVRLAFNLVKDPIPGTETFYTDGSCNRQSKEGKAGYITDRGK DKVKKLEQTTNQQAELEAFAMALTDSGPKVNIIVDSQYVMGIVASQPTESENKLVNQIIE EMTKKEAIYVAWVPAHKGIGGNQEVDHLVSQGIRQVL

[0056] HIV2 RT LOCUS amino acid sequence (SEQ ID NO. 83):

[0057] AVAKVEPIKIMLKPGKDGPKLRQWPLTKEKIEALKEICEKMEKEGQLEE APPTNPYNTPTFAIKKKDKNKWRMLIDFRELNKVTQDFTEIQLGIPHPAGLAKKRRITVL DVGDAYFSIPLHEDFRPYTAFTLPSVNNAEPGKRYIYKVLPQGWKGSPAIFQHTMRQVL EPFRKANKDVIIIQYMDDILIASDRTDLEHDRVILQLKELLNGLGFSTPDEKFQKDPPYHW MGYELWPTKWKLQKIQLPQKEIWTVNDIQKLVGVLNWAAQLYPGIKTKHLCRLIRGK MTLTEEVQWTELAEAELEENRIILSQEQEGHYYQEEKELEATVQKDQDNQWTYKIHQE DKILKVGKYAKVKNTHTNGIRLLAQVVQKIGKEALVIWGRIPKFHLPVEREIWEQWWD NYWQVTWIPDWDFVSTPPLVRLAFNLVGDPIPGAETFYTDGSCNRQSKEGKAGYVTDR GKDKVKKLEQTTNQQAELEAFAMALTDSGPKVNIIVDSQYVMGIVASQPTESESKIVNQ IIEEMIKKEAIYVAWVPAHKGIGGNQEVDHLVSQGIRQVL

[0058] The present disclosure does not require expensive machinery, and the methods are specifically designed to yield unambiguous results when determining if an individual is infected with SARS-CoV-2 virus.

[0059] The methodology presented herein is based on isothermal nucleic acid amplification and provides a significant advantage in relation to sensitivity and selectivity over other methods of detecting SARS-CoV-2 virus infection. Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is one example of an isothermal amplification technique that uses RNA as a template to generate complimentary DNA strands independently of any expensive thermocycler machinery. Loop-mediated isothermal amplification reactions have been described previously (Notomi et al., 28 Nucleic Acid Research, 2000; Mori and Notomi J Infect Chemother 15, 62-69, 2009). RT-LAMP requires carefully selected primers to exclusively amplify desired regions of a genome. For the present disclosure, molecular beacons were designed that anneal to RNA sequences unique to the SARS-CoV-2 (See Table 1 listed elsewhere herein). In some embodiments, the RT-LAMP reaction can be carried out with at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30 or more molecular beacons. In some embodiments, the disclosed methods contemplate using at least one nucleic acid sequence of SEQ ID NOs. 1-80. In some embodiments, the molecular beacons comprise at least one locked nucleic acid (LNA). In other embodiments, molecular beacons comprise LNA modifications and comprise at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77- 80.

[0060] While the compositions listed above are useful in RT-LAMP amplification of SARS-CoV-2virus nucleic acids, the present disclosure also encompasses the individual disclosed molecular beacons, the individual disclosed polymerase and the individual disclosed reverse transcriptase. Thus, one aspect of the invention encompasses any nucleic acid or any amino acid sequence as set forth in the compositions described herein. Furthermore, the compositions disclosed herein can be used in other assays such as, but not limited to, real-time PCR (RT-PCR), or multiplex PCR.

[0061] In one aspect, disclosed herein are methods for detecting a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in a sample from a subject. The method comprises, with molecular beacons, carrying out a reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on a reaction mixture comprising the sample; incubating the reaction mixture under polymerase reaction conditions to produce a reaction product comprising amplified SARS-CoV-2 nucleic acid; and, detecting the reaction product.

[0062] In another aspect, methods are also provided herein for detecting multiplexed detection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in two or more samples from two or more subjects. The methods comprise, with molecular beacons, carrying out within a same reaction mixture a reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on each of the two or more samples, incubating the reaction mixture under polymerase reaction conditions to produce a reaction product comprising amplified SARS-CoV-2 nucleic acid for each of the two or more samples; and, detecting the reaction product. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more samples can be multiplexed. In some embodiments, the samples are taken from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more subjects.

[0063] In further aspects, methods are also disclosed for monitoring a response to a medication in a subject need thereof. The methods comprising obtaining a first sample from the subject at a first time point; obtaining a second sample from the subject at a second time point following administration of the medication to the subject; determining an amount of SARS-CoV- 2 virus nucleic acids in the first and second samples, wherein the determining comprises performing, with molecular beacons, reverse transcription-based loop mediated isothermal amplification (RT-LAMP) (i) on a reaction mixture comprising the first sample and (ii) on a reaction mixture comprising the second sample; incubating the reaction mixtures (i) and (ii) under polymerase reaction conditions to produce a first and a second reaction product, respectively, comprising amplified SARS-CoV-2 nucleic acids for each of the first and second samples, respectively; and, comparing the amount of SARS-CoV-2 virus nucleic acids in the first and second reaction products, wherein a decrease in the amount of SARS-CoV-2 virus nucleic acids from the first reaction product relative to the second reaction product indicates a positive response to the medication.

[0064] In some embodiments, the medication comprises an antiviral therapy.

[0065] In some embodiments, the sample comprises a nasal, buccal, saliva or fecal sample. In other embodiments, the nasal sample comprises nasal cells or nasal secretion. In other embodiments, the buccal sample comprises buccal cells or saliva. The sample(s) useful for the disclosed methods is collected from a subject. In some embodiments, the subject is a mammal that can be a human or non-human mammal. In some embodiments, the subject is a human.

[0066] In some embodiments, the sample is not manipulated after collection. In some embodiments, the sample does not undergo nucleic acid extraction, purification and/or nucleic acid amplification prior to the RT-LAMP.

[0067] In some embodiments, detecting the reaction product comprises contacting the reaction product with an intercalating fluorescent dye. In some embodiments, detecting the reaction product comprises monitoring a fluorescent signal to allow quantitative and qualitative analysis of the presence and/or amount of SARS-CoV-2 virus nucleic acids. [0068] Kit

[0069] The present disclosure provides kits comprising the disclosed composition of molecular beacons, of polymerase and/or of reverse transcriptase; packaging for the compositions; and, instructions to use thereof.

[0070] In certain embodiments, the disclosed kits comprise a detection reagent that is suitable for detecting or monitoring SARS-CoV-2 virus nucleic acids. The detection reagents comprise for example fluorescent dyes associated with molecular beacons.

[0071] Aspects - 1

[0072] Aspect 1.1. A composition, comprising at least one molecular beacon that comprises at least one of nucleic acid sequences of SEQ ID NOs. 1-80.

[0073] Aspect 1.2. The composition of aspect 1.1 , wherein the composition comprises (1) a molecular beacon that comprises one nucleic acid sequence of SEQ ID NOs. 1-80, and (2) a molecular beacon that comprises another nucleic acid sequence of SEQ ID NOs. 1-80.

[0074] Aspect 1.3. A composition, comprising at least one molecular beacon that comprises at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and further comprises at least one locked nucleic acid (LNA).

[0075] Aspect 1.4. The composition of aspect 1.3, wherein the composition comprises (1) a molecular beacon that comprises one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and at least one LNA and (2) a molecular beacon that comprises another nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and at least one LNA.

[0076] Aspect 1.5. A DNA polymerase composition comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P.

[0077] Aspect 1.6. A reverse transcriptase composition comprising an amino acid sequence of SEQ ID NO. 82.

[0078] Aspect 1.7. A method for detecting a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in a sample from a subject, the method comprising: a) with molecular beacons, carrying out a reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on a reaction mixture comprising the sample; b) incubating the reaction mixture under polymerase reaction conditions to produce a reaction product comprising amplified SARS-CoV-2 nucleic acid; and, c) detecting the reaction product. [0079] Aspect 1.8. A method for multiplexed detection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in two or more samples from two or more subjects, the method comprising: a) with molecular beacons, carrying out within a same reaction mixture a reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on each of the two or more samples; b) incubating the reaction mixture under polymerase reaction conditions to produce a reaction product comprising amplified SARS-CoV-2 nucleic acid for each of the two or more samples; and, c) detecting the reaction product.

[0080] Aspect 1.9. A method of using a sample to monitor a response to a medication in a subject in need thereof, the method comprising: a) obtaining a first sample from the subject at a first time point; b) obtaining a second sample from the subject at a second time point following administration of the medication to the subject; c) determining an amount of SARS-CoV-2 virus nucleic acids in the first and second samples, wherein the determining comprises performing, with molecular beacons, reverse transcription-based loop mediated isothermal amplification (RT-LAMP) (i) on a reaction mixture comprising the first sample and (ii) on a reaction mixture comprising the second sample; d) incubating the reaction mixtures (i) and (ii) under polymerase reaction conditions to produce a first and a second reaction product, respectively, comprising amplified SARS- CoV-2 nucleic acids for each of the first and second samples, respectively; and, e) detecting and comparing the amount of SARS-CoV-2 virus nucleic acids in the first and second reaction products, wherein a decrease in the amount of SARS-CoV-2 virus nucleic acids from the first reaction product relative to the second reaction product indicates a positive response to the medication.

[0081] Aspect 1.10. The method of aspect 1.9, wherein the medication comprises an antiviral therapy.

[0082] Aspect 1.11. The method of any one of aspects 1.7- 1.10, wherein the molecular beacons comprise at least one of SEQ ID NOs. 1-80.

[0083] Aspect 1.12. The method of any one of aspects 1.7- 1.11, wherein the molecular beacons comprise at least one locked nucleic acid (LNA). [0084] Aspect 1.13. The method of any one of aspects 1.7- 1.12, wherein the molecular beacons comprise LNA modifications and comprise at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80.

[0085] Aspect 1.14. The method of any one of aspects 1.7- 1.13, wherein the reverse transcription reaction is performed using a RT comprising an amino acid sequence of SEQ ID NO. 82.

[0086] Aspect 1.15. The method of any one of aspects 1.7- 1.14, wherein the polymerase reaction is performed using a DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P.

[0087] Aspect 1.16. The method of any one of aspects 1.7- 1.15, wherein the sample comprises a nasal or a buccal sample.

[0088] Aspect 1.17. The method of aspect 1.16, wherein the buccal sample comprises saliva.

[0089] Aspect 1.18. The method of any one of aspects 1.7- 1.17, wherein the sample does not undergo nucleic acid purification and/or nucleic acid amplification prior to the RT- LAMP.

[0090] Aspect 1.19. The method of any one of aspects 1.7- 1.18, wherein the molecular beacons comprise one or more fluorescently labeled nucleic acids.

[0091] Aspect 1.20. The method of any one of aspects 1.7- 1.19, wherein detecting the reaction product comprises contacting the reaction product with an intercalating fluorescent dye.

[0092] Aspect 1.21. The method of any one of aspects 1.7- 1.20, wherein detecting the reaction product comprises monitoring a fluorescence.

[0093] Aspect 1.22. The method of any one of aspects 1.7- 1.21, wherein the subject is a mammal.

[0094] Aspect 1.23. The method of aspect 1.22, wherein the mammal is a human.

[0095] Aspect 1.24. A kit, comprising: the composition of at least one of aspects 1.1- 1.6; packaging for the composition; and, instructions to use thereof.

[0096] Aspects - II

[0097] Aspect II.1. A composition, comprising at least one molecular beacon that comprises at least one of nucleic acid sequences of SEQ ID NOs. 1-80. [0098] Aspect II.2. The composition of aspect II.1, wherein the composition comprises

(1) a molecular beacon that comprises one nucleic acid sequence of SEQ ID NOs. 1-80, and (2) a molecular beacon that comprises another nucleic acid sequence of SEQ ID NOs. 1-80.

[0099] Aspect II.3. The composition of aspect II.1 or aspect II.2 comprising at least one molecular beacon that comprises at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and further comprises at least one locked nucleic acid (LNA).

[00100] Aspect II.4. The composition of any one of aspects II.1- II.3, wherein the composition comprises (1) a molecular beacon that comprises one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and at least one LNA and

(2) a molecular beacon that comprises another nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and at least one LNA.

[00101] Aspect II.5. The composition of any one of aspects II.1- II.4, further comprising DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P.

[00102] Aspect II.6. The composition of any one of aspects II.1- II.5, further comprising a reverse transcriptase comprising an amino acid sequence of SEQ ID NO. 82.

[00103] Aspect II.7. A method for detecting a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in a sample from a subject, the method comprising: a) with molecular beacons, carrying out a reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on a reaction mixture comprising the sample; b) incubating the reaction mixture under polymerase reaction conditions to produce a reaction product comprising amplified SARS-CoV-2 nucleic acid; and, c) detecting the reaction product.

[00104] Aspect II.8. A The method of aspect II.7, wherein the method is for multiplexed detection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in two or more samples from two or more subjects in same reaction mixture.

[00105] Aspect II.9. The method of aspect II.7 or II.8 for monitoring a response to a medication in a subject in need thereof, the method further comprising: obtaining a first sample from the subject at a first time point; obtaining a second sample from the subject at a second time point following administration of the medication to the subject; and performing steps a)-c) for the first sample and the second sample. [00106] Aspect II.10. The method of any one of aspects II.7- II.9, further comprising comparing the amount of SARS-CoV-2 virus nucleic acids in the first and second reaction products, wherein a decrease in the amount of SARS-CoV-2 virus nucleic acids from the first reaction product relative to the second reaction product indicates a positive response to the medication.

[00107] Aspect II.11. The method of aspect II.9 or II.10, wherein the medication comprises an antiviral therapy.

[00108] Aspect II.12. The method of any one of aspects II.7- II.11, wherein the molecular beacons comprise at least one of SEQ ID NOs. 1-80.

[00109] Aspect II.13. The method of any one of aspects II.7- II.12, wherein the molecular beacons comprise at least one locked nucleic acid (LNA).

[00110] Aspect II.14. The method of any one of aspects II.7- II.13, wherein the molecular beacons comprise LNA modifications and comprise at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80.

[00111] Aspect II.15. The method of any one of aspects II.7- II.14, wherein the reverse transcription reaction is performed using a RT comprising an amino acid sequence of SEQ ID NO. 82.

[00112] Aspect II.16. The method of any one of aspects II.7- II.15, wherein the polymerase reaction is performed using a DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P.

[00113] Aspect II.17. The method of any one of aspects II.7- II.16, wherein the sample comprises a nasal or a buccal sample.

[00114] Aspect II.18. The method of aspect II.17, wherein the buccal sample comprises saliva.

[00115] Aspect II.19. The method of any one of aspects II.7- II.18, wherein the sample does not undergo nucleic acid purification and/or nucleic acid amplification prior to the RT- LAMP.

[00116] Aspect 11.20. The method of any one of aspects II.7- II.19, wherein the molecular beacons comprise one or more fluorescently labeled nucleic acids.

[00117] Aspect 11.21. The method of any one of aspects II.7- 11.20, wherein detecting the reaction product comprises contacting the reaction product with an intercalating fluorescent dye. [00118] Aspect 11.22. The method of any one of aspects II.7- 11.21, wherein detecting the reaction product comprises monitoring a fluorescence.

[00119] Aspect 11.23. The method of any one of aspects II.7- 11.22, wherein the subject is a mammal.

[00120] Aspect 11.24. The method of claim 11.23, wherein the mammal is a human.

[00121] Aspect 11.25. A composition comprising at least one molecular beacon that comprises at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and further comprises at least one locked nucleic acid (LNA).

[00122] Aspect 11.26. A DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P.

[00123] Aspect 11.27. A reverse transcriptase comprising an amino acid sequence of SEQ ID NO. 82.

[00124] Aspect 11.28. A kit, comprising: the composition of at least one of aspects II.1- II.6 or aspects 11.25- 11.27; packaging for the composition; and, instructions to use thereof.

[00125] Examples

[00126] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[00127] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[00128] Design of Molecular Beacons

[00129] Beacons were designed to detect product generated using previously published LAMP primer sets. To design beacons targeting the loop region of the LAMP amplification product, the forward inner primers (FIP) and backward inner primers (BIP) were mapped to the SARS-CoV-2 genome to find the entire forward and backward loop regions of the amplicon (potentially including regions outside the original loop forward (LF) and loop backward (LB) primers). Then, GC-rich subsequences were selected within these loops and selected bases for LNA modification based on the predicted change in melting temperature using a stepwise greedy heuristic of consecutively adding the LNA with the highest predicted Tm. Additional nucleotides were then added to the 5’ and 3’ ends to form a hairpin with predicted melting temperature between 60-65 °C. Where possible terminal bases of the target sequence were used as part of the hairpin. To allow easy and relatively affordable synthesis, beacons were kept shorter than 25 nt with 6 locked nucleic acids.

[00130] Design and purification of polymerases

[00131] Initially, the D434A mutant of the polA large fragment from Geobacillus stearothermophilus was chosen for LAMP due to its high strand displacement activity [20], Using strain DSM 13240, the polymerase coding sequence was amplified from genomic DNA, the D434A substitution was incorporated, and the construct was ligated into CDFDuet (Novagen) in-frame with an N-terminal hexahistidine tag. To explore alternative DNA polymerases for LAMP, R445A and R445P variants were generated, each of which results in disruption of the salt-bridge formed with Asp434 in the wild-type enzyme (Asp422 in pdb entry 1XWL). The polymerases were expressed in strain BL21(DE3) at 37°C for 3 h and purified using Talon (Clontech), heparin sepharose (GE), and MonoQ (GE) chromatography. Purified Bst-LF mutants were concentrated, glycerol added to 10%, and aliquots were flash frozen and stored at -80°C. Primer extension assays using Ml 3 DNA template and ³H-dTTP labeled dNTPs were used to establish specific activity as described for commercially prepared Bst (NEB).

[00132] To demonstrate that RT-LAMP can be performed using a reverse transcriptase generated in-house, a synthetic gene was first constructed for the HIV1 RT p66 (strain NL4-3) subunit containing substitutions expected to confer thermal stability (RTx; NEB). The p66 sequence was inserted into pET29b and the p51 subunit coding sequence was amplified by PCR and inserted in frame with an N-terminal hexahistidine tag in CDFDuet. As disclosed herein, an alternative novel RT (RT_Mod2, SEQ ID NO. 82) was produced using the disclosed technology with HIV2 RT as the template (Genbank AAB25033, amino acid sequence SEQ ID NO. 83 as listed elsewhere herein), where at least thirteen naturally occurring substitutions were incorporated. The full-length subunit was inserted into pCDFDuet and the smaller subunit was fused to a C-terminal hexahistidine tag after Thr436 in pETDuet. For both RTs, the subunits were co-expressed in BL21(DE3) and purified using Talon and heparin sepharose chromatography. The purified enzymes were concentrated, glycerol added to 10%, and aliquots were flash frozen and stored at -80°C. Primer extension assays using poly-A template and ³H- labeled dTTP were used to determine specific activity at 50°C as described for commercial RTx (NEB). [00133] RT-LAMP reaction mixtures

[00134] RT-LAMP reactions were prepared by mixing 7.5 ml commercial 2x LAMP master mix (NEB E1700L) or the presently disclosed LAMP mix (40 mM TrisHCl, pH 8.5, 20 mM (NH4)₂SO4, 100 mM KC1, 16 mM MgSO4, 0.2% Tween-20, 2.8 mM each dNTP, 16 pg/ml polA LF, and 2.6-7.7 pg/ml RT) with 1.5 ml of 1 Ox primer/beacon master mix (final concentration: 1.6 mM FIP/BIP primers, 0.2 mM F3/B3 region outer primers, 0.4 mM LF/LB primers, 0.05 mM beacon) and 6 ml of sample and/or water. For multiplexed LAMP reactions, the final total concentration of primers/beacons was maintained e.g. the individual primer/beacon concentrations were halved when two primer sets were added to the same reaction.

[00135] Assays using LAMP-BEAC

[00136] LAMP-BEAC reactions were performed at 63-65 °C with fluorescent quantification every 30 seconds on a ThermoFisher QuantStudio™ 5. Reactions typically completed within 45 minutes but for research purposes data was collected for additional time spans. The synthetic SARS-CoV-2 RNA used as a standard during assay development was obtained from Twist (MT007544.1). After reaction completion, for melt curve analysis, the reaction was heated to 95 °C for 5 minutes to inactivate any remaining enzyme, cooled to 25 °C (at a rate of 0.1 °C/sec) and then slowly heated to 95° C with fluorescence measured every degree.

[00137] RT-qPCR to characterize saliva samples

[00138] RNA was extracted from -140 ml saliva using the Qiagen QIAamp Viral RNA Mini Kit. The RT-qPCR assay used the CDC 2019-nCoV_Nl primer-probe set (2019-nCoV_Nl- F: GACCCCAAAATCAGCGAAAT (SEQ ID NO. 84); 2019-nCoV_Nl-R: TCTGGTTACTGCCAGTTGAATCTG (SEQ ID NO. 85); 2019_nCoV_Nl-P: FAM- ACCCCGCATTACGTTTGGTGGACC-IBFQ (SEQ ID NO. 86)). The RT-qPCR master mix contained: 8.5 ml dH₂O, 0.5 ml Nl-F (20 mM), 0.5 ml Nl-R (20 mM ), 0.5 ml Nl-P (5 mM ), 5.0 ml TaqMan™ Fast Virus 1-Step Master Mix per reaction. 5 ml of extracted RNA was added to 15 ml of prepared master mix for a final volume of 20 ml per reaction. Final concentrations of both 2019-nCoV_Nl-F and 2019-nCoV_Nl-R primers were 500nM and the final concentration of the 2019-nCoV_Nl-P probe was 125nM. The assay was performed using the ThermoFisher QuantStudio 5. The thermocycler conditions were: 5 minutes at 50°C, 20 seconds at 95°C, and 40 cycles of 3 seconds at 95°C and 30 seconds at 60°C.

[00139] Example 1: Designing molecular beacons for SARS-CoV-2 RT-LAMP

[00140] Several beacons were tested for detection of SARS-CoV-2 RNA in RT-LAMP reactions (Table 1). Optimization required identifying sequence designs that performed properly under the conditions of the RT-LAMP reaction, which is typically run at temperatures around 65°C. Function of the beacon requires that the hairpin remain mostly folded in the hairpin structure at this temperature, while still opening sufficiently often to allow annealing to the target RT-LAMP cDNA product. The annealed beacon-target cDNA duplex must then be sufficiently stable at 65°C to result in unquenching and an increase in fluorescence. To increase beacon affinity for use at higher temperatures, multiple dNTP positions within the target sequence of each beacon were substituted with locked nucleic acids [14], Locked nucleic acids reduce the conformational flexibility of dNTPs and make the free energy of nucleic acid annealing more favorable [16], Several beacons were tested for performance using previously reported RT- LAMP amplicons (Table 1).

[00141] Example 2: Testing LAMP-BEAC

[00142] An example of a successful beacon design is Penn-LF-beac (Table 1). The RT- LAMP amplicon targets the orflab coding region and was first reported by El-Tholoth and coworkers at the University of Pennsylvania (“Penn”)[8], The favored beacon was designed to target sequences within the forward DNA loop generated during LAMP; thus the beacon is designated Penn loop forward beacon, contracted to Penn-LF-beac.

[00143] Figures 2A-2D show use of the Penn-LF-beac system to detect synthetic SARS-CoV-2 RNA. Tests were carried out with commercial LAMP polymerase and reverse transcriptase preparations. In addition, to avoid possible supply chain problems and allow potential production of reagents in resource limited settings, novel DNA polymerase and reverse transcriptase enzymes were produced and purified, which were assayed in parallel with commercial preparations for some tests (described below).

[00144] To compare standard LAMP amplification with LAMP-BEAC, reactions were prepared containing both the SYTO 9 fluorescent dye (Fig. 2A), which detects bulk DNA by intercalation, and the Penn-LF-beac (Fig. 2B). Reaction products were detected at two wavelengths, allowing separate quantification of the bulk dye and the molecular beacon in single reactions. The standard RT-LAMP showed bulk DNA production at shorter times than with the water control, but the water control did amplify shortly after the positive samples. This spurious late amplification is commonly seen with RT-LAMP, though the mechanism is unclear. The primers may interact with each other to form products and launch amplification, or perhaps the reaction results from amplification of adventitious environmental DNA. In separate tests, synthesis of DNA products was shown to depend on addition of LAMP primers.

[00145] Reactions detected with the Penn-LF-beac showed more clear-cut discrimination (Fig. 2B). The positive samples showed positive signal after about the same amount of time as for the conventional RT-LAMP. However, no signal was detected for the negative water control. Lack of amplification in negative controls has been reproducible over multiple independent reactions (examples below).

[00146] The nature of the products could be assessed using thermal denaturation (Fig. 2C and 2D). Reactions were first cooled to allow full annealing of complementary DNA strands, then slowly heated while recording fluorescence intensity. The fluorescent signal of the intercalating dye started high but dropped with increasing temperature in all samples (Fig. 2C), consistent with denaturation of the duplex and release of the intercalating dye into solution. In contrast, the beacon’s fluorescent signal in the water controls started at low fluorescence (Fig. 2D), consistent with annealing of the beacon DNA termini to form the hairpin structure (Fig. 1). At temperatures above 70°C the fluorescence modestly increased, consistent with opening of the hairpin and reptation of the beacon as a random coil in solution. For reactions containing the RT- LAMP product and Penn-LF-beac, fluorescence values were high at lower temperatures, consistent with formation of the annealed duplex, then at temperature sufficient for denaturation, the fluorescence values fell to match those of the random coil (Fig. 2D). Thus the LAMP-BEAC assay generates strong fluorescence signals during LAMP amplification in the presence of target RNA but not in negative controls, and the thermal melting properties are consistent with formation of the expected products.

[00147] Example 3: Multiplex LAMP-BEAC assays

[00148] Additional LAMP-BEAC assays were developed to allow multiplex detection of SARS-CoV-2 RNA, and to allow parallel analysis of human RNA controls as a check on sample integrity, and so developed several additional beacons (Table 1). El-LB-beac recognizes an amplicon targeting the viral E gene, reported in Zhang et al., 2020, and Asle-LB-beac recognizes the SARS-CoV-2 Asle amplicon, reported in Rabe et al., 2020, targeting the orflab coding region. A positive control beacon, STATH-LB-beac, was also developed to detect a LAMP amplicon targeting the human STATH mRNA (Table 1). STATH was chosen because it is abundantly expressed in human saliva, and a tested RT-LAMP amplicon was available [17], [00149] To allow independent detection of each amplicon as a quadruplex assay, each beacon was labeled using fluorophores with different wavelengths of maximum emission. El- LB-beac was labeled with FAM and detected at 520 nm, STATH-LB-beac was labeled with hexachlorofluorescein (Hex) and detected at 587 nm, Asle-LB-beac was labeled with Tex615 and detected at 623 nm, and Penn-LF-beac was labeled with cyanine-5 (Cy5) and detected at 682 nm. [00150] As an example, the quadruplex LAMP-BEAC assay was tested with contrived samples, in which saliva was doped with synthetic SARS-CoV-2 RNA (Figs. 3A-3H). Prior to dilution, saliva was treated with TCEP and EDTA, followed by heating at 95°C, which inactivates both SARS-CoV-2 and cellular RNases[9], and so is part of the present sample processing pipeline. The STATH-LB-beac amplicon detected the human RNA control in all saliva samples (Fig. 3A).The El-LB-beac amplicon consistently detected SARS-CoV-2 RNA down to -250 copies per reaction (Fig. 3B), and the Asle-LB-beac amplicon detected SARS- CoV-2 RNA to -250 copies per reaction (Fig. 3C). The Penn-LF-beac amplicon was least sensitive, detecting SARS-CoV-2 RNA consistently only at -1000 copies per reaction (Fig. 3D); thus the Penn-LF-beac assay reports particularly high numbers of RNA copies.

[00151] Melt curve analysis was also carried out (Figs. 3 E-3H). Melt curve profiles were distinctive for each beacon, but the overall pattern included high fluorescence in the positive samples and low values in negative samples at lower temperatures, then convergence of positive and negative samples at high temperatures associated with full melting of the beacon and reptation in solution. The melt curve data for each beacon supported correct function and the expected structures of the amplification products.

[00152] Example 4: Assessing LAMP-BEAC performance on clinical saliva samples

[00153] Next the LAMP-BEAC assay was tested on a set of 20 saliva samples collected during surveillance for potential SARS-CoV-2 infection. Samples were from a clinical sample acquisition site, where subjects were tested by clinical nasopharyngeal (NP) swabbing and RT-qPCR, and also donated saliva for comparison. As controls, two fresh saliva samples were collected at the time of assay, and two negative controls containing water only were compared. Saliva samples were treated with TCEP and EDTA, and heated at 95°C for five minutes to inactivate RNase and SARS-CoV-2 [9], As an additional check, RNA was purified from the same set of saliva samples and RT-qPCR carried out using a CDC-recommend primer set, allowing investigation of possible differences between NP swabs and saliva as analytes.

[00154] The LAMP reaction was carried out using a multiplex with two SARS-CoV-2 LAMP-BEAC assays, Asle-LB-beac and Penn-LF-beac, or Asle-beac along with the human RNA-targeted STATH-LB-beac as a measure of sample integrity. Bulk DNA synthesis was also monitored using SYTO 9. The sample set was assayed twice to allow comparison of technical replicates (FIGs. 5A-5X). Melt curve analysis of the amplification products was consistent with the expected molecular structures as described above (FIGs. 5A-5X). Bulk DNA amplification was seen in all samples (FIGs. 5A-5X). [00155] Figure 4 compares the final maximum fluorescence values in the LAMP- BEAC assays to RT-qPCR quantitation on the same saliva samples. Of the clinical saliva samples, 19/22 were positive for the STATH RNA control. Three failed for unknown reasons. Of note is that the clinical samples had been stored for some time and frozen and thawed more than once, possibly leading to RNA degradation. The two fresh saliva samples were both positive for STATH RNA.

[00156] Ten of the saliva samples were called positive for SARS-CoV-2 RNA. The fresh SARS-CoV-2 negative saliva samples and the water controls were called negative as expected. Comparing the 10 positive samples to RT-qPCR on saliva showed that all samples with RT-qPCR calls of 100 or more viral RNA copies per microliter of saliva were identified as positive. An independent replication of the assay in another laboratory using LAMP-BEAC to analyze the same samples yielded closely similar results (FIGs. 7A-7B). Thus the RT-qPCR assay and LAMP-BEAC on saliva samples showed excellent agreement for the higher copy number samples.

[00157] A quadruplex LAMP-BEAC was then performed, using Penn-LF-beac, El- LB-beac, Asle-LB-beac and STATH-LB-beac, on these 20 samples plus an additional 56 saliva samples and compared the present results to clinical RT-qPCR on NP swabs obtained from the same patients to assess performance. Assuming that the results from NP swabs represent truth, then the LAMP-BEAC had a sensitivity of 0.57 and a specificity of 0.98. The great majority of disagreements between LAMP-BEAC and clinical assay corresponded to samples with low estimated copy numbers. For example, LAMP-BEAC detected all 10 samples with a RT-qPCR estimated copies per ml >1000. Thus, the LAMP-BEAC assays correlated perfectly with RT- qPCR on the same saliva samples at high RNA copies. Notably, the single saliva sample called positive by LAMP-BEAC but negative by clinical NP assay was also estimated at 2xl0⁵ viral RNA copies/ml by RT-qPCR. A recent study has documented differences between the loads of SARS-CoV-2 RNA at different body sites [18], including oral and nasal sites, potentially accounting at least in part for the observed differences.

[00158] The detection shown in Figures 4A-4C, using end point fluorescence values and not reaction progression curves, offers a simplified read out of reaction results. That is, advanced qPCR machines are not needed for quantification of product formation using LAMP- BEAC, but rather reaction end points can be used to read out results using a simpler fluorescent plate reader. This may help with bypassing possible supply chain bottlenecks associated with purchasing qPCR machines for SARS-CoV-2 assays. [00159] Example 5: Laboratory-based production of polymerases required for RT-LAMP

[00160] Polymerase enzymes are expensive and potentially subject to supply chain disruptions, as such novel reverse transcriptase and DNA polymerase enzymes were engineered and simple purification protocols were devised, allowing inexpensive local production of the required enzymes. HIV-2 reverse transcriptase and the polA large fragment from Geobacillus stearothermophilus were each engineered to contain several amino acid substitutions expected to stabilize enzyme folding at higher temperatures (RT) or improve strand displacement activity (Bst). Enzymes were purified and tested as described in the methods. Figures 6A-6P summarize results of side-by-side assays using lab-purified polymerases and commercial enzyme preparations, which indicate that the presently disclosed novel polymerase enzymes are at least as efficient as commercial preparations.

[00161] Example 6: Discussion and conclusion

[00162] Standard RT-LAMP is an attractive method for assay of SARS-CoV-2 RNA in patient samples due to the simplicity of the method and the use of a supply chain orthogonal to the clinical assay supply chain. However, conventional LAMP typically detects only the presence of amplified bulk DNA, and thus assays can be complicated by nonspecific amplification. Improved specificity can be achieved by sequence-specific detection, and multiple methods have been proposed [11, 12], However, some of these approaches are complicated by the need to open reaction tubes and manipulate products, creating severe danger of contamination between runs. Disclosed here are convenient methods for sequence-specific detection of SARS-CoV-2 RNA in unpurified saliva using molecular beacons — LAMP-BEAC — that do not require manipulation of reaction products but can be carried out in a multiplex format in a “single tube.”

[00163] The LAMP-BEAC method is not as sensitive as RT-qPCR on purified RNA, but it can be implemented inexpensively, potentially allowing frequent population screening. The reaction set up and incubation can be done in a couple of hours, allowing rapid turnaround. Thus the LAMP-BEAC assay meets the needs articulated by modeling studies for effective surveys of asymptomatic populations [10], To this end, the presently disclosed LAMP-BEAC was shown to work efficiently on inactivated saliva, providing an easily-collected analyte.

[00164] Comparison of LAMP-BEAC to RT-qPCR showed better concordance between the RT-qPCR assay carried out on the same saliva samples than for RT-qPCR carried out on eluates from NP swabs. For the assays on saliva, all samples with greater than 1000 viral RNA copies agreed between LAMP-BEAC and RT-qPCR, suggesting that both are similarly effective at identifying samples with high viral RNA copy numbers. The reason for divergence with some of the results for RT-qPCR on NP swabs is unknown — however, differences in viral RNA loads within patients at different body sites is well documented, possibly accounting for this difference[18]. One case where the two saliva assays called a sample as positive was instead negative on the NP sample, as well as several samples that were positive by NP and negative or low in both saliva assays, suggesting the differences can be in either direction, and that neither sample type is superior.

[00165] Recently Vogels et al. reported SalivaDirect, an RT-qPCR assay run on inactivated but unpurified saliva[19], SalivaDirect uses a duplex single-tube analytical method, with one amplicon targeting SARS-CoV-2 RNA and another targeting a human RNA. This parallels presently disclosed duplex LAMP-BEAC targeting viral RNA and human STATH RNA (Figures 4A-4C). The SalivaDirect method is rightly popular, however it is important to note that the present LAMP-BEAC method does not rely on commercial enzyme mixes, potentially providing more resilience to possible supply chain disruptions. In addition, LAMP- BEAC can be carried out as an end-point assay using a fluorescent plate reader to assess results, thus bypassing the need for quantitative real-time PCR machines.

[00166] Addressing the need for increased SARS-CoV-2 testing will likely require multiple well-designed assays ideally taking advantage of independent supply chains — LAMP- BEAC can contribute to meeting this need.

[00167] References

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[00171] 4. Lieberman JA, Pepper G, Naccache SN, Huang ML, Jerome KR, Greninger AL: Comparison of Commercially Available and Laboratory-Developed Assays for In Vitro Detection of SARS-CoV-2 in Clinical Laboratories. J Clin Microbiol 2020, 58.

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[00174] 7. Lee SH, Baek YH, Kim YH, Choi YK, Song MS, Ahn JY: One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV. Front Microbiol 2016, 7:2166.

[00175] 8. El-Tholoth M, Bau HH, Song J: A single and two stage, closed-tube, molecular test fo rthe 2019 novel coronavirus (COVID-19) at home, clinic, and points of entry, chemrxiv 2020.

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[00178] 11. Schmid-Burgk JL, Schmithausen RM, Li D, Hollstein R, Ben-Shmuel A, Israeli O, Weiss S, Paran N, Wilbeing G, Liebing J, et al: Lamp-Seq: population scale COVID- 19 diagnostics using combinatorial barcoding. bioRxiv 2020. [00179] 12. Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados A, Sotomayor-Gonzalez A, et al: CRISPR-Cas 12-based detection of SARS-CoV-2. Nat Biotechnol 2020, 38:870-874.

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[00182] 15. Liu W, Huang S, Liu N, Dong D, Yang Z, Tang Y, Ma W, He X, Ao D, Xu Y, et al: Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay. Sci Rep 2017, 7:40125.

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[00187] 20. : Piotrowski, Y., Gurung, M.K., Larsen, A. N. Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerases. BMC Mol Cell Biol. 2019 Aug 9;20(l):31 (doi: 10.1186/sl2860-019-0216-l).

Claims

What is Claimed:

1. A composition, comprising at least one molecular beacon that comprises at least one of nucleic acid sequences of SEQ ID NOs. 1-80.

2. The composition of claim 1, wherein the composition comprises (1) a molecular beacon that comprises one nucleic acid sequence of SEQ ID NOs. 1-80, and (2) a molecular beacon that comprises another nucleic acid sequence of SEQ ID NOs. 1-80.

3. The composition of claim 1 comprising at least one molecular beacon that comprises at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55- 59, 66-70, and 77-80 and further comprises at least one locked nucleic acid (LNA).

4. The composition of claim 3, wherein the composition comprises (1) a molecular beacon that comprises one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46- 49, 55-59, 66-70, and 77-80 and at least one LNA and (2) a molecular beacon that comprises another nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46- 49, 55-59, 66-70, and 77-80 and at least one LNA.

5. The composition of claim 1, further comprising DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P.

6. The composition of claim 1, further comprising a reverse transcriptase comprising an amino acid sequence of SEQ ID NO. 82.

7. A method for detecting a severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) nucleic acid in a sample from a subject, the method comprising: a) with molecular beacons, carrying out a reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on a reaction mixture comprising the sample; b) incubating the reaction mixture under polymerase reaction conditions to produce a reaction product comprising amplified SARS-CoV-2 nucleic acid; and, c) detecting the reaction product.

33 A The method of claim 7, wherein the method is for multiplexed detection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid in two or more samples from two or more subjects in same reaction mixture. The method of claim 7 for monitoring a response to a medication in a subject in need thereof, the method further comprising: obtaining a first sample from the subject at a first time point; obtaining a second sample from the subject at a second time point following administration of the medication to the subject; and performing steps a)-c) for the first sample and the second sample. The method of claim 9, further comprising comparing the amount of SARS-CoV-2 virus nucleic acids in the first and second reaction products, wherein a decrease in the amount of SARS-CoV-2 virus nucleic acids from the first reaction product relative to the second reaction product indicates a positive response to the medication. The method of claim 9, wherein the medication comprises an antiviral therapy. The method of any one of claims 7-11, wherein the molecular beacons comprise at least one of SEQ ID NOs. 1-80. The method of any one of claims 7-11, wherein the molecular beacons comprise at least one locked nucleic acid (LNA). The method of any one of claims 7-11, wherein the molecular beacons comprise LNA modifications and comprise at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80. The method of any one of claims 7-11, wherein the reverse transcription reaction is performed using a RT comprising an amino acid sequence of SEQ ID NO. 82. The method of any one of claims 7-11, wherein the polymerase reaction is performed using a DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P.

34 The method of any one of claims 7-11, wherein the sample comprises a nasal or a buccal sample. The method of claim 17, wherein the buccal sample comprises saliva. The method of any one of claims 7-11, wherein the sample does not undergo nucleic acid purification and/or nucleic acid amplification prior to the RT-LAMP. The method of any one of claims 7-11, wherein the molecular beacons comprise one or more fluorescently labeled nucleic acids. The method of any one of claims 7-11, wherein detecting the reaction product comprises contacting the reaction product with an intercalating fluorescent dye. The method of any one of claims 7-11, wherein detecting the reaction product comprises monitoring a fluorescence. The method of any one of claims 7-11, wherein the subject is a mammal. The method of claim 23, wherein the mammal is a human. A composition comprising at least one molecular beacon that comprises at least one nucleic acid sequence of SEQ ID NOs. 7-10, 17, 18, 25-27, 34-39, 46-49, 55-59, 66-70, and 77-80 and further comprises at least one locked nucleic acid (LNA). A DNA polymerase comprising an amino acid sequence of SEQ ID NO. 81, an amino acid sequence of SEQ ID NO. 81 with a mutation R445A, or an amino acid sequence of SEQ ID NO. 81 with a mutation R445P. A reverse transcriptase comprising an amino acid sequence of SEQ ID NO. 82. A kit, comprising: a. the composition of at least one of claims 1-6 or claims 25-27; b. packaging for the composition; and, c. instructions to use thereof.