Assay for detecting coronavirus infection 2019 (COVID-19)

Abstract

The present disclosure relates to materials and methods for amplifying and detecting 2019-Cov in a sample, including various combinations of amplification oligonucleotides and oligonucleotide probes. The disclosure also relates to oligonucleotide sequences, kits, and methods for detecting COVID-19.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 000,304, filed Mar. 26, 2020; U.S. Provisional Application No. 63 / 000,971, filed Mar. 27, 2020; U.S. Provisional Application No. 63 / 004,773, filed Apr. 3, 2020; U.S. Provisional Application No. 63 / 049,237, filed Jul. 8, 2020; and U.S. Provisional Application No. 63 / 155,599, filed Mar. 2, 2021, the entire contents of each of which are incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials A computer-readable nucleotide / amino acid sequence list, submitted concurrently with this application and identified as follows, is incorporated herein by reference in its entirety: one 5,136-byte ASCII (text) file named “2021-03-24_38391-601_SQL_ST25.txt,” created on Mar. 24, 2021.

[0003] This disclosure relates to methods for amplifying target nucleic acid sequences from SARS-CoV-2 to detect COVID-19.

Background Art

[0004] The novel coronavirus (SARS-CoV-2 (2019-nCoV)) emerged as a human pathogen causing fever, severe respiratory disease, and pneumonia in Hubei Province, China, in late 2019. This disease associated with SARS-CoV-2 was named COVID-19. This novel coronavirus is a member of the genus Betacoronavirus and is closely related to several bat coronaviruses and the severe acute respiratory syndrome coronavirus (SARS-CoV). However, unlike SARS-CoV, SARS-CoV-2 spreads rapidly among humans.

[0005] <As of the end of February 2021, more than 100 million cases of COVID-19 had been confirmed in over 200 countries, and complications from COVID-19 had been cited as a cause of death in more than 2.5 million individuals. [Overview of the Initiative] [Means for solving the problem]

[0006] (Summary of the invention) This disclosure provides reagents comprising oligonucleotides for amplifying and detecting the coronavirus SARS-CoV-2 in a sample. In some embodiments, the set of oligonucleotides comprises at least one first amplification oligonucleotide, at least one second amplification oligonucleotide, and at least one probe oligonucleotide. The probe oligonucleotide may include a detectable label (e.g., a fluorophore). In some embodiments, the set of oligonucleotides is intended for recombinase-polymerase amplification and detection of SARS-CoV-2 in a sample. In some embodiments, the reagent comprises a group of oligonucleotides comprising one or more sets of oligonucleotides.

[0007] In some embodiments, the set of oligonucleotides for recombinase-polymerase amplification and detection of SARS-CoV-2 in a sample includes a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 17, a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 19, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 18; or a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 20 or 21, a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25 or 26, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to any of SEQ ID NOs: 22-24; or a combination thereof, each probe oligonucleotide containing a detectable label. In some embodiments, the first amplification oligonucleotide contains a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 20, the second amplification oligonucleotide contains a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25, and the probe oligonucleotide contains a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 22 or 23. In some embodiments, the first amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 21, the second amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25 or 26, and the probe oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 24.

[0008] In some embodiments, the group of oligonucleotides for amplifying and detecting SARS-CoV-2 in a sample includes a first set of oligonucleotides, comprising a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 2, a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 4, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 6. In some embodiments, the group of oligonucleotides further includes a second set of oligonucleotides, comprising a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to either SEQ ID NO: 11 or 15, a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 3, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 5.

[0009] This disclosure also provides a method for detecting SARS-CoV-2 in a sample. The sample may include a nasal swab or brush, saliva, mucus, blood, serum, plasma, or feces.

[0010] In some embodiments, the method includes the steps of: contacting a sample with a set or group of oligonucleotides and amplification reagents described herein; amplifying one or more target SARS-CoV-2 nucleic acid sequences present in the sample using recombinase-polymerase amplification (RPA); hybridizing one or more oligonucleotide probes to one or more amplified target SARS-CoV-2 nucleic acid sequences; and detecting the hybridization of one or more probe oligonucleotide sequences to one or more amplified SARS-CoV-2 target nucleic acid sequences by measuring signals from detectable labels. In some embodiments, the method further includes the step of contacting first and second amplified oligonucleotides from the set of oligonucleotides with a recombinase agent. The presence of one or more signals from detectable labels may indicate the hybridization of one or more probe oligonucleotides to one or more amplified SARS-CoV-2 target nucleic acid sequences.

[0011] Amplification reagents may include polymerase; recombinase agents; recombinase loading proteins; single-chain binding proteins; nicking enzymes; helicases; resolvers; enzyme cofactors; buffers; deoxyribonucleotides; or ribonucleotide triphosphates; crowding agents; ATP, ATP analogs, or ATP production systems; or combinations thereof.

[0012] The Disclosure further provides a kit for detecting SARS-CoV-2 in a sample, comprising at least one set of oligonucleotides, any of the oligonucleotides disclosed herein, reagents for amplifying and detecting nucleic acid sequences, and / or instructions for use.

[0013] Other aspects and embodiments of this disclosure will become apparent from the following detailed description and accompanying drawings.

[0014] This patent or application file includes at least one drawing drawn in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Japan Patent Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0015] [Figure 1A] This graph shows the recombinase-polymerase amplification (RPA) of SARS-CoV-2 using combination 1 of the amplified oligonucleotide and probe oligonucleotide. [Figure 1B] This graph shows the recombinase-polymerase amplification (RPA) of SARS-CoV-2 using various combinations of amplified oligonucleotides and probe oligonucleotides. [Modes for carrying out the invention]

[0016] This disclosure is based, at least in part, on the development of a collection of oligonucleotide sequences that facilitate the rapid detection of COVID-19.

[0017] The terms “comprise(s),” “include(s),” “having,” “has,” “contain(s),” and their variations as used herein are intended to be open-ended transitional phrases, terms, and words that do not preclude the possibility of additional acts or structures. The singular forms “one,” “and,” and “the” include multiple referents unless the context expressly indicates otherwise. This disclosure also contemplates other embodiments “including,” “consisting of,” and “essentially consisting of,” the embodiments or elements presented herein, whether expressly or otherwise.

[0018] In the enumeration of numerical ranges in this specification, each intervening number having the same precision is also explicitly intended. For example, in the range 6 to 9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range 6.0 to 7.0, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly intended.

[0019] The terms “first” and “second” are used in this disclosure only in their relative sense. Unless otherwise noted, these terms are understood to be used merely for convenience in describing one or more embodiments. The terms “first” and “second” are used solely to distinguish one element from another, and the scope of rights to the disclosed technology should not be limited by these terms. For example, the first element may be designated as the second element, and similarly, the second element may be designated as the first element.

[0020] As used herein, the term “oligonucleotide” refers to a short nucleic acid sequence containing about 2 to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 nucleotides, or the range defined by any of the aforementioned values). As used herein, the terms “nucleic acid” and “polynucleotide” refer to any nucleotide, ribonucleotide (RNA), or deoxyribonucleotide (DNA) of any length in polymer form. These terms refer to the primary structure of a molecule and therefore include double-stranded and single-stranded DNA and double-stranded and single-stranded RNA. These terms include, as equivalents, analogues of either RNA or DNA made from nucleotide analogues, and modified polynucleotides, such as methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, but many other linkages are also known in the art (e.g., phosphorothioates, boranophosphates, etc.).

[0021] Oligonucleotides can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Oligonucleotides can be DNA, both genomic DNA and complementary DNA (cDNA), RNA, or hybrids, and nucleic acids may include combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Oligonucleotides can be obtained by chemical synthesis or by recombinant methods.

[0022] As used herein, the term “sequence identity percentage” refers to the percentage of a nucleotide or nucleotide analog in a nucleic acid sequence, or an amino acid in an amino acid sequence, that is identical to the corresponding nucleotide or amino acid in a reference sequence after two sequences have been aligned and, if necessary, gaps have been introduced to achieve the maximum identity percentage. Therefore, if the nucleic acid produced by this technique is longer than the reference sequence, any extra nucleotides in the nucleic acid that do not align with the reference sequence are not considered for determining sequence identity. Methods and computer programs for alignment, including BLAST, Align 2, and FASTA, are well known in the art.

[0023] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. The meaning and scope of terms shall be obvious, but in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0024] Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The section headings used herein are for purposes of organization only and are in no way to be construed as limiting the subject matter described.

[0025] 1. Amplification Oligonucleotides and Probe Oligonucleotides In one embodiment, the oligonucleotides described herein may be used for nucleic acid amplification (e.g., as primers) or as probes for nucleic acid hybridization and detection. As used herein, the terms "primer," "primer sequence," "primer oligonucleotide," and "amplification oligonucleotide" refer to an oligonucleotide that can act as a starting point for the synthesis of an extension product that is a complementary strand of a nucleic acid (any type of DNA or RNA) when placed under suitable amplification conditions (e.g., buffer, salts, temperature, and pH) in the presence of nucleotides and a nucleic acid polymerase (e.g., DNA-dependent or RNA-dependent polymerase). The amplification oligonucleotides of the present disclosure can be of any suitable size and preferably can contain, consist essentially of, or consist of about 15 to 50 nucleotides, preferably about 20 to 40 nucleotides. The oligonucleotides of the present disclosure can contain additional nucleotides in addition to those described herein. Depending on the type of amplification process used, the amplification oligonucleotides can contain, for example, nicking enzyme sites and upstream stabilizing regions (see, e.g., U.S. Patent No. 9,689,031, U.S. Patent No. 9,617,586, U.S. Patent No. 9,562,264, and U.S. Patent No. 9,562,263; each of which is incorporated herein by reference in its entirety).

[0026] The terms "probe", "probe array", and "probe oligonucleotide" refer to oligonucleotides that can selectively hybridize to at least a portion of a target sequence (e.g., a portion of an amplified target sequence) under appropriate hybridization conditions. Generally, a probe array can be specified as either "complementary" (e.g., complementary (+) to the coding strand or sense strand) or "reverse complementary" (e.g., complementary (-) to the antisense strand). The probes of the present disclosure can be of any suitable size, desirably, including, consisting essentially of, or consisting of about 10 to 50 nucleotides, preferably about 12 to 35 nucleotides.

[0027] As used herein, the terms "set", "primer set", and "primer and probe set" refer to two or more oligonucleotides that can prime together a target sequence or target nucleic acid of interest (e.g., a target sequence within SARS-CoV-2) and / or at least one probe that can detect the target sequence or target nucleic acid. In certain embodiments, the term "set" refers to a pair of oligonucleotides comprising a first oligonucleotide that hybridizes to the 5' end of the target sequence or target nucleic acid to be amplified and a second oligonucleotide that hybridizes to the complement of the target sequence or target nucleic acid to be amplified.

[0028] The sets of oligonucleotides described herein can be used to amplify and detect one or more target SARS-CoV-2 (2019-nCoV) sequences in a sample. The terms “target sequence” and “target nucleic acid” are used interchangeably herein and refer to specific nucleic acid sequences whose presence or absence is to be detected by the methods disclosed. In the context of this disclosure, a target sequence preferably includes a nucleic acid sequence to which one or more oligonucleotides hybridize and from which amplification begins. A target sequence may also include a probe hybridize region from which a probe can form a stable hybrid under appropriate amplification conditions. A target sequence may be single-stranded or double-stranded. A target SARS-CoV-2 sequence may be located within any part of the SARS-CoV-2 genome, for example, within a gene encoding a nucleocapsid (N) protein or a gene encoding RNA-dependent RNA polymerase (RDRP).

[0029] In some embodiments, the set includes a first amplified oligonucleotide, a second amplified oligonucleotide, and a probe oligonucleotide. In some embodiments, the set includes a first amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any of SEQ ID NOs. 1 and 10-16, a second amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO. 3, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO. 5; or a first amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO. 2, a second amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO. 4, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO. 6; or a first amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO. 7 The present invention includes a second amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 8, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 9; or a first amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 17, a second amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 19, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 18; or a first amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 20 or 21, a second amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25 or 26, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to any of SEQ ID NOs: 22 to 24; or a combination thereof.

[0030] In some embodiments, the set includes a first amplified oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to any of SEQ ID NOs: 1 and 10-16; a second amplified oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 3; and a probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 5; and a first amplified oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 2; a second amplified oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 4; and a probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 6.

[0031] In some embodiments, the set includes a first amplified oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 20, a second amplified oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25, and a probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 22 or 23.

[0032] In some embodiments, the set includes a first amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 21, a second amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25 or 26, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 24.

[0033] In some embodiments, the set comprises oligonucleotides for recombinase-polymerase amplification and detection of SARS-CoV-2 (2019-nCoV) in a sample, comprising: a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 17; a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 19; and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 18; or a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 20 or 21; a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25 or 26; and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to any of SEQ ID NOs: 22-24; or a combination thereof, each probe oligonucleotide comprising a detectable label. In some embodiments, the first amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 20, the second amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25, and the probe oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 22 or 23. In some embodiments, the first amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 21, the second amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25 or 26, and the probe oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 24.

[0034] In some embodiments, the group of oligonucleotides for amplifying and detecting SARS-CoV-2 (2019-nCoV) in a sample includes a first amplification oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 2, a second amplification oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 4, and a probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 6, wherein the probe oligonucleotide includes a detectable label.

[0035] In some embodiments, the group further includes a second set of oligonucleotides comprising a first amplified oligonucleotide having a nucleic acid sequence having at least 70% similarity to either SEQ ID NO: 11 or 15, a second amplified oligonucleotide having a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 3, and a probe oligonucleotide having a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 5, wherein the probe oligonucleotide includes a detectable label.

[0036] [Table 1]

[0037] [Table 2]

[0038] Any oligonucleotide described herein can be modified in any preferred manner to stabilize or enhance the binding affinity of the oligonucleotide to its target. For example, an oligonucleotide sequence described herein may contain one or more modified oligonucleotides. Furthermore, any sequence listed, including internal spacers or modifications, can be used without modification or spacers.

[0039] Any oligonucleotide described herein may include, for example, spacers, blocking groups, and modified nucleotides. Modified nucleotides are nucleotides or nucleotide triphosphates that differ in composition and / or structure from natural nucleotides and nucleotide triphosphates. Modifications include those of natural origin, resulting from modification by enzymes that modify nucleotides, such as methyltransferases. Modified nucleotides also include synthetic nucleotides or nucleotides of non-natural origin. For example, modified nucleotides include those having 2' modifications, such as 2'-O-methyl and 2'-fluoro. Other 2'-modified nucleotides are also known in the art and are described, for example, in U.S. Patent No. 9,096,897, which is incorporated in whole by reference herein. Modified nucleotides and nucleotide triphosphates used herein may be modified, for example, so that the modified nucleotide or nucleotide triphosphate protects the modified strand from cleavage by restriction enzymes when the modification is present on one strand of a double-stranded nucleic acid where a restriction endonuclease recognition site is located.

[0040] Blocking groups or polymerase-stopping molecules are chemical moieties that inhibit target sequence-independent nucleic acid polymerization by polymerase. Blocking groups can prevent an oligonucleotide from supporting template extension that utilizes a detectable oligonucleotide probe as a target, even though the oligonucleotide can bind to the target nucleic acid molecule. For example, the presence of one or more moieties that prevent polymerase progression can cause polymerase arrest, either in non-nucleic acid backbone addition to the oligonucleotide or due to stalling of the replication polymerase. Oligonucleotides having these moieties can prevent or reduce the non-canonical amplification of the probe during amplification reactions. Examples of blocking groups include alkyl groups, non-nucleotide linkers, phosphorothioates, alkanediol residues, peptide nucleic acids, and nucleotide derivatives lacking a 3'-OH group, such as cordycepin, spacer moieties, and damaged DNA bases. An example of a spacer is a C3 spacer. Spacers can be used, for example, within oligonucleotides and also, for example, at the ends to bind other groups, such as labels.

[0041] Any oligonucleotide sequences described herein include, are essentially, or may include a complement of any of the sequences disclosed herein. As used herein, the terms “complement” or “complementary sequence” refer to nucleic acid sequences that form a stable double helix having the oligonucleotides described herein by Watson-Crick base pairing rules, and typically share about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity with the disclosed oligonucleotides. The identity of nucleic acid sequences can be determined using any suitable mathematical algorithm or computer software known in the art, such as CLUSTAL-W, T-Coffee, and ALIGN (for nucleic acid and amino acid sequence alignment), the BLAST program (e.g., BLAST2.1, BL2SEQ, and later versions), and the FASTA program (e.g., FASTA3×, FASTM, and SSEARCH) (for sequence alignment and sequence similarity search).Sequence alignment algorithms are also discussed in, for example, Altschul et al., J. Molecular Biol., 215(3):403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009); Durbin et al. (eds.), Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7):951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge. These are disclosed in UK (1997), and each of them is incorporated herein by reference in its entirety.

[0042] The oligonucleotides described herein can be prepared using any suitable method, and various such methods are known in the art (e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 1989, 2. Supp. Ed., Cold Spring Harbour Laboratory Press: New York, NY; MAInnis (ed.), PCR Protocols. A Guide to Methods and Applications, Academic Press: New York, NY (1990); P. Tijssen, Hybridization with Nucleic Acid Probes - Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II), Elsevier Science (1993); MAInnis (ed.), PCR Strategies, Academic Press: New York, NY (1995); and FMAusubel (ed.), Short Protocols in Molecular Biology, John Wiley & See Sons:Secaucus, NJ (2002); Narang et al., Meth. Enzymol., 68: pp. 90-98 (1979); Brown et al., Meth. Enzymol., 68: pp. 109-151 (1979); and Belousov et al., Nucleic Acids Res., 25: pp. 3440-3444 (1997); each of these is incorporated herein by reference in its entirety. Oligonucleotide pairs can also be designed using various tools, such as the Primer-BLAST tool provided by the National Center of Biotechnology Information (NCBI).Oligonucleotide synthesis can be carried out using oligonucleotide synthesizers, for example, those commercially available from Perkin Elmer / Applied Biosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE), or Milligen (Bedford, MA). Alternatively, oligonucleotides can be custom-made and are available from various well-known commercial suppliers in the art, including, for example, Midland Certified Reagent Company (Midland, TX), Eurofins Scientific (Louisville, KY), and BioSearch Technologies, Inc. (Novato, CA). Oligonucleotides can be purified using any preferred method known in the art, for example, native acrylamide gel electrophoresis, anion exchange HPLC (see, e.g., Pearson et al., J. Chrom., 255: pp. 137-149 (1983); this is incorporated herein by reference), or reversed-phase HPLC (see, e.g., McFarland et al., Nucleic Acids Res., 7: pp. 1067-1080 (1979); this is incorporated herein by reference).

[0043] The sequences of oligonucleotides can be verified using any suitable sequencing method known in the art, including, but not limited to, chemical decomposition (see, e.g., Maxam et al., Methods of Enzymology, 65:499-560 (1980); this is incorporated herein by reference), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (see, e.g., Pieres et al., Nucleic Acids Res., 21:3191-3196 (1993); this is incorporated herein by reference), and mass spectrometry after combined alkaline phosphatase and exonuclease digestion (Wu et al., Anal. Biochem., 290:347-352 (2001); this is incorporated herein by reference).

[0044] 2. Detectable signs Any one or more oligonucleotide sequences described herein may include detectable labels so that one or more amplified oligonucleotides and / or probe oligonucleotides can be measured. In one embodiment, each of the probe oligonucleotide sequences described herein includes a detectable label. As used herein, the term “detectable label” refers to a portion or compound that produces a signal whose intensity is related to (e.g., proportional to) the amount of the substance bound to it. Any suitable detectable label can be used, many of which are known in the art, and which can be conjugated or linked to an oligonucleotide to detect the binding of the oligonucleotide to a target sequence. In one embodiment, the detectable label may be detected indirectly. An indirectly detectable label is typically a specific binding member used with a “conjugate” that is conjugated or coupled to a directly detectable label. Coupling chemistry for synthesizing such conjugates is well known in the art and is designed so that the specific binding properties of the specific binding member and the detectable properties of the label are not impaired. As used herein, the terms “specific binding member” and “conjugate” refer to two members of a binding pair, for example, two different molecules, where the specific binding member specifically binds to the polynucleotide of this disclosure, and the “conjugate” specifically binds to the specific binding member. The binding between the two members of a pair is typically chemical or physical in nature. Examples of such binding pairs include, but are not limited to, antigens and antibodies, avidin / streptavidin and biotin, haptens and hapten-specific antibodies, complementary nucleotide sequences, and enzyme cofactors / substrates and enzymes.

[0045] Preferably, each probe oligonucleotide sequence contains a detectable label. Each probe can be labeled with the same detectable label or with different detectable labels.

[0046] In some embodiments, the detectable label can be directly detected. Examples of such directly detectable labels include radioisotopes, fluorophores, chemiluminescent phosphodiphores, enzymes, colloidal particles, fluorescent microparticles, and insertion dyes (e.g., SYBR Green or ethidium bromide). In select embodiments, the detectable label can be a fluorophore, such as a fluorescein dye, polyhalofluorescein dye, hexachlorofluorescein dye, coumarin dye, rhodamine dye, cyanine dye, oxazine dye, thiazine dye, squalane dye, chelated lanthanide dye, azo dye, triphenylmethane dye, or BODIPY® dye. Examples of fluorophores include, but are not limited to, FAM(trademark), CAL-FLUOR(registered trademark), QUASAR(registered trademark), HEX(trademark), JOE(trademark), NED(trademark), PET(registered trademark), ROX(trademark), TAMRA(trademark), TET(trademark), TEXAS RED(registered trademark), and VIC(registered trademark). Those skilled in the art will understand that directly detectable labels may require additional components, such as substrates, trigger reagents, or light, to enable the detection of the label.Methods for labeling oligonucleotides, such as probes, are well known in the art, for example, LJ Kricka, Ann. Clin. Biochem., 39: pp. 114-129 (2002); van Gijlswijk et al., Expert Rev. Mol. Diagn., 1: pp. 81-91 (2001); Joos et al., J. Biotechnol., 35: pp. 135-153 (1994); Smith et al., Nucl. Acids Res., 13: pp. 2399-2412 (1985); Connoly et al., Nucl. Acids Res., 13: pp. 4485-4502 (1985); Broker et al., Nucl. Acids Res., 5: pp. 363-384 (1978); Bayer et al., Methods of Biochem. Analysis, 26: pp. 1-45 (1980); Langer et al., Proc. Natl. Acad. Sci. USA, 78: pp. 6633-6637 (1981); Richardson et al., Nucl. Acids Res., 11: pp. 6167-6184 (1983); Brigadi et al., Virol., 126: pp. 32-50 (1983); Tchen et al., Proc. Natl. Acad. Sci. USA, 81: pp. 3466-3470 (1984); Landegent et al., Exp. Cell Res., 15: pp. 61-72 (1984); AH Hopman et al., Exp. Cell This is described in Res., 169: pp. 357–368 (1987); and Temsamani et al., Mol. Biotechnol., 5: pp. 223–232 (1996), each of which is incorporated herein by reference in its entirety.

[0047] In some embodiments, any one or more of the oligonucleotides described herein may also include a quencher moiety. If the detectable label (e.g., a fluorophore) and the quencher moiety are held near the probe, for example at the end of the probe, the quencher moiety will interfere with the detection of the signal (e.g., fluorescence) from the detectable label. If the two moieties are physically separated, the signal will be detectable. The quencher may be selected from any suitable quencher known in the art, such as BLACK HOLE QUENCHER® 1 (BHQ-1®), BLACK HOLE QUENCHER® 2 (BHQ-2®), BLACK HOLE QUENCHER® 3 (BHQ-3®), IOWA BLACK® FQ, and IOWA BLACK® RQ. For example, an oligonucleotide probe may include a FAM fluorophore, CAL-FLUOR®, or QUASAR fluorophore and a BHQ-1 or BHQ-2 quencher.

[0048] The selection of a particular label and labeling technique depends on several factors, such as the ease and cost of the labeling method, the spectral interval between different detectable labels used, the desired amount of labeling in the sample, the effect of the detectable portion on the hybridization reaction (e.g., on the rate and / or efficiency of the hybridization process), the nature of the amplification method used, the nature of the detection system, and the nature and intensity of the signal produced by the detectable label.

[0049] 3. Methods for amplifying and detecting SARS-CoV-2 (2019-nCoV) This disclosure provides a method for detecting SARS-CoV-2 (2019-nCoV) in a sample. The method includes the steps of: contacting a sample with a set of oligonucleotides and amplification reagents disclosed herein; amplifying one or more target SARS-CoV-2 nucleic acid sequences present in the sample; hybridizing one or more oligonucleotide probes to one or more amplified target SARS-CoV-2 nucleic acid sequences; and detecting the hybridization of one or more probe oligonucleotide sequences to one or more amplified SARS-CoV-2 target nucleic acid sequences by measuring a signal from a detectable label. The description of the oligonucleotide set provided herein with respect to the aforementioned set of oligonucleotides also applies to this disclosed method.

[0050] The sample may be any suitable sample obtained from any suitable subject, typically mammals (e.g., dogs, cats, rabbits, mice, rats, goats, sheep, cows, pigs, horses, non-human primates, or humans). Preferably, the subject is human. The sample may be obtained from any suitable biological source, for example, a nasal swab or brush, or from a suitable physiological fluid, including but not limited to whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, lactation, ascites fluid, mucus, synovial fluid, peritoneal fluid, vaginal fluid, menstrual secretions, amniotic fluid, semen, feces, etc.

[0051] Samples can be obtained from the subject using routine techniques known to those skilled in the art, and the samples may be used directly as obtained from the biological source or after pretreatment to modify the properties of the sample. Such pretreatments include, for example, preparation of plasma from blood, dilution of viscous fluids, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, addition of reagents, and dissolution.

[0052] After obtaining a sample from the subject, the sample can be brought into contact with a set of oligonucleotides, including the amplified oligonucleotide and probe described herein, to form a reaction mixture. The reaction mixture is then placed under amplification conditions. As used herein, the term “amplification conditions” refers to conditions that promote the annealing and / or extension of the amplified oligonucleotide. Such conditions are well known in the art and depend on the amplification method selected. Amplification conditions encompass all reaction conditions, including but not limited to temperature and / or temperature cycling, buffers, salts, ionic strength, pH, etc.

[0053] Amplification of the SARS-CoV-2 nucleic acid sequence in a sample can be performed using any suitable nucleic acid sequence amplification method known in the art. In some embodiments, amplification methods include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), real-time PCR, transcription-mediated amplification (TMA), rolling circle amplification, nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated amplification (TMA), single-primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), and ligase chain reaction (LCR). In some embodiments, amplification of the SARS-CoV-2 (2019-nCoV) nucleic acid sequence is performed using isothermal amplification (e.g., RPA or NEAR). In some embodiments, amplification and detection of the SARS-CoV-2 nucleic acid sequence are performed using a point-of-care device (e.g., the ID NOW system (Abbott)).

[0054] In some embodiments, amplification of the SARS-CoV-2 nucleic acid sequence is performed using real-time PCR. As used herein, “real-time PCR” refers to a PCR method that measures the accumulation of amplified DNA products in real time as the reaction progresses, and quantifies the product after each cycle, unlike conventional PCR which detects the amplified DNA product by endpoint analysis. Real-time PCR is also known in the art as “quantitative PCR (qPCR).” Real-time detection of PCR products involves the use of non-specific fluorescent dyes and sequence-specific fluorescently labeled DNA probes that intercalate into any double-stranded DNA. Real-time PCR techniques and systems are known in the art (see, for example, Dorak, M. Tevfik, eds., Real-time PCR. Taylor & Francis (2007); and Fraga et al., "Real-time PCR," Current protocols essential laboratory techniques: 10-3 (2008); each of these is incorporated herein by reference in its entirety) and are commercially available from various suppliers (for example, the m2000rt REALTIME® PCR system (Abbott Molecular, Inc., Des Plaines, IL); the CFX real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA); and the TAQMAN® real-time PCR system (ThermoFisher Scientific, Waltham, MA)), and all of them can be used in the methods described herein.

[0055] A set of oligonucleotides useful for amplification may include a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to any of SEQ ID NOs: 1 and 10-16, a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 3, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 5; and / or a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 2, a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 4, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NOs: 6.

[0056] In select embodiments, isothermal amplification methods may rely on nickeling and extension reactions, or "nicking and extension amplification," to amplify shorter sequences in a faster timeframe than traditional amplification reactions. These methods may include, for example, reactions using only two amplification oligonucleotides, one or two nickeling enzymes and polymerases under isothermal conditions. A set of oligonucleotides useful for nickeling and extension amplification includes a first amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 7, a second amplification oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 8, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 9.

[0057] In nickeling and extension amplification, a target nucleic acid sequence having sense and antisense strands is brought into contact with a pair of amplification oligonucleotides. The first amplification oligonucleotide includes a nucleic acid sequence with a recognition region at its 3' end that is complementary to the 3' end of the target sequence antisense strand, a nickeling enzyme site upstream of the recognition region, and a stabilization region upstream of the nickeling enzyme site. The second amplification oligonucleotide includes a nucleotide sequence with a recognition region at its 3' end that is complementary to the 3' end of the target sequence sense strand, a nickeling enzyme site upstream of the recognition region, and a stabilization region upstream of the nickeling enzyme site. Two types of nickeling enzymes are prepared. One nickeling enzyme is nickelable at the nickeling enzyme site of the first amplification oligonucleotide but not within the target sequence. The other nickeling enzyme is nickelable at the nickeling enzyme site of the second amplification oligonucleotide but not within the target sequence. DNA polymerase is used under amplification conditions that involve the extension of multiple cycles of the amplified oligonucleotide, thereby generating double-stranded nickeling enzyme sites that are nickeled by nickeling enzymes to produce the amplified product. See, for example, U.S. Patent No. 9,689,031; U.S. Patent No. 9,617,586; U.S. Patent No. 9,562,264; and U.S. Patent No. 9,562,263, as well as U.S. Patent Application No. 15 / 467,893; U.S. Patent Application No. 15 / 600,951; and U.S. Patent Application No. 16 / 243 / 829. Each of these is incorporated herein by reference in its entirety.

[0058] In some embodiments, the ID NOW COVID-19 assay uses Nicking enzyme amplification (NEAR), an isothermal nucleic acid amplification technique, to target a highly conserved region of the RdRp gene in SARS-CoV-2 RNA. In some embodiments, the assay system includes a sample receiver containing elution / lysis buffer; a test base containing two sealed reaction tubes, each containing a lyophilized pellet; a transfer cartridge for transferring the eluted sample to the test base; and the ID NOW instrument. The ID NOW COVID-19 assay delivers positive results in just 5 minutes and negative results in 13 minutes, providing rapid COVID-19 results in a wide range of healthcare settings.

[0059] In some embodiments, the limit of detection (LOD) of the ID NOW COVID-19 POC assay is ≤125 copies / mL. In silico analysis revealed that all templates and probes have 100% homology to 957 SARS-CoV-2 sequences reported from 45 countries and 21 cities, as well as provinces in China, and predicted no significant cross-reactivity with other coronaviruses, influenza, RSV, and rhinoviruses, or other microorganisms causing common respiratory illnesses.

[0060] Clinical outcomes were assessed using 30 artificial samples containing SARS-CoV-2 RNA at known concentrations and 30 artificial negative samples. SARS-CoV-2 RNA was detected in all positive samples (positive agreement percentage 100% [CI, 88.6–100%]) and not detected in negative samples (negative agreement percentage 100% [CI, 88.6–100%]).

[0061] In select embodiments, the amplification of the SARS-CoV-2 nucleic acid sequence is performed using recombinase-polymerase amplification (RPA), which relies on the properties of the recombinase and related proteins, to insert single-stranded homologous DNA into double-stranded DNA, enabling sequence-specific priming of the DNA polymerase reaction.

[0062] A set of oligonucleotides useful for RPA includes a first amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 17, a second amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 19, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 18; or a first amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 20 or 21, a second amplified oligonucleotide containing a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25 or 26, and a probe oligonucleotide containing a nucleic acid sequence having at least 70% similarity to any of SEQ ID NOs: 22-24.

[0063] In some embodiments, the first amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 20, the second amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25, and the probe oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 22 or 23. In some embodiments, the first amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 21, the second amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 25 or 26, and the probe oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 24.

[0064] In RPA, a recombinase agent is brought into contact with the first and second amplification oligonucleotides to form a nucleoprotein. These nucleoproteins come into contact with the target sequence to form a first double-stranded structure in the first portion of the first strand and a double-stranded structure in the second portion of the second strand, so that the 3' ends of the first and second amplification oligonucleotides are oriented toward each other on the DNA containing the target sequence. The 3' ends of these amplification oligonucleotides in the nucleoprotein are extended by DNA polymerase to generate the first and second double-stranded nucleic acids and the first and second substitution strands of the nucleic acids. These steps are repeated until the desired amplification level is achieved.

[0065] Methods and materials useful for RPA of target nucleic acid sequences are known in the art, as described in U.S. Patent No. 7,270,981; U.S. Patent No. 8,460,875; U.S. Patent No. 7,399,590; U.S. Patent No. 7,666,598; U.S. Patent No. 8,030,000; U.S. Patent No. 8,426,134; U.S. Patent No. 8,945,845; U.S. Patent No. 9,663,820; U.S. Patent No. 10,329,603; U.S. Patent No. 10,329,602; U.S. Patent No. 8,017,339; See U.S. Patent No. 8,574,846; U.S. Patent No. 8,962,255; U.S. Patent No. 10,036,057; U.S. Patent No. 8,071,308; U.S. Patent No. 10,093,908; and U.S. Patent No. 8,637,253, as well as U.S. Patent Application No. 15 / 099,754; U.S. Patent Application No. 16 / 442,007; U.S. Patent Application No. 14 / 705,150; and U.S. Patent Application No. 16 / 155,133; each of these is incorporated herein by reference in whole. For example, suitable recombinase agents include Escherichia coli (E. coli) RecA protein, T4 uvsX protein, or any homologous protein or protein complex derived from any phylum. Other non-homologous recombinase agents may be used instead of RecA, for example, as RecT or RecO. Suitable recombinase-loading proteins include, for example, T4uvsY, E. coli recO, E. coli recR, and derivatives and combinations of these proteins. Suitable single-stranded DNA-binding proteins may be E. coli SSB or T4gp32, or derivatives or combinations of these proteins. The DNA polymerase may be a eukaryotic or prokaryotic polymerase. Examples of eukaryotic polymerases include pol-α, pol-β, pol-δ, pol-ε, and their derivatives and combinations.Examples of prokaryotic polymerases include E. coli DNA polymerase I Klenow fragment, bacteriophage T4 gp43 DNA polymerase, Bacillus stearothermophilus polymerase I large fragment, Phi-29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, E. coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E. coli DNA polymerase V, and their derivatives and combinations. Other components of RPA include ATP, ATP analogs, or systems for ATP regeneration (conversion of ADP to ATP). Such systems can utilize, for example, phosphocreatine and creatine kinase. ATP or ATP analogs may be ATP, ATP-γ-S, ATP-β-S, ddATP, or combinations thereof. The RPA reaction may also include a system for regenerating ADP from AMP and converting pyrophosphates to phosphates (pyrophosphates). Suitable crowding agents used in RPA include polyethylene glycol (PEG), dextran, and Ficol.

[0066] Following the amplification of one or more SARS-CoV-2 virus nucleic acid sequences present in a sample, the disclosed method may further include the step of hybridizing one or more probe oligonucleotide sequences disclosed herein to one or more amplified target SARS-CoV-2 (2019-nCoV) nucleic acid sequences.

[0067] Following the hybridization of one or more probe oligonucleotide sequences to one or more amplified target nucleic acid sequences, the method includes the step of detecting the hybridization of one or more probe oligonucleotide sequences to one or more amplified target nucleic acid sequences by measuring the signal from each of the detectable labels, wherein (i) the presence of one or more signals indicates the hybridization of one or more probe oligonucleotide sequences to one or more target SARS-CoV-2 nucleic acid sequences and the presence of SARS-CoV-2 in the sample, and (ii) the absence of signals indicates the absence of SARS-CoV-2 in the sample. Detection of signals from one or more probe oligonucleotide sequences can be carried out using a variety of well-known methodologies, depending on the type of detectable label. For example, detection can be carried out using solution real-time fluorescence or using a solid surface method.

[0068] 4. Treatment and monitoring of individuals identified as having SARS-CoV-2 Subjects identified as having SARS-CoV-2 according to the methods described herein can be treated, monitored (for the presence of SARS-CoV-2 nucleic acid determined in samples from the subject), treated and monitored, and / or monitored and treated using routine techniques known in the art. In some embodiments, the methods described herein further include the step of treating the subject if the presence of SARS-CoV-2 nucleic acid is determined in one or more samples obtained from the subject using the methods.

[0069] Treatment may take various forms depending on whether the patient is asymptomatic or experiencing mild, moderate, or severe symptoms of SARS-CoV-2 infection. For example, patients experiencing mild symptoms may have fever, cough (with or without sputum production), loss of appetite, fatigue, muscle pain, sore throat, shortness of breath, nasal congestion, headache, diarrhea, nausea, vomiting, or any combination thereof. Patients experiencing moderate symptoms may have a fever above 100.4°F lasting several days, chills, shortness of breath, lethargy, or any combination thereof. Such patients may also develop pneumonia. Patients experiencing severe infection may have shortness of breath, persistent chest pain or pressure, confusion, unresponsiveness, bluish lips or face, or any combination thereof. Such patients may develop severe pneumonia.

[0070] If the subject is asymptomatic or has mild symptoms, the subject may be treated with rest, sleep, keeping warm, fluid intake (e.g., continuous hydration), minimizing social contact with other subjects (e.g., isolation or continuous compulsory isolation, e.g., home isolation), or any combination thereof. Furthermore, the subject may be monitored to see if symptoms occur and / or worsen.

[0071] Subjects with moderate or severe symptoms of SARS-CoV-2 infection may be treated with one or more drugs (e.g., remdesivir), vaccines, convalescent plasma therapy (e.g., receiving plasma derived from blood collected from subjects who have overcome SARS-CoV-2 infection), respiratory support or assistance (e.g., receiving oxygen through nasal cannula, face mask, or invasive or non-invasive (e.g., intubation) assisted ventilation), or a combination thereof. Subjects receiving any of the above treatments may also be monitored using routine techniques known in the art.

[0072] 5. Vaccination In another embodiment, the disclosure relates to the use of the method described herein in connection with at least one vaccination and / or revaccination (e.g., further vaccination) of a subject against SARS-CoV-2 (2019-nCoV). In some embodiments, the method is used to detect the presence of SARS-CoV-2 nucleic acid in at least one sample obtained from a subject to determine whether or not at least one vaccine against SARS-CoV-2 (e.g., a first or initial vaccine, one or more further or supplemental vaccines, etc.) should or can be administered to the subject. In some embodiments, the subject being tested may be an unsensitized subject, such as one that has no immunity to SARS-CoV-2 or lacks immunologic immunity. In some embodiments, the subject may be a subject that has been previously vaccinated against SARS-CoV-2 but is unsensitized. In some embodiments, the subject may be a subject that is currently infected with SARS-CoV-2 but is asymptomatic or has mild symptoms and has never been previously vaccinated. In some embodiments, subjects may be individuals who are currently infected with SARS-CoV-2, are asymptomatic or have mild symptoms, and have previously received a SARS-CoV-2 vaccine. In some embodiments, subjects may be individuals who have recovered from a previous SARS-CoV-2 infection and have never previously received a SARS-CoV-2 vaccine. In some embodiments, subjects may be individuals who have recovered from a previous SARS-CoV-2 infection and have previously received a SARS-CoV-2 vaccine.

[0073] The method described above can be carried out regardless of the timing and / or severity of previous SARS-CoV-2 infections. Regardless of whether the administered vaccine is a first dose, a second dose (such as a booster), a third dose, or any further additional dose (such as a booster), if the presence of SARS-CoV-2 nucleic acid is detected in the sample using the method described herein, the subject must wait for a certain period (e.g., 30 days, 60 days, 90 days, etc.) before being administered at least one vaccine against SARS-CoV-2. Alternatively, if the presence of SARS-CoV-2 nucleic acid is not detected in the sample, at least one vaccine can be administered to the subject.

[0074] The phrase "at least one additional vaccine or at least one additional vaccination" or "at least one booster vaccine or at least one booster vaccination" implies a plan in which, after the first or current vaccine has been administered to the subject, an additional or additional vaccine or vaccination (e.g., N+1 (where N is the first or current vaccine + an additional or additional vaccine), N+2 (where N is the first vaccine + two additional vaccines), N+3, N+4, N+5, N+6, N+7, N+8, N+9, N+10 to N+N' (where N' is 1 to 1000, 1 to 500, 1 to 100)) is administered at a later point in time.

[0075] In another embodiment, the method described herein is used to detect the presence of SARS-CoV-2 nucleic acid in at least one sample obtained from a subject within a certain time frame after the subject has been administered at least one vaccine against SARS-CoV-2, in order to determine whether the subject should be administered at least one additional vaccine against SARS-CoV-2 (e.g., receive one or more boosters); and / or to monitor the subject after the administration of at least one vaccine against SARS-CoV-2. In some embodiments, the method includes the step of obtaining a sample within a certain time frame after the subject has been administered at least one vaccine against SARS-CoV-2. The time frame after administering at least one SARS-CoV-2 vaccine to the subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, This can be at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days, at least 40 days, at least 41 days, at least 42 days, at least 43 days, at least 44 days, at least 45 days, at least 46 days, at least 47 days, at least 48 days, at least 49 days, at least 50 days, and so on. In some embodiments, the sample is obtained about 7 to about 21 days after the subject has been administered at least one SARS-CoV-2 vaccine.Furthermore, in even more embodiments, the monitoring of the subject includes monitoring for post-vaccination overall symptoms or adverse events (e.g., fatigue or malaise, headache, dizziness or confusion, fever or chills, muscle, bone, joint or nerve symptoms, nausea, vomiting, diarrhea or other gastrointestinal symptoms, sleep changes, lymph node enlargement, skin / nail or facial changes, eye, ear, mouth or throat changes, cough, chest or respiratory symptoms, and / or memory or mood changes) after the subject has received one or more vaccines (e.g., after the first dose of the vaccine against SARS-CoV-2, after the second dose of the vaccine against SARS-CoV-2, etc.).

[0076] If SARS-CoV-2 nucleic acid is not detected in the sample using the methods described herein, at least one additional vaccine (e.g., one or more boosters) may be administered to the target. Alternatively, if SARS-CoV-2 nucleic acid is detected in the sample, at least one additional vaccine may or may not be administered to the target.

[0077] 6. Kit This disclosure also provides kits for amplifying and detecting SARS-CoV-2 (2019-nCoV) in a sample. The kits comprise at least one oligonucleotide described herein. In some embodiments, the kit comprises a set or group of oligonucleotides disclosed herein. The kits may further comprise reagents for amplifying and detecting nucleic acid sequences and instructions for use for amplifying and detecting SARS-CoV-2. The descriptions herein of oligonucleotides and sets of oligonucleotides relating to the said methods also apply to the same embodiments of the kits described herein. Many such reagents are described herein or otherwise known in the art and commercially available. Examples of reagents suitable for inclusion in the kit (in addition to the oligonucleotides described herein) include conventional reagents used in nucleic acid amplification reactions, such as one or more enzymes having polymerase activity, enzyme cofactors (e.g., magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, deoxyribonucleotides or ribonucleotide triphosphates (dNTPs / rNTPs; e.g., deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate), and labeling agents. Other reagents used in amplification reactions include nickeling enzymes, single-chain binding proteins, helicases, and resolvers.

[0078] The kit may include instructions for use of the amplification reagents and oligonucleotides described herein, for example, for processing test samples, extracting nucleic acid molecules, and / or performing tests, and for interpreting the results obtained. These instructions may be provided in print or electronically (e.g., available for viewing or acquisition via DVD, CD, or internet resources).

[0079] The kit can be supplied in solid (e.g., lyophilized) or liquid form. The various components of the kit of this disclosure may optionally include different containers (e.g., vials, ampoules, test tubes, flasks, or bottles) for each of the individual components (e.g., amplification oligonucleotides, probe oligonucleotides, or buffers). Each component is generally preferred to be supplied in a form dispensed or concentrated in its respective container. Other containers suitable for performing a particular step of an amplification / detection assay may also be provided. The individual containers are preferably kept tightly sealed for commercial sale.

[0080] The kit may further include a swab for obtaining a biological sample. In some embodiments, the kit includes reagents for accessing the biological sample and / or for extracting / isolating nucleic acids from the biological sample. [Examples]

[0081] 7. Examples [Example 1] The SARS-CoV-2 (2019-nCoV) nucleic acid sequence was amplified by PCR using various first amplification oligonucleotides together with a second amplification oligonucleotide containing SEQ ID NO: 3 and a probe oligonucleotide containing SEQ ID NO: 5.

[0082] The SARS-CoV-2 sample consisted of genomic RNA derived from the SARS-related coronavirus 2 isolate USA-WA1 / 2020 (BEI Resources). Genomic RNA was extracted from cell lysates and supernatant preparations from renal epithelial cells (Vero E6, ATCC® CRL-1586®) of Cercopithecus aethiops infected with the SARS-CoV-2 isolate USA-WA1 / 2020. The viral genomic RNA was present in the background of cellular nucleic acids and carrier RNA.

[0083] PCR cycling parameters:

[0084] [Table 3]

[0085] The PCR reaction mixture contained a COVID-19 primer set (primer set 1) containing SARS-CoV-2, SEQ ID NO: 3, and one of SEQ ID NOs: 1, 10, 12-14, or 16, as well as the probe oligonucleotide of SEQ ID NO: 5. Where indicated, an internal control nucleic acid and a primer set for the internal control were also included as a positive control. The reaction mixture also contained a reaction buffer, dNTPs, a reference dye, and DNA polymerase (rTth) at concentrations commonly used in the art.

[0086] Table 3 shows the results for each primer set. Both the SARS-CoV-2 target and the internal standard RNA were amplified under cycling conditions.

[0087] [Table 4]

[0088] An additional PCR reaction was performed, including a second COVID-19 primer set (primer set 2) along with the selected first COVID-19 primer set. The second COVID-19 primer set included SEQ ID NO: 2 and SEQ ID NO: 4. The reaction also included SEQ ID NO: 6 as a probe oligonucleotide. As shown in Table 4, primer set 2 showed a stronger signal (dRn) compared to any of primer sets 1, but the strongest overall signal was observed when primer set 1 was added to primer set 2.

[0089] [Table 5]

[0090] [Example 2] The SARS-CoV-2 (2019-nCoV) nucleic acid sequence was amplified using recombinase-polymerase amplification (TRA) with various combinations of amplification oligonucleotides and probe oligonucleotides. The combinations tested are shown in Table 5. Combination 1 targeted the target region, while combinations 2-5 targeted a secondary target region.

[0091] [Table 6]

[0092] Figures 1A and 1B show graphs of the dRn values ​​over time for each of the five combinations. The SARS-CoV-2 target was amplified under the reaction conditions for all combinations, and a detectable level of amplification occurred within 10 minutes of the start of the reaction. In combinations 1, 4, and 5, target amplification was detected between 4 and 6 minutes (Figures 1A and 1B).

[0093] [Example 3] Inclusivity was demonstrated by analyzing the sequences of SARS-CoV-2 primers and probes for homology to all full-length SARS-CoV-2 sequences available in GenBank as of April 28, 2020. A total of 1383 full-length SARS-CoV-2 genome sequences from 26 countries / regions (Australia, Brazil, China, Colombia, Czech Republic, France, Greece, Hong Kong, India, Iran, Israel, Italy, Malaysia, Nepal, Netherlands, Pakistan, Peru, South Africa, South Korea, Spain, Sri Lanka, Sweden, Taiwan, Turkey, USA, and Vietnam) were analyzed. 99.5% (1376 / 1383) showed 100% identity to all SARS-CoV-2 primer and probe sequences, while 0.5% (7 / 1383) contained a single mismatch for one of the primers or probes.

[0094] The comprehensiveness of the SARS-CoV-2 primers and probes was demonstrated by analyzing their respective sequences for homology to all full-length SARS-CoV-2 sequences available in the GISAID database as of May 5, 2020. A total of 14,964 full-length SARS-CoV-2 genome sequences from 81 countries / regions were analyzed; 1.1% (170 / 14,964) contained a single mismatch, 0.04% (6 / 14,964) contained two mismatches, and 0.007% (1 / 14,964) contained four mismatches.

[0095] [Example 4] The disclosed primer sets were evaluated using the Abbott Alinity m system with known SARS-CoV-2 positive, negative, and non-SARS-CoV-2 respiratory samples using real-time RT-PCR. As shown in Table 6, the detection limit of 100 copies / mL was determined by the dilution series of known positive samples. The limit of detection (LOD) was defined as the lowest detectable concentration of SARS-CoV-2 in which 95% or more of the total (true positive) replicas were positive as a result of the test.

[0096] [Table 7]

[0097] 109 pre-tested frozen nasopharyngeal swabs were re-tested using the real-time PCR assay used above for positive (PPA) and negative (NPA) agreement percentages (Table 7). The PPA rate was found to be 89.8% (53 / 59), and the NPA rate was found to be 98% (49 / 50).

[0098] [Table 8]

[0099] Clinical correlation, cross-reactivity, and detection limits (Table 8) were also evaluated using the Abbott m2000 real-time SARS-CoV-2 assay.

[0100] [Table 9]

[0101] A total of 104 samples were analyzed using both the Abbott m2000 real-time SARS-CoV-2 assay and the Alinity m SARS-CoV-2 real-time RT-PCR assay. The positive agreement percentage (PPA) between the two assays was 100% (47 / 47), and the negative agreement percentage (NPA) was 96.5% (55 / 57). The results are summarized in Tables 9 and 10.

[0102] [Table 10]

[0103] [Table 11]

[0104] [Example 5] The SARS-CoV-2 B.1.1.7 strain, first identified in the UK, is of greatest concern due to its observed increased transmissibility and association with spike gene mutations. While spike gene mutations primarily characterize the B.1.1.7 lineage, the presence of additional mutations throughout the genome could affect the performance of various diagnostic assays.

[0105] An initial in silico study of the B.1.1.7 strain in GISAID (N=1787, accessed December 21, 2020) did not reveal any lineage-defining mutations that would raise concerns about the performance of the primers, probes, and methods described herein. Viral cultures (BEI NR-54011, EPI_ISL_751801) were heat-inactivated at 65°C for 30 minutes and tested in dilution series. Multiple dilutions were detected within the expected range previously observed for other strains (Table 11). These results support the in silico prediction that the primers, probes, and methods can reliably detect the B.1.1.7 strain. These results are consistent with a recent assessment conducted by Public Health England.

[0106] Further evaluation was performed using the remaining patient nasopharyngeal swab samples. The genome sequences of all samples were completed, and they were confirmed to belong to the B.1.1.7 lineage. Due to limited supply, the remaining VTM (viral transport media) was diluted 5 to 62.5 times before testing. A sufficient amount of all samples were detected (Table 11).

[0107] [Table 12]

[0108] The B.1.351 strain was first identified in South Africa. Since then, it has spread to more than a dozen countries. Early reports indicate that this variant may evade neutralizing antibodies. The unique mutation profile of the B.1.351 strain is primarily characterized by spike gene mutations K417N, E484K, and N501Y, but the presence of additional mutations throughout the genome may affect the performance of various diagnostic assays.

[0109] An initial in silico study of the B.1.351 strain at GISAID (N=195, accessed December 27, 2020) did not reveal any strain-characterizing mutations that would raise concerns about the performance of the primers, probes, and methods described herein. Two virus cultures (BEI NR-54008, NR-54009) were thermally inactivated and tested in a dilution series, and detection was performed within the range previously observed in other strains (Table 12). These results supported the in silico prediction that the primers, probes, and methods can reliably detect the B.1.351 strain.

[0110] [Table 13]

[0111] The above-mentioned detailed description and accompanying examples are merely illustrative and should not be construed as limitations on the scope of the disclosure, and the scope of the disclosure is defined solely by the attached claims and their equivalents.

[0112] Various changes and modifications to the disclosed embodiments will be obvious to those skilled in the art and can be made without departing from their spirit and scope.

Claims

1. A set of oligonucleotides for the recombinase-polymerase amplification and detection of SARS-CoV-2 in a sample, (i) A first amplified oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 17, a second amplified oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 19, and a probe oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 18, (ii) A first amplified oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 20, a second amplified oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, and a probe oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 22, (iii) A first amplified oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 20, a second amplified oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, and a probe oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:

23. (iv) A first amplified oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 21, a second amplified oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, and a probe oligonucleotide comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:

24. (v) A first amplified oligonucleotide containing a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 21, a second amplified oligonucleotide containing a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 26, and a probe oligonucleotide containing a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 24, or those combinations Includes, Each probe oligonucleotide contains a detectable label. set.

2. The set according to claim 1, wherein the detectable label is a fluorophore.

3. A method for detecting SARS-CoV-2 in a sample, The step of contacting the sample with the oligonucleotide set and amplification reagent described in claim 1 or 2, A step of amplifying one or more target SARS-CoV-2 nucleic acid sequences present in a sample using recombinase-polymerase amplification. The steps include hybridizing one or more oligonucleotide probes to one or more amplified target SARS-CoV-2 nucleic acid sequences, and A step to detect the hybridization of one or more probe oligonucleotide sequences to one or more amplified SARS-CoV-2 target nucleic acid sequences by measuring the signal from a detectable label. Methods that include...

4. The method according to claim 3, wherein the presence of one or more signals from a detectable label indicates hybridization of one or more probe oligonucleotides to one or more amplified SARS-CoV-2 target nucleic acid sequences.

5. The method according to claim 3 or 4, further comprising the step of contacting first and second amplified oligonucleotides from a set of oligonucleotides with a recombinase agent.

6. The method according to any one of claims 3 to 5, wherein the amplification reagent is selected from the group consisting of polymerase; recombinase; recombinase loading protein; single-strand binding protein; buffer; deoxyribonucleotide; or ribonucleotide triphosphate; crowding agent; ATP, ATP analogues or ATP production systems; or combinations thereof.

7. The method according to any one of claims 3 to 6, wherein the sample comprises a nasal swab or brush, saliva, mucus, blood, serum, plasma, or feces.

8. A kit for detecting SARS-CoV-2 in a sample, comprising the set of oligonucleotides described in claim 1 or 2.

9. The kit according to claim 8, further comprising reagents for amplifying and detecting nucleic acid sequences and / or instructions for use.

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