CN116710570A - Assays for the detection of coronavirus disease 2019 (COVID-19) - Google Patents

Abstract

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

Description

Assays for the detection of coronavirus disease 2019 (COVID-19)

Cross References to Related Applications

This application claims U.S. Provisional Application No. 63/000,304, filed March 26, 2020, U.S. Provisional Application No. 63/000,971, filed March 27, 2020, U.S. Provisional Application No. 63/004,773, filed April 3, 2020 , U.S. Provisional Application No. 63/049,237, filed July 8, 2020, and U.S. Provisional Application No. 63/155,599, filed March 2, 2021, the contents of each of which are hereby incorporated by reference in their entirety middle.

Incorporation by Reference of Materials Submitted Electronically

Incorporated herein by reference in its entirety is the computer readable nucleotide/amino acid sequence listing filed herewith and identified as follows: Created on March 24, 2021 and named "2021-03-24_38391-601_SQL_ST25.txt" A 5136-byte ASCII (text) file of .

field

The present disclosure relates to methods for amplifying target nucleic acid sequences from SARS-CoV-2 and detecting COVID-19.

Background of the invention

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

As of the end of February 2021, more than 100 million cases of COVID-19 have been confirmed in more than 200 countries, and complications of COVID-19 have been cited as causing the death of more than 2.5 million individuals.

Summary of the invention

The present disclosure provides reagents, including 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. Probe oligonucleotides may comprise a detectable label (eg, a fluorophore). In some embodiments, the set of oligonucleotides is used for recombinase-polymerase amplification and detection of SARS-CoV-2 in a sample. In some embodiments, a reagent comprises a population of oligonucleotides comprising one or more sets of oligonucleotides.

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

In some embodiments, the group of oligonucleotides for amplifying and detecting SARS-CoV-2 in a sample comprises a first group of oligonucleotides comprising a group comprising the same sequence as SEQ ID NO: 2 have the first amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity, the second amplified oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID NO:4 and containing the nucleotide sequence with SEQ ID NO:4 NO: 6 Probe oligonucleotides having nucleic acid sequences with at least 70% similarity. In some embodiments, the group of oligonucleotides further comprises a second set of oligonucleotides comprising: a compound having at least 70% similarity to any one of SEQ ID NO: 11 and 15 The first amplified oligonucleotide of nucleic acid sequence, the second amplified oligonucleotide containing a nucleic acid sequence with at least 70% similarity to SEQ ID NO:3 and the second amplified oligonucleotide containing a nucleic acid sequence with at least 70% similarity to SEQ ID:5 Probe oligonucleotides for nucleic acid sequences.

The present disclosure also provides methods for detecting SARS-CoV-2 in a sample. Samples may comprise nasal swabs or brushes, saliva, mucus, blood, serum, plasma or feces.

In some embodiments, methods include: contacting a sample with a set or population of oligonucleotides as described herein and reagents for amplification; using recombinase-polymerase amplification (RPA) to amplify one or more target SARS-CoV-2 nucleic acid sequences present; make one or more oligonucleotide probes hybridize with one or more amplified target SARS-CoV-2 nucleic acid sequences; A labeled signal detects hybridization of one or more probe oligonucleotide sequences to one or more amplified SARS-CoV-2 target nucleic acid sequences. In some embodiments, the method further comprises contacting the first and second amplified oligonucleotides from the set of oligonucleotides with a recombinase agent. The presence of one or more signals from the detectable label can indicate hybridization of the one or more probe oligonucleotides to one or more amplified SARS-CoV-2 target nucleic acid sequences.

Reagents for amplification may include: polymerase; recombinase agent; recombinase-loaded protein; single-strand binding protein; nickase; or ribonucleoside triphosphates; a clustering agent; ATP, an ATP analog, or an ATP generating system; or a combination thereof.

The present disclosure further provides a kit for detecting SARS-CoV-2 in a sample comprising at least one oligonucleotide set, any oligonucleotide as disclosed herein, reagents for amplifying and detecting nucleic acid sequences, and /or instructions for use.

Other aspects and embodiments of the disclosure will be apparent from the following detailed description and accompanying drawings.

Brief description of the drawings

This 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.

Figures 1A and 1B are graphs of recombinase-polymerase amplification (RPA) of SARS-CoV-2 using various amplification and probe oligonucleotide combinations.

Detailed description of the invention

This disclosure is based at least in part on the development of a series of oligonucleotide sequences that facilitate the rapid detection of COVID-19.

As used herein, the terms "comprises", "including", "having", "has", "may", "containing" and variations thereof are intended not to exclude the possibility of additional acts or configurations open-ended transitional phrases, terms or words. The singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements presented herein, whether expressly stated or not.

For the recitation of numerical ranges herein, each intervening figure is expressly contemplated with the same degree of precision. For example, for the range 6-9, the numbers 7 and 8 are also considered in addition to 6 and 9, while for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

The terms "first" and "second" are used in this disclosure only in their relative sense. It should be understood that unless stated otherwise, these terms are used for convenience only in describing one or more embodiments. The terms "first" and "second" are only used to distinguish one element from another, and the scope of rights of the disclosed technology should not be limited by these terms. For example, a first element may be designated as a second element, and similarly a second element may be designated as a first element.

As used herein, the term "oligonucleotide" refers to an oligonucleotide comprising about 2 to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, A short nucleic acid sequence of 65, 70, 75, 80, 85, 90, 95, 99 or 100 nucleotides, or a range defined by any of the above values). The terms "nucleic acid" and "polynucleotide" as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA as well as double- and single-stranded RNA. These terms include as equivalents RNA or DNA analogs made from nucleotide analogs and modified polynucleotides, such as methylated and/or capped polynucleotides. Nucleic acids are typically joined by phosphate linkages to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (eg, phosphorothioate, boranephosphate, etc.).

An oligonucleotide can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Oligonucleotides can be DNA (both genomic and complementary DNA (cDNA)), RNA, or hybrids, where nucleic acids can contain combinations of deoxyribonucleotides and ribonucleotides, and include uracil, adenine, thymus A combination of bases including pyrimidine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Oligonucleotides can be obtained by chemical synthesis methods or by recombinant methods.

The term "percent sequence identity" as used herein refers to a nucleic acid sequence that is identical to the corresponding nucleotide or amino acid in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity The percentage of nucleotides or nucleotide analogs or amino acids in an amino acid sequence. Thus, if a nucleic acid according to the technique is longer than the reference sequence, additional 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 are well known in the art and include BLAST, Align 2 and FASTA.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

The preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present 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 organizational purposes only and should not be construed as limiting the described subject matter in any way.

1. Amplification and Probe Oligonucleotides

In one embodiment, the oligonucleotides described herein are useful in nucleic acid amplification (eg, primers) or as probes for nucleic acid hybridization and detection. The terms "primer", "primer sequence", "primer oligonucleotide" and "amplification oligonucleotide" as used herein refer to the presence of nucleotides and nucleic acid polymerizing agents (such as DNA-dependent or RNA-dependent Capable of serving as an initiation point for the synthesis of extension products that are complementary strands of nucleic acids (DNA or RNA of all types) when placed under appropriate amplification conditions (such as buffer, salt, temperature, and pH) of oligonucleotides. The amplification oligonucleotides of the present disclosure may be of any suitable size, and desirably comprise, consist essentially of, or consist of about 15-50 nucleotides, preferably about 20-40 nucleotides. The oligonucleotides of the present disclosure may also contain additional nucleotides in addition to those described herein. Depending on the type of amplification process employed, the amplification oligonucleotide may include, for example, a nickase site and an upstream stabilizing region (see, e.g., U.S. Pat. to this article).

The terms "probe", "probe sequence" and "probe oligonucleotide" refer to a probe that selectively hybridizes to at least a portion of a target sequence (eg, a portion of an amplified target sequence) under appropriate hybridization conditions. of oligonucleotides. Typically, probe sequences are identified as "complementary" (eg, complementary to the coding or sense strand (+)) or "reverse complementary" (eg, complementary to the antisense strand (-)). Probes of the present disclosure may be of any suitable size, and desirably comprise, consist essentially of, or consist of about 10-50 nucleotides, preferably about 12-35 nucleotides.

As used herein, the terms "set", "set of primers", "set of probes" and "set of primers and probes" refer to a set of primers or probes that together are capable of priming a target sequence of interest or a target nucleic acid (such as a target within SARS-CoV-2). sequence) and/or at least one probe capable of detecting a 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 The second oligonucleotide hybridizes to the target sequence or the complement of the target nucleic acid.

The oligonucleotide sets 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 a specific nucleic acid sequence whose presence or absence is to be detected by the disclosed methods. In the context of the present disclosure, a target sequence preferably comprises a nucleic acid sequence to which one or more oligonucleotides will hybridize and thereby initiate amplification. The target sequence may also include a region of the probe to which the probe may hybridize under appropriate amplification conditions to form a stable hybrid. The target sequence can be single-stranded or double-stranded. The SARS-CoV-2 sequence of interest can be located within any part of the SARS-CoV-2 genome, such as the gene encoding the nucleocapsid (N) protein or the gene encoding RNA-dependent RNA polymerase (RDRP).

In some embodiments, the set comprises a first amplification oligonucleotide, a second amplification oligonucleotide, and a probe oligonucleotide. In some embodiments, the group comprises at least 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%) of any one of SEQ ID NO: 1 and 10-16 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the first amplified oligonucleotide of the nucleic acid sequence of similarity, containing at least 70% similarity with SEQ ID NO:3 The second amplified oligonucleotide of the nucleotide sequence and the probe oligonucleotide containing the nucleotide sequence with at least 70% similarity with SEQ ID NO:5; or containing at least 70% similarity with SEQ ID NO:2 The first amplified oligonucleotide of a specific nucleic acid sequence, the second amplified oligonucleotide containing a nucleic acid sequence with at least 70% similarity with SEQ ID NO:4 and containing at least 70% with SEQ ID NO:6 The probe oligonucleotide of the nucleic acid sequence of % similarity; Or contain the first amplification oligonucleotide of the nucleic acid sequence that has at least 70% similarity with SEQ ID NO:7, contain and SEQ ID NO:8 have at least The second amplified oligonucleotide of the nucleic acid sequence of 70% similarity and the probe oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID NO:9; Or containing and SEQ ID NO:17 has The first amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity, the second amplified oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID: 19 and the second amplified oligonucleotide containing the nucleic acid sequence with SEQ ID NO: 18 There is the probe oligonucleotide of the nucleic acid sequence of at least 70% similarity; Or contain the first amplification oligonucleotide of the nucleic acid sequence that has at least 70% similarity with SEQ ID NO:20 or 21, contain and SEQ ID NO: 25 or 26 have the second amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity and contain any one in SEQ ID NO:22-24 and have the probe oligonucleotide of the nucleic acid sequence of at least 70% similarity nucleotide; or a combination thereof.

In some embodiments, the group comprises a first amplified oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to any one of SEQ ID NO: 1 and 10-16, comprising a sequence identical to SEQ ID NO: 3 There is the second amplification oligonucleotide of the nucleic acid sequence of at least 70% similarity and the probe oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID NO:5; And containing and SEQ ID NO: 2 have the first amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity, the second amplified oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID NO:4 and containing the nucleotide sequence with SEQ ID NO:4 :6 Probe oligonucleotides having nucleic acid sequences of at least 70% similarity.

In some embodiments, the group comprises a first amplified oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO:20, a nucleic acid comprising at least 70% similarity to SEQ ID NO:25 A second amplification oligonucleotide of sequence and a probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO:22 or 23.

In some embodiments, the group comprises a first amplified oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO:21, comprising at least 70% similarity to SEQ ID NO:25 or 26 The second amplification oligonucleotide of the nucleic acid sequence and the probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO:24.

In some embodiments, the set comprises an oligonucleotide for recombinase-polymerase amplification and detection of SARS-CoV-2 (2019-nCoV) in a sample comprising: The first amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity, the second amplified oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID NO:19 and the second amplified oligonucleotide containing the nucleic acid sequence with SEQ ID NO:19 18 has the probe oligonucleotide of the nucleic acid sequence of at least 70% similarity; Or contains the first amplification oligonucleotide of the nucleic acid sequence that has at least 70% similarity with SEQ ID NO:20 or 21, contains and SEQ ID NO:20 or 21 has the nucleic acid sequence of at least 70% similarity IDNO:25 or 26 has the second amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity and contains any one in SEQ ID NO:22-24 has the probe oligonucleotide with the nucleic acid sequence of at least 70% similarity Nucleotides; or combinations thereof, wherein each probe oligonucleotide comprises a detectable label. In some embodiments, the first amplification oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO:20, and the second amplification oligonucleotide comprises a nucleic acid sequence having at least 70% similarity to SEQ ID NO:25. Similar nucleic acid sequences, and probe oligonucleotides comprise nucleic acid sequences having at least 70% similarity to SEQ ID NO:22 or 23. In some embodiments, the first amplified oligonucleotide comprises a nucleic acid sequence with at least 70% similarity to SEQ ID NO:21, and the second amplified oligonucleotide comprises a nucleic acid sequence with at least 70% similarity to SEQ ID NO:25 or 26. A nucleic acid sequence having 70% similarity, and the probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO:24.

In some embodiments, the group of oligonucleotides for amplifying and detecting SARS-CoV-2 (2019-nCoV) in a sample comprises a first set of oligonucleotides comprising: containing the same sequence as SEQ ID NO: 2. The first amplified oligonucleotide with a nucleic acid sequence of at least 70% similarity, the second amplified oligonucleotide containing a nucleic acid sequence with at least 70% similarity with SEQ ID NO: 4 and the second amplified oligonucleotide with a sequence ID NO: 6 Probe oligonucleotides having nucleic acid sequences with at least 70% similarity, wherein the probe oligonucleotides comprise a detectable label.

In some embodiments, the group further comprises a second set of oligonucleotides comprising: a first amplified oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to any one of SEQ ID NO: 11 and 15 Nucleotide, the second amplified oligonucleotide containing a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 3 and the probe oligonucleotide containing a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 5 acid, wherein the probe oligonucleotide comprises a detectable label.

Table 1: Oligonucleotide sequences

sequence 5' to 3' length SEQ ID NO: 1 AAAGAAGAAGGCTGATGAAACT twenty two SEQ ID NO: 5 CCTTACCGCAGAGACAGAAGAAACAGC 27 SEQ ID NO: 3 TGGAGAAATCATCCAAATCTG twenty one SEQ ID NO: 2 TGCTTAGAATTATGGCCTCACT twenty two SEQ ID NO: 6 CGTGTTGTAGCTTGTCACACCGTTTCTA 28 SEQ ID NO: 4 ATACTTGAGCACACTCATTAG twenty one SEQ ID NO: 7 CGACTCCACACGGAGTCGCATTGTGCAAACTTT 33 SEQ ID NO: 8 CGACTCCACACGGAGTCGTGGGAACACTGTAGA 33 SEQ ID NO: 9 CTTTAATGTTTACTGTAGAGAATAAAACATTAAAG 35 SEQ ID NO: 10 AAAGAAGAACGCTGATGAAACT twenty two SEQ ID NO: 11 AAAGAAGAAGCCTGATGAAACT twenty two SEQ ID NO: 12 AAAGAAGAACGCTGATGAAAC twenty one SEQ ID NO: 13 GAGCCTAAAAAAGGACAAAAAGAAG twenty four SEQ ID NO: 14 GAGCCTAAATAGGACAAAAAGAAG twenty four SEQ ID NO: 15 GGCTGATGAAACTCAAGC 18 SEQ ID NO: 16 ATTCCCACCAACAGAGCCT 19

Table 2: Oligonucleotide sequences

Any of the oligonucleotides described herein can be modified in any suitable manner to stabilize or enhance the binding affinity of the oligonucleotide for its target. For example, an oligonucleotide sequence as described herein may comprise one or more modified oligonucleotides. Furthermore, any of the sequences listed which include internal spacers or modifications can be used without the modification or spacer.

Any of the oligonucleotides described herein may include, for example, spacers, blocking groups, and modified nucleotides. Modified nucleotides are nucleotides or nucleoside triphosphates that differ in composition and/or structure from natural nucleotides and nucleoside triphosphates. Modifications include naturally occurring ones resulting from modification by nucleotide-modifying enzymes, such as methyltransferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. For example, modified nucleotides include those with 2' modifications such as 2'-O-methyl and 2'-fluoro. Other 2'-modified nucleotides are known in the art and are described, for example, in U.S. Patent No. 9,096,897, which is incorporated herein by reference in its entirety. For example, the modified nucleotides or nucleoside triphosphates used herein may be modified in such a manner that when the modification is present on one strand of a double-stranded nucleic acid in which a restriction endonuclease recognition site exists, The modified nucleotide or nucleoside triphosphate protects the modified strand from cleavage by restriction enzymes.

A blocking group or polymerase-arresting molecule is a chemical moiety that inhibits target sequence-independent polymerization of a nucleic acid by a polymerase. Blocking groups enable oligonucleotides to bind target nucleic acid molecules, but do not support template extension using detectable oligonucleotide probes as targets. For example, the presence of one or more moieties that do not allow the polymerase to proceed may cause the polymerase to stop the addition of non-nucleic acid backbones to the oligonucleotide or by pausing a replicating polymerase. Oligonucleotides with these moieties prevent or reduce undesired amplification of the probe during the course of the amplification reaction. Examples of blocking groups include, for example, alkyl groups, non-nucleotide linkers, phosphorothioates, alkanediol residues, peptide nucleic acids, and nucleotide derivatives lacking a 3'-OH, including, for example, cordycepin, spacer moieties , damaged DNA bases, etc. Examples of spacers include, for example, C3 spacers. Spacers can be used, for example, within the oligonucleotide, and can also be used, for example, at the termini to attach other groups, such as labels.

Any of the oligonucleotide sequences described herein may comprise, consist essentially of, or consist of the complement of any of the sequences disclosed herein. The term "complement" or "complementary sequence" as used herein refers to a stable duplex with the oligonucleotides described herein via Watson-Crick base pairing rules, and generally with the disclosed oligonucleotides. 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 A nucleic acid sequence that is 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identical. Nucleic acid sequence identity can be determined using any suitable mathematical algorithm or computer software known in the art, such as CLUSTAL-W, T-Coffee and ALIGN (for alignment of nucleic acid and amino acid sequences) , BLAST programs (eg, BLAST 2.1, BL2SEQ and later versions) and FASTA programs (eg, FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also disclosed, for example, in 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 UK (1997), each of which is incorporated by reference in its entirety Incorporated into this article.

The oligonucleotides described herein can be prepared using any suitable method, various of which are known in the art (see, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 1989, 2. Supp. Ed., Cold Spring Harbor Laboratory Press: New York, N.Y.; M.A. Innis (Ed.), PCR Protocols. A Guide to Methods and Applications, Academic Press: New York, N.Y. (1990); P. Tijssen, Hybridization with Nucleic Acid Probes-Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II), Elsevier Science (1993); M.A. Innis (Ed.), PCR Strategies, Academic Press: New York, N.Y. (1995); and F.M. Ausubel (Ed.), Short Protocols in Molecular Biology, John Wiley & Sons: Secaucus, N.J. (2002); Narang et al., Meth. Enzymol., 68:90-98 (1979); Brown et al., Meth. Enzymol., 68:109-151 (1979); and Belousov et al. , Nucleic Acids Res., 25:3440-3444 (1997), each of which 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 performed on an oligonucleotide synthesizer such as those available from Perkin Elmer/Applied Biosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE) or Milligen (Bedford, MA). ) those that are commercially available. Alternatively, oligonucleotides can be custom made and obtained from various commercial sources well known in the art, including, for example, Midland Certified Reagent Company (Midland, TX), Eurofins Scientific (Louisville, KY), BioSearch Technologies, Inc. (Novato, CA) etc. Oligonucleotides can be purified using any suitable method known in the art, such as native acrylamide gel electrophoresis, anion exchange HPLC (see, e.g., Pearson et al., J. Chrom., 255:137-149 (1983 ), incorporated herein by reference) and reverse phase HPLC (see, eg, McFarland et al., Nucleic Acids Res., 7:1067-1080 (1979), incorporated herein by reference).

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

2. Detectable markers

Any one or more of the oligonucleotide sequences described herein may comprise a detectable label such that one or more amplification oligonucleotides and/or probe oligonucleotides can be measured. In one embodiment, each probe oligonucleotide sequence described herein comprises a detectable label. The term "detectable label" as used herein refers to a moiety or compound that produces a signal that is measurable and whose intensity correlates (eg, is proportional) to the amount of entity bound thereto. Any suitable detectable label that can be conjugated or linked to an oligonucleotide to detect binding of the oligonucleotide to a target sequence can be used, many of which are known in the art. In one embodiment, the detectable label can be detected indirectly. An indirect detectable label is typically a specific binding member used in conjunction with a "conjugate" that is linked or coupled to a directly detectable label. The coupling chemistry used to synthesize 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 remain intact. "Specific binding member" and "conjugate" as used herein refer to two members of a binding pair, e.g., two different molecules, wherein the specific binding member specifically binds to a polynucleotide of the disclosure, and " A "conjugate" specifically binds to a specific binding member. The bond between the two members of a pair is generally chemical or physical in nature. Examples of such binding pairs include, but are not limited to, antigen and antibody, avidin/streptavidin and biotin, haptens and hapten-specific antibodies, complementary nucleotide sequences, enzyme cofactors/substrates, and enzymes wait.

Each probe oligonucleotide sequence desirably includes a detectable label. Each probe can be labeled with the same detectable label or with different detectable labels.

In some embodiments, a detectable label is directly detectable. Such directly detectable labels include, for example, radioisotopes, fluorophores, chemiluminescent agents, enzymes, colloidal particles, fluorescent microparticles, intercalating dyes such as SYBR Green or ethidium bromide, and the like. In selected embodiments, the detectable label may be a fluorophore, such as a fluorescein family of dyes, a polyhalogenated fluorescein family of dyes, a hexachlorofluorescein family of dyes, a coumarin family of dyes, a rhodamine family of dyes, a cyanine family of dyes dye, Azine dyes, thiazine dyes, squaraine dyes, chelated lanthanide dyes, azo dyes, triphenylmethane dyes or /> family of dyes. Examples of fluorophores include, but are not limited to, FAM ^™ , /> HEX ^™ , JOE ^™ , NED ^™ , /> ROX ^™ , TAMRA ^™ , TET ^™ , TEXAS/> and /> Those skilled in the art will appreciate that directly detectable labels may require additional components, such as substrates, trigger reagents, light, etc., to enable detection of the label. Methods for labeling oligonucleotides, such as probes, are well known in the art and described, for example, in LJ Kricka, Ann. Clin. Biochem., 39:114-129 (2002); van Gijlswijk et al., Expert Rev. Mol. Diagn., 1:81-91 (2001); Joos et al., J. Biotechnol., 35:135-153 (1994); Smith et al., Nucl. Acids Res., 13:2399-2412 (1985); People such as Connoly, Nucl.Acids.Res., 13:4485-4502 (1985); People such as Broker, Nucl.Acids Res., 5:363-384 (1978); People such as Bayer, Methods of Biochem.Analysis, 26 :1-45 (1980); Langer et al., Proc.Natl.Acad.Sci.USA, 78:6633-6637 (1981); Richardson et al., Nucl.Acids Res., 11:6167-6184 (1983); People such as Brigati, Virol., 126:32-50 (1983); People such as Tchen, Proc.Natl.Acad.Sci.USA, 81:3466-3470 (1984); People such as Landegent, Exp.CellRes., 15: 61-72 (1984); AH Hopman et al., Exp.Cell Res., 169:357-368 (1987); and Temsamani et al., Mol.Biotechnol., 5:223-232 (1996), each of which is passed References are incorporated herein in their entirety.

In some embodiments, any one or more of the oligonucleotides described herein may also comprise a quencher moiety. When a detectable label (eg, a fluorophore) and a quencher moiety are held in close proximity (eg, at the end of a probe), the quencher moiety prevents a signal from the detectable label (eg, fluorescence) from being detected. When the two parts are physically separated, the signal becomes detectable. The quencher can be selected from any suitable quencher known in the art, such as BLACK HOLE BLACK HOLE/> 2/> BLACK HOLE/> IOWA/> FQ and IOWA/> RQ. For example, oligonucleotide probes may comprise FAM fluorophores, or QUASAR fluorophore and BHQ-1 or BHQ-2 quencher.

The choice of a particular label and labeling technique will depend on several factors, such as the ease and cost of the labeling method, the spectral spacing between the different detectable labels used, the desired quality of sample labeling, the detectable moiety's response to hybridization (e.g. for rate and/or efficiency of the hybridization process), the nature of the amplification method used, the nature of the detection system, the nature and intensity of the signal produced by the detectable label, etc.

3. Methods for amplification and detection of SARS-CoV-2 (2019-nCoV)

The present disclosure provides a method for detecting SARS-CoV-2 (2019-nCoV) in a sample. The method includes: contacting a sample with the set of oligonucleotides disclosed herein and reagents for amplification; amplifying one or more target SARS-CoV-2 nucleic acid sequences present in the sample; Acid probe hybridizes with the target SARS-CoV-2 nucleic acid sequence of one or more amplification; And by measuring the signal from detectable label, detect one or more probe oligonucleotide sequence and one or more amplified Hybridization of SARS-CoV-2 target nucleic acid sequences. The description herein of the oligonucleotides set forth in relation to the above-mentioned set of oligonucleotides also applies to the disclosed methods.

A sample can be obtained from any suitable subject, typically a mammal (eg, a dog, cat, rabbit, mouse, rat, goat, sheep, cow, pig, horse, non-human primate, or human). any suitable samples. Preferably, the subject is a human. Samples may be obtained from any suitable biological source (such as a nasal swab or nasal brush) or physiological fluid (including but not limited to whole blood, serum, plasma, interstitial fluid, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites, mucus, synovial fluid, peritoneal fluid, vaginal fluid, menstruation, amniotic fluid, semen, feces, etc.).

Samples can be obtained from a subject using routine techniques known to those skilled in the art, and can be used directly as obtained from a biological source, or after pretreatment to modify the properties of the sample. Such pretreatment may 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, lysis, and the like.

After a sample is obtained from a subject, the sample can be contacted with an oligonucleotide set comprising an amplification oligonucleotide and a probe as described herein to form a reaction mixture. The reaction mixture is then subjected to amplification conditions. The term "amplification conditions" as used herein refers to conditions that promote annealing and/or extension of amplified oligonucleotides. Such conditions are well known in the art and depend on the amplification method chosen. Amplification conditions include all reaction conditions including, but not limited to, temperature and/or temperature cycling, buffers, salts, ionic strength, pH, and the like.

Amplifying the SARS-CoV-2 nucleic acid sequence in the sample can be performed using any suitable nucleic acid sequence amplification method known in the art. In some embodiments, amplification includes, but is not limited to, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), real-time PCR, transcription-mediated amplification (TMA), rolling circle amplification, nucleic acid-based Sequence 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 nucleic acid sequence of SARS-CoV-2 (2019nCoV) is performed using isothermal amplification (eg, RPA or NEAR). In some embodiments, the amplification and detection of the SARS-CoV-2 nucleic acid sequence is performed using a point-of-care device such as the ID NOW system (Abbott).

In some embodiments, amplification of the SARS-CoV-2 nucleic acid sequence is performed using real-time PCR. "Real-time PCR" as used herein refers to a PCR method in which the accumulation of amplified product is measured in real time as the reaction proceeds and product quantification is performed after each cycle, unlike conventional PCR in which the amplified DNA product is detected in an endpoint analysis. forms a contrast. Real-time PCR is also known in the art as "quantitative PCR (qPCR)". Real-time detection of PCR products generally 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 e.g. Dorak, M. Tevfik, ed. Real-time PCR. Taylor & Francis (2007); and Fraga et al., "Real-time PCR," Current protocols essential laboratory techniques: 10-3 (2008), each of which is incorporated herein by reference in its entirety) and commercially available from various sources (e.g. m2000rtREALTIME ^™ PCR System (Abbott Molecular, Inc., Des Plaines, IL), CFX Real- Time PCRDetection Systems (Bio-Rad Laboratories, Inc., Hercules, CA) and TAQMAN ^™ Real-TimePCR System (ThermoFisher Scientific, Waltham, MA)), either of which can be used in the methods described herein.

The oligonucleotide group that is used for amplification can comprise the first amplified oligonucleotide containing the nucleotide sequence that any one has at least 70% similarity in SEQ ID NO:1 and 10-16, containing NO:3 has the second amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity and contains the probe oligonucleotide that has the nucleic acid sequence of at least 70% similarity with SEQ ID NO:5 and/or contains and SEQ ID NO:2 has the first amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity, the second amplified oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID NO:4 and A probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO:6.

In selected embodiments, isothermal amplification methods may rely on a nick and extension reaction - "nick and extension amplification" to amplify shorter sequences in a faster time frame than traditional amplification reactions. These methods may include, for example, reactions using only two amplification oligonucleotides, one or two nicking enzymes, and a polymerase under isothermal conditions. The oligonucleotide group that is used for nick and extension amplification can comprise the first amplification oligonucleotide containing the nucleic acid sequence that has at least 70% similarity with SEQ ID NO:7, contains and SEQ ID NO:8 has at least A second amplification oligonucleotide having a nucleic acid sequence of 70% similarity and a probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO:9.

In nick and extension amplification, a nucleic acid sequence of interest with sense and antisense strands is contacted with a pair of amplification oligonucleotides. The first amplification oligonucleotide comprises a nucleic acid sequence comprising: a recognition region at the 3' end complementary to the 3' end of the antisense strand of the target sequence, a nickase site upstream of the recognition region, and the nickase site point upstream of the stable zone. The second amplification oligonucleotide comprises a nucleotide sequence comprising: a recognition region at the 3' end complementary to the 3' end of the sense strand of the target sequence, a nickase site upstream of the recognition region, and the nick Stable region upstream of the enzyme site. Two nicking enzymes are provided. A nickase is capable of nicking at the nickase site of the first amplification oligonucleotide but not within the target sequence. Another nickase is capable of nicking at the nickase site of the second amplification oligonucleotide, but not within the target sequence. A DNA polymerase is used under conditions for amplification involving multiple extension cycles of the amplified oligonucleotide, thereby creating a double-stranded nickase site that is nicked by the nickase to generate the amplified oligonucleotide. increase product. See, eg, US Patent Nos.: 9,689,031, 9,617,586, 9,562,264, and 9,562,263, and US Patent Application Nos: 15/467,893, 15/600,951, and 16/243/829, each of which is incorporated herein by reference in its entirety.

In some embodiments, the ID NOW COVID-19 assay uses the Nickase Amplification Reaction (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 comprises: a sample receiver containing an elution/lysis buffer; a test substrate containing two sealed reaction tubes each containing lyophilized particles; a transfer cartridge for The eluted sample is transferred to the test substrate; and the ID NOW instrument. The ID NOW COVID-19 assay produces positive results in as little as 5 minutes and negative results in 13 minutes, providing rapid COVID-19 results in a wide range of healthcare settings.

In some embodiments, the ID NOW COVID-19 POC assay provides a lower limit of detection (LOD) of 125 copies/mL or less. Computer analysis found that all templates and probes shared 100% homology with 957 SARS-CoV-2 sequences reported from 45 countries and 21 provinces and cities in China, and were predicted to be 100% homologous to microorganisms that cause common respiratory diseases, including other coronaviruses. , influenza, RSV and rhinovirus) had no significant cross-reactivity.

Clinical performance was determined on 30 artificial samples containing known concentrations of SARS-CoV-2 RNA and 30 artificial negative samples. SARS-CoV-2 RNA was detected in all positive samples (percent positive 100% [CI, 88.6-100%]) but not in negative samples (percent negative 100% [CI, 88.6 -100%]).

In selected embodiments, amplification of the SARS-CoV-2 nucleic acid sequence is performed using recombinase-polymerase amplification (RPA), which relies on the properties of recombinase and associated proteins to invade double-stranded DNA with single-stranded homologous DNA, allowing sequence-specific priming of DNA polymerase reactions.

The oligonucleotide group that is used for RPA can comprise the first amplified oligonucleotide that contains the nucleotide sequence that has at least 70% similarity with SEQ ID NO:17, contains that there is at least 70% similarity with SEQ ID NO:19 The second amplification oligonucleotide of the nucleic acid sequence and the probe oligonucleotide containing the nucleic acid sequence with at least 70% similarity with SEQ ID NO:18; or containing at least 70% with SEQ ID NO:20 or 21 The first amplified oligonucleotide of the nucleic acid sequence of % similarity, the second amplified oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID NO:25 or 26 and the second amplified oligonucleotide containing the nucleic acid sequence with SEQ ID NO:22 - A probe oligonucleotide having a nucleic acid sequence of at least 70% similarity to any of the 24.

In some embodiments, wherein 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 % similarity to a nucleic acid sequence, 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 with at least 70% similarity to SEQ ID NO:21, and the second amplified oligonucleotide comprises a nucleic acid sequence with at least 70% similarity to SEQ ID NO:25 or 26. A nucleic acid sequence having 70% similarity, and the probe oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO:24.

In RPA, a recombinase agent is contacted with first and second amplification oligonucleotides to form a nucleoprotein. These nucleoproteins are contacted with target sequences to form a first double-stranded structure at a first portion of said first strand and a double-stranded structure at a second portion of said second strand, whereby said first amplified oligonucleotide The acid and the 3' ends of the second amplification oligonucleotide face each other on the DNA containing the target sequence. The 3' end of the amplification oligonucleotide in the nucleoprotein is extended by a DNA polymerase to produce first and second double-stranded nucleic acids, and first and second displaced nucleic acid strands. These steps are repeated until the desired level of amplification is achieved.

Methods and materials for RPA of target nucleic acid sequences are known in the art. See U.S. Patent Nos.: 7,270,981, 8,460,875, 7,399,590, 7,666,598, 8,030,000, 8,426,134, 8,945,845, 9,663,820, 10,329,603, 10,329,602, 8,017,339, 8,57 4,846, 8,962,255, 10,036,057, 8,071,308, 10,093,908, and 8,637,253, and U.S. Patent Application Nos.: 15/099,754, 16 /442,007, 14/705,150, and 16/155,133, each of which is incorporated herein by reference in its entirety. For example, suitable recombinant enzyme agents include E. coli RecA protein, T4 uvsX protein, or any homologous protein or protein complex from any phylum. Other non-homologous recombinase agents can be used in place of RecA, such as RecT or RecO. Suitable recombinase cargo proteins may include, for example, T4uvsY, E. coli recO, E. coli recR, and derivatives and combinations of these proteins. A suitable single-stranded DNA binding protein may be E. coli SSB or T4gp32 or a derivative or combination of these proteins. DNA polymerases can be eukaryotic or prokaryotic polymerases. Examples of eukaryotic polymerases include pol-α, pol-β, pol-δ, pol-ε, and derivatives and combinations thereof. Examples of prokaryotic polymerases include Escherichia 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 Bacillus subtilis Pol I, Escherichia coli DNA polymerase I, Escherichia coli DNA polymerase II, Escherichia coli DNA polymerase III, Escherichia coli DNA polymerase IV, Escherichia coli DNA polymerase V and derivatives and combinations thereof. Other components of RPA include ATP, ATP analogs or systems for ATP regeneration (conversion of ADP to ATP). Such systems may utilize, for example, phosphocreatine and creatine kinase. The ATP or ATP analog can be any one of ATP, ATP-γ-S, ATP-β-S, ddATP, or a combination thereof. RPA reactions may also include systems that regenerate ADP from AMP and systems that convert pyrophosphate to phosphoric acid (pyrophosphoric acid). Suitable clustering agents for use in RPA include polyethylene glycol (PEG), dextran and ficoll.

After amplifying one or more SARS-CoV-2 viral nucleic acid sequences present in the sample, the disclosed methods may further comprise combining one or more probe oligonucleotide sequences disclosed herein with one or more amplifying Target SARS-CoV-2 (2019-nCoV) nucleic acid sequence hybridization.

After one or more probe oligonucleotide sequences hybridize to one or more amplified target nucleic acid sequences, the method includes detecting the one or more probe oligonucleotide sequences by evaluating the signal from each detectable label acid sequence and one or more amplified target nucleic acid sequences, whereby (i) the presence of one or more signals indicates that one or more probe oligonucleotide sequences are compatible with one or more target SARS-CoV- 2 nucleic acid sequence hybridization and the presence of SARS-CoV-2 in the sample; and (ii) the absence of a signal indicating the absence of SARS-CoV-2 in the sample. Detection of signals from one or more probe oligonucleotide sequences can be performed using a variety of well-known methods depending on the type of detectable label. For example, detection can be performed using solution real-time fluorescence or using solid surface methods.

4. Treatment and monitoring of subjects identified as having SARS-CoV-2

Subjects identified as having SARS-CoV-2 according to the methods described herein may be treated, monitored (for example, by the presence of SARS-CoV-2 nucleic acid determined in a sample from the subject) using routine techniques known in the art ), treatment and monitoring and/or monitoring and treatment. In some embodiments, the methods described herein further comprise treating the subject when the presence of SARS-CoV-2 nucleic acid is determined in one or more samples obtained from the subject using the methods.

Treatment can take various forms, depending on whether the subject is asymptomatic or experiencing mild, moderate or severe symptoms of SARS-CoV-2 infection. For example, a subject experiencing mild symptoms will experience fever, cough (with or without sputum production), anorexia, malaise, muscle pain, sore throat, difficulty breathing, nasal congestion, headache, diarrhea, nausea, vomiting, or any combination thereof . Subjects experiencing moderate symptoms will experience fever above 100.4°F for several days, chills, shortness of breath, lethargy, or any combination thereof. Such subjects may also suffer from pneumonia. Subjects experiencing severe infection will experience difficulty breathing, persistent pain or pressure in the chest, confusion, inability to arouse, bluish lips or face, or any combination thereof. Such subjects may also suffer from severe pneumonia.

If the subject is asymptomatic or has mild symptoms, the subject can respond by resting, sleeping, keeping warm, consuming fluids (e.g., staying hydrated), minimizing social interaction with other subjects (e.g., keeping or isolation, such as at home), or any combination thereof. Additionally, the subject can be monitored for the onset and/or worsening of symptoms.

Subjects with moderate or severe symptoms of SARS-CoV-2 infection may receive one or more drugs (e.g., remdesivir), vaccines, convalescent plasma therapy (e.g., received from patients infected with SARS-CoV- 2 blood plasma collected from surviving subjects), respiratory support or assistance (such as receiving supplemental oxygen through nasal cannula, face mask, or non-invasive or invasive (such as intubation) ventilation) or a combination thereof. Subjects receiving any of the above treatments can also be further monitored using routine techniques known in the art.

5. Vaccination

In another embodiment, the present disclosure relates to the use of the methods 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 the subject should or can be administered at least one vaccine against SARS-CoV-2 (eg first or initial vaccine, one or more further or additional vaccines, etc.). In some embodiments, the tested subject may be naive, such that the subject does not have any immunity or lacks immunological immunity to SARS-CoV-2 and has not been previously vaccinated against SARS-CoV-3. In some embodiments, the subject may be naive despite having been previously vaccinated against SARS-CoV-2. In some embodiments, the subject is currently potentially infected with SARS-CoV-2, exhibits asymptomatic or mild symptoms, and lacks any previous vaccinations. In some embodiments, the subject is currently potentially infected with SARS-CoV-2, exhibits asymptomatic or mild symptoms, and has been previously vaccinated against SARS-CoV-2. In some embodiments, the subject may have recovered from a previous SARS-CoV-2 infection and has not been previously vaccinated against SARS-CoV-2. In some embodiments, the subject may have recovered from a previous SARS-CoV-2 infection and has been previously vaccinated against SARS-CoV-2.

The above methods can be implemented regardless of the timing and/or severity of prior SARS-CoV-2 infection. Using the methods described herein, if the presence of SARS-CoV-2 nucleic acid is detected in a sample, the subject may need to wait for a period of time (e.g., 30 days, 60 days, 90 days, etc.) -2 of the vaccine; whether the vaccine to be administered is the first dose of the vaccine, the second (eg, booster) dose, the third or any further additional (eg, booster) dose of the vaccine. Alternatively, if the presence of SARS-CoV-2 nucleic acid is not detected in the sample, the subject may be administered the vaccine at least once.

The phrase "at least one further vaccine or vaccination" or "at least one additional vaccine or vaccination" includes situations where at least one Additional or further vaccines or vaccinations (e.g. N+1 (where N is the first or current vaccine plus one additional or further vaccine), N+2 (where N is the first or current vaccine plus 2 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- 1000, 1-500, 1-100 integer)).

In another embodiment, the methods described herein are used to detect SARS-CoV-2 in at least one sample obtained from a subject within a time frame after at least one administration of a vaccine against SARS-CoV-2 to the subject The presence of nucleic acid to: determine whether the subject should be given at least one further vaccine against SARS-CoV-2 (e.g., receive one or more boosters); and/or after giving at least one vaccine against SARS-CoV-2 Subjects were monitored post-vaccination. In some embodiments, the method involves obtaining a sample within a time frame after at least one administration of the vaccine against SARS-CoV-2 to the subject. The time frame after at least one administration of the SARS-CoV-2 vaccine to the subject may be 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, 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, etc. In some embodiments, the sample is obtained within about 7 to about 21 days after at least one administration of the SARS-CoV-2 vaccine to the subject. Additionally, in still further embodiments, monitoring the subject involves following the subject's receipt of one or more vaccines (e.g., a first dose of a vaccine against SARS-CoV-2, a second dose of a vaccine against SARS-CoV-2 After vaccines, etc.) Monitor for post-vaccine symptoms or side effects (such as fatigue or malaise, headache, dizziness or lightheadedness, fever or chills, muscular, bone, joint or nervous symptoms, nausea, vomiting, diarrhea or other gastrointestinal symptoms, sleep changes in the lymph nodes, swollen lymph nodes, skin/nails or facial changes, eye, ear, mouth or throat changes, cough, chest or breathing symptoms and/or changes in memory or mood).

Using the methods described herein, if no SARS-CoV-2 nucleic acid is detected in the sample, at least one further vaccine (eg, one or more boosters) can be administered to the subject. Alternatively, at least one further vaccine may or may not be administered to the subject if SARS-CoV-2 nucleic acid is detected in the sample.

6. Kit

The present disclosure also provides a kit for amplifying and detecting SARS-CoV-2 (2019-nCoV) in a sample. The kit comprises at least one oligonucleotide as described herein. In some embodiments, a kit comprises a set or population of oligonucleotides described herein. The kit may further comprise reagents for amplifying and detecting nucleic acid sequences and instructions for amplifying and detecting SARS-CoV-2. The descriptions herein of oligonucleotides and sets of oligonucleotides set forth in relation to the methods described above also apply to those same aspects of the kits described herein. Many such agents are described herein or otherwise known in the art and are commercially available. Examples of suitable reagents for inclusion in the kit (in addition to the oligonucleotides described herein) include conventional reagents for nucleic acid amplification reactions, such as one or more enzymes with polymerase activity, enzyme cofactors (such as magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, deoxyribonucleoside triphosphates or ribonucleoside triphosphates (dNTP/rNTP; such as deoxyadenosine triphosphate, deoxyguanosine triphosphate Phosphate, deoxycytidine triphosphate and deoxythymidine triphosphate), blocking agents, labeling agents, etc. Additional reagents for amplification reactions include nicking enzymes, single-strand binding proteins, helicases, resolving enzymes, and the like.

Kits may comprise instructions for using the amplification reagents and oligonucleotides described herein, eg, for processing test samples, extracting nucleic acid molecules, and/or performing tests; and interpreting the results obtained. Instructions may be printed or provided electronically (eg DVD, CD or available for viewing or retrieval via Internet sources).

Kits may be provided in solid (eg lyophilized) or liquid form. The various components of the disclosed kits may optionally be contained in separate containers (e.g., vials, ampoules, test tubes, flask or bottle). Each ingredient is generally suitably presented as aliquots in its own container or in concentrated form. Other containers suitable for performing certain steps for the amplification/detection assays may also be provided. Individual containers are preferably kept closed and airtight for commercial sale.

The kit may further comprise a swab for obtaining a biological sample. In some embodiments, the kit comprises reagents for obtaining nucleic acid from a biological sample and/or extracting/isolated nucleic acid from a biological sample.

7. Example

Example 1

Amplification of the nucleic acid sequence of SARS-CoV-2 (2019-nCoV) by PCR using various first amplified oligonucleotides with a second amplified oligonucleotide comprising SEQ ID NO: 3 and a second amplified oligonucleotide comprising SEQ ID NO :5 probe oligonucleotides were performed.

The SARS-CoV-2 sample was genomic RNA from SARS-associated coronavirus 2, isolate USA-WA1/2020 (BEI Resources). Genomic RNA was obtained from African green monkey (Cercopithecus aethiops) kidney epithelial cells (Vero E6, CRL-1586 ^TM ) cell lysate and supernatant preparations. Viral genomic RNA is in the background of cellular nucleic acid and carrier RNA.

PCR cycling parameters:

The PCR reaction mixture comprises SARS-CoV-2, a COVID-19 primer set (primer set 1) comprising SEQ ID NO:3 and one of SEQ ID NO:1, 10, 12-14 or 16, and SEQ ID NO:5 probe oligonucleotides. Internal control nucleic acids and primer sets for internal controls were also included as positive controls (when stated). The reaction mixture also contains reaction buffer, dNTPs, reference dye, DNA polymerase (rTth) at concentrations commonly used in the art.

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

Table 3: PCR results for primer set 1 with different forward primers

^* Ct = cycle threshold

MR = maximum ratio

dRn = reported value minus baseline

An additional PCR reaction including a second COVID-19 primer set (Primer Set 2) was performed with the first COVID-19 primer set selected. The second COVID-19 primer set includes 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 gave a stronger signal (dRn) compared to any primer set 1, but the addition of primer set 1 to primer set 2 gave the strongest overall signal.

Table 4: PCR results for primer set 2 with primer set 1

*Ct = cycle threshold

MR = maximum ratio

dRn = reported value minus baseline

Example 2

Amplification of the SARS-CoV-2 (2019-nCoV) nucleic acid sequence was performed using recombinase-polymerase amplification (RPA) using various combinations of amplification oligonucleotides and probe oligonucleotides. The combinations tested are shown in Table 5. Combination 1 is for the target area, while combinations 2-5 are for the second target area.

Table 5: Combinations of Amplification and Probe Oligonucleotides for RPA

Combination number: FP RP probe positive signal 1 SEQ ID NO: 17 SEQ ID NO: 19 SEQ ID NO: 18 Y 2 SEQ ID NO: 20 SEQ ID NO: 25 SEQ ID NO: 22 Y 3 SEQ ID NO: 20 SEQ ID NO: 25 SEQ ID NO: 23 Y 4 SEQ ID NO: 21 SEQ ID NO: 25 SEQ ID NO: 24 Y 5 SEQ ID NO: 21 SEQ ID NO: 26 SEQ ID NO: 24 Y

Figures 1A and 1B show graphs of dRn values over time for each of the 5 combinations. For all combinations amplifying the SARS-CoV-2 target under reaction conditions, detectable levels of amplification occurred within 10 minutes of initiation of the reaction. For combinations 1, 4, and 5, target amplification was detected within 4-6 minutes (Fig. 1A-1B).

Example 3

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

Inclusion was further demonstrated by analyzing the homology of each SARS-CoV-2 primer and probe sequence 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 were analyzed; 1.1% (170/14,964) contained a single mismatch, 0.04% (6/14964) contained 2 mismatches, and 0.007% (1/14964) contained 4 mismatches.

Example 4

The disclosed primer sets were evaluated using real-time RT-PCR with the Abbott Alinity m system using known SARS CoV-2 positive, negative and non-SARS CoV-2 respiratory samples. As shown in Table 6, the dilution series of known positive samples established a detection limit of 100 copies/mL. The limit of detection (LOD) was defined as the lowest detectable concentration of SARS-CoV-2 for which greater than or equal to 95% of all (true positive) repeat tests were positive.

Table 6: LOD of SARS-CoV-2 Assay

copies/mL Positives/Tests Average CN detected% 400 10/10 37.52 100 200 10/10 38.87 100 100 19/20 39.78 95 50 9/10 40.20 90 25 2/10 39.05 20

109 previously tested frozen nasopharyngeal swabs were retested in the real-time PCR assay as used above for positive (PPA) and negative (NPA) percent agreement (Table 7). PPA was found to be 89.8% (53/59) and NPA was found to be 98% (49/50).

Table 7: NPA and PPA assayed for SARS-CoV-2

^a CN 27.84-31.43

^b CN 40.99

Clinical relevance, cross-reactivity, and detection limits were also assessed using the Abbott m2000 real-time SARS-CoV-2 assay (Table 8).

Table 8: m2000 real-time SARS-CoV-2 performance study

*Angeli et al., JVC, Validation and Validation of the Abbott RealTime SARS-CoV-2 Assay _Analytical and Clinical Performancc, May 2020, incorporated herein by reference.

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

Table 9: Evaluation of the Alinity m SARS-CoV-2 Assay

^a These samples had an Alinity m SARS-CoV-2 CN of 40.99

Table 10: NPA and PPA of Alinity m SARS-CoV-2 Assay

Example 5

The SARS-CoV-2 B.1.1.7 strain, first identified in the UK, is the most interesting to evaluate because of the observed link between increased transmissibility and mutations in the spike gene. Although the spike gene mutation primarily defines the B.1.1.7 lineage, the presence of additional mutations throughout the genome can affect the performance of various diagnostic assays.

Preliminary in silico examination of B.1.1.7 lineage sequences in GISAID (N = 1787, as accessed on December 21, 2020) revealed no lineage-defining mutations of concern to 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 min and tested in a dilution series. Multiple dilutions were tested within the expected range previously observed with other strains (Table 11). These results confirm in silico predictions that the present primers, probes and methods can reliably detect the B.1.1.7 strain and are consistent with recent assessments conducted by Public Health England.

Further evaluation was performed with the remaining patient nasopharyngeal swab samples. The genome sequences of all samples were completed and confirmed to belong to the B.1.1.7 lineage. Due to the limited remaining volume, the remaining VTM (Viral Transport Medium) was diluted 5-62.5x prior to testing. All samples were detected in sufficient numbers (Table 11).

Table 11. SARS-CoV-2 Assay Results

Sample ID Log GE/Test* TCID50/test result BEI 5.55 5.6 3/3 detected BEI 4.49 0.56 3/3 detected BEI 3.30 0.056 3/4 detected BEI 2.45 0.0112 0/3 detected 4 4.28 NA ^# detected 6 3.82 NA ^# detected 13 3.41 NA ^# detected

^# NA, not applicable, TCID ₅₀ not determined for patient samples

^* Calculated from a standard curve with an R2 value of ^0.99 log Genome Equivalent (GE)/test

The B.1.351 lineage was first identified in South Africa and has since spread to more than a dozen countries, with initial reports suggesting that the variant may escape neutralizing antibodies. The unique mutational spectrum of the B.1.351 lineage is mainly defined by the spike gene mutations K417N, E484K, and N501Y, however, the presence of additional mutations throughout the genome may affect the performance of various diagnostic assays.

Preliminary in silico examination of the B.1.351 lineage sequences in GISAID (N = 195, as accessed on December 27, 2020) did not reveal any lineage-defining mutations of concern for the performance of the primers, probes, and methods described herein. Two virus cultures (BEI NR-54008, NR-54009) were heat-inactivated, tested in a dilution series and within the range previously observed with other strains (Table 12). These results confirm in silico predictions that the present primers, probes and methods can reliably detect the B.1.351 lineage.

Table 12: SARS-CoV-2 Assay Results

^* Calculated Genome Equivalent (GE)/test from a GE/ml standard curve with an R2 value of ^0.99 and a unit conversion plot of TC1D50/mL versus GE/mL with an ^R2 value of 0.97

It should be understood that the foregoing detailed description and accompanying examples are illustrative only and should not be taken as limiting the scope of the present disclosure, which is defined only by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will become apparent to those skilled in the art and can be made without departing from the spirit and scope thereof.

<110> Ionian Technologies LLC

<120> Assays for the detection of coronavirus disease 2019 (COVID-19)

<130> ALERE-38391.206

<150> 63/155,599

<151> 2021-03-02

<150> 63/049,237

<151> 2020-07-08

<150> 63/004,773

<151> 2020-04-03

<150> 63/000,971

<151> 2020-03-27

<150> 63/000,304

<151> 2020-03-26

<160> 26

<170> PatentIn Version 3.5

<210> 1

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<212>DNA

<213> Artificial sequence

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<223> Synthetic

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aaagaagaag gctgatgaaa ct 22

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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tgcttagaat tatggcctca ct 22

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<212>DNA

<213> Artificial sequence

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<223> Synthetic

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tggagaaatc atccaaatct g 21

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<212>DNA

<213> Artificial sequence

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<223> Synthetic

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atacttgagcacactcatta g 21

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<212>DNA

<213> Artificial sequence

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<223> Synthetic

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ccttaccgca gagacagaag aaacagc 27

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<212>DNA

<213> Artificial sequence

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<223> Synthetic

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cgtgttgtag cttgtcacac cgtttcta 28

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<212>DNA

<213> Artificial sequence

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<223> Synthetic

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cgactccaca cggagtcgca ttgtgcaaac ttt 33

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<212>DNA

<213> Artificial sequence

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<223> Synthetic

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cgactccaca cggagtcgtg ggaacactgt aga 33

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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ctttaatgtt tactgtagag aataaaacat taaag 35

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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aaagaagaac gctgatgaaa ct 22

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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aaagaagaag cctgatgaaa ct 22

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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aaagaagaac gctgatgaaa c 21

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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gagcctaaaa aggacaaaaa gaag 24

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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gagcctaaat aggacaaaaa gaag 24

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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ggctgatgaa actcaagc 18

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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attcccacca acagagcct 19

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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taacatgctt agaattatgg cctcacttgt tc 32

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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cttgctcgca aacatacaac gtgttgtagc tgtcacaccg tttctat 47

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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tgaccatttc actcaatact tgagcacact c 31

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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aaagaagaag gctgatgaaa ctcaagcctt ac 32

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<211> 32

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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gctgatgaaa ctcaagcctt accgcagaga ca 32

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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agccttaccg cagagacaga agaaacagca aacttgactc ttcttcctg 49

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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cgcagagaca gaagaaacag caaactgtga ctctcttcct gctgcaga 48

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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agacagaaga aacagcaaac tgtgactctt cttctgctgc agatttgga 49

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<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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actgctcatg gattgttgca attgtttgga ga 32

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<211> 32

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic

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ttgagtcagc actgctcatg gattgttgca at 32

Claims (21)

1. The oligonucleotide group that is used for the recombinase-polymerase amplification and detection of SARS-CoV-2 in the sample, it comprises: Containing the first amplified oligonucleotide with the nucleotide sequence of at least 70% similarity with SEQ ID NO: 17, the second amplified oligonucleotide containing the nucleotide sequence with at least 70% similarity with SEQ ID NO: 19 acid and a probe oligonucleotide containing a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 18; or Containing the first amplified oligonucleotide with the nucleotide sequence of at least 70% similarity with SEQ ID NO: 20 or 21, the second amplified oligonucleotide containing the nucleotide sequence with at least 70% similarity with SEQ ID NO: 25 or 26 Amplifying oligonucleotides and probe oligonucleotides containing a nucleic acid sequence with at least 70% similarity to any one of SEQ ID NO: 22-24; or its combination, wherein each probe oligonucleotide comprises a detectable label. 2. The group of claim 1, wherein the first amplified oligonucleotide comprises a nucleic acid sequence having at least 70% similarity with SEQ ID NO: 20, and the second amplified oligonucleotide comprises a nucleic acid sequence with SEQ ID NO: 25 A nucleic acid sequence of at least 70% similarity, and said probe oligonucleotide comprises a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 22 or 23. 3. The group of claim 1, wherein the first amplified oligonucleotide comprises a nucleic acid sequence with at least 70% similarity with SEQ ID NO: 21, and the second amplified oligonucleotide comprises a sequence with SEQ ID NO: 25 or 26 has a nucleic acid sequence of at least 70% similarity, and the probe oligonucleotide comprises a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 24. 4. The set of any one of claims 1-3, wherein the detectable label is a fluorophore. 5. A method for detecting SARS-CoV-2 in a sample, comprising: contacting the sample with the set of oligonucleotides of any one of claims 1-4 and reagents for amplification; using recombinase-polymerase amplification to amplify 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 Hybridization of the one or more probe oligonucleotide sequences to the one or more amplified SARS-CoV-2 target nucleic acid sequences is detected by measuring the signal from the detectable label. 6. The method of claim 5, wherein the presence of one or more signals from said detectable label indicates that said one or more probe oligonucleotides are associated with said one or more amplified SARS-CoV- 2 Hybridization of target nucleic acid sequences. 7. The method of claim 5 or claim 6, further comprising contacting the first and second amplified oligonucleotides from the set of oligonucleotides with a recombinase agent. 8. The method of any one of claims 5-7, wherein said reagent for amplification is selected from the group consisting of: polymerase; recombinase; recombinase load protein; single-chain binding protein; A phosphate or ribonucleoside triphosphate; a clustering agent; ATP, an ATP analog, or an ATP generating system; or a combination thereof. 9. The method of any one of claims 5-8, wherein the sample comprises a nasal swab or brush, saliva, mucus, blood, serum, plasma or feces. 10. A test kit for detecting SARS-CoV-2 in a sample, comprising: at least one set of oligonucleotides according to any one of claims 1-4; or Any oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 17-26. 11. The kit of claim 10, further comprising reagents and/or instructions for the amplification and detection of nucleic acid sequences. 12. A group of oligonucleotides for amplification and detection of SARS-CoV-2 in a sample comprising: A first set of oligonucleotides, comprising: a first amplified oligonucleotide containing a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 2, containing at least 70% similarity to SEQ ID NO: 4 The second amplification oligonucleotide of the nucleic acid sequence and the probe oligonucleotide containing the nucleic acid sequence with at least 70% similarity to SEQ ID NO: 6, Wherein said probe oligonucleotide comprises a detectable label. 13. The group of claim 12, further comprising: A second group of oligonucleotides comprising: the first amplified oligonucleotide containing a nucleic acid sequence with at least 70% similarity to any one of SEQ ID NO: 11 and 15, containing the same sequence as SEQ ID NO: 3 There is the second amplified oligonucleotide of the nucleic acid sequence of at least 70% similarity and the probe oligonucleotide containing the nucleic acid sequence of at least 70% similarity with SEQ ID NO: 5, Wherein said probe oligonucleotide comprises a detectable label. 14. The population of claim 12 or claim 13, wherein the detectable label is a fluorophore. 15. A method for detecting SARS-CoV-2 in a sample comprising: contacting the sample with the population of oligonucleotides of any one of claims 12-14 and reagents for amplification; amplifying one or more target SARS-CoV-2 nucleic acid sequences present in said sample; hybridizing one or more oligonucleotide probes to one or more amplified target SARS-CoV-2 nucleic acid sequences; and Hybridization of the one or more probe oligonucleotide sequences to the one or more amplified SARS-CoV-2 target nucleic acid sequences is detected by measuring the signal from the detectable label. 16. The method of claim 15, wherein the presence of one or more signals from said detectable label indicates that said one or more probe oligonucleotides are associated with said one or more amplified SARS-CoV- 2 Hybridization of target nucleic acid sequences. 17. The method of claim 15 or claim 16, further comprising contacting the first and second amplified oligonucleotides from the set of oligonucleotides with a recombinase agent. 18. The method of any one of claims 15-17, wherein said reagents for amplification include: nickases, polymerases, single-strand binding proteins, recombinase agents, helicases, resolving enzymes, enzyme-assisted Factors, buffers, deoxyribonucleoside triphosphates or ribonucleoside triphosphates, or combinations thereof. 19. The method of any one of claims 15-18, wherein the sample comprises a nasal swab or brush, saliva, mucus, blood, serum, plasma or feces. 20. A test kit for detecting SARS-CoV-2 in a sample, comprising: at least one population of oligonucleotides according to any one of claims 12-14; or Any oligonucleotide comprising a nucleic acid sequence having at least 70% similarity to SEQ ID NO: 2-6, 11 and 15. 21. The kit of claim 20, further comprising reagents and/or instructions for the amplification and detection of nucleic acid sequences.