WO2023279042A2 - Compositions and methods for detection of severe acute respiratory syndrome coronavirus 2 variants - Google Patents

Compositions and methods for detection of severe acute respiratory syndrome coronavirus 2 variants Download PDF

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WO2023279042A2
WO2023279042A2 PCT/US2022/073285 US2022073285W WO2023279042A2 WO 2023279042 A2 WO2023279042 A2 WO 2023279042A2 US 2022073285 W US2022073285 W US 2022073285W WO 2023279042 A2 WO2023279042 A2 WO 2023279042A2
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primer
variant
seq
sequence
nucleotide sequence
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PCT/US2022/073285
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French (fr)
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WO2023279042A3 (en
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Isabelle PHILIPP
Wing-Sum LEE
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Siemens Healthcare Laboratory, Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is the virus that causes COVID-19 (coronavirus disease 2019), a respiratory illness.
  • SARS CoV-2 has also been referred to as 2019 novel coronavirus (2019-nCoV).
  • SARS CoV-2, and associated COVID-19 were declared a Public Health Emergency of International Concern in January 2020 by the World Health Organization, and a pandemic in March 2020. Detection, diagnosis, and treatment of SARS CoV-2 and viral variants thereof are important for addressing worldwide public health concerns.
  • the current disclosure describes, among other things, compositions, assays, and methods for detecting variants of a SARS CoV-2 virus.
  • the present disclosure encompasses a recognition that different SARS CoV-2 viral variants can be identified by particular sequence mutations (e.g., single nucleotide variants (SNVs)), or combinations thereof.
  • SNVs single nucleotide variants
  • the present disclosure provides the insight that a bubbled primer can be used to specifically detect SARS CoV- 2 mutations (e.g., SNVs).
  • SNVs single nucleotide variants
  • the present invention provides oligonucleotides that detect mutations in SARS CoV-2 genome.
  • the present disclosure provides SARS CoV-2 variant primers that are bubbled primers.
  • a SARS CoV-2 variant primer that specifically binds to a target nucleotide sequence.
  • a SARS CoV-2 variant primer includes: (i) an anchor, where the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence; (ii) a bridge, where the bridge is located immediately 3' of the anchor in the primer, and where the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; and (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of a target nucleotide sequence, which is 3' of the first portion of the target nucleotide sequence and comprises an interrogating nucleotide position, wherein the inter
  • a variant primer is 25 to 55 nucleotides long. In some embodiments, a variant primer is 30 to 40 nucleotides long. In some embodiments, a variant primer is composed of and/or comprises DNA. [0005] In some embodiments, the anchor of a variant primer is 15 to 40 nucleotides long. In some embodiments, the anchor is 20 to 35 nucleotides long. In some embodiments, the bridge of a variant primer is 4 to 10 nucleotides long. In some embodiments, the bridge is 6 to 8 nucleotides long. In some embodiments, the foot of a variant primer is 6 to 8 nucleotides long. In some embodiments, the foot is 7 nucleotides long.
  • a variant primer comprises a label or a tag.
  • the interrogating nucleotide of a variant primer is a nucleotide that corresponds to an amino acid substitution selected from: N501Y, E484K, K417N, A701V, and V1176F.
  • a target nucleotide sequence of a variant primer is a SARS CoV-2 genomic RNA sequence.
  • a target nucleotide sequence corresponds to a SARS CoV-2 S gene sequence or portion thereof.
  • a target nucleotide sequence contains one or more SNVs present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2).
  • a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha, beta, eta, gamma, iota, mu, omicron, theta, zeta, and C.1.2.
  • a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is an alpha variant, variant, beta variant, gamma variant, mu variant, theta variant, C.1.2 variant, or omicron variant.
  • the variant nucleotide sequence comprises a sequence that corresponds to an N501Y mutation. In some embodiments, the variant nucleotide sequence comprises A23063T nucleotide substitution. In some embodiments, the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 8, 20-22, and 29-35. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 8, 20-22, and 29-35. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 8, 20-22, and 29-35.
  • the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8, 20-22, and 29-35.
  • a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant or iota variant.
  • the variant nucleotide sequence comprises a sequence that corresponds to an A701V mutation.
  • the variant nucleotide sequence comprises a C23635T nucleotide substitution.
  • the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 12 and 84-86.
  • the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 12 and 84-86. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 12 and 84-86. In some embodiments, the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86. [0012] In some embodiments, a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a gamma variant, zeta variant, or theta variant.
  • the variant nucleotide sequence comprises a sequence that corresponds to a V1176F mutation. In some embodiments, the variant nucleotide sequence comprises a G25088T nucleotide substitution. In some embodiments, the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 16, 72-74, and 78-80. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 16, 72-74, and 78-80.
  • the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 16, 72-74, and 78-80. In some embodiments, the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16, 72-74, and 78-80.
  • a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, gamma variant, eta variant, or mu variant.
  • the variant nucleotide sequence comprises a sequence that corresponds to an E484 mutation.
  • the variant nucleotide sequence comprises an A22980G nucleotide substitution.
  • the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 38, 41-44, 48, and 50.
  • the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 38, 41-44, 48, and 50.
  • the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 38, 41-44, 48, and 50.
  • the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38, 41-44, 48, and 50.
  • a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, mu variant, or delta variant.
  • the variant nucleotide sequence comprises a sequence that corresponds to a K417N mutation.
  • the variant nucleotide sequence comprises a G22184T nucleotide substitution.
  • the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 51-55 and 62-64. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 51-55 and 62-64. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 51-55 and 62-64. In some embodiments, the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55 and 62-64.
  • primer pairs comprising a variant primer as described herein, and a complementary primer comprising or consisting of a nucleotide sequence that is complementary to a portion of a complementary strand sequence of the target nucleotide sequence.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 8 and 29-35, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 9 and 23-24.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 8 and 29-35, and (ii) a complementary primer comprising or consisting of a nucleotide that differs by no more than one nucleotide from according to any one of SEQ ID NOs: 9 and 23-24.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 20-22, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 25-28.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 20-22, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 25-28.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 20-22, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 25-28.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 12 and 84-86, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 13, 87, and 88.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 12 and 84-86, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 13, 87, and 88.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 16 and 72-73, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 17, 76, and 77.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 16 and 72-73, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 17, 76, and 77.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16 and 72-73, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17, 76, and 77.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 78-80, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 74-75.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 78-80, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 74-75.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 78-80, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 74-75.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 38 and 41-44, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 39-40 and 45-46.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 38 and 41-44, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 39-40 and 45-46.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38 and 41-44, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40 and 45-46.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from SEQ ID NO: 48 or 50, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from SEQ ID NO: 47 or 49.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from SEQ ID NO: 48 or 50, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from SEQ ID NO: 47 or 49.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to SEQ ID NO: 48 or 50, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to SEQ ID NO: 47 or 49.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 51-55, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 56-58.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 51-55, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 56-58.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 56-58.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 62-64, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 59-61.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 62-64, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 59-61.
  • a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 62-64, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61.
  • blocker pairs comprising (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence of SARS CoV-2; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of the target nucleotide sequence of SARS CoV-2, which is 3' of the first portion of the target nucleotide sequence and comprises a wild type nucleotide at an interrogating nucleotide position; and (iv) a modification at its 3
  • a wild type nucleotide at an interrogating nucleotide position is the nucleotide at that corresponding position of the genome of the Wuhan-Hu1 strain or USA- WA1/2020 strain.
  • a target nucleotide sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome but not a wild-type SARS CoV-2 genome, wherein the SARS CoV-2 variant is an alpha variant, variant, beta variant, gamma variant, mu variant, theta variant, C.1.2 variant, or omicron variant.
  • a modification that prevents extension of a blocker primer is or comprises: a PO 4 modification, an inverted 3'-3' linkage, an inverted dT, a C3 modification, or an amino-C7 modification. In some embodiments, a modification that prevents extension of a blocker primer a PO 4 modification or a C3 modification.
  • a blocker primer is 25 to 55 nucleotides long. In some embodiments, a blocker primer is 30 to 40 nucleotides long. In some embodiments, a blocker primer is composed of and/or comprises DNA. [0029] In some embodiments, the anchor of a blocker primer is 15 to 40 nucleotides long.
  • the anchor is 20 to 35 nucleotides long.
  • the bridge of a blocker primer is 4 to 10 nucleotides long. In some embodiments, the bridge is 6 to 8 nucleotides long.
  • the foot of a blocker primer is 6 to 8 nucleotides long. In some embodiments, the foot is 7 nucleotides long.
  • the wild type nucleotide at an interrogating nucleotide position is a nucleotide position that corresponds in a SARS CoV-2 genome that translates to a wild-type amino acid at a position selected from: N501, E484, K417, A701, and V1176.
  • the wild type nucleotide at an interrogating nucleotide position is a nucleotide that corresponds to a position in a SARS CoV-2 genome selected from: A23063, A22980, G22184, C23635, and G25088.
  • the interrogating nucleotide position of a blocker primer is a nucleotide position that corresponds in a SARS CoV-2 genome that translates to a wild-type amino acid at a position N501.
  • the interrogating nucleotide position of a blocker primer is a nucleotide position that corresponds in a SARS CoV-2 genome to A23063.
  • a blocker primer comprises or consists of a sequence according to SEQ ID NO: 10 or 37.
  • the interrogating nucleotide position of a blocker primer is a nucleotide position that corresponds in a SARS CoV-2 genome that translates to a wild-type amino acid at a position A701.
  • the interrogating nucleotide position of a blocker primer is a nucleotide position that corresponds in a SARS CoV-2 genome to C23635.
  • a blocker primer comprises or consists of a sequence according to SEQ ID NOs: 14 and 90-93.
  • the present disclosure provides primer sets that include one or more variant primers as described herein.
  • a primer set further comprises one or more complementary primers.
  • a primer set further comprises one or more probes.
  • a primer set further comprises a blocker primer as described herein.
  • variant primer and blocker primer sequences differ only at the nucleotide that anneals to the interrogating nucleotide position, and that the blocker primer comprises the modification that prevents extension.
  • a probe comprises or consists of a sequence that differs by no more than two nucleotides from a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89. In some embodiments, a probe comprises or consists of a sequence that differs by no more than one nucleotide from a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89. In some embodiments, a probe comprises or consists of a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24, and (iii) a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 7 or 36.
  • a primer set further comprises a blocker primer that comprises or consists of a sequence according to SEQ ID NO: 10 or 37.
  • a primer set that comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and (ii) a blocker primer that comprises or consists of a sequence according to SEQ ID NO: 10 or 37.
  • the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to a nucleotide of N501Y in the variant primer and a nucleotide that translates to a wild-type amino acid at a position N501 in the blocker primer.
  • the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to position 23063 in a SARS CoV-2 genome, with a T at this position in the variant sequence and an A at this position in the blocker sequence.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88, and (iii) a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 11 or 89.
  • a primer set further comprises a blocker primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and (ii) a blocker primer comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93.
  • the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to a nucleotide of A701V in the variant primer and a nucleotide that translates to a wild-type amino acid at a position A701 in the blocker primer.
  • the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to position 23635 in a SARS CoV-2 genome, with a T at this position in the variant sequence and an C at this position in the blocker sequence.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 16 and 72-73, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17, 76, and 77, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 15 or 81-83.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 78-80, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 74-75, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 15 or 81-83.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 38 and 41-44, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40 and 45-46, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 7 and 65-66.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to SEQ ID NO: 48 or 50, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to SEQ ID NO: 47 or 49, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 7 and 65- 66.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 56-58, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 67-71.
  • a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 62-64, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 67-71.
  • the present disclosure provides mixtures comprising variant primers as described herein and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence.
  • the present disclosure provides mixtures comprising primer pairs as described herein and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence. In some embodiments, the present disclosure provides mixtures comprising primer sets as described herein and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence. [0047] In some embodiments, the present disclosure provides methods that include: (i) contacting a sample from a subject with the variant primer, primer pair, or primer set as described herein, and amplifying a target nucleotide sequence from the sample. [0048] In some embodiments, the target nucleotide sequence is a SARS CoV-2 nucleotide sequence.
  • the method is to detect and/or identify a SARS CoV-2 variant in a sample comprising SARS CoV-2 nucleic acid.
  • the SARS CoV- 2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2).
  • the method is to detect and/or identify one or more nucleotide variants corresponding to one or more amino acid mutations in a sample comprising SARS CoV-2 nucleic acid.
  • the one or more mutations are selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F.
  • the one or more mutations are selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the one or more mutations comprise three or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. [0053] In some embodiments, the one or more mutations are selected from: N501Y, A701V, and V1176F. In some embodiments, the one or more mutations are at least three mutations that comprise at least N501Y, A701V, and V1176F.
  • the SARS CoV-2 in the sample is present in an amount that is less than 50,000 viral genome copies per milliliter (cp/mL), less than 40,000 cp/mL, less than 30,000 cp/mL or less than 20 cp/mL.
  • provided methods further comprise contacting a sample from the subject with a positive control primer set.
  • the positive control primer set comprises (i) oligonucleotides comprising or consisting of a sequence according to each of SEQ ID NOs: 1 to 3; and/or (ii) oligonucleotides comprising or consisting of a sequence according to each of SEQ ID NOs: 4 to 6.
  • provided methods further comprise performing whole genome sequencing on the sample.
  • methods of identifying a subject for treatment comprising (i) identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject, (ii) determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified, and (iii) administering therapy according the determined SARS CoV-2 variant strain(s), wherein the step of identifying one or more nucleotide sequence changes comprises contacting the sample with a variant primer, primer pair, or primer set as described herein.
  • nucleotide sequence changes e.g., SNVs
  • kits comprising a variant primer, primer pair, or primer set as described herein, and instructions for use.
  • a kit further comprises a positive control primer set.
  • the positive control primer set comprises (i) oligonucleotides comprising or consisting of a sequence according to each of SEQ ID NOs: 1 to 3; and/or (ii) oligonucleotides comprising or consisting of a sequence according to each of SEQ ID NOs: 4 to 6.
  • FIG. 1A depicts a schematic of the coding regions of the SARS CoV-2 genome with the genes encoding structural proteins labeled: spike (S), envelope (E), matrix (M), and nucleocapsid (N).
  • S spike
  • E envelope
  • M matrix
  • N nucleocapsid
  • FIG. 1B depicts a schematic of the SARS CoV-2 genome that labels genes within the ORF1ab region encoding certain non-structural proteins, as well as the general location of certain mutations associated with different SARS CoV-2 variants of interest (adapted from “Features, Evaluation, and Treatment of Coronavirus (COVID-19)”, Marco Cascella; Michael Rajnik; Arturo Cuomo; Scott C. Dulebohn; Raffaela Di Napoli. Statpearls, Jan 2021).
  • FIG.1C depicts a schematic of certain mutations within the S gene that are associated with particular SARS CoV-2 variants.
  • FIG.2 includes a plot of the fraction of positive samples that have been sequenced in different countries as of January 2021 (plot from Priya Singh, CSIR-CCMB).
  • the x-axis reflects the total number of COVID-19 positive cases in each country and the y-axis reflects the percent of cases that have been sequenced.
  • FIG.3 includes a schematic of an exemplary primer design as provided herein.
  • a primer includes an anchor sequence (brown), a bubble (also referred to as a bridge) (blue), and a foot sequence (red).
  • a primer can be referred to herein as a “bubbled” primer.
  • the present disclosure encompasses a recognition that a foot sequence should be short, such that a single mismatch at the terminal 3′ nucleotide destabilizes primer binding and prevent extension, enabling discrimination of different alleles.
  • “X” represents the interrogating nucleotide, which can be different in one or more variants. If a mismatch occurs at the interrogating nucleotide, primer binding will be destabilized and prevent extension.
  • a second primer can be used in a primer set with a bubbled primer.
  • a probe can be used, which is capable of hybridizing (e.g., is complementary to) to a sequence amplified using a primer set that includes a bubbled primer.
  • FIG.4 includes a schematic of an exemplary primer design as provided herein.
  • a primer set includes a wild-type (WT) primer and a variant primer.
  • WT primer is a primer that is capable of hybridizing with (e.g., is complementary to) a wild-type and/or reference sequence.
  • a variant primer is a primer that is capable of hybridizing with (e.g., is complementary to) a variant sequence.
  • a WT forward primer can have a modification (e.g.,PO 4 ) at its 3'-end to prevent extension from occurring.
  • Such a WT primer is also referred to as a “blocker” primer, and can compete with a variant primer that binds to a sequence ending with a certain nucleotide represented by “X” in FIG.4. Because the WT blocker primer can compete with a variant primer, it can suppress non-specific amplification of a wild-type allele by a variant primer.
  • a primer set can also include a third primer, which is used together with a WT blocker primer and a variant primer.
  • a third primer can be a reverse primer.
  • a probe can be used, which is capable of hybridizing with (e.g., is complementary to) a sequence amplified using a primer set as shown in FIG.4.
  • FIG.5 includes a schematic of an exemplary primer design as provided herein.
  • a primer set includes a forward primer and a reverse primer.
  • either the forward or reverse primer is capable of hybridizing with (e.g., is complementary to) a sequence that is present in a wild-type sequence, but which is not present (e.g., is in a deletion) in a variant sequence.
  • FIG. 6 includes a flow chart schematic that provides a process overview for development of an assay for detection of an exemplary SARS CoV-2 single nucleotide variant (SNV), N501Y, with high specificity.
  • SNV SARS CoV-2 single nucleotide variant
  • FIG.7 includes exemplary results of a screen of primer/probe sets for amplification of a N501Y mutation.
  • Panel (A) provides an amplification plot and Panel (B) provides the average expression (CT) and change in expression ( ⁇ CT) between WT and mutant templates.
  • FIG.8 includes exemplary results of a blocker screen for amplification of a N501Y mutation. Results depicted for an exemplary primer/probe set with increasing concentrations of blocker: 0 nM, 10 nM, 50 nM, 100 nM, 150 nM, and 300 nM. The corresponding amounts of amplification of N501Y mutant and wild-type templates are provided is provided in the table of Panel (B) [0070] FIG.
  • FIG. 9 includes results of an exemplary analytical sensitivity analysis for exemplary primer set for detection of a N501Y mutation, with and without a blocker primer.
  • Panel (A) provides a linear fit of the average CT vs. concentration for primer sets SNV- MutFor5/WTFor5-PO 4 /Rev4/Probe2 (“with blocker”) vs. SNV-MutFor5/Rev4/Probe2 (“without blocker”).
  • Panel (B) provides a table view of the results, with the concentration of template, average CT, and number of samples detected.
  • FIG. 10 includes exemplary results of primer/probe screens for (A) E484K mutation, (B) SA417 mutation, (C) BZ1176 mutation, and (D) SA701 mutation.
  • FIG. 11 includes exemplary results of a blocker screen analysis directed to the SA701 mutation.
  • FIG.12A and FIG.12B include exemplary results of primer/probe combinations for a multiplex assay for detecting N501Y, SA701, BZ1176, and a positive control.
  • FIG. 12A depicts exemplary results of combination 5, which includes N501Y with a FAM probe, SA701 with a CIV probe, BZ1176 with a TAMRA probe, and control with a Q670 probe.
  • FIG. 12B depicts exemplary results of combination 6, which includes SA701 with a FAM probe, N501Y with a CIV probe, BZ1176 with a TAMRA probe, and control with a Q670 probe. Both of these combinations generated a ⁇ CT within +/- 1 when compared with singleplex assays.
  • CERTAIN DEFINITIONS As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.
  • the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included. [0075] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value.
  • Allele refers to one of two or more existing genetic variants of a specific polymorphic genomic locus.
  • Detection agent refers to any element, molecule, functional group, compound, fragment or moiety that is detectable. In some embodiments, a detection agent is provided or utilized alone. In some embodiments, a detection agent is provided and/or utilized in association with (e.g., joined to) another agent.
  • detection agents include, but are not limited to: various ligands, radionuclides (e.g., 3 H, 14 C, 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99m Tc, 177 Lu, 89 Zr etc.), fluorescent dyes, chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.
  • Diagnostic test is a step or series of steps that is or has been performed to attain information that is useful in determining whether a patient has a disease, disorder or condition and/or in classifying a disease, disorder or condition into a phenotypic category or any category having significance with regard to prognosis of a disease, disorder or condition, or likely response to treatment (either treatment in general or any particular treatment) of a disease, disorder or condition.
  • diagnosis refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have or develop a disease, disorder or condition, state, staging or characteristic of a disease, disorder or condition as manifested in the subject, information related to the nature or classification of a tumor, information related to prognosis and/or information useful in selecting an appropriate treatment or additional diagnostic testing.
  • Selection of treatment may include the choice of a particular therapeutic agent or other treatment modality such as vaccination, antibody therapy, etc., a choice about whether to withhold or deliver therapy, a choice relating to dosing regimen (e.g., frequency or level of one or more doses of a particular therapeutic agent or combination of therapeutic agents), etc.
  • Hybridization refers to the physical property of single- stranded nucleic acid molecules (e.g., DNA or RNA) to anneal to complementary nucleic acid molecules. Hybridization can typically be assessed in a variety of contexts– including where interacting nucleic acid molecules are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).
  • hybridization can be detected by a hybridization technique, such as a technique selected from the group consisting of in situ hybridization (ISH), microarray, Northern blot, and Southern blot.
  • hybridization refers to 100% annealing between the single-stranded nucleic acid molecules and the complementary nucleic acid molecule. In some embodiments, annealing is less than 100% (e.g., at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% of a single-stranded nucleic acid molecule anneals to a complementary nucleic acid molecule.
  • Oligonucleotide refers to a molecule comprising two or more deoxyribonucleotides, ribonucleotides, and/or nucleotide analogs, the latter including nucleic acid analogs, such as isoguanosine, isocytosine, inosine, or deoxyinosine.
  • the length of the oligonucleotide will vary depending on the function of the oligonucleotide.
  • the oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof.
  • oligonucleotide is meant to encompass primers (both forward and reverse primers) and detection probes.
  • primer refers to an oligonucleotide which, whether purified from a nucleic acid restriction digest or produced synthetically, is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase, reverse transcriptase or the like, and at a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agents for polymerization.
  • the term “forward primer” refers to a primer that forms an extension product by binding in the 5′ to 3′ direction to the 3′ end of a strand of a nucleic acid analyte (e.g., denatured DNA).
  • sample refers to a biological sample obtained or derived from a subject (e.g., a human subject), as described herein.
  • a biological sample comprises biological tissue or fluid.
  • a bodily fluid is or comprises saliva, respiratory secretion, blood, and/or plasma.
  • a biological sample may comprise blood; blood cells; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; cerebrospinal fluid; lymph; tissue biopsy specimens; surgical specimens; other body fluids, secretions, and/or excretions; and/or cells therefrom.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • a primary biological sample is obtained by methods of collecting a body fluid and/or cells.
  • a sample of saliva from a subject by having the subject spit into a receptacle e.g., a tube.
  • the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample.
  • a “processed sample” may comprise, for example, nucleic acids or polypeptides extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription, isolation and/or purification of certain components (e.g., polynucleotide components, e.g., RNA and/or DNA).
  • components e.g., polynucleotide components, e.g., RNA and/or DNA.
  • SARS CoV-2 variant As used herein, the term “SARS CoV-2 variant” or “viral variant” or simply “variant” when used in reference to SARS CoV-2 (as will be apparent from context), is a SARS CoV-2 virus that includes one or more genetic changes relative to a SARS CoV-2 reference strain or one or more predominant viral variants already circulating in a population.
  • a SARS CoV-2 variant comprises one or more genomic mutations in an S gene sequence.
  • a SARS CoV-2 variant comprises one or more genomic mutations relative to a reference genome sequence or portion thereof.
  • a SARS CoV-2 variant comprises one or more genomic mutations in an S gene sequence relative to an S gene sequence of a reference genome.
  • a reference genome sequence corresponds to that of the Wuhan-Hu1 strain (the first genetic sequence identified) or USA-WA1/2020 strain (the first identified in the United States) or portion thereof.
  • a SARS CoV-2 variant comprises one or more genomic mutations relative to one or more predominant viral variants circulating in a population.
  • a SARS CoV-2 variant comprises one or more genomic mutations in an S gene sequence relative to an S gene sequence of a predominant viral variant circulating in a population.
  • Singleplex refers to a single assay that is not carried out simultaneously with any other assays.
  • a singleplex assay refers to a single reaction that is performed within a single well of the multiwell plate.
  • Singleplex assays include individual assays that are carried out sequentially.
  • Multiplex refers to multiple assays that are carried out simultaneously.
  • a multiplex assay refers to the number of target sites that the assay aims to identify. For example, a multiplex assay that is designed to identify two sites is termed a dualplex assay.
  • a multiplex assay performs multiple reactions within a single well of the multiwell plate.
  • subject refers to an organism, for example, a mammal (e.g., a human).
  • a human subject is an adult, adolescent, or pediatric subject.
  • a subject is at risk for a SARS CoV-2 viral infection.
  • a subject has been exposed or is suspected to have a COVID-19 infection.
  • a subject is susceptible to COVID-19; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing COVID-19.
  • a subject displays one or more symptoms of a SARS CoV-2 infection.
  • a subject does not display a particular symptom or characteristic of COVID-19.
  • a subject does not display any symptom or characteristic of COVID-19 (i.e., is asymptomatic).
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • Threshold value refers to a value (or values) that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay.
  • a threshold value can be determined based on one or more control samples. A threshold value can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold value can be a range of values. In some embodiments, a threshold value can be a value (or range of values) reported in the relevant field (e.g., a value found in a standard table).
  • SARS CoV-2 variant detection assays for identifying different human coronavirus SARS CoV-2 variants (e.g., N501Y, A701V, V1176F).
  • provided methods also detect a conserved genomic region of SARS CoV- 2 (e.g., ORF gene) which serves as a control.
  • SARS CoV-2 variant detection assays can allow sample selection and/or streamlining of downstream sequencing effort for different variants of SARS CoV-2, including currently major circulating variants.
  • a SARS CoV-2 variant detection assay that can be used, e.g., as research tools to help scientists and epidemiologists track mutation prevalence in a population as well as enable assessment of any potential impact on laboratory diagnostics, vaccines, and/or therapeutics.
  • a SARS CoV-2 variant detection assay provided herein will use a Reverse Transcription Polymerase Chain Reaction (RT-PCR) amplification process.
  • RT-PCR Reverse Transcription Polymerase Chain Reaction
  • a method as provided combines first reverse transcription (RT) of SARS CoV-2 RNA into DNA and a second step amplification of specific DNA targets using polymerase chain reaction (PCR).
  • a reverse transcriptase reverse transcribes a specific section of RNA into complimentary DNA (cDNA) and then a thermal-stable DNA polymerase amplifies the cDNA template.
  • a section is specified by a DNA oligonucleotide, called a primer.
  • the reverse transcription process is linear because it uses only the reverse strand primer.
  • the amplification process is exponential, using both forward and reverse strand primers.
  • the RT-PCR reaction includes polymerase, primers, a metal ion, deoxynucleoside triphosphates and buffer.
  • priming and replication will be accomplished by cycling a reaction mixture through two or more temperatures, such as an elevated temperature for denaturing the amplicon and a lower temperature or temperatures for annealing and extending the primer.
  • the present disclosure describes, inter alia, the design and characteristics of primers and probes useful for detecting and/or characterizing SARS CoV-2 (e.g., a multiplexed RT-PCR assay).
  • SARS CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) Variants
  • SARS CoV-2 is the virus that causes COVID-19, a respiratory illness that was declared a Public Health Emergency of International Concern in January 2020 by the World Health Organization, and a pandemic in March 2020.
  • SARS CoV-2 is an enveloped, positive-stranded RNA virus with a nucleocapsid.
  • the SARS CoV-2 genomic structure is organized in a +ssRNA of approximately 30 kb in length and with a 5′-cap structure and 3′-poly-A tail.
  • FIG.1A and FIG. 1B provide schematic representations of the SARS CoV-2 viral genome. As depicted in FIG.1A, the SARS CoV-2 genome includes from 5′ to 3′: a 5′ terminal sequence, ORF1ab (comprising ORF1a and ORF1b), ORFs encoding structural proteins: spike (S), envelope (E), matrix (M), nucleocapsid (N), and a 3′ terminal sequence.
  • the genomic schematic of FIG. 1B illustrates certain genes encoding non-structural proteins in the SARS CoV-2 genome (e.g., papain-like protease, 3CL-protease, replicase, and endoribonuclease), along with the spike protein (S) gene.
  • SARS CoV-2 As with all viruses, SARS CoV-2 accumulates random mutations during replication. Distinct SARS CoV-2 variant lineages formed as viruses circulating in particular regions evolved and increased in frequency.
  • SARS CoV-2 was first detected, a number of viral variants have emerged, some of which show higher transmissibility and/or may increase death rate. A number of different SARS CoV-2 variants have been identified to date.
  • SARS CoV-2 variants identified include, e.g., alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2).
  • a SARS CoV-2 variant is selected from alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), mu (B.1.621), and omicron (B.1.1.529), and/or a descendent lineage of any thereof.
  • a SARS CoV-2 variant is selected from alpha (B.1.1.7), beta (B.1.351), gamma (P.1), iota (B.1.526), mu (B.1.621), and omicron (B.1.1.529), theta (P.3), zeta (P.2), and/or a descendent lineage of any thereof.
  • the present disclosure provides methods and/or assays for detecting one or more mutations associated with a SARS CoV-2 variant.
  • FIG.1B also indicates certain mutations associated with SARS CoV-2 variants and the gene locus of these variants.
  • one or more mutations associated with a SARS CoV-2 variant are located in the S gene.
  • a SARS CoV-2 variant may include one or more mutations selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F.
  • a SARS CoV-2 variant may include one or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, one or more mutations to be detected are selected from N501Y, E484K, K417N, A701V, and V1176F. [0101] In some embodiments, a SARS CoV-2 variant may include two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, two or more mutations to be detected are selected from N501Y, E484K, K417N, A701V, and V1176F.
  • a SARS CoV-2 variant may include one or more mutations to be detected by a method and/or assay described herein (e.g., through detection of a nucleotide variant corresponding to an amino acid mutation). In some embodiments, one or more mutations to be detected are selected from N501Y, A701V, and V1176F. [0103] In some embodiments, the present disclosure provides methods and/or assays for detecting two or more mutations associated with a SARS CoV-2 variant (e.g., through detection of two or more nucleotide variants corresponding to the mutations). In some embodiments, two or more mutations to be detected are selected from N501Y, A701V, and V1176F.
  • the present disclosure provides methods and/or assays for detecting three or more mutations associated with a SARS CoV-2 variant (e.g., through detection of three or more nucleotide variants corresponding to the mutations).
  • three or more mutations to be detected include N501Y, A701V, and V1176F.
  • Some emerging SARS CoV-2 variants, e.g., N501Y variants, may have higher ability to escape the immune system or cause reinfection. Preliminary vaccine data show that the BioNtech (BNT162b2) and the Moderna (mRNA-1273) vaccine seem to have equivalent neutralizing titers to the N501 and Y501 viruses.
  • a SARS CoV-2 variant is or comprises a N501Y mutation, such as, beta (B.1.351), gamma (P.1), alpha (B.1.1.7), eta (B.1.525), and/or a descendent of any thereof.
  • a SARS CoV-2 variant is or comprises a A701V mutation, such as, beta (B.1.351) and iota (B.1.526), and/or descendent of any thereof.
  • a SARS CoV-2 variant is or comprises a V1176F mutation, such as, a gamma (P.1) variant, theta (P.3) variant, zeta (P.2) variant and/or a descendent of any thereof.
  • SARS CoV-2 variants are classified in different countries using differing nomenclature and standards. In the United States, SARS CoV-2 variants are classified based on their attributes and prevalence in the U.S. These U.S. classifications include (in order of increasing concern): variant being monitored (VBM), variant of interest (VOI), variant of Concern (VOC), and variant of high consequence (VOHC).
  • each variant classification includes the possible attributes of lower classes (for example, VOC includes the possible attributes of VOI).
  • VOC includes the possible attributes of VOI.
  • variant classification may be updated and/or changed (e.g., a variant may be reclassified) with emerging information on variant attributes and prevalence.
  • a variant is a VBM.
  • the CDC monitors all SARS CoV-2 variants circulating in the United States.
  • a VBM as a variant where (i) data indicates there is a potential or clear impact on approved or authorized medical countermeasures or that have been associated with more severe disease or increased transmission and (ii) this variant is no longer detected, or are circulating at very low levels, in the United States. In some embodiments, a VBM does not pose a significant and imminent risk to public health in the United States. In some embodiments, a VBM was previously classified as a VOI or VOC. [0110] In some embodiments, a variant is a VOI.
  • a VOI as a variant comprises specific genetic markers that have been associated with changes to receptor binding, reduced neutralization by antibodies generated against previous infection or vaccination, reduced efficacy of treatments, potential diagnostic impact, and/or predicted increase in transmissibility or disease severity.
  • a VOI comprises specific genetic markers that are predicted to affect transmission, diagnostics, therapeutics, and/or immune escape.
  • a VOI is a variant that is associated with an increased proportion of cases and/or unique outbreak clusters. [0111] In some embodiments, a variant is a VOC.
  • a VOC is a variant for which there is evidence of an increase in transmissibility, more severe disease (for example, increased hospitalizations or deaths), significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures.
  • a variant is a VOC and includes one or more characteristics of a VOI.
  • a VOC is further characterized by evidence of impact on diagnostics, treatments, or vaccines (e.g., widespread interference with diagnostic test targets, evidence of substantially decreased susceptibility to one or more class of therapies, evidence of significantly decreased neutralization by antibodies generated during previous infection or vaccination, evidence of reduced vaccine-induced protection from severe disease, evidence of increased transmissibility, and/or evidence of increased disease severity).
  • a variant is a VOHC.
  • a VOHC is a variant for which there is clear evidence that prevention measures or medical countermeasures (MCMs) have significantly reduced effectiveness relative to previously circulating variants.
  • MCMs medical countermeasures
  • a variant is a VOHC and includes one or more characteristics of a VOI and/or a VOC.
  • a VOHC is further characterized evidence of impact on MCMs (e.g., demonstrated failure of diagnostic test targets, evidence to suggest a significant reduction in vaccine effectiveness, a disproportionately high number of infections in vaccinated persons, very low vaccine-induced protection against severe disease, significantly reduced susceptibility to multiple therapeutics (e.g., approved and/or authorized therapeutics), and/or more severe clinical disease and increased hospitalizations.
  • Detecting SARS CoV-2 [0113] Current techniques for detecting SARS CoV-2 virus include antigen tests and Nucleic Acid Amplification Tests (NAATs). Antigen tests are immunoassays that detect the presence of a specific viral antigen, which implies current viral infection.
  • SARS CoV-2 antigen tests for SARS CoV-2 are less expensive than NAATs and provide faster results, and thus can be used in screening programs to quickly identify those who are likely to be contagious.
  • the SARS CoV-2 antigen tests currently authorized include point-of-care, laboratory-based, and self-tests.
  • antigen tests for SARS-CoV-2 are generally less sensitive than molecular test like real-time reverse transcription polymerase chain reaction (RT-PCR) and other nucleic acid amplification tests (NAATs), which detect and amplify the presence of viral nucleic acid.
  • RT-PCR real-time reverse transcription polymerase chain reaction
  • NAATs nucleic acid amplification tests
  • NAATs can produce positive results for weeks to months after initial infection and can detect levels of viral nucleic acid even when virus cannot be cultured, suggesting that the presence of viral nucleic acid may not always indicate contagiousness.
  • Antigen tests perform best in individuals with high viral loads (e.g., Ct values ⁇ 25-30, ⁇ 10E5/6 RNA copies/mL) 1 to 3 days prior to onset of symptoms and during the first 5 to 7 days of illness.
  • False negative results may occur with any molecular test for the detection of SARS- CoV-2, particularly if a mutation occurs in the part of the virus' genome assessed by that test.
  • a mutation e.g., a mutation associated with a SARS CoV-2 variant
  • mutations of SARS CoV-2 nucleoprotein gene can result in false-negative antigen test results despite high viral loads confirmed by other methods (e.g., NAAT).
  • NAAT e.g., NAAT
  • some assays have been designed to target highly conserved regions.
  • the FTD SARS CoV-2 Assay is a real time PCR assay that targets two highly conserved SARS CoV-2 genomic regions in ORF1ab and N gene.
  • SARS CoV-2 variants of concern VOCs
  • SNVs single nucleotide variants
  • the present disclosure encompasses a recognition that it would be useful to develop an assay with high specificity for particular SNVs.
  • developing an assay with high specificity for SNVs is more challenging than detecting sequence deletions or insertions (indels).
  • the present disclosure encompasses a recognition that reflex testing can facilitate rapid public health responses, including surge testing and strategic decision-making.
  • the present disclosure provides methods of reflex testing that can detect of one or more mutations that are indicative of a particular SARS CoV-2 variant.
  • Other manufacturers have also been developing reflex testing to identify SARS CoV-2 variants, such as the TaqPathTM COVID-19 Combo Kit (ThermoFisher Scientific) and the PhoenixDx ® SARS-CoV-2 Multiplex assay (Procomcure Biotech GmbH), such assays have limitations.
  • these assays are each directed to a single mutation assays that are multiplexed, e.g., in combination with wild type sequence and/or a positive control.
  • Provided Technologies for Detection of SARS CoV-2 Variants [0122]
  • reflex (genotyping) technologies e.g., assays, methods, compositions
  • detection of one or more mutations is indicative of a particular viral variant or variants.
  • the detection of one or more mutations is indicative of a SARS CoV-2 VBM, VOI, VOC, and/or VOHC.
  • a SARS CoV-2 variant assay provided herein detects a mutation in a SARS CoV-2 S gene.
  • a SARS CoV-2 variant assay provided herein detects a SARS CoV-2 mutation selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F.
  • a SARS CoV-2 variant assay detects one or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, a SARS CoV-2 variant assay detects two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, a SARS CoV-2 variant assay detects three or more mutations selected from N501Y, E484K, K417N, A701V, and V1176F. [0125] A listing of exemplary SARS CoV-2 variants associated with these SNVs is provided in Table 1 below.
  • a SARS CoV-2 variant detection assay detects nucleic acid changes that correspond to amino acid mutations N501Y, A701V, and/or V1176F.
  • Technologies for detecting SARS CoV-2 variants as provided herein may also include a control.
  • an assay for detecting SARS CoV-2 variants is a multiplex assay detecting one or more SNVs (e.g., SNVs with the S gene) and a SARS CoV-2 positive control that is targeted to the Nucleocapsid (N) gene or ORF1ab gene.
  • an assay for detecting SARS CoV-2 variants comprises a ROX reference dye.
  • a ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization in a real-time qPCR assay.
  • a ROX reference dye is present in an amount to generate a fluorescent signal in a range from about 50,000 to about 500,000.
  • a ROX reference dye is present in an amount to generate a fluorescent signal in a range from about 100,000 to about 250,000.
  • a ROX reference dye is present in an amount of 0.1X per reaction.
  • SARS CoV-2 Oligonucleotides [0130] In some embodiments, the present disclosure provides oligonucleotides for detecting and/or identifying one or more mutations associated with a SARS CoV-2 variant. In some embodiments, the present disclosure provides two or more oligonucleotides for detecting and/or identifying one or more mutations associated with a SARS CoV-2 variant. In some embodiments, two or more oligonucleotides comprise a variant primer (e.g., a bubbled primer) and second oligonucleotide. In some embodiments, a second oligonucleotide comprises a blocker primer.
  • a variant primer e.g., a bubbled primer
  • second oligonucleotide comprises a blocker primer.
  • a second oligonucleotide comprises a probe. In some embodiments, a second oligonucleotide is a complementary primer. In some embodiments, a variant primer is a forward primer and a complementary primer is a reverse primer. In some embodiments, a variant primer is a reverse primer and a complementary primer is a forward primer. [0131] In some embodiments, the present disclosure provides a primer set for detecting and/or identifying one or more mutations associated with a SARS CoV-2 variant (e.g., in a SARS CoV-2 S gene). In some embodiments, a primer set comprises a variant primer and a blocker primer. A primer set can also include a complementary primer, which is used together with a variant primer.
  • a variant primer is a forward primer
  • a complementary primer can be a reverse primer (and vice versa).
  • a probe can be used, which is capable of hybridizing with (e.g., is complementary to) a sequence amplified using a primer set as shown in FIG. 4.
  • a primer set comprises a variant primer, a complementary primer, and a probe.
  • a primer set comprises each of a variant primer, a complementary primer, a blocker primer, and a probe.
  • the present disclosure provides a primer set for a multiplex assay detecting one or more SNVs (e.g., SNVs with the S gene) and a SARS CoV-2 positive control that is targeted to the Nucleocapsid (N) gene or ORF1ab gene.
  • provided primer sets include a variant primer, a complementary primer, a first probe, as well as a positive control forward primer, positive control reverse primer, and a second probe.
  • provided primer sets include for each variant to be detected: a variant primer, a complementary primer, a probe, and optionally a blocker primer, and also includes a positive control forward primer, positive control reverse primer, and a probe for the positive control.
  • variant primers that are capable of hybridizing with (e.g., is complementary to) a variant sequence (e.g., a mutant sequence).
  • a variant sequence comprises a mutation that is a substitution (e.g., an SNV), a deletion, and/or insertion.
  • a variant primer is a bubbled primer.
  • a variant primer is a bubbled primer and comprises a sequence that is complementary to a mutation associated with one or more SARS CoV-2 variants.
  • the present disclosure provides one or more bubbled primers useful for detecting an SNV associated with a SARS CoV- 2 variant.
  • a variant primer is a bubbled primer that includes an anchor sequence, a bubble (also referred to as a bridge) sequence, and a foot sequence.
  • a foot sequence of a variant primer overlaps with (i.e., includes a nucleotide that is complementary to) the nucleotide of an SNV to be detected (referred to herein as the “interrogating nucleotide”).
  • the present disclosure encompasses a recognition that a foot sequence should be short, such that a single mismatch at the interrogating nucleotide destabilizes primer binding and prevent extension, enabling discrimination of different alleles.
  • a foot sequence is designed such that the SNV to be detected is positioned at the terminus of the foot sequence (e.g., in the last 3 nucleotides, last 2 nucleotides, or the terminal nucleotide of the foot sequence). In some embodiments, a foot sequence is designed such that the SNV to be detected corresponds to the terminal nucleotide of the foot sequence.
  • a variant primer that includes (i) an anchor, where the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence; (ii) a bridge (also referred to as a bubble), wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of a target nucleotide sequence; and (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of a target nucleotide sequence, which is 3' of the first portion of a target nucleotide sequence and comprises an interrogating nucleotide position.
  • an anchor where the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of
  • a target nucleotide sequence is a SARS CoV-2 variant nucleotide sequence.
  • a variant primer comprises is a bubbled primer having a length within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit.
  • the lower limit may be about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, or about 25 nucleotides.
  • a variant primer comprises is a bubbled primer comprising an anchor sequence, a bubble sequence, and a foot sequence, wherein the overall length of the primer is 25 to 55 nucleotides long. In some embodiments, a variant primer is 30 to 40 nucleotides long. [0138] In some embodiments, a variant primer comprises an anchor sequence, where the anchor is 15 to 40 nucleotides long.
  • an anchor sequence is 20 to 35 nucleotides long.
  • a variant primer comprises a bubble sequence (i.e., a bridge sequence) that is 4 to 10 nucleotides in length.
  • a bubble sequence i.e., a bridge sequence
  • a bubble sequence comprises a sequence that is 6 to 8 nucleotides in length.
  • a bubble sequence comprises a sequence that is 6 nucleotides in length.
  • a bubble sequence comprises a sequence that is 8 nucleotides in length.
  • a variant primer comprises a foot sequence comprises a sequence that is 6 to 8 nucleotides in length.
  • a foot sequence comprises a sequence that is 6 nucleotides in length. In some embodiments, a foot sequence comprises a sequence that is 7 nucleotides in length. In some embodiments, a foot sequence comprises a sequence that is 8 nucleotides in length. [0141] In some embodiments, a variant primer comprises a foot sequence and the interrogating nucleotide is located within the last 3 nucleotides of the foot sequence. In some embodiments, a variant primer comprises a foot sequence and the interrogating nucleotide is located within the last 2 nucleotides of the foot sequence.
  • a variant primer comprises a foot sequence and the interrogating nucleotide is located at the 3′ terminus of the foot sequence.
  • a variant primer is a forward primer.
  • a variant primer is a forward primer and includes in order from 5′ to 3′, an anchor sequence, a bubble or bridge sequence, and a foot sequence.
  • a variant primer is a bubbled primer that is a forward comprising in order 5′ to 3′, an anchor sequence, a bubble sequence that is 6 to 8 nucleotides in length, and a foot sequence with the interrogating nucleotide position at the 3′ terminus.
  • a variant primer is a reverse primer.
  • a variant primer is a reverse (antisense) primer and includes in order from 5′ to 3′, an antisense anchor sequence, a bubble sequence (i.e., a bridge sequence), and antisense foot sequence.
  • a variant primer is a bubbled primer that is a reverse comprising in order 5′ to 3′, an anchor sequence (i.e., an antisense anchor sequence), a bubble sequence (i.e., a bridge sequence) that is 6 to 8 nucleotides in length, and a foot sequence (i.e., an antisense foot sequence) with the interrogating nucleotide position at the 3′ terminus.
  • a complementary primer can be used in a primer set with a variant primer.
  • a probe can be used, which is capable of hybridizing (e.g., is complementary to) to a sequence amplified using a primer set that includes a bubbled primer.
  • Examples of commonly used probes used for RT-PCR assays include the following probes: TAQMAN ® probes, Molecular Beacons probes, SCORPIONS® probes, and SYBR® Green probes.
  • TAQMAN ® , Molecular Beacons, and SCORPIONS ® probes each have a fluorescent reporter dye (also called a “fluor”) attached to the 5′ end of the probes and a quencher moiety coupled to the 3′ end of the probes.
  • TAQMAN ® probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe; during PCR, when the polymerase replicates a template on which a TAQMAN ® probe is bound, the 5′-nuclease activity of the polymerase cleaves the probe thus, increasing fluorescence with each replication cycle.
  • Molecular Beacons which form a stem-loop structure when free in solution, remain intact during the amplification reaction. Molecular Beacons fluoresce during hybridization when the fluorescent dye and the quencher are separated. For signal measurement to be effective, the fluor and quencher must rebind in every cycle.
  • SCORPIONS® probes which maintain a stem-loop configuration in the unhybridized state, has at its 3′ end an additional sequence that is complementary to the extension product of the primer that is linked to the 5′ end of a specific primer via a non-amplifiable monomer.
  • a probe provided herein includes a TAQMAN ® probe selected from TAMRA, FAM, TET, and/or VIC.
  • a blocker primer can be used, which is capable of hybridizing (e.g., is complementary to) to a sequence includes the position to be interrogated, but comprises a different sequence, e.g., a wild type sequence or reference sequence.
  • a blocker primer is capable of hybridizing with (e.g., is complementary to) a wild- type sequence.
  • a blocker primer is capable of hybridizing with (e.g., is complementary to) a reference sequence.
  • a wild type or reference sequence comprises a sequence corresponding to that of the SARS CoV-2 Wuhan-Hu1 strain or USA- WA1/2020 strain.
  • a blocker primer comprises a modification to prevent extension from occurring.
  • a blocker primer is a forward primer and includes a modification to prevent extension at its 3'-end.
  • a blocker primer is a reverse primer and includes a modification to prevent extension at its 5'-end.
  • a blocker primer comprises a modification that blocks extension.
  • a blocker primer comprises a phosphate modification (e.g., a phosphate group or phosphate ester).
  • a blocker primer comprises a modification with an inverted 3'-3' linkage, an inverted dT, a C3, or amino-C7.
  • a blocker primer comprises a wild-type forward primer can have a PO 4 modification at its 3'-end to prevent extension from occurring. In some embodiments, a blocker primer comprises a wild-type forward primer can have a C3 modification at its 3'-end to prevent extension from occurring. [0153] In some embodiments, a blocker primer can compete with a variant primer that binds to a sequence ending with the position to be interrogated. The present disclosure encompasses a recognition that because a blocker primer can suppress non-specific amplification of a wild type allele by a variant primer.
  • blocker primers that include: (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence of SARS CoV-2; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of the target nucleotide sequence of SARS CoV-2, which is 3' of the first portion of the target nucleotide sequence and comprises a wild type nucleotide at an interrogating nucleotide position; and (iv) a modification at
  • the present disclosure encompasses a recognition that a foot sequence should be short, such that a single mismatch at the interrogating nucleotide destabilizes primer binding and prevent extension, enabling discrimination of different alleles.
  • a foot sequence is designed such that the interrogating nucleotide is positioned at the terminus of the foot sequence (e.g., in the last 3 nucleotides, last 2 nucleotides, or the terminal nucleotide of the foot sequence).
  • the interrogating the corresponds to the terminal nucleotide of the foot sequence.
  • the interrogating nucleotide is a wild type nucleotide that corresponds to the nucleotide at that position of the genome of the Wuhan-Hu1 strain or USA- WA1/2020 strain.
  • the wild type nucleotide at an interrogating nucleotide position is a nucleotide that translates to a wild-type amino acid at a position selected from: N501, E484, K417, A701, and V1176.
  • a blocker primer comprises is a bubbled primer having a length within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit.
  • the lower limit may be about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, or about 25 nucleotides.
  • the upper limit may be about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, or about 60 nucleotides.
  • a blocker primer comprises is a bubbled primer that comprises an anchor sequence, a bubble sequence, and a foot sequence, wherein the overall length of the blocker primer is 25 to 55 nucleotides long. In some embodiments, a blocker primer is 30 to 40 nucleotides long. [0159] In some embodiments, a blocker primer comprises an anchor sequence, where the anchor is 15 to 40 nucleotides long. In some embodiments, an anchor sequence is 20 to 35 nucleotides long. [0160] In some embodiments, a blocker primer comprises a bubble sequence (i.e., a bridge sequence) that is 4 to 10 nucleotides in length.
  • a bubble sequence i.e., a bridge sequence
  • a bubble sequence (i.e., a bridge sequence) comprises a sequence that is 6 to 8 nucleotides in length. In some embodiments, a bubble sequence comprises a sequence that is 6 nucleotides in length. In some embodiments, a bubble sequence comprises a sequence that is 8 nucleotides in length.
  • a blocker primer comprises a foot sequence that is 6 to 8 nucleotides in length. In some embodiments, a foot sequence is 6 nucleotides in length. In some embodiments, a foot sequence is 7 nucleotides in length. In some embodiments, a foot sequence is 8 nucleotides in length.
  • a blocker primer comprises a foot sequence and the interrogating nucleotide is located within the last 3 nucleotides of the foot sequence. In some embodiments, a blocker primer comprises a foot sequence and the interrogating nucleotide is located within the last 2 nucleotides of the foot sequence. In some embodiments, a blocker primer comprises a foot sequence and the interrogating nucleotide is located at the 3′ terminus of the foot sequence. [0163] In some embodiments, a blocker primer is a forward primer. In some embodiments, a blocker primer comprises a modification on its 3′ terminus that blocks extension.
  • a blocker primer comprises a phosphate modification on its 3′ terminus that blocks extension. In some embodiments, a blocker primer comprises an inverted dT modification on its 3′ terminus that blocks extension. In some embodiments, a blocker primer comprises an amino-C7 modification on its 3′ terminus that blocks extension.
  • An assay for detecting SARS CoV-2 variants as provided herein may also include a control. In some embodiments, an assay for detecting SARS CoV-2 variants is a multiplex assay detecting one or more SNVs and an internal control that is targeted to the Nucleocapsid (N) gene or ORF1ab gene as an internal control.
  • a positive control primer set targets (i.e., binds to) a sequence in the SARS CoV-2 ORF1ab amplicon.
  • a positive control primer set targets (i.e., binds to) a sequence in the SARS CoV-2 nucleocapsid amplicon.
  • Exemplary primer and probe sequences for the SARS CoV-2 internal control targeting amplicons in the N gene and ORF1ab gene are provided in the table below, which correspond to those of the FTD SARS CoV-2 Assay.
  • a positive control primer set comprises one or more primer and probe sequences of Table 2.
  • primer set that targets a nucleocapsid gene sequence
  • the primer set includes: (i) a forward primer that comprises a sequence of SEQ ID NO.1 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.1; (ii) a reverse primer that comprises a sequence of SEQ ID NO.3 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.3; and (iii) a probe that comprises a sequence of SEQ ID NO.2 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.2.
  • a positive control primer set targets a nucleocapsid gene sequence and comprises a forward primer sequence of SEQ ID NO:1, a reverse primer sequence of SEQ ID NO: 3, and a probe sequence of SEQ ID NO.2.
  • positive control primer set that targets a ORF1ab gene sequence, where the primer set includes: (i) a forward primer that comprises a sequence of SEQ ID NO.
  • a positive control primer set targets a ORF1ab gene sequence and comprises a forward primer sequence of SEQ ID NO:4, a reverse primer sequence of SEQ ID NO: 6, and a probe sequence of SEQ ID NO.5.
  • Exemplary Oligonucleotides the present disclosure provides primers and probes for detection of one or more SARS CoV-2 variants. In some embodiments, the present disclosure provides primer sets for detection of a SARS CoV-2 variant that includes a forward primer, a reverse primer, a probe and optionally a blocker primer. In some embodiments, a primer set includes one or more oligonucleotides as described in Table 3 below. [0174] Table 3: Exemplary SARS CoV-2 Oligonucleotides
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes: a forward primer that comprises a sequence of SEQ ID NO.8 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.8.
  • a primer set directed to a SNV associated with amino acid position 501 e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome
  • the primer set includes: a reverse primer that comprises a sequence of SEQ ID NO.9 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.9.
  • a primer set directed to a SNV associated with amino acid position 501 e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome
  • the primer set includes: a blocker primer that comprises a sequence of SEQ ID NO.10 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.10, and a 3′ modification that blocks extension.
  • a primer set directed to a SNV associated with amino acid position 501 e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome
  • the primer set includes: a probe that comprises a sequence of SEQ ID NO.7 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.7.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes: a forward primer that comprises a sequence of SEQ ID NO.12 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.12.
  • a primer set directed to a SNV associated with amino acid position 701 e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome
  • the primer set includes: a reverse primer that comprises a sequence of SEQ ID NO.13 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.13.
  • a primer set directed to a SNV associated with amino acid position 701 e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome
  • the primer set includes: a blocker primer that comprises a sequence of SEQ ID NO.14 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.14, and a 3′ modification that blocks extension.
  • a primer set directed to a SNV associated with amino acid position 701 e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome
  • the primer set includes: a probe that comprises a sequence of SEQ ID NO.11 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.11.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes: a forward primer that comprises a sequence of SEQ ID NO.16 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.16.
  • a primer set directed to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes: a reverse primer that comprises a sequence of SEQ ID NO.17 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.17.
  • a primer set directed to a SNV associated with amino acid position 1176 e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome
  • the primer set includes: a probe that comprises a sequence of SEQ ID NO.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 6 of Example 4 below.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer at listed in Table 6 of Example 4 below.
  • the primer set further comprises a probe that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.7 or SEQ ID NO. 36.
  • the primer set further comprises a blocker primer that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.10 or SEQ ID NO.37.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 6 of Example 4 below; (ii) a probe that comprises or consists of a sequence of SEQ ID NO.7 or SEQ ID NO.36; and (iii) a blocker primer that comprises or consists of a sequence of SEQ ID NO.10 or SEQ ID NO.37.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 6 of Example 4 below.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501, where the primer set includes a forward and a reverse primer at listed in Table 6 of Example 4 below.
  • the primer set further comprises a probe that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.7 or SEQ ID NO.36.
  • the primer set further comprises a blocker primer that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.10 or SEQ ID NO.37.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 484 (e.g., a SNV at nucleic acid position 22980 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 13 of Example 6 below.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 484 (e.g., a SNV at nucleic acid position 22980 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer at listed in Table 15 of Example 6 below.
  • the primer set further comprises a probe that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs.7, 65, and 66.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 484 (e.g., a SNV at nucleic acid position 22980 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 13 of Example 6 below; and (ii) a probe that comprises or consists of a sequence of any one of SEQ ID NOs.7, 65, or 66.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 484 (e.g., a SNV at nucleic acid position 22980 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 15 of Example 6 below; and (ii) a probe that comprises or consists of a sequence of SEQ ID NOs.7, 65, or 66.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 417 (e.g., a SNV at nucleic acid position 22184 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 14 of Example 6 below.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 417 (e.g., a SNV at nucleic acid position 22184 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer at listed in Table 15 of Example 6 below.
  • the primer set further comprises a probe that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs.67-71.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 417 (e.g., a SNV at nucleic acid position 22184 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 14 of Example 6 below; and (ii) probe that comprises or consists of a sequence of any one of SEQ ID NOs.67-71.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 417 (e.g., a SNV at nucleic acid position 22184 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 15 of Example 6 below; and (ii) probe that comprises or consists of a sequence of any one of SEQ ID NOs.67-71.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes a forward primer, a reverse primer, and a probe that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 18 of Example 6 below.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes a forward primer, a reverse primer, and a probe that each comprise or consist of a sequence listed in Table 18 of Example 6 below.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes a forward primer, a reverse primer, and a probe that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 19 of Example 6 below.
  • the primer set further comprises a blocker primer that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs.14 and 90-93.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes a forward primer, a reverse primer, and a probe that each comprise or consist of a sequence listed in Table 19 of Example 6 below.
  • a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 701, where the primer set includes a forward primer that comprises or consists of a sequence of SEQ ID NO.12, a reverse primer that comprises or consists of a sequence of SEQ ID NO.13 or 87, and a probe that comprises or consists of a sequence of SEQ ID NO.11.
  • the primer set further comprises a blocker primer that comprises or consists of a sequence of any one of SEQ ID NOs.14 and 90-93.
  • kits for detect and/or identify specific mutations in a sample e.g., a SARS CoV-2 positive sample.
  • detection of one or more mutations is indicative of a particular viral variant.
  • the detection of one or more mutations is indicative of a VBM, VOI, VOC, and/or VOHC.
  • methods to detect and/or identify a SARS CoV-2 variant in a sample are provided herein.
  • the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2).
  • a SARS CoV-2 variant is alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), mu (B.1.621), and omicron (B.1.1.529), and/or a descendent lineage of any thereof.
  • a SARS CoV-2 variant is alpha (B.1.1.7), beta (B.1.351), gamma (P.1), iota (B.1.526), mu (B.1.621), and omicron (B.1.1.529, BA lineage), theta (P.3), zeta (P.2), and/or a descendent lineage of any thereof.
  • a method of the present disclosure is capable of detecting SARS CoV-2 and/or particular variants at a low concentration. In some embodiments, a method of the present disclosure is capable of detecting one or more mutations associated with a SARS CoV-2 variant with a limit of detection that is less than 50,000 viral genome copies per milliliter (cp/mL), less than 40,000 cp/mL, less than 30,000 cp/mL or less than 20 cp/m. [0191] In some embodiments, the present disclosure provides methods and/or assays for detecting and/or identifying one or more mutations associated with a SARS CoV-2 variant.
  • one or more mutations to be detected and/or identified are selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F. In some embodiments, one or more mutations to be detected and/or identified are selected from: N501Y, E484K, K417N, A701V, and V1176F.
  • the nucleotide sequence changes to be detected and/or identified are SNVs that correspond with two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. [0193] In some embodiments, one or more mutations to be detected and/or identified are selected from N501Y, A701V, and V1176F. In some embodiments, provided methods further comprise a control. In some embodiments, provided methods can detect and/or identify one or more mutations associated with a SARS CoV-2 variant and also include a positive control (e.g., amplification of another SARS CoV-2 conserved region, e.g., in the N gene and/or the ORF1ab gene).
  • a positive control e.g., amplification of another SARS CoV-2 conserved region, e.g., in the N gene and/or the ORF1ab gene.
  • provided methods detect and/or identify one or more SARS CoV-2 mutations and a control in a single sample and/or well.
  • the present disclosure provides methods and/or assays for detecting and/or identifying two or more mutations associated with a SARS CoV-2 variant.
  • two or more mutations to be detected and/or identified are selected from N501Y, A701V, and V1176F.
  • provided methods can detect and/or identify two or more mutations (e.g., SARS CoV-2 SNVs) in a single sample and/or well.
  • provided methods further comprise a control.
  • provided methods can detect and/or identify two or more mutations associated with a SARS CoV-2 variant and also include a positive control (e.g., amplification of another SARS CoV-2 conserved region, e.g., in the N gene and/or the ORF1ab gene). In some embodiments, provided methods detect and/or identify two or more SARS CoV-2 mutations and a control in a single sample and/or well. [0195] In some embodiments, the present disclosure provides methods and/or assays for detecting and/or identifying three or more mutations associated with a SARS CoV-2 variant. In some embodiments, three or more mutations to be detected and/or identified include N501Y, A701V, and V1176F.
  • provided methods can detect and/or identify three or more mutations (e.g., SARS CoV-2 SNVs) in a single sample and/or well.
  • provided methods can detect and/or identify three or more mutations associated with a SARS CoV-2 variant and also include a positive control (e.g., amplification of another SARS CoV-2 conserved region, e.g., in the N gene and/or the ORF1ab gene).
  • provided methods detect and/or identify three or more SARS CoV-2 mutations and a control in a single sample and/or well.
  • an assay or method of the present disclosure is used and/or performed in combination with whole genome sequencing.
  • a method of the present disclosure and whole genome sequencing are performed in parallel. In some embodiments, a method of the present disclosure is performed, and if a SARS CoV-2 variant is identified, then whole genome sequencing is also performed. Diagnostic Tests for SARS CoV-2 [0197] The present disclosure provides diagnostic tests for SARS CoV-2 characterized by detection of SARS CoV-2 variants using SARS CoV-2 oligonucleotides as described herein and/or according to methods as described herein. [0198] In some embodiments, methods of detecting and/or diagnosing SARS CoV-2, and particularly identifying or characterizing the SARS CoV-2 variants, as taught by the present disclosure are improved methods as compared to standard techniques.
  • methods of the present disclosure include one or more of the following benefits: improved sensitivity for identifying SARS CoV-2 variants, improved specificity for identifying SARS CoV- 2 variants, improved accuracy for identifying SARS CoV-2 variants, reduced time to diagnosis for SARS CoV-2 variants, and/or reduced cost of screening patients for SARS CoV-2 variants.
  • methods of detecting and/or diagnosing SARS CoV-2 as taught by the present disclosure provide the advantage of an early screen for the presence of particular SARS CoV-2 variants in a sample.
  • technology disclosed herein e.g., disclosed oligonucleotides and/or methods
  • a diagnostic test as taught by the present disclosure can assist in the detection or diagnosis of SARS CoV-2 in a subject.
  • technology disclosed herein is used for diagnosing a subject with infection with a particular SARS CoV-2 variant.
  • a diagnostic test as taught by the present disclosure detects whether one or more SARS CoV-2 mutations are present in a sample obtained from a subject.
  • a sample obtained from a subject comprises a bodily fluid.
  • a bodily fluid is or comprises saliva, respiratory secretion (e.g., from a nasal swab), blood, and/or plasma.
  • a sample of respiratory secretion obtained from a subject for use in accordance with methods described herein is obtained by an anterior nares (nasal) swab, a mid-turbinate nasal swab, a nasopharyngeal swab, and/or an oropharyngeal swab.
  • a sample of saliva from a subject for use in accordance with methods described herein is obtained by having a subject spit into a receptacle (e.g., a tube).
  • a sample for use in the accordance with provided technologies is a sample of RNA isolated from a subject.
  • a sample is a sample of RNA isolated from a nasopharyngeal and/or oropharyngeal swab from a subject.
  • a subject is diagnosed with an infection with a particular SARS CoV-2 variant using a method described herein and then treated for COVID-19.
  • the treatment for COVID-19 may be selected and/or adjusted based on the SARS CoV-2 variant detected in a subject.
  • Methods of Use and Treatment [0204]
  • the present disclosure provides methods of and/or uses for identifying a subject for treatment, wherein the subject has been diagnosed infection with a particular SARS CoV-2 variant.
  • the treatment differs based on what particular SARS CoV-2 variant.
  • identifying a subject for treatment where the subject has been diagnosed infection with a SARS CoV-2 variant, said method or use comprising (i) identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject, (ii) determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified, and (iii) administering therapy according the determined SARS CoV-2 variant strain(s).
  • nucleotide sequence changes e.g., SNVs
  • the nucleotide sequence changes (e.g., SNVs) identified correspond with mutations selected from: one or more mutations selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F.
  • the nucleotide sequence changes are SNVs that correspond with mutations selected from: N501Y, E484K, K417N, A701V, and V1176F.
  • the nucleotide sequence changes are SNVs selected from: A23063T, A22980G, G22184T, C23635T, and G25088T. In some embodiments, the nucleotide sequence changes are SNVs that correspond with two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are two or more SNVs selected from: A23063T, A22980G, G22184T, C23635T, and G25088T.
  • the nucleotide sequence changes are SNVs that correspond to one or more mutations to be detected are selected from N501Y, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs selected from: A23063T, C23635T, and G25088T. In some embodiments, the nucleotide sequence changes are SNVs that correspond to each of the mutations N501Y, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond to each of A23063T, C23635T, and G25088T relative to a reference SARS CoV-2 genome.
  • an assay or method as described herein for identifying a subject infected with a SARS CoV-2 variant comprises identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject.
  • said assay or method for use further comprises determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified.
  • the method and/or use further comprises determining the predominant SARS CoV-2 variant strain(s) based on the nucleotide sequence changes identified in the samples from the population of subjects.
  • the nucleotide sequence changes (e.g., SNVs) identified correspond with mutations selected from: one or more mutations selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F.
  • the nucleotide sequence changes are SNVs that correspond with mutations selected from: N501Y, E484K, K417N, A701V, and V1176F.
  • the nucleotide sequence changes are SNVs that correspond with two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond to one or more mutations to be detected are selected from N501Y, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond to each of the mutations N501Y, A701V, and V1176F.
  • the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2).
  • a SARS CoV-2 variant is alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), mu (B.1.621), and omicron (B.1.1.529), and/or a descendent lineage of any thereof.
  • a SARS CoV-2 variant is alpha (B.1.1.7), beta (B.1.351), gamma (P.1), iota (B.1.526), mu (B.1.621), and omicron (B.1.1.529), theta (P.3), zeta (P.2), and/or a descendent lineage of any thereof.
  • kits comprising one or more oligonucleotides of the present disclosure and instructions for use (e.g., diagnostic use).
  • a kit of the disclosure is used for an in vitro diagnostic assay to diagnose SARS CoV-2 infection (e.g., with a particular variant).
  • a kit of the present disclosure comprises one or more variant primers described herein, along with instructions for use.
  • a kit includes one or more primer sets described herein.
  • a kit includes one or more probes.
  • a kit of the present disclosure comprises a primer set for detecting one or more variants of SARS CoV-2.
  • a kit of the present disclosure comprises a primer set for detecting one or more variants of SARS CoV-2 selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, a kit of the present disclosure further comprises a primer set for detecting a conserved region of SARS CoV-2 (i.e., a positive control). [0216] In some embodiments, a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 N501Y variant. In some embodiments, the kit comprises a variant primer that targets the N501Y genomic region and a complementary primer. In some embodiments, the kit comprises a blocker primer that blocks amplification of a N501 wt sequence.
  • kits of the present disclosure comprises a primer set for detecting a SARS CoV-2 E484K variant. In some embodiments, the kit comprises a variant primer that targets the E484K genomic region and a complementary primer. In some embodiments, the kit further comprises a probe. [0218] In some embodiments, a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 K417N variant. In some embodiments, the kit comprises a variant primer that targets the K417N genomic region and a complementary primer. In some embodiments, the kit further comprises a probe.
  • a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 A701V variant.
  • the kit comprises a variant primer that targets the A701V genomic region and a complementary primer.
  • the kit comprises a blocker primer that blocks amplification of a A701 wt sequence.
  • the kit further comprises a probe.
  • a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 V1176F variant.
  • the kit comprises a variant primer that targets the V1176F genomic region and a complementary primer.
  • the kit further comprises a probe.
  • kits of the disclosure can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and/or an agent for treating a condition or disorder described herein (e.g., COVID-19).
  • a kit further comprises a ROX reference dye.
  • a ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization in a real-time qPCR assay.
  • a ROX reference dye is present in an amount to generate a fluorescent signal in a range from about 50,000 to about 500,000.
  • kits for use in accordance with the present disclosure may include, a reference or control sample(s), instructions for processing samples, performing tests on samples, instructions for interpreting the results, buffers and/or other reagents necessary for performing tests.
  • kits for detection of one or more SARS CoV- 2 variants in a sample where the results of the kit are used to inform treatment.
  • a kit provided herein detects SNVs N501Y or E484K + control and K417N, A701V, V1176F + control.
  • Such a kit can detect five SARS CoV-2 variants B.1.1.7, B.1.525, B.1.351, B.1.526, and P.1 and differentiate four of these variants.
  • a SARS CoV-2 variant primer comprising: (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; and (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of a target nucleotide sequence, which is 3' of the first portion of the target nucleotide sequence and comprises an interrogating nucleotide position, wherein the interrogating nucleotide position corresponds to a SARS CoV-2 variant nucleotide.
  • Embodiment 2 The variant primer of embodiment 1, wherein the anchor is 15 to 40 nucleotides long, optionally wherein the anchor is 20 to 35 nucleotides long.
  • Embodiment 3 The variant primer of embodiment 1 or 2, wherein the bridge is 4 to 10 nucleotides long, optionally wherein the bridge is 6 to 8 nucleotides long.
  • Embodiment 4 The variant primer of any one of embodiments 1 to 3, wherein the foot is 6 to 8 nucleotides long, optionally wherein the foot is 7 nucleotides long.
  • Embodiment 6. The variant primer of any one of embodiments 1 to 5, wherein the primer is DNA.
  • Embodiment 7. The variant primer of any one of embodiments 1 to 6, wherein the primer comprises a label or a tag.
  • Embodiment 8. The variant primer of any one of embodiments 1 to 7, wherein the interrogating nucleotide is a nucleotide that corresponds to an amino acid substitution selected from: N501Y, E484K, K417N, A701V, and V1176F.
  • the variant primer of any one of embodiments 1 to 8, wherein the target nucleotide sequence is a SARS CoV-2 genomic RNA sequence.
  • Embodiment 10 The variant primer of any one of embodiments 1 to 8, wherein the target nucleotide sequence corresponds to a SARS CoV-2 S gene sequence or portion thereof.
  • Embodiment 11 The variant primer of any one of embodiments 1 to 8, wherein the target nucleotide sequence corresponds to a SARS CoV-2 S gene sequence or portion thereof.
  • Embodiment 12 The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha, beta, eta, gamma, iota, mu, omicron, theta, zeta, and C.1.2.
  • Embodiment 13 Embodiment 13.
  • Embodiment 14 The variant primer of embodiment 13, wherein the variant nucleotide sequence comprises a sequence that corresponds to an N501Y mutation.
  • Embodiment 15 The variant primer of embodiment 13 or 14, wherein the variant nucleotide sequence comprises A23063T nucleotide substitution.
  • Embodiment 17 The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant or iota variant.
  • Embodiment 18 The variant primer of embodiment 17, wherein the variant nucleotide sequence comprises a sequence that corresponds to an A701V mutation.
  • Embodiment 19 Embodiment 19.
  • Embodiment 20 The variant primer of any one of embodiments 1 to 10 or 17 to 19, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86.
  • Embodiment 21 The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a gamma variant, zeta variant, or theta variant.
  • Embodiment 22 Embodiment 22.
  • Embodiment 23 The variant primer of embodiment 21 or 22, wherein the variant nucleotide sequence comprises a G25088T nucleotide substitution.
  • Embodiment 24 The variant primer of any one of embodiments 1 to 10 or 21 to 23, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16, 72-74, and 78-80.
  • Embodiment 25 Embodiment 25.
  • Embodiment 26 The variant primer of embodiment 25, wherein the variant nucleotide sequence comprises a sequence that corresponds to an E484K mutation.
  • Embodiment 27 The variant primer of embodiment 25 or 26, wherein the variant nucleotide sequence comprises a A22980G nucleotide substitution.
  • Embodiment 28 Embodiment 28.
  • Embodiment 29 The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, mu variant, or delta variant.
  • Embodiment 30 The variant primer of embodiment 29, wherein the variant nucleotide sequence comprises a sequence that corresponds to a K417N mutation.
  • Embodiment 31 Embodiment 31.
  • Embodiment 32 The variant primer of any one of embodiments 1 to 10 or 29 to 31, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55 and 62-64.
  • Embodiment 33 A primer pair comprising: the variant primer of any one of embodiments 1 to 32, and a complementary primer comprising or consisting of a nucleotide sequence that is complementary to a portion of a complementary strand sequence of the target nucleotide sequence.
  • Embodiment 34 Embodiment 34.
  • a primer pair comprising: the variant primer of embodiment 16, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-28.
  • Embodiment 35 A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24.
  • Embodiment 36 Embodiment 36.
  • a primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 20-22, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 25-28.
  • Embodiment 37 A primer pair comprising: the variant primer of embodiment 20, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88.
  • a primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88.
  • Embodiment 39. A primer pair comprising: the variant primer of embodiment 24, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17 and 74-77.
  • Embodiment 40 Embodiment 40.
  • a primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16 and 72-73, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17, 76, and 77.
  • a primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 78-80, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 74-75.
  • a primer pair comprising: the variant primer of embodiment 28, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40, 45-47, and 49.
  • Embodiment 43 A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38 and 41-44, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40 and 45-46.
  • Embodiment 44 Embodiment 44.
  • a primer pair comprising: a variant primer comprises or consists of a sequence according to SEQ ID NO: 48 or 50, and a complementary primer comprising or consisting of a nucleotide sequence according to SEQ ID NO: 47 or 49.
  • Embodiment 45 A primer pair comprising: the variant primer of embodiment 32, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61 and 56-58.
  • Embodiment 46 Embodiment 46.
  • a primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 56-58.
  • a primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 62-64, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61.
  • a blocker primer comprising (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence of SARS CoV-2; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of the target nucleotide sequence of SARS CoV-2, which is 3' of the first portion of the target nucleotide sequence and comprises a wild type nucleotide at an interrogating nucleotide position; and (iv) a modification at its 3'-end to prevent extension from occurring.
  • Embodiment 49 The blocker primer of embodiment 48, wherein the wild type nucleotide at an interrogating nucleotide position is the nucleotide at that corresponding position of the genome of the Wuhan-Hu1 strain or USA-WA1/2020 strain.
  • Embodiment 50 The blocker primer of embodiment 48 or 49, wherein the target nucleotide sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome but not a wild-type SARS CoV-2 genome, wherein the SARS CoV-2 variant is an alpha variant, variant, beta variant, gamma variant, mu variant, theta variant, C.1.2 variant, or omicron variant.
  • Embodiment 51 Embodiment 51.
  • the blocker primer of any one of embodiments 48 to 50, wherein the modification that prevents extension is or comprises: a PO 4 modification, an inverted 3'-3' linkage, an inverted dT, a C3 modification, or an amino-C7 modification.
  • Embodiment 52 The blocker primer of any one of embodiments 48 to 51, wherein the modification that prevents extension is or comprises: a PO 4 modification or a C3 modification.
  • Embodiment 53 The blocker primer of any one of embodiments 48 to 52, wherein the anchor is 15 to 40 nucleotides long, optionally wherein the anchor is 20 to 35 nucleotides long.
  • Embodiment 54 Embodiment 54.
  • Embodiment 55 The blocker primer of any one of embodiments 48 to 54, wherein the foot is 6 to 8 nucleotides long, optionally wherein the foot is 7 nucleotides long.
  • Embodiment 56 The blocker primer of any one of embodiments 48 to 55, wherein the primer is 25 to 55 nucleotides long, optionally wherein the primer is 30 to 40 nucleotides long.
  • Embodiment 57 The blocker primer of any one of embodiments 48 to 56, wherein the primer is DNA.
  • Embodiment 58 The blocker primer of any one of embodiments 48 to 57, wherein the wild type nucleotide at an interrogating nucleotide position is a nucleotide that translates to a wild-type amino acid at a position selected from: N501, E484, K417, A701, and V1176.
  • Embodiment 59 The blocker primer of any one of embodiments 48 to 58, wherein the interrogating nucleotide position is a nucleotide that translates to a wild-type amino acid at a position N501.
  • Embodiment 60 Embodiment 60.
  • Embodiment 61 The blocker primer of any one of embodiments 48 to 58, wherein the interrogating nucleotide position is a nucleotide that translates to a wild-type amino acid at a position A701.
  • Embodiment 62 The blocker primer of embodiment 59, wherein the blocker primer comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93.
  • Embodiment 63 A primer set comprising: the variant primer of any one of embodiments 1 to 32, and a probe.
  • Embodiment 64 A primer set comprising: the primer pair of any one of embodiments 33 to 47, and a probe.
  • Embodiment 65 The primer set of embodiment 63 or 64, wherein the probe comprises or consists of a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89.
  • Embodiment 66 A primer set comprising: the primer pair of any one of embodiments 34-36, and a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 7 or 36.
  • Embodiment 67 Embodiment 67.
  • a primer set comprising: the primer pair of any one of embodiments 37-38, and a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 11 or 89.
  • Embodiment 68 A primer set comprising: the primer pair of any one of embodiments 39-41, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 15 or 81-83.
  • Embodiment 69 A primer set comprising: the primer pair of any one of embodiments 42-44, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 7 and 65-66.
  • Embodiment 70 A primer set comprising: the primer pair of any one of embodiments 42-44, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 7 and 65-66.
  • a primer set comprising: the primer pair of any one of embodiments 45-47, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 67-71.
  • Embodiment 71 A primer set comprising: the variant primer of any one of embodiments 1 to 32, and the blocker primer of any one of embodiments 48 to 62.
  • Embodiment 72 A primer set comprising: the primer pair of any one of embodiments 33 to 47, and the blocker primer of any one of embodiments 48 to 62.
  • Embodiment 73 Embodiment 73.
  • Embodiment 74 A primer set comprising: the variant primer of embodiment 16, and a blocker primer comprises or consists of a sequence according to SEQ ID NO: 10 or 37.
  • Embodiment 75 A primer set comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and a blocker primer comprises or consists of a sequence according to SEQ ID NO: 10 or 37.
  • Embodiment 76 Embodiment 76.
  • the primer set of embodiments 74 or 75 further comprising: a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24.
  • Embodiment 77 The primer set of any one of embodiments 74 to 76, wherein the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to a nucleotide of N501Y in the variant primer and a nucleotide that translates to a wild-type amino acid at a position N501 in the blocker primer.
  • Embodiment 78 Embodiment 78.
  • a primer set comprising: the variant primer of embodiment 20, and a blocker primer comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93.
  • a primer set comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and a blocker primer comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93.
  • Embodiment 80 The primer set of embodiments 74 or 75, further comprising: a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88.
  • Embodiment 81 The primer set of any one of embodiments 74 to 76, wherein the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to a nucleotide of A701V in the variant primer and a nucleotide that translates to a wild-type amino acid at a position A701 in the blocker primer.
  • Embodiment 82 A mixture comprising: the variant primer of any one of embodiments 1 to 32, and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence.
  • Embodiment 83 Embodiment 83.
  • Embodiment 85 A method comprising: contacting a sample from a subject with the variant primer of any one of embodiments 1 to 32, the primer pair of any one of embodiments 33 to 47, or the primer set of any one of embodiments 63 to 81; and amplifying a target nucleotide sequence from the sample.
  • Embodiment 86 The method of embodiment 85, wherein the target nucleotide sequence is a SARS CoV-2 nucleotide sequence.
  • Embodiment 87 The method of embodiment 85 or 86, wherein the method is to detect and/or identify a SARS CoV-2 variant in a sample comprising SARS CoV-2 nucleic acid.
  • Embodiment 88 Embodiment 88.
  • SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2). [0320] Embodiment 89.
  • Embodiment 90 The method of embodiment 89, wherein the one or more mutations are selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F.
  • Embodiment 91 Embodiment 91.
  • Embodiment 92 The method of embodiment 91, wherein the one or more mutations comprise three or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F.
  • Embodiment 93 The method of embodiment 89, wherein the one or more mutations are selected from: N501Y, A701V, and V1176F.
  • Embodiment 94 Embodiment 94.
  • Embodiment 95 The method of any one of embodiments 87 to 94, wherein the SARS CoV-2 in the sample is present in an amount that is less than 50,000 viral genome copies per milliliter (cp/mL), less than 40,000 cp/mL, less than 30,000 cp/mL or less than 20 cp/mL.
  • Embodiment 96 The method of any one of embodiments 85 to 95, wherein the method further comprises contacting the sample from the subject with a positive control primer set.
  • Embodiment 97 Embodiment 97.
  • Embodiment 98 The method of any one of embodiments 85 to 97, wherein the method further comprises performing whole genome sequencing on the sample. [0330] Embodiment 99.
  • a method of identifying a subject for treatment comprising (i) identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject, (ii) determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified, and (iii) administering therapy according to the determined SARS CoV-2 variant strain(s), wherein the step of identifying one or more nucleotide sequence changes comprises contacting the sample with the variant primer of any one of embodiments 1 to 32, the primer pair of any one of embodiments 33 to 47, or the primer set of any one of embodiments 63 to 81.
  • SNVs nucleotide sequence changes
  • a method of identifying a predominant SARS CoV-2 variant in a population of subjects comprising identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in samples from the population of subjects, wherein the identifying comprises contacting the samples with the variant primer of any one of embodiments 1 to 32, the primer pair of any one of embodiments 33 to 47, or the primer set of any one of embodiments 63 to 81.
  • Embodiment 101 comprising: the variant primer of any one of embodiments 1 to 32, the primer pair of any one of embodiments 33 to 47, or the primer set of any one of embodiments 63 to 81; and [0333] instructions for use.
  • Embodiment 102 The kit of embodiment 101, further comprising a positive control primer set.
  • Embodiment 103 The kit of embodiment 102, wherein the positive control primer set targets a sequence in the SARS CoV-2 Nucleocapsid (N) gene and/or ORF1ab gene.
  • EXEMPLIFICATION [0336] The present examples together describe development and characterization of assays for detecting SARS CoV-2 variants, e.g., particular sequence changes associated with such variants, including single nucleotide variants (SNVs). The following examples will describe the design, screening, and characterization of exemplary SARS CoV-2 SNVs.
  • SARS CoV-2 variants can include multiple mutations.
  • the B.1.1.7 (alpha) variant is defined by 23 mutations from the original SARS CoV-2 strain; these mutations include 4 deletions, 6 synonymous mutations, and 13 non-synonymous mutations.
  • the non- synonymous mutations include, among others, a series of mutations in the spike protein and a stop codon in ORF8, which are summarized in Table 4 below.
  • There are 6 synonymous mutations with 5 in ORF1ab (C913T, C5986T, C14676T, C15279T, C16175T), and one in the M gene (T26801C). This is an unusually large number of mutations in a single cluster.
  • Table 4 Lineage-defining protein altering mutations defining the B1.1.7 (alpha) SARS CoV-2 variant
  • a two-target assay may have better specificity for B1.1.7 and other variants (e.g., B1.351 in South Africa and P.1 in Brazil).
  • a two target assay for detecting B1.1.7 variant can target an SGF 3675-3677 deletion and an N501Y mutation.
  • An exemplary schematic for primer design for such a two target assay is provided in FIG.5 [0343] The N501Y single nucleotide variation (SNV) or mutation was selected as a target for assay development.
  • Example 2 Selecting SNVs for Identifying Other SARS CoV-2 Variants
  • This example describes selection of other mutations (e.g., SNVs) for detecting SARS CoV-2 variants.
  • SNVs e.g., SNVs
  • a listing of exemplary SARS CoV-2 mutations is provided in the Table # below. This table also describes the effects associated with these SNVs, identifies certain variants associated with these SNVs, and also proposes assay options.
  • Table 5 Exemplary SARS CoV-2 mutations and associated variants
  • Each of the exemplary mutations listed in Table 5 above is located in the SARS CoV-2 spike protein encoding region (S gene), as illustrated in FIG. 1C.
  • Option 3 Tube 1 N501Y or E484K + control and Tube 2 K417N, A701V, V1176F + control [0352]
  • four variants will be detected (B.1.1.7, B.1.351, B.1.526, and P.1) and 4 variants differentiated using N501Y in Tube 1.
  • Four variants will be detected (B.1.525, B.1.351, B.1.526, and P.1) and 4 variants differentiated using E484K in Tube 1.
  • the mutation N501Y was selected as an initial mutation to move forward with for development of an exemplary SARS CoV-2 variant detection assay.
  • Example 3 Primer Set Design
  • This example describes the design of primer set to achieve a highly selective and sensitive assay for detection of particular SAR CoV-2 mutation, e.g., a N501Y SNV mutation, among others.
  • Such a primer set includes a variant primer with a bubbled primer design.
  • a blocker primer directed to a wild- type sequence was also used.
  • an exemplary N501Y bubbled primer includes, in order from 5′ to 3′, an anchor sequence, a bubble or bridge sequence, and a foot sequence.
  • the foot sequence of the exemplary N501Y primer overlaps with (i.e., includes a sequence that is complementary to) the N501Y mutation (represented by “X” in the schematic).
  • the foot sequence was designed to be short, such that a single mismatch at the terminal 3′ nucleotide destabilizes primer binding and prevent extension, thereby enabling discrimination of different alleles (e.g., wild type and N501Y).
  • the exemplary N501Y bubbled primer was designed as part of a primer set that also includes a wild-type (WT) blocker primer, a reverse primer, and a probe, for example as depicted in FIG. 4.
  • WT wild-type
  • the exemplary WT blocker primer is capable of hybridizing with (e.g., is complementary to) a sequence encompassing the position to be interrogated, in this case N501.
  • the exemplary WT blocker primer also comprises a PO 4 modification at its 3'-end to prevent extension from occurring.
  • the present disclosure recognizes that such a WT blocker primer can compete with a variant primer and suppress non-specific amplification of a wild-type allele by a variant primer.
  • a primer set can also include a third primer, which is used together with a WT blocker primer and a variant primer.
  • a third primer can be a reverse primer.
  • a probe can be used, which is capable of hybridizing with (e.g., is complementary to) a sequence amplified using a primer set as shown in FIG.4.
  • FIG.5 An exemplary schematic for primer design for such a two target assay is provided in FIG.5
  • a number of bubbled primer designs were tested and screened that target the SGF 3675-3677 deletion in ORF1ab.
  • 8 bubbled primer combinations specific for ORF1ab SGF 3675-3677 deletion were screening using EVA green and RNA transcript at 1E+05 copies/reaction (data not shown), as described for N501Y screening above, 4 of which were selected to screen with probes.
  • bubbled primer/probe combinations directed to SGF 3675-3677 deletion in ORF1ab were not selective to mutant template.
  • the examples below describe the development and characterization of assays for detection of different SARS CoV-2 SNVs.
  • Example 4 Development and Characterization of Exemplary Assay for Detection of N501Y
  • This example describes development and characterization of an exemplary assay for detecting a SARS CoV-2 N501Y mutation.
  • a schematic flow chart overview of a process for a characterizing this exemplary SNV (N501Y) is provided in FIG.6.
  • In vitro transcription [0362] RNA transcripts of wild type and mutant variant sequences were generated from double-stranded gBlock templates obtained from IDT. RNA transcription was performed using a T7 in vitro transcription kit from gBlock templates.
  • Bubbled primers were screened using EVAgreen chemistry for RT-PCR to determine if bubbled primers generated signal from mutant templates. Specifically, 32 primer combinations were screened with N501Y mutant RNA transcript in decreasing amounts: 100,000 copies/reaction, 25,000 copies/reaction, 6,250 copies/reaction, and no template control (NTC). [0369] Table 7: N501Y Primer Combinations and Amplification Results
  • N501Y mutant RNA transcript lysed inactivated wild-type (WT) virus and combined N501Y mutant and WT template (1E+05 copies/reaction each). Lower amounts of N501Y mutant RNA template were also screened.
  • WT wild-type
  • Each of the above primer combinations were screened with the following probes: [0374] SNV-Probe 2-FAM TGGTGTTGGTTACCAACCATACAGAGT (SEQ ID NO.7) [0375] SNV-Probe 3-FAM TGGTTACCAACCATACAGAGTAGTAGTACT (SEQ ID NO.36) [0376] Primer and probe combinations tested were able to amplify N501Y mutant template.
  • Blocker Screening [0378] Primer/probe combinations were screened in combination with 6 different blocker primers using the following templates: N501Y mutant RNA transcript, lysed inactivated wild-type (WT) virus and combined N501Y mutant and WT template (1E+05 copies/reaction each). Blocker primers that are specific for a wild-type sequence were screened; screened blocker primers were forward primers that include a PO 4 modification at the 3'-end to prevent extension from occurring. Blocker primers were also tested at different concentrations (0 nM, 10 nM, 50 nM, 100 nM, 150 nM, and 300 nM). [0379] Exemplary results are depicted in FIG.8.
  • Wild-type blocker primers were able to block amplification of wild-type template in a concentration dependent manner.
  • Exemplary blocker primers were found to be effective at concentrations of 100 nM and above.
  • the following two primer/blocker/probe combinations: SNV-MutFor5/WTFor5-PO 4 /Rev4/Probe2 and SNV- MutFor5/WTFor6-PO 4 /Rev6/Probe2 were selected to move forward, using blocker primer at a concentration of 100 nM.
  • This example describes exemplary primer sets for detection of this SNV that include bubbled primers, a probe, a complementary primer, and a blocker primer, as well as describes characteristics thereof.
  • Example 5 Selection and Characterization of an Exemplary Control [0385]
  • This example describes development and characterization of exemplary primer sets for detecting a SARS CoV-2 virus for use as a positive control with technologies for detecting SARS CoV-2 variants described herein.
  • Exemplary primer sets for detecting SARS CoV-2 as a control are targeted to the Nucleocapsid (N) gene or ORF1ab gene.
  • the present example characterized primer sets of the FTD SARS CoV-2 Assay that target the N gene and ORF1ab gene as a control.
  • the exemplary positive control primers listed above were tested in combination with primers for detecting an exemplary variant (N501Y SNV) with different combinations of probes. Specifically, combinations of probes were tests for assays using N501Y SNV primers described above: SNV-Mut For 5 (SEQ ID NO.8), SNV-Rev 4 (SEQ ID NO.9), and SNV-WT For 5-PO 4 (SEQ ID NO.10), SNV-Probe 2 (SEQ ID NO.7) in combination with the N gene and ORF1 gene primers listed in Table 8 above. Specifically, the following 8 primer/probe combinations were tested as outlined in Table 9 below. [0389] Table 9 - Probe Combinations with Exemplary N501Y + Control Multiplex Assay
  • the exemplary N501Y and N gene primer/probe combinations were tested with the following templates: [0391]
  • the exemplary N501Y and ORF1ab gene primer/probe combinations were tested with the following templates: [0392] Each combinations described above were tested using templates at the following concentrations: 100,000 copies/reaction, 25,000 copies/reaction, 6,250 copies/reaction, and no template control (NTC). Exemplary results are provided in the tables below at 100,000 copies/reaction: [0393] Table 10: Difference of Expression N501Y and N gene Probe Combinations
  • Table 11 Difference of Expression N501Y and ORF1 gene Probe Combinations
  • Multiplex assays with primer/probe combinations that generated a difference of expression ⁇ CT within +/- 1 when compared with singleplex assay were selected. Accordingly, there were successful multiplex probe combinations for both N gene and ORF1 gene positive controls.
  • the ORF1 gene positive control had two different probe combinations that showed the ⁇ CT within +/- 1 CT difference (SP2_FAM/FTD-ORF_YY and SP2_CIV/FTD-ORF_FAM).
  • ORF1ab was selected as a positive control for a multiplex assay in combination with a N501Y SNV assay for further analysis.
  • blocker concentration were tested from +20% to -20% of control concentration for the exemplary multiplex assay using N501Y SNV primers: SNV-Mut For 5 (SEQ ID NO.8), SNV-Rev 4 (SEQ ID NO.9), and SNV-WT For 5-PO 4 (SEQ ID NO.10), SNV- Probe 2_FAM (SEQ ID NO. 7) and ORF1ab primers: FTD_ORF1_For (SEQ ID NO. 4), FTD_ORF1_Rev (SEQ ID NO. 6), and FTD-ORF_Probe_YY (SEQ ID NO. 5).
  • Example 6 Design and Characterization of Exemplary Assays for Detection of SNVs K417N, E484K, A701V and V1176F
  • This example describes development and characterization of exemplary assays for detecting SARS CoV-2 SNVs K417N, E484K, A701V and V1176F. The overview of the process for each of these was similar to that described in Example 4 and FIG. 6 above for the N501Y mutation.
  • RNA transcripts of wild type and mutant variant sequences were generated from double-stranded gBlock templates with the corresponding sequence as described in Example 4 above.
  • Bubbled Primer Screening [0408] The following variant primer pairs with a bubbled primer were screened for their ability to specifically detect mutant templates of E484K and K417N (bold indicates interrogated nucleotide), in Table 13 and Table 14, respectively. [0409] Table 13: Exemplary primers targeting a sequence that corresponds to SNV E484K [0410] Table 14: Exemplary primers targeting a sequence that corresponds to SNV K417N
  • RNA transcripts covering E484K and SA417 mutations and wild-type sequences were used as templates for screening. Bubbled primers were screened by using EVAgreen chemistry for RT-PCR to determine if bubbled primers generated signal from mutant template. We determined if bubbled primers generated signal from mutant templates. [0412] Specifically, 24 primer combinations of the above primers were screened for each of the E484K and K417N mutations. The linearity and PCR efficiency were also evaluated for selected primer combinations to move forward with for probe screening (data not shown). [0413] The following primers combinations were able to robustly amplify the E484K and K417N mutations.
  • Table 15 Primer Combinations for Amplification of E484K and K417N (SA417) [0415] Accordingly, this experiment demonstrates the successful design and screening of multiple bubbled primers that are able to specifically detect mutant templates of E484K and SA417.
  • Probe Screening [0417] Exemplary primer sets for detecting E484K, SA417, BZ1176 and SA701 SNVs were screened with different probes. Table # below provides probe sequences for E484K and K417N mutations, and primer and probe sequences for BZ1176 and SA701 mutations.
  • Table 16 Exemplary probe sequences for use in a primer set targeting a sequence that corresponds to a SNV associated with amino acid position 484 [0419]
  • Table 17 Exemplary probe sequences for use in a primer set targeting a sequence that corresponds to a SNV associated with amino acid position 417 [0420]
  • Table 18 Exemplary primer and probe sequences for use in a primer set targeting a sequence that corresponds to a SNV associated with amino acid position 1176
  • Table 19 Exemplary primer and probe sequences for use in a primer set targeting a sequence that corresponds to a SNV associated with amino acid position 701
  • RNA transcripts covering sequences corresponding to mutations E484K, SA417, BZ1176 and SA701 were used as template for screening and WT lysate was used to determine the specificity of assays to detect mutations.
  • each of the primer/probe combinations described Table 20 below were tested using templates at the following concentrations: 100,000 copies/reaction, 25,000 copies/reaction, 6,250 copies/reaction, and no template control (NTC).
  • Table 20 Exemplary primer/probe combinations:
  • Primer/probe combinations listed above were screened. Exemplary results are depicted in FIG.10. [0425] Each of the exemplary primer/probe combinations for E484K tested were able to amplify the E484K sequence; moreover, this amplification was highly specific as the wild-type sequence was not amplified in any of these combinations.
  • primer sets for the E484K mutation were selected for continued evaluation and characterization: 484-Mut For1/484- Rev3/Probe 3 (SEQ ID NO 38 / 45 / 66), 484-Mut For2/484-Rev3/Probe 2 (SEQ ID NO 41 / 45 / 65), and 484-Mut For3/484-Rev3/Probe 3 (SEQ ID NO 42 / 45 / 66).
  • each of the exemplary primer/probe combinations for SA K417N tested were able to amplify the SA K417N mutant sequence and this was also highly specific.
  • BZ1176-Mut F2/BZ1176-Rev2/BZ1176-Probe1 SEQ ID NO 16 / 17 / 15
  • BZ1176-Mut F2/BZ1176-Rev2/BZ1176-Probe2 SEQ ID NO 16 / 17 / 81
  • BZ1176- Mut F3/BZ1176-Rev1/BZ1176-Probe2 SEQ ID NO 73 / 76 / 81
  • BZ1176-Mut F3/BZ1176- Rev2/BZ1176-Probe1 SEQ ID NO 73 / 17 / 15
  • the exemplary primer/probe combinations for SA A701V listed above had mixed results for amplifying mutant sequence. Also, those sequences that amplified mutant sequence also amplified wild-type sequence. Accordingly, a wild-type blocker primer was employed to improve reaction specificity for amplification of the SA A701V mutation.
  • the following primer sets for the A701V mutation were selected for continued evaluation and characterization: SA701- Mut For5/SA701-Rev1/SA701-Probe1 (SEQ ID NO 12 / 13 / 11) and SA701-Mut For5/SA701- Rev4/SA701-Probe1 (SEQ ID NO 12 / 87 / 11).
  • Blockers for SA701 were screened with bubbled primers. RNA transcripts covering the SA701 mutation and WT lysate were used as templates to assay the blocker efficacy.
  • the primer/probe combinations of: SA701-Mut For5/SA701- Rev1/SA701-Probe1 (SEQ ID NO 12 / 13 / 11) and SA701-Mut For5/SA701-Rev4/SA701-Probe1 (SEQ ID NO 12 / 87 / 11) were screened in combination with one of each of the five following different blocker primers: [0432] SA701-WT For 1-PO 4 (SEQ ID NO.14) TCAATCCATCATTGCCTACACTACCATGGTTGGTGC (Sense) [0433] SA701-WT For 2-PO 4 (SEQ ID NO.90) CAATCCATCATTGCCTACACTACCATGGTTGGTGC (Sense) [0434
  • Example 7 Development and Characterization of an Exemplary Multiplex Assay Targeting N501Y, SA701, and V1176F [0439] This example describes development and characterization of an exemplary multiplex assay for detecting SARS CoV-2 N501Y, SA701 and BZ1176 mutations.
  • RNA transcripts covering these mutations and wild-type sequences were used as templates for screening, using template in decreasing amounts: 100,000 copies/reaction, 25,000 copies/reaction, 6,250 copies/reaction, and no template control (NTC).
  • NTC no template control
  • the present disclosure encompassed a recognition that a multiplex assay should generate a change in expression ( ⁇ CT) within +/- 1 when compared with the corresponding singleplex assays. Both of combination 5 and combination 6 generated a ⁇ CT within +/- 1 when compared with singlepex assays. Exemplary results are depicted in FIG. 12A and FIG.
  • Blockers As noted above, detection of the N501Y and SA701 mutations in an exemplary multiplex assay each included blocker primers.
  • a C3 modification was evaluated in the context of a blocker. It was found that a blocker with a C3 modification performed similarly to a PO 4 modification for both N501Y and SA701 mutations in the context of the exemplary multiplex assay.
  • LoD limit of detection
  • cp/mL copies per milliliter
  • CI confidence interval
  • this example describes the successful design and optimization of an exemplary multiplex assay that uses bubbled primer technology to detect multiple different SARS CoV-2 mutations and a positive control.
  • EQUIVALENTS [0459] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

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Abstract

The present disclosure describes the design and characterization of genomic assays for detecting SARS CoV-2 variants. The present disclosure provides compositions and methods associated with such assays.

Description

COMPOSITIONS AND METHODS FOR DETECTION OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 VARIANTS BACKGROUND [0001] Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is the virus that causes COVID-19 (coronavirus disease 2019), a respiratory illness. SARS CoV-2 has also been referred to as 2019 novel coronavirus (2019-nCoV). SARS CoV-2, and associated COVID-19, were declared a Public Health Emergency of International Concern in January 2020 by the World Health Organization, and a pandemic in March 2020. Detection, diagnosis, and treatment of SARS CoV-2 and viral variants thereof are important for addressing worldwide public health concerns. SUMMARY [0002] The current disclosure describes, among other things, compositions, assays, and methods for detecting variants of a SARS CoV-2 virus. The present disclosure encompasses a recognition that different SARS CoV-2 viral variants can be identified by particular sequence mutations (e.g., single nucleotide variants (SNVs)), or combinations thereof. The present disclosure provides the insight that a bubbled primer can be used to specifically detect SARS CoV- 2 mutations (e.g., SNVs). [0003] The present invention provides oligonucleotides that detect mutations in SARS CoV-2 genome. In some embodiments, the present disclosure provides SARS CoV-2 variant primers that are bubbled primers. In some embodiments, a SARS CoV-2 variant primer that specifically binds to a target nucleotide sequence. In some embodiments, a SARS CoV-2 variant primer includes: (i) an anchor, where the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence; (ii) a bridge, where the bridge is located immediately 3' of the anchor in the primer, and where the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; and (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of a target nucleotide sequence, which is 3' of the first portion of the target nucleotide sequence and comprises an interrogating nucleotide position, wherein the interrogating nucleotide position corresponds to a SARS CoV-2 variant nucleotide. [0004] In some embodiments, a variant primer is 25 to 55 nucleotides long. In some embodiments, a variant primer is 30 to 40 nucleotides long. In some embodiments, a variant primer is composed of and/or comprises DNA. [0005] In some embodiments, the anchor of a variant primer is 15 to 40 nucleotides long. In some embodiments, the anchor is 20 to 35 nucleotides long. In some embodiments, the bridge of a variant primer is 4 to 10 nucleotides long. In some embodiments, the bridge is 6 to 8 nucleotides long. In some embodiments, the foot of a variant primer is 6 to 8 nucleotides long. In some embodiments, the foot is 7 nucleotides long. [0006] In some embodiments, a variant primer comprises a label or a tag. [0007] In some embodiments, the interrogating nucleotide of a variant primer is a nucleotide that corresponds to an amino acid substitution selected from: N501Y, E484K, K417N, A701V, and V1176F. [0008] In some embodiments, a target nucleotide sequence of a variant primer is a SARS CoV-2 genomic RNA sequence. In some embodiments, a target nucleotide sequence corresponds to a SARS CoV-2 S gene sequence or portion thereof. In some embodiments, a target nucleotide sequence contains one or more SNVs present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2). [0009] In some embodiments, a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha, beta, eta, gamma, iota, mu, omicron, theta, zeta, and C.1.2. [0010] In some embodiments, a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is an alpha variant, variant, beta variant, gamma variant, mu variant, theta variant, C.1.2 variant, or omicron variant. In some embodiments, the variant nucleotide sequence comprises a sequence that corresponds to an N501Y mutation. In some embodiments, the variant nucleotide sequence comprises A23063T nucleotide substitution. In some embodiments, the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 8, 20-22, and 29-35. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 8, 20-22, and 29-35. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 8, 20-22, and 29-35. In some embodiments, the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8, 20-22, and 29-35. [0011] In some embodiments, a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant or iota variant. In some embodiments, the variant nucleotide sequence comprises a sequence that corresponds to an A701V mutation. In some embodiments, the variant nucleotide sequence comprises a C23635T nucleotide substitution. In some embodiments, the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 12 and 84-86. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 12 and 84-86. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 12 and 84-86. In some embodiments, the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86. [0012] In some embodiments, a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a gamma variant, zeta variant, or theta variant. In some embodiments, the variant nucleotide sequence comprises a sequence that corresponds to a V1176F mutation. In some embodiments, the variant nucleotide sequence comprises a G25088T nucleotide substitution. In some embodiments, the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 16, 72-74, and 78-80. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 16, 72-74, and 78-80. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 16, 72-74, and 78-80. In some embodiments, the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16, 72-74, and 78-80. [0013] In some embodiments, a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, gamma variant, eta variant, or mu variant. In some embodiments, the variant nucleotide sequence comprises a sequence that corresponds to an E484 mutation. In some embodiments, the variant nucleotide sequence comprises an A22980G nucleotide substitution. In some embodiments, the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 38, 41-44, 48, and 50. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 38, 41-44, 48, and 50. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 38, 41-44, 48, and 50. In some embodiments, the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38, 41-44, 48, and 50. [0014] In some embodiments, a target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, mu variant, or delta variant. In some embodiments, the variant nucleotide sequence comprises a sequence that corresponds to a K417N mutation. In some embodiments, the variant nucleotide sequence comprises a G22184T nucleotide substitution. In some embodiments, the variant primer sequence comprises a sequence with 1 to 6 nucleotide substitutions relative to any one of SEQ ID NOs: 51-55 and 62-64. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 51-55 and 62-64. In some embodiments, the variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 51-55 and 62-64. In some embodiments, the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55 and 62-64. [0015] In some embodiments, provided are primer pairs comprising a variant primer as described herein, and a complementary primer comprising or consisting of a nucleotide sequence that is complementary to a portion of a complementary strand sequence of the target nucleotide sequence. [0016] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 8 and 29-35, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 9 and 23-24. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 8 and 29-35, and (ii) a complementary primer comprising or consisting of a nucleotide that differs by no more than one nucleotide from according to any one of SEQ ID NOs: 9 and 23-24. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24. [0017] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 20-22, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 25-28. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 20-22, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 25-28. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 20-22, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 25-28. [0018] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 12 and 84-86, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 13, 87, and 88. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 12 and 84-86, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 13, 87, and 88. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88. [0019] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 16 and 72-73, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 17, 76, and 77. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 16 and 72-73, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 17, 76, and 77. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16 and 72-73, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17, 76, and 77. [0020] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 78-80, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 74-75. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 78-80, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 74-75. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 78-80, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 74-75. [0021] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 38 and 41-44, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 39-40 and 45-46. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 38 and 41-44, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 39-40 and 45-46. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38 and 41-44, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40 and 45-46. [0022] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from SEQ ID NO: 48 or 50, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from SEQ ID NO: 47 or 49. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from SEQ ID NO: 48 or 50, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from SEQ ID NO: 47 or 49. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to SEQ ID NO: 48 or 50, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to SEQ ID NO: 47 or 49. [0023] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 51-55, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 56-58. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 51-55, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 56-58. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 56-58. [0024] In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 62-64, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than two nucleotides from any one of SEQ ID NOs: 59-61. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 62-64, and (ii) a complementary primer comprising or consisting of a sequence that differs by no more than one nucleotide from any one of SEQ ID NOs: 59-61. In some embodiments, a primer pair comprises: (i) a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 62-64, and (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61. [0025] In some embodiments, provided are blocker pairs comprising (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence of SARS CoV-2; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of the target nucleotide sequence of SARS CoV-2, which is 3' of the first portion of the target nucleotide sequence and comprises a wild type nucleotide at an interrogating nucleotide position; and (iv) a modification at its 3'-end to prevent extension from occurring. [0026] In some embodiments, a wild type nucleotide at an interrogating nucleotide position is the nucleotide at that corresponding position of the genome of the Wuhan-Hu1 strain or USA- WA1/2020 strain. In some embodiments, a target nucleotide sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome but not a wild-type SARS CoV-2 genome, wherein the SARS CoV-2 variant is an alpha variant, variant, beta variant, gamma variant, mu variant, theta variant, C.1.2 variant, or omicron variant. [0027] In some embodiments, a modification that prevents extension of a blocker primer is or comprises: a PO4 modification, an inverted 3'-3' linkage, an inverted dT, a C3 modification, or an amino-C7 modification. In some embodiments, a modification that prevents extension of a blocker primer a PO4 modification or a C3 modification. [0028] In some embodiments, a blocker primer is 25 to 55 nucleotides long. In some embodiments, a blocker primer is 30 to 40 nucleotides long. In some embodiments, a blocker primer is composed of and/or comprises DNA. [0029] In some embodiments, the anchor of a blocker primer is 15 to 40 nucleotides long. In some embodiments, the anchor is 20 to 35 nucleotides long. In some embodiments, the bridge of a blocker primer is 4 to 10 nucleotides long. In some embodiments, the bridge is 6 to 8 nucleotides long. In some embodiments, the foot of a blocker primer is 6 to 8 nucleotides long. In some embodiments, the foot is 7 nucleotides long. [0030] In some embodiments, the wild type nucleotide at an interrogating nucleotide position is a nucleotide position that corresponds in a SARS CoV-2 genome that translates to a wild-type amino acid at a position selected from: N501, E484, K417, A701, and V1176. In some embodiments, the wild type nucleotide at an interrogating nucleotide position is a nucleotide that corresponds to a position in a SARS CoV-2 genome selected from: A23063, A22980, G22184, C23635, and G25088. [0031] In some embodiments, the interrogating nucleotide position of a blocker primer is a nucleotide position that corresponds in a SARS CoV-2 genome that translates to a wild-type amino acid at a position N501. In some embodiments, the interrogating nucleotide position of a blocker primer is a nucleotide position that corresponds in a SARS CoV-2 genome to A23063. In some embodiments, a blocker primer comprises or consists of a sequence according to SEQ ID NO: 10 or 37. [0032] In some embodiments, the interrogating nucleotide position of a blocker primer is a nucleotide position that corresponds in a SARS CoV-2 genome that translates to a wild-type amino acid at a position A701. In some embodiments, the interrogating nucleotide position of a blocker primer is a nucleotide position that corresponds in a SARS CoV-2 genome to C23635. In some embodiments, a blocker primer comprises or consists of a sequence according to SEQ ID NOs: 14 and 90-93. [0033] In some embodiments, the present disclosure provides primer sets that include one or more variant primers as described herein. In some embodiments, a primer set further comprises one or more complementary primers. In some embodiments, a primer set further comprises one or more probes. [0034] In some embodiments, a primer set further comprises a blocker primer as described herein. In some embodiments, variant primer and blocker primer sequences differ only at the nucleotide that anneals to the interrogating nucleotide position, and that the blocker primer comprises the modification that prevents extension. [0035] In some embodiments, a probe comprises or consists of a sequence that differs by no more than two nucleotides from a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89. In some embodiments, a probe comprises or consists of a sequence that differs by no more than one nucleotide from a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89. In some embodiments, a probe comprises or consists of a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89. [0036] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24, and (iii) a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 7 or 36. In some embodiments, a primer set further comprises a blocker primer that comprises or consists of a sequence according to SEQ ID NO: 10 or 37. [0037] In some embodiments, provided is a primer set that comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and (ii) a blocker primer that comprises or consists of a sequence according to SEQ ID NO: 10 or 37. In some embodiments, wherein the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to a nucleotide of N501Y in the variant primer and a nucleotide that translates to a wild-type amino acid at a position N501 in the blocker primer. In some embodiments, wherein the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to position 23063 in a SARS CoV-2 genome, with a T at this position in the variant sequence and an A at this position in the blocker sequence. [0038] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88, and (iii) a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 11 or 89. In some embodiments, a primer set further comprises a blocker primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93. [0039] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and (ii) a blocker primer comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93. In some embodiments, wherein the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to a nucleotide of A701V in the variant primer and a nucleotide that translates to a wild-type amino acid at a position A701 in the blocker primer. In some embodiments, wherein the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to position 23635 in a SARS CoV-2 genome, with a T at this position in the variant sequence and an C at this position in the blocker sequence. [0040] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 16 and 72-73, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17, 76, and 77, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 15 or 81-83. [0041] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 78-80, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 74-75, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 15 or 81-83. [0042] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 38 and 41-44, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40 and 45-46, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 7 and 65-66. [0043] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to SEQ ID NO: 48 or 50, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to SEQ ID NO: 47 or 49, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 7 and 65- 66. [0044] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 56-58, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 67-71. [0045] In some embodiments, a primer set comprises (i) a variant primer that comprises or consists of a sequence according to any one of SEQ ID NOs: 62-64, (ii) a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61, and (iii) a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 67-71. [0046] In some embodiments, the present disclosure provides mixtures comprising variant primers as described herein and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence. In some embodiments, the present disclosure provides mixtures comprising primer pairs as described herein and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence. In some embodiments, the present disclosure provides mixtures comprising primer sets as described herein and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence. [0047] In some embodiments, the present disclosure provides methods that include: (i) contacting a sample from a subject with the variant primer, primer pair, or primer set as described herein, and amplifying a target nucleotide sequence from the sample. [0048] In some embodiments, the target nucleotide sequence is a SARS CoV-2 nucleotide sequence. [0049] In some embodiments, the method is to detect and/or identify a SARS CoV-2 variant in a sample comprising SARS CoV-2 nucleic acid. In some embodiments, the SARS CoV- 2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2). [0050] In some embodiments, the method is to detect and/or identify one or more nucleotide variants corresponding to one or more amino acid mutations in a sample comprising SARS CoV-2 nucleic acid. [0051] In some embodiments, the one or more mutations are selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F. [0052] In some embodiments, the one or more mutations are selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the one or more mutations comprise three or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. [0053] In some embodiments, the one or more mutations are selected from: N501Y, A701V, and V1176F. In some embodiments, the one or more mutations are at least three mutations that comprise at least N501Y, A701V, and V1176F. [0054] In some embodiments, the SARS CoV-2 in the sample is present in an amount that is less than 50,000 viral genome copies per milliliter (cp/mL), less than 40,000 cp/mL, less than 30,000 cp/mL or less than 20 cp/mL. [0055] In some embodiments, provided methods further comprise contacting a sample from the subject with a positive control primer set. In some embodiments, the positive control primer set comprises (i) oligonucleotides comprising or consisting of a sequence according to each of SEQ ID NOs: 1 to 3; and/or (ii) oligonucleotides comprising or consisting of a sequence according to each of SEQ ID NOs: 4 to 6. [0056] In some embodiments, provided methods further comprise performing whole genome sequencing on the sample. [0057] In some embodiments, provided are methods of identifying a subject for treatment, comprising (i) identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject, (ii) determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified, and (iii) administering therapy according the determined SARS CoV-2 variant strain(s), wherein the step of identifying one or more nucleotide sequence changes comprises contacting the sample with a variant primer, primer pair, or primer set as described herein. [0058] In some embodiments, provided are methods of identifying a predominant SARS CoV-2 variant in a population of subjects, comprising identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in samples from the population of subjects, where the identifying comprises contacting the samples with a variant primer, primer pair, or primer set as described herein. [0059] In some embodiments, provided are kits comprising a variant primer, primer pair, or primer set as described herein, and instructions for use. In some embodiments, a kit further comprises a positive control primer set. In some embodiments, the positive control primer set comprises (i) oligonucleotides comprising or consisting of a sequence according to each of SEQ ID NOs: 1 to 3; and/or (ii) oligonucleotides comprising or consisting of a sequence according to each of SEQ ID NOs: 4 to 6. [0060] These and other features and advantages provided in the present disclosure will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWING [0061] The Drawing included herein, which is composed of the following Figures, is for illustration purposes only and not for limitation. [0062] FIG. 1A depicts a schematic of the coding regions of the SARS CoV-2 genome with the genes encoding structural proteins labeled: spike (S), envelope (E), matrix (M), and nucleocapsid (N). FIG. 1B depicts a schematic of the SARS CoV-2 genome that labels genes within the ORF1ab region encoding certain non-structural proteins, as well as the general location of certain mutations associated with different SARS CoV-2 variants of interest (adapted from “Features, Evaluation, and Treatment of Coronavirus (COVID-19)”, Marco Cascella; Michael Rajnik; Arturo Cuomo; Scott C. Dulebohn; Raffaela Di Napoli. Statpearls, Jan 2021). FIG.1C depicts a schematic of certain mutations within the S gene that are associated with particular SARS CoV-2 variants. [0063] FIG.2 includes a plot of the fraction of positive samples that have been sequenced in different countries as of January 2021 (plot from Priya Singh, CSIR-CCMB). The x-axis reflects the total number of COVID-19 positive cases in each country and the y-axis reflects the percent of cases that have been sequenced. Countries in blue sequenced less than 1% of total COVID-19 positive samples, countries in yellow sequenced 1 to 5% of total COVID-19 positive samples, and countries in green sequenced over 5% of samples of total COVID-19 positive samples. [0064] FIG.3 includes a schematic of an exemplary primer design as provided herein. As shown, in some embodiments, a primer includes an anchor sequence (brown), a bubble (also referred to as a bridge) (blue), and a foot sequence (red). Such a primer can be referred to herein as a “bubbled” primer. The present disclosure encompasses a recognition that a foot sequence should be short, such that a single mismatch at the terminal 3′ nucleotide destabilizes primer binding and prevent extension, enabling discrimination of different alleles. In FIG. 3, “X” represents the interrogating nucleotide, which can be different in one or more variants. If a mismatch occurs at the interrogating nucleotide, primer binding will be destabilized and prevent extension. In some embodiments, a second primer can be used in a primer set with a bubbled primer. For example, if a bubbled primer is a forward primer, then the second primer can be a reverse primer, and vice versa. In some embodiments, a probe can be used, which is capable of hybridizing (e.g., is complementary to) to a sequence amplified using a primer set that includes a bubbled primer. [0065] FIG.4 includes a schematic of an exemplary primer design as provided herein. In some embodiments, a primer set includes a wild-type (WT) primer and a variant primer. A WT primer is a primer that is capable of hybridizing with (e.g., is complementary to) a wild-type and/or reference sequence. A variant primer is a primer that is capable of hybridizing with (e.g., is complementary to) a variant sequence. As shown, a WT forward primer can have a modification (e.g.,PO4) at its 3'-end to prevent extension from occurring. Such a WT primer is also referred to as a “blocker” primer, and can compete with a variant primer that binds to a sequence ending with a certain nucleotide represented by “X” in FIG.4. Because the WT blocker primer can compete with a variant primer, it can suppress non-specific amplification of a wild-type allele by a variant primer. A primer set can also include a third primer, which is used together with a WT blocker primer and a variant primer. For example, if a WT blocker primer and a variant primer are forward primers, a third primer can be a reverse primer. In some embodiments, a probe can be used, which is capable of hybridizing with (e.g., is complementary to) a sequence amplified using a primer set as shown in FIG.4. [0066] FIG.5 includes a schematic of an exemplary primer design as provided herein. In some embodiments, a primer set includes a forward primer and a reverse primer. As shown, in some embodiments, either the forward or reverse primer is capable of hybridizing with (e.g., is complementary to) a sequence that is present in a wild-type sequence, but which is not present (e.g., is in a deletion) in a variant sequence. Accordingly, using a primer set as shown, an amplicon will be produced following amplification of a wild-type sequence, but an amplicon will not be produced from a variant sequence. In some embodiments, a probe can be used, which is capable of hybridizing with (e.g., is complementary to) a sequence amplified using a primer set as shown in FIG.5. In some embodiments, one or more (e.g., two) primer sets, as shown in FIG.5, can be used. [0067] FIG. 6 includes a flow chart schematic that provides a process overview for development of an assay for detection of an exemplary SARS CoV-2 single nucleotide variant (SNV), N501Y, with high specificity. [0068] FIG.7 includes exemplary results of a screen of primer/probe sets for amplification of a N501Y mutation. Panel (A) provides an amplification plot and Panel (B) provides the average expression (CT) and change in expression (ΔCT) between WT and mutant templates. [0069] FIG.8 includes exemplary results of a blocker screen for amplification of a N501Y mutation. Results depicted for an exemplary primer/probe set with increasing concentrations of blocker: 0 nM, 10 nM, 50 nM, 100 nM, 150 nM, and 300 nM. The corresponding amounts of amplification of N501Y mutant and wild-type templates are provided is provided in the table of Panel (B) [0070] FIG. 9 includes results of an exemplary analytical sensitivity analysis for exemplary primer set for detection of a N501Y mutation, with and without a blocker primer. Panel (A) provides a linear fit of the average CT vs. concentration for primer sets SNV- MutFor5/WTFor5-PO4/Rev4/Probe2 (“with blocker”) vs. SNV-MutFor5/Rev4/Probe2 (“without blocker”). Panel (B) provides a table view of the results, with the concentration of template, average CT, and number of samples detected. [0071] FIG. 10 includes exemplary results of primer/probe screens for (A) E484K mutation, (B) SA417 mutation, (C) BZ1176 mutation, and (D) SA701 mutation. Specifically, panel (A) depicts results for the primer set: 484-Mut For 3/484-Rev 3/484-Probe 3; panel (B) depicts results for the primer set: SA417-Mut For 3/SA417-Rev 1/SA417-Probe 1; panel (C) depicts results for the primer set: BZ1176-Mut F2/BZ1176-Rev2/BZ1176-Probe1; and panel (D) depicts results for the primer set: SA701-Mut For5/SA701-Rev1/SA701-Probe1. [0072] FIG. 11 includes exemplary results of a blocker screen analysis directed to the SA701 mutation. Specifically, provided an amplification plot for the primer set SA701-Mut For 5/SA701-Rev 1/SA701-Probe 1/SA701-WT For 1-PO4 with the following templates: Mutant, WT, Mutant+WT, and no template control (NTC). Templates were tested at a concentration of 1.0E5 and with blocker primer at a concentration of 200 nM. [0073] FIG.12A and FIG.12B include exemplary results of primer/probe combinations for a multiplex assay for detecting N501Y, SA701, BZ1176, and a positive control. FIG. 12A depicts exemplary results of combination 5, which includes N501Y with a FAM probe, SA701 with a CIV probe, BZ1176 with a TAMRA probe, and control with a Q670 probe. FIG. 12B depicts exemplary results of combination 6, which includes SA701 with a FAM probe, N501Y with a CIV probe, BZ1176 with a TAMRA probe, and control with a Q670 probe. Both of these combinations generated a ΔCT within +/- 1 when compared with singleplex assays. CERTAIN DEFINITIONS [0074] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included. [0075] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value. [0076] Allele: As used herein, the term “allele” refers to one of two or more existing genetic variants of a specific polymorphic genomic locus. [0077] Detection agent: The term “detection agent” as used herein refers to any element, molecule, functional group, compound, fragment or moiety that is detectable. In some embodiments, a detection agent is provided or utilized alone. In some embodiments, a detection agent is provided and/or utilized in association with (e.g., joined to) another agent. Examples of detection agents include, but are not limited to: various ligands, radionuclides (e.g., 3H, 14C, 18F, 19F, 32P, 35S, 135I, 125I, 123I, 64Cu, 187Re, 111In, 90Y, 99mTc, 177Lu, 89Zr etc.), fluorescent dyes, chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available. [0078] Diagnostic test: As used herein, “diagnostic test” is a step or series of steps that is or has been performed to attain information that is useful in determining whether a patient has a disease, disorder or condition and/or in classifying a disease, disorder or condition into a phenotypic category or any category having significance with regard to prognosis of a disease, disorder or condition, or likely response to treatment (either treatment in general or any particular treatment) of a disease, disorder or condition. Similarly, “diagnosis” refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have or develop a disease, disorder or condition, state, staging or characteristic of a disease, disorder or condition as manifested in the subject, information related to the nature or classification of a tumor, information related to prognosis and/or information useful in selecting an appropriate treatment or additional diagnostic testing. Selection of treatment may include the choice of a particular therapeutic agent or other treatment modality such as vaccination, antibody therapy, etc., a choice about whether to withhold or deliver therapy, a choice relating to dosing regimen (e.g., frequency or level of one or more doses of a particular therapeutic agent or combination of therapeutic agents), etc. Selection of additional diagnostic testing may include more specific testing for a given disease, disorder, or condition. [0079] Hybridization: The term “hybridization” refers to the physical property of single- stranded nucleic acid molecules (e.g., DNA or RNA) to anneal to complementary nucleic acid molecules. Hybridization can typically be assessed in a variety of contexts– including where interacting nucleic acid molecules are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell). In some embodiments, hybridization can be detected by a hybridization technique, such as a technique selected from the group consisting of in situ hybridization (ISH), microarray, Northern blot, and Southern blot. In some embodiments, hybridization refers to 100% annealing between the single-stranded nucleic acid molecules and the complementary nucleic acid molecule. In some embodiments, annealing is less than 100% (e.g., at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% of a single-stranded nucleic acid molecule anneals to a complementary nucleic acid molecule. [0080] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a molecule comprising two or more deoxyribonucleotides, ribonucleotides, and/or nucleotide analogs, the latter including nucleic acid analogs, such as isoguanosine, isocytosine, inosine, or deoxyinosine. The length of the oligonucleotide will vary depending on the function of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. As used herein, the term “oligonucleotide” is meant to encompass primers (both forward and reverse primers) and detection probes. [0081] Primer: As used herein, the term "primer" refers to an oligonucleotide which, whether purified from a nucleic acid restriction digest or produced synthetically, is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase, reverse transcriptase or the like, and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agents for polymerization. The term “forward primer” refers to a primer that forms an extension product by binding in the 5′ to 3′ direction to the 3′ end of a strand of a nucleic acid analyte (e.g., denatured DNA). The term “reverse primer” refers to a primer that forms an extension product by binding in the 3′ to 5′ direction to the 5′ end of a strand of a nucleic acid analyte (e.g., denatured DNA). [0082] Sample: As used herein, the term “sample” refers to a biological sample obtained or derived from a subject (e.g., a human subject), as described herein. In some embodiments, a biological sample comprises biological tissue or fluid. In some embodiments, a bodily fluid is or comprises saliva, respiratory secretion, blood, and/or plasma. In some embodiments, a biological sample may comprise blood; blood cells; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; cerebrospinal fluid; lymph; tissue biopsy specimens; surgical specimens; other body fluids, secretions, and/or excretions; and/or cells therefrom. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods of collecting a body fluid and/or cells. In some embodiments, a sample of respiratory secretion obtained from a subject by an anterior nares (nasal) swab, a mid-turbinate nasal swab, a nasopharyngeal swab, and/or an oropharyngeal swab. In some embodiments, a sample of saliva from a subject by having the subject spit into a receptacle (e.g., a tube). In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. A “processed sample” may comprise, for example, nucleic acids or polypeptides extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription, isolation and/or purification of certain components (e.g., polynucleotide components, e.g., RNA and/or DNA). [0083] SARS CoV-2 variant: As used herein, the term “SARS CoV-2 variant” or “viral variant” or simply “variant” when used in reference to SARS CoV-2 (as will be apparent from context), is a SARS CoV-2 virus that includes one or more genetic changes relative to a SARS CoV-2 reference strain or one or more predominant viral variants already circulating in a population. In some embodiments, a SARS CoV-2 variant comprises one or more genomic mutations in an S gene sequence. In some embodiments, a SARS CoV-2 variant comprises one or more genomic mutations relative to a reference genome sequence or portion thereof. In some embodiments, a SARS CoV-2 variant comprises one or more genomic mutations in an S gene sequence relative to an S gene sequence of a reference genome. In some embodiments, a reference genome sequence corresponds to that of the Wuhan-Hu1 strain (the first genetic sequence identified) or USA-WA1/2020 strain (the first identified in the United States) or portion thereof. In some embodiments, a SARS CoV-2 variant comprises one or more genomic mutations relative to one or more predominant viral variants circulating in a population. In some embodiments, a SARS CoV-2 variant comprises one or more genomic mutations in an S gene sequence relative to an S gene sequence of a predominant viral variant circulating in a population. [0084] Singleplex: As used herein, the term “singleplex” refers to a single assay that is not carried out simultaneously with any other assays. For example, within a multiwell plate, a singleplex assay refers to a single reaction that is performed within a single well of the multiwell plate. Singleplex assays include individual assays that are carried out sequentially. [0085] Multiplex: As used herein the term “multiplex” refers to multiple assays that are carried out simultaneously. As used herein, a multiplex assay refers to the number of target sites that the assay aims to identify. For example, a multiplex assay that is designed to identify two sites is termed a dualplex assay. Within a multiwell plate, a multiplex assay performs multiple reactions within a single well of the multiwell plate. [0086] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human). In some embodiments a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is at risk for a SARS CoV-2 viral infection. In some embodiments, a subject has been exposed or is suspected to have a COVID-19 infection. In some embodiments, a subject is susceptible to COVID-19; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing COVID-19. In some embodiments, a subject displays one or more symptoms of a SARS CoV-2 infection. In some embodiments, a subject does not display a particular symptom or characteristic of COVID-19. In some embodiments, a subject does not display any symptom or characteristic of COVID-19 (i.e., is asymptomatic). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered. [0087] Threshold value: As used herein, the term “threshold value” refers to a value (or values) that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay. A threshold value can be determined based on one or more control samples. A threshold value can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold value can be a range of values. In some embodiments, a threshold value can be a value (or range of values) reported in the relevant field (e.g., a value found in a standard table). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS [0088] This disclosure relates, inter alia, to SARS CoV-2 variant detection assays for identifying different human coronavirus SARS CoV-2 variants (e.g., N501Y, A701V, V1176F). In some embodiments, provided methods also detect a conserved genomic region of SARS CoV- 2 (e.g., ORF gene) which serves as a control. Currently, huge efforts are being made for genomic sequencing of SARS CoV-2 positive samples for variant identification. Provided SARS CoV-2 variant detection assays can allow sample selection and/or streamlining of downstream sequencing effort for different variants of SARS CoV-2, including currently major circulating variants. [0089] The present disclosure recognizes that ongoing surveillance of SARS CoV-2 variants is important for public health. The present disclosure provides SARS CoV-2 variant detection assays that can be used, e.g., as research tools to help scientists and epidemiologists track mutation prevalence in a population as well as enable assessment of any potential impact on laboratory diagnostics, vaccines, and/or therapeutics. [0090] In some embodiments, a SARS CoV-2 variant detection assay provided herein will use a Reverse Transcription Polymerase Chain Reaction (RT-PCR) amplification process. In some embodiments, a method as provided combines first reverse transcription (RT) of SARS CoV-2 RNA into DNA and a second step amplification of specific DNA targets using polymerase chain reaction (PCR). In some embodiments, a reverse transcriptase reverse transcribes a specific section of RNA into complimentary DNA (cDNA) and then a thermal-stable DNA polymerase amplifies the cDNA template. In some embodiments, a section is specified by a DNA oligonucleotide, called a primer. In some embodiments, the reverse transcription process is linear because it uses only the reverse strand primer. In some embodiments, the amplification process is exponential, using both forward and reverse strand primers. In some embodiments, the RT-PCR reaction includes polymerase, primers, a metal ion, deoxynucleoside triphosphates and buffer. In some embodiments, priming and replication will be accomplished by cycling a reaction mixture through two or more temperatures, such as an elevated temperature for denaturing the amplicon and a lower temperature or temperatures for annealing and extending the primer. [0091] The present disclosure describes, inter alia, the design and characteristics of primers and probes useful for detecting and/or characterizing SARS CoV-2 (e.g., a multiplexed RT-PCR assay). Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) Variants [0092] SARS CoV-2 is the virus that causes COVID-19, a respiratory illness that was declared a Public Health Emergency of International Concern in January 2020 by the World Health Organization, and a pandemic in March 2020. SARS CoV-2 is an enveloped, positive-stranded RNA virus with a nucleocapsid. The SARS CoV-2 genomic structure is organized in a +ssRNA of approximately 30 kb in length and with a 5′-cap structure and 3′-poly-A tail. FIG.1A and FIG. 1B provide schematic representations of the SARS CoV-2 viral genome. As depicted in FIG.1A, the SARS CoV-2 genome includes from 5′ to 3′: a 5′ terminal sequence, ORF1ab (comprising ORF1a and ORF1b), ORFs encoding structural proteins: spike (S), envelope (E), matrix (M), nucleocapsid (N), and a 3′ terminal sequence. The genomic schematic of FIG. 1B illustrates certain genes encoding non-structural proteins in the SARS CoV-2 genome (e.g., papain-like protease, 3CL-protease, replicase, and endoribonuclease), along with the spike protein (S) gene. [0093] As with all viruses, SARS CoV-2 accumulates random mutations during replication. Distinct SARS CoV-2 variant lineages formed as viruses circulating in particular regions evolved and increased in frequency. [0094] Since SARS CoV-2 was first detected, a number of viral variants have emerged, some of which show higher transmissibility and/or may increase death rate. A number of different SARS CoV-2 variants have been identified to date. For example, SARS CoV-2 variants identified include, e.g., alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2). [0095] In some embodiments, a SARS CoV-2 variant is selected from alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), mu (B.1.621), and omicron (B.1.1.529), and/or a descendent lineage of any thereof. [0096] In some embodiments, a SARS CoV-2 variant is selected from alpha (B.1.1.7), beta (B.1.351), gamma (P.1), iota (B.1.526), mu (B.1.621), and omicron (B.1.1.529), theta (P.3), zeta (P.2), and/or a descendent lineage of any thereof. [0097] In some embodiments, the present disclosure provides methods and/or assays for detecting one or more mutations associated with a SARS CoV-2 variant. FIG.1B also indicates certain mutations associated with SARS CoV-2 variants and the gene locus of these variants. [0098] In some embodiments, one or more mutations associated with a SARS CoV-2 variant are located in the S gene. [0099] In some embodiments, a SARS CoV-2 variant may include one or more mutations selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F. [0100] In some embodiments, a SARS CoV-2 variant may include one or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, one or more mutations to be detected are selected from N501Y, E484K, K417N, A701V, and V1176F. [0101] In some embodiments, a SARS CoV-2 variant may include two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, two or more mutations to be detected are selected from N501Y, E484K, K417N, A701V, and V1176F. [0102] In some embodiments, a SARS CoV-2 variant may include one or more mutations to be detected by a method and/or assay described herein (e.g., through detection of a nucleotide variant corresponding to an amino acid mutation). In some embodiments, one or more mutations to be detected are selected from N501Y, A701V, and V1176F. [0103] In some embodiments, the present disclosure provides methods and/or assays for detecting two or more mutations associated with a SARS CoV-2 variant (e.g., through detection of two or more nucleotide variants corresponding to the mutations). In some embodiments, two or more mutations to be detected are selected from N501Y, A701V, and V1176F. [0104] In some embodiments, the present disclosure provides methods and/or assays for detecting three or more mutations associated with a SARS CoV-2 variant (e.g., through detection of three or more nucleotide variants corresponding to the mutations). In some embodiments, three or more mutations to be detected include N501Y, A701V, and V1176F. [0105] Some emerging SARS CoV-2 variants, e.g., N501Y variants, may have higher ability to escape the immune system or cause reinfection. Preliminary vaccine data show that the BioNtech (BNT162b2) and the Moderna (mRNA-1273) vaccine seem to have equivalent neutralizing titers to the N501 and Y501 viruses. In some embodiments, a SARS CoV-2 variant is or comprises a N501Y mutation, such as, beta (B.1.351), gamma (P.1), alpha (B.1.1.7), eta (B.1.525), and/or a descendent of any thereof. [0106] In some embodiments, a SARS CoV-2 variant is or comprises a A701V mutation, such as, beta (B.1.351) and iota (B.1.526), and/or descendent of any thereof. [0107] In some embodiments, a SARS CoV-2 variant is or comprises a V1176F mutation, such as, a gamma (P.1) variant, theta (P.3) variant, zeta (P.2) variant and/or a descendent of any thereof. [0108] SARS CoV-2 variants are classified in different countries using differing nomenclature and standards. In the United States, SARS CoV-2 variants are classified based on their attributes and prevalence in the U.S. These U.S. classifications include (in order of increasing concern): variant being monitored (VBM), variant of interest (VOI), variant of Concern (VOC), and variant of high consequence (VOHC). Under this scheme, each variant classification includes the possible attributes of lower classes (for example, VOC includes the possible attributes of VOI). Given the continuous evolution of SARS CoV-2 variants and their impact on public health, variant classification may be updated and/or changed (e.g., a variant may be reclassified) with emerging information on variant attributes and prevalence. [0109] In some embodiments, a variant is a VBM. Currently, the CDC monitors all SARS CoV-2 variants circulating in the United States. In some embodiments, a VBM as a variant where (i) data indicates there is a potential or clear impact on approved or authorized medical countermeasures or that have been associated with more severe disease or increased transmission and (ii) this variant is no longer detected, or are circulating at very low levels, in the United States. In some embodiments, a VBM does not pose a significant and imminent risk to public health in the United States. In some embodiments, a VBM was previously classified as a VOI or VOC. [0110] In some embodiments, a variant is a VOI. In some embodiments, a VOI as a variant comprises specific genetic markers that have been associated with changes to receptor binding, reduced neutralization by antibodies generated against previous infection or vaccination, reduced efficacy of treatments, potential diagnostic impact, and/or predicted increase in transmissibility or disease severity. In some embodiments, a VOI comprises specific genetic markers that are predicted to affect transmission, diagnostics, therapeutics, and/or immune escape. In some embodiments, a VOI is a variant that is associated with an increased proportion of cases and/or unique outbreak clusters. [0111] In some embodiments, a variant is a VOC. In some embodiments, a VOC is a variant for which there is evidence of an increase in transmissibility, more severe disease (for example, increased hospitalizations or deaths), significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures. In some embodiments, a variant is a VOC and includes one or more characteristics of a VOI. In some embodiments, a VOC is further characterized by evidence of impact on diagnostics, treatments, or vaccines (e.g., widespread interference with diagnostic test targets, evidence of substantially decreased susceptibility to one or more class of therapies, evidence of significantly decreased neutralization by antibodies generated during previous infection or vaccination, evidence of reduced vaccine-induced protection from severe disease, evidence of increased transmissibility, and/or evidence of increased disease severity). [0112] In some embodiments, a variant is a VOHC. In some embodiments, a VOHC is a variant for which there is clear evidence that prevention measures or medical countermeasures (MCMs) have significantly reduced effectiveness relative to previously circulating variants. In some embodiments, a variant is a VOHC and includes one or more characteristics of a VOI and/or a VOC. In some embodiments, a VOHC is further characterized evidence of impact on MCMs (e.g., demonstrated failure of diagnostic test targets, evidence to suggest a significant reduction in vaccine effectiveness, a disproportionately high number of infections in vaccinated persons, very low vaccine-induced protection against severe disease, significantly reduced susceptibility to multiple therapeutics (e.g., approved and/or authorized therapeutics), and/or more severe clinical disease and increased hospitalizations. Detecting SARS CoV-2 [0113] Current techniques for detecting SARS CoV-2 virus include antigen tests and Nucleic Acid Amplification Tests (NAATs). Antigen tests are immunoassays that detect the presence of a specific viral antigen, which implies current viral infection. Most antigen tests for SARS CoV-2 are less expensive than NAATs and provide faster results, and thus can be used in screening programs to quickly identify those who are likely to be contagious. The SARS CoV-2 antigen tests currently authorized include point-of-care, laboratory-based, and self-tests. [0114] However, antigen tests for SARS-CoV-2 are generally less sensitive than molecular test like real-time reverse transcription polymerase chain reaction (RT-PCR) and other nucleic acid amplification tests (NAATs), which detect and amplify the presence of viral nucleic acid. However, NAATs can produce positive results for weeks to months after initial infection and can detect levels of viral nucleic acid even when virus cannot be cultured, suggesting that the presence of viral nucleic acid may not always indicate contagiousness. Antigen tests perform best in individuals with high viral loads (e.g., Ct values ≤ 25-30, ~10E5/6 RNA copies/mL) 1 to 3 days prior to onset of symptoms and during the first 5 to 7 days of illness. [0115] False negative results may occur with any molecular test for the detection of SARS- CoV-2, particularly if a mutation occurs in the part of the virus' genome assessed by that test. In some embodiments, a mutation (e.g., a mutation associated with a SARS CoV-2 variant) can result in changes to one or more viral proteins, which may also impact the performance of an antigen test. For example, mutations of SARS CoV-2 nucleoprotein gene can result in false-negative antigen test results despite high viral loads confirmed by other methods (e.g., NAAT). [0116] To reduce the probability of false negatives, some assays have been designed to target highly conserved regions. For example, the FTD SARS CoV-2 Assay, is a real time PCR assay that targets two highly conserved SARS CoV-2 genomic regions in ORF1ab and N gene. See Hennig et al., whitepaper entitled “Automation of the FTD SARS CoV-2 Assay” Nov.2020, which is incorporated by reference in its entirety. However, amplification of these regions in the ORF1ab and N genes do not differentiate between SARS CoV-2 variant strains. [0117] Current techniques to identify specific SARS-CoV-2 variants (e.g., VOCs) have used viral genome sequencing, such as whole viral genome sequencing or sequence of the S gene. While thorough, this approach is costly and only a small proportion of positive samples have been assigned to a particular variant lineage. The U.S. Centers for Disease Control and Prevention (“CDC”) is tracking emerging variants through genomic surveillance. As of January 25, 2021, the National SARS CoV-2 Strain Surveillance (NS3) system was scaled up to sequence 750 viral genomes per week. As of February 2, 2021, the Health Ministry of Israel reported 80 cases of the South African COVID-19 variant discovered. In addition, 16 pregnant women out of 20 screened were found to be infected with British variant. However, current sequencing methods are insufficient, as for the majority of countries, the percent of COVID-19 positive cases sequenced in the vast majority of countries is less than 5%, with most countries sequencing for less than 1% of cases. [0118] Whole genome sequencing of the SARS CoV-2 genome can be time consuming. This time delay in obtaining whole genome sequencing results can impede public health responses (e.g. contact tracing) and real-time calculation of the prevalence of different variants in a community. Accordingly, with the continual emergence of SARS CoV-2 variants of concern (VOCs), there is an ongoing need to develop assays that can detect particular variants with high specificity. [0119] Given that different SARS CoV-2 variants are characterized by one or more single nucleotide variants (SNVs), the present disclosure encompasses a recognition that it would be useful to develop an assay with high specificity for particular SNVs. However, developing an assay with high specificity for SNVs is more challenging than detecting sequence deletions or insertions (indels). [0120] The present disclosure encompasses a recognition that reflex testing can facilitate rapid public health responses, including surge testing and strategic decision-making. In some embodiments, the present disclosure provides methods of reflex testing that can detect of one or more mutations that are indicative of a particular SARS CoV-2 variant. [0121] Other manufacturers have also been developing reflex testing to identify SARS CoV-2 variants, such as the TaqPath™ COVID-19 Combo Kit (ThermoFisher Scientific) and the PhoenixDx® SARS-CoV-2 Multiplex assay (Procomcure Biotech GmbH), such assays have limitations. For example, these assays are each directed to a single mutation assays that are multiplexed, e.g., in combination with wild type sequence and/or a positive control. Provided Technologies for Detection of SARS CoV-2 Variants [0122] In some embodiments, provided herein are reflex (genotyping) technologies (e.g., assays, methods, compositions) to identify and/or detect specific mutations in a sample, e.g., a SARS CoV-2 positive sample. In some embodiments, detection of one or more mutations is indicative of a particular viral variant or variants. In some embodiments, the detection of one or more mutations is indicative of a SARS CoV-2 VBM, VOI, VOC, and/or VOHC. [0123] In some embodiments, a SARS CoV-2 variant assay provided herein detects a mutation in a SARS CoV-2 S gene. In some embodiments, a SARS CoV-2 variant assay provided herein detects a SARS CoV-2 mutation selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F. [0124] In some embodiments, a SARS CoV-2 variant assay detects one or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, a SARS CoV-2 variant assay detects two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, a SARS CoV-2 variant assay detects three or more mutations selected from N501Y, E484K, K417N, A701V, and V1176F. [0125] A listing of exemplary SARS CoV-2 variants associated with these SNVs is provided in Table 1 below. [0126] Table 1: Exemplary SARS CoV-2 mutations and associated variants
Figure imgf000032_0001
[0127] In some embodiments, a SARS CoV-2 variant detection assay detects nucleic acid changes that correspond to amino acid mutations N501Y, A701V, and/or V1176F. [0128] Technologies for detecting SARS CoV-2 variants as provided herein may also include a control. In some embodiments, an assay for detecting SARS CoV-2 variants is a multiplex assay detecting one or more SNVs (e.g., SNVs with the S gene) and a SARS CoV-2 positive control that is targeted to the Nucleocapsid (N) gene or ORF1ab gene. In some embodiments, provided is a multiplex assay for detecting one or more SARS CoV-2 variants and a control that detects a conserved region of the ORF1ab gene. [0129] In some embodiments, an assay for detecting SARS CoV-2 variants comprises a ROX reference dye. A ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization in a real-time qPCR assay. In some embodiments, a ROX reference dye is present in an amount to generate a fluorescent signal in a range from about 50,000 to about 500,000. In some embodiments, a ROX reference dye is present in an amount to generate a fluorescent signal in a range from about 100,000 to about 250,000. In some certain embodiments, a ROX reference dye is present in an amount of 0.1X per reaction. SARS CoV-2 Oligonucleotides [0130] In some embodiments, the present disclosure provides oligonucleotides for detecting and/or identifying one or more mutations associated with a SARS CoV-2 variant. In some embodiments, the present disclosure provides two or more oligonucleotides for detecting and/or identifying one or more mutations associated with a SARS CoV-2 variant. In some embodiments, two or more oligonucleotides comprise a variant primer (e.g., a bubbled primer) and second oligonucleotide. In some embodiments, a second oligonucleotide comprises a blocker primer. In some embodiments, a second oligonucleotide comprises a probe. In some embodiments, a second oligonucleotide is a complementary primer. In some embodiments, a variant primer is a forward primer and a complementary primer is a reverse primer. In some embodiments, a variant primer is a reverse primer and a complementary primer is a forward primer. [0131] In some embodiments, the present disclosure provides a primer set for detecting and/or identifying one or more mutations associated with a SARS CoV-2 variant (e.g., in a SARS CoV-2 S gene). In some embodiments, a primer set comprises a variant primer and a blocker primer. A primer set can also include a complementary primer, which is used together with a variant primer. For example, if a variant primer is a forward primer, a complementary primer can be a reverse primer (and vice versa). In some embodiments, a probe can be used, which is capable of hybridizing with (e.g., is complementary to) a sequence amplified using a primer set as shown in FIG. 4. In some embodiments, a primer set comprises a variant primer, a complementary primer, and a probe. In some embodiments, a primer set comprises each of a variant primer, a complementary primer, a blocker primer, and a probe. [0132] In some embodiments, the present disclosure provides a primer set for a multiplex assay detecting one or more SNVs (e.g., SNVs with the S gene) and a SARS CoV-2 positive control that is targeted to the Nucleocapsid (N) gene or ORF1ab gene. In some embodiments, provided primer sets include a variant primer, a complementary primer, a first probe, as well as a positive control forward primer, positive control reverse primer, and a second probe. In some embodiments, provided primer sets include for each variant to be detected: a variant primer, a complementary primer, a probe, and optionally a blocker primer, and also includes a positive control forward primer, positive control reverse primer, and a probe for the positive control. Variant Primers [0133] The present disclosure provides variant primers that are capable of hybridizing with (e.g., is complementary to) a variant sequence (e.g., a mutant sequence). In some embodiments, a variant sequence comprises a mutation that is a substitution (e.g., an SNV), a deletion, and/or insertion. In some embodiments, a variant primer is a bubbled primer. In some embodiments, a variant primer is a bubbled primer and comprises a sequence that is complementary to a mutation associated with one or more SARS CoV-2 variants. In some embodiments, the present disclosure provides one or more bubbled primers useful for detecting an SNV associated with a SARS CoV- 2 variant. [0134] In some embodiments, a variant primer is a bubbled primer that includes an anchor sequence, a bubble (also referred to as a bridge) sequence, and a foot sequence. In some embodiments, a foot sequence of a variant primer overlaps with (i.e., includes a nucleotide that is complementary to) the nucleotide of an SNV to be detected (referred to herein as the “interrogating nucleotide”). The present disclosure encompasses a recognition that a foot sequence should be short, such that a single mismatch at the interrogating nucleotide destabilizes primer binding and prevent extension, enabling discrimination of different alleles. In some embodiments, a foot sequence is designed such that the SNV to be detected is positioned at the terminus of the foot sequence (e.g., in the last 3 nucleotides, last 2 nucleotides, or the terminal nucleotide of the foot sequence). In some embodiments, a foot sequence is designed such that the SNV to be detected corresponds to the terminal nucleotide of the foot sequence. [0135] In some embodiments, provided is a variant primer that includes (i) an anchor, where the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence; (ii) a bridge (also referred to as a bubble), wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of a target nucleotide sequence; and (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of a target nucleotide sequence, which is 3' of the first portion of a target nucleotide sequence and comprises an interrogating nucleotide position. In some embodiments, a target nucleotide sequence is a SARS CoV-2 variant nucleotide sequence. [0136] In some embodiments, a variant primer comprises is a bubbled primer having a length within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit. In some embodiments, the lower limit may be about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, or about 25 nucleotides. In some embodiments, the upper limit may be about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, or about 60 nucleotides. [0137] In some embodiments, a variant primer comprises is a bubbled primer comprising an anchor sequence, a bubble sequence, and a foot sequence, wherein the overall length of the primer is 25 to 55 nucleotides long. In some embodiments, a variant primer is 30 to 40 nucleotides long. [0138] In some embodiments, a variant primer comprises an anchor sequence, where the anchor is 15 to 40 nucleotides long. In some embodiments, an anchor sequence is 20 to 35 nucleotides long. [0139] In some embodiments, a variant primer comprises a bubble sequence (i.e., a bridge sequence) that is 4 to 10 nucleotides in length. In some embodiments, a bubble sequence (i.e., a bridge sequence) comprises a sequence that is 6 to 8 nucleotides in length. In some embodiments, a bubble sequence comprises a sequence that is 6 nucleotides in length. In some embodiments, a bubble sequence comprises a sequence that is 8 nucleotides in length. [0140] In some embodiments, a variant primer comprises a foot sequence comprises a sequence that is 6 to 8 nucleotides in length. In some embodiments, a foot sequence comprises a sequence that is 6 nucleotides in length. In some embodiments, a foot sequence comprises a sequence that is 7 nucleotides in length. In some embodiments, a foot sequence comprises a sequence that is 8 nucleotides in length. [0141] In some embodiments, a variant primer comprises a foot sequence and the interrogating nucleotide is located within the last 3 nucleotides of the foot sequence. In some embodiments, a variant primer comprises a foot sequence and the interrogating nucleotide is located within the last 2 nucleotides of the foot sequence. In some embodiments, a variant primer comprises a foot sequence and the interrogating nucleotide is located at the 3′ terminus of the foot sequence. [0142] In some embodiments, a variant primer is a forward primer. In some embodiments, a variant primer is a forward primer and includes in order from 5′ to 3′, an anchor sequence, a bubble or bridge sequence, and a foot sequence. In some embodiments, a variant primer is a bubbled primer that is a forward comprising in order 5′ to 3′, an anchor sequence, a bubble sequence that is 6 to 8 nucleotides in length, and a foot sequence with the interrogating nucleotide position at the 3′ terminus. [0143] In some embodiments, a variant primer is a reverse primer. In some embodiments, a variant primer is a reverse (antisense) primer and includes in order from 5′ to 3′, an antisense anchor sequence, a bubble sequence (i.e., a bridge sequence), and antisense foot sequence. In some embodiments, a variant primer is a bubbled primer that is a reverse comprising in order 5′ to 3′, an anchor sequence (i.e., an antisense anchor sequence), a bubble sequence (i.e., a bridge sequence) that is 6 to 8 nucleotides in length, and a foot sequence (i.e., an antisense foot sequence) with the interrogating nucleotide position at the 3′ terminus. [0144] In some embodiments, a complementary primer can be used in a primer set with a variant primer. For example, if a variant primer is a forward primer, then the complementary primer can be a reverse primer, and vice versa. Probes [0145] In some embodiments, a probe can be used, which is capable of hybridizing (e.g., is complementary to) to a sequence amplified using a primer set that includes a bubbled primer. [0146] Examples of commonly used probes used for RT-PCR assays include the following probes: TAQMAN® probes, Molecular Beacons probes, SCORPIONS® probes, and SYBR® Green probes. TAQMAN®, Molecular Beacons, and SCORPIONS® probes each have a fluorescent reporter dye (also called a “fluor”) attached to the 5′ end of the probes and a quencher moiety coupled to the 3′ end of the probes. TAQMAN® probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe; during PCR, when the polymerase replicates a template on which a TAQMAN® probe is bound, the 5′-nuclease activity of the polymerase cleaves the probe thus, increasing fluorescence with each replication cycle. [0147] Unlike TAQMAN® probes, Molecular Beacons, which form a stem-loop structure when free in solution, remain intact during the amplification reaction. Molecular Beacons fluoresce during hybridization when the fluorescent dye and the quencher are separated. For signal measurement to be effective, the fluor and quencher must rebind in every cycle. SCORPIONS® probes, which maintain a stem-loop configuration in the unhybridized state, has at its 3′ end an additional sequence that is complementary to the extension product of the primer that is linked to the 5′ end of a specific primer via a non-amplifiable monomer. After extension of the SCORPIONS® primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop such that the fluor is no longer quenched and signal is seen. SYBR® Green probes binds double-stranded DNA and upon excitation emit light; thus as PCR product accumulates, fluorescence increases. [0148] In some embodiments, a probe provided herein includes a TAQMAN® probe selected from TAMRA, FAM, TET, and/or VIC. Blocker Primers [0149] In some embodiments, a blocker primer can be used, which is capable of hybridizing (e.g., is complementary to) to a sequence includes the position to be interrogated, but comprises a different sequence, e.g., a wild type sequence or reference sequence. In some embodiments, a blocker primer is capable of hybridizing with (e.g., is complementary to) a wild- type sequence. In some embodiments, a blocker primer is capable of hybridizing with (e.g., is complementary to) a reference sequence. In some embodiments, a wild type or reference sequence comprises a sequence corresponding to that of the SARS CoV-2 Wuhan-Hu1 strain or USA- WA1/2020 strain. [0150] In some embodiments, a blocker primer comprises a modification to prevent extension from occurring. In some embodiments, a blocker primer is a forward primer and includes a modification to prevent extension at its 3'-end. In some embodiments, a blocker primer is a reverse primer and includes a modification to prevent extension at its 5'-end. [0151] In some embodiments, a blocker primer comprises a modification that blocks extension. In some embodiments, a blocker primer comprises a phosphate modification (e.g., a phosphate group or phosphate ester). In some embodiments, a blocker primer comprises a modification with an inverted 3'-3' linkage, an inverted dT, a C3, or amino-C7. [0152] In some embodiments, a blocker primer comprises a wild-type forward primer can have a PO4 modification at its 3'-end to prevent extension from occurring. In some embodiments, a blocker primer comprises a wild-type forward primer can have a C3 modification at its 3'-end to prevent extension from occurring. [0153] In some embodiments, a blocker primer can compete with a variant primer that binds to a sequence ending with the position to be interrogated. The present disclosure encompasses a recognition that because a blocker primer can suppress non-specific amplification of a wild type allele by a variant primer. [0154] In some embodiments, provided are blocker primers that include: (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence of SARS CoV-2; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of the target nucleotide sequence of SARS CoV-2, which is 3' of the first portion of the target nucleotide sequence and comprises a wild type nucleotide at an interrogating nucleotide position; and (iv) a modification at its 3'-end to prevent extension from occurring. [0155] The present disclosure encompasses a recognition that a foot sequence should be short, such that a single mismatch at the interrogating nucleotide destabilizes primer binding and prevent extension, enabling discrimination of different alleles. In some embodiments, a foot sequence is designed such that the interrogating nucleotide is positioned at the terminus of the foot sequence (e.g., in the last 3 nucleotides, last 2 nucleotides, or the terminal nucleotide of the foot sequence). In some embodiments, the interrogating the corresponds to the terminal nucleotide of the foot sequence. [0156] In some embodiments, the interrogating nucleotide is a wild type nucleotide that corresponds to the nucleotide at that position of the genome of the Wuhan-Hu1 strain or USA- WA1/2020 strain. In some embodiments, the wild type nucleotide at an interrogating nucleotide position is a nucleotide that translates to a wild-type amino acid at a position selected from: N501, E484, K417, A701, and V1176. [0157] In some embodiments, a blocker primer comprises is a bubbled primer having a length within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit. In some embodiments, the lower limit may be about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, or about 25 nucleotides. In some embodiments, the upper limit may be about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, or about 60 nucleotides. [0158] In some embodiments, a blocker primer comprises is a bubbled primer that comprises an anchor sequence, a bubble sequence, and a foot sequence, wherein the overall length of the blocker primer is 25 to 55 nucleotides long. In some embodiments, a blocker primer is 30 to 40 nucleotides long. [0159] In some embodiments, a blocker primer comprises an anchor sequence, where the anchor is 15 to 40 nucleotides long. In some embodiments, an anchor sequence is 20 to 35 nucleotides long. [0160] In some embodiments, a blocker primer comprises a bubble sequence (i.e., a bridge sequence) that is 4 to 10 nucleotides in length. In some embodiments, a bubble sequence (i.e., a bridge sequence) comprises a sequence that is 6 to 8 nucleotides in length. In some embodiments, a bubble sequence comprises a sequence that is 6 nucleotides in length. In some embodiments, a bubble sequence comprises a sequence that is 8 nucleotides in length. [0161] In some embodiments, a blocker primer comprises a foot sequence that is 6 to 8 nucleotides in length. In some embodiments, a foot sequence is 6 nucleotides in length. In some embodiments, a foot sequence is 7 nucleotides in length. In some embodiments, a foot sequence is 8 nucleotides in length. [0162] In some embodiments, a blocker primer comprises a foot sequence and the interrogating nucleotide is located within the last 3 nucleotides of the foot sequence. In some embodiments, a blocker primer comprises a foot sequence and the interrogating nucleotide is located within the last 2 nucleotides of the foot sequence. In some embodiments, a blocker primer comprises a foot sequence and the interrogating nucleotide is located at the 3′ terminus of the foot sequence. [0163] In some embodiments, a blocker primer is a forward primer. In some embodiments, a blocker primer comprises a modification on its 3′ terminus that blocks extension. In some embodiments, a blocker primer comprises a phosphate modification on its 3′ terminus that blocks extension. In some embodiments, a blocker primer comprises an inverted dT modification on its 3′ terminus that blocks extension. In some embodiments, a blocker primer comprises an amino-C7 modification on its 3′ terminus that blocks extension. Control [0164] An assay for detecting SARS CoV-2 variants as provided herein may also include a control. In some embodiments, an assay for detecting SARS CoV-2 variants is a multiplex assay detecting one or more SNVs and an internal control that is targeted to the Nucleocapsid (N) gene or ORF1ab gene as an internal control. [0165] In some embodiments, a positive control primer set targets (i.e., binds to) a sequence in the SARS CoV-2 ORF1ab amplicon. [0166] In some embodiments, a positive control primer set targets (i.e., binds to) a sequence in the SARS CoV-2 nucleocapsid amplicon. [0167] Exemplary primer and probe sequences for the SARS CoV-2 internal control targeting amplicons in the N gene and ORF1ab gene are provided in the table below, which correspond to those of the FTD SARS CoV-2 Assay. In some embodiments, a positive control primer set comprises one or more primer and probe sequences of Table 2. [0168] Table 2: Primer and Probe Sequences for SARS CoV-2 Positive Control
Figure imgf000041_0001
[0169] In some embodiments, provided is positive control primer set that targets a nucleocapsid gene sequence, where the primer set includes: (i) a forward primer that comprises a sequence of SEQ ID NO.1 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.1; (ii) a reverse primer that comprises a sequence of SEQ ID NO.3 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.3; and (iii) a probe that comprises a sequence of SEQ ID NO.2 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.2. [0170] In some embodiments, a positive control primer set targets a nucleocapsid gene sequence and comprises a forward primer sequence of SEQ ID NO:1, a reverse primer sequence of SEQ ID NO: 3, and a probe sequence of SEQ ID NO.2. [0171] In some embodiments, provided is positive control primer set that targets a ORF1ab gene sequence, where the primer set includes: (i) a forward primer that comprises a sequence of SEQ ID NO. 4 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.4; (ii) a reverse primer that comprises a sequence of SEQ ID NO.6 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.6; and (iii) a probe that comprises a sequence of SEQ ID NO.5 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.5. [0172] In some embodiments, a positive control primer set targets a ORF1ab gene sequence and comprises a forward primer sequence of SEQ ID NO:4, a reverse primer sequence of SEQ ID NO: 6, and a probe sequence of SEQ ID NO.5. Exemplary Oligonucleotides [0173] In some embodiments, the present disclosure provides primers and probes for detection of one or more SARS CoV-2 variants. In some embodiments, the present disclosure provides primer sets for detection of a SARS CoV-2 variant that includes a forward primer, a reverse primer, a probe and optionally a blocker primer. In some embodiments, a primer set includes one or more oligonucleotides as described in Table 3 below. [0174] Table 3: Exemplary SARS CoV-2 Oligonucleotides
Figure imgf000042_0001
Figure imgf000043_0001
[0175] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes: a forward primer that comprises a sequence of SEQ ID NO.8 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.8. In some embodiments, provided is a primer set directed to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes: a reverse primer that comprises a sequence of SEQ ID NO.9 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.9. In some embodiments, provided is a primer set directed to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes: a blocker primer that comprises a sequence of SEQ ID NO.10 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.10, and a 3′ modification that blocks extension. In some embodiments, provided is a primer set directed to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes: a probe that comprises a sequence of SEQ ID NO.7 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.7. [0176] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes: a forward primer that comprises a sequence of SEQ ID NO.12 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.12. In some embodiments, provided is a primer set directed to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes: a reverse primer that comprises a sequence of SEQ ID NO.13 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.13. In some embodiments, provided is a primer set directed to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes: a blocker primer that comprises a sequence of SEQ ID NO.14 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.14, and a 3′ modification that blocks extension. In some embodiments, provided is a primer set directed to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes: a probe that comprises a sequence of SEQ ID NO.11 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.11. [0177] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes: a forward primer that comprises a sequence of SEQ ID NO.16 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.16. In some embodiments, provided is a primer set directed to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes: a reverse primer that comprises a sequence of SEQ ID NO.17 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.17. In some embodiments, provided is a primer set directed to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes: a probe that comprises a sequence of SEQ ID NO. 15 or a sequence that differs by no more than 2 nucleotides or no more than 1 nucleotide from SEQ ID NO.15. [0178] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 6 of Example 4 below. In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer at listed in Table 6 of Example 4 below. In some embodiments, the primer set further comprises a probe that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.7 or SEQ ID NO. 36. In some embodiments, the primer set further comprises a blocker primer that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.10 or SEQ ID NO.37. [0179] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 6 of Example 4 below; (ii) a probe that comprises or consists of a sequence of SEQ ID NO.7 or SEQ ID NO.36; and (iii) a blocker primer that comprises or consists of a sequence of SEQ ID NO.10 or SEQ ID NO.37. [0180] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501 (e.g., a SNV at nucleic acid position 23063 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 6 of Example 4 below. In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 501, where the primer set includes a forward and a reverse primer at listed in Table 6 of Example 4 below. In some embodiments, the primer set further comprises a probe that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.7 or SEQ ID NO.36. In some embodiments, the primer set further comprises a blocker primer that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.10 or SEQ ID NO.37. [0181] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 484 (e.g., a SNV at nucleic acid position 22980 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 13 of Example 6 below. In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 484 (e.g., a SNV at nucleic acid position 22980 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer at listed in Table 15 of Example 6 below. In some embodiments, the primer set further comprises a probe that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs.7, 65, and 66. [0182] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 484 (e.g., a SNV at nucleic acid position 22980 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 13 of Example 6 below; and (ii) a probe that comprises or consists of a sequence of any one of SEQ ID NOs.7, 65, or 66. In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 484 (e.g., a SNV at nucleic acid position 22980 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 15 of Example 6 below; and (ii) a probe that comprises or consists of a sequence of SEQ ID NOs.7, 65, or 66. [0183] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 417 (e.g., a SNV at nucleic acid position 22184 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 14 of Example 6 below. In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 417 (e.g., a SNV at nucleic acid position 22184 in a reference SARS CoV-2 genome), where the primer set includes a forward and a reverse primer at listed in Table 15 of Example 6 below. In some embodiments, the primer set further comprises a probe that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs.67-71. [0184] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 417 (e.g., a SNV at nucleic acid position 22184 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 14 of Example 6 below; and (ii) probe that comprises or consists of a sequence of any one of SEQ ID NOs.67-71. In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 417 (e.g., a SNV at nucleic acid position 22184 in a reference SARS CoV-2 genome), where the primer set includes (i) a forward primer and a reverse primer that each comprise or consist of a sequence listed in Table 15 of Example 6 below; and (ii) probe that comprises or consists of a sequence of any one of SEQ ID NOs.67-71. [0185] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes a forward primer, a reverse primer, and a probe that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 18 of Example 6 below. In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 1176 (e.g., a SNV at nucleic acid position 25088 in a reference SARS CoV-2 genome), where the primer set includes a forward primer, a reverse primer, and a probe that each comprise or consist of a sequence listed in Table 18 of Example 6 below. [0186] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes a forward primer, a reverse primer, and a probe that each comprise a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 19 of Example 6 below. In some embodiments, the primer set further comprises a blocker primer that comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs.14 and 90-93. [0187] In some embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 701 (e.g., a SNV at nucleic acid position 23635 in a reference SARS CoV-2 genome), where the primer set includes a forward primer, a reverse primer, and a probe that each comprise or consist of a sequence listed in Table 19 of Example 6 below. In some certain embodiments, provided is a primer set directed to a sequence that corresponds to a SNV associated with amino acid position 701, where the primer set includes a forward primer that comprises or consists of a sequence of SEQ ID NO.12, a reverse primer that comprises or consists of a sequence of SEQ ID NO.13 or 87, and a probe that comprises or consists of a sequence of SEQ ID NO.11. In some embodiments, the primer set further comprises a blocker primer that comprises or consists of a sequence of any one of SEQ ID NOs.14 and 90-93. Methods of Characterizing SARS CoV-2 Variants [0188] In some embodiments, provided herein are methods to detect and/or identify specific mutations in a sample, e.g., a SARS CoV-2 positive sample. In some embodiments, detection of one or more mutations is indicative of a particular viral variant. In some embodiments, the detection of one or more mutations is indicative of a VBM, VOI, VOC, and/or VOHC. In some embodiments, provided herein are methods to detect and/or identify a SARS CoV-2 variant in a sample. [0189] In some embodiments, the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2). In some embodiments, a SARS CoV-2 variant is alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), mu (B.1.621), and omicron (B.1.1.529), and/or a descendent lineage of any thereof. In some embodiments, a SARS CoV-2 variant is alpha (B.1.1.7), beta (B.1.351), gamma (P.1), iota (B.1.526), mu (B.1.621), and omicron (B.1.1.529, BA lineage), theta (P.3), zeta (P.2), and/or a descendent lineage of any thereof. [0190] In some embodiments, a method of the present disclosure is capable of detecting SARS CoV-2 and/or particular variants at a low concentration. In some embodiments, a method of the present disclosure is capable of detecting one or more mutations associated with a SARS CoV-2 variant with a limit of detection that is less than 50,000 viral genome copies per milliliter (cp/mL), less than 40,000 cp/mL, less than 30,000 cp/mL or less than 20 cp/m. [0191] In some embodiments, the present disclosure provides methods and/or assays for detecting and/or identifying one or more mutations associated with a SARS CoV-2 variant. [0192] In some embodiments, one or more mutations to be detected and/or identified are selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F. In some embodiments, one or more mutations to be detected and/or identified are selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the nucleotide sequence changes to be detected and/or identified are SNVs that correspond with two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. [0193] In some embodiments, one or more mutations to be detected and/or identified are selected from N501Y, A701V, and V1176F. In some embodiments, provided methods further comprise a control. In some embodiments, provided methods can detect and/or identify one or more mutations associated with a SARS CoV-2 variant and also include a positive control (e.g., amplification of another SARS CoV-2 conserved region, e.g., in the N gene and/or the ORF1ab gene). In some embodiments, provided methods detect and/or identify one or more SARS CoV-2 mutations and a control in a single sample and/or well. [0194] In some embodiments, the present disclosure provides methods and/or assays for detecting and/or identifying two or more mutations associated with a SARS CoV-2 variant. In some embodiments, two or more mutations to be detected and/or identified are selected from N501Y, A701V, and V1176F. In some embodiments, provided methods can detect and/or identify two or more mutations (e.g., SARS CoV-2 SNVs) in a single sample and/or well. In some embodiments, provided methods further comprise a control. In some embodiments, provided methods can detect and/or identify two or more mutations associated with a SARS CoV-2 variant and also include a positive control (e.g., amplification of another SARS CoV-2 conserved region, e.g., in the N gene and/or the ORF1ab gene). In some embodiments, provided methods detect and/or identify two or more SARS CoV-2 mutations and a control in a single sample and/or well. [0195] In some embodiments, the present disclosure provides methods and/or assays for detecting and/or identifying three or more mutations associated with a SARS CoV-2 variant. In some embodiments, three or more mutations to be detected and/or identified include N501Y, A701V, and V1176F. In some embodiments, provided methods can detect and/or identify three or more mutations (e.g., SARS CoV-2 SNVs) in a single sample and/or well. In some embodiments, provided methods can detect and/or identify three or more mutations associated with a SARS CoV-2 variant and also include a positive control (e.g., amplification of another SARS CoV-2 conserved region, e.g., in the N gene and/or the ORF1ab gene). In some embodiments, provided methods detect and/or identify three or more SARS CoV-2 mutations and a control in a single sample and/or well. [0196] In some embodiments, an assay or method of the present disclosure is used and/or performed in combination with whole genome sequencing. In some embodiments, a method of the present disclosure and whole genome sequencing are performed in parallel. In some embodiments, a method of the present disclosure is performed, and if a SARS CoV-2 variant is identified, then whole genome sequencing is also performed. Diagnostic Tests for SARS CoV-2 [0197] The present disclosure provides diagnostic tests for SARS CoV-2 characterized by detection of SARS CoV-2 variants using SARS CoV-2 oligonucleotides as described herein and/or according to methods as described herein. [0198] In some embodiments, methods of detecting and/or diagnosing SARS CoV-2, and particularly identifying or characterizing the SARS CoV-2 variants, as taught by the present disclosure are improved methods as compared to standard techniques. In some embodiments, methods of the present disclosure include one or more of the following benefits: improved sensitivity for identifying SARS CoV-2 variants, improved specificity for identifying SARS CoV- 2 variants, improved accuracy for identifying SARS CoV-2 variants, reduced time to diagnosis for SARS CoV-2 variants, and/or reduced cost of screening patients for SARS CoV-2 variants. [0199] In some embodiments, methods of detecting and/or diagnosing SARS CoV-2 as taught by the present disclosure provide the advantage of an early screen for the presence of particular SARS CoV-2 variants in a sample. [0200] In some embodiments, technology disclosed herein (e.g., disclosed oligonucleotides and/or methods) can be used for diagnosing COVID-19. In some embodiments, a diagnostic test as taught by the present disclosure can assist in the detection or diagnosis of SARS CoV-2 in a subject. In some embodiments, technology disclosed herein is used for diagnosing a subject with infection with a particular SARS CoV-2 variant. [0201] In some embodiments, a diagnostic test as taught by the present disclosure detects whether one or more SARS CoV-2 mutations are present in a sample obtained from a subject. In some embodiments, a sample obtained from a subject comprises a bodily fluid. In some embodiments, a bodily fluid is or comprises saliva, respiratory secretion (e.g., from a nasal swab), blood, and/or plasma. In some embodiments, a sample of respiratory secretion obtained from a subject for use in accordance with methods described herein is obtained by an anterior nares (nasal) swab, a mid-turbinate nasal swab, a nasopharyngeal swab, and/or an oropharyngeal swab. In some embodiments, a sample of saliva from a subject for use in accordance with methods described herein is obtained by having a subject spit into a receptacle (e.g., a tube). [0202] In some embodiments, a sample for use in the accordance with provided technologies is a sample of RNA isolated from a subject. In some embodiments, a sample is a sample of RNA isolated from a nasopharyngeal and/or oropharyngeal swab from a subject. [0203] In some embodiments, a subject is diagnosed with an infection with a particular SARS CoV-2 variant using a method described herein and then treated for COVID-19. In some embodiments, the treatment for COVID-19 may be selected and/or adjusted based on the SARS CoV-2 variant detected in a subject. Methods of Use and Treatment [0204] In some embodiments, the present disclosure provides methods of and/or uses for identifying a subject for treatment, wherein the subject has been diagnosed infection with a particular SARS CoV-2 variant. In some embodiments, the treatment differs based on what particular SARS CoV-2 variant. [0205] In some embodiments, provided are methods of and/or uses for identifying a subject for treatment, where the subject has been diagnosed infection with a SARS CoV-2 variant, said method or use comprising (i) identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject, (ii) determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified, and (iii) administering therapy according the determined SARS CoV-2 variant strain(s). In some embodiments, the nucleotide sequence changes (e.g., SNVs) identified correspond with mutations selected from: one or more mutations selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond with mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs selected from: A23063T, A22980G, G22184T, C23635T, and G25088T. In some embodiments, the nucleotide sequence changes are SNVs that correspond with two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are two or more SNVs selected from: A23063T, A22980G, G22184T, C23635T, and G25088T. [0206] In some embodiments, the nucleotide sequence changes are SNVs that correspond to one or more mutations to be detected are selected from N501Y, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs selected from: A23063T, C23635T, and G25088T. In some embodiments, the nucleotide sequence changes are SNVs that correspond to each of the mutations N501Y, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond to each of A23063T, C23635T, and G25088T relative to a reference SARS CoV-2 genome. [0207] In some embodiments, provided are uses of an assay or method as described herein for identifying a subject infected with a SARS CoV-2 variant. In some embodiments, said assay or method for use comprises identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject. In some embodiments, said assay or method for use further comprises determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified. [0208] In some embodiments, provided are methods of and/or uses for identifying the predominant SARS CoV-2 variant in a population of subjects, said method and/or use comprising identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV- 2 variant(s) in samples from the population of subjects. In some embodiments, the method and/or use further comprises determining the predominant SARS CoV-2 variant strain(s) based on the nucleotide sequence changes identified in the samples from the population of subjects. [0209] In some embodiments, the nucleotide sequence changes (e.g., SNVs) identified correspond with mutations selected from: one or more mutations selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond with mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond with two or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond to one or more mutations to be detected are selected from N501Y, A701V, and V1176F. In some embodiments, the nucleotide sequence changes are SNVs that correspond to each of the mutations N501Y, A701V, and V1176F. [0210] In some embodiments, the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2). In some embodiments, a SARS CoV-2 variant is alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), mu (B.1.621), and omicron (B.1.1.529), and/or a descendent lineage of any thereof. In some embodiments, a SARS CoV-2 variant is alpha (B.1.1.7), beta (B.1.351), gamma (P.1), iota (B.1.526), mu (B.1.621), and omicron (B.1.1.529), theta (P.3), zeta (P.2), and/or a descendent lineage of any thereof. [0211] In some embodiments, provided are methods of selecting a vaccine for a patient based on the predominant SARS CoV-2 variant in the population. [0212] In some embodiments, provided are methods of selecting a vaccine for a population of patients, wherein the predominant SARS CoV-2 variant in the population is identified using a method described herein. Kits [0213] Also provided by the present disclosure are kits comprising one or more oligonucleotides of the present disclosure and instructions for use (e.g., diagnostic use). In some embodiments, a kit of the disclosure is used for an in vitro diagnostic assay to diagnose SARS CoV-2 infection (e.g., with a particular variant). [0214] In some embodiments, a kit of the present disclosure comprises one or more variant primers described herein, along with instructions for use. In some embodiments, a kit of further includes one or more complementary primers. In some embodiments, a kit further includes one or more blocker primers. In some embodiments, a kit includes one or more primer sets described herein. In some embodiments, a kit includes one or more probes. [0215] In some embodiments, a kit of the present disclosure comprises a primer set for detecting one or more variants of SARS CoV-2. In some embodiments, a kit of the present disclosure comprises a primer set for detecting one or more variants of SARS CoV-2 selected from: N501Y, E484K, K417N, A701V, and V1176F. In some embodiments, a kit of the present disclosure further comprises a primer set for detecting a conserved region of SARS CoV-2 (i.e., a positive control). [0216] In some embodiments, a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 N501Y variant. In some embodiments, the kit comprises a variant primer that targets the N501Y genomic region and a complementary primer. In some embodiments, the kit comprises a blocker primer that blocks amplification of a N501 wt sequence. In some embodiments, the kit further comprises a probe. [0217] In some embodiments, a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 E484K variant. In some embodiments, the kit comprises a variant primer that targets the E484K genomic region and a complementary primer. In some embodiments, the kit further comprises a probe. [0218] In some embodiments, a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 K417N variant. In some embodiments, the kit comprises a variant primer that targets the K417N genomic region and a complementary primer. In some embodiments, the kit further comprises a probe. [0219] In some embodiments, a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 A701V variant. In some embodiments, the kit comprises a variant primer that targets the A701V genomic region and a complementary primer. In some embodiments, the kit comprises a blocker primer that blocks amplification of a A701 wt sequence. In some embodiments, the kit further comprises a probe. [0220] In some embodiments, a kit of the present disclosure comprises a primer set for detecting a SARS CoV-2 V1176F variant. In some embodiments, the kit comprises a variant primer that targets the V1176F genomic region and a complementary primer. In some embodiments, the kit further comprises a probe. [0221] In addition to the above, the composition of a kit of the disclosure can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and/or an agent for treating a condition or disorder described herein (e.g., COVID-19). [0222] In some embodiments, a kit further comprises a ROX reference dye. A ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization in a real-time qPCR assay. In some embodiments, a ROX reference dye is present in an amount to generate a fluorescent signal in a range from about 50,000 to about 500,000. In some embodiments, a ROX reference dye is present in an amount to generate a fluorescent signal in a range from about 100,000 to about 250,000. In some certain embodiments, a ROX reference dye is present in an amount of 0.1X per reaction. [0223] In certain embodiments, kits for use in accordance with the present disclosure may include, a reference or control sample(s), instructions for processing samples, performing tests on samples, instructions for interpreting the results, buffers and/or other reagents necessary for performing tests. [0224] In some embodiments, provided are kits for detection of one or more SARS CoV- 2 variants in a sample, where the results of the kit are used to inform treatment. For example, in some embodiments, a kit provided herein detects SNVs N501Y or E484K + control and K417N, A701V, V1176F + control. Such a kit can detect five SARS CoV-2 variants B.1.1.7, B.1.525, B.1.351, B.1.526, and P.1 and differentiate four of these variants. Certain References [0225] Investigation of novel SARS-COV-2 variant: 202012/01. Technical briefing 1, https://www.gov.uk/government/publications/investigation-of-novel-sars-cov-2-variant-variant- of-concern-20201201 [0226] Investigation of novel SARS-COV-2 variant: 202012/01. Technical briefing 2, https://www.gov.uk/government/publications/investigation-of-novel-sars-cov-2-variant-variant- of-concern-20201201 [0227] Rambaut A et al, A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology, Nature Microbiol. V5, Nov 2020, 1403-1407 [0228] Yang X et al, Genetic cluster analysis of SARS-CoV-2 and the identification of those responsible for the major outbreaks in various countries, Emerging Microbes & Infections, 9:1, 1287-1299. [0229] New evidence on VUI-202012/01 and review of the public health risk assessment“, Uni Edingburg and Sanger Institute public release [0230] European Centre for Disease Prevention and Control. Rapid increase of a SARS- CoV-2 variant with multiple spike protein mutations observed in the United Kingdom –20 December 2020. [0231] Schoenbrunner NJ et al, Covalent modification of primers improves PCR amplification specificity and yield, Biology Methods and Protocols, 2017, 1–10. EXEMPLARY EMBODIMENTS [0232] Embodiment 1. A SARS CoV-2 variant primer comprising: (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; and (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of a target nucleotide sequence, which is 3' of the first portion of the target nucleotide sequence and comprises an interrogating nucleotide position, wherein the interrogating nucleotide position corresponds to a SARS CoV-2 variant nucleotide. [0233] Embodiment 2. The variant primer of embodiment 1, wherein the anchor is 15 to 40 nucleotides long, optionally wherein the anchor is 20 to 35 nucleotides long. [0234] Embodiment 3. The variant primer of embodiment 1 or 2, wherein the bridge is 4 to 10 nucleotides long, optionally wherein the bridge is 6 to 8 nucleotides long. [0235] Embodiment 4. The variant primer of any one of embodiments 1 to 3, wherein the foot is 6 to 8 nucleotides long, optionally wherein the foot is 7 nucleotides long. [0236] Embodiment 5. The variant primer of any one of embodiments 1 to 4, wherein the primer is 25 to 55 nucleotides long, optionally wherein the primer is 30 to 40 nucleotides long. [0237] Embodiment 6. The variant primer of any one of embodiments 1 to 5, wherein the primer is DNA. [0238] Embodiment 7. The variant primer of any one of embodiments 1 to 6, wherein the primer comprises a label or a tag. [0239] Embodiment 8. The variant primer of any one of embodiments 1 to 7, wherein the interrogating nucleotide is a nucleotide that corresponds to an amino acid substitution selected from: N501Y, E484K, K417N, A701V, and V1176F. [0240] Embodiment 9. The variant primer of any one of embodiments 1 to 8, wherein the target nucleotide sequence is a SARS CoV-2 genomic RNA sequence. [0241] Embodiment 10. The variant primer of any one of embodiments 1 to 8, wherein the target nucleotide sequence corresponds to a SARS CoV-2 S gene sequence or portion thereof. [0242] Embodiment 11. The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2). [0243] Embodiment 12. The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha, beta, eta, gamma, iota, mu, omicron, theta, zeta, and C.1.2. [0244] Embodiment 13. The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is an alpha variant, variant, beta variant, gamma variant, mu variant, theta variant, C.1.2 variant, or omicron variant. [0245] Embodiment 14. The variant primer of embodiment 13, wherein the variant nucleotide sequence comprises a sequence that corresponds to an N501Y mutation. [0246] Embodiment 15. The variant primer of embodiment 13 or 14, wherein the variant nucleotide sequence comprises A23063T nucleotide substitution. [0247] Embodiment 16. The variant primer of any one of embodiments 1 to 15, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8, 20-22, and 29-35. [0248] Embodiment 17. The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant or iota variant. [0249] Embodiment 18. The variant primer of embodiment 17, wherein the variant nucleotide sequence comprises a sequence that corresponds to an A701V mutation. [0250] Embodiment 19. The variant primer of embodiment 17 or 18, wherein the variant nucleotide sequence comprises a C23635T nucleotide substitution. [0251] Embodiment 20. The variant primer of any one of embodiments 1 to 10 or 17 to 19, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86. [0252] Embodiment 21. The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a gamma variant, zeta variant, or theta variant. [0253] Embodiment 22. The variant primer of embodiment 21, wherein the variant nucleotide sequence comprises a sequence that corresponds to a V1176F mutation. [0254] Embodiment 23. The variant primer of embodiment 21 or 22, wherein the variant nucleotide sequence comprises a G25088T nucleotide substitution. [0255] Embodiment 24. The variant primer of any one of embodiments 1 to 10 or 21 to 23, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16, 72-74, and 78-80. [0256] Embodiment 25. The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, gamma variant, eta variant, or mu variant. [0257] Embodiment 26. The variant primer of embodiment 25, wherein the variant nucleotide sequence comprises a sequence that corresponds to an E484K mutation. [0258] Embodiment 27. The variant primer of embodiment 25 or 26, wherein the variant nucleotide sequence comprises a A22980G nucleotide substitution. [0259] Embodiment 28. The variant primer of any one of embodiments 1 to 10 or 25 to 27, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38, 41-44, 48, and 50. [0260] Embodiment 29. The variant primer of any one of embodiments 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, mu variant, or delta variant. [0261] Embodiment 30. The variant primer of embodiment 29, wherein the variant nucleotide sequence comprises a sequence that corresponds to a K417N mutation. [0262] Embodiment 31. The variant primer of embodiment 29 or 30, wherein the variant nucleotide sequence comprises a G22184T nucleotide substitution. [0263] Embodiment 32. The variant primer of any one of embodiments 1 to 10 or 29 to 31, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55 and 62-64. [0264] Embodiment 33. A primer pair comprising: the variant primer of any one of embodiments 1 to 32, and a complementary primer comprising or consisting of a nucleotide sequence that is complementary to a portion of a complementary strand sequence of the target nucleotide sequence. [0265] Embodiment 34. A primer pair comprising: the variant primer of embodiment 16, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-28. [0266] Embodiment 35. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24. [0267] Embodiment 36. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 20-22, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 25-28. [0268] Embodiment 37. A primer pair comprising: the variant primer of embodiment 20, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88. [0269] Embodiment 38. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88. [0270] Embodiment 39. A primer pair comprising: the variant primer of embodiment 24, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17 and 74-77. [0271] Embodiment 40. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16 and 72-73, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17, 76, and 77. [0272] Embodiment 41. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 78-80, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 74-75. [0273] Embodiment 42. A primer pair comprising: the variant primer of embodiment 28, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40, 45-47, and 49. [0274] Embodiment 43. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38 and 41-44, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40 and 45-46. [0275] Embodiment 44. A primer pair comprising: a variant primer comprises or consists of a sequence according to SEQ ID NO: 48 or 50, and a complementary primer comprising or consisting of a nucleotide sequence according to SEQ ID NO: 47 or 49. [0276] Embodiment 45. A primer pair comprising: the variant primer of embodiment 32, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61 and 56-58. [0277] Embodiment 46. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 56-58. [0278] Embodiment 47. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 62-64, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61. [0279] Embodiment 48. A blocker primer comprising (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence of SARS CoV-2; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of the target nucleotide sequence of SARS CoV-2, which is 3' of the first portion of the target nucleotide sequence and comprises a wild type nucleotide at an interrogating nucleotide position; and (iv) a modification at its 3'-end to prevent extension from occurring. [0280] Embodiment 49. The blocker primer of embodiment 48, wherein the wild type nucleotide at an interrogating nucleotide position is the nucleotide at that corresponding position of the genome of the Wuhan-Hu1 strain or USA-WA1/2020 strain. [0281] Embodiment 50. The blocker primer of embodiment 48 or 49, wherein the target nucleotide sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome but not a wild-type SARS CoV-2 genome, wherein the SARS CoV-2 variant is an alpha variant, variant, beta variant, gamma variant, mu variant, theta variant, C.1.2 variant, or omicron variant. [0282] Embodiment 51. The blocker primer of any one of embodiments 48 to 50, wherein the modification that prevents extension is or comprises: a PO4 modification, an inverted 3'-3' linkage, an inverted dT, a C3 modification, or an amino-C7 modification. [0283] Embodiment 52. The blocker primer of any one of embodiments 48 to 51, wherein the modification that prevents extension is or comprises: a PO4 modification or a C3 modification. [0284] Embodiment 53. The blocker primer of any one of embodiments 48 to 52, wherein the anchor is 15 to 40 nucleotides long, optionally wherein the anchor is 20 to 35 nucleotides long. [0285] Embodiment 54. The blocker primer of any one of embodiments 48 to 53, wherein the bridge is 4 to 10 nucleotides long, optionally wherein the bridge is 6 to 8 nucleotides long. [0286] Embodiment 55. The blocker primer of any one of embodiments 48 to 54, wherein the foot is 6 to 8 nucleotides long, optionally wherein the foot is 7 nucleotides long. [0287] Embodiment 56. The blocker primer of any one of embodiments 48 to 55, wherein the primer is 25 to 55 nucleotides long, optionally wherein the primer is 30 to 40 nucleotides long. [0288] Embodiment 57. The blocker primer of any one of embodiments 48 to 56, wherein the primer is DNA. [0289] Embodiment 58. The blocker primer of any one of embodiments 48 to 57, wherein the wild type nucleotide at an interrogating nucleotide position is a nucleotide that translates to a wild-type amino acid at a position selected from: N501, E484, K417, A701, and V1176. [0290] Embodiment 59. The blocker primer of any one of embodiments 48 to 58, wherein the interrogating nucleotide position is a nucleotide that translates to a wild-type amino acid at a position N501. [0291] Embodiment 60. The blocker primer of embodiment 59, wherein the blocker primer comprises or consists of a sequence according to SEQ ID NO: 10 or 37. [0292] Embodiment 61. The blocker primer of any one of embodiments 48 to 58, wherein the interrogating nucleotide position is a nucleotide that translates to a wild-type amino acid at a position A701. [0293] Embodiment 62. The blocker primer of embodiment 59, wherein the blocker primer comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93. [0294] Embodiment 63. A primer set comprising: the variant primer of any one of embodiments 1 to 32, and a probe. [0295] Embodiment 64. A primer set comprising: the primer pair of any one of embodiments 33 to 47, and a probe. [0296] Embodiment 65. The primer set of embodiment 63 or 64, wherein the probe comprises or consists of a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89. [0297] Embodiment 66. A primer set comprising: the primer pair of any one of embodiments 34-36, and a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 7 or 36. [0298] Embodiment 67. A primer set comprising: the primer pair of any one of embodiments 37-38, and a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 11 or 89. [0299] Embodiment 68. A primer set comprising: the primer pair of any one of embodiments 39-41, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 15 or 81-83. [0300] Embodiment 69. A primer set comprising: the primer pair of any one of embodiments 42-44, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 7 and 65-66. [0301] Embodiment 70. A primer set comprising: the primer pair of any one of embodiments 45-47, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 67-71. [0302] Embodiment 71. A primer set comprising: the variant primer of any one of embodiments 1 to 32, and the blocker primer of any one of embodiments 48 to 62. [0303] Embodiment 72. A primer set comprising: the primer pair of any one of embodiments 33 to 47, and the blocker primer of any one of embodiments 48 to 62. [0304] Embodiment 73. The primer set of embodiment 71 or 72, wherein the variant primer and the blocker primer sequences differ only at the nucleotide that anneals to the interrogating nucleotide position, and that the blocker primer comprises the modification that prevents extension. [0305] Embodiment 74. A primer set comprising: the variant primer of embodiment 16, and a blocker primer comprises or consists of a sequence according to SEQ ID NO: 10 or 37. [0306] Embodiment 75. A primer set comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and a blocker primer comprises or consists of a sequence according to SEQ ID NO: 10 or 37. [0307] Embodiment 76. The primer set of embodiments 74 or 75, further comprising: a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24. [0308] Embodiment 77. The primer set of any one of embodiments 74 to 76, wherein the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to a nucleotide of N501Y in the variant primer and a nucleotide that translates to a wild-type amino acid at a position N501 in the blocker primer. [0309] Embodiment 78. A primer set comprising: the variant primer of embodiment 20, and a blocker primer comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93. [0310] Embodiment 79. A primer set comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and a blocker primer comprises or consists of a sequence according to any one of SEQ ID NOs: 14 and 90-93. [0311] Embodiment 80. The primer set of embodiments 74 or 75, further comprising: a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88. [0312] Embodiment 81. The primer set of any one of embodiments 74 to 76, wherein the variant primer and the blocker primer comprise each comprise an interrogating nucleotide, which corresponds to a nucleotide of A701V in the variant primer and a nucleotide that translates to a wild-type amino acid at a position A701 in the blocker primer. [0313] Embodiment 82. A mixture comprising: the variant primer of any one of embodiments 1 to 32, and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence. [0314] Embodiment 83. A mixture comprising: the primer pair of any one of embodiments 33 to 47, and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence. [0315] Embodiment 84. A mixture comprising: the primer set of any one of embodiments 63 to 81, and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence. [0316] Embodiment 85. A method comprising: contacting a sample from a subject with the variant primer of any one of embodiments 1 to 32, the primer pair of any one of embodiments 33 to 47, or the primer set of any one of embodiments 63 to 81; and amplifying a target nucleotide sequence from the sample. [0317] Embodiment 86. The method of embodiment 85, wherein the target nucleotide sequence is a SARS CoV-2 nucleotide sequence. [0318] Embodiment 87. The method of embodiment 85 or 86, wherein the method is to detect and/or identify a SARS CoV-2 variant in a sample comprising SARS CoV-2 nucleic acid. [0319] Embodiment 88. The method of embodiment 87, wherein the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2). [0320] Embodiment 89. The method of embodiment 85 or 86, wherein the method is to detect and/or identify one or more mutations in a sample comprising SARS CoV-2 nucleic acid. [0321] Embodiment 90. The method of embodiment 89, wherein the one or more mutations are selected from: L18F, del69v70, D80A, delY144, D215G, L242_244L, R246I, K417N/T, N439K, E484K, N501Y, A570D, D614G, P681H, A701V, T716I, and V1176F. [0322] Embodiment 91. The method of embodiment 89, wherein the one or more mutations are selected from: N501Y, E484K, K417N, A701V, and V1176F. [0323] Embodiment 92. The method of embodiment 91, wherein the one or more mutations comprise three or more mutations selected from: N501Y, E484K, K417N, A701V, and V1176F. [0324] Embodiment 93. The method of embodiment 89, wherein the one or more mutations are selected from: N501Y, A701V, and V1176F. [0325] Embodiment 94. The method of embodiment 93, wherein the one or more mutations are at least three mutations that comprise at least N501Y, A701V, and V1176F. [0326] Embodiment 95. The method of any one of embodiments 87 to 94, wherein the SARS CoV-2 in the sample is present in an amount that is less than 50,000 viral genome copies per milliliter (cp/mL), less than 40,000 cp/mL, less than 30,000 cp/mL or less than 20 cp/mL. [0327] Embodiment 96. The method of any one of embodiments 85 to 95, wherein the method further comprises contacting the sample from the subject with a positive control primer set. [0328] Embodiment 97. The method of embodiment 96, wherein the positive control primer set targets a sequence in the SARS CoV-2 Nucleocapsid (N) gene and/or ORF1ab gene. [0329] Embodiment 98. The method of any one of embodiments 85 to 97, wherein the method further comprises performing whole genome sequencing on the sample. [0330] Embodiment 99. A method of identifying a subject for treatment, comprising (i) identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject, (ii) determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified, and (iii) administering therapy according to the determined SARS CoV-2 variant strain(s), wherein the step of identifying one or more nucleotide sequence changes comprises contacting the sample with the variant primer of any one of embodiments 1 to 32, the primer pair of any one of embodiments 33 to 47, or the primer set of any one of embodiments 63 to 81. [0331] Embodiment 100. A method of identifying a predominant SARS CoV-2 variant in a population of subjects, said method comprising identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in samples from the population of subjects, wherein the identifying comprises contacting the samples with the variant primer of any one of embodiments 1 to 32, the primer pair of any one of embodiments 33 to 47, or the primer set of any one of embodiments 63 to 81. [0332] Embodiment 101. A kit comprising: the variant primer of any one of embodiments 1 to 32, the primer pair of any one of embodiments 33 to 47, or the primer set of any one of embodiments 63 to 81; and [0333] instructions for use. [0334] Embodiment 102. The kit of embodiment 101, further comprising a positive control primer set. [0335] Embodiment 103. The kit of embodiment 102, wherein the positive control primer set targets a sequence in the SARS CoV-2 Nucleocapsid (N) gene and/or ORF1ab gene. EXEMPLIFICATION [0336] The present examples together describe development and characterization of assays for detecting SARS CoV-2 variants, e.g., particular sequence changes associated with such variants, including single nucleotide variants (SNVs). The following examples will describe the design, screening, and characterization of exemplary SARS CoV-2 SNVs. Example 1: Strategy for Selecting Mutations for Identifying B.1.1.7 and Other SARS CoV- 2 Variants [0337] SARS CoV-2 variants can include multiple mutations. For example, the B.1.1.7 (alpha) variant is defined by 23 mutations from the original SARS CoV-2 strain; these mutations include 4 deletions, 6 synonymous mutations, and 13 non-synonymous mutations. The non- synonymous mutations include, among others, a series of mutations in the spike protein and a stop codon in ORF8, which are summarized in Table 4 below. There are 6 synonymous mutations with 5 in ORF1ab (C913T, C5986T, C14676T, C15279T, C16175T), and one in the M gene (T26801C). This is an unusually large number of mutations in a single cluster. [0338] Table 4: Lineage-defining protein altering mutations defining the B1.1.7 (alpha) SARS CoV-2 variant
Figure imgf000070_0001
Figure imgf000071_0001
[0339] Developing an assay with high specificity for single nucleotide variants (SNVs) is more challenging than detecting deletions or insertions (indels). [0340] After assessment of all the clades classified in the GISAID database (https://www.gisaid.org/), and assessment of certain sequences in the NCBI nucleotide database, one characteristic deletion, with two adjacent SNVs, were found to be most specifically associated with the B1.1.7 (alpha) variant/lineage. [0341] In some embodiments, a two-target assay (possibly for two deleted regions) may achieve better specificity for B1.1.7, but not for other variants. [0342] A two-target assay (e.g., for 1 deleted region and 1 SNV) may have better specificity for B1.1.7 and other variants (e.g., B1.351 in South Africa and P.1 in Brazil). For example, a two target assay for detecting B1.1.7 variant can target an SGF 3675-3677 deletion and an N501Y mutation. An exemplary schematic for primer design for such a two target assay is provided in FIG.5 [0343] The N501Y single nucleotide variation (SNV) or mutation was selected as a target for assay development. Example 2: Selecting SNVs for Identifying Other SARS CoV-2 Variants [0344] This example describes selection of other mutations (e.g., SNVs) for detecting SARS CoV-2 variants. A listing of exemplary SARS CoV-2 mutations is provided in the Table # below. This table also describes the effects associated with these SNVs, identifies certain variants associated with these SNVs, and also proposes assay options. [0345] Table 5: Exemplary SARS CoV-2 mutations and associated variants
Figure imgf000072_0001
[0346] Each of the exemplary mutations listed in Table 5 above is located in the SARS CoV-2 spike protein encoding region (S gene), as illustrated in FIG. 1C. The K417N, E484K, N501Y are located in close proximity to one another. As such, the present disclosure recognizes that detection of these mutations cannot be combined in a single well. Different options for combinations of variants were considered. [0347] Option 1: Single tube / N501Y, A701V, V1176F + control
Figure imgf000072_0002
Figure imgf000073_0001
[0348] In an assay according to Option 1 as illustrated above, four variants will be detected (B.1.1.7, B.1.351, B.1.526, and P.1) and 4 variants differentiated. [0349] Option 2: Tube 1 N501Y or E484K + control and Tube 2 K417N, V1176F + control
Figure imgf000073_0002
Figure imgf000073_0003
[0350] In an assay according to Option 2 as illustrated above, three variants will be detected (B.1.1.7, B.1.351and P.1) and 3 variants differentiated using N501Y in Tube 1. Four variants will be detected (B.1.525, B.1.351, B.1.526, and P.1) and 2+1+1 variants differentiated using E484K in Tube 1. B.1.525 (UK new), and B.1.526 (New York) variants can be detected but not differentiated. [0351] Option 3: Tube 1 N501Y or E484K + control and Tube 2 K417N, A701V, V1176F + control
Figure imgf000074_0001
Figure imgf000074_0002
[0352] In an assay according to Option 3 as illustrated above, four variants will be detected (B.1.1.7, B.1.351, B.1.526, and P.1) and 4 variants differentiated using N501Y in Tube 1. Four variants will be detected (B.1.525, B.1.351, B.1.526, and P.1) and 4 variants differentiated using E484K in Tube 1. [0353] However, developing an assay with high specificity for one or more SNVs is more challenging than detecting deletions or insertions (indels). [0354] The mutation N501Y was selected as an initial mutation to move forward with for development of an exemplary SARS CoV-2 variant detection assay. Example 3: Primer Set Design [0355] This example describes the design of primer set to achieve a highly selective and sensitive assay for detection of particular SAR CoV-2 mutation, e.g., a N501Y SNV mutation, among others. Such a primer set includes a variant primer with a bubbled primer design. For certain SARS CoV-2 mutations, e.g., a N501Y SNV mutation, a blocker primer directed to a wild- type sequence was also used. A schematic of an exemplary bubbled primer design is provided in FIG. 3. As depicted, an exemplary N501Y bubbled primer includes, in order from 5′ to 3′, an anchor sequence, a bubble or bridge sequence, and a foot sequence. The foot sequence of the exemplary N501Y primer overlaps with (i.e., includes a sequence that is complementary to) the N501Y mutation (represented by “X” in the schematic). Moreover, the foot sequence was designed to be short, such that a single mismatch at the terminal 3′ nucleotide destabilizes primer binding and prevent extension, thereby enabling discrimination of different alleles (e.g., wild type and N501Y). [0356] The exemplary N501Y bubbled primer was designed as part of a primer set that also includes a wild-type (WT) blocker primer, a reverse primer, and a probe, for example as depicted in FIG. 4. The exemplary WT blocker primer is capable of hybridizing with (e.g., is complementary to) a sequence encompassing the position to be interrogated, in this case N501. The exemplary WT blocker primer also comprises a PO4 modification at its 3'-end to prevent extension from occurring. The present disclosure recognizes that such a WT blocker primer can compete with a variant primer and suppress non-specific amplification of a wild-type allele by a variant primer. [0357] A primer set can also include a third primer, which is used together with a WT blocker primer and a variant primer. For example, if a WT blocker primer and a variant primer are forward primers, a third primer can be a reverse primer. In some embodiments, a probe can be used, which is capable of hybridizing with (e.g., is complementary to) a sequence amplified using a primer set as shown in FIG.4. [0358] A two target assay for detecting a B1.1.7 variant was also tested that targeted an SGF 3675-3677 deletion and an N501Y mutation. An exemplary schematic for primer design for such a two target assay is provided in FIG.5 A number of bubbled primer designs were tested and screened that target the SGF 3675-3677 deletion in ORF1ab. Specifically, 8 bubbled primer combinations specific for ORF1ab SGF 3675-3677 deletion were screening using EVA green and RNA transcript at 1E+05 copies/reaction (data not shown), as described for N501Y screening above, 4 of which were selected to screen with probes. However, bubbled primer/probe combinations directed to SGF 3675-3677 deletion in ORF1ab were not selective to mutant template. [0359] The examples below describe the development and characterization of assays for detection of different SARS CoV-2 SNVs. Example 4: Development and Characterization of Exemplary Assay for Detection of N501Y [0360] This example describes development and characterization of an exemplary assay for detecting a SARS CoV-2 N501Y mutation. A schematic flow chart overview of a process for a characterizing this exemplary SNV (N501Y) is provided in FIG.6. [0361] In vitro transcription [0362] RNA transcripts of wild type and mutant variant sequences were generated from double-stranded gBlock templates obtained from IDT. RNA transcription was performed using a T7 in vitro transcription kit from gBlock templates. [0363] Sequence of gBlock with N501Y nucleotide change for B117, A570D, P681H, T716I-Mutant (22473-23492) 1020bp; M13 primer sites are underlined; T7 transcription start is in bold; nucleotide change for N501Y substitution is in bold and underlined.
Figure imgf000076_0001
Figure imgf000077_0001
[0364] Following in vitro transcription, dNTPs and genomic DNA were removed using a commercially available RNA clean up kit. The quantity of RNA transcript was determined by Nanodrop spectrophotometry and the size confirmed by gel electrophoresis. RNA transcript of the correct size were generated and used for subsequent oligonucleotide screening. [0365] Bubbled Primer Screening [0366] The following bubbled primers as listed in Table 6 were screened for their ability to specifically detect mutant templates of N501Y (bold indicates interrogated nucleotide (N501Y SNV). [0367] Table 6: Exemplary primers targeting a sequence that corresponds to SNV N501Y Name Sequence SEQ ID SNV-Mut Rev 1 AGTACTACTACTCTGTATGGTTGGTTTGGTTCACCATA 20 SNV-Mut Rev 2 AGAAAGTACTACTACTCTGTATGGTTGCATTGGTTCACCATA 21 SNV-Mut Rev 3 GCTGGTGCATGTAGAAGTTCAAATTGGTTCACCATA 22 SNV-Rev 4 AGTTGCTGGTGCATGTAGAA 9 SNV-Rev 5 GTTGCTGGTGCATGTAGAA 23 SNV-Rev 6 ACAGTTGCTGGTGCATGTAGA 24 SNV-Rev 7 AGTTGCTGGTGCATGTAGAA 9 SNV-For 1 CCGGTAGCACACCTTGTAAT 25 SNV-For 2 CACACCTTGTAATGGTGTTGAAG 26 SNV-For 3 TATCAGGCCGGTAGCACA 27 SNV-For 4 CCGGTAGCACACCTTGTAAT 28 SNV-Mut For 5 TTGTTACTTTCCTTTACAATCATATGGTCCTTCGCCCACTT 8 SNV-Mut For 6 GTTACTTTCCTTTACAATCATATGGTCCTTCGCCCACTT 29 SNV-Mut For 7 GTTACTTTCCTTTACAATCATATGGCGGTTGGTCCACTTA 30 SNV-Mut For 8 GCACACCTTGTAATGGTGTTGAAGGCGCGCCCCACTT 31 SNV-Mut For 9 CCTTGTAATGGTGTTGAAGGTTTGCGCGCCCCACTT 32 SNV-Mut For 10 TTGTTACTTTCCTTTACAATCATATGGTTCTTCGTCCACTTA 33 SNV-Mut For 11 GTTACTTTCCTTTACAATCATATGGTTCTTCGTCCACTTA 34 SNV-Mut For 12 TGTTACTTTCCTTTACAATCATATGTCGGTTGGCCCACTT 35 [0368] In vitro transcribed RNA transcripts with the N501Y mutation were used as template for screening. Bubbled primers were screened using EVAgreen chemistry for RT-PCR to determine if bubbled primers generated signal from mutant templates. Specifically, 32 primer combinations were screened with N501Y mutant RNA transcript in decreasing amounts: 100,000 copies/reaction, 25,000 copies/reaction, 6,250 copies/reaction, and no template control (NTC). [0369] Table 7: N501Y Primer Combinations and Amplification Results
Figure imgf000078_0001
Figure imgf000079_0001
[0370] This demonstrated that bubbled primers were able to strongly amplify mutant templates of N501Y. The linearity and PCR efficiency were also evaluated for selected primer combinations to move forward with for probe screening (data not shown). 8 primer combinations for N501Y were selected to move forward for probe screening, which were: SNV-MutFor5/SNV- Rev4, SNV-MutFor5/SNV-Rev5, SNV-MutFor5/SNV-Rev6, SNV-MutFor5/SNV-Rev7, SNV- MutFor6/SNV-Rev4, SNV-MutFor6/SNV-Rev5, SNV-MutFor6/SNV-Rev6, and SNV- MutFor6/SNV-Rev7. This experiment demonstrates the successful design of numerous bubbled primers for detection of the SNV associated with the SARS CoV-2 N501Y mutation. Exemplary bubbled primers that strongly amplified this SNV included forward primers, with a bubble sequence that is 6-8 nucleotides in length, and a nucleic acid to be interrogated in the last two nucleotides of a foot sequence. [0371] Probe Screening [0372] Probes for pairing with the selected bubbled primer combinations were screened to determine combinations for detection of N501Y mutation and for specificity thereto. Specifically, 16 bubbled primer/probe combinations were screened using the following templates: N501Y mutant RNA transcript, lysed inactivated wild-type (WT) virus and combined N501Y mutant and WT template (1E+05 copies/reaction each). Lower amounts of N501Y mutant RNA template were also screened. [0373] Each of the above primer combinations were screened with the following probes: [0374] SNV-Probe 2-FAM TGGTGTTGGTTACCAACCATACAGAGT (SEQ ID NO.7) [0375] SNV-Probe 3-FAM TGGTTACCAACCATACAGAGTAGTAGTACT (SEQ ID NO.36) [0376] Primer and probe combinations tested were able to amplify N501Y mutant template. Both the SNV-MutFor5/SNV-Rev4/Probe2 and SNV-MutFor5/SNV-Rev4/Probe3 combinations were found to have the strong performance, as did the MutFor5/Rev6/Probe2 combination. Exemplary results are depicted in FIG. 7. These bubbled primer and probe combinations were found to be highly sensitive and selective for mutant template, but still some amplification of WT template was observed. Accordingly, a wild-type blocker primer was employed to improve reaction specificity. [0377] Blocker Screening [0378] Primer/probe combinations were screened in combination with 6 different blocker primers using the following templates: N501Y mutant RNA transcript, lysed inactivated wild-type (WT) virus and combined N501Y mutant and WT template (1E+05 copies/reaction each). Blocker primers that are specific for a wild-type sequence were screened; screened blocker primers were forward primers that include a PO4 modification at the 3'-end to prevent extension from occurring. Blocker primers were also tested at different concentrations (0 nM, 10 nM, 50 nM, 100 nM, 150 nM, and 300 nM). [0379] Exemplary results are depicted in FIG.8. Wild-type blocker primers were able to block amplification of wild-type template in a concentration dependent manner. Exemplary blocker primers were found to be effective at concentrations of 100 nM and above. The following two primer/blocker/probe combinations: SNV-MutFor5/WTFor5-PO4/Rev4/Probe2 and SNV- MutFor5/WTFor6-PO4/Rev6/Probe2 were selected to move forward, using blocker primer at a concentration of 100 nM. [0380] WT For 5-PO4 (SEQ ID NO.10) TTGTTACTTTCCTTTACAATCATATGGTCCTTCGCCCACTA [0381] WT For 6-PO4 (SEQ ID NO.37) GTTACTTTCCTTTACAATCATATGGTCCTTCGCCCACTA [0382] Analytical Sensitivity [0383] Analytical sensitivity of the exemplary N501Y primer sets was assessed. Specifically, primer set combinations of SNV-MutFor5/Rev4/Probe2 with and without WTFor5- PO4 and SNV-MutFor5/Rev6/Probe2 with and without WTFor6-PO4 were screened using N501Y mutant RNA transcript at 9 different conc. from 1E+02 to 1E+07 copies/reaction. Exemplary results for SNV-MutFor5/WTFor5-PO4/Rev4/Probe2 vs. SNV-MutFor5/Rev4/Probe2 are depicted in FIG. 9. SNV-MutFor5/WTFor5-PO4/Rev4/Probe2 combination was found to be highly sensitive and specific for the N501Y SNV mutant. Inclusion of a blocker primer improved specificity and had only a modest decrease in PCR efficiency. [0384] This example demonstrated the successful design and selection of exemplary primer sets for highly sensitive and specific detection of a SARS CoV-2 SNV corresponding to the N501Y mutation. This example describes exemplary primer sets for detection of this SNV that include bubbled primers, a probe, a complementary primer, and a blocker primer, as well as describes characteristics thereof. Example 5: Selection and Characterization of an Exemplary Control [0385] This example describes development and characterization of exemplary primer sets for detecting a SARS CoV-2 virus for use as a positive control with technologies for detecting SARS CoV-2 variants described herein. Exemplary primer sets for detecting SARS CoV-2 as a control are targeted to the Nucleocapsid (N) gene or ORF1ab gene. Specifically, the present example characterized primer sets of the FTD SARS CoV-2 Assay that target the N gene and ORF1ab gene as a control. These exemplary primer sets are provided in Table 8 below. [0386] Table 8: Exemplary Primer and Probe Sequences for Positive Control
Figure imgf000082_0001
[0387] An in-house alignment of the 4,540 complete and high-coverage sequences classified as a Variant Under Investigation (“VUI”) 20202/1201 (B.1.1.7) in the GISAID global database was utilized to assess the level of risk associated with mutations that may occur in the ORF1ab and N gene regions. Both assay amplicons were found to be in highly conserved regions. This supports that these regions will detected in variant SARS CoV-2 strains just as well as wild- type SARS CoV-2 strain(s). Accordingly, these primer sets were tested in conjunction with an exemplary SARS CoV-2 multiplex assay for detecting one or more variants. [0388] The exemplary positive control primers listed above were tested in combination with primers for detecting an exemplary variant (N501Y SNV) with different combinations of probes. Specifically, combinations of probes were tests for assays using N501Y SNV primers described above: SNV-Mut For 5 (SEQ ID NO.8), SNV-Rev 4 (SEQ ID NO.9), and SNV-WT For 5-PO4 (SEQ ID NO.10), SNV-Probe 2 (SEQ ID NO.7) in combination with the N gene and ORF1 gene primers listed in Table 8 above. Specifically, the following 8 primer/probe combinations were tested as outlined in Table 9 below. [0389] Table 9 - Probe Combinations with Exemplary N501Y + Control Multiplex Assay
Figure imgf000083_0001
[0390] The exemplary N501Y and N gene primer/probe combinations were tested with the following templates:
Figure imgf000083_0002
[0391] The exemplary N501Y and ORF1ab gene primer/probe combinations were tested with the following templates:
Figure imgf000083_0003
[0392] Each combinations described above were tested using templates at the following concentrations: 100,000 copies/reaction, 25,000 copies/reaction, 6,250 copies/reaction, and no template control (NTC). Exemplary results are provided in the tables below at 100,000 copies/reaction: [0393] Table 10: Difference of Expression N501Y and N gene Probe Combinations
Figure imgf000084_0001
[0394] Table 11: Difference of Expression N501Y and ORF1 gene Probe Combinations
Figure imgf000084_0002
[0395] Multiplex assays with primer/probe combinations that generated a difference of expression ΔCT within +/- 1 when compared with singleplex assay were selected. Accordingly, there were successful multiplex probe combinations for both N gene and ORF1 gene positive controls. The ORF1 gene positive control had two different probe combinations that showed the ΔCT within +/- 1 CT difference (SP2_FAM/FTD-ORF_YY and SP2_CIV/FTD-ORF_FAM). [0396] ORF1ab was selected as a positive control for a multiplex assay in combination with a N501Y SNV assay for further analysis. [0397] Analysis of Primer/Probe Concentrations [0398] An exemplary multiplex assay using N501Y SNV primers: SNV-Mut For 5 (SEQ ID NO.8), SNV-Rev 4 (SEQ ID NO.9), and SNV-WT For 5-PO4 (SEQ ID NO.10), SNV-Probe 2_FAM(SEQ ID NO. 7) and ORF1ab primers: FTD_ORF1_For (SEQ ID NO. 4), FTD_ORF1_Rev (SEQ ID NO. 6), and FTD-ORF_Probe_YY (SEQ ID NO. 5), were tested at varying concentrations to evaluate optimal primer probe concentrations. Specifically, the following 9 combinations of concentrations were analyzed:
Figure imgf000085_0001
[0399] All primer/probe concentration combinations tested were able to amplify successfully. The combination of (i) primer at 400 nM and probe at 300 nM and (ii) primer at 300 nM and probe at 100 nM were selected for further analysis. [0400] Blocker Guardbanding [0401] Guardbanding is a method to test the reliability of results. For this blocker guardbanding analysis, blocker concentration were tested from +20% to -20% of control concentration for the exemplary multiplex assay using N501Y SNV primers: SNV-Mut For 5 (SEQ ID NO.8), SNV-Rev 4 (SEQ ID NO.9), and SNV-WT For 5-PO4 (SEQ ID NO.10), SNV- Probe 2_FAM (SEQ ID NO. 7) and ORF1ab primers: FTD_ORF1_For (SEQ ID NO. 4), FTD_ORF1_Rev (SEQ ID NO. 6), and FTD-ORF_Probe_YY (SEQ ID NO. 5). Templates assayed included: N501Y transcript only, N501Y + WT lysate, WT lysate only and N501Y transcript + ORF transcript. Primer/Probe/blocker concentrations tested were as outlined in Table 12 below: [0402] Table 12: Primer/Probe/Blocker concentrations assays for Guardbanding
Figure imgf000086_0001
[0403] The ΔCT between control and adjusted N501Y blocker was within +/- 1. Accordingly, the blocker guardbanding was successful. [0404] This example demonstrates the successful generation of a multiplex assay for detection of an exemplary SARS CoV-2 SNV, N501Y, and a positive viral control (targeting a conserved viral region). Example 6: Design and Characterization of Exemplary Assays for Detection of SNVs K417N, E484K, A701V and V1176F [0405] This example describes development and characterization of exemplary assays for detecting SARS CoV-2 SNVs K417N, E484K, A701V and V1176F. The overview of the process for each of these was similar to that described in Example 4 and FIG. 6 above for the N501Y mutation. [0406] RNA transcripts of wild type and mutant variant sequences were generated from double-stranded gBlock templates with the corresponding sequence as described in Example 4 above. [0407] Bubbled Primer Screening [0408] The following variant primer pairs with a bubbled primer were screened for their ability to specifically detect mutant templates of E484K and K417N (bold indicates interrogated nucleotide), in Table 13 and Table 14, respectively. [0409] Table 13: Exemplary primers targeting a sequence that corresponds to SNV E484K
Figure imgf000087_0001
[0410] Table 14: Exemplary primers targeting a sequence that corresponds to SNV K417N
Figure imgf000087_0002
Figure imgf000088_0001
[0411] RNA transcripts covering E484K and SA417 mutations and wild-type sequences were used as templates for screening. Bubbled primers were screened by using EVAgreen chemistry for RT-PCR to determine if bubbled primers generated signal from mutant template. We determined if bubbled primers generated signal from mutant templates. [0412] Specifically, 24 primer combinations of the above primers were screened for each of the E484K and K417N mutations. The linearity and PCR efficiency were also evaluated for selected primer combinations to move forward with for probe screening (data not shown). [0413] The following primers combinations were able to robustly amplify the E484K and K417N mutations. [0414] Table 15: Primer Combinations for Amplification of E484K and K417N (SA417)
Figure imgf000088_0002
[0415] Accordingly, this experiment demonstrates the successful design and screening of multiple bubbled primers that are able to specifically detect mutant templates of E484K and SA417. [0416] Probe Screening [0417] Exemplary primer sets for detecting E484K, SA417, BZ1176 and SA701 SNVs were screened with different probes. Table # below provides probe sequences for E484K and K417N mutations, and primer and probe sequences for BZ1176 and SA701 mutations. [0418] Table 16: Exemplary probe sequences for use in a primer set targeting a sequence that corresponds to a SNV associated with amino acid position 484
Figure imgf000089_0001
[0419] Table 17: Exemplary probe sequences for use in a primer set targeting a sequence that corresponds to a SNV associated with amino acid position 417
Figure imgf000089_0002
[0420] Table 18: Exemplary primer and probe sequences for use in a primer set targeting a sequence that corresponds to a SNV associated with amino acid position 1176
Figure imgf000089_0003
Figure imgf000090_0001
[0421] Table 19: Exemplary primer and probe sequences for use in a primer set targeting a sequence that corresponds to a SNV associated with amino acid position 701
Figure imgf000090_0002
[0422] RNA transcripts covering sequences corresponding to mutations E484K, SA417, BZ1176 and SA701 were used as template for screening and WT lysate was used to determine the specificity of assays to detect mutations. Specifically, each of the primer/probe combinations described Table 20 below were tested using templates at the following concentrations: 100,000 copies/reaction, 25,000 copies/reaction, 6,250 copies/reaction, and no template control (NTC). [0423] Table 20: Exemplary primer/probe combinations:
Figure imgf000091_0001
Figure imgf000092_0001
[0424] Primer/probe combinations listed above were screened. Exemplary results are depicted in FIG.10. [0425] Each of the exemplary primer/probe combinations for E484K tested were able to amplify the E484K sequence; moreover, this amplification was highly specific as the wild-type sequence was not amplified in any of these combinations. The following primer sets for the E484K mutation were selected for continued evaluation and characterization: 484-Mut For1/484- Rev3/Probe 3 (SEQ ID NO 38 / 45 / 66), 484-Mut For2/484-Rev3/Probe 2 (SEQ ID NO 41 / 45 / 65), and 484-Mut For3/484-Rev3/Probe 3 (SEQ ID NO 42 / 45 / 66). [0426] Similarly, each of the exemplary primer/probe combinations for SA K417N tested were able to amplify the SA K417N mutant sequence and this was also highly specific. Those SA K417N primer/probe combinations with a forward mutant (bubbled) primer all had no amplification of wild-type sequence, while those combinations that included a reverse mutant (bubbled) primer had low or no amplification of wild-type sequence. The following primer sets for the K417N mutation were selected for continued evaluation and characterization: SA417-Mut For 2/SA417-Rev 1/SA417-Probe 1 (SEQ ID NO 52 / 56 / 67), SA417-Mut For 3/SA417-Rev 1/SA417-Probe 1 (SEQ ID NO 53 / 56 / 67), and SA417-Mut For 3/SA417-Rev3/SA417-Probe 1 (SEQ ID NO 53 / 58 / 67). [0427] The almost all of the exemplary primer/probe combinations for BZ V1176F listed above were able to amplify the BZ V1176F mutant sequence. Moreover, this amplification was highly specific, with little or no amplification of a wild-type sequence with any of these combinations. The following primer sets for the V1176F mutation were selected for continued evaluation and characterization: BZ1176-Mut F2/BZ1176-Rev2/BZ1176-Probe1 (SEQ ID NO 16 / 17 / 15), BZ1176-Mut F2/BZ1176-Rev2/BZ1176-Probe2, (SEQ ID NO 16 / 17 / 81), BZ1176- Mut F3/BZ1176-Rev1/BZ1176-Probe2 (SEQ ID NO 73 / 76 / 81), and BZ1176-Mut F3/BZ1176- Rev2/BZ1176-Probe1 (SEQ ID NO 73 / 17 / 15). [0428] The exemplary primer/probe combinations for SA A701V listed above had mixed results for amplifying mutant sequence. Also, those sequences that amplified mutant sequence also amplified wild-type sequence. Accordingly, a wild-type blocker primer was employed to improve reaction specificity for amplification of the SA A701V mutation. The following primer sets for the A701V mutation were selected for continued evaluation and characterization: SA701- Mut For5/SA701-Rev1/SA701-Probe1 (SEQ ID NO 12 / 13 / 11) and SA701-Mut For5/SA701- Rev4/SA701-Probe1 (SEQ ID NO 12 / 87 / 11). [0429] Blocker Screening [0430] Blockers for SA701 were screened with bubbled primers. RNA transcripts covering the SA701 mutation and WT lysate were used as templates to assay the blocker efficacy. [0431] Specifically the primer/probe combinations of: SA701-Mut For5/SA701- Rev1/SA701-Probe1 (SEQ ID NO 12 / 13 / 11) and SA701-Mut For5/SA701-Rev4/SA701-Probe1 (SEQ ID NO 12 / 87 / 11) were screened in combination with one of each of the five following different blocker primers: [0432] SA701-WT For 1-PO4 (SEQ ID NO.14) TCAATCCATCATTGCCTACACTACCATGGTTGGTGC (Sense) [0433] SA701-WT For 2-PO4 (SEQ ID NO.90) CAATCCATCATTGCCTACACTACCATGGTTGGTGC (Sense) [0434] SA701-WT For 3-PO4 (SEQ ID NO.91) AGTCAATCCATCATTGCCTACACTACCATGGTTGGTGC [0435] SA701-WT For 4-PO4 (SEQ ID NO.92) TCAATCCATCATTGCCTACACTGCCATGGTTGGTGC (Sense) [0436] SA701-WT For 5-PO4 (SEQ ID NO.93) AGTCAATCCATCATTGCCTACACGGCCATGGTTGGTGC (Sense) [0437] Blocker was tested in increasing amounts: 0 nM, 100 nM, 150 nM, and 200 nM. The following templates were assessed: Mutant, WT, Mutant+WT, each a concentration of 1.0E5, and also a no template control (NTC). Exemplary results are depicted in FIG.11. [0438] All of the blockers tested were found to be effective at blocking the extension of WT amplification, and this effect was dose dependent. The following combination was selected for further development: SA701-Mut For 5/SA701-Rev 1/SA701-Probe 1/SA701-WT For 1-PO4, with blocker at a concentration of 200 nM. Example 7: Development and Characterization of an Exemplary Multiplex Assay Targeting N501Y, SA701, and V1176F [0439] This example describes development and characterization of an exemplary multiplex assay for detecting SARS CoV-2 N501Y, SA701 and BZ1176 mutations. Primer/probe sequences for detecting N501Y, SA701 and BZ1176 with primer sets as described in the preceding examples, and as summarized below: [0440] Primer set for variant detection corresponding to position 701: SA701-Mut For5/SA701-Rev1/SA701-Probe1/SA701-WTFor1-PO4 (SEQ ID NO 12 / 13 / 11 / 14) [0441] Primer set for variant detection corresponding to position 501: SNV- MutFor5/Rev4/Probe2/WTFor5-PO4 (SEQ ID NO 8 / 9 / 2 / 10) [0442] Primer set for variant detection corresponding to position 1176: BZ1176-Mut F2/BZ1176-Rev2/BZ1176-Probe1 (SEQ ID NO 16 / 17 / 15) [0443] These primer set combinations were test in combination with the N gene and ORF1 gene positive control primers described in Example 5. Specifically, the following six combinations of primers/probes as listed in Table 21 were tested. [0444] Table 21 - Primer/Probe Combinations for Exemplary Multiplex Assay
Figure imgf000095_0001
[0445] RNA transcripts covering these mutations and wild-type sequences were used as templates for screening, using template in decreasing amounts: 100,000 copies/reaction, 25,000 copies/reaction, 6,250 copies/reaction, and no template control (NTC). The present disclosure encompassed a recognition that a multiplex assay should generate a change in expression (ΔCT) within +/- 1 when compared with the corresponding singleplex assays. Both of combination 5 and combination 6 generated a ΔCT within +/- 1 when compared with singlepex assays. Exemplary results are depicted in FIG. 12A and FIG. 12B. [0446] Blockers [0447] As noted above, detection of the N501Y and SA701 mutations in an exemplary multiplex assay each included blocker primers. The blocker primers screened included a PO4 modification in order to block transcription, but other modifications to prevent extension can be used, such as inverted 3'-3' linkage, an inverted dT, a C3, or amino-C7. Specifically, a C3 modification was evaluated in the context of a blocker. It was found that a blocker with a C3 modification performed similarly to a PO4 modification for both N501Y and SA701 mutations in the context of the exemplary multiplex assay. This example supports that blocker primers with other modifications can be used in the context of multiplex assays of the present disclosure. [0448] Optimization [0449] The concentration of oligonucleotides (primer, probes, and blocker) for the multiplex assay were optimized. Specifically, the following concentrations were selected for an exemplary multiplex assay:
Figure imgf000096_0001
[0450] These examples confirm that there are range of concentrations of primers and probes useful in the context of a multiplex assay, with the above concentrations providing a useful exemplary embodiment. [0451] Guardbanding [0452] Guardbanding is a method to test the reliability of results. For this oligo guardbanding analysis, the oligo mix was tested by varying its volume by +/-10%. Training samples for UK, SA and BZ SARS CoV-2 variants are used as template, along with WT lysate. The results showed a CT difference within +/-1 CT after varying the oligo mix by +/-10%. Thus, the guardbanding was consider successful. [0453] Cross reactivity [0454] Three SARS-CoV-2 closely related templates were tested the cross reactivity of the exemplary SNV assays. No detection was observed in 3 SARS-CoV-2 closely related templates, confirming the specificity of the selected primer sets of the exemplary multiplex assay. [0455] Analytical Sensitivity [0456] The limit of detection for each of the different target channels was determined for the exemplary multiplex assay. The following analytical sensitivity was calculated for each:
Figure imgf000097_0001
LoD = limit of detection, cp/mL = copies per milliliter, CI = confidence interval [0457] These limit of detection values were verified using four templates: WT, Alpha, Beta and Gamma variants. These templates were diluted to 3xLoD, using four different commercially available elution buffers. Twenty replicates of each template were tested. There was 100% detection was observed in corresponding channels in each template with different elution buffers for all 20/20 replicates. Thus, the exemplary multiplex assay is highly sensitive and specific for characterization of each of the three mutations. [0458] In summary, this example describes the successful design and optimization of an exemplary multiplex assay that uses bubbled primer technology to detect multiple different SARS CoV-2 mutations and a positive control. EQUIVALENTS [0459] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

CLAIMS What is claimed is: 1. A SARS CoV-2 variant primer comprising: (i) an anchor, wherein the anchor comprises or consists of a nucleotide sequence that is complementary to a first portion of a target nucleotide sequence; (ii) a bridge, wherein the bridge is located immediately 3' of the anchor in the primer, and wherein the bridge comprises or consists of a nucleotide sequence that is not complementary to a portion of a target nucleotide sequence immediately 3' of the first portion of the target nucleotide sequence; and (iii) a foot, wherein the foot is located immediately 3' of the bridge in the primer, and wherein the foot comprises a nucleotide sequence that is complementary to a second portion of a target nucleotide sequence, which is 3' of the first portion of the target nucleotide sequence and comprises an interrogating nucleotide position, wherein the interrogating nucleotide position corresponds to a SARS CoV-2 variant nucleotide.
2. The variant primer of claim 1, wherein the anchor is 15 to 40 nucleotides long, optionally wherein the anchor is 20 to 35 nucleotides long.
3. The variant primer of claim 1 or 2, wherein the bridge is 4 to 10 nucleotides long, optionally wherein the bridge is 6 to 8 nucleotides long.
4. The variant primer of any one of claims 1 to 3, wherein the foot is 6 to 8 nucleotides long, optionally wherein the foot is 7 nucleotides long.
5. The variant primer of any one of claims 1 to 4, wherein the primer is 25 to 55 nucleotides long, optionally wherein the primer is 30 to 40 nucleotides long.
6. The variant primer of any one of claims 1 to 5, wherein the primer is DNA.
7. The variant primer of any one of claims 1 to 6, wherein the primer comprises a label or a tag.
8. The variant primer of any one of claims 1 to 7, wherein the interrogating nucleotide is a nucleotide that corresponds to an amino acid substitution selected from: N501Y, E484K, K417N, A701V, and V1176F.
9. The variant primer of any one of claims 1 to 8, wherein the target nucleotide sequence is a SARS CoV-2 genomic RNA sequence.
10. The variant primer of any one of claims 1 to 8, wherein the target nucleotide sequence corresponds to a SARS CoV-2 S gene sequence or portion thereof.
11. The variant primer of any one of claims 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha (B.1.1.7 and Q lineages), beta (B.1.351), gamma (P.1), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, C.1.2, lambda (C.37), mu (B.1.621), omicron (B.1.1.529 and BA lineages), theta (P.3), and zeta (P.2).
12. The variant primer of any one of claims 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is selected from: alpha, beta, eta, gamma, iota, mu, omicron, theta, zeta, and C.1.2.
13. The variant primer of any one of claims 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is an alpha variant, variant, beta variant, gamma variant, mu variant, theta variant, C.1.2 variant, or omicron variant.
14. The variant primer of claim 13, wherein the variant nucleotide sequence comprises a sequence that corresponds to an N501Y mutation.
15. The variant primer of claim 13 or 14, wherein the variant nucleotide sequence comprises A23063T nucleotide substitution.
16. The variant primer of any one of claims 1 to 15, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8, 20-22, and 29-35.
17. The variant primer of any one of claims 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant or iota variant.
18. The variant primer of claim 17, wherein the variant nucleotide sequence comprises a sequence that corresponds to an A701V mutation.
19. The variant primer of claim 17 or 18, wherein the variant nucleotide sequence comprises a C23635T nucleotide substitution.
20. The variant primer of any one of claims 1 to 10 or 17 to 19, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86.
21. The variant primer of any one of claims 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a gamma variant, zeta variant, or theta variant.
22. The variant primer of claim 21, wherein the variant nucleotide sequence comprises a sequence that corresponds to a V1176F mutation.
23. The variant primer of claim 21 or 22, wherein the variant nucleotide sequence comprises a G25088T nucleotide substitution.
24. The variant primer of any one of claims 1 to 10 or 21 to 23, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16, 72-74, and 78-80.
25. The variant primer of any one of claims 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, gamma variant, eta variant, or mu variant.
26. The variant primer of claim 25, wherein the variant nucleotide sequence comprises a sequence that corresponds to an E484K mutation.
27. The variant primer of claim 25 or 26, wherein the variant nucleotide sequence comprises a A22980G nucleotide substitution.
28. The variant primer of any one of claims 1 to 10 or 25 to 27, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38, 41-44, 48, and 50.
29. The variant primer of any one of claims 1 to 10, wherein the target sequence contains a single nucleotide variant present in a SARS CoV-2 variant genome, wherein the SARS CoV-2 variant is a beta variant, mu variant, or delta variant.
30. The variant primer of claim 29, wherein the variant nucleotide sequence comprises a sequence that corresponds to a K417N mutation.
31. The variant primer of claim 29 or 30, wherein the variant nucleotide sequence comprises a G22184T nucleotide substitution.
32. The variant primer of any one of claims 1 to 10 or 29 to 31, wherein the variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55 and 62-64.
33. A primer pair comprising: the variant primer of any one of claims 1 to 32, and a complementary primer comprising or consisting of a nucleotide sequence that is complementary to a portion of a complementary strand sequence of the target nucleotide sequence.
34. A primer pair comprising: the variant primer of claim 16, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-28.
35. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 8 and 29-35, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 9 and 23-24.
36. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 20-22, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 25-28.
37. A primer pair comprising: the variant primer of claim 20, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88.
38. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 12 and 84-86, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 13, 87, and 88.
39. A primer pair comprising: the variant primer of claim 24, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17 and 74-77.
40. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 16 and 72-73, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 17, 76, and 77.
41. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 78-80, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 74-75.
42. A primer pair comprising: the variant primer of claim 28, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40, 45-47, and 49.
43. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 38 and 41-44, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 39-40 and 45-46.
44. A primer pair comprising: a variant primer comprises or consists of a sequence according to SEQ ID NO: 48 or 50, and a complementary primer comprising or consisting of a nucleotide sequence according to SEQ ID NO: 47 or 49.
45. A primer pair comprising: the variant primer of claim 32, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61 and 56-58.
46. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 51-55, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 56-58.
47. A primer pair comprising: a variant primer comprises or consists of a sequence according to any one of SEQ ID NOs: 62-64, and a complementary primer comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 59-61.
48. A primer set comprising: the variant primer of any one of claims 1 to 32, and a probe.
49. A primer set comprising: the primer pair of any one of claims 33 to 47, and a probe.
50. The primer set of claim 48 or 49, wherein the probe comprises or consists of a sequence according to any one of SEQ ID NOs: 7, 11, 15, 36, 65-71, 81-83 and 89.
51. A primer set comprising: the primer pair of any one of claims 34-36, and a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 7 or 36.
52. A primer set comprising: the primer pair of any one of claims 37-38, and a probe comprising or consisting of a nucleotide sequence according to SEQ ID NO: 11 or 89.
53. A primer set comprising: the primer pair of any one of claims 39-41, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 15 or 81-83.
54. A primer set comprising: the primer pair of any one of claims 42-44, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 7 and 65-66.
55. A primer set comprising: the primer pair of any one of claims 45-47, and a probe comprising or consisting of a nucleotide sequence according to any one of SEQ ID NOs: 67-71.
56. A mixture comprising: the variant primer of any one of claims 1 to 32, and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence.
57. A mixture comprising: the primer pair of any one of claims 33 to 47, and a sample comprising a SARS CoV-2 nucleotide comprising the target nucleotide sequence.
58. A method comprising: contacting a sample from a subject with the variant primer of any one of claims 1 to 32 or the primer pair of any one of claims 33 to 47; and amplifying a target nucleotide sequence from the sample.
59. The method of claim 58, wherein the target nucleotide sequence is a SARS CoV-2 nucleotide sequence.
60. The method of claim 58 or 59, wherein the method is to detect and/or identify a SARS CoV-2 variant in a sample comprising SARS CoV-2 nucleic acid.
61. A method of identifying a subject for treatment, comprising (i) identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in a sample from the subject, (ii) determining a SARS CoV-2 variant strain(s) with the nucleotide sequence changes identified, and (iii) administering therapy according to the determined SARS CoV-2 variant strain(s), wherein the step of identifying one or more nucleotide sequence changes comprises contacting the sample with the variant primer of any one of claims 1 to 32, or the primer pair of any one of claims 33 to 47.
62. A method of identifying a predominant SARS CoV-2 variant in a population of subjects, said method comprising identifying one or more nucleotide sequence changes (e.g., SNVs) associated with a SARS CoV-2 variant(s) in samples from the population of subjects, wherein the identifying comprises contacting the samples with the variant primer of any one of claims 1 to 32 or the primer pair of any one of claims 33 to 47.
63. A kit comprising: the variant primer of any one of claims 1 to 32 or the primer pair of any one of claims 33 to 47; and instructions for use.
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CN116426617A (en) * 2023-04-25 2023-07-14 无锡市人民医院 High-sensitivity mutation detection system based on hairpin structure and enzyme digestion mechanism and application
US11911461B1 (en) 2023-05-05 2024-02-27 D4 Labs, Llc Adjuvanted vaccines containing modified S1 Spike protein of SARS-CoV-2 variant C.1.2 for subcutaneous administration and methods of use

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Publication number Priority date Publication date Assignee Title
CN116426617A (en) * 2023-04-25 2023-07-14 无锡市人民医院 High-sensitivity mutation detection system based on hairpin structure and enzyme digestion mechanism and application
CN116426617B (en) * 2023-04-25 2024-04-16 无锡市人民医院 High-sensitivity mutation detection system based on hairpin structure and enzyme digestion mechanism and application
US11911461B1 (en) 2023-05-05 2024-02-27 D4 Labs, Llc Adjuvanted vaccines containing modified S1 Spike protein of SARS-CoV-2 variant C.1.2 for subcutaneous administration and methods of use

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