Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) variants with spike protein mutations

Abstract

Methods are described for rapidly detecting the presence of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that contain mutations in the spike (S) protein gene in a biological or non-biological sample. The methods may include performing an amplification step, a hybridization step, and a detection step. Additionally, primers and probes that target SARS-CoV-2 variants that contain mutations in the S gene, as well as kits designed to detect SARS-CoV-2 variants that contain mutations in the S gene, are provided.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates to the field of viral diagnostics, and more particularly to the detection of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that contain mutations in the spike (S) protein gene. [Background technology]

[0002] 2. Background of the Invention Viruses of the Coronaviridae family have single-stranded, positive-sense RNA genomes ranging in length from 26 to 32 kilobases. Coronaviruses have been identified in several avian hosts, as well as in a variety of mammals, including camels, bats, masked palm civets, mice, dogs, and cats. Novel mammalian coronaviruses are now being identified regularly. For example, the HKU2-related coronavirus of bat origin was responsible for a fatal acute diarrheal syndrome in pigs in 2018.

[0003] Among the several coronaviruses that are pathogenic for humans, most are associated with mild clinical symptoms, with two notable exceptions: severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), a novel betacoronavirus that emerged in November 2002 in Guangdong province in southern China and caused over 8000 human infections and 774 deaths in 37 countries between 2002 and 2003; and Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), first detected in Saudi Arabia in 2012 and responsible for 2494 laboratory-confirmed infections and 858 deaths since September 2012, 38 of whom died after a single visit to South Korea.

[0004] In late December 2019, several patients with viral pneumonia were found to be epidemiologically linked to a market in Wuhan, Hubei province, China, where some non-aquatic animals such as birds and rabbits were also sold prior to the outbreak. A novel human-transmitting coronavirus, initially named 2019 novel coronavirus (2019-nCoV), was identified using next-generation sequencing. This novel coronavirus is classified in the Coronaviridae family, genus Betacoronavirus and subgenus Sarbecovirus, and is described in "Genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding" by Lu, R.et al., Lancet, 2020, Vol.395, p.565-574. On February 11, 2020, the World Health Organization (WHO) announced the official name of the virus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0005] Several types of research and product development strategies are being pursued to combat the spread of SARS-CoV-2 and reduce morbidity and mortality associated with COVID-19. These include the development of mRNA, viral vector, and protein subunit vaccines, small molecule antivirals, immunomodulators, and other non-pharmaceutical interventions. Vaccines that have been researched and developed thus far have focused primarily on the viral envelope glycoprotein (spike, S) and have been notably successful thus far. In addition, monoclonal antibodies targeting S have been developed as antiviral drugs and are an effective treatment when administered soon after infection or symptom onset. Monoclonal antibodies can also be administered to uninfected individuals to prevent infection by SARS-CoV-2.

[0006] Like all RNA viruses, SARS-CoV-2 is prone to evolve in response to external selection pressures due to its error-prone RNA-dependent RNA polymerase and large population size. Although coronaviruses have a proofreading function as part of the replicase complex, their high replication rate in each host and in the vast population of infected people leads to the generation of a huge pool of viral variants from which more fit variants can emerge. It is almost certain that strong but incomplete replication inhibition, which can occur in infected individuals with partial immunity or those treated with a single anti-S monoclonal antibody, will lead to the selection of SARS-CoV-2 variants with escape mutations in S, with higher replication fitness than the wild-type virus, in a population of susceptible hosts. Similarly, if a naturally occurring variant had an increased ability to spread in an immunologically naive population, it could outcompete the wild-type virus in a relatively short period of time.

[0007] More than a year after the start of the SARS-CoV-2 pandemic, uncontrolled global transmission and significant viral evolution have given rise to hundreds of variants. Some of these variants, considered variants of concern (VOCs), e.g., UK (B.1.1.17), South Africa (B.1.351), Brazil (P.1 / B.1.1.248), and variants of interest (VOI) US [B.1.526 (NY) and B.1.427 / B.1.429 (California, Ohio)], may be more transmissible and / or affect treatment or vaccine response. Epidemiological and virological evaluations have confirmed that more transmissible VOCs are now emerging independently, and the first reports from the UK in December 2020 were soon followed by reports from South Africa, which showed that a different lineage could spread rapidly and become the dominant lineage within weeks. Although the significance of the mutation has yet to be fully determined, genomic data showing rapid replacement of other lineages suggests that this lineage may be associated with increased transmissibility. A variant from Brazil emerged in early December 2020 and by mid-January 2021 had already caused a large resurgence in cases. Concerns regarding vaccines and associated spike-borne SARS-CoV-2 adaptation have prompted epidemiological and surveillance efforts for VOC selection. Key mutations found in these emerging strains (del69-70, N501Y, E484K) have been used to track the spread of these most concerning strains. Mutations in the SARS-CoV-2 spike gene render the virus highly transmissible. Therefore, there is a need in the art for rapid, reliable, specific, and sensitive methods to detect SARS-CoV-2 variants, especially those containing the 69-70 deletion (del69-70) and mutations at N501Y and E484K in the spike protein gene. Summary of the Invention

[0008] The present disclosure relates to a method for rapid detection of the presence or absence of SARS-CoV-2 variants with mutations in the spike protein in a biological or non-biological sample, for example, multiplex detection of SARS-CoV-2 variants by qualitative or quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) in a single test tube. The embodiments include a method for detection of SARS-CoV-2 variants with one or more mutations including del69-70, N501Y, and E484K, comprising performing a reverse transcription step and at least one cycling step that may include an amplification step and a hybridization step. Additionally, the present disclosure includes primers, probes, and kits designed to detect SARS-CoV-2 variants in a single tube.

[0009] In one aspect, a method is provided for detecting a SARS-CoV-2 variant having a mutation in the spike protein in a sample, the method comprising: performing an amplification step, if SARS-CoV-2 nucleic acid is present in the sample, comprising contacting the sample with a primer set to generate an amplification product; performing a hybridization step, comprising contacting the amplification product with one or more detectable probes; and detecting the presence of the amplification product, wherein detection of the amplification product indicates the presence of the SARS-CoV-2 variant in the sample; the primer set is selected from the group consisting of SEQ ID NOs: 1-5. The method comprises the steps of: a first primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 7-14 or its complement; and a second primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 7-14 or its complement; the one or more detectable probes comprising or consisting of a third oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 16-25 or its complement; the spike protein mutation is selected from a 69-70 deletion (del69-70), an N501Y mutation, or an E484K mutation, or a combination thereof. In one embodiment, the step is performed in the presence of one or more blocking oligonucleotide probes. In a further embodiment, the one or more blocking oligonucleotide probes comprise or consist of an oligonucleotide sequence of SEQ ID NOs: 37, 38, or 39, or any combination thereof.

[0010] In another aspect, a multiplex method for detecting SARS-CoV-2 variants having a mutation in the spike protein in a sample is provided, the method comprising: performing an amplification step, if a nucleic acid of SARS-CoV-2 is present in the sample, comprising contacting the sample with at least two primer sets to generate a first amplification product and a second amplification product; performing a hybridization step, comprising contacting the amplification products with at least two detectable probes that hybridize to the first amplification product and the second amplification product generated by the at least two primer sets; and detecting the presence of at least one amplification product, wherein the presence of the at least one amplification product indicates the presence of a SARS-CoV-2 variant in the sample; the first primer set comprises a forward primer comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 1, and a second primer set comprising a detectable probe that hybridizes to the first amplification product and the second amplification product generated by the at least two primer sets; a reverse primer comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 8, and a second primer set comprising a forward primer comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 2, and a reverse primer comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 9, 10 or 11; a first detectable probe hybridizing to a first amplification product generated by the first primer set comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 16-17, or a complement thereof; a second detectable probe hybridizing to a second amplification product generated by the second primer set comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 18-20, or a complement thereof; the mutation in the spike protein is selected from a 69-70 deletion (del69-70), an N501Y mutation, or an E484K mutation, or a combination thereof. In one embodiment, the process is performed in the presence of one or more blocking oligonucleotide probes comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 37, 38 or 39, or any combination thereof.

[0011] As used herein, the SARS-CoV-2 variant is selected from a 69-70 deletion (del69-70), an N501Y mutation or an E484K mutation in the spike protein as a result of the respective mutations and deletions in the S gene, or a combination thereof. In some embodiments, the first or second detectable probe specifically hybridizes to an S gene sequence that causes a 69-70 deletion in SARS-CoV-2. In some embodiments, the first or second detectable probe specifically hybridizes to an S gene sequence that causes an N501Y mutation in SARS-CoV-2. In some embodiments, the first or second detectable probe specifically hybridizes to an S gene sequence that causes an E484K mutation in SARS-CoV-2. In one embodiment, the one or more blocking probes comprise or consist of an oligonucleotide sequence that perfectly matches the wild-type S gene sequence at amino acids 69-70, or at amino acid 484, or at amino acid 501 of the spike protein. In some embodiments, one or more blocking probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 37, 38 or 39, and combinations thereof. In further embodiments, a primer set is provided that amplifies a specific nucleic acid sequence from the non-structural open reading frame (ORF1a / b) of SARS-CoV-2, and a detectable probe that hybridizes to and detects the ORF1a / b amplification product generated by the primer set. In one embodiment, the primer set comprises a forward primer that comprises or consists of the oligonucleotide sequence of SEQ ID NO: 6, and a reverse primer that comprises or consists of the oligonucleotide sequence of SEQ ID NO: 15; the detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO: 36, or its complement.

[0012] In one embodiment, the primer set for amplification of SARS-CoV-2 variants comprises a first primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 1, a second primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 7 or 8, and a detectable probe comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 16 or 17 or its complement. In another embodiment, the first primer comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 2, the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 9-11, and the detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO: 18-20 or its complement. In one embodiment, the first primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 1, the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 8, and the detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO: 17 or its complement. In another embodiment, the first primer comprises or consists of the oligonucleotide sequence of SEQ ID NO:2, the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO:10, and the detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO:19, or a complement thereof. In yet another embodiment, the first primer comprises or consists of the oligonucleotide sequence of SEQ ID NO:2, the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO:10, and the detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO:20, or a complement thereof.

[0013] Other aspects of the disclosure provide oligonucleotides comprising or consisting of a sequence of nucleotides selected from SEQ ID NOs: 1-39, or a complement thereof, having 100 or fewer nucleotides. In another aspect, the disclosure provides oligonucleotides comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) to any of SEQ ID NOs: 1-39, or a complement thereof, having 100 or fewer nucleotides. Generally, these oligonucleotides can be primer nucleic acids, probe nucleic acids, etc. in these embodiments.

[0014] In certain embodiments, the oligonucleotide has 40 or less nucleotides (e.g., 35 or less nucleotides, 30 or less nucleotides, 25 or less nucleotides, 20 or less nucleotides, 15 or less nucleotides, etc.). In some embodiments, the oligonucleotide comprises at least one modified nucleotide, for example, to change the stability of nucleic acid hybridization compared to unmodified nucleotides. The oligonucleotide optionally comprises at least one label and optionally at least one quencher moiety.

[0015] In one embodiment, the amplification may use a polymerase enzyme with 5' to 3' nuclease activity. Thus, the donor fluorescent moiety and the acceptor moiety, e.g., a quencher, may be within 5 to 20 nucleotides (e.g., within 8 or 10 nucleotides) of each other along the length of the probe. In another embodiment, the probe includes a nucleic acid sequence that allows for the formation of a secondary structure. The formation of such a secondary structure may result in spatial proximity between the first and second fluorescent moieties. According to this method, the second fluorescent moiety on the probe may be a quencher.

[0016] In one embodiment, a detectable probe for detecting SARS-CoV-2 variants may be labeled with a fluorescent dye that acts as a reporter. The probe may also have a second dye that acts as a quencher. The reporter dye allows for detection and identification of the amplified SARS-CoV-2 target by being measured at a defined wavelength. The fluorescent signal of the intact probe is suppressed by the quencher dye. During the amplification step of PCR, hybridization of the probe to a specific single-stranded DNA template results in cleavage by the 5' to 3' nuclease activity of the DNA polymerase, resulting in separation of the reporter and quencher dyes and generation of a fluorescent signal. With each PCR cycle, the amount of cleaved probe increases, and the cumulative signal of the reporter dye increases accordingly. Optionally, one or more additional probes (e.g., an internal reference control or other targeted probes (e.g., other viral nucleic acids) can also be labeled with a reporter fluorescent dye that is unique and different from the fluorescent dye label associated with the SARS-CoV-2 probe. In such cases, the individual reporter dyes are measured at defined wavelengths, allowing simultaneous detection and discrimination of the amplified SARS-CoV-2 target and the one or more additional probes.

[0017] The present disclosure also provides methods for detecting the presence or absence of SARS-CoV-2 variants or SARS-CoV-2 nucleic acids containing mutations or deletions in the spike protein gene in a biological sample from an individual. These methods can be used to detect the presence or absence of SARS-CoV-2 variants or SARS-CoV-2 with spike gene mutations or deletions in nasopharyngeal (NSP) and oropharyngeal swab samples for use in diagnostic tests. Additionally, other types of samples can be evaluated by those skilled in the art using the same tests to detect SARS-CoV-2 variants or SARS-CoV-2 spike gene mutations and deletions. Such methods generally include performing a reverse transcription step and at least one cycling step including an amplification step and a dye-binding step. Typically, the amplification step includes contacting the sample with multiple pairs of oligonucleotide primers to generate one or more amplification products if nucleic acid molecules are present in the sample, and the dye-binding step includes contacting the amplification products with a double-stranded DNA-binding dye. Such methods also include detecting the presence or absence of binding of a double-stranded DNA binding dye to the amplification product, the presence of binding indicating the presence of SARS-CoV-2 variants or SARS-CoV-2 spike gene mutations and deletions in the sample, and the absence of binding indicating the absence of SARS-CoV-2 variants or SARS-CoV-2 spike gene mutations and deletions in the sample. A representative double-stranded DNA binding dye is ethidium bromide. Other nucleic acid binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YO-YO® and SYBR® Green. Additionally, such methods may also include determining the melting temperature between the amplification product and the double-stranded DNA binding dye, which confirms the presence or absence of SARS-CoV-2 variants or SARS-CoV-2 nucleic acid mutations and deletions.

[0018] In a further embodiment, a kit is provided for detecting mutations in one or more spike genes from SARS-CoV-2 variants. The kit may include one or more primer sets specific for amplifying gene targets; and one or more detectable oligonucleotide probes specific for detecting the amplification products.

[0019] In one embodiment, the kit may include donor and corresponding acceptor moieties, such as probes already labeled with other fluorescent moieties or dark quenchers, or may include fluorophore moieties for labeling the probes. The kit may also include nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. The kit may also include a package insert and instructions for using the primers, probes, and fluorophore moieties to detect the presence or absence of mutations and deletions in the SARS-CoV-2 spike gene in a sample.

[0020] In one aspect, a method is provided for allele-specific amplification of a target sequence, present in a sample in the form of several variant sequences, the method comprising providing a blocking oligonucleotide comprising a 5' end, a 3' end, and at least one nucleotide that is a locked nucleic acid (LNA), wherein the blocking oligonucleotide is fully complementary to a wild-type (WT) sequence when it hybridizes to form a first complex having a first melting temperature (Tm), and the blocking oligonucleotide is fully complementary to a wild-type (WT) sequence when it hybridizes to form a second complex having a second melting temperature (Tm). or a plurality of nucleotides, the blocking oligonucleotide is partially non-complementary to the target mutant (MT) sequence, the first Tm being higher than the second Tm, and the blocking oligonucleotide is blocked at the 3' end to prevent extension; the amplifying step is performed at a temperature higher than the second Tm but lower than the first Tm, and the amplifying step includes, if WT and / or target MT sequences are present in the sample, contacting the sample with a primer set to generate an amplification product, and the blocking oligonucleotide becomes non-hybridizable to the target MT sequence during the amplifying step but remains hybridized to the WT sequence and inhibits amplification of the WT sequence.

[0021] In another aspect, a kit is provided for allele-specific amplification of a target sequence present in the form of several variant sequences, the kit comprising a primer set; a blocking oligonucleotide comprising a 5' end, a 3' end, and at least one nucleotide that is a locked nucleic acid (LNA), wherein the blocking oligonucleotide is fully complementary to the wild type (WT) sequence when hybridized to form a first complex having a first melting temperature (Tm), and the blocking oligonucleotide is partially non-complementary at one or more nucleotides to the target mutant (MT) sequence when hybridized to form a second complex having a second melting temperature (Tm), and the first Tm is higher than the second Tm.

[0022] In another aspect, there is provided an oligonucleotide for performing allele-specific amplification of a target sequence, present in the form of several variant sequences, the oligonucleotide comprising a 5' end and a 3' end that is blocked at the 3' end to prevent extension, the sequence being fully complementary to a wild type (WT) sequence when hybridizing to form a first complex having a first melting temperature (Tm) and being partially non-complementary at one or more nucleotides to a target mutant (MT) sequence when hybridizing to form a second complex having a second melting temperature (Tm), the first Tm being higher than the second Tm; and at least one nucleotide is a locked nucleic acid (LNA).

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0024] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims. [Brief description of the drawings]

[0025] [Figure 1] The spike protein gene deletions and mutations of the present disclosure and their locations within the SARS-CoV-2 genome are shown. ORF, open reading frame; S, spike protein; RBD, receptor binding domain. [Diagram 2] FIG. 1 shows growth curves generated from the SARS-CoV-2 variants test described in Example 4 using Zeptometrix wild-type SARS-CoV-2 genomic RNA at the indicated levels in a multiplex PCR test with detection in the coumarin channel. [Diagram 3] 1 shows growth curves generated from the SARS-CoV-2 variant testing described in Example 4 using mutant transcripts containing both the E484K and N501Y mutations at the indicated levels in a multiplex PCR test with detection in the FAM channel (left) and HEX channel (right). [Figure 4] Growth curves generated from the SARS-CoV-2 variant testing described in Example 4 using Twist synthetic control transcripts carrying both the N501Y mutation and the 69-70 deletion at the indicated levels in a multiplex PCR test with detection in the HEX channel (left) and JA270 channel (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Invention Diagnosis of SARS-CoV-2 infection (both wild type and variants) by nucleic acid amplification provides a method for rapid, accurate, reliable, specific, and sensitive detection of viral infection. Described herein is a real-time reverse transcriptase PCR assay for detection of SARS-CoV-2 variants with mutations in the spike protein gene in non-biological or biological samples. Primers and probes for detecting SARS-CoV-2 variants are provided, as are articles of manufacture or kits containing such primers and probes. Compared with other methods, the high specificity and sensitivity of real-time PCR for detection of SARS-CoV-2 variants, as well as the improved features of real-time PCR, including sample containment and real-time detection of amplification products, make the implementation of this technology feasible for routine diagnosis of SARS-CoV-2 infections in clinical laboratories as well as infections of SARS-CoV-2 variants. Furthermore, this technology can be used for in vitro diagnosis as well as prognosis. This SARS-CoV-2 variant detection assay can also be multiplexed in parallel with other assays for the detection of other nucleic acids, e.g., influenza virus, SARS-CoV, MERS-CoV.

[0027] The SARS-CoV-2 genome is a positive-sense single-stranded RNA molecule 29,903 bases long (as shown in GenBank accession number NC_045512) with the following gene order (5' to 3'): replicase ORF1ab (21,291 bases with 16 predicted nonstructural proteins essential for viral replication and viral assembly), spike (S gene, 3,822 bases encoding the spike protein involved in binding to cellular receptors), ORF3ab (828 bases long), envelope (E gene, 228 bases encoding the envelope protein), membrane (M gene, 669 bases encoding the membrane protein), and nucleocapsid (N gene, 1260 bases encoding the nucleocapsid protein that forms a complex with the genomic RNA). In addition, there is a noncoding region of 265 bases at the 5' end and a noncoding region of 229 bases at the 3' end.

[0028] The S gene encodes the spike protein, also called S protein or surface glycoprotein, a transmembrane glycosylated protein consisting of 1273 amino acids that assembles as a homotrimer to form the spike that protrudes from the SARS-CoV-2 viral envelope. The spike protein mediates viral entry into the host cell by first binding to the host receptor via the receptor-binding domain (RBD) in the S1 subunit and then fusing the viral and host membranes via the S2 subunit. Similar to SARS-CoV, SARS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2) as its host receptor that binds to the viral S protein. The RBD of the SARS-CoV-2 spike protein has been characterized as a region of approximately 200 amino acids from residues 331 to 524 (or from residues 333 to 527 in other reports).

[0029] Recent findings report the emergence of S gene variants that exhibit greater infectivity, higher viral loads and potentially increased lethality, coupled with reduced neutralization by antibodies generated by vaccines using wild-type S targets. These variants of concern (VOCs) are UK B1.1.7 (69-70del, N501Y, among others), South African B.1.351 (K417N, E484K and N501Y) and Brazilian B.1.1.28 (E484K, N501Y) and P1. These variants were detected by sequencing samples after PCR-based detection. The locations of the 69-70del, N501Y and E484K mutations, as well as the commonly observed D614G mutation, are shown in Figure 1.

[0030] The present disclosure includes oligonucleotide primers and fluorescently labeled hydrolysis probes that hybridize to the spike protein gene of the SARS-CoV-2 genome for specific identification of SARS-CoV-2 variants, for example, using TaqMan® amplification and detection technology. The oligonucleotides hybridize specifically to the S gene. Because having oligonucleotides that hybridize to multiple locations in the genome is advantageous for improving sensitivity compared to targeting only a single locus, the present disclosure also provides oligonucleotide primers and hydrolysis probes that hybridize to other regions in the SARS-CoV-2 genome (e.g., the ORF1ab gene).

[0031] The disclosed method may include performing a reverse transcription step and at least one cycling step that includes amplifying one or more portions of a gene target of a nucleic acid molecule from a sample using one or more primer pairs. As used herein, "SARS-CoV-2 primer(s)" refers to an oligonucleotide primer that specifically anneals to a nucleic acid sequence found in the SARS-CoV-2 genome and initiates DNA synthesis therefrom under appropriate conditions to generate a respective amplification product. Examples of nucleic acid sequences found in the SARS-CoV-2 genome include nucleic acids within the ORF1ab, S, ORF3ab, E, M and N genes and other predicted ORF regions. Each of the SARS-CoV-2 primers considered anneals to a target region such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. If one or more nucleic acids are present in the sample, one or more amplification products are generated, and thus the presence of one or more amplification products indicates the presence of SARS-CoV-2 in the sample. The amplification products should contain a nucleic acid sequence complementary to one or more detectable probes for SARS-CoV-2. As used herein, "SARS-CoV-2 probe(s)" refers to an oligonucleotide probe that specifically anneals to a nucleic acid sequence found in the SARS-CoV-2 genome. Each cycling step includes an amplification step, a hybridization step, and a detection step (wherein the sample is contacted with one or more detectable SARS-CoV-2 probes to detect the presence or absence of SARS-CoV-2 in the sample).

[0032] As used herein, the term "amplifying" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., a nucleic acid molecule derived from the SARS-CoV-2 genome). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending from the primer to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and an appropriate buffer and / or cofactors (e.g., MgCl2 and / or KCl) for optimal activity of the polymerase enzyme.

[0033] The term "primer" as used herein is known to those skilled in the art and refers to oligomeric compounds, primarily oligonucleotides, but also modified oligonucleotides that are capable of "priming" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group at which further "nucleotides" can be attached by a template-dependent DNA polymerase that establishes a 3' to 5' phosphodiester linkage, thereby using a deoxynucleoside triphosphate, thereby releasing pyrophosphate.

[0034] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. "Hybridization conditions" typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0035] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0036] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., the enzyme catalyzes the formation of primer extension products complementary to the template and does not irreversibly denature when subjected to elevated temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, non-thermostable polymerases can also be used in PCR assays, provided the enzyme is replenished as necessary.

[0037] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0038] The term "extension" or "lengthening" when used in reference to a nucleic acid refers to when an additional nucleotide (or other similar molecule) is incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide-incorporating biocatalyst, such as a polymerase, which typically adds a nucleotide to the 3' end of the nucleic acid.

[0039] The terms "identical" or percent "identity" in the context of two or more nucleic acid sequences refer to two or more sequences or subsequences that have the same or a specified percentage of identical nucleotides when compared and aligned for maximum correspondence, e.g., as determined using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, e.g., Altschul et al. (1990) "Basic local alignment search tool," J. Mol. Biol. 215:403-410; Gish et al. (1993) "Identification of protein coding regions by database similarity search," Nature Genet. 3:266-272; Madden et al. (1996) "Applications of network BLAST server," Meth. Enzymol. 266:131-141; Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402; and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation," Genome Res. 7:649-656.

[0040] "Modified nucleotide" in the context of oligonucleotides refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide that provides desired properties to the oligonucleotide. Exemplary modified nucleotides that may be substituted in the oligonucleotides described herein include, for example, t-butylbenzyl, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-O-methylribo-U, 2'-O-methylribo-C, N4-ethyl-dC, N6-methyl-dA, and the like. Many other modified nucleotides that can be substituted in an oligonucleotide are mentioned herein or are otherwise known in the art. In certain embodiments, the modified nucleotide substitutions modify the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. Nucleoside modifications can also include moieties that increase the stringency of hybridization or increase the melting temperature of the oligonucleotide probe. For example, nucleotide molecules can be modified with an additional bridge connecting the 2' and 4' carbons to result in "locked nucleic acid (LNA)" nucleotides that are resistant to cleavage by nucleases (as described in U.S. Patent No. 6,268,490 to Imanishi et al. and U.S. Patent No. 6,794,499 to Wengel et al.). To further illustrate, certain modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer-dimer formation, etc.), increase the yield of the intended target amplicon, etc. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611.Other modified nucleotide substitutions may alter the stability of the oligonucleotide or provide other desirable characteristics.

[0041] SARS-CoV-2 Detection The present disclosure provides methods for detecting SARS-CoV-2 variants with mutations in the spike protein, for example, by amplifying a portion of the nucleic acid sequence of the SARS-CoV-2 S gene. The nucleic acid sequence of the SARS-CoV-2 genome is available (e.g., GenBank Accession No. NC_045512, where the S gene is located at nucleotide positions 21563 to 25384). In particular, primers and probes for amplifying and detecting mutations and deletions target sequences in the SARS-CoV-2 S gene are provided by embodiments of the present disclosure.

[0042] For detection of SARS-CoV-2 VOCs, primers that amplify the S gene and probes that specifically detect mutations and deletions in the S gene are provided. SARS-CoV-2 nucleic acids other than those exemplified herein can also be used to detect SARS-CoV-2 variants in a sample. For example, functional variants can be evaluated for specificity and / or sensitivity by those of skill in the art using routine methods. Representative functional variants can include, for example, one or more deletions, insertions and / or substitutions in the SARS-CoV-2 nucleic acids disclosed herein.

[0043] More specifically, oligonucleotide embodiments include nucleic acids having sequences selected from SEQ ID NOs: 1-5, 7-14, and 16-25, or the complements of SEQ ID NOs: 1-5, 7-14, and 16-25, respectively. In some embodiments, oligonucleotide probes are provided that block detection of the wild type (e.g., wild type residues 69-70, E484, N501) selected from SEQ ID NOs: 37-39.

[0044] [Table 1]

[0045] [Table 2]

[0046] [Table 3-1] [Table 3-2]

[0047] [Table 4]

[0048] In one embodiment, the SARS-CoV-2 primer sets and probes described above are used to provide detection of SARS-CoV-2 in biological samples suspected of containing a SARS-CoV-2 variant (Tables 1-4). The primer sets and probes may comprise or consist of primers and probes specific for the nucleic acid sequences of SARS-CoV-2, including or consisting of the nucleic acid sequences of SEQ ID NOs: 1-5, 7-14, 16-25, and 37-39.

[0049] As detailed above, the primer (and / or probe) may be chemically modified, i.e., the primer and / or probe may contain modified nucleotides or non-nucleotide compounds. Thus, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modifications, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate or phosphate-like moiety, or a combination thereof. For example, a "label" can be attached to the base portion of a "nucleotide" to obtain a "modified nucleotide". A natural base in a "nucleotide" can also be replaced by, for example, a 7-deazapurine, thereby also obtaining a "modified nucleotide". The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analog") differs from a natural nucleoside by some modifications in the manner as outlined above for "modified nucleotide" (or "nucleotide analog").

[0050] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acids encoding SARS-CoV-2 targets, e.g., nucleic acid molecules encoding another portion of SARS-CoV-2, can be designed using computer programs such as, for example, OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Important features in designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size of the amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of the primer pair, and the length of each primer (i.e., primers should be long enough to anneal and initiate synthesis with sequence specificity, but not so long that fidelity is compromised during oligonucleotide synthesis). Typically, oligonucleotide primers are 8-50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).

[0051] In addition to the primer set, the method may use one or more probes to detect the presence or absence of SARS-CoV-2 variants. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that contains a specific nucleotide sequence that allows it to specifically (i.e., preferentially) hybridize to the "target nucleic acid" under a given stringency defined by design or selection, if the SARS-CoV-2 (target) nucleic acid is present. A "probe" may be referred to as a "detection probe," which means that it detects the target nucleic acid.

[0052] In some embodiments, the disclosed SARS-CoV-2 probes can be labeled with at least one fluorescent label. In one embodiment, the SARS-CoV-2 probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher. In one embodiment, the probe comprises or consists of a fluorescent moiety and the nucleic acid sequence comprises or consists of SEQ ID NOs: 21-26.

[0053] The design of oligonucleotides used as probes can be done in a similar manner to the design of primers. Embodiments may use a single probe or a probe pair for detection of the amplification product. Depending on the embodiment, the probe(s) used may include at least one label and / or at least one quencher moiety. Like primers, the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15-40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0054] The constructs may include vectors, each of which includes one of the SARS-CoV-2 primer and probe nucleic acid molecules. The constructs may be used, for example, as a control template nucleic acid molecule. Vectors suitable for use are commercially available and / or are produced by recombinant nucleic acid technology methods routine in the art. SARS-CoV-2 nucleic acid molecules may be obtained, for example, by chemical synthesis, direct cloning from SARS-CoV-2, or by nucleic acid amplification.

[0055] Constructs suitable for use in the methods typically include, in addition to a nucleic acid molecule of SARS-CoV-2 (e.g., a nucleic acid molecule comprising one or more of the sequences of SEQ ID NOs: 1-5, 7-14, and 16-25), a sequence encoding a selectable marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, and an origin of replication. The choice of vector system typically depends on several factors, including, but not limited to, the choice of host cell, replication efficiency, selectability, inducibility, and ease of recovery.

[0056] Constructs containing nucleic acid molecules of SARS-CoV-2 can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant and animal cells. Prokaryotic hosts can include E. coli, Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into host cells using any technique generally known to those skilled in the art.For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and virus-mediated nucleic acid transfer are common methods for introducing nucleic acid into host cells.In addition, naked DNA can be delivered directly into cells (see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466).

[0057] Polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides (e.g., SEQ ID NOs: 1-20) that can act as initiation points for nucleic acid synthesis within the nucleic acid sequence of SARS-CoV-2 described. Primers can be purified from restriction digests by conventional methods or produced synthetically. Primers are preferably single-stranded for maximum efficiency in amplification, although primers can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0058] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method, including physical, chemical, or enzymatic means. One method of separating the strands of a nucleic acid involves heating until the nucleic acid is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the salt concentration of the buffer, as well as the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C, depending on the characteristics of the reactants, such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes and 30 seconds, or 1.5 minutes).

[0059] When the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence. The annealing temperature is typically about 35°C to about 65°C, (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds; about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes the activity of the polymerase, i.e., a temperature sufficient for extension to occur from the annealed primers to generate products complementary to the template nucleic acid. The temperature should be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., the temperature for extension is generally in the range of about 40°C to about 80°C (e.g., about 50°C to about 70°C; about 60°C)). The extension time can be from about 10 seconds to about 5 minutes (eg, from about 30 seconds to about 4 minutes; from about 1 minute to about 3 minutes; from about 1 minute 30 seconds to about 2 minutes).

[0060] The genomes of retroviruses or RNA viruses, such as SARS-CoV-2, as well as other flaviviruses, are composed of ribonucleic acid, i.e., RNA. In such cases, the template nucleic acid, RNA, must first be transcribed into complementary DNA (cDNA) through the action of the enzyme reverse transcriptase. Using the RNA template and a short primer complementary to the 3' end of the RNA, reverse transcriptase directs the synthesis of first strand cDNA, which can then be used directly as a template for the polymerase chain reaction.

[0061] The PCR assay may use nucleic acids of SARS-CoV-2, such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified; it may be a trace fraction of a complex mixture, such as the nucleic acids of SARS-CoV-2 contained in human cells. Nucleic acid molecules of SARS-CoV-2 may be extracted from biological samples by routine techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids may be obtained from any number of sources, for example, plasmids, or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0062] Oligonucleotide primers (e.g., SEQ ID NOs: 1-20) are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction typically contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0063] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing, and extension can be repeated as many times as necessary to generate a desired amount of amplification product corresponding to the target SARS-CoV-2 nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection use, the number of cycling steps can depend, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps may be required to amplify the target sequence enough for detection. Generally, the cycling steps are repeated at least about 20 times, but can be repeated 40, 60, or even 100 times.

[0064] Fluorescence Resonance Energy Transfer (FRET) FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance from each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. Typically, the donor transfers energy to the acceptor when excited by light irradiation of a suitable wavelength. Typically, the acceptor re-emits the transferred energy in the form of light irradiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non-fluorescent donor moiety (see, for example, U.S. Pat. No. 7,741,467).

[0065] In one example, an oligonucleotide probe contains a donor fluorescent moiety (e.g., HEX) and a corresponding quencher (e.g., BlackHole), which may or may not be fluorescent, that dissipates the transferred energy in a form other than light. The probe may include a Quencher™ (BHQ). When the probe is intact, energy transfer typically occurs between the donor and acceptor moieties such that the fluorescent emission from the donor fluorescent moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example by the 5' to 3' nuclease activity of Taq polymerase, such that the fluorescent emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Pat. Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0066] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at specific positions determined by the complementarity of the oligonucleotide probes to the SARS-CoV-2 target nucleic acid sequence. When the oligonucleotide probes hybridize to the nucleic acid of the amplification product at the appropriate positions, they generate a FRET signal. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0067] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor fluorescence emission in a specific range), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed with an argon ion laser, a high-intensity mercury (Hg) arc lamp, a xenon lamp, a fiber optic light source, or other high-intensity light source appropriately filtered for excitation of the desired range.

[0068] As used herein with respect to a donor and a corresponding acceptor moiety, "corresponding" refers to an acceptor fluorescent moiety or a dark quencher that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. This allows efficient non-irradiation energy transfer between them.

[0069] Fluorescent donors and corresponding acceptor moieties are generally selected for (a) high efficiency Förster energy transfer; (b) large final Stokes shift (>100 nm); (c) shift of emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) shift of emission to a wavelength higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety may be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and its fluorescence emission has good overlap with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety may be selected that has a high extinction coefficient, a high quantum yield, its excitation has good overlap with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0070] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0071] The donor and acceptor fluorescent moieties may be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important since the linker arm may affect the distance between the donor and acceptor fluorescent moieties. The length of the linker arm may be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, the linker arm is from about 10 Å to about 25 Å. The linker arm may be of the type described in WO84 / 03285. WO84 / 03285 also discloses methods of attaching a linker arm to a particular nucleotide base and methods of attaching a fluorescent moiety to the linker arm.

[0072] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to produce, for example, LC Red 640-labeled oligonucleotides. Linkers frequently used to couple donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (derived from fluorescein-NHS-ester, e.g., CX-Fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPG, which requires coupling of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0073] SARS-CoV-2 Detection The present disclosure provides a method for detecting the presence or absence of SARS-CoV-2 variants having mutations (including deletions and insertions) of the spike protein in a biological or non-biological sample. The provided method avoids the problems of sample contamination, false negatives, and false positives. The method includes performing a reverse transcription step, at least one cycling step including amplifying a portion of the SARS-CoV-2 S gene nucleic acid molecule from the sample using one or more pairs of SARS-CoV-2 primers, and a FRET detection step. The multiple cycling steps are preferably performed in a thermocycler. To specifically detect the presence of the S gene mutation of SARS-CoV-2, the method can be performed using primers and probes for the SARS-CoV-2 S gene, and detection of SARS-CoV-2 indicates the presence of the SARS-CoV-2 variant in the sample.

[0074] As described herein, the amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products and therefore the presence or absence of SARS-CoV-2 variants. TaqMan® technology utilizes, for example, one single-stranded hybridization probe labeled with one fluorescent dye (e.g., HEX) and one quencher (e.g., BHQ), which may or may not be fluorescent. When the first fluorescent moiety is excited with light of an appropriate wavelength, the absorbed energy is transferred to a second fluorescent moiety or dark quencher according to the principles of FRET. The second moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded during the subsequent extension stage, for example, by the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for carrying out the methods described herein to detect the presence or absence of SARS-CoV-2 variants in a sample.

[0075] Molecular beacons in combination with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and a fluorescent label is typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows for the formation of a secondary structure (e.g., a hairpin). The formation of a secondary structure within the probe results in both fluorescent moieties being in spatial proximity when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other, so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0076] Another common format of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the light source of the LightCycler® instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., LightCycler®-Red640 (LC Red640) or LightCycler®-Red705 (LC Red705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of SARS-CoV-2 genomes). When amplification of the SARS-CoV-2 target nucleic acid occurs and an amplification product is generated, the hybridizing step results in a detectable signal based on FRET between the members of the probe pair.

[0077] Generally, the presence of FRET indicates the presence of SARS-CoV-2 in the sample, and the absence of FRET indicates the absence of SARS-CoV-2 in the sample. However, inadequate specimen collection, shipping delays, improper shipping conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results.

[0078] Representative biological samples that may be used in the practice of the method include, but are not limited to, respiratory specimens (nasopharyngeal and oropharyngeal swabs), urine, fecal specimens, blood specimens, plasma, skin swabs, wound swabs, blood cultures, skin, and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. The biological sample may be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release nucleic acids of SARS-CoV-2, or in some cases, the biological sample may be directly contacted with PCR reaction components and appropriate oligonucleotides.

[0079] Melting curve analysis is an additional step that can be included in the cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), defined as the temperature at which one half of a DNA duplex separates into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature(s) of the SARS-CoV-2 probe from the SARS-CoV-2 amplification product can confirm the presence or absence of SARS-CoV-2 in the sample.

[0080] During each thermocycler run, control samples may be cycled as well. A positive control sample may amplify a target nucleic acid control template (other than the amplification product of the target gene described), for example, using control primers and a control probe. A positive control sample may also amplify, for example, a plasmid construct containing a target nucleic acid molecule. Such a plasmid control may be amplified internally (e.g., within the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect the intended target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reaction. Each thermocycler run may also include, for example, a negative control lacking target template DNA. The negative control may measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, the control reactions can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate extension, as well as the ability of probes to hybridize with sequence specificity and FRET to occur.

[0081] In some embodiments, the method includes steps to avoid contamination. For example, enzymatic methods utilizing uracil-DNA glycosylase to reduce or eliminate contamination between one thermocycler run and the next are described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313.

[0082] The method can be implemented using conventional PCR combined with FRET technology. In one embodiment, LightCycler® instrument is used. The following patent applications describe the real-time PCR used with LightCycler® technology: WO97 / 46707, WO97 / 46714, and WO97 / 46712.

[0083] The LightCycler® may be operated using a PC workstation and may utilize the Window NT operating system. Signals from samples are obtained as the machine sequentially positions the capillaries on the optical unit. The software may display the fluorescent signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number may be continuously updated for all samples. The data generated may be stored for further analysis.

[0084] As an alternative to FRET, the amplification products can be detected using double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Double-stranded DNA binding dyes such as nucleic acid intercalating dyes can also be used. When double-stranded DNA binding dyes are used, a melting curve analysis is usually performed to confirm the presence of the amplification products.

[0085] Those skilled in the art will understand that other nucleic acid or signal amplification methods can also be used. Examples of such methods include, but are not limited to, branched DNA signal amplification, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), strand displacement amplification (SDA), or smart amplification process version 2 (SMAP2).

[0086] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0087] Manufactured products / kits The embodiments of the present disclosure further provide an article of manufacture or kit for detecting SARS-CoV-2 variants with mutations in the spike protein. The article of manufacture may include primers and probes used to detect the S gene target of SARS-CoV-2 along with suitable packaging materials. Representative primers and probes for specific detection of S gene mutations of SARS-CoV-2 can hybridize to SARS-CoV-2 target nucleic acid molecules. In addition, the kit may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to SARS-CoV-2 S gene target nucleic acid molecules are provided.

[0088] The article of manufacture may also include one or more fluorescent moieties for labeling the probe, or the probe provided with the kit may be labeled. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling the SARS-CoV-2 probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0089] The article of manufacture may also include a package insert or packaging label with instructions for using the SARS-CoV-2 primers and probes to detect SARS-CoV-2 variants with spike protein mutations in a sample. The article of manufacture may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0090] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the invention as claimed. EXAMPLES

[0091] The following examples and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0092] Example 1 Assay Description A real-time reverse transcription-polymerase chain reaction (RT-PCR) test was developed on the cobas® 6800 / 8800 system for the qualitative and discriminatory detection of SARS-CoV-2 mutations N501Y, del69-70 and E484K in e.g. nasal and nasopharyngeal swab specimens from known SARS-CoV-2 infected patients to support understanding of variant epidemiology for Population Health Management. Mutation-specific PCR assays may be used as reflex testing of SARS-CoV-2 positive samples to identify known mutations of concern as part of a surveillance strategy for SARS-CoV-2 variants. The assay was strategically designed to drive specificity for the detection of point mutations (E484K and N501Y) using hydrolysis (TaqMan®) probes incorporating locked nucleic acid (LNA) chemistry to increase the melting temperature (Tm) to enable single base mutation detection. Detection of the 6-base deletion in del69-70 could be performed using a conventional TaqMan® probe. The assay also included three dyeless wild-type (wt) probes for del69-70, E484K, and N501Y, acting as blocking oligonucleotide probes. The assay was performed under competitive conditions in the presence of both fluorescently labeled mutant probes and wt dyeless probes, so that mismatched probes could be inhibited from binding by stable binding of the exact match probe. In one embodiment, the blocking oligonucleotide probes incorporate LNA to further increase the Tm difference between the perfect match and the one-base (or more) mismatched sequences. The test also included a SARS-CoV-2 wild-type specific ORF1a / b assay using coumarin-labeled probes as a control.

[0093] Nucleic acids from patient samples and added RNA internal control molecules (same as existing RNA QS reagents) are extracted simultaneously. Viral nucleic acids are released by the addition of proteinase and lysis reagents to the sample. The released nucleic acids bind to the silica surface of added magnetic glass particles. Unbound material and impurities such as denatured proteins, cell debris and potential PCR inhibitors are removed in subsequent wash reagent steps and the purified nucleic acids are eluted from the magnetic glass particles with elution buffer at elevated temperature.

[0094] Example 2: Selection of primer and probe oligonucleotides The master mix contains fluorescently labeled detection probes specific for the mutations E484K, N501Y, del69-70 of the S gene, and additionally the wild type ORF1a / b gene. The RNA internal control detection probe was also labeled with Cy5.5 dye, which acts as a reporter. Each probe also contained a second dye, which acts as a quencher. PCR primers to amplify the region of interest were designed for the target region. Thus, we started the study with a single-well assay design to detect SARS-CoV-2 variants containing mutations in the S gene using: (i) the non-structural open reading frame (ORF1a / b) in the genome of SARS-CoV-2 in the coumarin channel; (ii) the S gene E484K mutation in the FAM channel; (iii) the S gene N501K mutation in the HEX channel; (iv) the 69-70 deletion of the S gene in the JA270 channel. We performed a bioinformatics analysis of various assays that could be multiplexed with the RNA internal control oligonucleotides used for the detection of process controls to screen the initial assays for performance. To increase assay specificity, a competing unlabeled wild-type oligonucleotide (i.e., blocking probe) was included in the master mix of E484, N501, and wt69-70. Selected combinations of primer sets and probes are shown in Table 5.

[0095] [Table 5]

[0096] Example 3: PCR assay reagents and conditions Real-time PCR detection of SARS-CoV-2 (both wild type and variants) was performed using the cobas® 6800 / 8800 system platform (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of amplification reagents are shown below.

[0097] [Table 6]

[0098] The table below shows a typical thermal profile used in a PCR amplification reaction.

[0099] [Table 7]

[0100] The Pre-PCR program included incubations at 55°C, 60°C and 65°C for initial denaturation and reverse transcription of the RNA template. Incubating at three temperatures has the advantageous effect that at lower temperatures even slightly mismatched target sequences (such as genetic variants of an organism) are transcribed, while at higher temperatures the formation of RNA secondary structures is suppressed, thus resulting in more efficient transcription. The PCR cycling is divided into two runs, both of which apply a one-stage setup (combining annealing and extension). The first five cycles at 55°C allow for increased inclusiveness by preamplifying the slightly mismatched target sequences, while the 45 cycles of the second run increase specificity by using an annealing / extension temperature of 58°C.

[0101] Example 4: Performance evaluation Evaluation of components, workflow and assay reagents for SARS-CoV-2 variant testing was performed using cobas® 6800 reagents. Linearized recombinant plasmids were tested with assay oligonucleotides to evaluate performance. In vitro transcripts were also generated to evaluate assay performance using synthetic RNA. Nucleic acid quantification was performed using Qubit with DNA and RNA standards. Plasmid DNA and transcripts were serially diluted in MultiPrep Specimen Diluent Buffer (also known as Bulk Generic Specimen Diluent) and used for assay performance studies. An internal control oligonucleotide (Generic Internal Control, GIC) was included for evaluation with both linearized DNA and RNA transcripts. Experiments were performed on the analytical cycler of the cobas® 6800 system. Nasopharyngeal (NSP) samples were obtained from patients presenting with upper respiratory tract symptoms using agglutination swabs and collected in Universal Viral Transport Medium (3 mL). A modified sample preparation workflow (Process and Elute, PnE) was used on the cobas® 6800 System to process either 300 or 400 μL of NSP samples to prepare nucleic acid eluates. These eluates followed the same NSP sample preparation process on the cobas® 6800 and contain gIC armored RNA (QS RNA control) to act as an internal sample processing control. The eluates were then used in testing with SARS-CoV-2 assays with amplification and detection on the LC480 and / or cobas® 6800 analytical cyclers.

[0102] A multiplex PCR assay was then performed, testing the primer and probe oligonucleotides listed in Table 5 in a single reaction. Test samples (n=2) included Zeptometrix SARS-CoV-2 wild-type genomic RNA tested at concentrations of 1e6 to 1e1 copies / PCR, a mutant transcript containing both the E484K and N501Y mutations tested at concentrations of 1e10 to 1e1 copies / PCR, and a Twist synthetic control transcript with both the N501Y mutation and the 69-70 deletion tested at concentrations of 1e6 to 1e1 copies / PCR. All experiments were performed on the devised NPS, and the results of these tests are shown in Figure 2, Figure 3, and Figure 4. The data showed robust growth curves and PCR efficiencies over a wide dynamic range, with transcripts detected up to 10 copies per PCR reaction for all targets.

[0103] Example 5: Determination of analytical sensitivity (detection limit) For the determination of analytical sensitivity, six SARS-CoV-2 virus stocks were used: two isolates each prepared at the University of Zurich (UZ1 isolate: P.2 lineage, clade 20B with E484K; UZ2 isolate: B.1 lineage, clade 20A with N501Y) and the University of Frankfurt (UF1 isolate: B.1.351 lineage, clade 20H / 501Y.V2 with E484K and N501Y; and UF2 isolate: B.1.1.7 lineage, clade 20I / 501Y.V1 with N501Y and del69 / 70). Labor Berlin used two isolates kindly provided by the National Consultation Laboratory for Coronaviruses at the Institute of Virology, Medical University at the Charite Berlin (LB1 isolate: B.1.351 lineage, clade 20H / 501Y.V2 with E484K and N501Y; and LB2 isolate: B.1.1.7 lineage, clade 20I / 501Y.V1 with N501Y and del69 / 70).

[0104] The titer of the viral stock was determined using the cobas® SARS-CoV-2 Assay for use with the cobas® 6800 / 8800 System, which reports cycle threshold (Ct) values. The first international standard of the World Health Organization (WHO) for SARS-CoV-2 ribonucleic acid (RNA; NIBSC National Institute of Biological Standards and Control code 20 / 146) was also tested in this assay at two different concentrations (3.7 and 5.7 log IU / ml), allowing the conversion of the Ct of unknown samples to international units (IU) based on the linear regression of the standard curve (log10 IU / mL=12.66-0.297*Ct). Four to seven dilutions of each of six different viral isolates were prepared in CPM (cobas® PCR Media) or UTM-based mock matrix (UTM, 50,000 human peripheral blood mononuclear cells / mL, 0.05% mucin) to generate a panel containing at least four concentrations: approximately 3x, 1x, 0.3x, and 0.1x the expected limit of detection (LoD). Each panel member was tested in 21 replicates. Hit rate analysis was used to determine the LoD (the concentration that yields at least 95% positive results) and reported in IU / mL.

[0105] The lowest virus concentration tested that gave at least 95% positive results for each locus, as well as the corresponding mean cycle thresholds for the SCI controls, are shown in Table 8. For E484K, the limit of detection (LoD) determined by this method was between 180 and 620 IU / mL for the three different isolates tested. For N501Y, the LoD was between 270 and 720 IU / mL (five isolates), whereas for the deletions of codons 69 and 70, it was 80 or 92 IU / mL. The LoD for the SCI positive control targets was between 18 and 80 IU / mL.

[0106] [Table 8]

[0107] Example 6: Determining Accuracy Using Clinical Samples To determine the accuracy of the SARS-CoV-2 variant test, specimens containing SARS-CoV-2 with or without one or more of the three targeted loci were tested at four sites. The presence or absence of mutations was established by sequencing of S using standard Sanger-based methods (University of Zurich) or next-generation methods (Labor Berlin, University Hospital of Regensburg and Bioscientia, Ingelheim; see Supplementary Methods). A total of 273 isolates were included. As the standard Sanger-based method used at the University of Zurich does not cover the deletion of codons 69–70, these samples were excluded from the analysis of that locus. All samples were RT-PCR positive using a variety of commercially available or laboratory-developed tests. Various specimen types were included, including nasal, nasopharyngeal and oropharyngeal swabs, bronchoalveolar lavage fluid, tracheal secretions, and respiratory washes in diverse media (e.g., water, saline, Universal Transport Medium, cobas PCR Medium) (Table 9).

[0108] [Table 9]

[0109] SCI control reactions for all 273 isolates were positive, indicating the presence of viral RNA in the samples. A total of 20 specimens in which E484K was present were tested by sequencing (Table 10). All were reactive with the E484K probe (sensitivity: 100%). Conversely, 252 specimens without a substitution at position 484 were not reactive with E484K (specificity: 100%). One specimen was missing sequence data at position 484. Similar results were obtained for N501Y (108 with substitution, 164 without substitution; one specimen was missing sequence data) and deletions at codons 69 and 70 (99 with deletion, 157 without deletion; 17 specimens were missing sequence data in this region) and are summarized in Table 10. No false positive or false negative results were observed.

[0110] [Table 10]

[0111] Example 7: Determination of analytical specificity (interfering organisms) Specificity was assessed using samples engineered to contain one of 17 different viruses (target concentration: 105 units / mL in a UTM-based mock matrix), eight bacteria (106 units / mL), or Pneumocystis jirovecii (106 units / mL). The 17 viruses tested were adenovirus, enterovirus, human coronavirus 229E, HKU1, NL63, and OC43, human metapneumovirus, influenza A and B viruses, MERS-coronavirus, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus, human rhinovirus, and SAR-coronavirus. The eight bacteria were Bordetella pertussis, Chlamydia pneumoniae, Haemophilus influenzae, Legionella pneumophila, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Streptococcus pyogenes, and Streptococcus pneumoniae. No signal was observed for any of the targeted mutations using either SCI or samples containing potentially cross-reactive organisms.

[0112] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made. For example, all of the techniques and devices described above may be used in various combinations.

Claims

1. 1. A method for detecting a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant having a mutation in a spike protein in a biological sample, comprising: - if SARS-CoV-2 nucleic acid is present in the sample, performing an amplification step comprising contacting the sample with a primer set and a polymerase enzyme having 5' to 3' nuclease activity to generate an amplification product; - carrying out a hybridization step comprising contacting said amplification products with one or more detectable probes; and - detecting the presence of said amplification product, wherein detection of said amplification product indicates the presence of said SARS-CoV-2 variant in said sample. Including, the primer set comprises a first primer comprising a first oligonucleotide sequence of SEQ ID NO:1, and a second primer comprising a second oligonucleotide sequence of SEQ ID NO:8; the one or more detectable probes comprise a third oligonucleotide sequence of SEQ ID NO: 17, or a complement thereof; the hybridizing step, or both the amplifying step and the hybridizing step, are performed in the presence of one or more blocking oligonucleotide probes, one of which has an oligonucleotide sequence that perfectly matches the wild-type spike protein sequence at amino acids 69-70; The method, wherein the spike protein mutation is a 69-70 deletion (del69-70).

2. 2. The method of claim 1, wherein the hybridizing step comprises contacting the amplification products with one or more detectable probes labeled with a donor fluorescent moiety and a corresponding acceptor moiety, and the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, the presence of fluorescence indicating the presence of a SARS-CoV-2 variant in the sample.

3. A set of primers comprising a first primer comprising an oligonucleotide sequence of SEQ ID NO:2 and a second primer comprising an oligonucleotide sequence of SEQ ID NO:10, and a detectable probe comprising an oligonucleotide sequence of SEQ ID NO:20 or its complement; wherein the hybridizing step, or both the amplifying and hybridizing steps, are performed in the presence of one or more additional blocking oligonucleotide probes, one of which has an oligonucleotide sequence that perfectly matches the wild-type spike protein sequence at amino acid position 484, and the mutation in the spike protein is an E484K mutation; and / or a set of primers comprising a first primer comprising the oligonucleotide sequence of SEQ ID NO:2 and a second primer comprising the oligonucleotide sequence of SEQ ID NO:10, and a detectable probe comprising the oligonucleotide sequence of SEQ ID NO:19 or its complement; wherein the hybridizing step, or both the amplifying and hybridizing steps, are performed in the presence of one or more additional blocking oligonucleotide probes, one of said one or more blocking oligonucleotide probes having an oligonucleotide sequence that perfectly matches the wild-type spike protein sequence at amino acid position 501, and the spike protein mutation is a N501Y mutation. The method of claim 1 or 2, further comprising:

4. The method described in any one of claims 1 to 3, wherein the one or more blocking oligonucleotide probes comprise an oligonucleotide sequence of SEQ ID NO: 37, 38 or 39, or any combination thereof.

5. 5. The method of any one of claims 1 to 4, further comprising providing a primer set that amplifies a specific nucleic acid sequence from the non-structural open reading frame (ORF1a / b) of SARS-CoV-2, and a detectable probe that hybridizes to and detects the ORF1a / b amplification product generated by the primer set.

6. The method of claim 5, wherein the primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:6 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:15; and the detectable probe comprises the oligonucleotide sequence of SEQ ID NO:

36.

7. 1. A multiplex method for detecting SARS-CoV-2 variants having a mutation in the spike protein in a biological sample, comprising: - if said SARS-CoV-2 nucleic acid is present in said sample, performing an amplification step comprising contacting said sample with at least two primer sets to generate a first amplification product and a second amplification product; - carrying out a hybridization step comprising contacting said amplification products with at least two detectable probes that hybridize to said first amplification product and said second amplification product generated by said at least two primer sets; and - detecting the presence of at least one of the first amplification product and the second amplification product, wherein the presence of the at least one amplification product indicates the presence of the SARS-CoV-2 variant in the sample. Including, The first primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:1, and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:7 or 8; the second primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:2, and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:9, 10, or 11; a first detectable probe that hybridizes to the first amplification product generated by the first primer set comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOs:16-17, or a complement thereof; a second detectable probe that hybridizes to the second amplification product generated by the second primer set comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOs:18-20, or a complement thereof; the hybridizing step, or both the amplifying step and the hybridizing step, are performed in the presence of one or more blocking oligonucleotide probes, one of which has an oligonucleotide sequence that perfectly matches a spike protein sequence that is wild-type at amino acids 69-70, wild-type at amino acid 484, and / or wild-type at amino acid 501; The multiplex method, wherein the mutation in the spike protein is selected from a 69-70 deletion (del69-70), a N501Y mutation, or a E484K mutation, or a combination thereof.

8. The method described in claim 7, wherein the one or more blocking probes comprise an oligonucleotide sequence of SEQ ID NO: 37, 38 or 39, or any combination thereof.

9. 9. The method of claim 7 or 8, further comprising providing a primer set that amplifies a specific nucleic acid sequence from the nonstructural open reading frame (ORF1a / b) gene of SARS-CoV-2, and a detectable probe that hybridizes to and detects the ORF1a / b amplification product generated by the primer set.

10. 10. The method of claim 9, wherein the primer set for amplifying the ORF1a / b gene comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:6 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:15, and the detectable probe comprises the oligonucleotide sequence of SEQ ID NO:36 or its complement.

11. The first primer set for amplifying the SARS-CoV-2 nucleic acid comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:1 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:8, and the first detectable probe comprises the oligonucleotide sequence of SEQ ID NO:17 or a complement thereof; 11. The method of any one of claims 7 to 10, wherein the second primer set for amplifying a nucleic acid of SARS-CoV-2 comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:2 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:10, and the second detectable probe comprises the oligonucleotide sequence of SEQ ID NO:19 or a complement thereof.

12. 12. The method of any one of claims 7 to 11, wherein a third detectable probe is provided that hybridizes to the second amplification product generated by the second primer set, the third detectable probe comprising an oligonucleotide sequence of SEQ ID NO: 20 or a complement thereof.

13. 1. A kit for detecting mutations in one or more spike protein (S) genes from SARS-CoV-2 variants, comprising: a first primer comprising a first oligonucleotide sequence of SEQ ID NO:1; a second primer comprising a second oligonucleotide sequence of SEQ ID NO: 8; - a detectably labeled probe comprising an oligonucleotide sequence of SEQ ID NO: 17 or a complement thereof, the detectably labeled probe being configured to hybridize to an amplicon generated by said first primer and said second primer, the detectably labeled probe comprising a donor fluorescent moiety and a corresponding acceptor moiety; and a blocking oligonucleotide probe having an oligonucleotide sequence that perfectly matches the wild-type spike protein sequence at amino acids 69-70; Including the kit.

14. The method further comprising the steps of: a first primer comprising the oligonucleotide sequence SEQ ID NO:2; a second primer comprising the oligonucleotide sequence SEQ ID NO: 10; - a detectably labeled probe comprising the oligonucleotide sequence of SEQ ID NO: 20 or its complement; and - a blocking oligonucleotide probe having an oligonucleotide sequence that perfectly matches the wild-type spike protein sequence at amino acid position 484, The kit of claim 13 , comprising:

15. - a detectably labeled probe comprising the oligonucleotide sequence of SEQ ID NO: 19 or its complement; and a blocking oligonucleotide probe having an oligonucleotide sequence that perfectly matches the wild-type spike protein sequence at amino acid position 501; The kit of claim 14, further comprising:

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