Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants having 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 the Invention The present disclosure relates to the field of virus diagnosis, and more particularly to the detection of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) containing mutations in the spike (S) protein gene.

Background Art

[0002] Background of the Invention Viruses of the family Coronaviridae have a single-stranded positive-sense RNA genome with a length in the range of 26 to 32 kilobases. Coronaviruses have been identified in several avian hosts as well as in various mammals including camels, bats, masked palm civets, mice, dogs and cats. Novel mammalian coronaviruses are currently being identified regularly. For example, the bat-derived HKU2-related coronavirus was the cause of a fatal acute diarrhea syndrome in pigs in 2018.

[0003] Among several coronaviruses pathogenic to 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 Guangdong Province, southern China, in November 2002, and caused more than 8,000 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, which was the cause of 2,494 confirmed infections and 858 deaths in laboratories 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 birds and several non-aquatic animals, including rabbits, had also been sold prior to the outbreak. A novel human infectious coronavirus, initially named 2019-nCoV, was identified using next-generation sequencing. This novel coronavirus is classified as belonging to the Coronaviridae family, Betacoronavirus genus, and Salvecovirus subgenus, as described in Lu, R. et al., Lancet, 2020, Vol. 395, pp. 565-574, "Genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding." On February 11, 2020, the World Health Organization (WHO) officially named the virus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0005] To combat the spread of SARS-CoV-2 and reduce COVID-19-related morbidity and mortality, several types of research and product development strategies are being pursued. These include the development of mRNA, viral vectors, and protein subunit vaccines, small molecule antiviral drugs, immunomodulators, and other non-pharmaceutical interventions. Vaccines developed and researched to date have primarily focused on the viral envelope glycoprotein (spike, S) and have been remarkably successful. Furthermore, monoclonal antibodies targeting S have been developed as antiviral drugs and are an effective treatment when administered immediately 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 tends to evolve in response to external selection pressures due to its error-prone RNA-dependent RNA polymerase and large population size. While coronaviruses possess proofreading function as part of their replicase complex, their high replication rate in each host and in the vast population of infected individuals leads to the generation of a huge pool of viral variants in which more fit variants can emerge. Potent but incomplete replication inhibition, which can occur in infected individuals with partial immunity or those treated with a single anti-S monoclonal antibody, almost certainly leads to the selection of SARS-CoV-2 variants with S escape mutations that have higher replication fit than the wild-type virus in a population of susceptible hosts. Similarly, if a naturally occurring variant has an increased ability to spread in an immunologically naive population, it could outperform the wild-type virus in a relatively short period.

[0007] More than a year has passed since the start of the SARS-CoV-2 pandemic, and uncontrolled global transmission and significant viral evolution have resulted in hundreds of variants. Some of these variants, considered to be VOCs (Variants of Concern), such as those in the UK (B.1.1.17), South Africa (B.1.351), Brazil (P.1 / B.1.1.248), and VOCs of Interest (VOIs) US [B.1.526 (NY) and B.1.427 / B.1.429 (California, and Ohio)], are more transmissible and / or may affect treatment or vaccine response. Epidemiological and virological assessments have confirmed that more transmissible VOCs are now emerging independently, with reports from South Africa indicating a rapidly spreading, distinct lineage becoming dominant within weeks, shortly after the initial report from the UK in December 2020. While the significance of the mutation has not yet been fully determined, genomic data showing rapid replacement of other lineages suggest 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-scale resurgence in cases. Concerns regarding vaccines and related spike-borne SARS-CoV-2 adaptations necessitated epidemiological and surveillance efforts for VOC selection. Key mutations found in these emerging strains (del69-70, N501Y, E484K) are being used to track the spread of these most concerning strains. Mutations in the SARS-CoV-2 spike gene make the virus highly transmissible. Therefore, there is a need in the art for rapid, reliable, specific, and highly sensitive methods to detect SARS-CoV-2 variants, particularly those including the 69-70 deletion (del69-70) and mutations in the spike protein gene at N501Y and E484K. [Overview of the project]

[0008] This disclosure relates to a rapid method for detecting the presence or absence of SARS-CoV-2 variants having spike protein mutations in living or non-living samples, 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. Embodiments include a method for detecting SARS-CoV-2 variants having one or more mutations, including del69-70, N501Y, and E484K, comprising carrying out a reverse transcription step and at least one cycling step which may include an amplification step and a hybridization step. Furthermore, this disclosure includes primers, probes, and kits designed to detect SARS-CoV-2 variants in a single tube.

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

[0010] In another embodiment, a multiplex method is provided for detecting SARS-CoV-2 variants having a spike protein mutation in a sample, the method comprising: an amplification step, if the nucleic acid of SARS-CoV-2 is present in the sample, comprising contacting the sample with at least two primer sets to produce a first amplification product and a second amplification product; a hybridization step, comprising contacting the amplification product with at least two detectable probes that hybridize to the first amplification product and the second amplification product produced by the at least two primer sets; and detection of the presence of at least one amplification product, wherein the presence of at least one amplification product indicates the presence of a SARS-CoV-2 variant in the sample; the first primer set comprising a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 1, and SEQ ID NO: 7 or The first set of primers comprises a reverse primer containing or consisting of 8 oligonucleotides, the second set of primers comprises a forward primer containing or consisting of the oligonucleotide sequence of SEQ ID NO: 2, and a reverse primer containing or consisting of the oligonucleotide sequence of SEQ ID NO: 9, 10, or 11; the first detectable probe that hybridizes to the first amplification product produced by the first set of primers comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 16-17 or its complement; the second detectable probe that hybridizes to the second amplification product produced by the second set of primers comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 18-20; the spike protein mutation is selected from 69-70 deletions (del69-70), N501Y mutations, or E484K mutations, or combinations thereof. In one embodiment, the step is carried out in the presence of one or more blocking oligonucleotide probes containing or consisting of the oligonucleotide sequence of SEQ ID NOs: 37, 38, or 39, or any combination thereof.

[0011] In this specification, SARS-CoV-2 variants are selected from 69-70 deletions (del69-70), N501Y mutations, or E484K mutations of the spike protein as a result of each mutation and deletion of the S gene, or a combination thereof. In some embodiments, a first or second detectable probe specifically hybridizes to the S gene sequence that causes the 69-70 deletion in SARS-CoV-2. In some embodiments, a first or second detectable probe specifically hybridizes to the S gene sequence that causes the N501Y mutation in SARS-CoV-2. In some embodiments, a first or second detectable probe specifically hybridizes to the S gene sequence that causes the E484K mutation in SARS-CoV-2. In one embodiment, one or more blocking probes contain or consist of oligonucleotide sequences that perfectly match the wild-type S gene sequence at amino acids 69-70, or at amino acid 484 or 501 of the spike protein. In some embodiments, one or more blocking probes include or consist of the oligonucleotide sequences of SEQ ID NOs. 37, 38, or 39, and combinations thereof. Further embodiments provide a primer set for amplifying specific nucleic acid sequences derived from the unstructured open reading frame (ORF1a / b) of SARS-CoV-2, and a detectable probe for hybridizing to and detecting the ORF1a / b amplification product generated by the primer set. In one embodiment, the primer set includes a forward primer containing or consisting of the oligonucleotide sequence of SEQ ID NOs. 6, and a reverse primer containing or consisting of the oligonucleotide sequence of SEQ ID NOs. 15; the detectable probe contains or consists of the oligonucleotide sequence of SEQ ID NOs. 36 or its complement.

[0012] In one embodiment, a primer set for amplifying a SARS-CoV-2 variant includes a first primer containing or comprising the oligonucleotide sequence of SEQ ID NO: 1, a second primer containing or comprising the oligonucleotide sequence of SEQ ID NO: 7 or 8, and a detectable probe containing or comprising the oligonucleotide sequence of SEQ ID NO: 16 or 17 or its complement. In another embodiment, the first primer contains or comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 2, the second primer contains or comprises the oligonucleotide sequences of SEQ ID NOs: 9-11, and the detectable probe contains or comprises the oligonucleotide sequences of SEQ ID NOs: 18-20 or their complements. In one embodiment, the first primer contains or comprises the oligonucleotide sequence of SEQ ID NO: 1, the second primer contains or comprises the oligonucleotide sequence of SEQ ID NO: 8, and the detectable probe contains or comprises 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 its complement. 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 its complement.

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

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

[0015] In one embodiment, amplification may be performed using a polymerase enzyme having 5'-to-3' nuclease activity. Thus, the donor fluorescent moiety and acceptor moiety, e.g., the quencher, may be within 5-20 nucleotides (e.g., 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 the quencher.

[0016] In one embodiment, a detectable probe for detecting SARS-CoV-2 variants may be labeled with a fluorescent dye acting as a reporter. The probe may also have a second dye acting as a quencher. The reporter dye, by being measured at a specified wavelength, enables the detection and identification of amplified SARS-CoV-2 targets. The fluorescent signal of the intact probe is suppressed by the quencher dye. During the PCR amplification step, hybridization of the probe to a specific single-stranded DNA template results in 5'-to-3' nuclease activity cleavage by DNA polymerase, leading to the separation of the reporter and quencher dyes and the generation of fluorescent signals. 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., internal reference controls or other targeted probes (e.g., other viral nucleic acids)) may also be labeled with a reporter fluorescent dye distinct from the specific fluorescent dye labeling associated with the SARS-CoV-2 probe. In such cases, the individual reporter dyes are measured at a defined wavelength, enabling simultaneous detection and identification of the amplified SARS-CoV-2 target and one or more additional probes.

[0017] This disclosure also provides methods for detecting the presence or absence of SARS-CoV-2 variants or SARS-CoV-2 nucleic acids, including mutations or deletions in the spike protein gene, in biological samples derived from an individual. These methods may be used for diagnostic testing to detect the presence or absence of SARS-CoV-2 variants or SARS-CoV-2 with mutations or deletions in the spike gene in nasopharyngeal (NSP) and oropharyngeal swab samples. Furthermore, those skilled in the art may use the same tests to evaluate other types of samples to detect SARS-CoV-2 variants or mutations and deletions in the SARS-CoV-2 spike gene. Such methods generally involve performing a reverse transcription step and at least one cycling step, including an amplification step and a dye-binding step. Typically, the amplification step, if nucleic acid molecules are present in the sample, involves contacting the sample with multiple pairs of oligonucleotide primers to produce one or more amplification products, and the dye-binding step involves 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 indicates the presence of a SARS-CoV-2 variant or mutations and deletions of the SARS-CoV-2 spike gene in the sample, while the absence of binding indicates the absence of a SARS-CoV-2 variant or mutations and deletions of the SARS-CoV-2 spike gene in the sample. A typical 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. Furthermore, such methods may also include determining the melting temperature between the amplification product and the double-stranded DNA-binding dye to confirm the presence or absence of a SARS-CoV-2 variant or mutations and deletions of the SARS-CoV-2 nucleic acid.

[0018] In a further embodiment, a kit is provided for detecting mutations in one or more spike genes derived from SARS-CoV-2 variants. The kit may comprise one or more primer sets specific to the amplification of a gene target; and one or more detectable oligonucleotide probes specific to the detection of the amplification product.

[0019] In one embodiment, the kit may include a probe already labeled with a donor and corresponding acceptor moiety, such as another fluorescent moiety or dark quencher, or a fluorophore moiety for labeling the probe. The kit may also include a nucleoside triphosphate, 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, probe, and fluorophore moiety to detect the presence or absence of mutations and deletions in the SARS-CoV-2 spike gene in a sample.

[0020] In one embodiment, 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 which is a locked nucleic acid (LNA), wherein the blocking oligonucleotide is fully complementary to the wild-type (WT) sequence when hybridizing to form a first complex having a first melting temperature (Tm), and the blocking oligonucleotide is one when hybridizing to form a second complex having a second melting temperature (Tm). Alternatively, in multiple nucleotides, the blocking oligonucleotide is partially non-complementary to the target mutant (MT) sequence, the first Tm is higher than the second Tm, and the blocking oligonucleotide is blocked at the 3' end and its extension is inhibited; the amplification step is carried out at a temperature higher than the second Tm but lower than the first Tm, and the amplification step includes contacting the sample with a primer set to produce an amplification product, if the WT sequence and / or target MT sequence are present in the sample, the blocking oligonucleotide no longer hybridizes to the target MT sequence during the amplification step but remains hybridized to the WT sequence and inhibits the amplification of the WT sequence.

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

[0022] In another embodiment, oligonucleotides are provided for carrying out allele-specific amplification of a target sequence, which exist in the form of several variant sequences, the oligonucleotide comprising a 5' end and a 3' end that is blocked at the 3' end and prevents elongation, the sequence being fully complementary to the wild-type (WT) sequence when hybridizing to form a first complex having a first melting temperature (Tm), and being partially complementary to the target variant (MT) sequence in one or more nucleotides 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 being a locked nucleic acid (LNA).

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains. Similar or equivalent methods and materials may be used in the practice or testing of this subject, but preferred methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative and not intended to limit the scope.

[0024] 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 become apparent from the drawings, the mode for carrying out the invention, and the claims.

Brief Description of the Drawings

[0025] [Figure 1] Shows deletions and mutations of the spike protein gene of the present disclosure and their positions within the SARS-CoV-2 genome. ORF, open reading frame; S, spike protein; RBD, receptor binding domain. [Figure 2] Growth curves generated from the SARS-CoV-2 variant tests described in Example 4 using Zeptometrix wild-type SARS-CoV-2 genomic RNA at the indicated levels in a multiplex PCR test using detection in the coumarin channel. [Figure 3] Growth curves generated from the SARS-CoV-2 variant tests described in Example 4 using mutant transcripts containing both the E484K and N501Y mutations at the indicated levels in a multiplex PCR test using detection in the FAM channel (left) and the HEX channel (right). [Figure 4] Growth curves generated from the SARS-CoV-2 variant tests described in Example 4 using Twist synthetic control transcripts having both the N501Y mutation and the 69-70 deletion at the indicated levels in a multiplex PCR test using detection in the HEX channel (left) and the JA270 channel (right). [[ID=|19]]

Mode for Carrying Out the Invention

[0026] Detailed Description of the Invention Nucleic acid amplification-based diagnosis of SARS-CoV-2 infection (both wild-type and variant) provides a method for rapid, accurate, reliable, specific, and sensitive detection of viral infection. A real-time reverse transcription PCR assay for detecting SARS-CoV-2 variants with spike protein gene mutations in non-living or living samples is described herein. Primers and probes for detecting SARS-CoV-2 variants are provided, as are manufactured products or kits containing such primers and probes. Compared to other methods, the high specificity and sensitivity of real-time PCR for detecting SARS-CoV-2 variants, as well as the improved features of real-time PCR including sample containment and real-time detection of amplified products, make the implementation of this technology feasible for the routine diagnosis of not only SARS-CoV-2 infection but also SARS-CoV-2 variant infection in clinical laboratories. Furthermore, this technology can be used for in vitro and prognostic diagnosis. This SARS-CoV-2 variant detection assay can also be multiplexed in parallel with other assays for the detection of other nucleic acids, such as influenza virus, SARS-CoV, and MERS-CoV.

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

[0028] The S gene encodes the spike protein, also known as the S protein or surface glycoprotein, a transmembrane glycosylated protein composed of 1273 amino acids that assemble as a homotrimer to form the spike that protrudes from the SARS-CoV-2 viral envelope. The spike protein mediates viral entry into host cells by first binding to a host receptor via the receptor-binding domain (RBD) within the S1 subunit, and then fusing the viral membrane with the host membrane via the S2 subunit. Similar to SARS-CoV, SARS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2) as its host receptor for the viral S protein. The RBD of the SARS-CoV-2 spike protein is 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 exhibiting greater infectivity, higher viral load, and potentially increased lethality, coupled with reduced neutralization by antibodies produced by vaccines using wild-type S targets. These VOC variants include UK B1.1.7 (in particular, 69-70del, N501Y), South Africa B.1.351 (K417N, E484K, and N501Y), and Brazil B.1.1.28 (E484K, N501Y) and P1. These variants were detected by sequencing of 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] This disclosure includes, for example, oligonucleotide primers and fluorescently labeled hydrolysis probes that hybridize to the spike protein gene of the SARS-CoV-2 genome for the specific identification of SARS-CoV-2 variants using TaqMan® amplification and detection techniques. The oligonucleotides specifically hybridize to the S gene. Since having oligonucleotides that hybridize to multiple locations within the genome is advantageous for improved sensitivity compared to targeting only a single locus, this disclosure also provides oligonucleotide primers and hydrolysis probes that hybridize to other regions within the SARS-CoV-2 genome (e.g., the ORF1ab gene).

[0031] The disclosed method may include a reverse transcription step and at least one cycling step, which involves 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 primers” refers to oligonucleotide primers that specifically anneal to nucleic acid sequences found in the SARS-CoV-2 genome and, under appropriate conditions, initiate DNA synthesis therefrom to produce their respective amplification products. Examples of nucleic acid sequences found in the SARS-CoV-2 genome include the ORF1ab gene, S gene, ORF3ab gene, E gene, M gene, and N gene, and nucleic acids within other predicted ORF regions. Each of the SARS-CoV-2 primers under consideration anneals to a target region such that at least a portion of each amplification product contains the nucleic acid sequence corresponding to the target. If one or more nucleic acids are present in the sample, one or more amplification products will be produced, and therefore, the presence of one or more amplification products indicates the presence of SARS-CoV-2 in the sample. The amplification products should contain nucleic acid sequences complementary to one or more detectable probes for SARS-CoV-2. As used herein, “SARS-CoV-2 probes” refers to oligonucleotide probes that specifically anneal to nucleic acid sequences found in the SARS-CoV-2 genome. Each cycling step comprises an amplification step, a hybridization step, and a detection step (in which the sample comes into contact with one or more detectable SARS-CoV-2 probes for the detection of the presence or absence of SARS-CoV-2 in the sample).

[0032] As used herein, the term “amplify” 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 the primers to the template nucleic acid at a temperature below the primer melting temperature, and enzymatically extending from the primers to produce an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphate, a DNA polymerase enzyme (e.g., Platinum® Taq), and appropriate buffers and / or cofactors (e.g., MgCl2 and / or KCl) for optimal activity of the polymerase enzyme.

[0033] As used herein, the term “primer” is known to those skilled in the art and refers to oligomeric compounds, primarily oligonucleotides, but also to modified oligonucleotides that can “prime” DNA synthesis by template-dependent DNA polymerases, namely, for example, the 3' end of an oligonucleotide provides a free 3'-OH group, to which a “nucleotide” can be further attached by a template-dependent DNA polymerase that establishes a phosphodiester linkage from 3' to 5', thereby utilizing a deoxynucleoside triphosphate and releasing pyrophosphate.

[0034] The term "hybridize" refers to the annealing of one or more probes to the amplification product. "Hybridization conditions" typically include a temperature lower than the melting temperature of the probes but that avoids nonspecific hybridization of the probes.

[0035] The term "5'-to-3' nuclease activity" typically refers to the activity of nucleic acid polymerases involved in nucleic acid chain synthesis, where nucleotides are removed from the 5' end of the nucleic acid chain.

[0036] The term "thermally stable polymerase" refers to a polymerase enzyme that is thermally stable, meaning the enzyme catalyzes the formation of primer extension products complementary to the template and does not irreversibly denature when subjected to high temperatures for the time required to cause denaturation of the double-stranded template nucleic acid. Generally, synthesis begins at the 3' end of each primer and proceeds along the template strand in the 5' to 3' direction. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, if necessary, non-thermally unstable polymerases can still be used in PCR assays, provided the enzymes are supplemented.

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

[0038] When used in relation to nucleic acids, the terms "extension" or "lengthening" refer to the incorporation of additional nucleotides (or other similar molecules) into the nucleic acid. For example, nucleic acids can be arbitrarily extended by biocatalysts that incorporate nucleotides, such as polymerases, which typically add nucleotides to the 3' end of nucleic acids.

[0039] In the context of two or more nucleic acid sequences, the term “identical” or “identity” percentage refers to two or more sequences or subsequences that, when compared and aligned to the greatest extent possible, for example, using one of the sequence comparison algorithms available to those skilled in the art or by visual inspection, have identical or a specific percentage of identical nucleotides. An exemplary algorithm suitable for determining sequence identity percentage and sequence similarity is the BLAST program, for example, see 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 This is described in Res.7:649-656.

[0040] In the context of oligonucleotides, a "modified nucleotide" refers to a change in an oligonucleotide sequence in which at least one nucleotide is replaced by a different nucleotide that provides the oligonucleotide with a desired property. Examples of 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, and N6-methyl-dA. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or otherwise known in the art. In certain embodiments, a modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. Nucleoside modifications may also include a moiety that increases the stringency of hybridization or a moiety that increases the melting temperature of the oligonucleotide probe. For example, a nucleotide molecule may be modified with an additional bridge connecting the 2' and 4' carbons, resulting in a “locked nucleic acid (LNA)” nucleotide that is resistant to cleavage by nucleases (as described in U.S. Patent No. 6,268,490 by Imanishi et al. and U.S. Patent No. 6,794,499 by Wengel et al.). Further, certain modified nucleotide substitutions may, in some embodiments, reduce nonspecific nucleic acid amplification (e.g., minimizing primer dimer formation, etc.) and 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] Detection of SARS-CoV-2 This disclosure provides a method for detecting SARS-CoV-2 variants having spike protein mutations by, for example, 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 number NC_045512, where the S gene is located at nucleotides 21563 to 25384). Specifically, primers and probes for amplifying and detecting mutation and deletion target sequences of the SARS-CoV-2 S gene are provided by embodiments of this disclosure.

[0042] For the detection of SARS-CoV-2 VOCs, primers for amplifying the S gene and probes for specifically detecting mutations and deletions in the S gene are provided. Other SARS-CoV-2 nucleic acids not exemplified herein may 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 skilled in the art using routine methods. Representative functional variants may include, for example, one or more deletions, insertions, and / or substitutions in the SARS-CoV-2 nucleic acids disclosed herein.

[0043] More specifically, each embodiment of the oligonucleotide comprises a nucleic acid having a sequence selected from SEQ ID NOs: 1-5, 7-14, and 16-25, or complements of SEQ ID NOs: 1-5, 7-14, and 16-25. In some embodiments, oligonucleotide probes are provided that block the detection of the wild type (e.g., wild type residues at positions 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 above-described SARS-CoV-2 primer set and probe are used to provide detection of SARS-CoV-2 in a biological sample suspected of containing a SARS-CoV-2 variant (Tables 1-4). The primer set and probe may include or consist of primers and probes specific to the nucleic acid sequence of SARS-CoV-2, containing or comprising the nucleic acid sequences of SEQ ID NOs. 1-5, 7-14, 16-25, and 37-39.

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

[0050] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acids encoding SARS-CoV-2 targets, such as nucleic acid molecules encoding other parts of SARS-CoV-2, can be designed using computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Key features in designing oligonucleotides to be used as amplification primers include, but are not limited to, appropriately sized amplification products to facilitate detection (e.g., by electrophoresis), similar melting temperatures for members of the primer pair, and the length of each primer (i.e., primers must be long enough to anneal with sequence specificity and initiate synthesis, but not so long as to reduce accuracy during oligonucleotide synthesis). Typically, oligonucleotide primers are 8 to 50 nucleotides long (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).

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

[0052] In some embodiments, the disclosed SARS-CoV-2 probes may be labeled with at least one fluorescent label. In one embodiment, the SARS-CoV-2 probe may 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 includes or comprises a fluorescent moiety, and the nucleic acid sequence includes or comprises SEQ ID NOs. 21-26.

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

[0054] Each construct may contain a vector, each containing one of the SARS-CoV-2 primer and probe nucleic acid molecules. The construct can be used, for example, as a control template nucleic acid molecule. Suitable vectors are commercially available and / or prepared by recombinant nucleic acid techniques common in the art. SARS-CoV-2 nucleic acid molecules can be obtained, for example, by chemical synthesis, direct cloning from SARS-CoV-2, or nucleic acid amplification.

[0055] Suitable constructs for use in the method typically include a SARS-CoV-2 nucleic acid molecule (e.g., a nucleic acid molecule containing one or more sequences from SEQ ID NOs: 1-5, 7-14, and 16-25), as well as sequences encoding selectable markers (e.g., antibiotic resistance genes) for selecting the desired construct and / or transformant, and an origin of replication. The selection of a vector system usually depends on several factors, including but not limited to host cell selection, replication efficiency, selectivity, inducibility, and ease of recovery.

[0056] Constructs containing the nucleic acid molecule of SARS-CoV-2 can be grown in host cells. As used herein, the term host cell means prokaryotes and eukaryotes, including, for example, yeast, plant, and animal cells. Examples of prokaryotic hosts include Escherichia coli (E. coli), Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Examples of eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, and 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 constructs can be introduced into host cells using any technique commonly 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 acids into host cells. Furthermore, naked DNA can be delivered directly to cells (see, for example, U.S. Patents 5,580,859 and 5,589,466).

[0057] Polymerase chain reaction (PCR) U.S. Patents 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Useful primers in some embodiments include oligonucleotides (e.g., SEQ ID NOs. 1-20) that can act as starting points for nucleic acid synthesis within the described SARS-CoV-2 nucleic acid sequence. Primers can be purified from restriction digests by conventional methods or can be synthesized. While single-stranded primers are preferred for maximum amplification efficiency, 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 achieved by any suitable denaturation method, including physical, chemical, or enzymatic means. One method for separating nucleic acid strands involves heating until the nucleic acid is predominantly denatured (e.g., denaturation exceeding 50%, 60%, 70%, 80%, 90%, or 95%). The heating conditions required to denaturate the template nucleic acid depend, for example, the salt concentration of the buffer, as well as the length and nucleotide composition of the nucleic acid being denatured, but are typically in the range of 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 30 seconds, or 1.5 minutes).

[0059] If the double-stranded template nucleic acid is 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 around 35°C to 65°C (e.g., around 40°C to 60°C, or around 45°C to 50°C). The annealing time can be around 10 seconds to 1 minute (e.g., around 20 seconds to 50 seconds; or around 30 seconds to 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient for extension to occur from the annealed primers and produce a complementary product to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but should not be so high as to denature the extension product from its complementary template (e.g., the temperature for extension is generally in the range of around 40°C to 80°C (e.g., around 50°C to 70°C; or around 60°C)). The duration of elongation can range from approximately 10 seconds to approximately 5 minutes (for example, approximately 30 seconds to approximately 4 minutes; approximately 1 minute to approximately 3 minutes; approximately 1 minute 30 seconds to approximately 2 minutes).

[0060] The genomes of retroviruses or RNA viruses, such as SARS-CoV-2, and 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) via the action of the enzyme reverse transcriptionase. The reverse transcriptionase directs the synthesis of the first strand of cDNA using the RNA template and a short primer complementary to the 3' end of the RNA, which can then be used directly as a template for polymerase chain reaction.

[0061] PCR assays can use SARS-CoV-2 nucleic acids, such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified; it can be a trace fraction of a complex mixture, such as SARS-CoV-2 nucleic acid found in human cells. SARS-CoV-2 nucleic acid molecules can be extracted from biological samples using 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 can be obtained from any number of sources, such as plasmids, or from 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 that induce primer extension. For example, the chain extension reaction product typically includes 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 product usually contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more of their analogues.

[0063] The newly synthesized chains form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of chain separation, annealing, and extension can be repeated as many times as necessary to produce the desired amount of amplification product corresponding to the target SARS-CoV-2 nucleic acid molecule. Limiting factors of the reaction are the amounts of primers, thermostable enzymes, and nucleoside triphosphates present in the reactants. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection applications, the number of cycling steps may depend, for example, on the properties of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps may be required to amplify a sufficient target sequence 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. Patents 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescence moiety and a corresponding acceptor fluorescence moiety are positioned within a certain distance of each other, energy transfer occurs between the two fluorescence moieties, which can be visualized or otherwise detected and / or quantified. Typically, the donor transfers energy to the acceptor when excited by irradiation with light of a suitable wavelength. Typically, the acceptor re-emits the transferred energy in the form of irradiation with light of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via biomolecules that substantially contain a non-fluorescent donor moiety (see, for example, U.S. Patent 7,741,467).

[0065] For example, an oligonucleotide probe may consist of a donor fluorescent moiety (e.g., HEX) and a corresponding quencher (e.g., BlackHole) that may or may not be fluorescent and dissipates the transferred energy in a form other than light. This may include Quencher® (BHQ). When the probe is intact, energy transfer typically occurs between the donor and acceptor moieties so that fluorescence emission from the donor moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplified product is cleaved by the 5'-to-3' nuclease activity of, for example, Taq polymerase, so that fluorescence emission from the donor moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patents 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 Includes Black (trademark) (Integrated DNA Tech., Inc., Coralville, Iowa) and BlackBerry (trademark) Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0066] In another example, two oligonucleotide probes, each containing a fluorescent moiety, may hybridize to an amplification product at a specific location 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 location, a FRET signal is generated. The hybridization temperature can range from approximately 35°C to approximately 65°C for approximately 10 seconds to approximately 1 minute.

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

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

[0069] Fluorescent donor and corresponding acceptor moieties are generally selected for (a) highly efficient Foerster energy transfer; (b) a large final Stokes shift (>100 nm); (c) a shift of emission to the red portion of the visible spectrum (>600 nm) as much as possible; and (d) a shift of emission to wavelengths higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescence moiety may be selected that has its maximum excitation wavelength near the 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 fluorescence moiety. A corresponding acceptor fluorescence 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 fluorescence 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, β-phycoerythrin, 9-acrididine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrene butyrate, and derivatives of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Typical acceptor fluorescence moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, lysamine 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). Donor and acceptor fluorescence moieties can be obtained, for example, from Molecular Probes (Junction City, Oregon) or Sigma Chemical Co. (St. Louis, Mexico).

[0071] The donor and acceptor fluorescent moieties can be attached to a suitable probe oligonucleotide via linker arms. The length of each linker arm is important because the linker arms can affect the distance between the donor and acceptor fluorescent moieties. The length of a linker arm may be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, linker arms are approximately 10 Å to 25 Å. The linker arms may be of the type described in WO84 / 03285. WO84 / 03285 also discloses methods for attaching linker arms to specific nucleotide bases and methods for attaching fluorescent moieties to linker arms.

[0072] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing aminolinkers (e.g., C6-aminophosphoramidites 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 (from FITC, e.g., fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide linkers (from fluorescein-NHS-ester, e.g., CX-fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPGs that require coupling of fluorescein-NHS-ester after oligonucleotide synthesis.

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

[0074] As described herein, the amplification product may be detected using a labeled hybridization probe that utilizes FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of the amplification product, and therefore the presence or absence of the SARS-CoV-2 variant. TaqMan® technology utilizes a single-strand hybridization probe labeled with, for example, 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 principle of FRET. The second moiety is generally the 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 step by, for example, 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, excitation of the first fluorescent moiety in the absence of a quencher may allow for the detection of fluorescence emission from the first fluorescent moiety. For example, the ABI PRISM® 7700 sequence detection system (Applied Biosystems) is suitable for performing the method described herein to detect the presence or absence of SARS-CoV-2 variants in a sample using TaqMan® technology.

[0075] Molecular beacons, combined with FRET, can also be used to detect the presence of amplified products using real-time PCR. Molecular beacon technology uses hybridization probes labeled with a first and a second fluorescent moiety. The second fluorescent moiety is typically a quencher, and the fluorescent label is usually located at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow for the formation of secondary structures (e.g., hairpins). As a result of the formation of secondary structures within the probe, both fluorescent moieties are spatially close when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplified product), the secondary structure of the probe is disrupted, and the fluorescent moieties separate from each other, thereby allowing the emission of the first fluorescent moiety to be detected after excitation with light of the appropriate wavelength.

[0076] Another common format of FRET technology utilizes two hybridization probes. Each probe may be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within the target DNA molecule (e.g., the amplification product). The donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the light source of a LightCycler® instrument. During FRET, fluorescein transfers its energy to the acceptor fluorescent moiety, e.g., LightCycler®-Red640 (LC Red640) or LightCycler®-Red705 (LC Red705). The acceptor fluorescent moiety then emits longer wavelength light, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can only occur if 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 may correlate 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 amplified product is generated, the hybridization process yields a detectable signal based on FRET between 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 sample collection, delayed transport, inappropriate transport 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] Typical 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 specimens are known to those skilled in the art. Biological specimens may be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release the nucleic acid of SARS-CoV-2, or in some cases, the biological specimens may be in direct contact with PCR reaction components and appropriate oligonucleotides.

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

[0080] While each thermocycler is running, control samples may also be cycled. Positive control samples may amplify target nucleic acid control templates (other than the amplified product of the target gene described) using, for example, control primers and probes. Positive control samples may also amplify plasmid constructs containing the target nucleic acid molecule, for example. Such plasmid controls may be amplified internally (e.g., within the sample) or in separate samples 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 amplification, hybridization, and / or FRET reaction. Each thermocycler run may also include, for example, a negative control lacking the target template DNA. Negative controls can be used to measure contamination. This ensures that the system and reagents do not produce false-positive signals. Thus, the reaction of the control can be easily determined, for example, the ability of the primers to anneal with sequence specificity and initiate extension, as well as the ability of the probes to hybridize with sequence specificity and for FRET to occur.

[0081] In some embodiments, the method includes a step to avoid contamination. For example, an enzymatic method utilizing uracil-DNA glycosylase to reduce or eliminate contamination between the operation of a thermocycler and the following is described in U.S. Patents No. 5,035,996, No. 5,683,896 and No. 5,945,313.

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

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

[0084] As an alternative to FRET, the amplification product 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, may also be used. When double-stranded DNA-binding dyes are used, melting curve analysis is typically performed to confirm the presence of the amplification product.

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

[0086] Embodiments of this disclosure are not limited to the configuration of one or more commercially available devices.

[0087] Manufactured products / kits Embodiments of this disclosure further provide a product or kit for detecting SARS-CoV-2 variants having spike protein mutations. The product may include primers and probes used to detect the SARS-CoV-2 S gene target, along with appropriate packaging materials. Representative primers and probes for the specific detection of SARS-CoV-2 S gene mutations can hybridize to SARS-CoV-2 target nucleic acid molecules. Furthermore, 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 for amplifying and hybridizing to SARS-CoV-2 S gene target nucleic acid molecules are provided.

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

[0089] The product may also include a package insert or packaging label with instructions for using SARS-CoV-2 primers and probes to detect SARS-CoV-2 variants having spike protein mutations in a sample. The product 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 this disclosure are further described in the following examples, which do not limit the scope of the invention as described in the claims. [Examples]

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

[0092] Example 1: Description of the assay A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed on the cobas® 6800 / 8800 system for the qualitative and identification of SARS-CoV-2 mutations N501Y, del69-70, and E484K in, for example, nasal and nasopharyngeal swab samples from known SARS-CoV-2 infected patients, supporting the understanding of variant epidemiology for population health management. The mutation-specific PCR assay can be used as a reflex test of SARS-CoV-2 positive samples to identify known mutations of concern as part of a SARS-CoV-2 variant surveillance strategy. The assay was strategically designed to enable single-nucleotide mutation detection by increasing the melting temperature (Tm) using a hydrolysis (TaqMan®) probe incorporating locked nucleic acid (LNA) chemistry, thereby promoting specificity for the detection of point mutations (E484K and N501Y). Detection of the six-nucleotide deletion in del69-70 could be performed using a conventional TaqMan® probe. The assay also included three dye-free 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 the fluorescently labeled mutant probes and the wt dye-free probes, so that the mismatched probes could be inhibited from binding by the stable binding of the precisely matched probes. In one embodiment, the blocking oligonucleotide probes incorporated LNA to further increase the Tm difference between perfectly matched and one-nucleotide (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 the patient sample and the added RNA internal control molecule (same as the existing RNA QS reagent) are extracted simultaneously. Viral nucleic acids are released by adding proteinase and lysis reagents to the sample. The released nucleic acids bind to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured proteins, cell debris, and potential PCR inhibitors, are removed in a subsequent washing reagent step, and the purified nucleic acids are eluted from the magnetic glass particles at high temperature using elution buffer.

[0094] Example 2: Selection of primer and probe oligonucleotides The master mix contains fluorescently labeled detection probes specific to the S gene mutations E484K, N501Y, del69-70, and also to the wild-type ORF1a / b gene. An RNA internal control detection probe was also labeled with a Cy5.5 dye acting as a reporter. Each probe also contained a second dye acting as a quencher. PCR primers were designed for target regions to amplify the desired region. Thus, we began our research with single-well assay designs to detect SARS-CoV-2 variants, including S gene mutations, using: (i) unstructured open reading frames (ORF1a / b) in the SARS-CoV-2 genome in a coumarin channel; (ii) the S gene E484K mutation in a FAM channel; (iii) the S gene N501K mutation in a HEX channel; and (iv) the 69-70 deletion of the S gene in a JA270 channel. Bioinformatics analysis of various assays, which could be multiplexed with RNA internal control oligonucleotides used for process control detection, was performed to screen initial assays for performance. To enhance the specificity of the assay, the master mixes of E484, N501, and wt69-70 were combined with competing unlabeled wild-type oligonucleotides (i.e., blocking probes). The 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 variant) was performed using the cobas® 6800 / 8800 system platform (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of the amplification reagents are shown below.

[0097] [Table 6]

[0098] The table below shows typical thermal profiles used in PCR amplification reactions.

[0099] [Table 7]

[0100] The pre-PCR program included incubation at 55°C, 60°C, and 65°C for initial denaturation and reverse transcription of the RNA template. Incubating at the three temperatures has the advantage that lower temperatures transcribe slightly mismatched target sequences (such as genetic variants of an organism), while higher temperatures suppress the formation of RNA secondary structures, thus resulting in more efficient transcription. The PCR cycling was divided into two measurements, each applying a single-step setting (combining annealing and extension). The first five cycles at 55°C allow for improved inclusivity by pre-amplifying slightly mismatched target sequences, while the 45 cycles of the second measurement increase specificity by using an annealing / extension temperature of 58°C.

[0101] Example 4: Performance Evaluation The components, workflow, and assay reagents for SARS-CoV-2 variant testing were evaluated using cobas® 6800 reagents. Linear recombinant plasmids were tested and evaluated using assay oligonucleotides. In vitro transcripts were also generated to evaluate the performance of assays 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 in evaluations using both linearized DNA and RNA transcripts. Experiments were performed using an analytical cycler of the cobas® 6800 system. Nasopharyngeal (NSP) samples were obtained from patients presenting with upper respiratory tract symptoms using agglutinating swabs and collected in Universal Viral Transport Medium (3 mL). A modified sample preparation workflow (process and elution, PnE) was used with the cobas® 6800 system to prepare nucleic acid eluates by processing either 300 or 400 μL of NSP samples. These eluates contained gIC-armored RNA (QS RNA control) acting as an internal sample processing control, following the same NSP sample preparation process in the cobas® 6800. The eluates were then used in a SARS-CoV-2 assay with amplification and detection in an LC480 and / or cobas® 6800 analytical cycler.

[0102] Next, multiplex PCR assays were performed, testing the primers and probe oligonucleotides listed in Table 5 in single reactions. Test samples (n=2) included Zeptometrix SARS-CoV-2 wild-type genomic RNA tested at concentrations of 1e6–1e1 copies / PCR, mutant transcripts containing both E484K and N501Y mutations tested at concentrations of 1e10–1e1 copies / PCR, and a Twist synthetic control transcript containing both the N501Y mutation and 69–70 deletions tested at concentrations of 1e6–1e1 copies / PCR. All experiments were performed using the designed NPS, and the results of these tests are shown in Figures 2, 3, and 4. The data demonstrated robust growth curves and PCR efficiency 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) Six SARS-CoV-2 virus stocks were used to determine the analytical sensitivity. Two isolates were prepared from each stock 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 generously provided by the National Consultation Laboratory for Coronaviruses at the Institute of Virology, Medical University in Berlin (LB1 isolate: lineage B.1.351, clade 20H / 501Y.V2 with E484K and N501Y; and LB2 isolate: lineage B.1.1.7, 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 in use with the cobas® 6800 / 8800 system, which reports cycle threshold (Ct) values. The World Health Organization's (WHO) first international standard 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 for the conversion of the Ct of unknown samples to international units (IU) based on 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 monocytes / mL, 0.05% mucin) to generate a panel containing at least four of the following concentrations: approximately 3x, 1x, 0.3x, and 0.1x above the expected limit of detection (LoD). Each panel member was tested 21 times. The LoD (concentration that yields at least a 95% positive result) was determined using hit rate analysis and reported in IU / mL.

[0105] Table 8 shows the lowest viral concentration tested for each locus that yielded at least 95% positive results, as well as the corresponding mean cycle threshold for SCI controls. 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 (for five isolates), but for deletions at codons 69 and 70, it was 80 or 92 IU / mL. The LoD for SCI-positive control targets was between 18 and 80 IU / mL.

[0106] [Table 8]

[0107] Example 6: Determination of accuracy using clinical specimens 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 target loci were tested at four sites. The presence or absence of mutations was established by S sequencing using standard Sanger-based methods (University of Zurich) or next-generation methods (Labor Berlin, University Hospital of Regensburg, and Bioscientia, Ingelheim; see Supplemental Methods). A total of 273 isolates were included. Since the standard Sanger-based method used at the University of Zurich does not cover deletions at codons 69–70, these specimens were excluded from analysis of that locus. All specimens were RT-PCR positive using various commercially available or laboratory-developed tests. A variety of specimen types were included, including nasal, nasopharyngeal, and oropharyngeal swabs, bronchoalveolar lavage fluid, tracheal secretions, and respiratory lavage fluid in various media (water, saline, universal transport medium, cobas PCR medium, etc.) (Table 9).

[0108] [Table 9]

[0109] SCI control reactions were positive for all 273 isolates, indicating the presence of viral RNA in the samples. Sequence analysis tested a total of 20 samples containing E484K (Table 10). All were reactive to the E484K probe (sensitivity: 100%). Conversely, 252 samples without substitution at position 484 were unreactive to E484K (specificity: 100%). One sample lacked sequence data at position 484. Similar results were obtained for N501Y (108 samples with substitution, 164 without substitution; one sample lacked sequence data) and deletions at codons 69 and 70 (99 samples with deletions, 157 without deletions; 17 samples lacked sequence data in this region), 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 evaluated using samples designed to contain one of 17 different viruses (target concentration: 10⁵ units / mL in a UTM-based simulated matrix), 8 types of 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 targeted mutations using either SCI or samples containing potentially cross-reactive organisms.

[0112] While the invention described above has been explained in some detail for the purpose of clarifying and understanding it, it will be apparent to those skilled in the art that various modifications can be made to the form and details. For example, all the techniques and apparatus described above can be used in various combinations.

Claims

1. A method for detecting SARS-CoV-2 variants having spike protein mutations in biological samples, - If the nucleic acid of SARS-CoV-2 is present in the sample, an amplification step is performed, which includes contacting the sample with a primer set and a polymerase enzyme having 5'-to-3' nuclease activity to produce an amplification product; - Performing a hybridization step which includes contacting the amplified product with one or more detectable probes; and - Detecting the presence of the amplification product, wherein the detection of the amplification product indicates the presence of the SARS-CoV-2 variant in the sample. Includes, The primer set comprises a first primer containing the first oligonucleotide sequence of SEQ ID NO: 1, and a second primer containing the second oligonucleotide sequence of SEQ ID NO: 8; The one or more detectable probes include the third oligonucleotide sequence of SEQ ID NO: 17 or its complement; A method wherein the hybridizing step, or both the amplification step and the hybridizing step, are carried out in the presence of one or more blocking oligonucleotide probes that are blocked at the 3' end, thereby preventing the elongation of the one or more blocking oligonucleotide probes, one of which has an oligonucleotide sequence that is in complete agreement with the wild-type spike protein sequence at amino acids 69-70, and comprises at least one nucleotide that is a locked nucleic acid (LNA), and the mutation in the spike protein is a 69-70 deletion (del69-70).

2. The method according to claim 1, wherein the hybridizing step includes contacting the amplified product with one or more detectable probes labeled with a donor fluorescent portion and a corresponding acceptor portion, and the detection step includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent portion and the acceptor portion 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 containing the oligonucleotide sequence of SEQ ID NO: 2 and a second primer containing the oligonucleotide sequence of SEQ ID NO: 10, and a detectable probe containing the oligonucleotide sequence of SEQ ID NO: 20 or its complement, Herein, the hybridization step, or both the amplification step and the hybridization step, are carried out 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 spike protein mutation is the E484K mutation; and / or A set of primers comprising a first primer containing the alkyl sequence of SEQ ID NO: 2 and a second primer containing the alkyl sequence of SEQ ID NO: 10, and a detectable probe containing the alkyl sequence of SEQ ID NO: 19 or its complement. Here, the hybridization step, or both the amplification step and the hybridization step, are carried out 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 the 501st amino acid position, and the spike protein mutation is an N501Y mutation. The method according to claim 1 or 2, further comprising:

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

5. The method according to any one of claims 1 to 4, further comprising providing a primer set for amplifying specific nucleic acid sequences derived from the unstructured open reading frame (ORF1a / b) of SARS-CoV-2, and a detectable probe for hybridizing to and detecting the ORF1a / b amplification product generated by the primer set.

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

36.

7. A multiplex method for detecting SARS-CoV-2 variants having mutations in the spike protein in a biological sample, - If the nucleic acid of SARS-CoV-2 is present in the sample, an amplification step is performed, which includes contacting the sample with at least two primer sets to produce a first amplification product and a second amplification product; - A hybridization step is performed which includes contacting the amplification product 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 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. Includes, The first primer set comprises a forward primer containing the oligonucleotide sequence of SEQ ID NO: 1 and a reverse primer containing the oligonucleotide sequence of SEQ ID NO: 7 or 8; the second primer set comprises a forward primer containing the oligonucleotide sequence of SEQ ID NO: 2 and a reverse primer containing 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 or its complement selected from the group consisting of SEQ ID NOs: 16-17; a second detectable probe that hybridizes to the second amplification product generated by the second primer set comprises an oligonucleotide sequence or its complement selected from the group consisting of SEQ ID NOs: 18-20; The hybridization step, or both the amplification step and the hybridization step, are carried out in the presence of one or more blocking oligonucleotide probes that are blocked at the 3' end, thereby preventing the elongation of the 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, and comprises at least one nucleotide that is a locked nucleic acid (LNA). A multiplexing method in which the mutation in the spike protein is selected from 69-70 deletion (del69-70), N501Y mutation, E484K mutation, or a combination thereof.

8. The method according to claim 7, wherein the one or more blocking probes include the oligonucleotide sequence of SEQ ID NO: 37, 38, or 39, or any combination thereof.

9. The method according to claim 7 or 8, further comprising providing a primer set for amplifying specific nucleic acid sequences derived from the unstructured open reading frame (ORF1a / b) genes of SARS-CoV-2, and a detectable probe for hybridizing to and detecting the ORF1a / b amplification product generated by the primer set.

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

11. The first primer set for amplifying the nucleic acid of SARS-CoV-2 comprises a forward primer containing the oligonucleotide sequence of SEQ ID NO: 1 and a reverse primer containing the oligonucleotide sequence of SEQ ID NO: 8, and the first detectable probe comprises the oligonucleotide sequence of SEQ ID NO: 17 or its complement. The method according to any one of claims 7 to 10, wherein the second primer set for amplifying the 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. The method according to 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 the oligonucleotide sequence of SEQ ID NO: 20 or a complement thereof.

13. A kit for detecting mutations in one or more spike protein (S) genes derived from SARS-CoV-2 variants, - A first primer containing the first oligonucleotide sequence of SEQ ID NO: 1; - A second primer containing the second oligonucleotide sequence of Sequence ID No. 8; - A detectably labeled probe comprising the oligonucleotide sequence or complement of Sequence ID No. 17, configured to hybridize to an amplicon generated by the first primer and the second primer, and comprising a donor fluorescent moiety and a corresponding acceptor moiety; and - A blocking oligonucleotide probe that will be blocked at its 3' end, thereby preventing elongation, and comprising at least one nucleotide that is a locked nucleic acid (LNA), wherein the blocking oligonucleotide probe further has an oligonucleotide sequence that is in complete agreement with the wild-type spike protein sequence at amino acids 69-70, A kit that includes this.

14. Further comprising a second primer set, the second primer set is - A first primer containing the oligonucleotide sequence of SEQ ID NO: 2; - A second primer containing the oligonucleotide sequence of SEQ ID NO: 10; - A detectably labeled probe containing 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 according to claim 13, including the following:

15. - A detectably labeled probe containing 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 according to claim 14, further comprising:

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