WO2022157389A1 - Composition and method for detecting sars-cov-2 voc 202012/01 - Google Patents

Abstract

The invention relates to novel primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2 VOC 202012/01, in particular by detecting the c5388a mutation (leading to A1708D) using primers and detectably-labelled probes for the wt and the mut allele, respectively, in PCR.

Description

COMPOSITION AND METHOD FOR DETECTING SARS-COV-2 VOC 202012/01

Field of the Invention

The invention relates to novel primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2.

Background to the Invention

Coronaviruses are members of the subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales. A novel coronavirus, 2019-nCoV or COVID-19, capable of infecting humans was identified in Wuhan, China and then led to a worldwide pandemic. Based on its phylogenetic relationships and genomic structures, the virus belongs to genera Betacoronavirus and has close similarities to severe acute respiratory syndrome-related coronaviruses (SARS-CoV). The virus uses ACE2 as the entry receptorlike SARS-CoV. These similarities had led the Coronavirus Study Group of the International Committee on Taxonomy of Viruses to term the virus SARS-CoV-2. Since the virus is novel, new methods are required to test for the virus and distinguish it from other similar coronaviruses. The sequence of SARS- Cov-2 has been published under accession no. MN908947 (https://www.ncbi.nlm.nih.gov/nuccore/MN908947.3'). Sequence information from around the world nevertheless indicates that numerous variants of the virus exist.

As is common with RNA viruses, variants of the strain have started to emerge. Multiple SARS-CoV-2 variants are circulating globally. Several new variants emerged in the last quarter of 2020, most notably:

In the United Kingdom (UK), a new variant of SARS-CoV-2 carrying the N501Y mutation (known as 20I/501Y.V1, VOC 202012/01, or B.l.1.7) emerged with an unusually large number of mutations. This variant has since been detected in numerous countries around the world, including the United States (US) and Canada and many European countries. This SARS-CoV-2 variant has a mutation in the receptor binding domain (RBD) of the spike protein at position 501, where amino acid asparagine (N) has been replaced with tyrosine (Y). The shorthand for this mutation is N501Y. It has been associated with increased transmissibility. This variant also has several other mutations, including: 69/70 deletion: occurred spontaneously many times and likely leads to a conformational change in the spike protein P681H: near the S1/S2 furin cleavage site, a site with high variability in coronaviruses. This mutation has also emerged spontaneously multiple times. ORF8 stop codon (Q27stop): mutation in ORF8, the function of which is unknown. A second variant carrying the N501Y mutation has emerged. This variant was first identified in Nelson Mandela Bay, South Africa, in samples dating back to the beginning of October 2020, and cases have since been detected outside of South Africa and is known as B.1.351 lineage (a.k.a. 20H/501 Y. V2). This variant has multiple mutations in the spike protein, including K417N, E484K, N501Y. Unlike the B.1.1.7 lineage detected in the UK this variant does not contain the deletion at 69/70. It too has been associated with increased transmissibility.

A third variant has also been identified in Japan and Brazil: P. l lineage (a.k.a. 20J/501Y. V3 The P.l lineage contains 17 unique amino acid changes and 3 deletions. This variant contains three mutations in the spike protein receptor binding domain: K417T, E484K, and N501Y.There is evidence to suggest that some of the mutations in the P.l variant may affect its transmissibility and antigenic profile, which may affect the ability of antibodies generated through a previous natural infection or through vaccination to recognize and neutralize the virus.

As the pandemic and the infections with these new variants continue to evolves it is important to have detection methods which can discriminate between these new variants.

Summary of the Invention

The inventors have surprisingly identified new primers, probes, kits and methods for determining the presence or absence of the variant of SARs-CoV-2 VOC 202012/01, also known as the UK variant and the presence or absence of a unique mutation within that variant in a variety of samples. The new primers, probes, kits and methods target the unique mutation in the nsp3 gene in SARs-CoV-2. The nsp3 gene encodes the non-structural protein 3 located within Open Reading Frame lab (ORF lab) and can distinguish the UK variant from other SARS-CoV-2 variants carrying the N501Y mutation.

The invention in a first aspect provides:

A pair of primers for determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a sample, wherein the pair of primers is selected from:

(a) a forward primer comprising a polynucleotide having the sequence shown in SEQ ID NO: 1, or a variant thereof having at least about 75% homology to SEQ ID NO: 1 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length, or

(b) the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6, or a variant thereof having at least about 75% homology to SEQ ID NO: 6 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7 or a variant thereof having at least about 75% homology to SEQ ID NO: 7 based on sequence identity over its entire length.

Preferably the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 and preferably, the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2.

Two complementary DNA (cDNA) amplicon amplified from SARS-CoV-2 cDNAby the pair of primers of the invention - the first carrying a mutation encoding for the C5388A mutation, the second encoding a region from the wild type (WT) nsp3 antigen.

A polynucleotide probe (e.g. SEQ ID NO: 4, 9, 11 or 13) which specifically hybridises to the WT cDNA amplicon of the invention; and a second probe (e.g. SEQ ID NO: 3, 8, 10 or 12) which specifically hybridises to the C5388A amplicon.

Preferably the polynucleotide probes for determining the presence or absence of a SARS-CoV-2 variant containing the C5388A mutation or a WT in a sample, wherein the polynucleotide probes comprises the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 or a variant thereof having at least about 80% homology to SEQ ID NO: 3 and SEQ ID NO: 4 based on sequence identity over its entire length.

A cDNA amplicon amplified from SARS-CoV-2 cDNA and comprising a sequence (a) to which the sequence shown in SEQ ID NO: 3 or 4 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3 or 4.

A target cDNA polynucleotide comprising a sequence (a) to which the sequence shown in SEQ ID NO: 4 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3 or A target cDNA polynucleotide comprising a sequence (a) to which the sequence shown in SEQ ID NO: 3 or 4 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 4.

A kit for determining the presence or absence of a SARS-CoV-2 VOC 202012/01 or another SARS-CoV- 2 variant in a sample, comprising (a) a pair of primers of the invention and (b) two polynucleotide probes of the invention.

A kit for determining the presence or absence of a SARS-CoV-2 VOC 202012/01 in a sample, comprising (a) a pair of primers of the invention and (b) a single polynucleotide probe of the invention selected from SEQ ID NO: 3, 8, 10 or 12.

A method of determining the presence or absence of a SARS-CoV-2 VOC 202012/01 or another SARS- CoV-2 variant in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using a pair of primers of the invention and detecting the cDNA amplicon amplified by the primers, if present, and thereby determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in the sample.

A method of determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS- CoV-2 variant in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using a pair of primers that amplifies a cDNA amplicon of the invention and a polynucleotide probe of the invention and thereby determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in the sample.

A method of determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS- CoV-2 variant in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using a kit of the invention and thereby determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in the sample.

A method for measuring the titre of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a sample, wherein the method comprises conducting a real time RT-PCR method of the invention and, if SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant is present, evaluating the cycle quantification (Cq) value and thereby measuring the titre of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant.

A method of determining whether or not a patient is infected with SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant, wherein the method comprises conducting a method of the invention on a sample from the patient and thereby determining whether or not a patient is infected with SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant.

A method of treating SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a patient identified as being infected with SARS-CoV-2 N501Y mutant using a method of the invention, comprising administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment and thereby treating the SARS-CoV-2 infection in the patient.

A method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method of the invention, comprising administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment and thereby treating the SARS-CoV-2 infection in the patient.

Description of the Figures

Figure 1: Left: Amplification of A1708D primer set 1 and probe set 3 as two singleplex reactions. Right: Scatter plot of end point RFU (relative fluorescence units in the final cycle of qPCR) for A1708D primer set 1 and probe set 3 as two Singleplex reactions.

Figure 2: Left: Amplification of A1708D primer set 1 and probe set 3 as a multiplex reaction. Right: Scatter plot of end point RFU for A1708D primer set 1 and probe set 3 as a multiplex reaction.

Figure 3: Left: Amplification of A1708D primer set 2 and probe set 3 two singleplex reactions. Right: Scatter plot of end point RFU for A1708D primer set 2 and probe set 3 two singleplex reactions.

Figure 4: Left: Amplification of A1708D primer set 2 and probe set 3 as a multiplex reaction. Right- Scatter plot of end point RFU for A1708D primer set 2 and probe set 3 as a multiplex reaction. Figure 5: a bar graph of the end point RFU comparing single plex and multiplex assay with A1708D primer sets 1 and 2.

Figure 6: amplification for the A1708D probe optimisation.

Figure 7: amplification of the assay mix containing A1708D primer set 2 with OneStep Lyophilised master mix and oasig OneStep 2x qPCR master mix.

Figure 8: the standard curve of the A1708D multiplex assay using gBlock amounts of 10^A4, 10^A3, 10^A2 and 10^Al copies per reaction for both the WT and SNP positive control templates.

Description of the Sequence Listing

In the table above, +X (such as +G) is a LNA nucleotide

SEQ ID NO: 1 shows the preferred forward nsp3 gene primer used in the Example SEQ ID NO: 2 shows the preferred reverse nsp3 gene primer used in the Example. SEQ ID NO: 3 shows the A1708D SNP probe used in the Example.

SEQ ID NO: 4 shows the A1708D WT probe used in the Example.

SEQ ID NO: 5 shows the A1708D mutant cDNA amplified in the Example.

SEQ ID NO: 18 shows the A1708D wild type cDNA amplified in the Example.

SEQ ID NO: 6- 13 show alternative primers and probes.

PCR amplicon.

SEQ ID NO: 14 shows the Lactococcus lactis (L. lactis) forward primer.

SEQ ID NO: 15 shows the L. lactis reverse primer.

SEQ ID NO: 16 shows the L. lactis probe 1.

SEQ ID NO: 17 shows the L. lactis probe 2.

Detailed Description of the Invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a primer” includes one, two or more primers, reference to “a probe” includes one, two or more probes, reference to “a sample” includes one, two or more such samples, reference to “a patient” includes one, two or more such patients, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Terminology In all instances herein, SARS-CoV-2 is interchangeable with 2019-nCoV or COVID-19. The term SARS-CoV-2 VOC202012 is also known as 201/501 Y.V1, VOC 202012/01, or B. 1.1.7.

In all instances herein, determining is interchangeable with detecting. The new primers, probes, kits and methods of the invention may be for detecting the presence or absence of SARS-CoV-2VOC 202012/01 in a sample or patient. Also, in all instances, the new primers, probes, kits and methods of the invention may be for determining/detecting/identifying whether or not a sample contains or comprises SARS-CoV- 2 VOC 202012/01 or whether it contains or comprises another strain or variant of SARS-CoV-2. In certain embodiments, the sample has been prior tested and been shown to contain SARS-CoV-2 and the invention therefore is directed in certain embodiments to determining/detecting/identifying whether or not the SARS-CoV-2 in the sample contains or comprises SARS-CoV-2 VOC 202012/01.

Advantages of the invention

The inventors have surprisingly identified new primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a variety of samples. The compositions and methods have several advantages.

First, the new primers, probes, kits and methods are capable of detecting the presence of SARS-CoV-2 VOC 202012/01 with a high degree of sensitivity.

The new primers, probes, kits and methods do not cross react with other human viruses, such as other human coronaviruses. The new primers, probes, kits and methods discriminate between the presence of VOC 202012/01 and other SARS-CoV-2 variants and wild type SARS Cov-2. The new primers, probes, kits and methods therefore have a very low false positive rate that is around 95% accurate, more preferably 97, 98 or 99% accurate.

Third, the new primers, probes, kits and methods are capable of measuring the SARS-CoV-2 titre in sample and distinguishing between a low SARS-CoV-2 titre and a high SARS-CoV-2 titre. This is particularly helpful in determining the level of infection in patients and allowing physicians to tailor their treatments to those levels.

Primers of the invention The present invention provides a pair of primers for determining the presence or absence of SARS-CoV- 2 VOC 202012/01 in a sample.

The forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 75% homology to SEQ ID NO: 1 based on sequence identity over its entire length.

The reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length.

Alternative primers are provided as SEQ ID NO: 6 (forward) and SEQ ID NO: 7 (reverse).

A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The nucleotides can be naturally occurring or artificial.

A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2’0-methyl, 2’ methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and the nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The nucleosides are most preferably adenosine, guanosine, uridine and cytidine

The nucleotides are typically ribonucleotides or deoxy ribonucleotides. The nucleotides are preferably deoxyribonucleotides. The nucleotides typically contain a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5’ or 3’ side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5- hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxy cytidine monophosphate (dCMP), deoxy cytidine diphosphate (dCDP) and deoxy cytidine triphosphate (dCTP), 5-methyl-2’ -deoxy cytidine monophosphate, 5 -methyl-2’ -deoxy cytidine diphosphate, 5-methyl-2’ -deoxy cytidine triphosphate, 5-hydroxymethyl-2’-deoxycytidine monophosphate, 5-hydroxymethyl-2’-deoxycytidine diphosphate and 5-hydroxymethyl-2’- deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP. The nucleotides are preferably selected from dAMP, dTMP, dGMP or dCMP.

The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2’amino pyrimidines (such as 2’-amino cytidine and 2’-amino uridine), 2’-hyrdroxyl purines (such as , 2’-fluoro pyrimidines (such as 2’ -fluorocytidine and 2’fluoro uridine), hydroxyl pyrimidines (such as 5’-a-P-borano uridine), 2’-O-methyl nucleotides (such as 2’-O-methyl adenosine, 2’-O-methyl guanosine, 2’-O-methyl cytidine and 2’-O-methyl uridine), 4’-thio pyrimidines (such as 4’-thio uridine and 4’-thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2’-deoxy uridine, 5-(3-aminopropyl)-uridine and l,6-diaminohexyl-N-5-carbamoylmethyl uridine) .

One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag. The label may be any suitable label which allows the polynucleotide to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵1, ³⁵S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin. The nucleotides in the polynucleotide may be atached to each other in any manner. The nucleotides may be linked by phosphate, 2’0-methyl, 2’ methoxy-ethyl, phosphorami date, methylphosphonate or phosphorothioate linkages. The nucleotides are typically atached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. The polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.

The polynucleotide may be DNA or RNA. In the context of the invention, a primer polynucleotide that is DNA has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 if all of the instances of dAMP, dTMP, dGMP and dCMP are replaced with AMP, UMP, GMP and CMP respectively. In other words, the DNA nucleotides are replaced with their corresponding RNA nucleotides and T is replaced with U.

The polynucleotide is preferably DNA. At certain points along the polynucleotide there can also be bridged nucleic acid (BNA).

The polynucleotide may be single stranded or double stranded. The primer polynucleotide is preferably single stranded.

Any of the polynucleotides discussed herein, such as the primers, probes, cDNA amplicons and target cDNA sequences, may be isolated, substantially isolated, purified or substantially purified. The polynucleotide is isolated or purified if it is completely free of any other components, such as buffer, other polynucleotides, virus material or cells. The polynucleotide is substantially isolated or substantially purified if it is only mixed with carriers or diluents, such as buffers or excipients, which will not interfere with its intended use, such as in a RT-PCR assay. The primer polynucleotides are not naturally occurring.

The primer of the invention may comprise a variant sequence based on SEQ ID NO: 1 or 2, or in the alternative SEQ ID NO: 6 or 7.. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2, 6 or 7 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 1 or 2, 6 or 7. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2, 6 or 7 and which retains its ability to specifically hybridise to a sequence that is complementary to SEQ ID NO: 1 or 2, 6 or 7. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2, 6 or 7 and which retains its ability to amplify the complementary DNA (cDNA) amplicon amplified from SARS-CoV-2 cDNA by SEQ ID NO: 1 and 2 (i.e. SEQ ID NO: 5 and 18). The term "amplified" refers to the process of making multiple copies of the polynucleotide, such as cDNA, from a single polynucleotide or fewer polynucleotides.

A variant “specifically hybridises” to its target sequence when it hybridises with preferential or high affinity to the partner but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other polynucleotides, especially other sequences in SARS-CoV-2. Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995)). Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCl and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCl, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0. 1650 M Na+) SSC at 55 °C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37 °C, followed by a wash in 0. IX (0.0165 M Na+) SSC at 60 °C. A variant “specifically hybridises” if it hybridises to its partner with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other polynucleotides. More preferably, the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other polynucleotides. Preferably, the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for a polynucleotide which differs from its partner by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The variant typically hybridises to its target sequence with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C. Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.

The variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides. The variant sequence is typically the same length as SEQ ID NO: 1 or 2, but may be longer or shorter. A variant of SEQ ID NO: 1 or 2 is preferably at least 17 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length.

Over the entire length of the sequence of SEQ ID NO: 1 or 2, 6 or 7 a variant sequence preferably has at least about 94%, This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 1 or 2, 6 or 7.

Methods of measuring homology based on sequence identity or identity are known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology or identity (e.g. used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387- 395).

The PILEUP and BLAST algorithms can also be used to calculate identity, homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

A variant is typically identical to SEQ ID NO: 1 or 2 over at least about 17, at least about 18, at least about 19, at least about 20, at least about 21 or at least about 22 consecutive nucleotides.

Each polynucleotide in the primer may be any length. The forward primer is preferably at least 17 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length. The reverse primer is at least 17 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length.

The forward primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 2.

The forward primer preferably is the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably is the exact sequence shown in SEQ ID NO: 2.

The forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 2.

The pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above. The pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 2. The pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above.

The pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 2. The pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is the exact sequence shown in SEQ ID NO: 2.

The pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 2.

In alternative embodiments the invention provides primers as set forth in SEQ ID NO: 6 and 7 or variants thereof.

Primers having any of the sequences above may be manufactured using standard techniques. Custom primers having specific sequences are commercially available from various suppliers (such as Biolegio). cDNA amplicons of the invention

The invention also provides a complementary DNA (cDNA) amplicon amplified from SARS-CoV-2 VOC 202012/01 cDNA by the pair of primers of the invention. The invention also provides a cDNA amplicon amplified from SARS-CoV-2 cDNA and comprising or consisting of a sequence (a) to which the sequence shown in SEQ ID NO: 3 or Sequence ID no 8 specifically hybridises and/or (b) which is the exact sequence SEQ ID NO: 5. Specific hybridisation is defined above with reference to the primers of the invention. The cDNA amplicon is preferably amplified from SARS-CoV-2 cDNA and comprises or consists of a sequence which is the exact sequence shown in SEQ ID NO: 5.

SARS-CoV-2 VOC 202012/01 cDNA is cDNA produced by the reverse transcription of SARS-CoV-2 VOC 202012/01 RNA. The sequence of SARS-CoV-2 cDNA is published (see above) and methods for reverse transcribing it are known in the art and discussed in the Example. Once the SARS-CoV-2 cDNA has been reversed transcribed, the pair of primers of the invention may be used to amplify the cDNA amplicon of the invention using polymerase chain reaction (PCR). Methods for conducting PCR using primers are known in the art and discussed below and in the Example. A cDNA amplicon is amplified from SARS-CoV-2 VOC 202012/01 cDNA if it comprises a portion/part of the SARS-CoV-2 VOC 202012/01 cDNA. In other words, it comprises a portion/part of the sequence of the SARS-CoV-2 VOC 202012/01 cDNA.

The term "amplified" refers to the process of making multiple copies of the cDNA from a single polynucleotide or fewer polynucleotides. An amplicon is the cDNA that results from amplification. In the context of the invention, primers, such as the primers of the invention, may be used to amplify a part of the SARS-CoV-2 cDNA resulting in the shorter cDNA amplicons of the invention. These amplicons typically contain the cDNA target sequence being used in the context of the invention.

The cDNA amplicon is typically a polynucleotide comprising nucleotides selected from dAMP, dTMP, dGMP or dCMP as discussed above. The cDNA may be double stranded or single stranded. The cDNA is preferably single stranded.

The cDNA amplicon may be any length, such as from about 20 to about 250 nucleotides in the length, such as from about 25 to about 200, from about 30 to about 180 or from about 40 to about 150 nucleotides in length. The cDNA amplicon is preferably 113 nucleotides in length.

The cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 5.

The cDNA amplicon may be isolated, substantially isolated, purified or substantially purified as discussed above. The cDNA amplicon is not naturally occurring.

Polynucleotide probes of the invention

The invention also provides polynucleotide probes (SNP probe) for determining the presence or absence of SARS-CoV-2 VOC 202012/01 in a sample for example, SEQ ID NO: 3. In an embodiment the invention provides a second probe for the determining the presence of other SARS-CoV-2 variants, for example as provided in SEQ ID NO: 4. In one embodiment, the polynucleotide SNP probe specifically hybridises to the cDNA amplicon of the invention. The polynucleotide probe comprises or consists of a sequence which specifically hybridises to the cDNA amplicon of the invention. The polynucleotide probe preferably comprises or consists of a sequence which specifically hybridises to a target sequence on the cDNA amplicon of the invention. The target sequence may be any length, such as from 17 to 32 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 32 nucleotides in length.

Polynucleotides and specific hybridisation are defined above with reference to the primers of the invention. The probe polynucleotide may be any of the polynucleotides discussed above. The polynucleotide probe is preferably DNA. The polynucleotide probe is preferably single stranded DNA. The polynucleotide probe may be any length, such as from 17 to 32 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length. The polynucleotide probe is preferably the same length as the target sequence it detects. The target sequence is preferably specific for SARS-CoV-2. The target sequence is preferably found/present in SARS-CoV-2 VOC 202012/01 cDNA but is not found in the RNA of any other virus or human virus or others SARS-CoV-2 variants. The target sequence is preferably found/present in SARS-CoV-2 cDNA but is not found in the RNA of any other virus or human virus or in any human RNA. The target sequence is preferably found/present in the cDNA amplicon of the invention. The target sequence is preferably the sequence shown in SEQ ID NO: 5.

In another embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 3 or 8 or 10 or a variant thereof having at least about 80% homology to SEQ ID NO: 3 or 8 or 10 based on sequence identity over its entire length. Preferably the probe consists of SEQ ID NO: 3. Polynucleotides are defined above with reference to the primers of the invention. The probe polynucleotide may be any of the polynucleotides discussed above. The polynucleotide probe is preferably DNA. The polynucleotide probe is preferably single stranded DNA. The polynucleotide probe may be any length, such as from 18 to 31 nucleotides in length, such as 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 nucleotides in length. Additionally, in an embodiment, a probe directed to wild type SARS-CoV-2 is provided. Such polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 4, 9, 11 or 13 or a variant thereof having at least about 80% homology to SEQ ID NO: 4, 9, 11 or 13 based on sequence identity over its entire length. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 3, 8, 10 or 12 and which retains its ability to specifically hybridise to the target sequence in SEQ ID NO: 5. Specific hybridisation is discussed above. The variant sequence must not recognise or hybridise to any cDNA sequence from any other virus.

The variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides. The variant sequence is typically the same length as SEQ ID NO: 3 8, 10 or 12 but may be longer or shorter. A variant of SEQ ID NO: 3, 8, 10 or 12 is preferably at least 18 nucleotides in length, such as 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 nucleotides in length.

Over the entire length of the sequence of SEQ ID NO: 3 and SEQ ID NO: 4, a variant sequence preferably has at least about 94%, This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

The polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3, 8, 10 or 12. Most preferably the polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3.

The polynucleotide probes based on any one of SEQ ID NOs: 3, 4 and 8-13 preferably consist of or comprise a combination of DNA and LNA nucleotides. The polynucleotide probes preferably comprise one or more LNA nucleotides amongst DNA nucleotides. A locked nucleic acid (LNA) nucleotide, also known as a bridged nucleic acid (BNA) nucleotide or inaccessible RNA nucleotide, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. This structure can be attributed to the increased stability against enzymatic degradation.

The polynucleotide probe may comprise/contain any number of LNA nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more LNA nucleotides. The one or more LNA nucleotides are preferably adenine (A), thymine (T), guanine (G) or cytosine (C) containing LNA nucleotides.

In a preferred embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 3 or a variant thereof as defined above wherein the nucleotides at one or more of positions 11 to 13 of SEQ ID NO: 3 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 3 or the variant thereof are DNA nucleotides. Preferably, all of the nucleotides at positions 11 to 13 of SEQ ID NO: 3 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 3 or the variant thereof are DNA nucleotides. This may be shown TGGTGAAGCT+G+A+TAACTT (where +X is a LNA nucleotide).

In another preferred embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 4 or a variant thereof as defined above wherein the nucleotides at one or more of positions 11 to 13 of SEQ ID NO: 4 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 4 or the variant thereof are DNA nucleotides. Preferably, all of the nucleotides at positions 11 to 13 of SEQ ID NO: 4 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 4 or the variant thereof are DNA nucleotides. This may be shown TGGTGAAGCT+G+C+TAACTT (where +X is a LNA nucleotide).

In a preferred embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 8 or a variant thereof as defined above wherein the nucleotide at position 12 of SEQ ID NO: 8 or the corresponding position in the variant are a LNA nucleotide and the remaining nucleotides in SEQ ID NO: 8 or the variant thereof are DNA nucleotides. This may be shown TGGTGAAGCTG+ATAACTT (where +X is a LNA nucleotide).

In another preferred embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 9 or a variant thereof as defined above wherein the nucleotide at position 12 of SEQ ID NO: 9 or the corresponding position in the variant is a LNA nucleotides and the remaining nucleotides in SEQ ID NO: 9 or the variant thereof are DNA nucleotides. This may be shown TGGTGAAGCTG+CTAACTT (where +X is a LNA nucleotide).

In another preferred embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 10 or a variant thereof as defined above wherein the nucleotides at one or more of positions 12 to 13 of SEQ ID NO: 10 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 10 or the variant thereof are DNA nucleotides. Preferably, all of the nucleotides at positions 12 to 13 of SEQ ID NO: 10 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 10 or the variant thereof are DNA nucleotides. This may be shown TGGTGAAGCTG+A+TAACTT (where +X is a LNA nucleotide). In another preferred embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 11 or a variant thereof as defined above wherein the nucleotides at one or more of positions 12 and 13 of SEQ ID NO: 11 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 11 or the variant thereof are DNA nucleotides. Preferably, all of the nucleotides at positions 12 and 13 of SEQ ID NO: 11 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 11 or the variant thereof are DNA nucleotides. This may be shown TGGTGAAGCTG+C+TAACTT (where +X is a LNA nucleotide).

In another preferred embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 12 or a variant thereof as defined above wherein the nucleotides at one or more of positions 9 to 12 of SEQ ID NO: 12 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 12 or the variant thereof are DNA nucleotides. Preferably, all of the nucleotides at positions 9 to 12 of SEQ ID NO: 12 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 12 or the variant thereof are DNA nucleotides. This may be shown GTGAAGCT+G+A+T+AACT (where +X is a LNA nucleotide).

In another preferred embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 13 or a variant thereof as defined above wherein the nucleotides at one or more of positions 8 to 11 of SEQ ID NO: 13 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 13 or the variant thereof are DNA nucleotides. Preferably, all of the nucleotides at positions 8 to 11 of SEQ ID NO: 13 or the corresponding positions in the variant are LNA nucleotides and the remaining nucleotides in SEQ ID NO: 13 or the variant thereof are DNA nucleotides. This may be shown TGAAGCT+G+C+T+AACT (where +X is a LNA nucleotide).

The polynucleotide probe may be isolated, substantially isolated, purified or substantially purified as discussed above. The polynucleotide probe is not naturally occurring.

The polynucleotide probe is preferably DNA probe, a TaqMan probe, a molecular beacon or a scorpion probe. DNA probes hybridise to the, typically complementary, target sequence and then can be detected. TaqMan probes are known in the art and are polynucleotides that have a fluorescent dye attached to the 5' end and a quencher to the 3' end. The polymerase used in PCR cleaves hybridised probes freeing the fluorescent dye from quenching such it can be detected. Molecular beacon probes are known in the art and are similar to TaqMan probes except (rather than using cleavage to separate the dye from the quencher) hybridisation to the target sequence separates the dye from the quencher. Scorpion probes are known the art and are similar to molecular beacons except the 3' end also contains a sequence that is complementary to the extension product of the primer on the 5' end which opens the probe on hybridisation and allows the dye to be detected. The polynucleotide probe is preferably a TaqMan probe.

The polynucleotide probes of the invention are preferably detectably-labelled. Suitable detectable labels are known in the art. The detectable labels are preferably fluorescent molecules or dyes, such as fluorescein derivatives. Suitable fluorescent molecules or dyes include, but are not limited to, 6- carboxyfluorescein (FAM), 6-Carboxyl-X-Rhodamine (ROX), 2'-chloro-7'phenyl-l,4-dichloro-6- carboxy-fluorescein (VIC®), Hexachloro-Fluorescein (HEX) and tetrachlorofluorescein (TET). A suitable quencher for use with these dyes in TaqMan, molecular beacon or scorpion probes is tetramethylrhodamine (TAMRA) or Black Hole Quencher 1 (BHQ1).

The detectable labels are most preferably 6-carboxyfluorescein (FAM) and Hexachloro-Fluorescein (HEX). In an embodiment of the invention the detection of SARS CoV-2 VOC 202012/01 is detected as a FAM signal. Polynucleotide probes are also available from commercial sources (such as Biolegio or Eurogentec).

Target cDNA sequence (amplicon)

The invention also provides a target cDNA polynucleotide comprising or consisting of a sequence (a) to which the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4. Specific hybridisation is discussed above. The target cDNA may be any length. It is typically 26 nucleotides in length. The target cDNA polynucleotide may be double stranded or single stranded. The target cDNA polynucleotide is preferably single stranded. The target cDNA polynucleotide may be isolated, substantially isolated, purified or substantially purified as discussed above. The target cDNA polynucleotide is not naturally occurring.

Kits of the invention The invention also provides a kit for determining the presence or absence of SARS-CoV-2 in a sample. The kit comprises a pair of primers of the invention. The kit also comprises a pair of polynucleotide probes of the invention or a single polynucleotide probe of the invention selected from SEQ ID NO: 3, 8, 10 or 12. Any of the primers and probes discussed above may be used. The primers and probes may be isolated, substantially isolated, purified or substantially purified as discussed above.

The kit preferably further comprises a positive control polynucleotide comprising or consisting of a cDNA amplicon of the invention and/or a target cDNA polynucleotide of the invention. These may form the basis of a positive control for RT-PCR. Any of the cDNA amplicons and cDNA target sequences discussed above may be used. The kit preferably comprises a polynucleotide comprising or consisting of the sequence shown in SEQ ID NO: 5. The cDNA polynucleotides may be isolated, substantially isolated, purified or substantially purified as discussed above. The kit preferably contains a negative control, such as DNAase-free and RNAase-free water.

The kit of the invention preferably further comprises a RNA Internal Extraction Control (IEC). The kit more preferably comprises a set of RNA IEC primers and probe. The RNA IEC primers and probe are preferably DNA. DNA primers and probes are discussed above. The kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the methods below to be carried out. Such reagents or instruments include, but are not limited to, one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a patient (such as a vessel or an instrument comprising a needle or a swab) or tubes in which quantitative reactions can be done. The kit may, optionally, comprise instructions to enable the kit to be used in the methods of the invention or details regarding which patients may be tested. The kit may further comprise any of the reagents required to conduct RT-PCR discussed below, including a reverse transcriptase and/or heat-stable DNA polymerase.

Method of determining the presence or absence of the SARS-CoV-2 VOC 202112/01 variant in a sample

The invention provides various methods of determining the presence or absence of SARS-CoV-2 VOC 202112/01_in a sample. All of the methods comprise conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample. Methods for conducting RT-PCR are well known in the art and any suitable conditions may be used. Reverse transcription involves the use of the enzyme reverse transcriptase to convert viral RNA into cDNA. In the context of the invention, PCR involves using the primers to amplify a specific portion of the cDNA, namely the cDNA amplicon, which typically contains the target cDNA of interest.

Methods for reverse transcription are well known in the art and reverse transcriptases are commercially available (e.g. Superscript® II reverse transcriptase (Invitrogen) and Affinity script (Agilent)). Methods typically construct cDNA from random cDNA hexamers included in the reaction mixture.

PCR involves amplifying a cDNA amplicon using a heat-stable DNA polymerase and a pair of forward and reverse polynucleotide primers. Because the newly synthesized cDNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. Many polymerase chain methods are known to those of skill in the art and may be used in the methods of the invention. For example, cDNA can be subjected to 20 to 40 cycles of amplification in a thermocycler as follows: 95°C for 30 sec, 52° to 60°C for 1 min, and 72°C for 1 min, with a final extension step of 72°C for 5 min. For another example, cDNA can be subjected to 20 to 40 polymerase chain reaction cycles in a thermocycler at a denaturing temperature of 95°C for 30 sec, followed by varying annealing temperatures ranging from 54°C to 58°C for 1 min, an extension step at 70°C for 1 min, with a final extension step at 70°C for 5 min. Heat stable DNA polymerases are commercially available (such as GoTaq G2 (Promega)).

The RT-PCR assay may be conducted using the conditions set out in the Example. The reverse transcriptase is preferably Affinity script (Agilent). The DNA polymerase is preferably GoTaq G2 (Promega). Both enzymes are preferably present in an Oasig one step master mix. The composition of Oasig onestep master mix can be found in the Example. The reaction mix is preferably lOul Oasig onestep master mix resuspended in 525ul Oasig resuspension buffer, lul primer/probe mix, 4ul of DNA/RNAase free water and from 5ul to 500ul sample. The concentrations of the primers and probe are preferably 18pmol/ul forward primer, 18pmol/ul reverse primer, 3pmol/ul wild type probe and 5pmol/ul in the primer/probe mix. The reaction conditions are preferably (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.

The RT-PCR method is preferably real time RT-PCR. This method is known in the art. In one embodiment, the method comprises using a pair of primers of the invention and detecting the cDNA amplicon amplified by the primers if present. Any method may be used to detect the cDNA amplicon. For instance, the method may use SYBR Green to detect the cDNA amplicon. When the SYBR Green binds to the cDNA, it emits light and the intensity of the fluorescence increases as the cDNA amplicons accumulate. This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding. The method preferably uses a polynucleotide probe of the invention which specifically hybridises to the cDNA amplicon. The probe may be any of those discussed above. The use of the probe allows the specific cDNA amplicon to be identified if present. The probe is preferably a TaqMan probe.

In another embodiment, the method comprises using a pair of primers that amplifies a cDNA amplicon comprising (or consisting of) a sequence (a) to which the sequence shown in SEQ ID NO: 3 and 4 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3 if present. The cDNA amplicon may be any of those discussed above. The cDNA amplicon preferably comprises or consists of SEQ ID NO: 5 or 18. The method also comprises using a polynucleotide probe of the invention which comprises the sequence shown in SEQ ID NO: 3 and 4 (WT & SNP)or a variant thereof as defined above to detect the cDNA amplicon if present. The polynucleotide probes may be any of those discussed above. The probes are preferably TaqMan probes. The polynucleotide WT probe specifically hybridises to cDNA amplicon if a SARS-CoV-2 strain other than VOC 202012/01 is present. The polynucleotide SNP probe specifically hybridises to cDNA amplicon if the SARS-CoV-2 VOC 202012/01 variant is present. Detection of the cDNA wild type amplicon indicates that the sample contain a SARS-CoV-2 strain or variant other than VOV202012/01 (z.e. indicates the presence of WT SARS-CoV-2 in the sample). Detection of the cDNA SNP amplicon indicates that the sample contain the SARS-CoV-2 VOC 202012/01 variant (i.e. indicates the presence of the SARS-CoV-2 VOC 202012/01 variant in the sample). A lack of detection of the cDNA amplicon indicates that the sample does not contain SARS-CoV-2 (i.e. indicates the absence of SARS-CoV-2 from the sample).

In another embodiment, the method comprises using a kit of the invention. The kit comprises a pair of primers of the invention and a pair of polynucleotide probes of the invention. The kit may also comprise a pair of primers of the invention and a single polynucleotide probe of the invention selected from SEQ ID NO: 3, 8, 10 or 12. The kit may comprise any of the primers and probes discussed above. The pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 1 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 2. The polynucleotide probes preferably comprises or consists of the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4. The pair of primers amplifies a cDNA amplicon of the invention and then this is specifically detected using the polynucleotide probes of the invention. Detection of the cDNA amplicon indicates that the sample contain SARS-CoV-2, either the VOC variant or another strain/variant (i.e. indicates the presence of SARS-CoV-2 either the VOC variant or another strain/variant in the sample). A lack of detection of the cDNA amplicon indicates that the sample does not contain SARS-CoV-2 (i.e. indicates the absence of SARS-CoV-2 from the sample).

In all embodiments, detection of the cDNA amplicon (i.e. the presence of the cDNA amplicon) indicates that the sample contain SARS-CoV-2 (i.e. indicates the presence of SARS-CoV-2 in the sample). A lack of detection of the cDNA amplicon (i.e. the absence of the cDNA amplicon) indicates that the sample does not contain SARS-CoV-2 (i.e. indicates the absence of SARS-CoV-2 from the sample). Detection of multiple copies of the cDNA amplicon (i.e. the presence of multiple copies of the cDNA amplicon) indicates that the sample contain SARS-CoV-2 (i.e. indicates the presence of SARS-CoV-2 in the sample). A lack of detection of multiple copies of the cDNA amplicon (i.e. the absence of multiple copies of the cDNA amplicon) indicates that the sample does not contain SARS-CoV-2 (i.e. indicates the absence of SARS-CoV-2 from the sample).

In all embodiments, the method preferably comprises (a) reverse transcribing any RNA in the sample, (b) contacting any cDNA produced in (a) with the pair of primers under conditions which allow the primers to amplify their cDNA amplicon and (c) determining the presence or absence of the cDNA amplicon. Step (c) preferably uses a polynucleotide probe as described above.

The RT-PCR assay is preferably a one-step RT-PCR assay conducted in one tube or vessel. Such one- step reactions are routine in the art.

In all the embodiments, the method preferably does not comprise extracting RNA from the sample before conducting the RT-PCR assay. In some instances, the methods may comprise extracting any RNA from the sample before conducting the RT-PCR assay. Suitable kits for doing this are commercial available (such as QIAamp® virus RNA mini kit (Qiagen)).

The sample may be any sample. The invention is typically carried out on a sample that is known to contain or SARS-CoV-2. Alternatively, the invention may be carried out on any sample whose SARS- CoV-2 status is unknown to confirm the presence or absence of SARS-CoV-2. The sample may be a biological sample. The sample may be obtained from or extracted from any organism or microorganism. The organism or microorganism may be archaeal, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista. The sample may be obtained from or extracted from any virus. Typically, the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep, fish, chickens or pigs or may alternatively be pets such as cats or dogs. Alternatively, the sample may be of plant origin, such as a sample obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton. The sample may be derived from human or animal food.

The sample is preferably a fluid sample. The sample typically comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus, amniotic fluid, blood, plasma or serum. The sample is preferably a nasopharyngeal sample, a saliva sample or a blood sample. Typically the swab is derived from a nasopharyngeal swab.

The sample may be a non-biological sample. Any non-biological sample can be tested. The non- biological sample is preferably a fluid sample. Examples of non-biological samples include, but are not limited to, surgical fluids, water such as drinking water, sea water or river water, reagents for laboratory tests and wet swabs of surfaces or materials.

The sample is typically processed prior to being used in the invention, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be tested immediately upon being taken. The sample may also be typically stored prior to assay, preferably below -70°C.

Methods of measuring the SARS-CoV-2 titre

The invention also provides methods for measuring the SARS-CoV-2 titre in a sample. The method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of SARS-CoV-2 in a sample as discussed above may be used. The quantitative method uses real time RT-PCR. If SARS-CoV-2 is present, the method comprises evaluating the cycle quantification (Cq) value and thereby measuring the SARS-CoV-2 titre. The method preferably comprises evaluating the Cq value against standard Cq values generated from standard dilution curves.

In real time RT-PCR, the threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels. The threshold level is preferably 10% of the end point fluorescence. Autocalling may be used to set the threshold level.

Method of diagnosis of the invention

The invention also provides a method of determining whether or not a patient is infected with the SARS- CoV-2 VOC 202012/01 variant or not. The invention therefore relates to the diagnosis of SARS-CoV-2 infection. The diagnostic method of the invention may be carried out in conjunction with other assays or genetic tests. The method comprises conducting a method of the invention for determining the presence or absence of SARS-CoV-2 VOC 202012/01 on a sample from the patient. Any of the methods discussed above may be used. The presence of SARS-CoV-2 in the sample indicates the presence of SARS-CoV- 2 in the patient. The presence of SARS-CoV-2 VOC 202012/01 in the sample indicates the patient is infected with SARS-CoV-2 VOC 202012/01 or another variant. The absence of SARS-CoV-2 from the sample typically indicates the absence of SARS-CoV-2 in the patient. The absence of SARS-CoV-2 from the sample typically indicates the patient is not infected with SARS-CoV-2. The absence of SARS- CoV-2 from the sample may indicate that the particular sample from the patient does not contain SARS- CoV-2 and does not necessarily mean the patient is not infected. The diagnostic method preferably uses a nasopharyngeal sample. The absence of SARS-CoV-2 from these samples does typically indicate the patient is not infected with SARS-CoV-2.

Typically, the patient displays the symptoms of SARS-CoV-2, i.e. the patient is known or expected to be infected with SARS-CoV-2. The patient may be asymptomatic, i.e. the patient’s SARS-CoV-2 status is unknown or the patient is expected not to be infected with SARS-CoV-2. The patient may be susceptible to, or at risk from, infection with SARS-CoV-2. The patient is may have underlying health conditions which make infection with SARS-CoV-2 particularly serious.

The patient is generally a human patient. The patient may be a fetus, a newborn, an infant, a juvenile or an adult. Therapeutic methods of the invention

The invention provides a method of treating SARS-CoV-2 in a patient identified as being infected with SARS-CoV-2 using a method of the invention. The method comprises administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment.

The invention also provides a method of treating SARS-CoV-2 in a patient. The method comprises (a) identifying the patient as being infected with SARS-CoV-2 using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment.

The invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being infected with SARS-CoV-2 using a method of the invention. The invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being infected with SARS-CoV-2 using a method of the invention and (b) administering the substance of composition to the patient. The substance or composition is preferably an anti-SARS-CoV-2 substance or composition.

The invention also provides a method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method of the invention. The method comprises administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment. The invention also provides a method of treating SARS-CoV-2 in a patient comprising (a) identifying the patient as having a high SARS-CoV-2 titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment. In a preferred method, the high SARS-CoV-2 titre is about 2 x 10³ virions/pl or higher. In this method, a Cq value of about 28.99 or lower indicates the sample has a high SARS-CoV-2 titre.

In a preferred method, a high SARS-CoV-2 titre is about 2 x 10⁵ virions/pl or higher. In this method, a Cq value of about 23.99 or lower indicates the sample has a high SARS-CoV-2 titre.

The invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method of the invention. The invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being having a high SARS-CoV-2 titre using a method of the invention and (b) administering the substance or composition to the patient. The substance or composition is preferably an anti-SARS-CoV-2 substance or composition

Any treatment, substance or composition may be used in the invention. Suitable anti-SARS-CoV-2 treatments, substances and compositions are well known. The anti-SARS-CoV-2 treatment, substance or composition may inhibit viral entry into cells by inhibiting ACE2 receptors, such as ACE2 receptor antibodies or arbidol, or inhibiting TMPRSS2, such as camosat mesylate. The anti-SARS-CoV-2 treatment, substance or composition may inhibit 3 -chymotrypsin-like protease, such as lopinavir or darunavir. The anti-SARS-CoV-2 treatment, substance or composition may inhibit viral replication by inhibiting viral RNA-dependent RNA polymerase (RdRP), such as ribavirin, remdesivir or favipiravir. The anti-SARS-CoV-2 treatment, substance or composition may be an anti-SARS-CoV-2 small interfering RNA (siRNA) designed to inhibit entry of the virus into cells and/or inhibit viral replication by targeting SARS-CoV-2 genes involved in these processes. Some patients infected with SARS-Cov- 2, especially patients with a high SARS-Cov-2 titre, suffer serious immune responses, including cytokine storms. IL-6 has been shown to be partly responsible for such cytokine storms. The anti-SARS-CoV-2 treatment, substance or composition may be an anti-IL-6 therapy or antibody, such as tocilizumab or sarilumab. The anti-SARS-CoV-2 treatment, substance or composition may be any anti-inflammatory and inhibitor of immune responses.

The invention concerns administering to the patient a therapeutically effective amount of the treatment, substance or composition to the patient. A therapeutically effective amount is an amount which ameliorates one or more symptoms of the SARS-CoV-2 infection. A therapeutically effective amount is preferably a number which abolishes one or symptoms of the SARS-CoV-2 infection. A therapeutically effective amount may cure or abolish the SARS-CoV-2 infection. Suitable amounts are discussed in more detail below.

The composition of the invention may be administered to any suitable patient. Suitable patients are discussed above with reference to the diagnostic embodiments of the invention.

The invention may be used in combination with other means of, and substances for, treating the disease or disorder or providing pain relief In some cases, the treatment, substance or composition may be used in combination with existing treatments for the SARS-CoV-2 infection including intensive care treatment and the use of ventilators. The treatment, substance or composition of the invention may be formulated using any suitable method. Formulation with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the composition to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19^th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

The treatment, substance or composition may be administered by any route. Suitable routes include, but are not limited to, enteral or parenteral routes such as via buccal, anal, pulmonary, intravenous, intraarterial, intramuscular, intraperitoneal, intraarticular, topical and other appropriate routes. If the lungs are being treated, the treatment, substance or composition is preferably administered by inhalation.

Pharmaceutical compositions may be prepared together with a physiologically acceptable carrier or diluent. The treatment, substance or composition may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions typically take the form of solutions or suspensions and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.

In addition, if desired, the pharmaceutical compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness. The composition preferably comprises human serum albumin.

One suitable carrier or diluents is Plasma-Lyte A®. This is a sterile, nonpyrogenic isotonic solution for intravenous administration. Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C6H1 lNaO7); 368 mg of Sodium Acetate Trihydrate, USP (C2H3NaO2»3H2O); 37 mg of Potassium Chloride, USP (KC1); and 30 mg of Magnesium Chloride, USP (MgC12»6H2O). It contains no antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

If the treatment, substance or composition is a siRNA, the most preferred pharmaceutically acceptable carrier or diluent is a pharmaceutically acceptable transfection reagent. A pharmaceutical acceptable transfection reagent is suitable for administration to patients. The pharmaceutical acceptable transfection reagent may be liposomes, preferably cationic liposomes, polymers, preferably cationic polymers, and dendrimers. The pharmaceutical acceptable transfection reagent is more preferably a pharmaceutically acceptable PEI transfection reagent, such as a linear PEI transfection reagent. The most preferred pharmaceutical acceptable transfection reagent is GMP in v/vo-jetPEI®.

The composition is administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective. The quantity to be administered depends on the subject to be treated, capacity of the patient’s immune system and the degree of treatment desired. Precise amounts required to be administered may depend on the judgement of the practitioner and may be peculiar to each patient.

Any suitable dose of the treatment, substance or composition may be administered to a patient. The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.01 to 50 mg per kg of body weight, according to the activity of the specific substance, the age, weight and conditions of the subject to be treated and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. For example, from about 0.01 to about 50mg per kg of patient of sRNA may administered, such as from about 0.05 to about 40, from about 0. 1 to about 30, from about 0.5 to about 20, from about 1 to about 10 or from about 2 to about 5 mg per kg. At least about 0.01 mg per kg of patient may administered, such as at least about 0.05, at least about 0.1, at least about 0.5, at least about 1, at least about 2, at least about 5, at least about 10, at least about 20, at least at least about 30 or at least about 40 mg per kg.

These doses may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily. Other suitable regular intervals include, but are not limited to, every day, every week, every fortnight or every month.

The following Example illustrates the invention.

Detailed Description of the Figures

Figure 1. Left: Amplification of the two A1708D templates (WT and SNP) by the WT and SNP assays, using primer set 1, as two single pl ex assays. Blue=FAM(WT assay), Green=HEX(SNP assay). Right: Allelic discrimination of the WT and SNP template by the two assays. Orange=amplification by the WT assay, Blue= Amplification by the SNP assay. Black=NTC/Amplifi cation is too low to discriminate allele (NTC or undetermined).

Figure 2. Left: Amplification of the two A1708D templates (WT and SNP) by the WT and SNP probes as a multiplex using primer set 1. Blue=FAM(WT assay), Green=HEX(SNP assay). Right: Allelic discrimination of the WT and SNP template. Orange=amplification by the WT assay, Blue= Amplification by the SNP assay. Black=NTC/undetermined.

Figure 3. Left: Primer set 2 amplification of the two A1708D templates (WT and SNP) by the WT and SNP assays as two singleplex assays (mixes 4 and 5). Blue=FAM(WT assay), Green=HEX(SNP assay). Right: Allelic discrimination of the WT and SNP template by the two assays. Orange=amplification by the WT assay, Blue=Amplification by the SNP assay. Black=NTC/undetermined. Figure 4. Left: Primer set 2 amplification of the two templates (WT and SNP) by the WT and SNP probes as a multiplex. Blue=FAM(WT assay), Green=HEX(SNP assay). Right: Allelic discrimination of the WT and SNP template. Orange=amplification by the WT assay, Blue=Amplification by the SNP assay. Black= Amplification is too low to discriminate allele. (NTC or end RFU low).

Figure 5. End RFU for each assay as a single plex and as a multiplex, with each template (WT and SNP) as a sample. Assays for primer set one (Left) and primer set two (right).

Figure 6. Left: Probe optimisation amplification of the two A1708D templates (WT and SNP) by the WT and SNP probes as a multiplex. Blue=FAM(WT assay), Green=HEX(SNP assay). Right: Allelic discrimination of the WT and SNP template. Orange=amplification by the WT assay, Blue= Amplification by the SNP assay. Black=NTC/undetermined

Figure 7. Amplification using the A1708D multiplex assay with OneStep Lyophilised master mix and oasig™ OneStep 2X RT-qPCR Master Mix

Figure 8. Amplification of the two A1708D templates (WT and SNP), serially diluted from 10⁴ to 10¹ copies per reaction, by the WT and SNP probes as a multiplex. Blue=FAM(WT assay), Green=HEX(SNP assay).

Examples

Reagents, instruments and associated consumables are shown in Table 2.

Table 1: Reagents, instruments and associated consumables

C5388A is the nucleotide position and bases for the A1708D mutation

Oasig Onestep components

• dNTPs (lOOmM)

• goTAQ G2

• Affinity Script

• SSB4 (lOmg/ml (T7 single stranded binding protein)

• IM Trizma pH8.5

• Excipients - Component buffer

• Trizma® hydrochloride

• Trizma® base

• Potassium chloride

• EGTA

• Magnesium chloride

• Ammonium sulphate

• DNAse/RNase free water

• Glycerol

• D-Mannitol

• Sucrose

• DNTPs lOOmM

• 10% TritonX-100

• RNAse/DNAse free water

• osrHSA (recombinant human serum albumin)

Primer selection

Probe selection is not part of this example. Alternative probes not used in the final kit are shown in SEQ

ID 8-13. Primers used are SEQ ID 1 & 2 and 6 & 7

All stages for primer/probe selection were run using the following: Reaction mixes

All reaction mixes contained per reaction: lOpL OneStep Lyophilised Master Mix

4pL of RNase/DNase free H₂O

1 pL of 20x primer probe mix

Cycling conditions for Primer set 2-all qPCR runs were done on a CFX96 Real Time PCR Detection System (Bio-Rad)

*Acquisition occurred at the end of this step.

Cycling conditions for Primer set 1

This thermal profile was used in error for Primer set 1 single plex and multiplex in the primer selection only. It does not affect the performance in any way because gBlocks are DNA. Since the fluorescent signal has plateaued long before cycle 40, it will also not affect the relative fluorescent units (RFU) in the final cycle.

Channels:

Fluorescence for the WT assay is -detected in the FAM channel. > > /nm (emission) 518 Fluorescence for the SNP assay is detected in the HEX channel. > /nm (emission) 554 Primer set selection

Template All samples contained synthetic oligo (single stranded DNA) (WT or SNP) of unknown final reaction concentration.

From the stock, the following dilution was made:

• IpL of the stock was added to 999pL of H₂O.

• lOpL of this was added to 290 pL H₂O.

• 5pL of this was added to 495 pL H₂O.

• 50pL of this was added to 450pL H₂O.

• This was used as the sample.

Two 20x stock mix of primers/probes was made containing:

Once 15pL of the reaction mix was added to each reaction well, 5pL of synthetic oligo was added to the reaction as a sample. For No Template Controls (NTCs), 5pL of RNase/DNase free H₂O was added.

Results for Primer Set 1

Table 2: Mean of 3 Cq values of amplification of the two A1708D templates (WT, SNP) by the WT and SNP assays as two single plex assays, (Mixes 1 and 2) and a multiplex assay (Mix 3).

Table 3: Mean of 3 end point (final cycle) RFU values from the amplification of the two A1708D templates (WT, SNP) by the WT and SNP assays containing Primer set 1 as two single plex assays (mixes 1 and 2) and a multiplex (mix 3).

The results show that the fluorescence is much lower with the SNP assay amplification than the WT amplification. There is also cross reactivity between the SNP assay and the WT template in the single plex reaction but in the multiplex, this cross reactivity is not seen.

Results for Primer Set 2

Table 4: Mean of 3 Cq values of amplification of the two A1708D templates (WT, SNP) by the WT and SNP assays as two single plex and a multiplex using primer set 2.

| SNP

| 306.06

| 8468.17 | -47.90

| 7405.38

|

Table 5: Mean of 3 end point RFU values for amplification of the two A1708D templates (WT, SNP) by the WT and SNP assays as two single plex assay (mixes 4 and 5) and a multiplex (Mix 6) using Primer set 2.

For primer set 2 again there was cross reactivity of the SNP assay with the WT template which was not see in the multiplex assay. Both single plex and multiplex showed low fluorescence with the SNP assay

Conclusion: With both primer sets, the amplification of the SNP template with the SNP assay gave very low fluorescence, less that half that of the WT assay. Primer set 1 was chosen to be used in the A1708D kit.

Probe Optimisation

Template

All samples contained gBlock with a final reaction concentration of 10⁴ copies/reaction:

The following gBlock templates: G870(WT), G871(SNP), were each resuspended in 500pL of template preparation buffer to give a concentration of 10⁶ copies/reaction. From this the following dilution was made:

• lOpL of the resuspension was added to 990pL of template buffer. This was used as the 10⁴ copies/reaction sample.

A 20x stock mix of primers/probes was made containing:

Once 15pL of the reaction mix was added to each reaction well, 5pL of gBlock was added to the reaction as a sample. For NTCs, 5pL of RNase/DNase free H₂O was added.

Cycling conditions:

The cycling conditions for all stages were as follows:

*Acquisition occurred at the end of this step.

All other parameters remained the same.

The results are shown in Tables 6 and 7 and Figure 6.

Table 6: Mean Cq values of amplification of the two templates (WT, SNP) by the WT and SNP assays as a multiplex. *one repeat excluded due to pipetting error.

Table 7: Mean of 3 end point RFU values for the amplification of the two A1708D templates (WT, SNP) by the WT and SNP assays as a multiplex.

The probe concentration optimisation has reduced the fluorescence from the wild type assay and increased the fluorescence in the SNP assay so that they are more similar in end point RFU values. The difference in Cq between this optimisation and the primer selection is that single stranded oligo of unknown concertation was used as template for the primer selection and gBlock (synthetic double stranded DNA) at a known concentration was used for the optimisation.

Bridging to oasig™ OneStep 2X RT-qPCR Master Mix Lyophilised

This was done to ensure compatibility with a standard master mix

Reaction mixes:

All reaction mixes contained per reaction: lOpL OneStep Lyophilised Master Mix 4pL of RNase/DNase free H₂O

1 pL of 20x primer probe mix or, all reaction mixes contained per reaction: lOpL oasig™ OneStep 2X RT-qPCR Master Mix Lyophilised

4pL of RNase/DNase free H₂O

1 pL of 20x primer probe mix

Template

All samples contained gBlock containing the amplicon sequence of either the SNP template (G871) or the WT template (G870), at 10⁴ copies/reaction.

Primer/probe mix and cycling conditions as before.

The results are shown in Table 7 and Figure 7.

Table 8: Mean Cq values of amplification of the two templates (WT, SNP) by the WT and SNP assays as a multiplex using either OneStep Lyophilised Master Mix or oasig™ OneStep 2X RT-qPCR Master Mix Lyophilised. *one repeat was N/A

Standard Curve

All stages for primer/probe selection were run using the following:

Reaction mixes:

All reaction mixes contained per reaction: lOpL OneStep Oasig Lyophilised Master Mix

4pL of RNase/DNase free H₂O 1 pL of 20x primer probe mix

Cycling conditions: As previously stated for the probe optimisation

A 20x stock mix of primers/probes was made containing:

Once 15pL of the reaction mix was added to each reaction well, 5pL of gBlock was added to the reaction as a sample. For NTCs, 5pL of RNase/DNase free H₂O was added.

Template

All samples contained gBlock containing the amplicon sequence of either the SNP template (G871) or the WT template (G870).

A serial dilution was performed for each gBlock template: G870 (WT template) and G871 (SNP template).

Each gBlock was resuspended in 500pL of template preparation buffer to give a concentration of 10⁶ copies/reaction. From this the following dilution was made:

• lOpL of the resuspension was added to 990pL of template buffer. This was used as the 10⁴ copies/reaction sample (2x10³ copies/pl).

• 50pL of this dilution was added to 450pL of template buffer. This was used as the 10³ copies/reaction sample (2x10² copies/pl).

• 50pL of this dilution was added to 450pL of template buffer. This was used as the 10² copies/reaction sample 2xl0³ copies/pl).

• 50pL of this dilution was added to 450pL of template buffer. This was used as the 10¹ copies/reaction sample (2x10° copies/pl). The results are shown in Tables 9 and 10 and Figure 8

Table 9. Mean Cq values of the amplification of the serial dilutions of the A1708D WT and SNP gB locks in the multiplex probe assay. WT is FAM labelled and the SNP is HEX labelled. * Cq later than 40.

Table 10: PCR Efficiency and R² value of the A1708D multiplex assay.

Both the WT and the SNP assays have a limit of detection of 10 copies/reaction, PCR efficiency in the accepted range on 90-110% and both standard curves are linear. A cut off at a Cq of 40 was applied. Any Cq above this value is considered a negative result.

Claims

43 CLAIMS

1. A pair of primers for determining the presence or absence of SARS-CoV-2 VOC 202012/01 in a sample, wherein the pair of primers is selected from:

(a) a forward primer comprising a polynucleotide having the sequence shown in SEQ ID NO: 1, or a variant thereof having at least about 75% homology to SEQ ID NO: 1 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length, or

(b) the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6, or a variant thereof having at least about 75% homology to SEQ ID NO: 6 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7 or a variant thereof having at least about 75% homology to SEQ ID NO: 7 based on sequence identity over its entire length.

2. A pair of primers according to claim 1, wherein the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2.

3. A pair of primers according to claim 1 or 2, wherein the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6 and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7.

4. A pair of primers according to any preceding claim, wherein the SARS-CoV-2 variant is VOC 202012/01 (United Kingdom).

5. A pair of primers according to any preceding claim, wherein the SARS-CoV-2 VOC 202012/01 comprises a C5388A mutation in the non-structural protein 3 (nsp3) within the open reading frame lab (ORF lab).

6. A complementary DNA (cDNA) amplicon amplified from SARS-CoV-2 cDNA by the pair of primers according to any preceding claim. 44

7. A set of two polynucleotide probes, of which the first probe specifically hybridises to the cDNA amplicon of the VOC 202012/01 variant and the second probe specifically hybridises to the cDNA of all other known variants.

8. A set as claimed in claim 7 comprising two polynucleotide probes for determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a sample, wherein (a) a first probe is selected from the group comprising SEQ ID NO: 3, 8, 10 and 12, or a variant thereof having at least about 80% homology thereto based on sequence identity over its entire length and (b) a second probe selected from the group comprising SEQ ID NO: 4, 9, 11 and 13, or a variant thereof having at least about 80% homology thereto based on sequence identity over its entire length.

9. The set according to claim 7 or 8, wherein the first probe comprises the sequence shown in SEQ ID NO: 3 and the second probe comprises the sequence shown in SEQ ID NO: 4.

10. The set according to claim 7 to 9, wherein the first probe comprises the sequence shown in SEQ ID NO: 8 and the second probe comprises the sequence shown in SEQ ID NO: 9.

11. The set 7 to 10, wherein the first probe comprises the sequence shown in SEQ ID NO: 10 and the second probe comprises the sequence shown in SEQ ID NO: 11.

12. The set according to claim 7 to 11, wherein the first probe comprises the sequence shown in SEQ ID NO: 12 and the second probe comprises the sequence shown in SEQ ID NO: 13.

13. The set according to any one of claims 7 to 12, wherein each probe is a DNA probe, aTaqMan probe, a molecular beacon or a scorpion probe.

14. The set according to any one of claims 8 to 13, wherein the probe is detectably -labelled.

15. The set according to claim 14, wherein the detectable label is a fluorescent molecule or dye, such as Fluorescein amidite (FAM) or Hexachloro- Fluorescein (HEX).

16. A cDNA amplicon amplified from SARS-CoV-2 cDNA wherein the cDNA amplicon comprises a sequence (a) to which the sequence shown in SEQ ID NO: 3, 4, 8, 9, 10, 11, 12 or 13 45 specifically hybridises and/or (b) to which is complementary to the sequence shown in SEQ ID NO: 3, 4, 8, 9, 10, 11, 12 or 13.

17. A target cDNA polynucleotides comprising a sequence (a) to which the sequence shown in SEQ ID NO: 3, 4, 8, 9, 10, 11, 12 or 13 specifically hybridises and/or (b) to which is complementary to the sequence shown in SEQ ID NO: 3, 4, 8, 9, 10, 11, 12 or 13.

18. A kit for determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a sample, comprising (a) a pair of primers according to claim 1 to 3 and (b) two polynucleotide probes according to any one of claims 7 to 15.

19. A kit according to claim 18, wherein the kit comprises (a) a pair of primers according to claim 2 and (b) two polynucleotide probes according to claim 9.

20. A kit for determining the presence or absence of SARS-Cov-2 VOC 202012/01 variant in a sample, comprising (a) a pair of primer according to claim 1 to 3 and (b) a single polynucleotide probe according to SEQ ID NO: 3, 8, 10 or 12.

21. A kit according to claim 18 to 20, wherein the kit further comprises (a) a positive control polynucleotide comprising a cDNA amplicon according to claim 3 or 10 and/or a target cDNA polynucleotide according to claim 17 and/or (b) a negative control.

22. A method of determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using a pair of primers according to claim 1 to 3 and detecting the cDNA amplicon amplified by the primers, if present, and thereby determining the presence or absence of SARS-CoV-2 VOC 202012/01 in the sample.

23. A method of determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using a pair of primers that amplifies a cDNA amplicon according to claim 17 and two polynucleotide probes according to claim 8 to 15 and thereby determining the presence or absence of SARS-CoV-2 VOC 202012/01 in the sample.

24. A method of determining the presence or absence of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using a kit according to claim 18 to 21 and thereby determining the presence or absence of SARS-CoV-2 VOC 202012/01 in the sample.

25. A method according to any one of claims 22 to 24, wherein the method comprises (a) reverse transcribing any RNA in the sample, (b) contacting any cDNA produced in (a) with the pair of primers under conditions which allow the primers to amplify their cDNA amplicon and (c) determining the presence or absence of the cDNA amplicon.

26. A method according to any one of claims 22 to 25, wherein the RT-PCR assay is a one-step RT-PCR assay conducted in one tube or vessel.

27. A method according to any one of claims 22 to 26, wherein the method comprises extracting any RNA from the sample before conducting the RT-PCR assay.

28. A method according to any one of claims 22 to 27, wherein the sample is a human sample.

29. A method according to any one of claims 22 to 28, wherein the sample is a nasopharyngeal sample, a saliva sample or a blood sample.

30. A method for measuring the titre of SARS-CoV-2 VOC 202012/01 another SARS-CoV-2 variant in a sample, wherein the method comprises conducting a real time RT-PCR method according to any one of claims 22 to 29 and, if SARS-CoV-2 VOC 202012/01 is present, evaluating the cycle quantification (Cq) value and thereby measuring the titre of SARS-CoV-2 VOC 202012/01 another SARS-CoV-2 variant.

31. A method of determining whether or not a patient is infected with SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant, wherein the method comprises conducting a method according to any one of claims 22 to 29 on a sample from the patient and thereby determining whether or not a patient is infected with SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant.

32. A method of measuring the titre of SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a patient, wherein the method comprises conducting a method according to claim 30 on a sample from the patient and thereby measuring the viral titre in the patient.

33. A method according to claim 31 or 32, wherein the method comprises taking a sample from the patient before conducting the RT-PCR assay.

34. A method of treating SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a patient identified as being infected with SARS-CoV-2 using a method according to claim 31, comprising administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment and thereby treating the SARS-CoV-2 infection in the patient.

35. A method of treating SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant in a patient identified as having a high SARS-CoV-2 titre using a method according to claim 32, comprising administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment and thereby treating the SARS-CoV-2 VOC 202012/01 or another SARS-CoV-2 variant infection in the patient.