JP2024102396A - METHOD FOR DETECTING VARIANT SARS-CoV-2 - Google Patents

Abstract

To provide a method for allowing specific and easy detection of variant SARS-CoV-2, thereby solving the problems in which: variants of SARS-CoV-2 have been difficult to distinguish from the original virus by virus detection methods constructed in the prior art; and it has been possible to comprehensively detect variations by sequencing the viral genomes, but not by a method that can be implemented quickly or easily, e.g., because a high-speed sequencer is required.SOLUTION: An oligonucleotide is found which is useful in comparing and examining genomic RNA sequences of SARS-CoV-2 and its variant strain and detecting viral variant strain with specifically mutated spike protein by nucleic acid amplification. Also constructed herein is a method for detecting variant SARS-CoV-2 using the oligonucleotide.SELECTED DRAWING: None

Description

The present invention relates to an oligonucleotide used for detecting mutant SARS-CoV-2, a method for detecting mutant SARS-CoV-2 using the oligonucleotide, and a kit for use in the detection method.

Among infectious diseases caused in humans by bacteria or viruses, there are some that are exclusively localized, and others that have the potential to spread widely regardless of geographical conditions. Infectious diseases that are transmitted between humans fall into the latter category, and depending on their infectiousness and the severity of the symptoms exhibited by infected patients, they can become a social problem. There are many infectious diseases that have caused large-scale epidemics and influenced subsequent history, such as smallpox, plague, and influenza (Spanish flu).

Even today, when methods for treating and preventing many infectious diseases have been developed, there are still infectious diseases that require continued caution, and there is also the risk of emerging infectious diseases. SARS coronavirus (SARS-CoV), discovered in 2003, was found to be a virus that causes severe respiratory illness. Furthermore, a new coronavirus, SARS-CoV-2, emerged in 2019 and has spread worldwide in 2020.

Measures to combat infectious diseases include the development and dissemination of therapeutic drugs, as well as the improvement of hygienic environments. In addition, identifying the presence of pathogens and infected individuals, taking early isolation measures, and blocking the route of infection are extremely important in preventing the spread of infectious diseases. Methods for detecting the above-mentioned SARS-CoV-2 have already been developed, including nucleic acid testing methods that target viral RNA (e.g., Non-Patent Document 1), antigen testing methods that target viral proteins, and antibody testing methods that use blood samples from people suspected of infection. Currently, nucleic acid testing methods are the mainstream for virus testing, mainly from the perspective of detection sensitivity. Meanwhile, multiple mutant viruses (mutant strains) have emerged from the virus, and it has been reported that these have different properties from the original virus (e.g., Non-Patent Documents 2 and 3).

These mutant strains are difficult to distinguish from the original virus using the virus detection methods developed to date, posing problems in the epidemiological study of viral mutations. Furthermore, mutant strains may not be detectable by virus detection systems developed based on information about the virus before mutation, or the detection sensitivity may be reduced. It is possible to comprehensively detect mutations by deciphering the base sequence of the viral genome, but this is not a rapid or easy method to implement, as it requires a high-speed sequencer.

As described above, there is a need for a method that can specifically and easily detect mutated SARS-CoV-2.

Japanese Journal of Infectious Diseases (2020), 73(4), 320-322 Science (2021), 372(6538), eabg3055 Cell Mol. Immunol. (2021), 18(4), 1058-1060

Many of the mutant SARS-CoV-2 strains have multiple mutations in the spike protein. Three representative lineages have been reported based on the country of origin: UK VOC-202012/01 (B.1.1.7), South Africa 501Y. V2 (B.1.351), and Brazil 501Y. V3 (P.1). It has been pointed out that mutant strains may increase the infectiousness of infection and may evade the immune system, weakening the effectiveness of vaccines. Table 1 shows information on the mutations occurring in the spike protein of these mutant strains, as described in a report by the National Institute of Infectious Diseases (dated April 7, 2021).

On the other hand, the mutant B. 1.617 lineage has the L452R, D614G, and P681R mutations in the spike protein in common (National Institute of Infectious Diseases, report dated May 12, 2021). Many mutant strains of this lineage have been detected in India, and are said to be showing a higher growth rate than previous epidemic strains. B. 1.617.1 and B. 1.617.3, which are part of the lineage, have the E484Q mutation, and B. 1.617.2 has the T478K mutation (European Centre for Disease Prevention and Control, report dated May 24, 2021).

New mutant strains are reported from time to time. In a report by the European Centre for Disease Prevention and Control dated August 5, 2021, several mutant strains, including those mentioned above, are listed as "Variant of Concern (VOC)" and "Variant of Interest (VOI)". Among these, the C. 37 strain (characteristically having L452Q and F490S mutations in the spike protein) described as VOI in the above report has many infected people in South America. Furthermore, the B. 1.621 strain described as VOI in a report by the European Centre for Disease Prevention and Control dated October 14, 2021 has R346K, E484K, N501Y, D614G and P681H mutations in the spike protein. In addition, the World Health Organization (WHO) has labeled mutant strains that are spreading globally with Greek letter labels (WHO labels). The WHO labels given to the above strains are shown below.
B. 1.1.7: Alpha
501Y. V2 (B.1.351): Beta
501Y. V3 (P.1): Gamma
B. 1.617.2:Delta
C. 37: Lambda
B. 1.621: Mu

It is believed that mutations in the receptor binding domain (RBD; positions 319-541) of the spike protein may affect the infectivity of SARS-CoV-2 and its reactivity with antibodies. For this reason, it is particularly important to understand the status of transmission of SARS-CoV-2 mutants with mutations in N501, E484, L452, T478, F490, etc. in the above region, as well as mutant SARS-CoV-2 mutants with mutations in amino acids close to the cleavage site (R685-S686) by Furin-like proteases.

The inventors compared and examined the genomic RNA sequences of SARS-CoV-2 and its mutant strains, and discovered oligonucleotides that are useful for detecting virus mutants with specific mutations in the spike protein by nucleic acid amplification methods. Furthermore, they constructed a method for detecting mutant SARS-CoV-2 using the oligonucleotides, thereby completing the present invention.

The present invention is outlined below.
[1] An oligonucleotide for use in detecting mutant SARS-CoV-2, comprising:
(a) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 35 or a sequence complementary to said sequence;
(b) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 36 or a sequence complementary to said sequence;
(c) an oligonucleotide comprising the base sequence shown in SEQ ID NO:61 or a sequence complementary to said sequence;
(d) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 88 or a sequence complementary to said sequence;
(e) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 113 or a sequence complementary to said sequence;
(f) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 137 or a sequence complementary to said sequence;
(g) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 138 or a sequence complementary to said sequence;
(h) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 162 or a sequence complementary to said sequence, and (i) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 163 or a sequence complementary to said sequence;
An oligonucleotide selected from:
[2] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 4, 8, 12, 14, and 16, or a sequence complementary to said sequence.
[3] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 24, 27, 30, 32, 34, 37, 44, 45, and 46, or a sequence complementary to said sequence.
[4] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 53, 54, 55, 56, and 57, or a sequence complementary to said sequence.
[5] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 68, 69, 70, 71, 81, 82, 83, 84, and 85, or a sequence complementary to said sequence.
[6] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 95, 96, 97, 98, 99, and 100, or a sequence complementary to said sequence.
[7] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 122, 123, and 124, or a sequence complementary to said sequence.
[8] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 131, 132, and 133, or a sequence complementary to said sequence.
[9] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 152, 153, 154, 155, and 156, or a sequence complementary to said sequence.
[10] The oligonucleotide according to [1], which consists of a base sequence selected from SEQ ID NOs: 157, 158, 159, 160, and 161, or a sequence complementary to said sequence.
[11] The oligonucleotide according to [1], which is labeled with a fluorescent substance and a quencher.
[12] The oligonucleotide according to [1], to which a minor groove binder (MGB) is attached.
[13] The oligonucleotide according to [1], which comprises a bridged nucleic acid (BNA).
[14] A method for detecting a mutant SARS-CoV-2 in a sample, comprising:
(1) synthesizing a DNA or a fragment thereof complementary to the SARS-CoV-2 genome contained in a sample; and (2) detecting a base sequence or a part thereof encoding a mutant spike protein contained in the DNA or a fragment thereof obtained in the step (1) using an oligonucleotide according to any one of [1] to [13].
The method according to claim 1, further comprising:
[15] The method according to [14], wherein step (1) further comprises a step of amplifying the synthesized DNA fragment.
[16] The method described in [14], wherein in step (2), detection of the base sequence encoding the mutant spike protein or a part thereof is carried out by decomposition of an oligonucleotide hybridized to the DNA or a fragment thereof.
[17] A kit for detecting a mutant SARS-CoV-2 in a sample, comprising:
(1) An oligonucleotide according to any one of (1) to (13) above, and (2) a kit comprising a reagent for synthesizing a DNA or a fragment thereof complementary to the SARS-CoV-2 genome.
[18] The kit according to [17], further comprising a reagent for amplifying a DNA fragment complementary to the SARS-CoV-2 viral genome.
[19] The kit of [17], comprising a primer pair used for amplifying a DNA fragment complementary to the SARS-CoV-2 viral genome.

The present invention provides a method for quickly detecting mutant SARS-CoV-2 that has N501Y, E484K, E484Q, L452R, L452Q, T478K, F490S, P681H or P681R mutations in the spike protein using a nucleic acid amplification method.

N501Y mutation detection E484K mutation detection E484K mutation detection E484K mutation detection Simultaneous detection of N501Y and E484K mutations

"Oligonucleotides of the present invention"

The present invention provides an oligonucleotide for detecting mutant SARS-CoV-2, specifically a mutant virus having a mutation selected from N501Y, E484K, E484Q, L452R, L452Q, T478K, F490S, P681H, or P681R in the spike protein of SARS-CoV-2. In this specification, "mutant SARS-CoV-2" refers to a spike protein (mutant spike protein) with an amino acid sequence different from that of the spike protein of Wuhan strain SARS-CoV-2, and a SARS-CoV-2 strain (mutant strain) that retains genomic RNA containing a base sequence that codes for the protein, without particularly limiting the present invention. Note that in this specification, Wuhan strain SARS-CoV-2 may be referred to as wild type.

In this specification, the N501Y mutation refers to a substitution of the 501st asparagine (N) with tyrosine (Y) in the amino acid sequence (YP_009724390.1) of the spike protein of the Wuhan strain SARS-CoV-2, which is published in GeneBank under the accession number NC_045512.2. Here, this mutation is caused by a change from AAT, the codon corresponding to the asparagine, to TAT in the region encoding the spike protein of the SARS-CoV-2 genome RNA. Therefore, a mutant strain having the N501Y mutation can be detected by detecting a base sequence in which the first base of the triplet is changed from A to T.

In this specification, the E484K mutation refers to a substitution of the 484th glutamic acid (E) with lysine (K) in the amino acid sequence of the spike protein of SARS-CoV-2. Here, this mutation is caused by a change of the codon GAA corresponding to the glutamic acid to AAA in the region coding for the spike protein of SARS-CoV-2 genomic RNA. Therefore, by detecting a base sequence in which the first base of the triplet is changed from G to A, a mutant strain having the E484K mutation can be detected. The E484Q mutation refers to a substitution of the 484th glutamic acid with glutamine (Q). Here, this mutation is caused by a change of the GAA to CAA in the region coding for the spike protein of SARS-CoV-2 genomic RNA. Therefore, by detecting a base sequence in which the first base of the triplet is changed from G to C, a mutant strain having the E484Q mutation can be detected.

In this specification, the L452R mutation refers to the substitution of leucine (L) at position 452 in the amino acid sequence of the spike protein of SARS-CoV-2 with arginine (R). Here, this mutation is caused by a change of the codon CTG corresponding to the leucine to CGG in the region encoding the spike protein of the SARS-CoV-2 virus genome RNA. Therefore, a mutant strain having the L452R mutation can be detected by detecting a base sequence in which the second base of the triplet is changed from T to G. In addition, the L452Q mutation refers to the substitution of leucine at position 452 with glutamine. Here, this mutation is caused by a change of CTG to CAG in the region encoding the spike protein of the SARS-CoV-2 virus genome RNA. Therefore, a mutant strain having the L452Q mutation can be detected by detecting a base sequence in which the second base of the triplet is changed from T to G.

In this specification, the T478K mutation refers to a substitution of threonine (T) at position 478 in the amino acid sequence of the spike protein of SARS-CoV-2 with lysine. Here, this mutation is caused by a change from ACA, the codon corresponding to the E, to AAA in the region of the SARS-CoV-2 genomic RNA that codes for the spike protein. Therefore, by detecting a base sequence in which the second base of the triplet is changed from C to A, a mutant strain having the T478K mutation can be detected.

In this specification, the F490S mutation refers to a substitution of phenylalanine (F) at position 490 in the amino acid sequence of the spike protein of SARS-CoV-2 with serine (S). Here, this mutation is caused by a change from TTT, the codon corresponding to the phenylalanine, to TCT in the region of the SARS-CoV-2 genomic RNA that codes for the spike protein. Therefore, by detecting a base sequence in which the second base of the triplet is changed from T to C, a mutant strain having the F490S mutation can be detected.

In this specification, the P681H mutation refers to the substitution of the 681st proline (P) with histidine (H) in the amino acid sequence of the spike protein of SARS-CoV-2. Here, this mutation is caused by the change of the codon corresponding to the proline, CCT, to CAT in the region coding for the spike protein of the SARS-CoV-2 virus genome RNA. Therefore, by detecting a base sequence in which the second base of the triplet is changed from C to A, a mutant strain having the P681H mutation can be detected. In addition, the P681R mutation refers to the substitution of the 681st proline with arginine. Here, this mutation is caused by the change of the CCT to CGT in the region coding for the spike protein of the SARS-CoV-2 virus genome RNA. Therefore, by detecting a base sequence in which the second base of the triplet is changed from C to G, a mutant strain having the P681R mutation can be detected.

The oligonucleotide of the present invention capable of detecting a SARS-CoV-2 mutant strain having an N501Y mutation is an oligonucleotide having a base sequence in which the codon at position N501 in the region encoding the spike protein of SARS-CoV-2 genomic RNA is changed to a codon corresponding to tyrosine, or a sequence complementary to the base sequence. An example of the oligonucleotide is an oligonucleotide having the base sequence shown in SEQ ID NO: 35 or a sequence complementary to the base sequence. Although not limiting the present invention, examples of the oligonucleotide include those having a chain length of 13 bases or more. The chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

The oligonucleotide of the present invention capable of detecting a mutant strain having the E484K mutation is an oligonucleotide having a base sequence in which the codon at position E484 in the region encoding the spike protein of the SARS-CoV-2 genomic RNA has been changed to a codon corresponding to lysine, or a sequence complementary to the base sequence. An example of the oligonucleotide is an oligonucleotide having the base sequence shown in SEQ ID NO: 36 or a sequence complementary to the base sequence. Although not intended to limit the present invention, examples of the oligonucleotide include those having a chain length of 13 bases or more. The chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

The oligonucleotide of the present invention capable of detecting mutant strains having the E484Q mutation is an oligonucleotide having a base sequence in which the codon at position E484 in the region encoding the spike protein of SARS-CoV-2 genomic RNA has been changed to a codon corresponding to glutamine, or a sequence complementary to the base sequence. An example of the oligonucleotide is an oligonucleotide having the base sequence shown in SEQ ID NO:61 or a sequence complementary to the sequence. Although not intended to limit the present invention, examples of the oligonucleotide include those having a chain length of 13 bases or more. The chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

The oligonucleotide of the present invention capable of detecting mutant strains having the L452R mutation is an oligonucleotide having a base sequence in which the codon at position L452 in the region encoding the spike protein of SARS-CoV-2 genomic RNA has been changed to a codon corresponding to arginine, or a sequence complementary to the base sequence. An example of the oligonucleotide is an oligonucleotide having the base sequence shown in SEQ ID NO:88 or a sequence complementary to the base sequence. Although not intended to limit the present invention, examples of the oligonucleotide include those having a chain length of 13 bases or more. The chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

The oligonucleotide of the present invention capable of detecting a mutant strain having the T478K mutation is an oligonucleotide having a base sequence in which the codon corresponding to position T478 in the region encoding the spike protein of SARS-CoV-2 genomic RNA has been changed to a codon corresponding to lysine, or a sequence complementary to the base sequence. An example of the oligonucleotide is an oligonucleotide having the base sequence shown in SEQ ID NO: 113 or a sequence complementary to the sequence. Although not intended to limit the present invention, examples of the oligonucleotide include those having a chain length of 13 bases or more. The chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

The oligonucleotide of the present invention capable of detecting mutant strains having the L452Q mutation is an oligonucleotide having a base sequence in which the codon at position L452 in the region encoding the spike protein of SARS-CoV-2 genomic RNA has been changed to a codon corresponding to glutamine, or a sequence complementary to the base sequence. Examples of the oligonucleotide include an oligonucleotide having the base sequence shown in SEQ ID NO: 138 or a sequence complementary to the sequence. Although not limiting the present invention, examples of the oligonucleotide include those having a chain length of 13 bases or more. The chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

The oligonucleotide of the present invention capable of detecting mutant strains having the F490S mutation is an oligonucleotide having a base sequence in which the codon at position F490 in the region encoding the spike protein of SARS-CoV-2 genomic RNA has been changed to a codon corresponding to serine, or a sequence complementary to the base sequence. An example of the oligonucleotide is an oligonucleotide having the base sequence shown in SEQ ID NO: 137 or a sequence complementary to the sequence. Although not intended to limit the present invention, examples of the oligonucleotide include those having a chain length of 13 bases or more. The chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

The oligonucleotide of the present invention capable of detecting mutant strains having the P681H mutation is an oligonucleotide having a base sequence in which the codon corresponding to position P681 in the region encoding the spike protein of SARS-CoV-2 genomic RNA has been changed to a codon corresponding to histidine, or a sequence complementary to the base sequence. An example of the oligonucleotide is an oligonucleotide having the base sequence shown in SEQ ID NO: 162 or a sequence complementary to the base sequence. Although not intended to limit the present invention, examples of the oligonucleotide include those having a chain length of 9 bases or more. The chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

Furthermore, the oligonucleotide of the present invention capable of detecting mutant strains having the P681R mutation is an oligonucleotide having a base sequence in which the codon corresponding to position P681 in the region encoding the spike protein of SARS-CoV-2 genomic RNA has been changed to a codon corresponding to arginine, or a sequence complementary to the base sequence. An example of the oligonucleotide is an oligonucleotide having the base sequence shown in SEQ ID NO: 163 or a sequence complementary to the sequence. Although not intended to limit the present invention, examples of the oligonucleotide include those having a chain length of 11 bases or more. Furthermore, the chain length of the oligonucleotide is 30 bases or less, preferably 25 bases or less, and more preferably 23 bases or less.

Naturally, the oligonucleotide of the present invention specifically hybridizes with nucleic acid derived from the genomic RNA of the mutant strain. Here, "specifically hybridizes" means that the oligonucleotide of the present invention can hybridize with nucleic acid derived from the genomic RNA of the mutant strain under conditions where it does not hybridize with nucleic acid derived from the genomic RNA of the parent strain (wild type), and can distinguish between nucleic acid derived from the mutant strain and nucleic acid derived from the parent strain.

The oligonucleotide of the present invention can be used as a probe for detecting nucleic acid derived from mutant SARS-CoV-2, for example, DNA (cDNA) complementary to the genomic RNA of the mutant strain, or a fragment thereof. Therefore, by appropriately selecting the chain length and base sequence of the oligonucleotide and the label to be added depending on the detection method used to detect the mutant strain, it is possible to design a probe that can efficiently detect mutant SARS-CoV-2.

As a technique for detecting a target nucleic acid using a probe, in addition to the classical hybridization method, various methods are known, such as the TaqMan method, the cycling probe method, and the molecular beacon method. The TaqMan method is a method in which a probe hybridized with a target nucleic acid is degraded in parallel with nucleic acid amplification by PCR, and is characterized by using a DNA polymerase having 5'-3' nuclease activity for nucleic acid amplification. The probe used in the cycling probe method contains RNA in its molecule, and is cleaved by ribonuclease H when hybridized with a target nucleic acid. The probe used in the molecular beacon method forms a double strand in its molecule, and when hybridized with a target nucleic acid, the intramolecular double strand structure is dissolved. By applying an appropriate label, any of these probes can be used to detect a target nucleic acid using the generation of a signal (e.g., fluorescence) derived from the label as an indicator.

Hereinafter, an explanation will be given taking as an example the oligonucleotide of the present invention used as a TaqMan probe. The oligonucleotide of the present invention can be suitably used in the method, and its sequence and chain length can be adjusted as appropriate. Although not particularly limiting the present invention, oligonucleotides having a base sequence selected from SEQ ID NOs: 4, 8, 12, 14, and 16 that can be used to detect N501Y mutant strains, oligonucleotides having a base sequence selected from SEQ ID NOs: 24, 27, 30, 32, 34, 37, 44, 45, and 46 that can be used to detect E484K mutant strains, oligonucleotides having a base sequence selected from SEQ ID NOs: 53, 54, 55, 56, and 57 that can be used to detect E484Q mutant strains, oligonucleotides having a base sequence selected from SEQ ID NOs: 68, 69, 70, 71, 81, 82, 83, 84, and 85 that can be used to detect L452R mutant strains, oligonucleotides having a base sequence selected from SEQ ID NOs: 95, 96, 97, 98, 99, 10 The oligonucleotides having a base sequence selected from SEQ ID NOs: 131, 132, and 133 and usable for detecting a T478K mutant strain, the oligonucleotides having a base sequence selected from SEQ ID NOs: 131, 132, and 133 and usable for detecting an L452Q mutant strain, the oligonucleotides having a base sequence selected from SEQ ID NOs: 122, 123, and 124 and usable for detecting an F490S mutant strain, the oligonucleotides having a base sequence selected from SEQ ID NOs: 152, 153, 154, 155, and 156 and usable for detecting a P681H mutant strain, and the oligonucleotides having a base sequence selected from SEQ ID NOs: 157, 158, 159, 160, and 161 and usable for detecting a P681R mutant strain are preferred embodiments of the present invention. Furthermore, although this invention is not particularly limited, the 3' end of these oligodeoxynucleotides may be modified to prevent an extension reaction by DNA polymerase from the end. Examples of the modification include labeling with a fluorescent substance or a quenching substance.

The oligonucleotide of the present invention is not particularly limited to the present invention, but is usually composed of deoxyribonucleotides (DNA) and can be synthesized by a method well known to those skilled in the art. The oligonucleotide of the present invention may contain nucleotides other than DNA, such as RNA, nucleotides having non-natural bases (e.g., deoxyuridine, inosine, 7-deazaguanosine, 7-deazaadenosine, etc.), and nucleotides containing ribose with a bridged structure (bridged nucleic acid; BNA), as long as the oligonucleotide does not lose the property of hybridizing with the nucleic acid derived from the mutant SARS-CoV-2 to be detected. BNA is an RNA analog having a structure in which the oxygen atom at the 2' position of ribose is bridged with the carbon atom at the 4' position, and 2',4'-BNA (LNA), 3'-amino-2',4'-BNA, etc. are known. There is no particular limit to the content of nucleotides other than DNA, and it may be appropriately set in consideration of the stability and specificity of hybridization. For example, up to about half of all nucleotides can be such nucleotides.

In order to detect hybridization of the target nucleic acid (nucleic acid derived from the mutant genomic RNA) and the oligonucleotide of the present invention, the oligonucleotide of the present invention may be appropriately labeled. The oligonucleotide labeled with both a fluorescent substance and a quenching substance with an appropriate distance between them does not emit fluorescence as it is, but when the distance between the fluorescent substance and the quenching substance increases due to decomposition, cleavage, etc. of the oligonucleotide, fluorescence is emitted. There are no particular limitations on the fluorescent substance and quenching substance used, but examples of the fluorescent substance include 6-FAM, VIC, HEX, ROX, TET, TAMRA, Cy3, Cy5, etc., and examples of the quenching substance include DABCYL, Eclipse (registered trademark), TAMRA, BHQ (registered trademark, black hole quencher), etc. By appropriately combining these, a double-labeled oligonucleotide of the present invention can be prepared. There are no particular limitations on the positions at which the fluorescent substance and the quenching substance are added, as long as they are appropriately spaced apart. For example, the fluorescent substance and the quencher may be added to both ends of the oligonucleotide of the present invention, or either or both of them may be added to a position other than the end.

The oligonucleotide of the present invention may have a minor groove binder (MGB) added thereto. MGB is a substance that has the property of entering the minor groove of double-stranded DNA, and the Tm value of the double-stranded nucleic acid formed with a nucleic acid having a complementary sequence of an oligonucleotide having an MGB added thereto is higher than that of an oligonucleotide having no MGB added thereto (for example, WO96/32496). Since a difference in one base of the hybridizing nucleic acid causes a large difference in the Tm value of an MGB-modified oligonucleotide, the target nucleic acid can be detected with reduced background. In general, MGB has a crescent-shaped three-dimensional structure and a molecular weight of about 150 to about 2000 daltons. Examples of MGBs include netropsin, distamycin, distamycin A, lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, prenyl, CC-1065, Hoechst 33258, 4'-6-diamidino-2-phenylindole (DAP1), CDPI3, and derivatives thereof. There are no particular limitations on the position at which the MGB is added in the oligonucleotide of the present invention, but examples include the 3' end and 5' end.

"The method for detecting mutant SARS-CoV-2 of the present invention"

The present invention provides a method for detecting mutant SARS-CoV-2 in a sample using the oligonucleotide of the present invention.

The method for detecting mutant SARS-CoV-2 of the present invention is a method for detecting mutant SARS-CoV-2 having a mutation selected from N501Y, E484K, E484Q, L452R, L452Q, T478K, F490S, P681H and P681R in the spike protein. Specifically, a DNA complementary to the SARS-CoV-2 genome contained in a sample or a fragment of the complementary DNA is synthesized, and then this is contacted with an oligonucleotide of the present invention. DNA containing a base sequence encoding the mutant spike protein or a part thereof hybridizes with the oligonucleotide of the present invention, thus making it possible to determine whether a mutant strain is present in a sample.

The method of the present invention can detect SARS-CoV-2 mutants that have at least one of the mutations selected from N501Y, E484K, E484Q, L452R, L452Q, T478K, F490S, P681H, and P681R, and can also detect mutant SARS-CoV-2 mutants that have other mutations.

SARS-CoV-2 is an RNA virus, and the virus particle retains an RNA genome. DNA having a sequence complementary to the RNA genome, i.e., cDNA or a fragment thereof, is synthesized by reverse transcription using the genomic RNA as a template. The reverse transcription reaction can be carried out using a reaction solution commonly used in reverse transcription reactions, which contains a reverse transcriptase and appropriate primers. The cDNA or cDNA fragments synthesized by this process may be converted to double-stranded DNA by a method well known to those skilled in the art.

As the reverse transcriptase, a reverse transcriptase derived from Moloney murine leukemia virus (MMLV) or a mutant thereof, a reverse transcriptase derived from avian myeloblastosis virus (AMV) or a mutant thereof, a DNA polymerase having reverse transcription activity (Tth DNA polymerase, Bca DNA polymerase, etc.) or a mutant thereof, but is not limited thereto. Examples of the mutants include mutants with improved heat resistance, and mutants with reduced or lost nuclease activity (ribonuclease H activity, etc.). Many different reverse transcriptases are commercially available, and they can also be used in the method of the present invention.

Primers used in the reverse transcription reaction can be either those complementary to a specific sequence on the viral genome or those with random sequences. Although this invention is not particularly limited, it is preferable to use primers designed to synthesize cDNA corresponding to a region on the viral genome that corresponds to the region that codes for the spike protein, preferably a region that contains codons corresponding to N501, E484, L452, T478, F490, or P681.

In a preferred embodiment of the present invention, in order to improve the sensitivity of virus detection, a cDNA fragment containing a region to which the oligonucleotide of the present invention can hybridize is amplified. There is no limitation on the nucleic acid amplification method used in this step, and known methods such as PCR and LAMP can be used. The nucleic acid amplification method is carried out using cDNA or a cDNA fragment synthesized by reverse transcription using the viral genomic RNA as a template, and generates a nucleic acid (DNA) fragment containing a mutation site as an amplified product.

In a preferred embodiment of the present invention, the PCR method is used as the nucleic acid amplification method. The PCR method is widely used as a nucleic acid detection technique in which a reaction solution containing one or more primer pairs, a heat-resistant DNA polymerase, and dNTPs as main components is treated with a temperature cycler. Examples of the heat-resistant DNA polymerase that can be used include Taq polymerase derived from Thermus bacteria, Tth polymerase, and their mutants, Pfu polymerase derived from thermophilic archaea, KOD polymerase, and their mutants. PCR can also be performed by mixing multiple types of DNA polymerase. Many heat-resistant DNA polymerases suitable for PCR are commercially available, and they can also be used in the method of the present invention.

The synthesis of cDNA fragments and their amplification can be carried out in a series of reactions. Although not limiting to the present invention, one-step RT-PCR, in which reverse transcription and PCR are carried out in one reaction vessel, is a preferred embodiment of the method of the present invention. Various RT-PCR reaction solutions are known, including those that use different enzymes, and many are also commercially available in the form of kits. When carrying out the method of the present invention using one-step RT-PCR, a solution containing two enzymes, reverse transcriptase and a thermostable DNA polymerase, may be used, or a reaction solution containing only a thermostable DNA polymerase with reverse transcription activity (e.g., Tth DNA polymerase) may be used.

The primer pair used for amplifying a cDNA fragment in the method of the present invention is not particularly limited in its sequence, so long as it is designed to amplify a cDNA fragment containing a region to which the oligonucleotide of the present invention can hybridize. By adding such a pair of primers to a one-step RT-PCR reaction solution, one of the primers also functions as a primer for cDNA synthesis. The primers used in the present invention are designed taking into consideration possible mutations that may exist in the region on the viral genome RNA corresponding to the cDNA fragment to be amplified. For example, if there is a concern about the presence of base substitution, a primer can be designed with the base portion as a mixed base, or multiple primers corresponding to the wild-type genome RNA sequence and the genome RNA sequence in which base substitution has occurred can be prepared and used in combination, thereby obtaining an amplified cDNA fragment regardless of the presence or absence of base substitution. In particular, the present invention is not limited to a specific example. For detection of a mutant strain having an N501Y mutation, a primer pair consisting of a forward primer having a base sequence selected from SEQ ID NOs: 1, 5, 9, 17, 19, 21 and a reverse primer having a base sequence selected from SEQ ID NOs: 2, 6, 10, 18, 20, 25 is used. For detection of a mutant strain having an E484K or E484Q mutation, a primer pair consisting of a forward primer having a base sequence selected from SEQ ID NOs: 21, 28, 9, 17 and a reverse primer having a base sequence selected from SEQ ID NOs: 22, 25, 6, 10, 18 is used. For detection of a mutant strain having an L452R mutation or an L452Q mutation, a primer pair consisting of a forward primer having a base sequence selected from SEQ ID NOs: 64, 66, 75, 77, 79, 125, 127, 129 and a reverse primer having a base sequence selected from SEQ ID NOs: 65, 67, 76, 78, 80, 126, 128, 130 is used. For detection of mutant strains having a T478K mutation, a primer pair consisting of a forward primer having a base sequence selected from SEQ ID NOs: 91, 93, 107, 109, 28, 64 and a reverse primer having a base sequence selected from SEQ ID NOs: 92, 94, 108, 110, 6, 65 is preferred; for detection of mutant strains having a F490S mutation, a primer pair consisting of a forward primer having a base sequence selected from SEQ ID NOs: 114, 115, 116 and a reverse primer having a base sequence selected from SEQ ID NOs: 117, 118, 119 is preferred; and for detection of mutant strains having a P681H mutation or P681R, a primer pair consisting of a forward primer having a base sequence selected from SEQ ID NOs: 139, 140, 141, 142 and a reverse primer having a base sequence selected from SEQ ID NOs: 143, 144, 145, 146 is preferred. For example, the region of the SARS-CoV-2 genome to which the primer having the base sequence of SEQ ID NO:5 anneals may contain base substitutions related to the known mutations S477N and T478K. For this reason, when using the primer of SEQ ID NO:5, the method of the present invention may be carried out in combination with the primers of SEQ ID NO:62 and SEQ ID NO:63, which correspond to the RNA sequence having the above-mentioned two types of base substitutions.

Furthermore, when RT-PCR is performed in the presence of an appropriately modified oligonucleotide of the present invention, the target nucleic acid, i.e., a cDNA fragment derived from the genome of the mutant strain, can be detected in parallel with DNA amplification. By preparing an oligonucleotide of the present invention designed to be suitable for the TaqMan method, cycling probe method, molecular beacon method, etc., and adjusting the composition of the reaction solution to be suitable for the detection means, quantitative RT-PCR can be performed to optically monitor the amplification of the target nucleic acid over time.

In a preferred embodiment of the present invention, a method for detecting mutant SARS-CoV-2 in real time by one-step RT-PCR is provided. In this method, a reaction solution is prepared containing reverse transcriptase and a thermostable DNA polymerase (or a thermostable DNA polymerase having reverse transcription activity), at least one primer pair, dNTPs, an oligonucleotide of the present invention labeled with a fluorescent substance and a quencher, and other components necessary for RT-PCR, and a sample is added to the reaction solution. The reaction solution is kept at a temperature suitable for the reverse transcription reaction, and then transferred to a temperature cycle reaction to amplify the cDNA fragment. During the temperature cycle reaction, fluorescence corresponding to the amount of amplification of the cDNA fragment is emitted from the reaction solution, and the presence of mutant SARS-CoV-2 in the sample can be confirmed using this as an indicator.

Multiple mutations in SARS-CoV-2 can be detected by a single RT-PCR. Such a reaction system (multiplex RT-PCR) is well known to those skilled in the art. By labeling each of the oligonucleotides of the present invention corresponding to the mutations to be detected with a fluorescent substance having a different maximum fluorescence wavelength, the type of mutation occurring in SARS-CoV-2 present in the sample can be known based on which fluorescent substance the fluorescence emitted by the reaction solution is derived from. For example, when an RT-PCR reaction solution containing both an oligonucleotide for detecting an N501Y mutation and an oligonucleotide for detecting an E484K mutation is used, a mutant strain having an N501Y mutation, a mutant strain having an E484K mutation, and a mutant strain having both an N501Y mutation and an E484K mutation in the sample can be detected at once. In addition, an RT-PCR reaction solution containing an oligonucleotide capable of detecting each of multiple amino acid substitutions that may occur in a certain amino acid residue and an oligonucleotide corresponding to the wild-type sequence can perform typing of the mutation of the amino acid residue in a single reaction. In this way, the present invention also provides a multiplex RT-PCR system for detecting multiple mutations in a viral genome in a single reaction.

The detection method of the present invention using multiplex RT-PCR is carried out using a reaction solution containing a plurality of oligonucleotides of the present invention, a reverse transcription primer and an amplification primer pair for generating and amplifying a cDNA fragment derived from the SARS-CoV-2 genome containing a mutation site corresponding to the oligonucleotide. A plurality of combinations of reverse transcription primer and amplification primer pair may be used for each mutation to be detected, or a single combination capable of generating and amplifying a cDNA fragment containing multiple mutation sites may be used. As described above, one of the amplification primer pair may also serve as the reverse transcription primer.

In the detection method of the present invention using PCR, amplification and detection of a positive control nucleic acid may be further combined in order to examine the influence of PCR inhibitors and the like in the sample. The positive control nucleic acid may be a nucleic acid derived from a gene (e.g., a housekeeping gene, etc.) that is present in the sample and is different from the gene to be detected. When a gene present in the sample is used as a positive control nucleic acid, a primer pair for amplifying an arbitrary region of the gene is used in combination with a detection probe. An artificial nucleic acid may also be prepared and added to the sample in advance, and may be, for example, a nucleic acid having the same base sequence as the amplified region of the target nucleic acid and non-target nucleic acid or a nucleic acid having a different base sequence from these. When an artificial nucleic acid is used as a positive control nucleic acid, it may be amplified using a primer pair used for amplifying the target nucleic acid and non-target nucleic acid depending on the base sequence, or a primer pair different from the amplified region of the target nucleic acid and non-target nucleic acid may be used. A probe that can selectively detect these positive control nucleic acids is used for detecting the positive control nucleic acid. By detecting these positive control nucleic acids simultaneously with the detection of the target nucleic acid of the present invention, it is possible to confirm whether there is any abnormality in the amplification in the detection system of the present invention, and to perform semi-quantitative analysis of the initial amount of the target nucleic acid by comparing the amplification curves.

Furthermore, the detection method of the present invention may be carried out using a reaction solution containing known components useful for PCR. The components are not particularly limited, but examples thereof include surfactants, proteins (bovine serum albumin, gelatin, nucleic acid binding proteins, etc.), amphoteric substances (betaine, etc.), acidic polymeric substances, PCNA (Proliferating Cell Nuclear Antigen), etc.

The conditions for the reverse transcription reaction and PCR in one-step RT-PCR (reaction temperature, holding time, number of thermal cycles, etc.) may be set appropriately. For example, the reaction may be performed under the conditions recommended in a commercially available RT-PCR kit (SARS-CoV-2 detection kit, etc.) or modified conditions. It is well known to those skilled in the art that the reaction conditions should be set taking into consideration the base sequence and chain length of the oligonucleotide of the present invention, the base sequence and chain length of the primers used, the chain length of the DNA fragment to be amplified, etc.

When multiple PCR tests are performed in parallel, if the amplified DNA fragments produced after the reaction are mixed with other reaction solutions before the reaction, erroneous test results will be produced. A method for preventing such contamination of reaction solutions is known. When uracil-N-glycosylase (UNG) is applied to DNA in which uracil (U) has been incorporated into the chain, the DNA is cleaved at the uracil position. DNA fragments amplified using a PCR reaction solution containing dUTP contain uracil, but if this amplified DNA fragment is mixed with other reaction solutions (reaction solutions containing dUTP and heat-labile UNG) before the reaction is performed, the mixed DNA can be decomposed and lose its function as a template by keeping the reaction solution at a temperature at which UNG acts before the start of the reaction. Since UNG is inactivated by the subsequent temperature cycle of PCR, decomposition of the DNA that has incorporated U does not occur during amplification. In the detection method of the present invention, cross-contamination can also be prevented by using a reaction solution containing dUTP and heat-labile UNG.

There is no particular limitation on the sample to which the method of the present invention is applied. All samples suspected of containing mutant SARS-CoV-2, such as biological samples and environmental samples, are subject to the method of the present invention. The biological samples are not particularly limited, but examples include oral scrapings, pharyngeal swabs, nasal swabs, nasopharyngeal swabs, nasal aspirates, sputum, bronchial lavage fluid, alveolar lavage fluid, rectal swabs, various body fluids (saliva, blood, cerebrospinal fluid, sweat), tissues, urine, or fecal suspensions. Environmental samples include environmental water (seawater, river water, lake water, sewage, domestic wastewater, industrial wastewater, etc.), samples obtained by wiping the surface of an object with a swab, etc., and suspensions of materials collected from the air. These samples may be subjected to the method of the present invention as is, or may be used after simple treatment (heat treatment, dilution, concentration, removal of insoluble matter, solubilization treatment, cell lysis treatment, denaturation or decomposition of contaminating proteins, etc.) or purification of nucleic acid. The treatment method is selected taking into consideration the amount of nucleic acid contained in the sample and the properties and amount of contaminants. Each of the above samples may be subjected to the detection method of the present invention individually, or multiple samples may be mixed and then subjected to the detection method of the present invention.

"Kit of the present invention"

The present invention provides a kit for use in the method for detecting mutant SARS-CoV-2 of the present invention.

The kit of the present invention is characterized by including the oligonucleotide of the present invention and a reagent for synthesizing DNA complementary to the SARS-CoV-2 genome.

Examples of "reagents for synthesizing DNA complementary to the SARS-CoV-2 genome" include reverse transcriptase or a primer for cDNA synthesis. The kit of the present invention includes, for example, a kit containing the oligonucleotide of the present invention and a primer used for synthesizing cDNA using the SARS-CoV-2 genome as a template, a kit containing the oligonucleotide of the present invention, the primer for cDNA synthesis, and a reverse transcriptase. The latter may further contain other components used in preparing the reverse transcription reaction solution (e.g., buffer components, divalent metal salts, dNTPs, etc.). Each of these can be used as described in the detection method of the present invention.

Furthermore, the kit of the present invention may contain reagents for amplifying cDNA. Although this is not a limitation of the present invention, the kit may contain, for example, reagents for amplifying cDNA by PCR, i.e., a heat-stable DNA polymerase, a primer pair designed to amplify cDNA corresponding to the region on the viral genome that codes for the spike protein, and various components for preparing a reaction solution for PCR.

In a preferred embodiment of the present invention, a kit is provided that contains various reagents for synthesizing cDNA fragments from a viral genome, amplifying the cDNA fragments, and detecting a target nucleic acid in one reaction vessel by one-step RT-PCR. The kit contains an oligonucleotide of the present invention that is designed and labeled so as to optically detect the target nucleic acid, a reverse transcriptase and a heat-stable DNA polymerase (or a heat-stable DNA polymerase having reverse transcription activity), at least one pair of primers, and various components for preparing a reaction solution for RT-PCR (buffer components, divalent metal salts, dNTPs, etc.). Furthermore, the kit may contain multiple types of oligonucleotides of the present invention, or a kit that contains an oligonucleotide of the present invention and an oligonucleotide for detecting other mutations. Examples of one embodiment of the present invention include a multiplex RT-PCR kit that can detect both the N501Y mutation and the E484K mutation, and a typing kit for mutations at positions E484, L452, P681, etc.

The various components described above may be stored in separate containers and configured to prepare a reaction solution for RT-PCR when used, or may be included in the kit as a mixture of multiple components. The present invention also includes kits containing all of the components necessary for the reaction other than the sample, and containing premixed reagents that can prepare a reaction solution simply by mixing with an appropriately treated and/or diluted sample. Furthermore, the kit of the present invention may contain reagents and instruments used for sample treatment and nucleic acid purification from the sample, a positive control that serves as an indicator for determining the presence of a reaction inhibitor, and primers and probes for amplifying and detecting the positive control.

The kit of the present invention may contain known components useful for PCR. The components are not particularly limited, but examples include surfactants, proteins (bovine serum albumin, gelatin, nucleic acid binding proteins, etc.), amphoteric substances (betaine, etc.), acidic polymeric substances, PCNA, etc.

The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples.

Example 1
Primers and probes for detecting N501Y mutant strains Detection of N501Y mutant strains by the detection method of the present invention was examined. First, sets #1 to #8 were constructed, each consisting of a forward primer (containing F in its name), a reverse primer (containing R in its name), and a probe (two types, one for detecting wild-type SARS-CoV-2 and one for detecting mutant SARS-CoV-2) as shown in Table 2. MGB was added to the 3' end of the probe, and the 5' end was labeled with FAM and the 3' end was labeled with BHQ (registered trademark) 1.

As test samples, synthetic single-stranded RNA having the sequence of the wild-type SARS-CoV-2 virus genome (product name: Twist Synthetic SARS-CoV-2 RNA Control 2 (MN908947.3), manufactured by Twist Bioscience) and synthetic single-stranded RNA having the sequence of the mutant (including N501Y mutation and E484K mutation) SARS-CoV-2 virus genome (product name: Twist Synthetic SARS-CoV-2 RNA Control 16 (B.1.351, EPI_ISL_678597), manufactured by Twist Bioscience) were prepared. These two synthetic RNAs are referred to as wild-type RNA and mutant RNA, respectively, in this specification. As a negative control, RNase Free H ₂ O was prepared.

1. Wild-type RNA and mutant RNA were added to RNase Free H ₂ O to a final concentration of 5000 copies/μl to prepare a test sample solution. For detection of the test sample, a component of a commercially available product named SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A) was used. That is, 1 μl of the above-mentioned test sample solution was mixed with 15 μl of RT-qPCR Mix containing enzymes and substrates, a forward primer (final concentration 0.2 μM), a reverse primer (final concentration 0.2 μM), and a probe (final concentration 0.2 μM; either for wild-type RNA detection or for mutant RNA detection), and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared with RNase Free H ₂ O. The reagent Solution A included in the kit was not used in this test because it was an unpurified sample pretreatment reagent.

2. Reaction solutions were prepared for each set shown in Table 2, and tests were performed in duplicate to improve experimental accuracy.

The thermal cycler used was Thermal Cycler Dice (registered trademark) Real Time System III (Cy5) with PC (Takara Bio, product #TP990). The PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Table 3 and Figure 1 (amplification curves for each reaction).

Results: As a result of the investigation, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #2 and #5 had small Ct values and high S/N ratios for both wild-type and mutant RNA detection, and it was found that the single base substitution at N501 could be distinguished with high sensitivity. In addition, no amplification products were confirmed in any of the reaction solutions in which a negative control was added instead of the sample.

Example 2
Primer-probe for detecting E484K mutant strain Five sets of primer-probes, #9, #10, #11, #12, and #13, were synthesized in the same manner as in Example 1, and a test was performed on the detection of mutant RNA having E484K mutation. Among these, the combination of primer-probes included in the sets #10, #11, and #12, which were judged to have better performance than the other sets, was changed, and new primer-probe sets #14 and #15 were constructed. The reaction confirmation was performed for these two sets by the same operation as in Example 1. The base sequences of the forward primer, reverse primer, and 3'-end MGB-labeled probe included in the primer-probe set used in the above test are shown in Table 4. In addition, the wild-type RNA detection probe included in both sets was labeled with FAM at the 5' end and BHQ (registered trademark) 1 at the 3' end, and the wild-type RNA detection probe was labeled with VIC at the 5' end and BHQ (registered trademark) 1 at the 3' end. As test samples, the same RNA as in Example 1 was used for wild-type RNA and mutant-type RNA, and serially diluted solutions were prepared in RNase Free H ₂ O to final concentrations of 5000 copies/μl, 500 copies/μl, and 50 copies/μl. RNase Free H ₂ O was also prepared as a negative control.

A one-step RT-PCR reaction solution containing each sample was prepared in the same manner as in Example 1. The thermal cycler used was an Applied Biosystems (registered trademark) 7500Fast Real-time PCR System (Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Table 5 and Figure 2 (amplification curves for each reaction).

Results: As a result of the investigation, it was confirmed that RNA corresponding to the probe was detected in both sets. In particular, the Ct value of the reaction #15 was small for both wild-type RNA detection and mutant RNA detection, and the S/N ratio was high, indicating that the single base substitution in E484K could be distinguished with high sensitivity. In addition, no amplification products were confirmed in any of the reaction solutions in which a negative control was added instead of the sample.

Example 3
Detection using primers and probes for detecting E484K mutant strains using LNA (Locked Nucleic Acid) technology was examined. First, sets #16, #17, #18, #19, #20, and #21 consisting of forward primers (those containing F in the name), reverse primers (those containing R in the name), and probes (two types for detecting wild-type SARS-CoV-2 and mutant SARS-CoV-2) shown in Table 6 were constructed. In addition, some nucleotides of the probes were replaced with LNA. The probe for detecting the wild type was labeled at its 5' end with FAM and at its 3' end with BHQ (registered trademark) 1. The probe for detecting the mutant type was labeled at its 5' end with Cy5 and at its 3' end with BHQ (registered trademark) 2.

The test samples and reagents were prepared in the same manner as in Example 1. RNase Free H ₂ O was prepared as a negative control.

A one-step RT-PCR reaction solution containing each sample was prepared in the same manner as in Example 2. The thermal cycler used was a QuantStudio (registered trademark) 5 Real-time PCR System (Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Tables 7 and 8 and in Figures 3 and 4 (amplification curves for each reaction).

Results: As a result of the investigation, it was confirmed that RNA corresponding to the probe was detected in all sets. In particular, the Ct value of the reaction #18 was small for both wild-type RNA detection and mutant RNA detection, and the S/N ratio was high, indicating that the single base substitution in E484K could be distinguished with high sensitivity. In addition, no amplification products were confirmed in any of the reaction solutions in which a negative control was added instead of the sample.

Example 4
Multiplex PCR detection using primers and probes for detecting N501Y mutation and primers and probes for detecting E484K mutant strains was examined. First, sets #22, #23, #24, and #25 were constructed, each consisting of a forward primer (containing F in its name), a reverse primer (containing R in its name), and a probe (for detecting wild-type SARS-CoV-2, for detecting mutant SARS-CoV-2, wild-type) shown in Tables 9 and 10. All sets contain N1 and N2, which are primer-probe sets for detecting SARS-CoV-2 genomic RNA, and oligonucleotides of SEQ ID NOs: 21 and 25, which are primer pairs for synthesizing and amplifying cDNA containing regions corresponding to N501 and E484. In addition, the 5' end of the probe for detecting the E484K mutant was labeled with Cy5 and the 3' end was labeled with BHQ (registered trademark) 2, and some nucleotides of some probes were replaced with LNA. The probe for detecting the N501Y mutation was labeled at its 5' end with FAM and at its 3' end with BHQ (registered trademark) 1, and further had MGB added to its 3' end. The probes for detecting N1 and N2 were labeled at their 5' end with HEX and at their 3' end with BHQ (registered trademark) 1, respectively.

A series of dilutions containing both wild-type RNA and mutant RNA at final concentrations of 5000 copies/μl, 500 copies/μl, and 50 copies/μl were prepared using RNase Free H ₂ O to prepare test specimen solutions. For detection of the test specimen, a component of a commercially available product named SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A) was used. That is, 1 μl of the above-mentioned test specimen solution was mixed with 15 μl of RT-qPCR Mix containing enzymes and substrates, each primer at a final concentration of 0.2 μM, and each probe at a final concentration of 0.2 μM, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared with RNase Free H ₂ O. The reagent Solution A included in the kit was an unpurified sample pretreatment reagent and was not used in this test.

The reaction solution thus prepared was subjected to one-step RT-PCR. The thermal cycler used was a QuantStudio (registered trademark) 5 Real-time PCR System (Thermo Fisher Scientific). The reaction conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 58°C for 30 seconds. The results are shown in Table 11 and Figure 5 (amplification curves for each reaction).

As shown in Table 11 and Figure 5, wild-type, N501Y mutant, and E484K mutant RNAs were detected in all sets. There was no significant difference in the Ct values for each set, and the S/N ratio was also high, indicating that all sets are useful for detecting mutant SARS-CoV-2.

Example 5
Primer and probe for detecting E484Q and K mutants The detection of E484Q and E484K mutants by the detection method of the present invention was examined. First, the forward primer of SEQ ID NO: 21 and the reverse primer of SEQ ID NO: 25, as shown in Table 4, were synthesized. Next, sets #26 to #30 consisting of these primer pairs and three types of probes containing LNA in the sequence (for detecting wild-type SARS-CoV-2 with "W" in the name, for detecting E484K mutation with "M" in the name, and for detecting E484Q mutation with "Q" in the name), as shown in Table 12, were constructed. The 5' end of the probe for detecting wild-type SARS-CoV-2 was labeled with FAM and the 3' end with BHQ (registered trademark) 1. The 5' end of the probe for detecting E484K mutation was labeled with Cy5 and the 3' end with BHQ (registered trademark) 2, respectively. The 5' end of the probe for detecting the E484Q mutation was labeled with HEX and the 3' end with BHQ (registered trademark)1.

As test specimens, synthetic single-stranded RNA having the sequence of wild-type SARS-CoV-2 virus genome RNA (E484E-CONTROL_RNA_Wild: SEQ ID NO: 58), synthetic single-stranded RNA having the sequence of mutant (E484K) SARS-CoV-2 virus genome RNA (E484K-CONTROL_RNA_Mut: SEQ ID NO: 59), and synthetic single-stranded RNA having the sequence of mutant (E484Q) SARS-CoV-2 virus genome RNA (E484Q-CONTROL_RNA_Mut: SEQ ID NO: 60) were used. These single-stranded RNAs were prepared by constructing a plasmid DNA incorporating a double-stranded DNA corresponding to the base sequence, and performing an in vitro transcription reaction using this as a template. These three types of RNA are referred to as E484E_RNA, E484K_RNA, and E484Q_RNA, respectively, in this specification. As a negative control, RNase Free H ₂ O was prepared.

E484E_RNA, E484K_RNA, and E484Q_RNA were added to RNase Free H ₂ O at 5000 copies/μl, 500 copies/μl, 50 copies/μl, or 5000 copies/μl, 500 copies/μl, and 50 copies/μl to prepare test sample solutions. For detection of the test sample, components of a commercially available product named SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A) were used. That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), a forward primer (final concentration 0.2 μM), a reverse primer (final concentration 0.2 μM), and three types of probes (final concentration 0.2 μM each) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

A reaction solution containing each set of primers and probes shown in Table 12 was prepared with the above composition. Tests were performed in duplicate to improve experimental accuracy.

Thermal cycler used was a QuantStudio (registered trademark) 5 real-time PCR system (Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Table 13.

Results: As a result of the study, the multiplex RT-PCR system that simultaneously detects E484E_RNA (wild type), E484K_RNA, and E484Q_RNA was able to detect wild type and mutant type (E484K or E484Q) RNA, respectively, and it was shown that typing at position 484, that is, discrimination between wild type (E), K, or Q, is possible using the reaction solution. #28 and #30 showed particularly good results. Furthermore, no amplification products were confirmed in any of the reaction solutions in which a negative control was added instead of the sample.

Example 6
Primer/probe for detecting L452R mutant strain Detection of L452R mutant strain by the detection method of the present invention was examined. First, the forward primer (containing "F" in the name) and reverse primer (containing "R" in the name) described in Table 14 were synthesized. Next, sets #31 to #71 consisting of these primer pairs and probes containing LNA in the sequence (either for detecting wild-type SARS-CoV-2 containing "W" in the name or for detecting mutant SARS-CoV-2 containing "M" in the name) described in Tables 15 and 16 were constructed. The 5' end of the probe for detecting wild-type SARS-CoV-2 was labeled with FAM and the 3' end with BHQ (registered trademark) 1, respectively. The 5' end of the probe for detecting mutant SARS-CoV-2 was labeled with Cy5 and the 3' end with BHQ (registered trademark) 2, respectively.

As test specimens, synthetic single-stranded RNA (L452R-CONTROL_RNA_Wild: SEQ ID NO: 89) having the sequence of wild-type SARS-CoV-2 virus genomic RNA and synthetic single-stranded RNA (L452R-CONTROL_RNA_Mut: SEQ ID NO: 90) having the sequence of mutant (L452R) SARS-CoV-2 virus genomic RNA were used. These single-stranded RNAs were prepared by constructing a plasmid DNA incorporating a double-stranded DNA corresponding to the base sequence and performing an in vitro transcription reaction using this as a template. These two RNAs are referred to as wild-type RNA452 and mutant RNA452, respectively, in this specification. RNase Free H ₂ O was prepared as a negative control.

Wild-type RNA452 and mutant-type RNA452 were each added to RNase Free H ₂ O to a final concentration of 5000 copies/μl to prepare a test sample solution. For detection of the test sample, components of a commercially available product name SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A) were used. That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, etc., 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), a forward primer (final concentration 0.2 μM), a reverse primer (final concentration 0.2 μM), and a probe (final concentration 0.2 μM; for detecting wild-type RNA or mutant-type RNA) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

Reaction solutions containing the primer-probe sets shown in Tables 15 and 16 were prepared with the above composition. Tests were performed in duplicate to improve experimental accuracy.

The thermal cycler used was a QuantStudio (registered trademark) 5 Real-time PCR System (manufactured by Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Tables 17 and 18.

Results As a result of the investigation, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #33, #36, #40, #43, #47, #51, #53, #65, and #69 were able to detect wild-type RNA or mutant RNA with small Ct values and high fluorescence intensity, respectively. In other words, it was found that the oligonucleotides of SEQ ID NOs: 83 and 70 can distinguish the single base substitution in the L452R mutation with high sensitivity. In addition, no amplification products were confirmed in all reaction solutions in which a negative control was added instead of a sample.

Example 7
Primer-probe for detecting L452R mutant strain Four sets of primer-probes, #72, #73, #74, and #75, shown in Table 19, were constructed and tested for detection of mutant RNA having L452R mutation. In the table, the names of the primers include "F" or "R". These sets include both a probe for detecting wild-type SARS-CoV-2 and a probe for detecting mutant SARS-CoV-2 (the name of the probe for detecting wild-type SARS-CoV-2 includes "W" and the name of the probe for detecting mutant SARS-CoV-2 includes "M"). The probe for detecting wild-type RNA was labeled at the 5' end with Cy5 and at the 3' end with BHQ (registered trademark) 2, respectively, and the probe for detecting mutant RNA was labeled at the 5' end with FAM and at the 3' end with BHQ (registered trademark) 1, respectively. The RT-PCR reaction solution was prepared in the same manner as in Example 1 except that it contained two types of probes, and the reaction was confirmed. As test samples, the same RNA as in Example 6 was used for wild-type RNA452 and mutant RNA452, and serially diluted solutions were prepared in RNase Free H ₂ O to final concentrations of 5000 copies/μl, 500 copies/μl, 50 copies/μl, and 5 copies/μl. RNase Free H ₂ O was also prepared as a negative control. Furthermore, the RT-PCR conditions were 52° C. for 5 minutes, 95° C. for 10 seconds, followed by 45 cycles of 95° C. for 5 seconds and 58° C. for 30 seconds. The results are shown in Table 20.

Results: As a result of the investigation, no inconvenience was found due to the multiplex RT-PCR system that simultaneously detects wild-type RNA and mutant RNA. As a probe for detecting mutant RNA, the oligonucleotide of SEQ ID NO: 70 showed better results than the oligonucleotide of SEQ ID NO: 68, and as a probe for detecting wild-type RNA, the oligonucleotide of SEQ ID NO: 73 showed better results than the oligonucleotide of SEQ ID NO: 72. Furthermore, no amplification products were confirmed in any of the reaction solutions in which a negative control was added instead of the sample.

Example 8
Primer/probe for detecting T478K mutant strain The detection of T478K mutant strain by the detection method of the present invention was examined. First, the forward primer (containing "F" in the name) and reverse primer (containing "R" in the name) described in Table 21 were synthesized. Next, sets #76 to #148 consisting of these primer pairs and probes containing LNA in the sequence (either for detecting wild-type SARS-CoV-2 containing "W" in the name or for detecting mutant SARS-CoV-2 containing "M" in the name) described in Tables 22, 23, and 24 were constructed. The 5' end of the probe for detecting wild-type SARS-CoV-2 was labeled with FAM and the 3' end with BHQ (registered trademark) 1, respectively. The 5' end of the probe for detecting mutant SARS-CoV-2 was labeled with Cy5 and the 3' end with BHQ (registered trademark) 2, respectively.

As test specimens, synthetic single-stranded RNA (T478K-CONTROL_RNA_Wild: SEQ ID NO: 111) having the sequence of wild-type SARS-CoV-2 virus genome RNA and synthetic single-stranded RNA (T478K-CONTROL_RNA_Mut: SEQ ID NO: 112) having the sequence of mutant (T478K) SARS-CoV-2 virus genome RNA were used. These single-stranded RNAs were prepared by constructing a plasmid DNA incorporating a double-stranded DNA corresponding to the base sequence and performing an in vitro transcription reaction using this as a template. These two RNAs are referred to as wild-type RNA478 and mutant RNA478, respectively, in this specification. RNase Free H ₂ O was prepared as a negative control.

Wild-type RNA478 and mutant RNA478 were added to RNase Free H ₂ O to a final concentration of 5000 copies/μl to prepare a test sample solution. For detection of the test sample, components of a commercially available product name SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A) were used. That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, etc., 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), a forward primer (final concentration 0.2 μM), a reverse primer (final concentration 0.2 μM), and a probe (final concentration 0.2 μM; for detecting wild-type RNA or mutant-type RNA) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

Reaction solutions containing the primer-probe sets shown in Tables 22, 23, and 24 were prepared with the above composition. Tests were performed in duplicate to improve experimental accuracy.

The thermal cycler used was a QuantStudio (registered trademark) 5 Real-time PCR System (Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Tables 25, 26, and 27.

Results As a result of the study, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #101, #103, #106, #109, #112, #113, #115, #118, #121, #124, #127, #133, #139, and #145 were able to detect wild-type RNA or mutant RNA with small Ct values and high fluorescence intensity, respectively. In other words, it was found that the oligonucleotides of SEQ ID NOs: 95, 97, and 100 can distinguish single base substitution in the T478K mutation with high sensitivity. In addition, no amplification products were confirmed in all reaction solutions in which a negative control was added instead of a sample.

Example 9
Primer-probe sets for detecting T478K mutant strains Nine primer-probe sets #149, #150, #151, #152, #153, #154, #155, #156, and #157 shown in Table 28 were constructed, and tests were performed on the detection of mutant RNA having the T478K mutation. These sets include both a probe for detecting wild-type SARS-CoV-2 and a probe for detecting mutant SARS-CoV-2 (the name of the wild-type detection probe includes "W" and the name of the mutant detection probe includes "M"). The wild-type RNA detection probe was labeled at the 5' end with FAM and at the 3' end with BHQ (registered trademark) 1, and the mutant RNA detection probe was labeled at the 5' end with Cy5 and at the 3' end with BHQ (registered trademark) 2. The RT-PCR reaction solution was prepared in the same manner as in Example 7, except that it contained two types of probes, and the reaction was confirmed. Wild-type RNA478 and mutant RNA478 were used as test samples, and serially diluted solutions were prepared in RNase Free H ₂ O to final concentrations of 5000 copies/μl, 500 copies/μl, 50 copies/μl, and 5 copies/μl. RNase Free H ₂ O was also prepared as a negative control. Furthermore, the RT-PCR conditions were 52° C. for 5 minutes, 95° C. for 10 seconds, followed by 45 cycles of 95° C. for 5 seconds and 58° C. for 30 seconds. The results are shown in Table 28.

Example 10
The detection of the F490S mutant strain by the detection method of the present invention was examined. First, the forward primer (containing "F" in the name) and reverse primer (containing "R" in the name) described in Table 30 were synthesized. Next, sets #158 to #202 consisting of these primer pairs and probes containing LNA in the sequence (either for detecting wild-type SARS-CoV-2 containing "W" in the name or for detecting mutant SARS-CoV-2 containing "M" in the name) described in Tables 31 and 32 were constructed. The 5' end of the probe for detecting wild-type SARS-CoV-2 was labeled with FAM and the 3' end with BHQ (registered trademark) 1, respectively. The 5' end of the probe for detecting mutant SARS-CoV-2 was labeled with Cy5 and the 3' end with BHQ (registered trademark) 2, respectively.

As test specimens, synthetic single-stranded RNA (WILD_RNA_CONTROL2: SEQ ID NO: 134) having the sequence of wild-type SARS-CoV-2 virus genome RNA and synthetic single-stranded RNA (F490S_RNA_CONTROL_RNA_Mut: SEQ ID NO: 136) having the sequence of mutant (F490S) SARS-CoV-2 virus genome RNA were used. These single-stranded RNAs were prepared by constructing a plasmid DNA incorporating a double-stranded DNA corresponding to the base sequence and performing an in vitro transcription reaction using this as a template. These two RNAs are referred to as wild-type RNA2 and mutant RNAF490S, respectively, in this specification. RNase Free H ₂ O was also prepared as a negative control.

A test sample solution was prepared by adding wild-type RNA2 and mutant RNAF490S to RNase Free H ₂ O to a final concentration of 5000 copies/μl. The test sample was detected using components of a commercially available product called SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A). That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, etc., 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), a forward primer (final concentration 0.2 μM), a reverse primer (final concentration 0.2 μM), and a probe (final concentration 0.2 μM; for detecting wild-type RNA or mutant-type RNA) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

Reaction solutions containing the primers and probes of each set shown in Tables 31 and 32 were prepared with the above composition. Tests were performed in duplicate to improve experimental accuracy.

The thermal cycler used was a QuantStudio (registered trademark) 5 Real-time PCR System (Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Tables 33 and 34.

Results As a result of the investigation, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #174, #175, #176, #179, #180, #181, #184, #185, and #186 were able to detect wild-type RNA2 or mutant RNAF490S with small Ct values and high fluorescence intensity, respectively. In other words, it was found that the oligonucleotides of SEQ ID NOs: 121, 122, and 123 can distinguish single base substitutions in F490S mutations with high sensitivity. In addition, no amplification products were confirmed in any of the reaction solutions in which a negative control was added instead of a sample.

Example 11
Primer-probe sets for detecting F490S mutant strains Four sets of primer-probe sets #203, #204, #205, and #206 shown in Table 35 were constructed, and tests were performed on the detection of mutant RNA having the F490S mutation. These sets include both a probe for detecting wild-type SARS-CoV-2 and a probe for detecting mutant SARS-CoV-2 (the name of the wild-type detection probe includes "W" and the name of the mutant detection probe includes "M"). The wild-type RNA detection probe was labeled at the 5' end with FAM and at the 3' end with BHQ (registered trademark) 1, and the mutant RNA detection probe was labeled at the 5' end with Cy5 and at the 3' end with BHQ (registered trademark) 2. The RT-PCR reaction solution was prepared in the same manner as in Example 10, except that it contained two types of probes, and the reaction was confirmed. Wild-type RNA2 or mutant RNAF490S was used as the test specimen, and serially diluted solutions were prepared in RNase Free H ₂ O to final concentrations of 5000 copies/μl, 500 copies/μl, 50 copies/μl, and 5 copies/μl. RNase Free H ₂ O was also prepared as a negative control. Furthermore, the RT-PCR conditions were 52° C. for 5 minutes, 95° C. for 10 seconds, followed by 45 cycles of 95° C. for 5 seconds and 58° C. for 30 seconds. The results are shown in Table 36.

Results As a result of the investigation, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #204, #205, and #206 were able to detect wild-type RNA2 or mutant RNAF490S with small Ct values and high fluorescence intensity, respectively. In other words, it was found that the oligonucleotides of SEQ ID NOs: 122 and 123 can distinguish single base substitution in F490S mutation with high sensitivity. In addition, no amplification products were confirmed in all reaction solutions in which a negative control was added instead of a sample.

Example 12
Primer and probe for detecting L452Q and R mutants The detection of L452Q and L452R mutants by the detection method of the present invention was examined. First, forward and reverse primers of SEQ ID NOs: 125 to 130 were synthesized, respectively, to obtain primer pairs #1 to #3 in Table 37. Next, sets #207 to #236 consisting of these primer pairs and three types of probes containing LNA in the sequence (for detecting wild-type SARS-CoV-2 containing "W" in the name, for detecting L452Q mutation containing "Q" in the name, and for detecting L452R mutation containing "M4, M6 or M7" in the name) as described in Table 38 were constructed. The 5' end of the probe for detecting wild-type SARS-CoV-2 was labeled with FAM, and the 3' end with BHQ (registered trademark) 1, respectively. The 5' end of the probe for detecting L452R mutation was labeled with Cy5, and the 3' end with BHQ (registered trademark) 2, respectively. The 5' end of the probe for detecting the L452Q mutation was labeled with HEX and the 3' end with BHQ (registered trademark)1.

As test specimens, wild-type RNA2 used in Example 10, mutant RNA452 used in Example 6, and synthetic single-stranded RNA having the sequence of mutant (L452Q) SARS-CoV-2 virus genome RNA (L452Q_RNA_CONTROL_RNA_Mut: SEQ ID NO: 134) were used. The RNA of SEQ ID NO: 134 was prepared by constructing a plasmid DNA incorporating a double-stranded DNA corresponding to the base sequence, and performing an in vitro transcription reaction using this as a template. This RNA is referred to as mutant RNAL452Q in this specification. RNase Free H ₂ O was also prepared as a negative control.

Wild-type RNA2, mutant RNA452, and mutant RNAL452Q were each added to RNase Free H ₂ O to a final concentration of 5000 copies/μl to prepare a test sample solution. For detection of the test sample, components of a commercially available product name SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A) were used. That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, etc., 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), a forward primer (final concentration 0.2 μM), a reverse primer (final concentration 0.2 μM), and a probe (final concentration 0.2 μM; for detecting wild-type RNA or mutant-type RNA) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

A reaction solution containing each set of primers and probes shown in Table 38 was prepared with the above composition. Tests were performed in duplicate to improve experimental accuracy.

The thermal cycler used was a QuantStudio (registered trademark) 5 Real-time PCR System (Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Table 39.

Results As a result of the study, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #207, #210, #213, #217, #220, #223, #227, #230, and #233 were able to detect wild-type RNA2, mutant RNA452, and mutant RNAL452Q with small Ct values and high fluorescence intensity. In other words, it was found that the oligonucleotides of SEQ ID NOs: 131, 71, and 72 can distinguish single base substitutions in mutations with high sensitivity. In addition, no amplification products were confirmed in all reaction solutions in which a negative control was added instead of a sample.

Example 13
Primer probes for detecting L452Q and R mutants The detection of L452Q and L452R mutants by the detection method of the present invention was examined. First, a set of #237 to #252 consisting of the primer pair #1 described in Table 38 prepared in Example 12 and three types of probes containing LNA in the sequence described in Table 40 (for detecting wild-type SARS-CoV-2 containing "W" in the name, for detecting L452Q mutation containing "Q" in the name, and for detecting L452R mutation) was constructed. The 5' end of the probe for detecting wild-type SARS-CoV-2 was labeled with FAM and the 3' end with BHQ (registered trademark) 1. The 5' end of the probe for detecting L452R mutation was labeled with Cy5 and the 3' end with BHQ (registered trademark) 2, respectively. The 5' end of the probe for detecting the L452Q mutation was labeled with HEX and the 3' end with BHQ (registered trademark)1.

As test samples, wild-type RNA2, mutant RNA452, and mutant RNAL452Q used in Example 12 were used. As a negative control, RNase Free H ₂ O was prepared.

Wild-type RNA2, mutant RNA452, and mutant RNAL452Q were added to RNase Free H ₂ O at concentrations of 50 copies/μl, 500 copies/μl, and 5000 copies/μl to prepare test sample solutions. Components of a commercially available product named SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A) were used to detect the test samples. That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), forward primer (final concentration 0.8 μM) and reverse primer (final concentration 0.8 μM), and three types of probes (final concentrations of 0.2 μM (1×) or 0.4 μM (2×) respectively) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

A reaction solution containing each set of primers and probes listed in Table 40 was prepared with the above composition. Tests were performed in duplicate to improve experimental accuracy.

Thermal cycler used was a QuantStudio (registered trademark) 5 Real-time PCR System (Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 58°C for 30 seconds. The results are shown in Table 41.

Results: As a result of the study, the multiplex RT-PCR system that simultaneously detects wild-type RNA2, mutant RNA452, and mutant RNAL452Q was able to detect wild-type and mutant (L452R or L452Q) RNAs, respectively, and it was shown that typing of the L452 position, that is, discrimination between wild-type (L), R, or Q, is possible using the reaction solution. #237 and #238 showed particularly good results. Furthermore, no amplification products were confirmed in any of the reaction solutions in which a negative control was added instead of the sample.

Example 14
Primer and probe for detecting P681H and R mutants The detection of P681H and P681R mutants by the detection method of the present invention was examined. First, forward and reverse primers of SEQ ID NOs: 139 to 146 were synthesized, and two pairs of primer pairs were selected from them and listed in Table 42 as #1 to #2. Next, sets of #147 to #161 consisting of these primer pairs and a total of 15 probes containing LNA in the sequence (for detecting wild-type SARS-CoV-2 with "W" in the name, for detecting P681H mutation with "H" in the name, and for detecting P681R mutation with "R" in the name) listed in Table 43 were constructed. The 5' end of the probe for detecting wild-type SARS-CoV-2 was labeled with FAM and the 3' end with BHQ (registered trademark) 1. The 5' end of the probe for detecting the P681H mutation was labeled with Cy5 and the 3' end with BHQ (registered trademark) 2. The 5' end of the probe for detecting the P681R mutation was labeled with HEX and the 3' end with BHQ (registered trademark) 1.

As test specimens, wild-type single-stranded RNA (SEQ ID NO: 164) having the spike protein region sequence of the SARS-CoV-2 virus genome and various single-stranded RNAs having mutations corresponding to P681H and P681R were prepared. The wild-type single-stranded RNA was prepared by artificially synthesizing double-stranded DNA corresponding to the base sequence and inserting it into the multicloning site (Nhe I-Xba I) of the plasmid pVAX1 by a known method, and then performing an in vitro transcription reaction using this recombinant plasmid as a template. In addition, RNAs having the P681H mutation (CCT, the codon corresponding to the 681st proline, is converted to CAT) and the P681R mutation (CCT, the codon corresponding to the 681st proline, is converted to CGT) were prepared by introducing each mutation into the DNA of SEQ ID NO: 164 and then by the same method as the wild-type RNA. These RNAs are referred to herein as wild-type RNAP681, mutant RNAP681H, and mutant RNAP681R. As a negative control, RNase Free H ₂ O was prepared.

A test sample solution was prepared by adding wild-type RNAP681, mutant RNAP681H, and mutant RNAP681R to RNase Free H ₂ O to a final concentration of 5000 copies/μl. The test sample was detected using components of a commercially available product called SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A). That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, etc., 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), a forward primer (final concentration 0.2 μM), a reverse primer (final concentration 0.2 μM), and a probe (final concentration 0.2 μM; for detecting wild-type RNA or mutant-type RNA) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

Reaction solutions containing the primer-probe sets shown in Table 43 in the above composition were prepared and tests were carried out.

The thermal cycler used was a QuantStudio (registered trademark) 5 Real-time PCR System (Thermo Fisher Scientific). The RT-PCR conditions were 52°C for 5 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The results are shown in Table 44.

Results As a result of the study, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #248, #251, #252, #256, #257, #262, #263, #266, #267, #271, and #272 were able to detect wild-type RNAP681, mutant RNAP681H, and mutant RNAP681R with small Ct values and high fluorescence intensity. In other words, it was found that the oligonucleotides of SEQ ID NOs: 149, 150, 153, 154, 158, and 159 can distinguish single base substitutions in mutations with high sensitivity. In addition, no amplification products were confirmed in all reaction solutions in which a negative control was added instead of a sample.

Example 15
Primers and probes for detecting P681H and P681R mutant strains Detection of the P681H mutant strain and the P681R mutant strain by the detection method of the present invention was examined. First, a primer pair in Table 45 was prepared using the forward and reverse primers of SEQ ID NOs: 139 to 146 synthesized in Example 14.

Next, sets #275 to #322 were constructed, each consisting of probes selected from these primer pairs and the fluorescently labeled probes prepared in Example 14 (for detecting wild-type SARS-CoV-2 with a name containing "W", for detecting the P681H mutation with a name containing "H", and for detecting the P681R mutation with a name containing "R").

A test sample solution was prepared by adding wild-type RNAP681, mutant RNAP681H, and mutant RNAP681R to RNase Free H ₂ O to a final concentration of 5000 copies/μl. The test sample was detected using components of a commercially available product called SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A). That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, etc., 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), a forward primer (final concentration 0.2 μM), a reverse primer (final concentration 0.2 μM), and a probe (final concentration 0.2 μM; for detecting wild-type RNA or mutant-type RNA) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

The test samples used were the wild-type RNAP681, mutant RNAP681H, and mutant RNAP681R used in Example 14. RNase Free H ₂ O was prepared as a negative control.

Reaction solutions containing the primer-probe sets shown in Tables 46, 47, and 48 were prepared with the above composition. Tests were performed in duplicate to improve experimental accuracy.

The reaction was carried out using the same thermal cycler and under the same RT-PCR conditions as in Example 14. The results are shown in Tables 49, 50, 51, and 52.

Results As a result of the study, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #275, #276, #287, #288, #323, #324, #335, and #336 were able to detect wild-type RNAP681, mutant RNAP681H, and mutant RNAP681R with small Ct values and high fluorescence intensity. As in Example 14, it was found that the oligonucleotides of SEQ ID NOs: 153, 154, 158, and 159 can distinguish single base substitutions in mutations with high sensitivity. In addition, no amplification products were confirmed in all reaction solutions in which a negative control was added instead of a sample.

Example 16
Typing of Position P681 Typing of position P681 by the detection method of the present invention was examined. First, sets #275 to #370 were constructed, each consisting of primer pair #9 prepared in Example 15 and shown in Table 45, and probes (P681-Wild-LNA-7, P681H-Mut-LNA-6 or P681H-Mut-LNA-7, P681R-Mut-LNA-6) prepared in Example 14.

The test samples used were the wild-type RNAP681, mutant RNAP681H, and mutant RNAP681R used in Example 14. RNase Free H ₂ O was prepared as a negative control.

Wild-type RNA2, mutant RNA452, and mutant RNAL452Q were added to RNase Free H ₂ O at concentrations of 50 copies/μl, 500 copies/μl, and 5000 copies/μl to prepare test sample solutions. Components of a commercially available product named SARS-CoV-2 Direct Detection RT-qPCR Kit (manufactured by Takara Bio, product #RC300A) were used to detect the test samples. That is, 1 μl of the above-mentioned test sample solution, 15 μl of RT-qPCR Mix containing enzymes and substrates, 1.2 μl of heat-treated Solution A, 0.6 μl of ROX Reference Dye II (50X), forward primer (final concentration 0.8 μM) and reverse primer (final concentration 0.8 μM), and three types of probes (final concentrations of 0.2 μM (1×) or 0.4 μM (2×) respectively) were mixed, and a one-step RT-PCR reaction solution with a final volume of 30 μl was prepared using RNase Free H ₂ O.

A reaction solution containing each set of primers and probes shown in Table 53 was prepared with the above composition. Tests were performed in duplicate to improve experimental accuracy.

The reaction was carried out using the same thermal cycler and under the same RT-PCR conditions as in Example 14. The results are shown in Table 54.

Results As a result of the investigation, it was confirmed that RNA corresponding to the probe was detected in all combinations. In particular, the reactions of sets #371 and #372 were able to detect wild-type RNAP681, mutant RNAP681H, and mutant RNAP681R with small Ct values and high fluorescence intensity. In other words, it was found that the oligonucleotides of SEQ ID NOs: 150, 154, and 158 can distinguish a single base substitution at position P681 with high sensitivity. In addition, no amplification products were confirmed in any of the reaction solutions in which a negative control was added instead of a sample.

The technology of the present invention can specifically amplify mutant genes and detect them with high sensitivity, making it possible to detect mutant SARS-CoV-2. This method is useful in a wide range of fields, including genetic engineering, biology, and medicine.

SEQ ID NO1:N501Y_1Fv2.Position 15 "R" is A or G.
SEQ ID NO2: N501Y_1R.
SEQ ID NO3: N501-1c-MGB(WT).
SEQ ID NO4: 501Y-1c-MGB(FAM).
SEQ ID NO5: N501Y-MGB-F1.
SEQ ID NO6: N501Y-MGB-R1-E484K-3R.
SEQ ID NO7: N501Y-MGB-P-wild1.
SEQ ID NO8: N501Y-MGB-P-mut1.
SEQ ID NO9: N501Y-MGB-F2-E484K-4F.
SEQ ID NO10: N501Y-MGB-R2-2-E484K-4R.
SEQ ID NO11: N501Y-MGB-P-wild2.
SEQ ID NO12: N501Y-MGB-P-mut2.
SEQ ID NO13: N501Y-MGB-P-wild3.
SEQ ID NO14: N501Y-MGB-P-mut3(FAM).
SEQ ID NO15: N501Y-MGB-P-wild4.
SEQ ID NO16: N501Y-MGB-P-mut4(FAM).
SEQ ID NO17: JP-MGB-F1-E484K-5F.
SEQ ID NO18: JP-MGB-R1-E484K-5R.
SEQ ID NO19: JP-MGB-F2.
SEQ ID NO20: JP-MGB-R2.
SEQ ID NO21: E484K-1F-E484K-2F.
SEQ ID NO22: E484K-1R.
SEQ ID NO23: E484_FAM-MGB1.
SEQ ID NO24: 484K_VIC-MGB1.
SEQ ID NO25: E484K-2R.
SEQ ID NO26: E484_FAM-MGB2.
SEQ ID NO27: 484K_VIC-MGB2.
SEQ ID NO28: E484K-3F.
SEQ ID NO29: E484_FAM-MGB3.
SEQ ID NO30: 484K_VIC-MGB3.
SEQ ID NO31: E484_FAM-MGB4.
SEQ ID NO32: 484K_VIC-MGB4.
SEQ ID NO33: E484_FAM-MGB5.
SEQ ID NO34: 484K_VIC-MGB5.
SEQ ID NO35: N501Y-MGB-P_12_BASE.
SEQ ID NO36: E484K-MGB-P_13_BASE.
SEQ ID NO37: TBD-LNA-M2.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO38: E484K-LNA-W1(TBD).Locked Nucleic Acid (LNA) Probe.
SEQ ID NO39: E484K-LNA-W2(TBD).Locked Nucleic Acid (LNA) Probe.
SEQ ID NO40: E484K-LNA-W3(TBD).Locked Nucleic Acid (LNA) Probe.
SEQ ID NO41: E484K-LNA-W4(TBD).Locked Nucleic Acid (LNA) Probe.
SEQ ID NO42: E484K-LNA-W5(TBD).Locked Nucleic Acid (LNA) Probe.
SEQ ID NO43: E484K-LNA-W6(TBD).Locked Nucleic Acid (LNA) Probe.
SEQ ID NO44: E484K_MGB_3.
SEQ ID NO45: E484K(mut)_LNA2.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO46: TBD-LNA-mut-3.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO47: 2019-nCoV_N1-F.
SEQ ID NO48: 2019-nCoV_N1-R.
SEQ ID NO49:2019-nCoV_N1-P_HEX.
SEQ ID NO50: 2019-nCoV_N2-F.
SEQ ID NO51: 2019-nCoV_N2-R.
SEQ ID NO52:2019-nCoV_N2-P_HEX.
SEQ ID NO53: E484Q-LNA-1_P_HEX.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO54: E484Q-LNA-2_P_HEX.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO55: E484Q-LNA-4_P_HEX.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO56: E484Q-LNA-5_P_HEX.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO57: E484Q-LNA-6_P_HEX.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO58: E484E-CONTROL_RNA_Wild.
SEQ ID NO59: E484K-CONTROL_RNA_Mut.
SEQ ID NO60: E484Q-CONTROL_RNA_Mut.
SEQ ID NO61: E484Q-P_13_BASE.
SEQ ID NO62: N501Y-MGB-F(S477N).
SEQ ID NO63: N501Y-MGB-F(T478K).
SEQ ID NO64: L452R-S477N-F1.
SEQ ID NO65: L452R-S477N-R1.
SEQ ID NO66: L452R-S477N-F2.
SEQ ID NO67: L452R-S477N-R2.
SEQ ID NO68: L452R-LNA-M1.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO69:L452R-LNA-M2.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO70:L452R-LNA-M3.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO71:L452R-LNA-M4.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO72:L452R-LNA-W1.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO73:L452R-LNA-W2.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO74:L452R-LNA-W3.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO75: L452R-F3.
SEQ ID NO76: L452R-R3.
SEQ ID NO77: L452R-F4.
SEQ ID NO78: L452R-R4.
SEQ ID NO79: L452R-F5.
SEQ ID NO80: L452R-R5.
SEQ ID NO81: L452R-LNA-M5.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO82:L452R-LNA-M6.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO83:L452R-LNA-M7.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO84:L452R-LNA-M8.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO85:L452R-LNA-M9.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO86:L452R-LNA-W4.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO87:L452R-LNA-W5.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO88: L452R-LNA_BASE9.
SEQ ID NO89: L452R-CONTROL_RNA_Wild.
SEQ ID NO90: L452R-CONTROL_RNA_Mut.
SEQ ID NO91: T478K-F1.
SEQ ID NO92: T478K-R1.
SEQ ID NO93: T478K-F1.
SEQ ID NO94: T478K-R2.
SEQ ID NO95:T478K-LNA-M1.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO96:T478K-LNA-M2.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO97: T478K-LNA-M3.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO98: T478K-LNA-M4.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO99: T478K-LNA-M5.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO100：T478K-LNA-M6.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO101：T478K-LNA-W1.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO102: T478K-LNA-W2.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO103: T478K-LNA-W3.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO104：T478K-LNA-W4.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO105:T478K-LNA-W5.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO106:T478K-LNA-W6.Locked Nucleic Acid (LNA) Probe.
SEQ ID NO107: T478K F3.
SEQ ID NO108: T478K R3.
SEQ ID NO109: T478K F4.
SEQ ID NO110: T478K R4.
SEQ ID NO111: T478K-CONTROL_RNA_Wild.
SEQ ID NO112: T478K-CONTROL_RNA_Mut.
SEQ ID NO113:T478K-LNA_BASE6.
SEQ ID NO114: F490S-F1.
SEQ ID NO115: F490S-F2.
SEQ ID NO116: F490S-F3.
SEQ ID NO117: F490S_LNA_R4.
SEQ ID NO118: F490S_LNA_R5.
SEQ ID NO119: F490S_LNA_R6.
SEQ ID NO120:F490S_LNA_Wild_2P.
SEQ ID NO121: F490S_LNA_Wild_4P.
SEQ ID NO122:F490S_LNA_Mut_1P.
SEQ ID NO123:F490S_LNA_Mut_2P.
SEQ ID NO124:F490S_LNA_Mut_7P.
SEQ ID NO125: L452Q-F1.
SEQ ID NO126: L452Q-R1.
SEQ ID NO127: L452Q-F2.
SEQ ID NO128: L452Q-R2.
SEQ ID NO129: L452Q-F3.
SEQ ID NO130: L452Q-R3.
SEQ ID NO131: L452Q-LNA-Mut-1.
SEQ ID NO132: L452Q-LNA-Mut-2.
SEQ ID NO133: L452Q-LNA-Mut-3.
SEQ ID NO134: WILD_RNA_CONTROL2.
SEQ ID NO135: L452Q_RNA_CONTROL_RNA_Mut.
SEQ ID NO136: F490S_RNA_CONTROL_RNA_Mut.
SEQ ID NO137:F490S-LNA_BASE7.
SEQ ID NO138:L452QR-LNA_BASE9.
SEQ ID NO139: P681_F2.
SEQ ID NO140: P681_F3.
SEQ ID NO141: P681_F4.
SEQ ID NO142: P681_F5.
SEQ ID NO143: P681_R1.
SEQ ID NO144: P681_R2.
SEQ ID NO145: P681_R3.
SEQ ID NO146: P681_R4.
SEQ ID NO147:P681-Wild-LNA-1.
SEQ ID NO148:P681-Wild-LNA-5.
SEQ ID NO149:P681-Wild-LNA-6.
SEQ ID NO150:P681-Wild-LNA-7.
SEQ ID NO151:P681-Wild-LNA-10.
SEQ ID NO152:P681H-Mut-LNA-1.
SEQ ID NO153:P681H-Mut-LNA-6.
SEQ ID NO154:P681H-Mut-LNA-7.
SEQ ID NO155:P681H-Mut-LNA-8.
SEQ ID NO156:P681H-Mut-LNA-9.
SEQ ID NO157:P681R-Mut-LNA-2.
SEQ ID NO158: P681R-Mut-LNA-6.
SEQ ID NO159: P681R-Mut-LNA-7.
SEQ ID NO160:P681R-Mut-LNA-8.
SEQ ID NO161:P681R-Mut-LNA-9.
SEQ ID NO162:P681H-LNA_BASE9.
SEQ ID NO163:P681R-LNA_BASE10.
SEQ ID NO164: P681_WILD_RNA_3860.

Claims (19)

An oligonucleotide for use in detecting mutant SARS-CoV-2, comprising:
(a) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 35 or a sequence complementary to said sequence;
(b) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 36 or a sequence complementary to said sequence;
(c) an oligonucleotide comprising the base sequence shown in SEQ ID NO:61 or a sequence complementary to said sequence;
(d) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 88 or a sequence complementary to said sequence;
(e) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 113 or a sequence complementary to said sequence;
(f) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 137 or a sequence complementary to said sequence;
(g) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 138 or a sequence complementary to said sequence;
(h) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 162 or a sequence complementary to said sequence, and (i) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 163 or a sequence complementary to said sequence;
An oligonucleotide selected from: The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 4, 8, 12, 14, and 16, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 24, 27, 30, 32, 34, 37, 44, 45, and 46, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 53, 54, 55, 56, and 57, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 68, 69, 70, 71, 81, 82, 83, 84, and 85, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 95, 96, 97, 98, 99, and 100, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 122, 123, and 124, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 131, 132, and 133, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 152, 153, 154, 155, and 156, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which comprises a base sequence selected from SEQ ID NOs: 157, 158, 159, 160, and 161, or a sequence complementary to said sequence. The oligonucleotide according to claim 1, which is labeled with a fluorescent substance and a quencher. The oligonucleotide of claim 1, to which a minor groove binder (MGB) is attached. The oligonucleotide of claim 1, which contains a bridged nucleic acid (BNA). 1. A method for detecting a mutant SARS-CoV-2 in a sample, comprising:
(1) synthesizing a DNA or a fragment thereof complementary to the SARS-CoV-2 genome contained in a sample; and (2) detecting a base sequence or a part thereof encoding a mutant spike protein contained in the DNA or a fragment thereof obtained in the step (1) by using the oligonucleotide according to any one of claims 1 to 13.
The method according to claim 1, further comprising: The method according to claim 14, wherein step (1) further comprises a step of amplifying the synthesized DNA fragment. The method according to claim 14, wherein in step (2), the detection of the base sequence encoding the mutant spike protein or a part thereof is carried out by decomposition of an oligonucleotide hybridized to the DNA or a fragment thereof. A kit for detecting a mutant SARS-CoV-2 in a sample, comprising:
A kit comprising: (1) an oligonucleotide according to any one of claims 1 to 13; and (2) a reagent for synthesizing a DNA complementary to the SARS-CoV-2 genome or a fragment thereof. The kit according to claim 17, further comprising a reagent for amplifying a DNA fragment complementary to the SARS-CoV-2 viral genome. The kit of claim 17, comprising a primer pair used to amplify a DNA fragment complementary to the SARS-CoV-2 viral genome.

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