WO2022202728A1 - プライマー対、塩基配列変異の判定方法及び塩基配列の変異判定用キット - Google Patents

プライマー対、塩基配列変異の判定方法及び塩基配列の変異判定用キット Download PDF

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WO2022202728A1
WO2022202728A1 PCT/JP2022/012939 JP2022012939W WO2022202728A1 WO 2022202728 A1 WO2022202728 A1 WO 2022202728A1 JP 2022012939 W JP2022012939 W JP 2022012939W WO 2022202728 A1 WO2022202728 A1 WO 2022202728A1
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strand
mutation
primer
base
type
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French (fr)
Japanese (ja)
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幸信 林田
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富士フイルム株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the present invention relates to a primer pair, a method for determining nucleotide sequence mutation, and a kit for determining nucleotide sequence mutation.
  • Gene mutation may be involved in one of the causes of individual differences in disease susceptibility, drug efficacy, side effect susceptibility, and the like. Therefore, research is underway to search for a treatment method suitable for the constitution, focusing on the difference in this gene arrangement. Gene mutations can also serve as genetic markers for many diseases. That is, elucidation of gene mutation is clinically important, and therefore establishment of an analytical method capable of detecting gene mutation is desired.
  • nucleic acid sequencing includes, for example, nucleic acid sequencing, RFLP (restriction enzyme cleavage polymorphism), ASP (allele-specific primer), ASO (allele-specific oligoprobe), single base A decompression method and the like are known.
  • RFLP restriction enzyme cleavage polymorphism
  • ASP allele-specific primer
  • ASO allele-specific oligoprobe
  • PCR polymerase chain reaction
  • Patent Document 1 Also known is a method of detecting gene mutation by performing PCR using a forward primer in which some or all of the phosphodiester bonds of nucleotides are phosphorothioated.
  • Patent Document 1 ⁇ a primer containing one or more chemically modified nucleotides, bases, or phosphodiester bonds so that the nucleotide chain extending from the primer is substantially resistant to exonuclease activity,'' Using "a primer comprising a nucleotide sequence having a sense sequence immobilized on a solid support and a sequence complementary thereto", a step of amplifying a target nucleotide sequence to generate an amplification product, based on the result Methods are disclosed for detecting and determining whether a target nucleotide is homozygous or heterozygous.
  • the present invention has been made in view of the above circumstances, and provides a primer pair used for accurate determination of genetic mutation, an accurate method for determining genetic mutation, and a nucleotide sequence mutation used for accurate determination of genetic mutation.
  • the object is to provide a determination kit.
  • both the forward primer and the reverse primer have phosphorothioated phosphodiester bonds on the 3' side of 1 to 4 consecutive nucleotides upstream from the second base from the 3' end.
  • the inventors found that the correct base sequence can be detected and determined, and have completed the present invention.
  • the present invention typically includes the following inventions.
  • a forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is phosphorothioated (S)
  • S phosphorothioated
  • a primer pair with a reverse primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base toward the 5' side is S-converted.
  • Primer pair M A primer that anneals to one strand (second strand) of a double-stranded nucleic acid having a mutation, anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side.
  • a forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is S-formed
  • a primer that anneals to the other strand (first strand) of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and anneals the complementary sequence of the mutation sequence of the first strand to the 3' end side A primer pair of a reverse primer having a phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed
  • Primer pair W A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to a region containing the base sequence in the wild-type second
  • primer pair M The primer pair according to [2] above, wherein the mutation in the base sequence of the primer pair M is a base substitution or insertion, and the primer pair M is as follows: A primer that anneals to the second strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the second strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the second strand, and has 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side.
  • a primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the first strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the first strand, and is continuous from the second base from the 3' end toward the 5' side.
  • primer pair M is as follows: The primer pair according to claim 2, wherein the mutation in the nucleotide sequence of the primer pair M is a deletion of a base, and the primer pair M is: A primer that anneals to the second strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and attaches a sequence complementary to the 5'-side nucleotide sequence adjacent to the deletion of the second strand to the 3' end.
  • the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the second strand, and extends from the second base from the 3' end to the 5' side a forward primer in which the phosphodiester bond on the 3′ side of consecutive 1 to 4 nucleotides is S-formed;
  • primer pair W is: A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to the region containing the base sequence in the wild-type second strand, and placing a complementary sequence of the base sequence of the wild-type second strand corresponding to the mutation of the mutant second strand on the 3' end side and the other nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the nucleotide sequence of the wild-type second strand corresponding to the mutation of the mutant second strand, a forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 'end toward the 5' side is S-formed; A primer that anneals to the other strand (wild-type second strand) and correspond
  • a pair of reverse primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-converted.
  • Any of the above [1] to [5], wherein the phosphodiester bond on the 3' side of three consecutive nucleotides from the 2nd base from the 3' end of the primer toward the 5' side is S-ified.
  • nucleic acid of the test sample as a template, a nucleic acid amplification reaction is performed using the primer pair according to any one of [1] to [7] above to detect the reaction product, and the resulting detection results are also reported. and a method for determining a mutation in a base sequence, which determines a mutation in the base sequence of the nucleic acid.
  • a nucleic acid amplification reaction is performed using the primer pair according to any one of the above [2] to [7], and the reaction product is detected. Based on the detection results obtained, the mutation in the base sequence of the nucleic acid is determined.
  • the determination method includes the following steps: (1) Using the nucleic acid of the test sample as a template, performing a nucleic acid amplification reaction using the primer pair M according to any one of [2] to [4] and [6] to [7] above, detecting the product; (2) Using the nucleic acid of the same test sample as that used in the step (1) above as a template, and using the primer pair W according to any one of [2], [5] to [7] above. a step of performing a nucleic acid amplification reaction and detecting a reaction product; (3) A step of judging a mutation in the base sequence of the nucleic acid of the test sample based on the detection results obtained in the above steps (1) and (2).
  • a nucleotide sequence mutation determination kit comprising the primer pair according to any one of [1] to [7] above.
  • nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made. According to the method for determining nucleotide sequence mutations of the present invention, nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made. According to the method for determining nucleotide sequence mutations using the kit of the present invention, nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of non-S-conjugated primers obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers obtained in Example 1, in which one nucleotide is S-converted.
  • 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which two bases are S-modified obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which 3 bases are S-modified obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which 4 bases are S-modified obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which 4 bases are S-modified obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers having 5 nucleotides in S, obtained in Example 1.
  • FIG. Amplification obtained by performing real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, targeting the PiS mutation in the genomic DNA of COLO201, A549, HepG2, and MCF7 obtained in Example 2.
  • curve. Amplification obtained by performing real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, targeting the PiZ mutation in the genomic DNA of COLO201, A549, HepG2, and MCF7 obtained in Example 3. curve.
  • FIG. 4 is a model of amplification curves of wild-type, homozygous, or heterozygous PiS mutations obtained in Example 4.
  • FIG. 6 is a model of amplification curves for wild-type, homozygous, or heterozygous PiZ mutations obtained in Example 5.
  • FIG. Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, with the PiS mutation in the genomic DNA derived from the oral swab obtained in Example 6 as the detection target.
  • Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, with the PiS mutation in saliva-derived genomic DNA obtained in Example 6 as the detection target.
  • Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, with the PiZ mutation in the genomic DNA derived from the oral swab obtained in Example 7 as the detection target.
  • Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, with the PiZ mutation in saliva-derived genomic DNA obtained in Example 7 as a detection target.
  • FIG. 2 shows an amplification curve obtained by real-time PCR using the PKD1 detection primer pair or the PKLR detection primer pair of the present invention, targeting gene mutations.
  • the PKD1 gene obtained in Example 8 was confirmed to be wild-type and the PKLR gene was of a mutant type.
  • Fig. 2 shows amplification curves obtained by real-time PCR using the PKD1 detection primer pair or the PKLR detection primer pair of the present invention, targeting mutations in the PKD1 gene and the PKLR gene in genomic DNA.
  • Fig. 10 is an amplification curve obtained by real-time PCR using the primer pair for VPS13B detection of the present invention, with the VPS13B mutation in canine genomic DNA obtained in Example 9 as the detection target.
  • 10 shows an amplification curve obtained by real-time PCR using the SN501Y mutation detection primer pair of the present invention, with the SN501Y mutation of the SARS-CoV-2 virus-derived genomic RNA obtained in Example 10 as the detection target.
  • 10 shows an amplification curve obtained by real-time PCR using the E484K mutation detection primer pair of the present invention, with the SARS-CoV-2 virus-derived genomic RNA and the E484K mutation as detection targets, obtained in Example 10.
  • a nucleotide sequence in which no nucleotide sequence mutation occurs is referred to as a "wild-type nucleotide sequence" with respect to the target gene whose nucleotide sequence mutation is to be detected.
  • the term “mutation” refers to a base sequence different from that of the wild type of the target gene whose base sequence mutation is to be detected, or having the above-mentioned base sequence. Examples of “mutation” include base substitution, deletion, insertion, and the like with respect to the wild-type base sequence of the gene whose base sequence mutation is to be detected.
  • the number of mutated bases is not particularly limited. 1 to 4 base mutations, preferably 1 to 3 base mutations, more preferably 1 mutation are included per mutation.
  • the base sequence of the gene to be detected when the base sequence of the gene to be detected does not have mutations, the base sequence is said to be wild-type.
  • the nucleotide sequence of the gene to be detected has mutations, the nucleotide sequence is said to be of a mutant type.
  • genotypes are determined in the present invention
  • a case where both base sequences of alleles are wild type is referred to as wild type.
  • Heterozygosity is when one of the alleles contains the mutation.
  • homozygous when both nucleotide sequences of alleles contain the same mutation, it is called homozygous. "Mutation" in the present invention includes this heterozygous and homozygous genotype.
  • PKD1 c. 9864C>A
  • PKLR c. 693 + 304G>A
  • VPS13B g.4411950_4411953delGTTT
  • MFSD8 HEXB
  • GLB1 etc.
  • viruses for example, SARS-CoV-2, N501Y (A23063T), E484K ( G23012A) and the like.
  • target genes for detecting and determining nucleotide sequence mutations include, for example, BRAF, Pis, PiZ, etc. are mentioned.
  • complementary base refers to a base that is complementary to a certain base.
  • complementary strand refers to a nucleic acid strand having a base sequence complementary to a given nucleic acid strand.
  • complementary sequence refers to a base sequence that is complementary to a given base sequence.
  • nucleotide sequence includes both a single base sequence and a multiple base sequence.
  • FIG. S1 shows a mutation in which one strand of a wild-type double-stranded nucleic acid and one strand of a mutant double-stranded nucleic acid are substituted for a base sequence X with a base sequence Y for a gene.
  • Figure 10 illustrates a chain with
  • mutant sequence represents the base sequence of Y of the nucleic acid with mutation.
  • nucleotide sequence corresponding to the mutation refers to the X nucleotide sequence of the wild-type nucleic acid.
  • the term “mutation-containing region” refers to the region (*) of the nucleotide sequence containing Y in the nucleic acid having the mutation.
  • a “region containing a (wild-type) base sequence corresponding to a mutation” refers to a region (+) of a base sequence containing X in a wild-type nucleic acid.
  • FIG. S2 shows one strand of a wild-type double-stranded nucleic acid and one strand of a mutant-type double-stranded nucleic acid for a gene, wherein the base sequence Y is between the base sequence X1 and the base sequence X2.
  • mutant sequence represents the base sequence of Y of the nucleic acid with mutation.
  • nucleotide sequence corresponding to the mutation represents the nucleotide sequence of X1 and/or X2 of the wild-type nucleic acid.
  • the term “mutation-containing region” refers to the region (*) of the nucleotide sequence containing Y in the nucleic acid having the mutation.
  • a region containing a (wild-type) base sequence corresponding to a mutation refers to a base sequence region (+) containing X1 and X2 of a wild-type nucleic acid.
  • FIG. S3 shows one strand of a wild-type double-stranded nucleic acid and one strand of a mutant-type double-stranded nucleic acid for a gene, wherein the nucleotide sequence Y between the nucleotide sequence X1 and the nucleotide sequence X2 is It is a diagram illustrating the case of having a missing mutation.
  • the “mutant sequence” represents the consecutive base sequences of X1 and X2.
  • Wild-type nucleotide sequence corresponding to mutation refers to the Y nucleotide sequence of the wild-type nucleic acid.
  • mutant-containing region refers to a region (*) of a base sequence containing a mutant sequence in a nucleic acid chain having a mutation.
  • a “region containing a (wild-type) base sequence corresponding to a mutation” refers to a region (+) of a base sequence containing Y in a wild-type nucleic acid.
  • Nucleic acids include DNA and RNA, preferably DNA.
  • the abbreviations commonly used in the field of the present invention (adenine “A” or “a”, guanine “G” or “g”, cytosine “C” or “c”) ”, and thymine as “T” or “t”).
  • Primer pair of the present invention is "A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is phosphorothioated (S-formed), and the 3' end A pair of reverse primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base to the 5' side is S-converted".
  • Forward primer is hereinafter referred to as "F primer”.
  • reverse primer is described as “R primer”.
  • the F primer and R primer according to the primer pair of the present invention are composed of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side of the phosphodiester bond moiety on the 3' side.
  • One of the two oxygen atoms of the residue is substituted with a sulfur atom, so-called phosphorothioated (also described herein as “S”).
  • the number of S-modified nucleotides in the primer according to the present invention is preferably 1 to 4, more preferably 2 to 4, and particularly preferably 3.
  • the number of S-nucleotides in the F primer and the number of S-nucleotides in the R primer may be the same or different, but are preferably the same.
  • the length of the primer according to the present invention is preferably 10 bases or more, which is considered to be the number of bases required to maintain specificity as a primer sequence, and more preferably 20 bases or more. Examples include 20 to 60 bases.
  • a well-known method may be used to synthesize an S-primer into which a phosphorothioate bond has been introduced.
  • a phosphorothioate bond is added to the required site by a known method. should be introduced.
  • a phosphorothioate bond is introduced into the oligonucleotide instead of a normal phosphodiester bond by performing an oxidation treatment with an appropriate S-conjugation reagent (phosphorothioate reagent). can be done.
  • Beaucage's reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide), TETD/Acetonitrile (TETD: tetraethylthiuram disulfide), and the like are known as sulfide reagents.
  • S-primers are also available through the custom service of vendors.
  • Preferred primer pairs of the present invention include, for example, primer pair M and primer pair W below.
  • Primer pair M A primer that anneals to one strand (second strand) of a double-stranded nucleic acid having a mutation, anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side.
  • an F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides from the second base from the 3′ end toward the 5′ side is S-converted;
  • a primer that anneals to the other strand (first strand) of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and anneals the complementary sequence of the mutation sequence of the first strand to the 3' end side A primer pair of R primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-converted,
  • Primer pair W A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to a region containing the base sequence in the wild-
  • the "double-stranded nucleic acid" in primer pair M and primer pair W of the present invention includes DNA. Also included are RNA and its complementary strand, and cDNA obtained by reverse transcription using RNA as a template and its complementary strand.
  • one strand (second strand) of a double-stranded nucleic acid having a nucleotide sequence mutation and "one strand (second strand) of a wild-type double-stranded nucleic acid )”, the description of “one strand (second strand)”, and “the other strand of the double-stranded nucleic acid having a mutation (first strand)” and “the other side of the wild-type double-stranded nucleic acid
  • the description of "the other strand (first strand)” in relation to "the strand (first strand)” simply means that it refers to one strand and the other strand of a double-stranded nucleic acid, and the first Strand and secondary strand each have no particular meaning.
  • primer that anneals to one strand (second strand) of a double-stranded nucleic acid means a complementary strand (first This means that the primer has the same base sequence as the base sequence of the (strand).
  • the "primer that anneals to the other strand (first strand) of the double-stranded nucleic acid” means the complementary strand (first strand) of the region of the first strand to which the primer anneals. It means that the primer has the same base sequence as the base sequence of the second strand).
  • the "3' terminal side" refers to a region containing several nucleotides including the 3' terminal nucleotide, preferably a region containing 1 to 4 nucleotides.
  • the F primer associated with the primer pair M of the present invention has a base sequence of several, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 nucleotides including the 3′ terminal nucleotide of the base sequence. It preferably has a sequence complementary to the mutated sequence of the second strand of the mutated double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the mutated base of the second strand.
  • the R primer associated with the primer pair M of the present invention has a base sequence of several, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 nucleotides including the 3' terminal nucleotide.
  • it has the complementary sequence of the mutant sequence of the strand.
  • the base of the 3' terminal nucleotide is the complementary base of the mutated base of the first strand.
  • nucleotide bases including the 3' terminal nucleotide are mutated. It preferably has a sequence complementary to the nucleotide sequence in the second strand of the wild-type double-stranded nucleic acid that corresponds to the mutation in the second strand of the double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the base in the second strand of the wild-type double-stranded nucleic acid corresponding to the mutation.
  • nucleotide bases including the 3' terminal nucleotide are mutated. It preferably has a sequence complementary to the nucleotide sequence in the first strand of the wild-type double-stranded nucleic acid corresponding to the mutation in the first strand of the double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the base in the first strand of the wild-type double-stranded nucleic acid corresponding to the mutation.
  • the above-described primer pair M of the present invention provides an amplification product in a nucleic acid amplification reaction using this primer pair when the nucleotide sequence of the nucleic acid of the test sample has a mutation.
  • a primer pair that does not have a mutation does not yield an amplification product in a nucleic acid amplification reaction using this primer pair. That is, the primer pair M of the present invention is a primer pair for mutation detection.
  • the primer pair W of the present invention is a primer pair that gives an amplification product in a nucleic acid amplification reaction using this primer pair when the base sequence of the nucleic acid in the test sample is of the wild type. That is, the primer pair W of the present invention is a wild-type detection primer pair that is wild-type with respect to the mutation to be detected.
  • primer pair M and primer pair W of the present invention will be described in detail below.
  • primer pair M of the present invention examples include a primer pair (M-1) for detecting nucleotide sequence substitution or insertion mutations, or a primer pair (M-2) for detecting nucleotide deletion mutations.
  • Primer pair (M-1) of the present invention for detecting nucleotide sequence substitution or insertion is as follows.
  • a primer that anneals to the second strand of a double-stranded nucleic acid having a substitution or insertion mutation anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side
  • the other nucleotide sequence is the same as the complementary sequence of the 3'-side nucleotide sequence adjacent to the mutation of the second strand, and is continuous from the second base from the 3'-end toward the 5'-side.
  • a primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the first strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the first strand, and is continuous from the second base from the 3' end toward the 5' side.
  • a primer pair of R primers in which the phosphodiester bond on the 3' side of the nucleotide is S-sylated.
  • Substitution or insertion mutations in the base sequence to be detected are preferably substitutions or insertions of 1 to 4 bases. Substitutions or insertions of 1-3 bases are preferred. Substitution or insertion of a single base is more preferred.
  • a primer that anneals to the second strand of a double-stranded nucleic acid having a substitution or insertion mutation, anneals to the region containing the mutation, and 1 to 4 consecutive nucleotides including the 3' terminal nucleotide of the second strand It is the same as the complementary sequence of the mutant sequence, and the other base sequence is the same as the complementary sequence of the 3' side base sequence adjacent to the mutation in the second strand, from the second base from the 3' end an F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides toward the 5′ side is S-formed;
  • a primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and 1 to 4 consecutive nucleotides including the 3' terminal nucleotide are the mutation of the first strand It is the same as the complementary sequence of the sequence, and the rest of the nu
  • a primer that anneals to the second strand of a double-stranded nucleic acid having a single-base substitution or insertion mutation anneals to the region containing the mutation, and has a 3′ terminal nucleotide that is complementary to the mutated base of the second strand.
  • the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the second strand, from the second base from the 3' end to the 5' side
  • An F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-formed
  • the other nucleotide sequence is the same as the complementary sequence of the 3′ side nucleotide sequence adjacent to the mutation of the first strand, and is continuous from the second base from the 3′ end toward the 5′ side.
  • a primer pair of R primers in which the phosphodiester bonds on the 3′ sides of the four nucleotides are S-sylated.
  • FIG. A illustrates the case where the number of S-nucleotides in each primer is three.
  • FIG. A(1) A representation of the base sequences of the first and second strands of a wild-type double-stranded nucleic acid.
  • Figure A (2) Primer pair for detecting single base substitution
  • Figure A (2) shows that the bases marked ⁇ in the wild-type base sequence are substituted from guanine (g) to adenine (a) in the first strand.
  • An example of a primer pair (M-1) for detecting a single-base substitution mutation in which cytosine (c) is substituted with thymine (t) in the second strand is shown.
  • the F primer has at its 3′ end a, the complementary base of the mutated base (t) of the second strand.
  • the other nucleotide sequences are the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the mutated nucleotide t of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed.
  • the R primer has t at its 3' end, which is the complementary base of the mutated base (a) of the first strand.
  • the other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated nucleotide a of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • Figure A (3) Primer pair for detecting double-base substitution
  • An example of a primer pair (M-1) that detects a 2-base mutation in which the chain is substituted from ca to tg is shown.
  • the F primer has at its 3' end ca, the complementary sequence of the mutated sequence (tg) of the second strand.
  • the other nucleotide sequence is the same as the complementary sequence (tgtgattgg) of the 3'-side nucleotide sequence (ccaatcaca) adjacent to the mutated sequence tg of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgg) from the second base from the 3' end of the F primer toward the 5' side (cgg) is S-formed.
  • the R primer has at its 3' end tg, the complementary sequence of the mutant sequence (ca) of the first strand.
  • the other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to ca, which is the mutant sequence of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the R primer toward the 5' side (tgt) is S-formed. .
  • An example of a primer pair (M-1) that detects a 3-base substitution mutation in which cac is replaced with tga in the strand is shown.
  • the F primer has at its 3' end tca, the complement of the second strand mutant sequence (tga).
  • the other nucleotide sequence is the same as the complementary sequence (tgtgattg) of the 3′-side nucleotide sequence (caatcaca) adjacent to the mutated sequence tga of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctg) from the second base from the 3' end of the F primer toward the 5' side (ctg) is S-formed.
  • the R primer has at its 3' end tga, the complementary sequence of the first strand mutant sequence (tca).
  • the other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated sequence tca of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
  • FIG. B exemplifies the case where the number of S-nucleotides in each primer is three.
  • FIG. B(1) A representation of the base sequences of the first and second strands of a wild-type double-stranded nucleic acid.
  • Figure B (2) Primer pair for detecting single base insertion Figure B (2) shows that t is inserted at the position marked ⁇ in the first strand and marked ⁇ in the second strand against the wild-type base sequence.
  • An example of a primer pair (M-1) for detecting a single-base insertion mutation in which a is inserted at the position of is shown.
  • the F primer has t at its 3' end, which is the complementary base of base (a) inserted in the second strand.
  • the other nucleotide sequences are the same as the complementary sequence (tgtgattggtg) of the 3'-side nucleotide sequence (caccaatcaca) adjacent to the mutated nucleotide a of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) from the second base g from the 3' end of the F primer toward the 5' side is S-formed.
  • the R primer has at its 3' end a, the complementary base of the base (t) inserted in the first strand.
  • the other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated nucleotide t of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • Figure B (3) Primer pair for detecting double base insertion Figure B (3) shows that ta is inserted at the position marked ⁇ in the first strand and marked ⁇ in the second strand against the wild-type base sequence.
  • An example of a primer pair (M-1) for detecting a 2-base insertion mutation in which ta is inserted at the position of is shown.
  • the F primer has at its 3' end ta, the complement of ta inserted in the second strand.
  • the other nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the 3′-side nucleotide sequence (caccaatcaca) adjacent to the mutant sequence ta of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the F primer toward the 5' side (tgt) is S-formed.
  • the R primer has at its 3' end ta, the complementary sequence of ta inserted in the first strand.
  • the other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutant sequence ta of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the R primer toward the 5' side (tgt) is S-formed. .
  • Figure B (4) Primer pair for detecting 3-base insertion
  • a tag is inserted at the position marked ⁇ in the first strand against the wild-type base sequence, and marked ⁇ in the second strand.
  • An example of a primer pair (M-1) for detecting a 3-base insertion mutation in which cta is inserted at the position of is shown.
  • the F primer has tag at its 3' end, which is the complementary sequence of cta inserted in the second strand.
  • the other nucleotide sequences are the same as the complementary sequence (tgtgattggtg) of the nucleotide sequence (caccaatcaca) on the 3' side adjacent to the mutant sequence cta of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (atg) from the second base from the 3' end of the F primer toward the 5' side (atg) is S-formed.
  • the R primer has cta at its 3' end, which is the complementary sequence of the tag inserted in the first strand.
  • the other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to tag, which is the mutant sequence of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tcg) from the second base from the 3' end of the R primer toward the 5' side (tcg) is S-formed. .
  • primer pair M-1 of the present invention for example, PiS mutation, which is a single base substitution mutation of human ⁇ 1-antitrypsin gene (genomic DNA GenBank Accession No. NG_008290.1, mRNA GenBank Accession No. NM_000295.5)
  • the mutation detection primer pair (primer pair M-1 of the present invention, corresponding to the case of FIG. A(2) above) will be described below as an example.
  • PiS mutation which is a gene mutation of human ⁇ 1-antitrypsin, is the following single base substitution.
  • the human PiS mutation is a single-base substitution in which the 863rd adenine (A) in the coding DNA is replaced with thymine (T), and as a result, the 288th Glu in the ⁇ 1-antitrypsin amino acid sequence is replaced with Val. Mutation.
  • the primer pair of the present invention for detecting this single base substitution, the strand in which A is replaced with T in the double-stranded DNA of the ⁇ 1-antitrypsin gene is defined as the first strand.
  • the strand of the above double-stranded DNA in which T in the same portion of the base is replaced with A is referred to as the second strand.
  • the * mark indicates the position where the phosphodiester bond is S-converted.
  • PiS mutation detection primer pair (M-1) for example, the following are designed.
  • the F primer of the PiS mutation detection primer pair has T at the 3′ end, which is the complementary base of the mutated base (A) of the second strand of the double-stranded nucleic acid having the PiS mutation.
  • Other nucleotide sequences are designed to be the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the mutated nucleotide (A) of the second strand.
  • the R primer of the pair of primers for detecting PiS mutation type has, at its 3' end, A, which is the complementary base to the base (T) of the first strand of the double-stranded nucleic acid having the PiS mutation.
  • nucleotide sequences are designed to be the same as the 3' nucleotide sequence adjacent to the mutated nucleotide (T) of the first strand. Furthermore, in the example of the above primer pair, the 2nd to 4th nucleotides from the 3' end are nucleotides whose phosphodiester bonds are S-converted.
  • the F primer has the same 3′ terminal base as the mutated base (T) of the PiS gene, and the other base sequence is 5 from the mutated base (T) of the first strand of the double-stranded nucleic acid having the PiS mutation. It is designed to be the same as the base sequence facing the ' side.
  • the R primer has the same 3′ terminal base as the complementary base (A) of the mutated base (T) of the PiS gene, and the other base sequence is the same as the first strand of the double-stranded nucleic acid having the PiS mutation.
  • the 2nd to 4th nucleotides from the 3'-end are nucleotides whose phosphodiester bonds are S-converted.
  • primer pair M of the present invention include the following mutation detection for detecting PiZ mutation (single nucleotide mutation, c.1096G>A p.Glu366Lys), which is another genetic mutation of human ⁇ 1-antitrypsin. Primer pairs are included.
  • primer pair M-1 of the present invention include those listed in Table 1 below.
  • Primer pair (M-2) of the present invention for detecting nucleotide sequence defects is as follows. A primer that anneals to the second strand of a double-stranded nucleic acid having a deletion mutation, anneals to the region containing the deletion, and anneals to the 5'-side nucleotide sequence adjacent to the deletion of the second strand.
  • the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the second strand, from the second base from the 3' end toward the 5' side
  • An F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-formed A primer that anneals to the first strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and attaches a sequence complementary to the 5' base sequence adjacent to the deletion of the first strand to the 3' end.
  • the other base sequence is the same as the complementary sequence of the salt sequence on the 3' side adjacent to the deletion of the first strand, from the second base from the 3' end toward the 5' side
  • a pair of R primers in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-converted.
  • “Annealing to the deletion-containing region" of the primer pair (M-2) of the present invention means binding to at least bases on both ends (5' side and 3' side) of the deletion position.
  • Deletion mutations in the base sequence to be detected are preferably deletions of 1 to 4 bases. A deletion of 1 to 3 bases is more preferred. A single base deletion is more preferred.
  • FIG. C shows three examples of primer pairs (M-2) that detect mutations in which one base is deleted at the position of the arrow in the wild-type base sequence.
  • the first strand lacks the arrow g and the second strand lacks the arrow c.
  • blanks in the base sequences indicate that bases are missing, and in fact, the 5' and 3' bases of the blanks are continuous. Note that FIG. C illustrates a case where the number of S-nucleotides in each primer is three.
  • FIG. C(1) A representation of the base sequences of the wild-type first and second strands.
  • the F primer has c at its 3' end which is the complementary base of the 5' base (g) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed.
  • the R primer has at its 3' end a, the complementary base of the 5' base (t) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • the F primer has at its 3' end ca, the complementary sequence of the 5' nucleotide sequence (tg) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctg) from the second base from the 3' end of the F primer toward the 5' side (ctg) is S-formed. .
  • the R primer has at its 3' end ac, the complementary sequence of the 5' nucleotide sequence (gt) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (agt) from the second base from the 3' end of the R primer toward the 5' side (agt) is S-formed. .
  • the F primer has cat at its 3' end, which is the complementary sequence of the 5' nucleotide sequence (atg) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (act) from the second base from the 3' end of the F primer toward the 5' side (act) is S-formed. .
  • the R primer has at its 3' end acc, the complementary sequence of the 5' nucleotide sequence (ggt) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cag) from the second base from the 3' end of the R primer toward the 5' side (cag) is S-formed. .
  • FIG. D shows three examples of primer pairs (M-2) for detecting mutations in which 4 bases marked with ⁇ are deleted from the wild-type base sequence.
  • the first strand lacks ggtg marked with ⁇
  • the second strand lacks cacc marked with ⁇ .
  • a blank in the base sequence indicates that a base is missing, and the 5'-side and 3'-side bases of the blank are actually continuous. Note that FIG. D illustrates a case where the number of S-nucleotides in each primer is three.
  • FIG. D(1) A representation of the base sequences of the wild-type first and second strands.
  • Figure D (2) Primer pair-1 for detecting 4-base deletion
  • the F primer has c at its 3' end which is the complementary base of the 5' base (g) adjacent to the second strand defect.
  • the rest of the base sequence is the same as the complementary sequence (tgtgatt) of the base sequence (aatcaca) on the 3' side adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tta) from the second base from the 3' end of the F primer toward the 5' side (tta) is S-formed.
  • the R primer has at its 3' end a, the complementary base of the 5' base (t) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • the F primer has at its 3' end ca, the complementary sequence of the 5' nucleotide sequence (tg) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgatt) of the 3′-side nucleotide sequence (aatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctt) from the second base from the 3' end of the F primer toward the 5' side (ctt) is S-formed. .
  • the R primer has at its 3' end aa, which is the complementary sequence of the 5' nucleotide sequence (tt) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (agt) from the second base from the 3' end of the R primer toward the 5' side (agt) is S-formed. .
  • Figure D Primer pair for detecting 4-base deletion -3
  • the F primer has cat at its 3' end, which is the complementary sequence of the 5' nucleotide sequence (atg) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgatt) of the 3'-side nucleotide sequence (aatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (act) from the second base from the 3' end of the F primer toward the 5' side (act) is S-formed. .
  • the R primer has at its 3' end aat, which is the complementary sequence of the 5' nucleotide sequence (att) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (aag) from the second base from the 3' end of the R primer toward the 5' side (aag) is S-formed. .
  • a primer pair for detecting a 2-base deletion or a 3-base deletion can also be designed by a similar method.
  • primer pair M-2 of the present invention include the following.
  • a 4-base deletion in the canine VPS13B gene is known, and the following primer pair is designed as a primer pair for detecting the mutation.
  • VPS13B mutation detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGC*T*T*A-3' (SEQ ID NO: 73)
  • primer pair M-2 of the present invention include those listed in Table 2 below.
  • the primer pair M of the present invention is shown in FIG. ), and the primer pair of Figure D(2) are preferred. More preferred are the primer pairs of Figure A(2) and Figure B(2).
  • primer pair W of the present invention examples include a wild-type detection primer pair (W-1) used for detecting substitution or insertion mutation of a base sequence, or a wild-type detection primer pair (W-1) used for detecting base deletion mutation.
  • Type-detecting primer pair (W-2) can be mentioned.
  • Primer pair (W-1) of the present invention is as follows.
  • the rest of the nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the wild-type second strand nucleotide sequence corresponding to the mutation of the mutant second strand.
  • an F primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed;
  • the other nucleotide sequence is the same as the complementary sequence of the 3'-side nucleotide sequence adjacent to the wild-type first strand nucleotide sequence corresponding to the mutation of the mutant first strand.
  • a pair of R primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucle
  • substitution or insertion mutation of the base sequence to be detected is preferably a substitution or insertion of 1 to 4 bases. Substitutions or insertions of 1-3 bases are preferred. Substitution or insertion of a single base is more preferred.
  • primer pair W-1 of the present invention are preferred.
  • an F primer A primer that anneals to the first strand of the wild-type double-stranded nucleic acid (wild-type first strand), and replaces the first strand of the mutant double-stranded nucleic acid (mutant first strand) or Annealed to a region containing a nucleotide sequence in the wild-type first strand corresponding to the insertion mutation, and 1 to 4 consecutive nucleotides including the 3′ terminal nucleotide correspond to the mutation in the mutant first strand and the other nucleotide sequence is adjacent to the wild-type first strand nucleotide sequence corresponding to the mutation in the mutant first strand.
  • wild-type first strand that anneals to the region containing the nucleotide sequence in the wild-type first strand corresponding to the substitution or insertion mutation, and the 3′ terminal nucleotide corresponds to the mutation in the mutant-type first strand and the other base sequence is complementary to the base sequence on the 3' side adjacent to the base sequence of the wild-type first strand corresponding to the mutation of the first strand of the mutant type
  • the primer pair (W-1) of the present invention which is a wild-type detection primer pair used for detecting substitution of base sequences, will be described with reference to FIG. A2 below.
  • FIG. A2 exemplifies the case where the number of S-nucleotides in each primer is three.
  • FIG. A2(1) shows an example of the primer pair (W-1) of the present invention used for wild type detection when detecting a single nucleotide substitution.
  • the detection target is a single base substitution in which the wild-type base sequence is replaced with a by g in the first strand, and c is replaced by t in the second strand. indicates when The F primer is the second strand of a wild-type double-stranded nucleic acid (wild It has at its 3' end the complementary base (g) of c, which is the base of the second strand of the type.
  • the other nucleotide sequence is complementary to the nucleotide sequence (accaatcaca) on the 3′ side adjacent to c, which is the nucleotide of the wild-type second strand corresponding to the mutated nucleotide (t) of the second strand of the mutant type. Same as array (tgtgattggt).
  • array (tgtgattggt) the nucleotide of the wild-type second strand corresponding to the mutated nucleotide (t) of the second strand of the mutant type.
  • array tgtgattggt
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
  • the R primer is the first strand of the wild-type double-stranded nucleic acid (wild It has at its 3' end the complementary base (c) of g, which is the base of the first strand of the type).
  • the other nucleotide sequence is complementary to the nucleotide sequence (catccgc) on the 3′ side adjacent to g, which is the base of the wild-type first strand corresponding to the mutated nucleotide (a) of the first strand of the mutant type. Same as array (gcggatg).
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
  • FIG. A2(2) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting a two-base substitution.
  • W-1 primer pair
  • FIG. A2 shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting a two-base substitution.
  • tg is substituted with ca in the first strand
  • ca is substituted with tg in the second strand.
  • the F primer is the second strand of a wild-type double-stranded nucleic acid ( It has a complementary sequence (tg) of ca, which is the nucleotide sequence of the wild-type second strand), at its 3' end.
  • the other nucleotide sequence is the 3′-side nucleotide sequence (ccaatcaca) adjacent to ca, which is the nucleotide sequence of the wild-type second strand corresponding to the mutant nucleotide sequence (tg) of the second strand of the mutant type. is the same as the complementary sequence (tgtgattgg) of
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
  • the R primer is the first strand of a wild-type double-stranded nucleic acid corresponding to the mutated base sequence (ca) of the first strand of the mutant double-stranded nucleic acid having the above two base substitutions (mutant first strand). It has a complementary sequence (ca) of tg, which is the base sequence of (wild-type first strand), at its 3' end.
  • the other base sequence is the base sequence (catccgc) on the 3' side adjacent to tg, which is the base sequence of the wild-type first strand corresponding to the mutant base sequence (ca) of the first strand of the mutant type.
  • FIG. A2(3) shows an example of the primer pair (W-1) of the present invention, which is used for wild-type detection when detecting mutations in which three bases are substituted.
  • detection target is 3 base substitutions in which gtg is substituted with tca in the first strand and cac is substituted with tga in the second strand.
  • the F primer is a wild-type double-stranded nucleic acid second strand ( It has a complementary sequence (gtg) of cac, which is the nucleotide sequence of the wild-type second strand), at its 3' end.
  • the other base sequence is the base sequence (caatcaca) on the 3' side adjacent to cac, which is the base sequence of the wild-type second strand corresponding to the mutant base sequence (tga) of the second strand of the mutant type. is the same as the complementary sequence (tgtgattg) of
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
  • the R primer is the first strand of a wild-type double-stranded nucleic acid corresponding to the mutated nucleotide sequence (tca) of the first strand of the mutant double-stranded nucleic acid having the three base substitutions (mutant first strand). It has a gtg complementary sequence (cac), which is the base sequence of (wild-type first strand), at its 3' end. The other base sequence is the base sequence (catccgc) on the 3' side adjacent to the base sequence gtg of the wild-type first strand corresponding to the mutant base sequence (tca) of the first strand of the mutant type.
  • Wild-type detection primer pair (W-1) used for insertion detection The primer pair (W-1) of the present invention, which is a wild-type detection primer pair used for detecting insertion of a nucleotide sequence will be explained based on the following diagram B2.
  • FIG. B2 exemplifies the case where the number of S-nucleotides in each primer is three.
  • FIG. B2(1) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when single-base insertion is detected.
  • FIG. B2(1) shows the case of detecting a single base insertion in which t is inserted in the first strand and a is inserted in the second strand at the positions indicated by the arrows in the wild-type base sequence.
  • the F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end c, the complementary base of the 5' base (g) adjacent to the (strand) position.
  • the other nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the 3′-side nucleotide sequence (caccaatcaca) adjacent to the position of the wild-type second strand corresponding to the insertion mutation of the mutant second strand. be.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) extending from the second base from the 3' end of the F primer toward the 5' side (gtg) is S-formed. .
  • the R primer is the first strand of the wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the insertion mutation of the first strand of the mutant double-stranded nucleic acid having the single-base insertion (mutant first strand). It has at its 3' end g, the complementary base of the 3' base (c) adjacent to the 1 strand) position. and the other base sequence is the 3′-side base (c) adjacent to the 3′-side base (c) (adjacent to the position of the wild-type first strand corresponding to the insertion mutation of the mutant first strand). It is the same as the complementary sequence (cgcggatg) of the base sequence (atccgc).
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
  • FIG. B2(2) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting double base insertion.
  • FIG. B2(2) shows a case in which 2-base insertions in which ta is inserted in the first strand and ta is inserted in the second strand at the positions indicated by arrows in the wild-type base sequence are detected.
  • the F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end gc, which is the complementary sequence of the base sequence (gc) flanking the position of the strand).
  • the other nucleotide sequence is complementary to the nucleotide sequence (accaatcaca) on the 3′ side adjacent to the nucleotide sequence (gc) flanking the position of the wild-type second strand corresponding to the insertion mutation of the second strand of the mutant type. Same as array (tgtgattggt).
  • array (tgtgattggt) the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) extending from the second base from the 3' end of the F primer toward the 5' side (gtg) is S-formed. .
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first It has at its 3' end gc, which is the complementary sequence of the base sequence (gc) flanking the position of the strand).
  • the other nucleotide sequence is the complement of the nucleotide sequence (atccgc) on the 3' side adjacent to the nucleotide sequence (gc) flanking the position of the wild-type first strand corresponding to the insertion mutation of the first strand of the mutant type. Same as array (cggatg).
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ggt) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • FIG. B2(3) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting double base insertion.
  • W-1 the primer pair of the present invention used for wild-type detection when detecting double base insertion.
  • ta is inserted in the first strand at the position of the arrow, and 2 base insertions in which ta is inserted in the second strand are to be detected.
  • the F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end ca, which is a complementary sequence of gt, which is the 5' base sequence of the (strand) position.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the nucleotide sequence (caccaatcaca) on the 3' side adjacent to the position of the wild-type second strand corresponding to the insertion mutation of the mutant second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgt) from the second base from the 3' end of the F primer toward the 5' side (cgt) is S-formed. .
  • the R primer is the first strand of the wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the insertion mutation of the first strand of the mutant double-stranded nucleic acid having the single-base insertion (mutant first strand). It has at its 3' end g, the complementary base of the 3' base (c) adjacent to the 1 strand) position. And other nucleotide sequences are the 3′ side adjacent to the 3′ side base (c) (adjacent to the position of the wild-type first strand corresponding to the insertion mutation of the mutant first strand). It is the same as the complementary sequence (cgcggatg) of the nucleotide sequence (atccgc). In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • primer pair W-1 of the present invention for example, a wild-type detection primer pair (primer pair W-1 of the present invention, FIG. A2 This corresponds to the case of (2).) will be described below as an example.
  • PiS mutation which is a gene mutation of ⁇ 1-antitrypsin.
  • the primer pair W-1 of the present invention which is a wild-type detection primer pair used for detecting this single base substitution
  • the double-stranded DNA of the ⁇ 1-antitrypsin gene in which A was replaced with T was used. is the first strand.
  • the strand of the above double-stranded DNA in which T in the same portion of the base is replaced with A is referred to as the second strand.
  • the * mark indicates the position where the phosphodiester bond is S-converted.
  • PiS wild-type detection primer pair (W-1) for example, the following are designed.
  • the F primer of the wild-type detection primer pair used for PiS mutation detection is a wild-type corresponding to the mutated base (A) of the second strand of the double-stranded nucleic acid having a PiS mutation (second strand of the mutant type). It has A, which is the complementary base of the base (T) of the second strand (wild-type second strand) of the double-stranded nucleic acid, at the 3′ end.
  • Other nucleotide sequences are the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the wild-type second strand nucleotide (T) corresponding to the mutant second strand nucleotide (A).
  • the R primer of the primer pair is the first strand of a wild-type double-stranded nucleic acid ( It has a T at the 3′ end, which is the complementary base of base (A) of wild-type first strand).
  • Other base sequences are designed to be the same as the base sequence on the 3' side adjacent to the wild-type first-strand base (A) corresponding to the mutant first-strand base (T). be done.
  • the 2nd to 4th nucleotides from the 3' end are nucleotides whose phosphodiester bonds are S-converted.
  • the F primer has the same 3′ terminal base as the wild-type base (A) corresponding to the mutated base (T) of the PiS gene, and the other base sequence is the wild-type double-stranded nucleic acid of the PiS gene. It is designed to have the same nucleotide sequence as the 5′ side of the wild-type nucleotide (A) corresponding to the above-mentioned mutated nucleotide (T) of the first strand.
  • the R primer has the same 3′ terminal base (A) as the complementary base (A) of the wild-type base (T) corresponding to the mutated base (T) of the PiS gene, and the other base sequence is the wild-type base (T) of the PiS gene. designed to be the same as the complementary sequence of the base sequence toward the 3′ side adjacent to the wild-type base (A) corresponding to the mutated base (T) of the first strand of the double-stranded nucleic acid of the type be.
  • the 2nd to 4th nucleotides from the 3'-end are nucleotides whose phosphodiester bonds are S-converted.
  • primer pair W of the present invention examples include a wild-type detection primer pair used for detecting PiZ mutation (single nucleotide mutation), which is another gene mutation of ⁇ 1-antitrypsin.
  • primer pair W-1 of the present invention include those listed in Table 3 below.
  • Primer pair (W-2) of the present invention which is a pair of primers for wild-type detection used for detecting deletion of nucleotide sequences, is as follows.
  • a primer that anneals to the second strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a defect in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) A complementary sequence of the base sequence of the wild-type second strand that anneals to the region containing the base sequence in the wild-type second strand and corresponds to the deletion of the mutant-type second strand, or the wild-type first strand It has a complementary sequence of the base sequence of the second strand and the base sequence of the 5' side adjacent to it at the 3' end side, and the other base sequence is the wild-type second strand corresponding to the deletion of the second strand of the mutant type.
  • an F primer in which the phosphodiester bond on the side is S-ified A primer that anneals to the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) and corresponds to a defect in the first strand of a mutant-type double-stranded nucleic acid (mutant-type first strand)
  • the first strand of the wild type has a sequence complementary to the base sequence of the first strand and the base sequence of the 5' side adjacent thereto at the 3' end side, and the other base sequence is the first strand
  • Wild-type detection primer pair (W-2) used for detecting 1-base deletion The primer pair of the present invention, which is a wild-type detection primer pair used for detecting 1-base deletion in a nucleotide sequence ( W-2) will be explained with reference to Figure C2 below.
  • FIG. C2 shows a case where a 1-base deletion is targeted for detection, in which the first strand lacks the arrow g and the second strand lacks the arrow c in the wild-type base sequence.
  • FIG. C2 shows the case where the number of S-nucleotides in each primer is 3.
  • Figure C2 Primer pair for detecting 1 base deletion (wild type) -1
  • the F primer is the second strand of the wild-type double-stranded nucleic acid (wild-type second strand) corresponding to the deletion of the second strand of the mutant double-stranded nucleic acid having a single base deletion (mutant second strand). It has at its 3′ end the complementary base (g) of c, which is the base of the double strand).
  • the other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand.
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3′ end the complementary base (c) of g, which is the base of the first strand).
  • the other nucleotide sequence is a complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the wild-type first strand corresponding to the deletion of the mutant-type first strand. is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • Figure C2 Primer pair for detecting 1 base deletion (wild type) -2
  • the F primer is the second strand of the wild-type double-stranded nucleic acid (wild-type second strand) corresponding to the deletion of the second strand of the mutant double-stranded nucleic acid having a single base deletion (mutant second strand). It has at its 3′ end the complementary base (g) of c, which is the base of the double strand).
  • the other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand.
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3' end a complementary sequence (ca) of the base g of the first strand) and the adjacent 5' base (t).
  • the other nucleotide sequence is a complementary sequence (gcggatgc) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the second strand of the wild type corresponding to the deletion of the first strand of the mutant type. is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgt) from the second base from the 3' end of the R primer toward the 5' side (cgt) is S-formed. .
  • Figure C2 Primer pair for detecting 1 base deletion (wild type) -3
  • the F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end a complementary sequence (gc) of the base c of the chain) and the adjacent 5' base (g).
  • the other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand.
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3′ end the complementary base (c) of g, which is the base of the first strand).
  • the other nucleotide sequence is the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the wild-type first strand corresponding to the deletion of the mutant-type first strand. is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
  • Wild-type detection primer pair (W-2) used for detecting 4-base deletion The primer pair of the present invention, which is a wild-type detection primer pair used for detecting 4-base deletion in a nucleotide sequence ( W-2) will be explained based on the following diagram D2.
  • the first strand lacks ggtg marked with ⁇
  • the second strand lacks cacc marked with ⁇ . show.
  • FIG. D2 a blank in the base sequence indicates that a base is missing, and the 5'-side and 3'-side bases of the blank are actually continuous.
  • FIG. D2 shows the case where the number of S-nucleotides in each primer is three.
  • Figure D2 Primer pair for detecting 4-base deletion
  • the F primer is a wild-type double-stranded nucleic acid corresponding to the deletion of the second strand of a mutant double-stranded nucleic acid having a 4-base deletion (mutant second strand). It has at its 3′ end a complementary sequence (ggtc) of cacc, which is the base sequence of the second strand (wild-type second strand).
  • the other nucleotide sequence is a complementary sequence (tgtgatt ) is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first It has at its 3' end the complementary sequence (cacc) of ggtg, which is the base sequence of the ggtg chain).
  • the other nucleotide sequence is a complementary sequence (gcggatg ) is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cac) from the second base from the 3' end of the R primer to the 5' side (cac) is S-formed. .
  • a primer pair W-2 for detecting a 2-base deletion or a 3-base deletion can also be designed by a similar method, although not explained using the figures.
  • primer pair W-2 of the present invention include the following.
  • the following primer pair is designed as a wild-type detection primer pair for detecting the above-described 4-base deletion in the canine VPS13B gene.
  • the primer pairs shown in Figure A2(2), Figure B2(2), Figure C2(2), and Figure D2(2) are preferable. Considering the ease of determination of nucleotide sequence mutations, the primer pairs of Figure A2(2) and Figure B2(2) are more preferable.
  • primer pair W-2 of the present invention include those listed in Table 4 below.
  • the method for determining a nucleotide sequence mutation of the present invention comprises "Using the nucleic acid of the test sample as a template, a nucleic acid amplification reaction is performed using the primer pair of the present invention (for example, primer pair M or/and primer pair W) to detect the reaction product, and the resulting detection result is also reported. and a method for determining a mutation in a base sequence, which determines a mutation in the base sequence of the nucleic acid.” is.
  • Test sample used in the method for determining a nucleotide sequence mutation of the present invention includes oral swabs (oral swabs), nasal swabs, nasopharyngeal swabs, pharyngeal swabs, saliva, Body fluids such as ascites, pleural effusion, nerve root fluid, lymphatic fluid, cerebrospinal fluid, digestive fluid, intratracheal aspirate, pharyngeal mucus, sputum, transbronchial sample, bronchial lavage, various clinical materials such as pleural effusion, plasma, serum, total Blood samples such as blood, tissue sections, tissue lavage fluids, feces, urine and other biological samples, cultured cells, cell supernatants, cell lysates, or pretreated samples such as heat-treated samples and those adjusted by dilution or concentration, etc., as necessary.
  • oral swabs oral swabs
  • nasal swabs nasal swabs
  • the specimen from which the test sample according to the present invention is obtained is not limited to animals, plants, viruses, etc., as long as it contains the nucleic acid of the gene to be detected.
  • Examples of animals from which the test sample according to the present invention can be obtained include mammals (humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, bats, mice, rats, etc.), Examples include reptiles and the like.
  • Viruses from which test samples according to the present invention can be obtained include SARS-CoV-2 and the like.
  • test sample nucleic acid or “test sample-derived nucleic acid” used for detecting gene mutation according to the present invention, a nucleic acid extracted and purified from the above-described test sample, or a nucleic acid amplification detection system, etc. It may be an amplified nucleic acid. Nucleic acids may be double-stranded or single-stranded, and may be DNA or RNA. In the case of RNA, cDNA obtained by reverse transcription may be used as the nucleic acid.
  • the nucleic acid of the test sample used in the method for determining the nucleotide sequence of the present invention may be isolated and purified from the test sample by methods commonly used in this field.
  • a nucleic acid amplification reaction commonly performed in this field may be performed, except that the primer pair of the present invention is used.
  • the primer pair of the present invention it is hybridized with the nucleic acid in the sample, and nucleic acid amplification by DNA polymerase [for example, PCR (polymerase chain reaction), LAMP (Loop-mediated Isothermal Amplification) method, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, LCR (ligase chain reaction) method, SDA (strand displacement amplification) method] to extend the primer.
  • DNA polymerase for example, PCR (polymerase chain reaction), LAMP (Loop-mediated Isothermal Amplification) method, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, LCR (ligase chain reaction) method, SDA (strand displacement amplification) method
  • the conditions, operation method, etc. of the nucleic acid amplification reaction may be
  • the "method for detecting a reaction product obtained by a nucleic acid amplification reaction” includes, for example, the intercalator method, the TaqMan TM real-time PCR method, the MGB Eclipse Probe System method, the Molecular Beacons Probe Technology method, the LUX Fluorogenic Primer method, Quenching probe-PCR (QP) method, a method based on electrophoresis of the obtained primer extension product after performing nucleic acid amplification reaction, a method based on the result, a method obtained by performing nucleic acid amplification reaction using labeled primers
  • QP Quenching probe-PCR
  • a variety of detection methods are included, including methods that measure labeling of primer extension products.
  • real-time PCR using a known intercalator e.g., EvaGreen TM (manufactured by Biotium), SYBR TM Green I (manufactured by Molecular Probe), etc.
  • a known intercalator e.g., EvaGreen TM (manufactured by Biotium), SYBR TM Green I (manufactured by Molecular Probe), etc.
  • Fluorescence intensity derived from the intercalator is detected.
  • nucleic acid amplification reaction is performed using the purified nucleic acid derived from the test sample as a template, and the mutation to be detected in the nucleic acid is used. Amplify the region containing (1st PCR). Then, using the amplified product as a template, a nucleic acid amplification reaction using the primer pair of the present invention may be performed.
  • Method for determining mutation As a method for determining mutation, 1) A method for determining whether the nucleotide sequence of a nucleic acid in a test sample is wild-type, homozygous mutation, or heterozygous mutation; or 2) Determining whether the nucleotide sequence of a nucleic acid in a test sample is wild-type or mutant. how to, is mentioned. Each determination method will be described below.
  • a determination method comprising the following steps: (i) using the nucleic acid of the test sample as a template, performing a nucleic acid amplification reaction using the primer pair M of the present invention, and detecting the reaction product; (ii) a step of performing a nucleic acid amplification reaction using the primer pair W of the present invention using the nucleic acid of the same test sample as that used in step (i) above as a template, and detecting the reaction product; (iii) A step of judging a mutation in the base sequence of the nucleic acid of the test sample based on the detection results obtained in the above steps (i) and (ii). ] is mentioned.
  • Primer pair M of the present invention and primer pair W of the present invention are as described in the section " ⁇ 1. Primer pair of the present invention>", and preferred examples and specific examples are also the same.
  • the method of carrying out the nucleic acid amplification reaction using each primer pair in the above steps (i) and (ii) may be carried out according to the method described in the section "(2) Nucleic acid amplification reaction", which is preferable. Examples, specific examples, etc. are the same. Either step (i) or step (ii) may be performed first, and the order of the steps does not matter.
  • Step (iii) may be performed by performing steps (i) and (ii) to obtain an amplification curve, respectively, and comparing the amplification curve with a control amplification curve obtained by the method described below. good.
  • Determining method based on amplification curve Determining whether the nucleotide sequence of the nucleic acid of the test sample is a wild type, homozygous mutation, or heterozygous mutation (that is, determining genotype)
  • a method of making determination based on an amplification curve of fluorescence intensity measured in a nucleic acid amplification reaction there is a method of making determination based on an amplification curve of fluorescence intensity measured in a nucleic acid amplification reaction.
  • a method for judging by comparing an amplification curve obtained using a test sample-derived nucleic acid as a template with a control amplification curve will be described below as an example.
  • An oligonucleotide (wild-type control) containing the nucleotide sequence corresponding to the above mutation is designed and prepared.
  • Primer pairs of the present invention> using a primer pair M for detecting a mutation to be detected and a primer pair W for wild-type detection corresponding to the same mutation to be detected, and using a control of the combination shown in Table 5 below as a template. , prepare a sample control amplification curve using the primer pairs of the invention.
  • a nucleic acid amplification reaction is performed using the primer pair W of the present invention to obtain an amplification curve (amplification curve 1-1'). Further, using the same wild-type control as a template, a similar nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 1-2'). Then, the resulting amplification curve 1-1' and amplification curve 1-2' are plotted in one graph, and this graph is used as a wild-type control.
  • a nucleic acid amplification reaction is performed using the primer pair W of the present invention to obtain an amplification curve (amplification curve 2-1'). Further, using the same mutant type control as a template, a similar nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 2-2'). Then, the obtained amplification curve 2-1' and amplification curve 2-2' are represented in one graph, and this graph is used as a heterozygous control.
  • a mixture of the same wild-type control and the same mutant control as above is used as a template, and the primer pair W of the present invention is used to carry out a nucleic acid amplification reaction to obtain an amplification curve (amplification curve 3-1').
  • the same nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 3-2' ).
  • the obtained amplification curve 3-1' and amplification curve 3-2' are plotted in one graph, and this graph is used as a homozygous control. Wild-type and mutant controls are equal in weight in the templating mixture.
  • the amplification curve 1-1' rises earlier than the amplification curve 1-2'. That is, the amplification curve 1-1' is detected earlier than the amplification curve 1-2'.
  • the amplification curve 2-1' has a slower rise than the amplification curve 2-2'. That is, the amplification curve 2-1' is detected later than the amplification curve 2-2'.
  • the rises of the amplification curves 3-1' and 3-2' are comparable (detected to the same extent), i.e., the amplification curves 3-1' and 3-2' are detected with similar rapidity, and the two amplification curves are close to each other.
  • amplification curve 1 Creation of an amplification curve using a nucleic acid derived from a test sample as a template
  • a nucleic acid derived from a test sample as a template Using the same primer pair W of the present invention as used, a nucleic acid amplification reaction is performed to obtain an amplification curve (amplification curve 1).
  • a nucleic acid amplification reaction is performed using the same primer pair M of the present invention as used in the above "1-a-1) Creation of amplification curve of object", An amplification curve is obtained (amplification curve 2).
  • the obtained amplification curve 1 and amplification curve 2 are shown in one graph.
  • nucleotide sequence mutation The relationship between the amplification curve 1 and the amplification curve 2 obtained using the nucleic acid derived from the test sample obtained in 1-a-2) above as a template is Compare with the wild-type control, heterozygous control, and homozygous control obtained in a-1). If the relationship between the obtained amplification curves 1 and 2 is close to that of the wild-type control, the gene to be detected in the test sample is determined to be wild-type. If the relationship between the obtained amplification curves 1 and 2 is close to that of the heterozygous control, it is determined that the gene to be detected in the test sample is heterozygous. If the relationship between the obtained amplification curves 1 and 2 is close to that of the homozygous control, it is determined that the gene to be detected in the test sample is homozygous mutation.
  • the gene to be detected in the test sample is wild
  • the gene to be detected in the test sample is a heterozygous mutation
  • the reaction product obtained in step (i) above is detected at a cycle number close to the reaction product obtained in step (ii) above, the test sample The gene to be detected is determined to be a homozygous mutation.
  • the gene to be detected in the test sample is a homozygous mutation
  • the Ct value or Tm value obtained in the detection of the step (i) above is the Ct value obtained in the detection of the step (ii)
  • the gene to be detected in the test sample is determined to be a heterozygous mutation.
  • the method of determining the genotype whether the base sequence of the nucleic acid of the test sample according to the present invention is a wild type, homozygous mutation, or heterozygous mutation, ⁇ 1- by a real-time PCR detection system using an intercalator A method for determining the genotype of PiS, which is an antitrypsin gene mutation, will be described as an example.
  • a purified DNA sample is obtained from a test sample (eg, human blood, saliva, swab, etc.) by a known method.
  • a test sample eg, human blood, saliva, swab, etc.
  • a nucleic acid amplification reaction is performed using the following primer pair and the resulting purified DNA sample as a template (1st PCR) to amplify the region containing the PiS mutation in the purified DNA to obtain an amplified product.
  • F primer 5'-CTGCTGATGAAATACCTGGGCAATG-3' (SEQ ID NO: 25)
  • R primer 5'-GGTTGGGGAATCACCTTCTGTCTTC-3' (SEQ ID NO: 26)
  • real-time PCR is performed, for example, as follows, using the following wild-type detection primer pair and PiS mutation type detection primer pair.
  • each of the F primer and the R primer of the PiS mutation type detection primer pair or the wild type detection primer pair of the present invention an intercalator (for example, EvaGreen TM (manufactured by Biotium), SYBR TM Green I (Molecular Probe etc.), 0.2 mM dNTPs, 0.1 to 2 units (U)/mL of DNA polymerase, respectively, in a buffer or aqueous solution containing a purified DNA sample or 1st PCR amplification product (10 pg) purified from the test sample ⁇ 10 ng) to make the reaction mixture for PCR.
  • an intercalator for example, EvaGreen TM (manufactured by Biotium), SYBR TM Green I (Molecular Probe etc.
  • 0.2 mM dNTPs 0.1 to 2 units (U)/mL of DNA polymerase
  • an oligonucleotide having a partial nucleotide sequence of the ⁇ 1-antitrypsin gene containing the mutated nucleotide sequence of the PiS mutation (PiS mutant control: for example, an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 54), and ⁇ 1- Oligonucleotide having a wild-type nucleotide sequence of ⁇ 1-antitrypsin gene (PiS wild-type control, for example, an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 53, including a nucleotide sequence corresponding to PiS mutation in the antitrypsin gene ).
  • PiS wild type detection primer pair SEQ ID NO: 35-SEQ ID NO: 36
  • PiS mutation detection primer pair SEQ ID NO: 37 - SEQ ID NO: 38
  • a wild-type control is used as a template, and a nucleic acid amplification reaction is performed using a PiS wild-type detection primer pair to obtain an amplification curve.
  • a nucleic acid amplification reaction is performed using a PiS mutant detection primer pair to obtain an amplification curve.
  • the two amplification curves obtained are merged into one figure, which is the wild-type control figure.
  • a nucleic acid amplification reaction is performed using a PiS wild-type detection primer pair to obtain an amplification curve.
  • a nucleic acid amplification reaction is performed using a PiS mutant detection primer pair to obtain an amplification curve.
  • the two amplification curves obtained are combined into one figure, which is the figure of the homozygous control.
  • a mixture of PiS wild-type control and PiS mutant control is used as a template, and a primer pair for PiS wild-type detection is used to perform a nucleic acid amplification reaction to obtain an amplification curve.
  • a primer pair for PiS wild-type detection is used to perform a nucleic acid amplification reaction to obtain an amplification curve.
  • the amplification curve obtained by carrying out nucleic acid amplification reaction using a pair of primers for detecting PiS mutation is combined into one figure, and this figure is used as a hetero This is a diagram of the mating type control.
  • the amplification curves obtained using the test sample-derived nucleic acids are compared with the heterozygous control, wild-type control, and homozygous control graphs.
  • An amplification curve obtained using a nucleic acid derived from a test sample and using a PiS wild-type detection primer pair, and an amplification curve obtained using a nucleic acid derived from a test sample and using a PiS mutation type detection primer pair is closest to that of the heterozygous control
  • the PiS gene to be detected in the test sample is determined to be heterozygous. If the relationship between the amplification curves is the closest to that of the homozygous control, the PiS gene to be detected in the test sample is determined to be homozygous mutation. If the relationship between the amplification curves is closest to that of the wild-type control, the PiS gene to be detected in the test sample is determined to be wild-type.
  • a nucleic acid amplification reaction is performed using the primer pair M of the present invention and/or the primer pair W of the present invention, and using nucleic acid derived from a test sample as a template.
  • the reaction product is detected in the nucleic acid amplification reaction using the primer pair M of the present invention, but the reaction product is detected using the primer pair W of the present invention. No or very little reaction product is detected in nucleic acid amplification reactions.
  • the nucleotide sequence of the gene to be detected in the test sample is of the wild type, no or almost no reaction product is detected in the nucleic acid amplification reaction using the primer pair M of the present invention.
  • a reaction product is detected in the nucleic acid amplification reaction using the primer pair W of the present invention.
  • real-time PCR is performed using the primer pair M of the present invention, an intercalator, and a purified DNA sample purified from a test sample as a template. Then, the fluorescence intensity derived from the intercalator that intercalates with the amplified product is measured.
  • Real-time PCR is performed in the same manner as in the case of using the primer pair M of the present invention except that the primer pair W of the present invention is used, and the fluorescence intensity derived from the intercalator is measured.
  • the method for determining whether the nucleic acid sequence of a test sample according to the present invention is wild-type or mutant the method for determining the N501Y mutation of the SARS-CoV-2 virus will be described.
  • purified total RNA is obtained from the SARS-CoV-2 virus by a known method, and using the obtained single-stranded RNA as a template, reverse transcription is performed by a conventional method to obtain cDNA.
  • a nucleic acid amplification reaction is performed using the following primer pair and the resulting purified cDNA as a template (1st PCR) to amplify the region containing the N501Y mutation in the cDNA to obtain an amplified product.
  • F primer 5'-CTATCAGGCCGGTAGCACACCTTG-3' (SEQ ID NO: 75)
  • R primer 5'-CCACAAACAGTTGCTGGTGCATGTAG-3' (SEQ ID NO: 76)
  • real-time PCR is performed, for example, by the intercalator method, using the following N501Y wild-type detection primer pair and N501Y mutant-type detection primer pair.
  • ⁇ N501Y wild type detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*A-3' (SEQ ID NO: 79) R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*T-3' (SEQ ID NO: 80) ⁇ N501Y mutation detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*T-3' (SEQ ID NO: 81) R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*A-3' (SEQ ID NO: 82)
  • reaction products were not detected or hardly detected, but the primer pair for detecting N501Y wild type (primer pair of the present invention If a reaction product is obtained in the nucleic acid amplification reaction using pair W), it is determined that the nucleotide sequence of the N501Y gene to be detected in the test sample is of the wild type without mutation.
  • primer pair M and primer pair W are used in the method for determining mutation of the present invention
  • the F primer of primer pair M and the F primer of primer pair W to be used are "complementary sequence of the mutated nucleotide sequence of the second strand ” are preferably the same.
  • the R primer of the primer pair M and the R primer of the primer pair W to be used preferably have the same base sequence except for the sequence corresponding to the "complementary sequence of the mutated base sequence of the first strand.”
  • primer pair M and primer pair W when primer pair M and primer pair W are used, it is preferable that the number of S-nucleotides in the F primer of primer pair M and the F primer of primer pair W to be used is the same. Similarly, the number of s-nucleotides in the R primer of primer pair M and the R primer of primer pair W to be used is preferably the same.
  • the kit of the present invention is a kit for determining whether the base sequence of a nucleic acid in a test sample is wild-type, homozygous mutation, or heterozygous mutation, or for determining whether it is wild-type or mutant.
  • nucleotide sequence mutation determination kit examples include those containing the primer pair of the present invention.
  • Preferred are those containing the primer pair M of the present invention and/or the primer pair W of the present invention.
  • Those containing the primer M of the present invention and the primer W of the present invention are preferred.
  • the primer pair of the present invention, the primer pair M of the present invention, and the primer pair W of the present invention according to the kit of the present invention are as described in the section " ⁇ 1. Primer pair of the present invention>", and preferred examples , specific examples, etc. are also the same.
  • the primer pair of the present invention may be in the form of a solution-state test solution such as a suspension suspended in an appropriate buffer, or may be a frozen product or a lyophilized product. good too.
  • concentration of the primer pair of the present invention in the test solution, the solvent of the test solution, and the like may be appropriately set within the range usually used in this field.
  • the kit of the present invention may contain necessary amounts of reagents necessary for carrying out a nucleic acid amplification reaction.
  • reagents necessary for carrying out a nucleic acid amplification reaction for example, in addition to the primer pair of the present invention, nucleoside triphosphate, nucleic acid synthetase, PCR buffer and the like may be further provided.
  • nucleic acid synthetase examples include DNA polymerase, RNA polymerase, reverse transcriptase, and the like.
  • buffers used as the PCR buffer include Tris buffer, phosphate buffer, Veronal buffer, borate buffer, Good's buffer, etc. All the buffers used in the case are listed, and the pH thereof is not particularly limited.
  • kit of the present invention may optionally contain substrates (dNTPs, rNTPs, etc.) suitable for the enzyme, such as EvaGreen TM (manufactured by Cosmo Bio Co., Ltd.), SYBR TM Green I (manufactured by Molecular Probe). , ethidium bromide, fluorene, and other double-stranded intercalators, and labeled detection substances such as FAM and TAMRA.
  • substrates dNTPs, rNTPs, etc.
  • substrates dNTPs, rNTPs, etc.
  • EvaGreen TM manufactured by Cosmo Bio Co., Ltd.
  • SYBR TM Green I manufactured by Molecular Probe
  • labeled detection substances such as FAM and TAMRA.
  • stabilizers, preservatives, etc. which do not inhibit the stability of coexisting reagents and the like, and which do not inhibit PCR
  • the kit of the present invention contains an oligonucleotide (wild-type control) having the base sequence of the gene (which may be a partial sequence), including the mutated base sequence of the gene to be detected, and/or the wild-type base of the gene.
  • An oligonucleotide (mutant type control) containing the sequence sequence and the base sequence corresponding to the mutation may be included.
  • Kits of the invention may also include sample amplification curves for determining genotype (wild-type, heterozygous, or homozygous).
  • the kit of the present invention may comprise the necessary amount of the primer pair used in the 1st PCR and the reagents necessary for the 1st PCR.
  • the kit of the present invention includes a nucleic acid amplification reaction procedure using the primers of the present invention, a wild-type control and a mutant control to determine the genotype (wild-type, heterozygous, or homozygous).
  • the above-mentioned "instruction” means the instruction manual, package insert, pamphlet (leaflet), etc. of the above-mentioned kit in which the features, principles, operation procedures, judgment procedures, etc. of the above-mentioned method are substantially described in sentences, diagrams, etc. means
  • kit of the present invention may be equipped with means (for example, cotton swabs, etc.) for collecting test samples.
  • Example 1 Detection of Single Nucleotide Mutation in Human BRAF Gene
  • a single nucleotide substitution (c1799T>A(V600E)) in the human BRAF gene of the present invention was detected by the following method.
  • TIG-3 human fetal normal fibroblasts, JCRB cell bank
  • COLO201 human colon adenocarcinoma cells, ATCC No. CCL-224
  • A375 melanoma cells, ATCC No. CRL-1619
  • Primer pair The following primer pair was designed and synthesized. In addition, in the nucleotide sequence of each primer, the * mark indicates the position where the phosphodiester bond is S-converted.
  • Primer pair without S-merization/primer pair for wild-type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGT-3' (SEQ ID NO: 1) R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTTCA-3' (SEQ ID NO: 2) ⁇ Primer pair for mutation type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGA-3' (SEQ ID NO: 3) R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTTCT-3' (SEQ ID NO: 4)
  • a single-nucleotide mutation in the human BRAF gene (c1799T>A (V600E)) is a mutation of T to A at position 1799 in the nucleotide sequence of the human BRAF gene.
  • the mutation detection primer pair used in Example 1 is The R primer has the complementary base T of the mutated base A of the first strand at the 3' end.
  • the rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the first strand.
  • the F primer of the pair of primers for detecting mutation type has a complementary base A of the mutated base T of the second strand at the 3' end.
  • the rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
  • EvaGreen TM (manufactured by Biotium): fluorescent reagent for quantitative real-time PCR, manufactured by Cosmo Bio Co., Ltd.
  • KOD FX Neo (manufactured by Toyobo Co., Ltd.): 1 U/ ⁇ L
  • real-time PCR 20 ⁇ L of the reaction solution for PCR prepared in i) above is placed in the wells of a 96-well reaction plate (Microamp Optical 96-well reaction plate, manufactured by Applied Biosystems Japan Co., Ltd.) and subjected to TaqMan TM PCR thermal cycler detection.
  • Real-time PCR was performed using a device (ABI 7500, manufactured by Applied Biosystems Japan). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen TM was measured.
  • FIGS. 1 to 6 show the results using a primer pair of primers that are not S-conjugated.
  • FIG. 2 shows the results of using a primer pair of primers in which one nucleotide is S-converted.
  • FIG. 3 shows the results of using a primer pair of primers in which 2 bases are S-converted.
  • FIG. 4 shows the results of using a primer pair of primers in which 3 nucleotides are S-converted.
  • FIG. 5 shows the results of using a primer pair of primers in which 4 bases are S-converted.
  • FIG. 6 shows the results of using a primer pair of primers in which 5 nucleotides are S-converted.
  • FIGS. 1 to 6 (1) shows the results of using TIG-3 genomic DNA as a template, (2) shows the results of using COLO201 genomic DNA as a template, and (3) shows the results of A375 genomic DNA. are used as templates.
  • the amplification curve W shows the results of using the wild-type detection primer pair
  • the amplification curve M shows the results of using the mutation-type detection primer pair.
  • TIG-3 cells do not have mutations in the BRAF gene.
  • COLO201 cells have a heterozygous mutation c1799T>A in the BRAF gene.
  • A375 cells have a homozygous c1799T>A mutation in the BRAF gene.
  • the amplification curve using the wild type detection primer pair for the wild type shows the mutation type detection primer pair It rose earlier (earlier detection) than the amplification curve used.
  • the heterozygous type ((2) COLO201)
  • the amplification curve using the wild-type detection primer pair and the amplification curve using the mutant detection primer pair were close to each other.
  • the homozygous type ((3) A375) used the wild type detection primer pair in the amplification curve using the mutant type detection primer pair. It rose ahead of the amplification curve (detected earlier).
  • the heterozygous type and the homozygous type can be clearly distinguished and determined by performing detection using a primer pair of primers in which 2 bases are S-converted or a primer pair of primers in which 3 bases are S-converted. rice field.
  • Example 2 Mutation detection of human ⁇ 1-antitrypsin-1 (PiS) Humans with ⁇ 1-antitrypsin deficiency are known to have the following single base substitutions in the gene. PiS : c.863A>T p.Glu288Val PiZ : c.1096G>A p.Glu366Lys In Example 2, PiS mutation was detected using the primer pair of the present invention.
  • Genomic DNA extraction COLO201 human colon cancer cells, ATCC No. CCL-224
  • A549 human lung cancer cells, ATCC No. CCL-185
  • HepG2 human liver cancer cells, ATCC No. HB-8065
  • a culture solution of MCF7 human breast cancer cells, ATCC No. HTB-22
  • genomic DNA in the collected cells was extracted according to the protocol of the kit.
  • Single base mutation of PiS in ⁇ 1-antitrypsin deficiency is a single nucleotide substitution in which A at position 863 of the nucleotide sequence of ⁇ 1-antitrypsin gene is mutated to T. .
  • the R primer of the primer pair has the complementary base A of the mutated base T of the first strand at the 3' end.
  • the rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the first strand.
  • the F primer of the PiS mutation detection primer pair has the complementary base T of the mutated base A of the second strand at the 3' end.
  • the rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
  • the F primer of the PiS mutation detection primer pair used here has the mutated base T of the first strand at the 3' end.
  • the rest of the base sequence of the F primer is the same as the base sequence 5' from the mutated base T in the base sequence of the first strand.
  • the R primer of the PiS mutation detection primer pair has the complementary base A of the mutated base T of the first strand at the 3' end.
  • the rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence 3' from the mutated base T of the first strand.
  • the obtained amplification curve is shown in FIG. In FIG. 7, (1) is the result of using COLO201 genomic DNA as a template, (2) is the result of using HepG2 genomic DNA as a template, and (3) is the result of using A549 genomic DNA as a template. and (4) show the results using MCF7 genomic DNA as a template, respectively.
  • the amplification curve W shows the results using the wild-type detection primer pair
  • the amplification curve M shows the results using the PiS mutation type detection primer pair.
  • COLO201 (Fig. 7 (1)) and A549 (Fig. 7 (3)) Amplification curve using a wild-type detection primer pair and the amplification curves using the primer pair for mutation type detection were close to each other. From this, COLO201 and A549 could be determined to have heterozygous PiS mutations.
  • the amplification curve using the wild-type detection primer pair is the amplification using the mutation type detection primer pair. It rose earlier than the curve (detected earlier). From the above, the HepG2 cells and MCF7 cells were determined to be wild-type without PiS mutation.
  • Example 3 Mutation detection of ⁇ 1-antitrypsin-2 (PiZ) In Example 3, PiZ mutation was detected using the primer pair of the present invention.
  • genomic DNA COLO201 human colon cancer cells, ATCC No. CCL-224
  • A549 human lung cancer cells, ATCC No. CCL-185
  • HepG2 human liver cancer cells, ATCC No. HB-8065
  • a culture solution of MCF7 human breast cancer cells, ATCC No. HTB-22
  • genomic DNA in the collected cells was extracted according to the protocol of the kit.
  • Primer pair for PCR amplification of genomic DNA The following primer pair was designed and synthesized.
  • ⁇ PiZ wild type detection primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCG*A*C*G-3' (SEQ ID NO: 49)
  • ⁇ PiZ mutation detection primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCG*A*C*A-3' (SEQ ID NO: 51)
  • R primer: 5'-CCCCAGCAGCTTCAGTCCCTTT*C*T*T-3' SEQ ID NO: 52
  • the ⁇ 1-antitrypsin deficiency PiZ nucleotide mutation (PiZ: c.1096G>A p.Glu366Lys) is a single nucleotide substitution in which the G at position 1096 in the nucleotide sequence of the ⁇ 1-antitrypsin gene is mutated to A.
  • the R primer of the primer pair has the complementary base T of the mutated base A of the first strand at the 3' end.
  • the rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base A of the first strand.
  • the F primer of the PiZ mutation detection primer pair has the complementary base A of the mutated base T of the second strand at the 3' end.
  • the rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
  • the amplification curve W shows the results of using the wild-type detection primer pair
  • the amplification curve M shows the results of using the mutation-type detection primer pair.
  • Example 4 PiS single nucleotide mutation detection (1) Preparation of sample (control DNA) A partial nucleotide sequence of a region containing the base at position 863 of the human ⁇ 1-antitrypsin gene, and the base corresponding to position 863 is the mutated base T , an oligonucleotide having the base sequence (SEQ ID NO: 54) was designed and synthesized. This oligonucleotide was used as PiS mutant control DNA.
  • an oligonucleotide having a nucleotide sequence (SEQ ID NO: 53), which is a partial nucleotide sequence of a region containing the 863rd nucleotide of the human ⁇ 1-antitrypsin gene and in which the nucleotide corresponding to the 863rd nucleotide is wild-type A, was designed. and synthesized. This oligonucleotide served as the PiS wild-type control DNA.
  • S-primer pair The same S-primer pair as used in real-time PCR in Example 2 (3) (PiS wild-type detection primer pair: SEQ ID NO: 35-SEQ ID NO: 36 primer pair, PiS mutation type detection A primer pair for SEQ ID NO: 37-SEQ ID NO: 38) was used.
  • PCR reaction solution having the following composition was prepared. Amount of DNA control added: 10 pg for PiS wild-type control DNA only 10 pg for PiS mutant control DNA only 5 pg each for PiS mutant control DNA and PiS wild type control
  • Real-time PCR 20 ⁇ L of the reaction solution for PCR prepared in 1) above is placed in wells of a 96-well reaction plate (Hard-Shell TM Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad), Real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen TM was measured.
  • CFX-96 real-time PCR device
  • FIG. 9(1) shows the results using only PiS wild-type control DNA as a sample. This result is a model for the wild-type PiS gene.
  • FIG. 9(2) shows the results of using a mixed solution of PiS mutant control DNA and PiS wild-type control DNA as a sample. This result is a model for heterozygous PiS mutations.
  • FIG. 9(3) shows the results using only the PiS mutant control DNA as a sample. This result is a model for homozygous PiS mutations.
  • the amplification curve W shows the results using the wild-type detection primer pair
  • the amplification curve M shows the results using the PiS mutation type detection primer pair.
  • the appearance of the amplification curve differs depending on the wild type, homozygous mutation, and heterozygous mutation. It can be seen that whether the ⁇ 1-antitrypsin gene of the subject to be tested in the test sample is wild-type, homozygous PiS mutation, or heterozygous PiS mutation can be discriminated.
  • Example 5 PiZ single nucleotide mutation detection (1) sample preparation A nucleotide sequence ( An oligonucleotide having SEQ ID NO: 56) was designed and obtained by standard methods. This oligonucleotide was used as the PiZ mutant control DNA. In addition, an oligonucleotide having a nucleotide sequence (SEQ ID NO: 55), which is a partial nucleotide sequence of a region containing the 1096th nucleotide of the human ⁇ 1-antitrypsin gene and in which the nucleotide corresponding to the 1096th nucleotide is a wild-type G, is designed. and obtained by a conventional method. This oligonucleotide served as the PiZ wild-type control DNA.
  • SEQ ID NO: 55 which is a partial nucleotide sequence of a region containing the 1096th nucleotide of the human ⁇ 1-antitrypsin gene and in which the nucleotide corresponding to
  • S-primer pair The same S-primer pair as used in real-time PCR in Example 3 (4) (primer pair for wild type detection: primer pair of SEQ ID NO: 49-SEQ ID NO: 50, for detecting PiZ mutation type Primer pair: primer pair of SEQ ID NO:51-SEQ ID NO:52) was used.
  • PCR reaction solution having the following composition was prepared. Amount of DNA control added: 10 pg for PiZ wild-type control DNA only 10 pg for PiZ mutant control DNA only 5 pg each for PiZ mutant control DNA and PiZ wild type control
  • Real-time PCR 20 ⁇ L of the reaction solution for PCR prepared in 1) above is placed in the wells of a 96-well reaction plate (Microamp Optical 96-well Reaction Plate, Applied Biosystems Japan), and a thermal cycler for TaqMan TM PCR detection is used.
  • Real-time PCR was performed using a device (ABI 7500, manufactured by Applied Biosystems Japan). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen TM was measured.
  • FIG. 10(1) shows the results using only PiZ wild-type control DNA as a sample. This result is a model for the wild-type PiZ gene.
  • FIG. 10(2) shows the results of using a mixed solution of PiZ mutant control DNA and PiZ wild-type control DNA as a sample. This result is a model for heterozygous PiZ mutations.
  • FIG. 10(3) shows the results of using only the PiZ mutant control DNA as a sample. This result is a model for homozygous PiZ mutations.
  • the amplification curve W shows the results using the wild-type detection primer pair
  • the amplification curve M shows the results using the PiZ mutation type detection primer pair.
  • the appearance of the amplification curve differs depending on the wild type, homozygous mutation, and heterozygous mutation. It can be seen that whether the ⁇ 1-antitrypsin gene of the test sample is a wild type, a PiZ homozygous mutation, or a PiZ heterozygous mutation can be discriminated.
  • Example 6 Detection of PiS 1 base mutation using human oral swab and saliva sample (1) Preparation of sample 10 ⁇ L of human oral swab (purchased from Busicom Japan) was placed in a tube and treated at 95° C. for 6 minutes in a hot water bath. ⁇ 10 ⁇ L of human saliva (Japanese) was placed in a tube and treated in a hot water bath at 95°C for 6 minutes.
  • PCR amplification 20 ⁇ L of the PCR reaction solution prepared in 2) above was added to a 96-well reaction plate (Hard-Shell TM Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad). It was placed in wells and real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). PCR was carried out by incubating at 98°C for 20 seconds, followed by 36 cycles of reaction at 98°C for 8 seconds and 68°C for 20 seconds.
  • CFX-96 real-time PCR device
  • FIGS. 11 and 12 show the results when mouthwash was used as a sample.
  • FIG. 11 shows the results when mouthwash was used as a sample.
  • FIG. 11 shows the results of mouthwash was used as a sample.
  • FIG. 11 shows the results of saliva was used as a sample.
  • FIG. 12 shows the results when saliva was used as a sample.
  • FIG. 12 shows the results of four specimens, respectively.
  • W indicates the amplification curve obtained using the PiS wild-type detection primer pair
  • M indicates the amplification curve obtained using the PiS mutation type detection primer pair.
  • specimens (1), (2), and (4) were determined to be wild-type (wild-type) with respect to the PiS mutation.
  • the amplification curve W and the amplification curve M rose close to each other (detected at about the same speed), and had the same pattern as in FIG. 9(2). Therefore, specimen (3) was determined to be a heterozygous PiS mutation.
  • specimens (1) to (4) were determined to be wild-type (wild-type) with respect to the PiS mutation.
  • Example 7 PiZ Single Nucleotide Mutation Detection Using Human Oral Wipes and Saliva Samples (1) Preparation of Samples Using the same human oral swabs and human saliva as used in Example 6, pretreatment by heat treatment was performed in the same manner.
  • PCR amplification 20 ⁇ L of the reaction mixture for PCR prepared in 2) above was poured into the wells of a 96-well reaction plate (Hard-Shell TM Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad). , and PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). PCR was carried out by incubating at 98°C for 20 seconds, followed by 36 cycles of reaction at 98°C for 8 seconds and 68°C for 20 seconds.
  • CFX-96 real-time PCR device
  • FIGS. 13 and 14 show the results when a mouthwash was used as a sample.
  • FIG. 13 shows the results when a mouthwash was used as a sample.
  • FIG. 13 shows the results of four specimens, respectively.
  • Fig. 14 shows the results when saliva was used as a sample.
  • (1) to (4) show the results of four specimens, respectively.
  • W indicates the amplification curve obtained using the PiZ wild-type detection primer pair
  • M indicates the amplification curve obtained using the PiZ mutation type detection primer pair.
  • specimens (1) to (4) were all determined to be wild-type for the PiZ mutation.
  • specimens (1) to (4) were all determined to be wild-type for the PiZ mutation.
  • Example 8 Detection of Nucleotide Sequence Mutations in Cats In cats, the following single-nucleotide substitutions in genes are known.
  • PKD1 c.9864C>A
  • PKLR c.693 + 304G > A
  • the primer pairs of the present invention were used to detect feline PKD1 and PKLR gene mutations.
  • Example 2 (2) Preparation of PCR reaction mixture and 1st-PCR
  • the sample-derived genomic DNA was used as a template to prepare a PCR reaction solution and perform PCR amplification to obtain a PCR amplification product.
  • a region containing base 9864 of the PKD1 gene is amplified.
  • a region containing bases at positions 693+304 of the PKLR gene is amplified.
  • ⁇ PKD1 wild type detection primer pair F primer: 5'-GTCCAGCGGGCCACCTGT*T*G*C-3' (SEQ ID NO: 61) R primer: 5'-CAGGAAGAGGCAGACGAGGAGG*A*C*G-3' (SEQ ID NO: 62) ⁇ PKD1 mutation detection primer pair F primer: 5'-GTCCAGCGGGCCACCTGT*T*G*A-3' (SEQ ID NO: 63) R primer: 5'-CAGGAAGAGGCAGACGAGGAGG*A*C*T-3' (SEQ ID NO: 64) ⁇ PKLR wild type detection primer pair F primer: 5'-CCCCGTGCCCCCGTCCC*A*C*G-3' (SEQ ID NO: 65) R primer: 5'-GTCAGGGGCGAGCCGGGGGCAGA*G*T*C-3' (SEQ ID NO: 66) ⁇ PKLR mutation detection primer pair F primer: 5'-CCCCGTGCCCCCGTCCC*A*C*A
  • FIGS. 15 and 16 Results The obtained amplification curves are shown in FIGS. 15 and 16.
  • FIG. 15 shows the results of using, as a template, genomic DNA derived from cats in which the PKD1 gene and PKLR gene have been confirmed to be wild-type.
  • Fig. 16 shows the results of using, as a template, genomic DNA derived from cats in which the PKD1 gene was confirmed to be wild-type and the PKLR gene was confirmed to be mutant.
  • the amplification curve W shows the results using the wild-type detection primer pair
  • the amplification curve M shows the results using the PKD1 mutation detection primer pair or the PKLR mutation detection primer pair.
  • a primer pair for detecting the PKD1 wild-type is used.
  • the amplification curve using the PKD1 mutation type rose earlier than the amplification curve using the primer pair for detecting the PKD1 mutation (detected earlier).
  • the amplification curve using the PKLR wild-type detection primer pair rose earlier than the amplification curve using the PKLR mutation type detection primer pair (earlier detection was done). Based on the above, it was determined that the subject cat was a wild-type cat with no PKD1 mutation or PKLR mutation.
  • the PKD1 gene is wild-type, and when genomic DNA derived from a cat that has been confirmed to have a PKLR gene mutation is used as a template (Fig. 16), as is clear from Fig. 16 (1), a PKD1 wild-type detection primer The amplification curve using the pair rose earlier (detected earlier) than the amplification curve using the primer pair for detecting the PKD1 mutation type. On the other hand, as is clear from FIG. 15(2), the amplification curve using the PKLR wild-type detection primer pair and the amplification curve using the PKLR mutation type detection primer pair were close to each other. From the above, it was determined that the subject cat was wild-type with respect to PKD1, but had a PKLR mutation, and that the mutation was heterozygous.
  • Example 9 Detection of Nucleotide Sequence Mutations in Dogs Dogs are known to have a 4-nucleotide deletion mutation that lacks GTTT at positions 4411950-4411953 of the VPS13B gene (g.4411950_4411953delGTTT).
  • the primer pair of the present invention was used to detect canine VPS13B gene mutation.
  • Primer pair for PCR amplification of genomic DNA The following primer pair was designed and synthesized.
  • ⁇ VPS13B wild type detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGCTTG*T*T-3' (SEQ ID NO: 71) R primer: 5'-GTCTACTGGTTCGTTTCTGAGGCTGATAA*A*C*A-3' (SEQ ID NO: 72) ⁇ VPS13B mutation detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGC*T*T*A-3' (SEQ ID NO: 73) R primer: 5'-GTCTACTGGTTCGTTTCTGAGGCTGAT*A*A*G-3' (SEQ ID NO: 74)
  • amplification curve W shows the results using the wild-type detection primer pair
  • amplification curve M shows the results using the VPS13B mutation detection primer pair.
  • the amplification curve W and the amplification curve M using samples derived from Chihuahua and seeds are overlapped, so they appear to be one.
  • Example 10 Determination of N501Y and E484K Mutations of SARS-CoV-2 Virus We focused on the following mutations of SARS-CoV-2 virus.
  • N501Y mutation Mutation identified in British, South African, and Brazilian variants. It is a single nucleotide substitution of A23063T in the nucleotide sequence of the SARS-CoV-2 virus (GenBank, Accession No. MN908947.3). That is, it is a mutation in which the 23063rd base of the RNA of the SARS-CoV-2 virus is mutated from A to T, and a mutation in which the 501st amino acid asparagine of the spike protein is mutated to tyrosine.
  • the N501Y mutant of the SARS-CoV-2 virus causes protein mutations in the receptor-binding portion of the spike protein, resulting in increased binding affinity with the human and mouse receptor ACE2.
  • E484K mutation A mutation identified in the South African variant and the Brazilian variant. It is a single nucleotide substitution of G23012A in the nucleotide sequence of the SARS-CoV-2 virus. That is, the 23012th base of the SARS-CoV-2 virus RNA is mutated from G to A, and the 484th amino acid glutamic acid of the spike protein is mutated to lysine.
  • the E484K variant of the SARS-CoV-2 virus may neutralize antibodies generated by vaccination.
  • F primer for E484K mutation 5'-CCAGATGATTTTACAGGCTGCGTTATAGC-3' (SEQ ID NO: 77)
  • R primer 5'-CAAACAGTTGCTGGTGCATGTAGAAGTTC-3' (SEQ ID NO: 78)
  • This 1st-PCR amplifies the region containing the mutated portion of each mutation in the nucleic acid of the SARS-CoV-2 viral gene.
  • the region containing the portion corresponding to each mutation portion is amplified.
  • N501Y mutation detection/N501Y wild type detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*A-3' (SEQ ID NO: 79) R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*T-3' (SEQ ID NO: 80) ⁇ N501Y mutation detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*T-3' (SEQ ID NO: 81) R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*A-3' (SEQ ID NO: 82)
  • E484K mutation detection/E484K wild type detection primer pair F primer: 5'-CGGTAGCACACCTTGTAATGGTG*T*T*G-3' (SEQ ID NO: 83) R primer: 5'-CCATATGATTGTAAAGGAAAGTAACAATTAAAACC*T*T*C-3' (SEQ ID NO: 84) ⁇ E484K mutation detection primer pair F primer: 5'-CGGTAGCACACCTTGTAATGGTG*T*T*A-3' (SEQ ID NO: 85) R primer: 5'-CCATATGATTGTAAAGGAAAGTAACAATTAAAACC*T*T*T-3' (SEQ ID NO: 86)
  • ii) real-time PCR That is, 20 ⁇ L of the PCR reaction solution prepared in i) above was placed in the wells of a 96-well reaction plate (Hard-Shell TM Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad).
  • Real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). That is, after incubating at 94°C for 2 minutes, reaction at 98°C for 5 seconds and at 68°C for 10 seconds was repeated for 40 cycles. Then, fluorescence intensity derived from EvaGreen TM was measured.
  • FIG. 18 shows the results of detecting the N501Y mutation.
  • FIG. 19 shows the results of detecting the E484K mutation.
  • FIGS. 18 and 19 (1) the results obtained using DNA having the base sequence of the wild strain of the SARS-CoV-2 virus, (2) the N501Y mutation of the SARS-CoV-2 virus or Results obtained using DNA having the base sequence of the E484K mutation are shown respectively.
  • W indicates the results obtained using the wild-type detection primer pair
  • M indicates the results obtained using the mutation-type detection primer pair.

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