Gene mutation assessment kit

Abstract

To identify gene mutations in MYD88 and / or CD79B simultaneously.SOLUTION: The present invention comprises: a primer set for MYD88 gene mutations, and a primer set for CD79B gene mutations; as well as a mutant-type probe for MYD88 gene, a wild-type probe for MYD88 gene, a mutant-type probe for CD79B gene, and a wild-type probe for CD79B gene.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a gene mutation evaluation kit comprising a primer set for amplifying a nucleic acid region containing a mutation for a predetermined gene mutation and a probe for detecting the amplified nucleic acid fragment.

Background Art

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoma subtype and can occur in any organ where physiologically mature B cells are present. DLBCL has been found to be a collection of heterogeneous disease groups with diverse molecular genetic backgrounds. The diagnosis of DLBCL is made based on the image of large B-cell-derived tumor cells proliferating diffusely in the biopsy tissue.

[0003] In recent years, genetic classification based on gene mutations in DLBCL has been clarified. Specifically, it is classified into four subtypes: MCD subtype (L265P mutation of MYD88 and CD79B mutation), BN2 subtype (BCL6 fusion and NOTCH2 mutation), N1 subtype (NOTCH1 mutation), and EZB subtype (EZH2 mutation and BCL2 translocation). Among these, for DLBCL of the MCD subtype, as described in Non-Patent Document 1 and Patent Document 1, it has been suggested that the R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) combined with ibrutinib, a kind of BTK inhibitor, is effective.

[0004] Furthermore, according to the WHO classification (2017), lymphoplasmacytic lymphoma (LPL) is defined as a lymphoid tumor in which small B-cell lymphocytes, lymphocytes with a tendency to differentiate into plasma cells, and plasma cells are mixed, regardless of the presence or absence of IgM-type M protein. Waldenstrom's macroglobulinemia (WM) is defined as a subset of LPL accompanied by bone marrow infiltration and IgM-type M proteinemia. And mutations in the MYD88 gene are observed in about 90% of WM / LPL patients, and identifying MYD88 gene mutations is useful for a definitive diagnosis.

[0005] By the way, the gene mutations as described above generally mean a state in which a gene has some kind of abnormality either congenitally or acquiredly. Examples of gene mutations include a state in which the bases constituting DNA are substituted, deleted, or added. Here, a gene is composed of a coding region that encodes a protein and non-coding regions such as an expression control region. Gene mutations exist in both the coding region and the non-coding region. Among them, gene mutations in the coding region include missense mutations in which the encoded amino acid changes, nonsense mutations in which the codon encoding a predetermined amino acid becomes a stop codon, and silent mutations in which the encoded amino acid does not change.

[0006] Gene mutations are the cause of genetic diseases and cancers, and are also related to the efficacy of specific drugs. In addition, gene mutations are related to physical constitutions such as obesity. Therefore, identifying specific gene mutations (also referred to as genotyping) is an indispensable technique for diagnosing genetic diseases and knowing the efficacy of drugs.

[0007] As genotyping methods, for example, in addition to DNA sequencing, there are the SSCP (Single Strand Conformation Polymorphism) method, the RFLP (Restriction Fragment Length Polymorphism) method, the PCR (Polymerase Chain Reaction) method, the AFLP (Amplified Fragment Length Polymorphism) method, the ASO (Allele Specific Oligonucleotide) probe method, methods for detecting binding to DNA microarrays and DNA beads, and so on. Among these techniques, in the method using a DNA microarray, the DNA microarray usually has mutant probes and wild-type probes fixed on a carrier for the gene mutations to be detected.

[0008] In the method using a DNA microarray, first, a region having a gene mutation to be detected is amplified by a nucleic acid amplification reaction using a primer having a fluorescent label. Then, a hybridization reaction is performed between the nucleic acid fragment having the fluorescent label, the mutant probe, and the wild-type probe. When the amplified nucleic acid fragment contains a mutant type, fluorescence is observed from the mutant probe. Therefore, by observing the fluorescence from the mutant probe and the wild-type probe, genotyping of the gene mutation can be performed.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] By the way, a technique for simultaneously identifying gene mutations in MYD88 and / or gene mutations in CD79B described above is not known. If this becomes possible, for example, the MCD subtype in DLBCL can be easily identified. Therefore, an object of the present invention is to provide a kit for gene mutation evaluation that can simultaneously identify gene mutations in MYD88 and / or gene mutations in CD79B.

Means for Solving the Problems

[0012] The method for detecting gene mutations according to the present invention that has achieved the above object includes the following.

[0013] (1) A kit for gene mutation evaluation used for the diagnosis of diseases related to gene mutations in the MYD88 gene and / or gene mutations in the CD79B gene, comprising a primer set for MYD88 gene mutation that amplifies a nucleic acid region containing a gene mutation in the MYD88 gene and a primer set for CD79B gene mutation that amplifies a nucleic acid region containing a gene mutation in the CD79B gene, a mutant probe for the MYD88 gene related to the gene mutation in the MYD88 gene, a wild-type probe for the MYD88 gene, a mutant probe for the CD79B gene related to the gene mutation in the CD79B gene, and a wild-type probe for the CD79B gene.

[0014] (2) The gene mutation evaluation kit according to (1), wherein the gene mutation in the MYD88 gene is a substitution mutation of leucine at position 265 to proline, and the gene mutation in the CD79B gene is a missense mutation and / or nonsense mutation of tyrosine residue at position 196 and a silent mutation of the tyrosine residue.

[0015] (3) The primer set for MYD88 gene mutation is a set consisting of a forward primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 and a reverse primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 78, and the primer set for CD79B gene mutation is a set consisting of a forward primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 85 and a reverse primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 86, which is characterized in the gene mutation evaluation kit described in (1).

[0016] (4) The reverse primer for the MYD88 gene has a label and is at a concentration 2 to 6 times that of the forward primer for the MYD88 gene, and the forward primer for the CD79B gene has a label and is at a concentration 5 to 10 times that of the reverse primer for the CD79B gene, which is characterized in the gene mutation evaluation kit described in (3).

[0017] (5) The mutant probe for the MYD88 gene, the wild-type probe for the MYD88 gene, the mutant probe for the CD79B gene, and the wild-type probe for the CD79B gene contain the nucleotide sequences shown in Table 1, which is characterized in the gene mutation evaluation kit described in (1).

Table 1

[0018] (6) The gene mutation evaluation kit described in (1) further comprises a blocking nucleic acid for MYD88 that hybridizes to a nucleic acid fragment having a wild type among the nucleic acid fragments amplified by the primer set for MYD88 gene mutation, and / or a blocking nucleic acid for CD79B that hybridizes to a nucleic acid fragment having a wild type among the nucleic acid fragments amplified by the primer set for CD79B gene mutation.

[0019] (7) The blocking nucleic acid for MYD88 consists of the nucleotide sequence of SEQ ID NO: 89, and the blocking nucleic acid for CD79B consists of the nucleotide sequence of SEQ ID NO: 95, which is characterized in the gene mutation evaluation kit described in (6).

[0020] (8) The blocking nucleic acid for MYD88 is prepared so as to be in the range of 0 to 500 nM in the hybridization solution, and the blocking nucleic acid for CD79B is prepared so as to be in the range of 125 to 1000 nM in the hybridization solution, and the gene mutation evaluation kit according to (6).

Effects of the Invention

[0021] According to the gene mutation evaluation kit of the present invention, gene mutations in MYD88 and gene mutations in CD79B can be simultaneously identified. By using the gene mutation evaluation kit according to the present invention, based on gene mutations in MYD88 and / or gene mutations in CD79B, for example, the MCD subtype in DLBCL can be easily identified.

Brief Description of the Drawings

[0022]

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Best Mode for Carrying Out the Invention

[0023] Hereinafter, the gene mutation evaluation kit according to the present invention will be described in detail. The gene mutation evaluation kit simultaneously identifies at least a gene mutation in the MYD88 gene and a gene mutation in the CD79B gene. Therefore, the gene mutation evaluation kit can be used for the diagnosis of diseases associated with gene mutations in the MYD88 gene and / or the CD79B gene. The MYD88 gene and the CD79B gene are genes encoding proteins related to the NF-κB signaling pathway, and it is considered that the NF-κB pathway is constantly activated by gene mutations in these genes, leading to the occurrence of lymphoma and the like. For example, the MYD88 L265P mutation is frequently observed in lymphoplasmacytic lymphoma (LPL) / Waldenstrom macroglobulinemia (WM). In addition, the MYD88 L265P mutation and the CD79B Y196 mutation are frequently observed in diffuse large B-cell lymphoma (DLBCL).

[0024] Here, the gene mutation evaluation kit includes a primer set for amplifying a nucleic acid region containing a gene mutation and a probe set for detecting the amplified nucleic acid fragment. That is, the gene mutation evaluation kit includes a primer set for MYD88 gene mutation for amplifying a nucleic acid region containing a gene mutation in the MYD88 gene and a primer set for CD79B gene mutation for amplifying a nucleic acid region containing a gene mutation in the CD79B gene. In addition, the gene mutation evaluation kit includes a probe set consisting of a mutant probe for the MYD88 gene and a wild-type probe for the MYD88 gene related to the gene mutation in the MYD88 gene, and a probe set consisting of a mutant probe for the CD79B gene and a wild-type probe for the CD79B gene related to the gene mutation in the CD79B gene.

[0025] MYD88 is a cytoplasmic adapter protein that plays a central role in innate and adaptive immune responses and functions as an essential signaling molecule in the interleukin-1 and Toll-like receptor signaling pathways. MYD88 is also known as IMD68, MYD88D, and WM1. A gene mutation in the MYD88 gene is a missense mutation that results in proline due to a base substitution in the codon (CTG) encoding leucine located at the 265th position from the N-terminus. The missense mutation is caused by a mutation (794T>C) in which the 794th thymine from the 5' end of the region encoding MYD88 is replaced by cytosine.

[0026] On the other hand, CD79B exists as a heterodimeric complex with CD79A and associates with cell surface immunoglobulins to form the B cell antigen receptor. CD79B is also known as B29, Ig-β, and AGM6. A gene mutation in the CD79B gene means three types of gene mutations, namely, a missense mutation that results in a different amino acid, a nonsense mutation that results in a stop codon, and a silent mutation in which the encoded amino acid remains tyrosine, due to a base substitution in the codon (TAC) encoding tyrosine located at the 196th position from the N-terminus.

[0027] Here, the missense mutations in the CD79B gene are mutations in which the 589th thymine from the 5' end of the region encoding CD79B is replaced by cytosine (this mutation is described as 589T>C; hereinafter, A: adenine, G: guanine, C: cytosine, T: thymine, and the same applies), 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T. The nonsense mutations in the CD79B gene are 591C>G and 591C>A. Furthermore, the silent mutation in the CD79B gene is 591C>T.

[0028] The mutations for thymine at position 196 in these CD79B genes are summarized in Table 2. In Table 2, for the wild-type sequence, it is denoted as W in the type column, M1 - M6 in the same column are missense mutations, M7 and M8 are nonsense mutations, and M9 is a silent mutation.

[0029]

Table 2

[0030] 1) Primer set The primer set included in the gene mutation evaluation kit according to the present invention includes a primer set for MYD88 gene mutation that amplifies a nucleic acid region containing a gene mutation in the MYD88 gene, and a primer set for CD79B gene mutation that amplifies a nucleic acid region containing a gene mutation in the CD79B gene.

[0031] The primer set for MYD88 gene mutation and the primer set for CD79B gene mutation are designed based on the fact that, as shown in the examples described later, a desired amount of nucleic acid fragment is amplified by each primer set in the same reaction system, and there is little non-specific amplification. Specifically, when performing a nucleic acid amplification reaction using a plasmid containing the nucleic acid region to be amplified as a template and when performing a nucleic acid amplification reaction using genomic DNA containing the nucleic acid region to be amplified as a template, a primer set is designed such that the amount (molar concentration) of the obtained nucleic acid fragment is close. Also, after performing the nucleic acid amplification reaction, a primer set with little non-specific amplification can be designed by detecting the nucleic acid fragments contained in the reaction solution by a method such as electrophoresis.

[0032] As an example, the primer set for MYD88 gene mutation can be a set consisting of a forward primer for the MYD88 gene having the nucleotide sequence of SEQ ID NO: 79 (5’-CAGGTGCCCATCAGAAG-3’) and a reverse primer for the MYD88 gene having the nucleotide sequence of SEQ ID NO: 78 (5’-CGCAGACAGTGATGAACC-3’), and the primer set for CD79B gene mutation can be a set consisting of a forward primer for the CD79B gene having the nucleotide sequence of SEQ ID NO: 85 (5’-CAGGATGACAGCAAGGC-3’) and a reverse primer for the CD79B gene having the nucleotide sequence of SEQ ID NO: 86 (5’-CAACCACACCAGCAGATAG-3’).

[0033] The primer sets defined by these nucleotide sequences are designed such that the Tm values of the forward primer and the reverse primer are close to each other. Therefore, within a range where the difference in the Tm values between the forward primer and the reverse primer does not become large, the nucleotide sequence can be one with one or more nucleotides added to the 5'-end and / or 3'-end, or one with one or more nucleotides deleted from the 5'-end and / or 3'-end.

[0034] The Tm value of the primer can be calculated by the nearest neighbor method or software applying the nearest neighbor method. The difference in the Tm values between the forward primer and the reverse primer is, for example, within 3°C, preferably within 2.5°C, more preferably within 2.0°C, still more preferably within 1.5°C, still more preferably within 1.0°C, and still more preferably within 0.5°C.

[0035] The base length of the primer can usually be 17 to 25 bases. Therefore, when adding one or more bases to the 5' end and / or 3' end of the specific base sequence described above, it is preferable to be within this range. Also, the one or more bases to be added can be determined based on the base sequences of the MYD88 gene (NCBI Gene ID: 4615) and the CD79B gene (NCBI Gene ID: 974) registered in known databases.

[0036] In addition, it is preferable that both the primer set for MYD88 gene mutation and the primer set for CD79B gene mutation have a label on either the forward primer or the reverse primer. By having a label on the forward primer or the reverse primer, a nucleic acid fragment having the label can be amplified. Although not particularly limited, the reverse primer for the MYD88 gene in the primer set for MYD88 gene mutation can have a label, and the forward primer for the CD79B gene in the primer set for CD79B gene mutation can have a label.

[0037] Here, the label is not particularly limited, but it means binding a labeling substance such as a radioisotope, a fluorescent dye, or an organic compound such as digoxigenin (DIG) or biotin to the above-mentioned primer. As the labeling substance, various conventionally known labeling substances can be appropriately selected and used. In particular, it is preferable to use fluorescent dyes such as indocyanine compounds (IC5-OSu, IC5-PE-maleimide, IC3-OSu, and IC3-PE-maleimide), BDP, cyanine dyes, fluorescein dyes, pyrene dyes, and rhodamine dyes, which can be detected simply and with high sensitivity.

[0038] Furthermore, the ratio (concentration ratio) of the forward primer to the reverse primer included in the primer set for MYD88 gene mutation is not particularly limited, but it is preferably a ratio such that the one with a label is larger than the one without a label. For example, when the reverse primer has a label, the ratio of the reverse primer to the forward primer (reverse primer concentration: forward primer concentration) can be 15:10, preferably 20:10, more preferably 30:10, and most preferably 40:10. Incidentally, the said ratio (reverse primer concentration: forward primer concentration) may be 50:10 or 60:10. That is, when the reverse primer has a label, the concentration of the reverse primer can be 1.5 to 6 times that of the forward primer, particularly preferably 2 to 6 times, more preferably 3 to 5 times, and most preferably 4 times. The most excellent fluorescence intensity value was achieved at this time. By setting such a ratio, the fluorescence intensity derived from the amplification product obtained with the primer set for MYD88 gene mutation can be increased.

[0039] In addition, the ratio (concentration ratio) of the forward primer to the reverse primer included in the primer set for CD79B gene mutation is not particularly limited, but it is preferably a ratio such that the primer with a label is larger than the primer without a label. For example, when the forward primer has a label, the ratio of the forward primer to the reverse primer (forward primer concentration: reverse primer concentration) can be 15:10, can be 20:10, can be 30:10, can be 40:10, and can be 50:10. Further, the ratio is preferably 60:10, and more preferably 70:10. Furthermore, the ratio can be 80:10, can be 90:10, and can be 100:10. That is, when the forward primer has a label, the concentration of the forward primer can be 1.5 to 10 times that of the reverse primer, particularly preferably 5 to 10 times, more preferably 6 to 7 times, and most preferably 7 times. By setting such a ratio, the fluorescence intensity derived from the amplification product obtained with the primer set for CD79B gene mutation can be increased.

[0040] 2) Probe The probe set included in the gene mutation evaluation kit according to the present invention includes a probe set related to gene mutation in the MYD88 gene and a probe set related to gene mutation in the CD79B gene. Each probe set includes a mutant probe corresponding to the mutant type in the gene mutation to be detected and a wild-type probe corresponding to the wild-type in the gene mutation. When the gene mutation to be detected has multiple mutant patterns, multiple mutant probes corresponding to all of the multiple mutant patterns may be prepared, or one or more mutant probes corresponding to a specific mutant type among the multiple mutant patterns may be prepared.

[0041] Hereinafter, each probe set will be described. The mutant probes and wild-type probes included in these probe sets are preferably nucleic acids, more preferably DNA. DNA includes both double-stranded and single-stranded, but preferably single-stranded DNA. The mutant probes and wild-type probes can be obtained, for example, by chemically synthesizing them using a nucleic acid synthesizer. As the nucleic acid synthesizer, devices called DNA synthesizers, fully automatic nucleic acid synthesizers, nucleic acid automatic synthesizers, etc. can be used.

[0042] Probe set related to gene mutation of MYD88 gene The probe set related to the gene mutation of the MYD88 gene can be designed based on the base sequence of the nucleic acid fragment amplified by the nucleic acid amplification reaction using the above-described primer set for MYD88 gene mutation. That is, the mutant probes and wild-type probes included in the probe set can be designed as base sequences complementary to the region containing the site of the gene mutation contained in the amplified nucleic acid fragment. As described above, in the gene mutation of the MYD88 gene, the wild-type has a T at the 794th base, and the mutant type has a C at the 794th base in the gene mutation. Therefore, the wild-type probe and the mutant probe will contain the base corresponding to this 794th base.

[0043] The wild-type probe and the mutant probe may be designed such that only the base corresponding to the gene mutation is different and the rest are all the same, but they may also be designed to have different base lengths from each other, or the 3'-end and / or 5'-end may be different. The wild-type probe and the mutant probe are not particularly limited, but for example, they can have a base length of 10 to 30 bases, and preferably a base length of 15 to 25 bases. Also, in the wild-type probe and the mutant probe, the position of the base corresponding to the gene mutation is preferably the center position when the bases constituting the wild-type probe and the mutant probe are regarded as a character string. Note that the center of the character string means including the case where it is shifted by one in the 5'-end or 3'-end direction for the wild-type probe and the mutant probe consisting of an even number of bases.

[0044] More specifically, for the nucleic acid fragment obtained by nucleic acid amplification reaction using a set of a forward primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 (5'-CAGGTGCCCATCAGAAG-3') and a reverse primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 78 (5'-CGCAGACAGTGATGAACC-3'), a wild-type probe for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 2 (5'-AGCGACTGATCCCCAT-3') and a mutant probe for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 6 (5'-GAAGCGACCGATCCC-3') can be designed. According to these wild-type probe and mutant probe for the MYD88 gene, since the fluorescence intensity derived from the amplified nucleic acid fragment is high and the resolution is high, the gene mutation of the test subject can be detected with high precision. Here, the resolution means the difference in the determination value for each sample calculated according to the following formula 1 by amplifying nucleic acid fragments using samples with different mutation ratios, detecting each nucleic acid fragment with a wild-type probe and a mutant probe. As samples with different mutation ratios, a 100% wild-type sample and a 5% mutant sample can be used. Formula 1: [Signal intensity from mutant probe] / ([Signal intensity from wild-type probe] + [Signal intensity from mutant probe])

[0045] Probe set related to gene mutation of CD79B gene The probe set related to the gene mutation of the CD79B gene can be designed based on the nucleotide sequence of the nucleic acid fragment amplified by the nucleic acid amplification reaction using the primer set for CD79B gene mutation described above. That is, the mutant probe and the wild-type probe included in the probe set can be designed as nucleotide sequences complementary to the region containing the site of the gene mutation contained in the amplified nucleic acid fragment. As described above, in the wild type of the gene mutation of the CD79B gene, the 589th base is T, the 590th base is A, and the 591st base is C. In addition, in the mutant type of the gene mutation, as shown in Table 2 above, there are missense mutations shown as M1 to M6, nonsense mutations shown as M7 and M8, and silent mutations shown as M9.

[0046] The wild-type probe and the mutant probe may be designed such that only the bases corresponding to the gene mutation are different and the rest are all the same, but they may also be designed to have different base lengths or different 3'-ends and / or 5'-ends. The wild-type probe and the mutant probe are not particularly limited, but for example, they can have a length of 10 to 30 bases, and preferably a length of 15 to 25 bases. In addition, in the wild-type probe and the mutant probe, the position of the base corresponding to the gene mutation is preferably the center position of the character string when the bases constituting the wild-type probe and the mutant probe are regarded as a character string. Note that the center of the character string means including the case where it is shifted by one in the 5'-end or 3'-end direction for the wild-type probe and the mutant probe consisting of an even number of bases.

[0047] Regarding gene mutations in the CD79B gene, mutant probes are designed for each of the nine mutant types described above. More specifically, for the nucleic acid fragment obtained by nucleic acid amplification reaction using a set of a forward primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 85 (5’-CAGGATGACAGCAAGGC-3’) and a reverse primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 86 (5’-CAACCACACCAGCAGATAG-3’), although not particularly limited, probes consisting of the nucleotide sequences shown in Table 3 can be designed. In the probe nucleotide sequences described in Table 3, the portion corresponding to the codon encoding the 196th amino acid from the N-terminus is underlined.

[0048]

Table 3

[0049] In the following description, mutant probes for detecting missense mutations (M1 to M6) and nonsense mutations (M7 and M8) may be referred to as the first probes, and probes for detecting silent mutations (M9) may be referred to as the second probes.

[0050] According to the wild-type probes and mutant probes designed for gene mutations of the CD79B gene shown in Table 3, since the fluorescence intensity derived from the amplified nucleic acid fragment is high and the resolution is high, gene mutations of the test subject can be detected with high precision. Here, the resolution means the difference in the determination values for each sample calculated according to the following formula 1 by amplifying nucleic acid fragments using samples with different mutation ratios, detecting each nucleic acid fragment with wild-type probes and mutant probes. As samples with different mutation ratios, a 100% wild-type sample and a 5% mutant sample can be used. Formula 1: [Signal intensity from mutant probe] / ([Signal intensity from wild-type probe] + [Signal intensity from mutant probe])

[0051] DNA chip (DNA microarray) The wild-type probe and the mutant probe designed as described above are preferably used in the form of a microarray (for example, a DNA chip) by immobilizing their 5'-ends on a carrier. At this time, the microarray has a first probe, a second probe, and a wild-type probe for the gene mutation to be examined. Note that the microarray may have a wild-type probe and a mutant probe for the gene mutation of the MYD88 gene and the gene mutation of the CD79B gene, respectively. Alternatively, a microarray having a wild-type probe and a mutant probe for the gene mutation of the MYD88 gene and a microarray having a wild-type probe and a mutant probe for the gene mutation of the CD79B gene may be prepared.

[0052] As the material of the carrier, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten, and their compounds, and conductive materials such as carbon typified by graphite and carbon fiber; silicon materials typified by single-crystalline silicon, amorphous silicon, silicon carbide, silicon oxide, and silicon nitride, and composite materials of these silicon materials typified by SOI (silicon-on-insulator); inorganic materials such as glass, quartz glass, alumina, sapphire, ceramics, forsterite, and photosensitive glass; organic materials such as polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene copolymer, polyphenylene oxide, and polysulfone. The shape of the carrier is not particularly limited, but is preferably flat.

[0053] As the carrier, preferably a carrier having a carbon layer and a chemical modification group on its surface is used. Carriers having a carbon layer and a chemical modification group on their surface include those having a carbon layer and a chemical modification group on the surface of a substrate, and those having a chemical modification group on the surface of a substrate made of a carbon layer. As the material of the substrate, those known in the art can be used, and there is no particular limitation, and the same materials as those listed as the above carrier materials can be used.

[0054] In a microarray, a carrier having a fine flat plate-like structure is preferably used. The shape is not limited, such as rectangular, square, and round, but usually, those with sides of 1 to 75 mm, preferably those with sides of 1 to 10 mm, more preferably those with sides of 3 to 5 mm are used. Since it is easy to manufacture a carrier having a fine flat plate-like structure, it is preferable to use a substrate made of a silicon material or a resin material, and particularly preferable is a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon. Single crystal silicon includes those in which the direction of the crystal axis changes slightly in some parts (sometimes referred to as mosaic crystals), and those containing atomic-scale disorder (lattice defects).

[0055] The carbon layer formed on the substrate is not particularly limited, but it is preferable to use synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (e.g., diamond-like carbon), amorphous carbon, any of carbon-based substances (e.g., graphite, fullerene, carbon nanotube), their mixtures, or those laminated. Also, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide, etc. may be used. Here, soft diamond generally refers to an incomplete diamond structure which is a mixture of diamond and carbon such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. The carbon layer is excellent in chemical stability and can withstand reactions in subsequent introduction of chemical modification groups and binding to the analyte. It is advantageous in that the binding has flexibility because it binds to the analyte by electrostatic interaction, is transparent to the detection system UV because it has no UV absorption, and can be energized during electroblotting. Also, it is advantageous in that there is little non-specific adsorption in the binding reaction with the analyte. As described above, a carrier in which the substrate itself is made of a carbon layer may be used.

[0056] The formation of the carbon layer can be carried out by known methods. For example, microwave plasma CVD (Chemical vapor deposit) method, ECRCVD (Electric cyclotron resonance chemical vapor deposit) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) sputtering method, ionized evaporation method, arc evaporation method, laser evaporation method, EB (Electron beam) evaporation method, resistance heating evaporation method, etc. can be mentioned.

[0057] In the high-frequency plasma CVD method, the raw material gas (methane) is decomposed by glow discharge generated between electrodes by high frequency, and a carbon layer is synthesized on the substrate. In the ionization deposition method, using thermoelectrons generated by a tungsten filament, the raw material gas (benzene) is decomposed and ionized, and a carbon layer is formed on the substrate by a bias voltage. A carbon layer may be formed by the ionization deposition method in a mixed gas composed of 1 to 99% by volume of hydrogen gas and the remaining 99 to 1% by volume of methane gas.

[0058] In the arc evaporation method, an arc discharge is caused in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), generating a plasma of carbon atoms from the cathode, and applying a bias voltage more negative than the evaporation source to the substrate to accelerate carbon ions in the plasma toward the substrate, thereby forming a carbon layer.

[0059] In the laser evaporation method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd:YAG laser (pulse oscillation) light to melt it and depositing carbon atoms on a glass substrate.

[0060] When forming a carbon layer on the surface of the substrate, the thickness of the carbon layer is usually about a monolayer to 100 μm. If it is too thin, the surface of the underlying substrate may be locally exposed. Conversely, if it is too thick, the productivity deteriorates. Therefore, it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

[0061] By introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed, the probe can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited. Examples include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group.

[0062] The introduction of an amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet rays in ammonia gas or by plasma treatment. Alternatively, it can be carried out by irradiating the carbon layer with ultraviolet rays in chlorine gas for chlorination and then irradiating with ultraviolet rays in ammonia gas. Or it can also be carried out by reacting the carbon layer chlorinated in polyvalent amine gases such as methylenediamine and ethylenediamine.

[0063] The introduction of a carboxyl group can be carried out, for example, by reacting an appropriately selected compound with the carbon layer aminated as described above. Examples of the compounds used for introducing a carboxyl group include halocarboxylic acids represented by the formula: X-R1-COOH (wherein X represents a halogen atom and R1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms), such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; dicarboxylic acids represented by the formula: HOOC-R2-COOH (wherein R2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyvalent carboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; keto acids or aldehyde acids represented by the formula: R3-CO-R4-COOH (wherein R3 represents a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms and R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms); monohalides of dicarboxylic acids represented by the formula: X-OC-R5-COOH (wherein X represents a halogen atom and R5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as succinic acid monochloride, malonic acid monochloride; and acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, butanetetracarboxylic anhydride.

[0064] The introduction of epoxy groups can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Alternatively, it can be obtained by reacting an organic peracid with the carbon-carbon double bond contained in the carbon layer. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, trifluoroperacetic acid, and the like.

[0065] The introduction of formyl groups can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

[0066] The introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

[0067] The active ester group means an ester group having an electron-withdrawing group with high acidity on the alcohol side of the ester group to activate the nucleophilic reaction, that is, an ester group with high reaction activity. It is an ester group having an electron-withdrawing group on the alcohol side of the ester group and being more activated than an alkyl ester. The active ester group has reactivity with groups such as amino groups, thiol groups, and hydroxyl groups. More specifically, phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. are known as active ester groups having much higher activity than alkyl esters, etc. More specifically, examples of the active ester group include, for example, p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2,3-dicarboximide group, etc., and in particular, the N-hydroxysuccinimide group is preferably used.

[0068] The introduction of the active ester group can be carried out, for example, by subjecting the carboxyl group introduced as described above to active esterification with a dehydrating condensing agent such as cyanamide or carbodiimide (e.g., 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and a compound such as N-hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the terminal of the hydrocarbon group through an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).

[0069] A wild-type probe or a mutant probe is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted onto a carrier by a spotter device or the like, whereby a microarray in which the wild-type probe and the mutant probe are immobilized on the carrier can be manufactured. Alternatively, the spotting solution may be manually spotted with a micropipettor.

[0070] After spotting, it is preferable to perform incubation to allow the reaction of the wild-type probe and the mutant probe to bind to the carrier. The incubation is usually carried out at a temperature of -20 to 100°C, preferably 0 to 90°C, and usually for 0.5 to 16 hours, preferably 1 to 2 hours. The incubation is desirably carried out under an atmosphere of high humidity, for example, under conditions of a humidity of 50 to 90%. Following the incubation, it is preferable to perform washing using a washing solution (e.g., 50 mM TBS / 0.05% Tween 20, 2×SSC / 0.2% SDS solution, ultrapure water, etc.) to remove the DNA that has not bound to the carrier.

[0071] 3) Blocking nucleic acid Blocking nucleic acid is used to prevent the nucleic acid fragment corresponding to the wild type among the nucleic acid fragments amplified using the primer set described above from non-specifically hybridizing to the mutant probe, and has a base sequence that specifically hybridizes to the nucleic acid fragment corresponding to the wild type. The nucleic acid for blocking can be designed based on the base sequence of the nucleic acid fragment amplified using the primer set described above. Specifically, the blocking nucleic acid is designed to have a base sequence complementary to at least the region containing the position of the gene mutation (wild type) among the nucleic acid fragments amplified using the primer set described above.

[0072] The blocking nucleic acid can be mixed with a reaction solution containing wild-type and mutant nucleic acid fragments, and the resulting mixed solution can be brought into contact with the DNA chip described above to allow the hybridization reaction between the mutant nucleic acid fragment and the mutant probe to proceed. Alternatively, a reaction solution containing wild-type and mutant nucleic acid fragments and a solution containing the blocking nucleic acid can be mixed on the DNA chip to simultaneously allow specific hybridization between the mutant nucleic acid fragment and the mutant probe to proceed. In either case, by specifically hybridizing the blocking nucleic acid to the wild-type nucleic acid fragment, it is possible to prevent the wild-type nucleic acid fragment from non-specifically hybridizing to the mutant probe.

[0073] In the blocking nucleic acid, the position of the gene mutation (wild type) to be detected is not particularly limited. Also, the blocking nucleic acid is not particularly limited, but preferably has a length of 60% or more relative to the base length of the mutant probe. Further, the blocking nucleic acid preferably has a length of 140% or less relative to the base length of the mutant probe. For example, if the length of the mutant probe is 25 bases, the base length of the blocking nucleic acid is preferably 15 to 35 bases.

[0074] Furthermore, if the blocking nucleic acid has bases complementary to the wild-type bases for the gene mutation to be detected, it may contain bases mismatched (non-complementary bases) to the wild-type nucleic acid fragment in other regions. When the blocking nucleic acid is 15 bases in length, the number of mismatched bases can be 1 to 3, preferably 1 to 2. When the nucleic acid for blocking is 25 bases in length, the number of mismatched bases can be 1 to 5, preferably 1 to 4.

[0075] In addition, if the non-specific hybridization between the wild-type nucleic acid fragment and the mutant probe is slight, the blocking nucleic acid may not be used. However, if the non-specific hybridization between the wild-type nucleic acid fragment and the mutant probe is at a level that does not pose a problem, it is unnecessary. When using the blocking nucleic acid, its concentration is not particularly limited. For example, it can be appropriately set according to the concentration of the non-target nucleic acid and / or the target nucleic acid, or according to the primer concentration.

[0076] More specifically, as the blocking nucleic acid, it is possible to design a blocking nucleic acid for MYD88 gene mutation that is effective when detecting the gene mutation in the MYD88 gene described above, and a blocking nucleic acid for CD79B gene mutation that is effective when detecting the gene mutation in the CD79B gene described above. For example, when using a set of a forward primer for MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 (5'-CAGGTGCCCATCAGAAG-3') and a reverse primer for MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 78 (5'-CGCAGACAGTGATGAACC-3'), the nucleotide sequence of SEQ ID NO: 89 can be designed as the blocking nucleic acid for MYD88 gene mutation. Also, when using a set of a forward primer for CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 85 (5'-CAGGATGACAGCAAGGC-3') and a reverse primer for CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 86 (5'-CAACCACACCAGCAGATAG-3'), the nucleotide sequence of SEQ ID NO: 95 can be designed as the blocking nucleic acid for CD79B gene mutation.

[0077] In particular, when using a set of a forward primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 (5'-CAGGTGCCCATCAGAAG-3') and a reverse primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 78 (5'-CGCAGACAGTGATGAACC-3'), the non-specific hybridization between the amplified wild-type nucleic acid fragment and the mutant-type probe for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 6 (5'-GAAGCGACCGATCCC-3') is slight, and a blocking nucleic acid is not necessarily required. That is, the blocking nucleic acid for the MYD88 gene mutation may not be used (blocking nucleic acid concentration is 0 nM). However, in order to detect the mutant-type nucleic acid fragment more precisely, it is preferable to use the blocking nucleic acid for the MYD88 gene mutation consisting of the nucleotide sequence of SEQ ID NO: 89. In this case, the concentration of the blocking nucleic acid for the MYD88 gene mutation is not particularly limited, but can be 0 to 500 nM, preferably 10 to 450 nM, preferably 20 to 400 nM, preferably 30 to 350 nM, preferably 40 to 300 nM, preferably 50 to 250 nM, preferably 60 to 200 nM, preferably 70 to 150 nM. As an example, the concentration of the blocking nucleic acid for the MYD88 gene mutation can be used as 125 nM.

[0078] In addition, when using a set of a forward primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 85 (5'-CAGGATGACAGCAAGGC-3') and a reverse primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 86 (5'-CAACCACACCAGCAGATAG-3'), nonspecific hybridization occurs between the amplified wild-type nucleic acid fragment and the mutant probe for the CD79B gene shown in Table 3. Therefore, it is preferable to use a blocking nucleic acid for CD79B gene mutation consisting of the nucleotide sequence of SEQ ID NO: 95. In this case, the concentration of the blocking nucleic acid for CD79B gene mutation is not particularly limited, but can be 125 to 1000 nM, and preferably 500 to 750 nM. As an example, the concentration of the blocking nucleic acid for CD79B gene mutation can be used at 750 nM.

[0079] 4) Method for detecting gene mutation As described above, the gene mutation evaluation kit identifies gene mutations in the CD79B gene and gene mutations in the MYD88 gene, and can be used for the diagnosis of diseases related to these gene mutations. Specifically, when these gene mutations are detected, they can be classified into the MCD subtype in diffuse large B-cell lymphoma (DLBCL). Cancer Cell 39, 1643-1653, December 13, 2021 suggests that when classified into the MCD subtype in DLBCL, the R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) combined with ibrutinib, a kind of BTK inhibitor, is effective. Therefore, by identifying the MCD type of DLBCL using the above-described gene mutation evaluation kit, information useful for determining the treatment strategy can be provided.

[0080] Hereinafter, the procedure for identifying gene mutations in the MYD88 gene and gene mutations in the CD79B gene using a gene mutation evaluation kit will be specifically described. In the following description, a form using a microarray having wild-type probes and mutant probes for gene mutations in the MYD88 gene and wild-type probes and mutant probes for gene mutations in the CD79B gene will be adopted.

[0081] By using the above-described microarray, for example, gene mutations in the MYD88 gene and gene mutations in the CD79B gene in a subject to be diagnosed can be identified.

[0082] Specifically, when examining gene mutations, it includes a step of extracting DNA from a sample derived from a subject to be diagnosed, a step of amplifying a region containing the gene mutation to be examined using the extracted DNA as a template, a step of hybridizing the amplified nucleic acid fragment with wild-type probes and mutant probes using the above-described microarray, and a step of detecting signals from the wild-type probes and mutant probes. In the above hybridization step, the "wild-type probes and mutant probes" means wild-type probes and mutant probes for gene mutations in the MYD88 gene and wild-type probes and mutant probes for gene mutations in the CD79B gene. Also, in the above signal detection step, the "wild-type probes and mutant probes" similarly means wild-type probes and mutant probes for gene mutations in the MYD88 gene and wild-type probes and mutant probes for gene mutations in the CD79B gene.

[0083] The subject to be diagnosed is usually a human and is not particularly limited by race or the like. In particular, it is preferably a Mongoloid, more preferably an East Asian, and most preferably a Japanese. Also, the subject to be diagnosed can be a patient suspected of having a disease related to the gene mutation to be examined. The sample derived from the subject to be diagnosed is not particularly limited. For example, it includes blood-related samples (blood, serum, plasma, etc.), lymph fluid, feces, cancer cells, tissue, or crushed materials and extracts of organs.

[0084] First, DNA is extracted from a sample collected from the subject to be diagnosed. The extraction means is not particularly limited. For example, DNA extraction methods using phenol / chloroform, ethanol, sodium hydroxide, CTAB, etc. can be used.

[0085] Next, an amplification reaction is performed using the obtained DNA as a template to amplify the region containing the gene mutation to be examined. As the amplification reaction, polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, etc. can be applied. As described above, the amplified nucleic acid fragment can be labeled by labeling the primer used in the nucleic acid amplification reaction. That is, the amplified nucleic acid fragment can be labeled by adding a labeling substance to either the forward primer or the reverse primer included in the primer set for CD79B gene mutation, or either the forward primer or the reverse primer included in the primer set for MYD88 gene mutation.

[0086] This reaction system is a reaction system containing a buffer, a heat-resistant DNA polymerase, a primer set for MYD88 gene mutation and a primer set for CD79B gene mutation, a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate added with a fluorescent label, etc.), a nucleotide triphosphate, and magnesium chloride, etc., which are necessary for nucleic acid amplification and labeling.

[0087] The nucleic acid fragment to be amplified depends on the design of the primer set for MYD88 gene mutation and the primer set for CD79B gene mutation. For example, it is preferably 1 kbp or less, more preferably 800 bp or less, still more preferably 500 bp or less, and particularly preferably 350 bp or less.

[0088] Perform a hybridization reaction between the amplified nucleic acid obtained as described above and the wild-type probe and mutant probe immobilized on the carrier, detect the signals from the wild-type probe and mutant probe based on the labeling of the hybridized nucleic acid fragments, and the gene mutation in the subject to be diagnosed can be examined.

[0089] The signal from the label can be quantified by, for example, when using a fluorescent label, detecting the fluorescent signal using a fluorescence scanner and analyzing this with image analysis software. The hybridization reaction is preferably carried out under stringent conditions. Stringent conditions refer to conditions under which specific hybrids are formed and non-specific hybrids are not formed. For example, after performing a hybridization reaction at 47 - 52 °C (specifically 51.5 °C) for 15 - 90 minutes (specifically 30 minutes), washing 30 times under the conditions of 0.1×SSC / 0.1% SDS, 25 °C, and then washing 80 times under the conditions of 1×SSC, 25 °C. When the probe has a short chain length, it is more preferable to lower the hybridization temperature, and when the chain length is long, it is more preferable to raise the hybridization temperature. It goes without saying that as the salt concentration increases, the hybridization temperature with specificity increases, and conversely, as the salt concentration decreases, the hybridization temperature with specificity decreases.

[0090] Also, when using a microarray equipped with a mutant probe and a wild-type probe for the above-described gene mutation, the gene mutation can be examined using the signal intensities from these mutant probe and wild-type probe. Specifically, measure the signal intensities in the mutant probe and the wild-type probe respectively, and calculate a determination value for evaluating the signal intensity derived from the mutant probe. As an example of calculating the determination value, for example, the method of evaluating the mutant corresponding to the mutant probe using Equation 1: [Signal intensity derived from the mutant probe] / ([Signal intensity derived from the wild-type probe] + [Signal intensity derived from the mutant probe]) can be mentioned.

[0091] Then, the determination value calculated by the above formula 1 is compared with a predetermined threshold value (cut-off value). When the determination value exceeds the threshold value, it is determined that the amplified nucleic acid contains a gene mutation (mutant type) corresponding to the mutant probe. When the determination value is below the threshold value, it is determined that the amplified nucleic acid does not contain the gene mutation (mutant type).

[0092] Here, the threshold value is not particularly limited. For example, it can be defined based on the determination value calculated by the above formula 1 using a sample for which it is confirmed that the gene mutation is wild type. More specifically, a plurality of determination values can be calculated using a plurality of samples for which it is confirmed that the gene mutation is wild type, and the value of the average value + 3σ (σ: standard deviation) can be used as the threshold value. In addition, the value of the average value + 2σ or the average value + σ can also be used as the threshold value.

[0093] As described above, for each of the gene mutations of the MYD88 gene and the CD79B gene, by using a microarray equipped with mutant probes and wild-type probes, it is possible to accurately identify whether the gene mutation is of the mutant type or the wild-type.

[0094] In particular, when using the primer set for MYD88 gene mutations consisting of the forward primer for MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 (5'-CAGGTGCCCATCAGAAG-3') and the reverse primer for MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 78 (5'-CGCAGACAGTGATGAACC-3'), and the primer set for CD79B gene mutations consisting of the forward primer for CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 85 (5'-CAGGATGACAGCAAGGC-3') and the reverse primer for CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 86 (5'-CAACCACACCAGCAGATAG-3'), the target nucleic acid fragments can be obtained stably and in a high amplification amount, and non-specific amplification based on non-specific hybridization of the forward primer and / or reverse primer can be suppressed low. As a result, by using these primer sets, each gene mutation can be identified with more excellent sensitivity.

[0095] In addition, for the nucleic acid fragment obtained using the set of the forward primer for MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 (5'-CAGGTGCCCATCAGAAG-3') and the reverse primer for MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 78 (5'-CGCAGACAGTGATGAACC-3') described above, by using the wild-type probe for MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 2 (5'-AGCGACTGATCCCCAT-3') and the mutant-type probe for MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 6 (5'-GAAGCGACCGATCCC-3'), the gene mutation of the MYD88 gene in the subject to be diagnosed can be identified with high precision.

[0096] Incidentally, regarding gene mutations in the CD79B gene, for nucleic acid fragments obtained using a set of a forward primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 85 (5'-CAGGATGACAGCAAGGC-3') and a reverse primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 86 (5'-CAACCACACCAGCAGATAG-3'), a first probe which is a mutant probe for detecting missense mutations (M1 to M6) and nonsense mutations (M7 and M8), and a second probe which is a mutant probe for detecting silent mutations (M9) have been designed.

[0097] In this case, as follows, it is possible to identify whether the gene mutation of the CD79B gene is wild type or mutant type. In particular, in the present invention, when it is detected by the second probe that the gene mutation is a silent mutation, it can be determined that the gene mutation is wild type.

[0098] Specifically, when using a microarray comprising the first probe, the second probe, and a wild-type probe, gene mutations in the CD79B gene can be examined using the signal intensities from these first probe, second probe, and wild-type probe. That is, the signal intensities in the first probe, second probe, and wild-type probe are measured respectively, and a determination value for evaluating the signal intensity derived from the first probe or the second probe is calculated. As an example of calculating the determination value, for example, Equation 1: [Signal intensity derived from the first probe] / ([Signal intensity derived from the wild-type probe]+[Signal intensity derived from the first probe]) is used to evaluate the missense mutation and / or nonsense mutation corresponding to the first probe, and Equation 2: [Signal intensity derived from the second probe] / ([Signal intensity derived from the wild-type probe]+[Signal intensity derived from the second probe]) is used to evaluate the silent mutation corresponding to the second probe.

[0099] Then, the determination value calculated by the above formula 1 is compared with a predetermined threshold value (cutoff value). When the determination value exceeds the threshold value, it is determined that the amplified nucleic acid contains a missense mutation and / or a nonsense mutation corresponding to the first probe for the gene mutation. When the determination value is lower than the threshold value, it is determined that the amplified nucleic acid does not contain the missense mutation and / or the nonsense mutation. Also, the determination value calculated by the above formula 2 is compared with a predetermined threshold value (cutoff value). When the determination value exceeds the threshold value, it is determined that the amplified nucleic acid contains a silent mutation corresponding to the second probe for the gene mutation. When the determination value is lower than the threshold value, it is determined that the amplified nucleic acid does not contain the silent mutation.

[0100] And when it is determined that a silent mutation is included based on the above formula 2, it is determined that the gene mutation of the CD79B gene to be tested is wild type. Also, when it is determined that neither a missense mutation nor a nonsense mutation is included based on the above formula 1 and no silent mutation is included based on the above formula 2, it is also determined that the gene mutation to be tested is wild type. Thus, by using a microarray comprising a first probe, a second probe, and a wild-type probe for detecting the gene mutation of the CD79B gene, it is possible to accurately identify whether the gene mutation is a mutant type of a missense mutation and / or a nonsense mutation or a wild type.

[0101] As described above, by identifying whether the gene mutation of the MYD88 gene is wild type or mutant type and whether the gene mutation of the CD79B gene is wild type or mutant type for the subject to be diagnosed, it is possible to provide effective information for diagnosing whether it is the MCD type in diffuse large B-cell lymphoma (DLBCL).

Example

[0102] Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0103] <Array Design> In this example, for gene mutations in the MYD88 gene and gene mutations in the CD79B gene, a primer set for amplifying the region containing the gene mutation, a probe for detecting the wild type or mutant type contained in the nucleic acid fragment amplified by the primer set, and a blocking nucleic acid corresponding to the wild type for the gene mutation of the MYD88 gene and the gene mutation of the CD79B gene were designed.

[0104] The probes corresponding to the gene mutations of the MYD88 gene designed in this example are shown in Table 4. In the "Wild type (W) / Mutant type (M)" column of Table 4, W is described for the probe corresponding to the wild type, and M is described for the probe corresponding to the mutant type.

[0105]

Table 4

[0106] The probes corresponding to the gene mutations of the CD79B gene designed in this example are shown in Tables 5 and 6. In the "Wild type (W) / Mutant type (M)" columns of Tables 5 and 6, W is described for the probe corresponding to the wild type, and M1 to M9 are described for the probes corresponding to the mutant types. The notations of M1 to M9 follow Table 3.

[0107]

Table 5

[0108]

Table 6

[0109] In addition, the primer set for amplifying the region containing the gene mutation of the MYD88 gene designed in this example is shown in Table 7.

[0110]

Table 7

[0111] Furthermore, Table 8 shows a primer set for amplifying a region containing a gene mutation of the CD79B gene designed in this example.

[0112]

Table 8

[0113] Furthermore, Table 9 shows a blocking nucleic acid corresponding to the wild type in the gene mutation of the MYD88 gene designed in this example.

[0114]

Table 9

[0115] Furthermore, Table 10 shows a blocking nucleic acid corresponding to the wild type in the gene mutation of the CD79B gene designed in this example.

[0116]

Table 10

[0117] <Examination of Optimal Mutant Probes> Regarding the probes described in Tables 4 to 6, the fluorescence intensity was measured using a 100% mutation model specimen or a 5% mutation model specimen. Specifically, a DNA chip immobilized with each of the above probes was set in a gene analyzer BIOSHOT HT-32 (manufactured by Toyo Kohan Co., Ltd.) to perform a hybridization reaction with the model specimen. A DNA chip needle for fixing the DNA chip, and further, after the hybridization reaction of the DNA chip, a washing solution (0.1×SSC / 0.1% SDS solution, room temperature), a rinse solution (1×SSC solution, room temperature), and a detection solution for fluorescence detection (1×SSC solution, room temperature) for washing excess reagents were set in the BIOSHOT HT-32, and the operation was started.

[0118] By means of automatic operation, while the reaction solution in the designated PCR tube was heated to 51.5°C, the DNA chip needle was inserted, and the hybridization reaction was carried out for 30 minutes. After the hybridization reaction, the DNA chip needle was quickly immersed in the cleaning solution tank, and stirred 30 times so that the DNA chip entered and exited the water surface of the cleaning solution. The DNA chip needle after cleaning was immersed in the rinsing solution tank, and stirred 80 times so that the DNA chip entered and exited the water surface of the rinsing solution.

[0119] The DNA chip needle after rinsing was slowly immersed in the detection solution tank, and excited light was captured by the CCD camera for 2 seconds by irradiating with a single-wavelength laser of 640 nm. Then, a determination value was calculated from the measured fluorescence intensity value according to the following formula. [Determination value] = [Fluorescence intensity of mutant probe] / ([Fluorescence intensity of mutant probe] + [Fluorescence intensity of wild-type probe]) Note that the 100% mutant model specimen means a sample in which the ratio of mutant type to wild type in the gene mutation to be detected is 100:0. Also, the 5% mutant model specimen means a sample in which the ratio of mutant type to wild type in the gene mutation to be detected is 5:95.

[0120] The results of measuring the fluorescence intensity values when using the 100% mutant model specimen are shown in Figure 1, the results of measuring the fluorescence intensity values when using the 5% mutant model specimen are shown in Figure 2, and the results of measuring the determination value from the results shown in Figure 2 are shown in Figure 3. Note that in Figures 1 to 3, for comparison, the results of calculating the fluorescence intensity values from each probe using a wild-type sample in which the ratio of mutant type to wild type in the gene mutation to be detected is 0:100 are also shown together.

[0121] The specificities of the mutant probe and the wild-type probe were confirmed from the fluorescence intensity values shown in Figure 1. As a result, it was found that excellent fluorescence intensity values were obtained for all the tested mutant probes and wild-type probes. Also, for all the tested mutant probes and wild-type probes, the cross-reaction was suppressed to about 1 / 2 or less of the specific fluorescence intensity value, and it was judged that the target gene mutations (mutant type, wild type) could be distinguished.

[0122] Also, from the results shown in FIG. 1, regarding the silent mutation 591C>T indicated by M9 among the gene mutations of the CD79B gene, a strong fluorescence intensity value (47125) was detected in the M8 probe (CD79B_M8_v3-2). If the detection of the silent mutation M9 is not attempted using the mutant probe, there is a possibility that a sample having the silent mutation of M9 may be erroneously identified as an M8 mutation. Therefore, by surely identifying the silent mutation using the mutant probe corresponding to M9, which is a silent mutation, when identifying the gene mutation of the CD79B gene, false determination can be prevented. At this time, when the silent mutation is identified, it is determined as "wild type". As a result, it is possible to precisely identify whether the gene mutation of the CD79B gene is a mutant type or a wild type, and very effective information can be provided regarding diseases related to the gene mutation of the CD79B gene.

[0123] On the other hand, FIG. 2 shows the fluorescence intensity values of the mutant probe and the wild-type probe when using a sample with a low mutation ratio. Even for a sample with a low mutation ratio, by obtaining a fluorescence intensity value equal to or higher than a desired value, the target gene mutation can be detected with high precision. For example, assuming that a fluorescence intensity value of 10,000 or more is preferable, it can be seen from the results shown in FIG. 2 that sufficient fluorescence intensity values have not been obtained for CD79B_M8_v3-3 and CD79B_M8_v4-3.

[0124] In addition, FIG. 3 shows the determination values calculated from the fluorescence intensity values shown in FIG. 2 according to the above formula. As can be understood from the formula for calculating the determination value, it shows that the higher the value of the determination value, the better the separation ability by the mutant probe and the wild-type probe. Also, it can be said that the lower the determination value calculated for the mutant probe when using the wild-type sample, the better the separation ability. For example, based on the criterion that the determination value calculated for the mutant probe when using the wild-type sample is 0.05 or less, it can be seen from the results shown in FIG. 3 that CD79B_M4_v3-2, CD79B_M4_v4-4, and CD79B_M6-v4-3 do not have sufficient separation ability.

[0125] As a result of comprehensively considering the above-described results, it was found that for the gene mutation of the MYD88 gene, it is preferable to adopt MYD88_M_v2-1 as the mutant probe. Also, for the gene mutations (9 types of M1 to M9) of the CD79B gene, it was found that it is preferable to adopt CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-1.

[0126] <Examination of the Optimal Wild-Type Probe> First, a wild-type probe for detecting the wild type in the gene mutation of the MYD88 gene was examined. The fluorescence intensity values and determination values from the wild-type probe and the mutant probe (v2-1) were calculated when using the tested wild-type samples and the 5% mutant model specimens. Also, the fluorescence intensity values and determination values were calculated in the same manner using genomic DNA (gDNA-1) instead of the wild-type samples and the 5% mutant model specimens.

[0127] The results are shown in Fig. 4. When selecting a wild-type probe, it is better that the fluorescence intensity value is higher when using genomic DNA, the resolution is high (i.e., the difference between the mutant determination value and the wild-type determination value is large), and the determination value calculated by the above formula from the fluorescence intensity value when using a wild-type sample is low. These were set as the conditions required for the wild-type probe. Specifically, a criterion was set such that the fluorescence intensity value from the wild-type probe was 10,000 or more when using genomic DNA, the resolution was the highest among the tested wild-type probes (the difference between the mutant determination value and the wild-type determination value was the largest), and the determination value when using a wild-type sample was 0.05 or less. As a result, v2-1 with the highest resolution could be selected as the optimal wild-type probe.

[0128] Next, for the gene mutation of the CD79B gene, a wild-type probe for detecting the wild type was examined. The examination method and evaluation criteria were in accordance with the method for selecting a wild-type probe in the gene mutation of the MYD88 gene described above. As the mutant probes, CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-1 selected above were used.

[0129] The results of measuring the fluorescence intensity values are shown in Fig. 5, and the results of calculating the determination values from the fluorescence intensity values are shown in Fig. 6. As can be seen from Figs. 5 and 6, considering comprehensively that the fluorescence intensity value is high when using genomic DNA, the resolution is high, and the determination value is low when using a wild-type sample, v3-1 could be selected as the optimal wild-type probe.

[0130] <Performance Evaluation of the Selected Mutant Probes and Wild-Type Probes> Using the mutant probes (MYD88_M_v2-1) and wild-type probes (MYD88_W_v2-1) selected for gene mutations of the MYD88 gene, and the mutant probes (CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2 and CD79B_M9-1) and wild-type probes (CD79B_W_v3-1) selected for gene mutations of the CD79B gene, gene mutations in genomic DNA were identified. As genomic DNA, three types were used: gDNA-1, gDNA-2 and gDNA-3. gDNA-1 is genomic DNA derived from a healthy individual. gDNA-2 is genomic DNA derived from a non-Hodgkin lymphoma patient. gDNA-3 is genomic DNA derived from lymphoplasmacytic lymphoma.

[0131] The results of identifying each gene mutation using these mutant probes and wild-type probes are shown in Fig. 7. As shown in Fig. 7, for gDNA-1, it was identified that the gene mutation of the MYD88 gene was not a mutant type and the gene mutation of the CD79B gene was not a mutant type. Also, for gDNA-2, it was identified that the gene mutation of the MYD88 gene was a mutant type (L265P) and the gene mutation of the CD79B gene was a mutant type (Y196D, M3). Also, for gDNA-3, it was identified that the gene mutation of the MYD88 gene was a mutant type (L265P) and the gene mutation of the CD79B gene was not a mutant type.

[0132] To verify the results when using the mutant probe and wild-type probe shown in Fig. 7, for the three types of genomic DNAs (gDNA-1, gDNA-2, and gDNA-3) tested, gene mutations in the MYD88 gene and gene mutations in the CD79B gene were confirmed by the direct sequencing method. As a result, it was confirmed that both the gene mutation in the MYD88 gene and the gene mutation in the CD79B gene in gDNA-1 were wild-type. Also, it was confirmed that the gene mutation in the MYD88 gene in gDNA-1 was a mutant type of L265P, and the gene mutation in the CD79B gene was a mutant type of Y196D (M3). Furthermore, it was confirmed that the gene mutation in the MYD88 gene in gDNA-3 was a mutant type of L265P, and the gene mutation in the CD79B gene was wild-type.

[0133] The identification results of each gene mutation using the mutant probe and wild-type probe selected as described above were completely consistent with the results confirmed by the direct sequencing method. From this, it was shown that the mutant probe and wild-type probe selected as described above can accurately detect each gene mutation with high precision even in actual use scenarios.

[0134] <Primer Set Design> Regarding the primer set for amplifying the region containing the gene mutation of the CD79B gene shown in Tables 7 and 8 and the primer set for amplifying the region containing the gene mutation of the MYD88 gene, seven combinations ([1] to [7]) shown in Fig. 8 were tested, and the optimal primer set was selected based on the amplification amount of the target nucleic acid fragment and the amplification amount of non-specific nucleic acid amplification.

[0135] First, a reaction solution was prepared using a wild-type model specimen or genomic DNA (gDNA) as a template. Using the prepared PCR reaction solution, the thermal cycle of PCR was carried out at 95°C for 5 minutes, followed by 30 seconds at 95°C, 30 seconds at a predetermined annealing temperature (e.g., 59°C), and 45 seconds at 72°C as one cycle. Then, it was carried out at 72°C for 10 minutes, and finally maintained at 4°C. The annealing temperature was 56°C, 58°C, 60°C, 62°C, or 64°C.

[0136] The relationship between the nucleic acid amplification amount and the annealing temperature is shown in Fig. 9. As can be seen from Fig. 9, in order to amplify the region containing the gene mutation of the MYD88 gene and the region containing the gene mutation of the CD79B gene in the same manner for both the wild-type model specimen and genomic DNA (multiplex amplification), it was the combination of [5], [6], and [7].

[0137] Also, Fig. 10 shows the results of confirming by electrophoresis the nucleic acid fragments contained in the reaction solution after PCR performed with the combination of [5], [6], and [7]. As shown in Fig. 10, when the combination of [7] was used, a plurality of nucleic acid fragments due to non-specific amplification other than the target nucleic acid fragment could be confirmed. From this result, it was found that the combination of [5] and [6] was superior to the combination of [7].

[0138] Next, a reaction solution was prepared using a 5% mutant model specimen or genomic DNA (gDNA-1) as a template. PCR was similarly performed using the reaction solution, and the fluorescence intensity based on the amplified fragment was measured. The results are shown in Fig. 11. As shown in Fig. 11, when the combination of [5] was used, it was found that the fluorescence intensity from the probe corresponding to the mutant type in the gene mutation of the MYD88 gene was lower compared to other combinations.

[0139] Then, regarding the combination of [6] and [7], when the mixing ratio of the labeled primer and the unlabeled primer was changed, the change in the fluorescence intensity value was examined. The results are shown in Fig. 12. As shown in Fig. 12, in the case of the combination of [6], the fluorescence intensity increased as the mixing ratio of the labeled primer increased, and a fluorescence intensity value corresponding to the ratio of the labeled primer was obtained. In contrast, in the case of the combination of [7], the tendency of the change in the ratio of the labeled primer and the fluorescence intensity value did not match. This was considered to be due to non-specific amplification observed when the combination of [7] was used. From these results, it became clear that it is preferable to multiplex-amplify the region containing the gene mutation of the CD79B gene and the region containing the gene mutation of the MYD88 gene by the combination of [6].

[0140] <Examination of primer ratio (labeled vs unlabeled)> In each of the primer sets for amplifying the region containing the gene mutation of the CD79B gene and the primer set for amplifying the region containing the gene mutation of the MYD88 gene, the ratio of the labeled primer and the unlabeled primer was changed, and how the fluorescence intensity value varied was examined, and the optimal ratio of the labeled primer and the unlabeled primer was examined. In this example, in the primer set for amplifying the region containing the gene mutation of the CD79B gene, the forward primer is the labeled primer (CD79B_Fw-4), and the reverse primer is the unlabeled primer (CD79B_Rv-4). Also, in the primer set for amplifying the region containing the gene mutation of the MYD88 gene, the forward primer is the unlabeled primer (MYD88_Fw-5-3), and the reverse primer is the labeled primer (MYD88_Rv-5).

[0141] In this example, as shown in FIG. 13, the final concentrations of the labeled primers (CD79B_Fw-4, MYD88_Rv-5) and unlabeled primers (CD79B_Rv-4, MYD88_Fw-5-3) contained in the PCR reaction solution were prepared, and PCR was similarly performed. Note that as the template, a mutant 5% model specimen and genomic DNA were used. The results when using a primer set for amplifying the region containing the gene mutation of the MYD88 gene are shown in FIG. 14, and the results when using a primer set for amplifying the region containing the gene mutation of the CD79B gene are shown in FIG. 15.

[0142] As can be seen from FIG. 14, excellent fluorescence intensity values were achieved in the range of 2-fold (i.e., labeled primer: unlabeled primer = 20:10) to 6-fold compared with the unlabeled primer for the final concentration of the labeled primer for amplifying the region containing the gene mutation of the MYD88 gene. In particular, more excellent fluorescence intensity values were achieved in the range of 3-fold to 5-fold, and the most excellent fluorescence intensity value was achieved at 4-fold.

[0143] Also, as can be seen from FIG. 15, excellent fluorescence intensity values were achieved in the range of 5-fold to 10-fold compared with the unlabeled primer for the final concentration of the labeled primer for amplifying the region containing the gene mutation of the CD79B gene. In particular, more excellent fluorescence intensity values were achieved in the range of 6-fold to 7-fold, and the most excellent fluorescence intensity value was achieved at 7-fold.

[0144] <Examination of Optimal Blocking Nucleic Acid> For the blocking nucleic acids related to the gene mutations of the MYD88 gene shown in Table 9 and the blocking nucleic acids related to the gene mutations of the CD79B gene shown in Table 10, the optimal blocking nucleic acids were selected based on their function as blocking nucleic acids. The blocking nucleic acid has the function of specifically hybridizing to the wild-type nucleic acid fragment among the nucleic acid fragments amplified from the region containing the gene mutation and preventing the wild-type nucleic acid fragment from non-specifically hybridizing to the mutant probe. Therefore, when selecting the optimal blocking nucleic acid, the non-specific hybridization between the wild-type nucleic acid fragment and the mutant probe in the presence of the blocking nucleic acid can be judged based on the fluorescence intensity of the mutant probe. Also, whether the blocking nucleic acid inhibits the specific hybridization between the mutant nucleic acid fragment and the mutant probe in the presence of the blocking nucleic acid can be judged based on the fluorescence intensity of the mutant probe.

[0145] Regarding the blocking nucleic acid related to the gene mutation of the MYD88 gene shown in Table 9, hybridization between the wild-type model specimen and the mutant probe (MYD88_M_v2-1) was carried out in the presence of the blocking nucleic acid (125 nM or 500 nM), and the fluorescence intensity of the mutant probe was measured. Also, hybridization between the 100% mutant model specimen and the mutant probe (MYD88_M_v2-1) was carried out in the presence of the blocking nucleic acid, and the fluorescence intensity of the mutant probe was measured. The results are shown in Figure 16.

[0146] As can be seen from Figure 16, non-specific hybridization between the wild-type model specimen and the mutant probe could be suppressed in all the tested blocking nucleic acids. However, for v1 and v2-1 among the blocking nucleic acids, it was revealed that they inhibited the specific hybridization between the 100% mutant model specimen and the mutant probe. Therefore, using the 5% mutant model specimen, it was verified which of v2-2 and v2-3 functions better as a blocking nucleic acid.

[0147] That is, in the presence of v2-2 or v2-3 (125 nM or 500 nM), hybridization was performed between the 5% mutation model specimen and the wild-type probe (MYD88_W_v2-1) and the mutant probe (MYD88_M_v2-1), and the fluorescence intensities of the wild-type probe and the mutant probe were measured. Further, based on the above formula, a determination value was calculated from the measured fluorescence intensity values. The results of measuring the fluorescence intensity are shown in FIG. 17, and the results of calculating the determination value are shown in FIG. 18.

[0148] As can be seen from FIGS. 17 and 18, when v2-2 was used, the fluorescence intensity of the wild-type probe was lower compared to the case when v2-3 was used, and it was revealed that it was superior in function as a blocking nucleic acid compared to v2-3. From these results, among those shown in Table 9, v2-2 was selected as the optimal blocking nucleic acid for the blocking nucleic acid related to the gene mutation of the MYD88 gene. Further, from these results, it was found that even when no blocking nucleic acid related to the gene mutation of the MYD88 gene was used (i.e., in the case of 0 nM), the determination value when using the wild-type model specimen and the determination value when using the 5% mutation model specimen were sufficiently separated. From the above results, it was found that the concentration of the blocking nucleic acid related to the gene mutation of the MYD88 gene is preferably 0 to 500 nM.

[0149] Next, regarding the blocking nucleic acid related to the gene mutation of the CD79B gene shown in Table 10, in the presence of the blocking nucleic acid, hybridization was performed between the wild-type model specimen or the 5% mutation model specimen and the wild-type probe (CD79B_W_v3-1) and the mutant probes (CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-2), the fluorescence intensities from the wild-type probe and the mutant probes were measured, and a determination value was calculated according to the above formula. The results are shown in FIG. 19.

[0150] As shown in Fig. 19, when using a wild-type model specimen, for all mutant probes with a determination value of 0.05 or less, it was only the case when using the blocking nucleic acids of v4-1 and v4-2. For the blocking nucleic acids of v4-1 and v4-2, the determination values and fluorescence intensity values for each mutant probe calculated using a wild-type model specimen or a mutant 5% model specimen are shown in Figs. 20 and 21, respectively. As can be seen from Figs. 20 and 21, when comparing the determination values for v4-1 and v4-2, both were almost equivalent, but when comparing the fluorescence intensity values for v4-1 and v4-2, it was found that v4-2 showed a higher value. From this result, among those shown in Table 10, v4-2 was selected as the optimal blocking nucleic acid as the blocking nucleic acid related to the gene mutation of the CD79B gene.

[0151] <Examination of Blocking Nucleic Acid Concentration> Regarding v4-2 selected as the blocking nucleic acid related to the gene mutation of the CD79B gene, hybridization was performed with a wild-type model specimen or a mutant 5% model specimen and wild-type probes (CD79B_W_v3-1) and mutant probes (CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-2) in the presence of blocking nucleic acids at various concentrations, the fluorescence intensity from the wild-type probes and mutant probes was measured, and the determination value was calculated according to the above formula. The results are shown in Fig. 22.

[0152] As shown in FIG. 22, when the concentration of the blocking nucleic acid was 125 nM, there were multiple mutant probes with a determination value exceeding 0.05 when using the wild-type model specimen. From this result, it was shown that the concentration of the blocking nucleic acid is preferably higher than 125 nM. Also, when the concentration of the blocking nucleic acid was set to 500 nM, 750 nM, or 1000 nM, the results of measuring the fluorescence intensity from each mutant probe when using the 5% mutant model specimen are shown in FIG. 23. From this result, it was found that when the concentration of the blocking nucleic acid was 1000 nM, the fluorescence intensity value was lower compared to the cases where the concentration was 500 nM or 750 nM. From these results, it was suggested that it is preferable to set the concentration of the blocking nucleic acid to 500 nM or 750 nM.

[0153] To confirm the above results, the determination values of each mutant probe when using the wild-type model specimen or the 5% mutant model specimen were calculated when the concentration of the blocking nucleic acid was 500 nM or 750 nM. The results are shown in FIG. 24. As can be seen from FIG. 24, in any case where the concentration of the blocking nucleic acid was 500 nM or 750 nM, the determination value when using the wild-type model specimen and the determination value when using the 5% mutant model specimen were sufficiently separated, and it was found that the wild-type and each mutant type could be accurately detected. In particular, when the concentration of the blocking nucleic acid was 750 nM, the determination value when using the wild-type model specimen and the determination value when using the 5% mutant model specimen were more significantly separated compared to the case where the concentration was 500 nM.

[0154] From the above results, it was found that the concentration of the blocking nucleic acid can be in the range of 125 nM to 1000 nM, and it is preferably 500 to 750 nM. As an example, it was found that the concentration of the blocking nucleic acid is most preferably 750 nM.

Claims

1. A kit for gene mutation evaluation used for the diagnosis of diseases associated with gene mutations in the MYD88 gene and / or gene mutations in the CD79B gene, comprising: A primer set for MYD88 gene mutation that amplifies a nucleic acid region containing a gene mutation in the MYD88 gene and a primer set for CD79B gene mutation that amplifies a nucleic acid region containing a gene mutation in the CD79B gene; A mutant probe for the MYD88 gene related to the gene mutation in the MYD88 gene, a wild-type probe for the MYD88 gene, a mutant probe for the CD79B gene related to the gene mutation in the CD79B gene, and a wild-type probe for the CD79B gene.

2. The gene mutation in the MYD88 gene is a substitution mutation of leucine at position 265 to proline, and the gene mutation in the CD79B gene is a missense mutation and / or nonsense mutation of tyrosine residue at position 196 and a silent mutation of the tyrosine residue. The kit for gene mutation evaluation according to Claim 1.

3. The primer set for MYD88 gene mutation is a set of a forward primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 and a reverse primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO:

78. The primer set for CD79B gene mutation is a set of a forward primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO: 85 and a reverse primer for the CD79B gene consisting of the nucleotide sequence of SEQ ID NO:

86. The kit for gene mutation evaluation according to Claim 1.

4. The reverse primer for the MYD88 gene has a label and has a concentration 2 to 6 times that of the forward primer for the MYD88 gene. The forward primer for the CD79B gene has a label and has a concentration 5 to 10 times that of the reverse primer for the CD79B gene. The kit for gene mutation evaluation according to Claim 3.

5. The mutant probe for the MYD88 gene, the wild-type probe for the MYD88 gene, the mutant probe for the CD79B gene, and the wild-type probe for the CD79B gene contain the nucleotide sequences shown in Table 1. The kit for gene mutation evaluation according to Claim 1. 【Table 1】

6. The kit for gene mutation evaluation according to claim 1, further comprising a MYD88 blocking nucleic acid that hybridizes to a nucleic acid fragment having a wild type among the nucleic acid fragments amplified by the primer set for the MYD88 gene mutation, and / or a CD79B blocking nucleic acid that hybridizes to a nucleic acid fragment having a wild type among the nucleic acid fragments amplified by the primer set for the CD79B gene mutation.

7. The kit for gene mutation evaluation according to claim 6, wherein the MYD88 blocking nucleic acid consists of the nucleotide sequence of SEQ ID NO: 89, and the CD79B blocking nucleic acid consists of the nucleotide sequence of SEQ ID NO:

95.

8. The kit for gene mutation evaluation according to claim 6, wherein the MYD88 blocking nucleic acid is prepared to be in the range of 0 to 500 nM in the hybridization solution, and the CD79B blocking nucleic acid is prepared to be in the range of 125 to 1000 nM in the hybridization solution.

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