Genetic mutation evaluation kit

Abstract

The present invention simultaneously identifies genetic mutations in MYD88 and / or genetic mutations in CD79B.　The present invention comprises: a primer set for MYD88 gene mutations and a primer set for CD79B gene mutations; and a mutant probe for the MYD88 gene together with a wild-type probe for the MYD88 gene, and a mutant probe for the CD79B gene together with a wild-type probe for the CD79B gene.

Description

Gene mutation evaluation kit

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

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma and can arise in any organ where mature B cells are physiologically present. DLBCL is known to be a heterogeneous group of diseases with diverse molecular genetic backgrounds. The diagnosis of DLBCL is based on the finding of diffuse proliferation of large B-cell-derived tumor cells in biopsy tissue.

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

[0004] Furthermore, lymphoplasmacytic lymphoma (LPL) is defined as a lymphoid neoplasm consisting of small B-cell lymphocytes, lymphocytes with a tendency to differentiate into plasma cells, and plasma cells, with or without IgM M-protein, according to the WHO classification (2017). Waldenstrom's macroglobulinemia (WM) is defined as a subset of LPL associated with bone marrow infiltration and IgM M-proteinemia. Mutations in the MYD88 gene are found in approximately 90% of WM / LPL patients, and identifying MYD88 mutations is useful for definitive diagnosis.

[0005] In a broad sense, the above-mentioned genetic mutation refers to a state in which a gene has undergone some kind of congenital or acquired abnormality. Examples of genetic mutations include substitution, deletion, or addition of bases constituting DNA. Here, a gene is composed of a coding region that encodes a protein and a non-coding region such as an expression control region. Gene mutations can occur in both coding and non-coding regions. In particular, genetic mutations present in coding regions include missense mutations that change the encoded amino acid, nonsense mutations that convert a codon encoding a specific amino acid into a stop codon, and silent mutations that do not change the encoded amino acid.

[0006] Gene mutations are the cause of hereditary diseases and cancer, and are also related to the efficacy of certain drugs. Gene mutations are also related to physical conditions such as obesity. Therefore, identifying specific gene mutations (also known as genotyping) is an essential technique for diagnosing hereditary diseases and understanding the efficacy of drugs.

[0007] Genotyping methods include, for example, DNA sequencing, SSCP (Single Strand Conformation Polymorphism), RFLP (Restriction Fragment Length Polymorphism), PCR (Polymerase Chain Reaction), AFLP (Amplified Fragment Length Polymorphism), ASO (Allele Specific Oligonucleotide) probes, and methods that detect binding to DNA microarrays or DNA beads. Among these techniques, methods that use DNA microarrays typically include mutant and wild-type probes immobilized on a carrier for the gene mutation to be detected.

[0008] In the method using a DNA microarray, first, a region containing a gene mutation to be detected is amplified by a nucleic acid amplification reaction using fluorescently labeled primers. Then, a hybridization reaction is carried out between the fluorescently labeled nucleic acid fragment and a mutant probe and a wild-type probe. If the amplified nucleic acid fragment contains a mutant, fluorescence is observed from the mutant probe. Therefore, by observing the fluorescence from the mutant probe and the wild-type probe, gene mutations can be genotyped.

[0009] Special Publication No. 2017-523188

[0010] Cancer Cell 39, 1643-1653, December 13, 2021

[0011] However, no technique for simultaneously identifying the above-mentioned MYD88 gene mutation and / or CD79B gene mutation is known, and if this technique were possible, it would be possible to easily identify, for example, the MCD subtype in DLBCL. Therefore, an object of the present invention is to provide a gene mutation evaluation kit that can simultaneously identify MYD88 gene mutation and / or CD79B gene mutation.

[0012] The method for detecting a gene mutation according to the present invention, which achieves the above-mentioned object, includes the following.

[0013] (1) A gene mutation evaluation kit used for diagnosing a disease associated with a gene mutation in the MYD88 gene and / or a gene mutation in the CD79B gene, the gene mutation evaluation kit comprising: a primer set for MYD88 gene mutations that amplifies a nucleic acid region containing a gene mutation in the MYD88 gene; a primer set for CD79B gene mutations that amplifies a nucleic acid region containing a gene mutation in the CD79B gene; a MYD88 gene mutant probe and a MYD88 gene wild-type probe relating to a gene mutation in the MYD88 gene; and a CD79B gene mutant probe and a CD79B gene wild-type probe relating to a gene mutation in the CD79B gene.

[0014] (2) A kit for evaluating gene mutations according to (1), characterized in that the gene mutation in the MYD88 gene is a substitution mutation of leucine at position 265 with proline, and the gene mutation in the CD79B gene is a missense mutation and / or a nonsense mutation in the tyrosine residue at position 196 and a silent mutation in the tyrosine residue.

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

[0016] (4) The kit for evaluating gene mutations according to (3), wherein the reverse primer for the MYD88 gene is labeled and has a concentration 2 to 6 times higher than that of the forward primer for the MYD88 gene, and the forward primer for the CD79B gene is labeled and has a concentration 5 to 10 times higher than that of the reverse primer for the CD79B gene.

[0017] (5) A kit for evaluating gene mutations according to (1), characterized in that 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 base sequences shown in Table 1.

[0018] (6) A kit for evaluating gene mutations described in (1), characterized in that it further contains a blocking nucleic acid for MYD88 that hybridizes to a wild-type nucleic acid fragment among the nucleic acid fragments amplified with the primer set for MYD88 gene mutations, and / or a blocking nucleic acid for CD79B that hybridizes to a wild-type nucleic acid fragment among the nucleic acid fragments amplified with the primer set for CD79B gene mutations.

[0019] (7) A kit for evaluating gene mutations according to (6), characterized in that the blocking nucleic acid for MYD88 has the base sequence of SEQ ID NO: 89, and the blocking nucleic acid for CD79B has the base sequence of SEQ ID NO: 95.

[0020] (8) The kit for evaluating gene mutations according to (6), wherein the MYD88 blocking nucleic acid is prepared in a concentration range of 0 to 500 nM in the hybridization solution, and the CD79B blocking nucleic acid is prepared in a concentration range of 125 to 1000 nM in the hybridization solution. This specification incorporates the disclosure of Japanese Patent Application No. 2023-216494, from which the present application claims priority.

[0021] The gene mutation evaluation kit according to the present invention allows for simultaneous identification of gene mutations in MYD88 and CD79B. Use of the gene mutation evaluation kit according to the present invention allows for easy identification of, for example, the MCD subtype in DLBCL based on gene mutations in MYD88 and / or CD79B.

[0022] 1 is a characteristic diagram showing the results of measuring the fluorescence intensity values ​​of various probes designed in the examples when a 100% mutation model specimen was used. FIG. 2 is a characteristic diagram showing the results of measuring the fluorescence intensity values ​​of various probes designed in the examples when a 5% mutation model specimen was used. FIG. 3 is a characteristic diagram showing the determination values ​​calculated from the fluorescence intensity values ​​of various probes designed in the examples when a 5% mutation model specimen was used. FIG. 4 is a characteristic diagram showing the results of measuring the fluorescence intensity values ​​of a wild-type probe (gene mutation of MYD88 gene) designed in the examples when a wild-type sample, a 5% mutation model specimen, or genomic DNA was used, and calculating the determination value. FIG. 5 is a characteristic diagram showing the results of measuring the fluorescence intensity values ​​of a wild-type probe (gene mutation of CD79B gene) designed in the examples when a wild-type sample, a 5% mutation model specimen, or genomic DNA was used. FIG. 6 is a characteristic diagram showing the determination values ​​calculated from the fluorescence intensity values ​​of a wild-type probe (gene mutation of CD79B gene) designed in the examples when a wild-type sample, a 5% mutation model specimen, or genomic DNA was used. FIG. 7 is a characteristic diagram showing the results (determination values) of detecting a gene mutation in genomic DNA using various probes selected in the examples. 1 is a characteristic diagram showing the combinations of a primer set for amplifying a region containing a gene mutation in the CD79B gene and a primer set for amplifying a region containing a gene mutation in the MYD88 gene, which were used in the Examples.

[0034] FIG. 1 is a characteristic diagram showing the relationship between the amount of nucleic acid amplified and the annealing temperature for each primer set combination.

[0035] FIG. 1 is a photograph showing the results of electrophoresis of nucleic acid fragments contained in the reaction solution for primer set combinations [5], [6], and [7].

[0036] FIG. 1 is a characteristic diagram showing the results of PCR performed with primer set combinations [5], [6], and [7], and detecting the amplified fragments by the fluorescence intensity of the wild-type probe and the mutant-type probe.

[0037] FIG. 1 is a characteristic diagram showing the results of measuring fluorescence intensity for primer set combinations [6] and [7], changing the mixing ratio of labeled primers to unlabeled primers.

[0038] FIG. 1 is a characteristic diagram showing the concentration ratio of the forward primer and the reverse primer contained in the primer sets used in the Examples.1 is a characteristic diagram showing the relationship between the concentration ratio of the forward primer and the reverse primer contained in the primer set (MYD88 gene) tested in the Examples and the fluorescence intensity value. FIG. 2 is a characteristic diagram showing the relationship between the concentration ratio of the forward primer and the reverse primer contained in the primer set (CD79B gene) tested in the Examples and the fluorescence intensity value. FIG. 3 is a characteristic diagram showing the results of measuring the fluorescence intensity from the mutant probe when using the blocking nucleic acid (MYD88 gene) designed in the Examples. FIG. 4 is a characteristic diagram showing the results of measuring the fluorescence intensity from the wild-type probe and the mutant probe when using v2-2 or v2-3 of the blocking nucleic acids (MYD88 gene) designed in the Examples. FIG. 5 is a characteristic diagram showing the determination value calculated from the fluorescence intensity measured with the wild-type probe and the mutant probe when using v2-2 or v2-3 of the blocking nucleic acids (MYD88 gene) designed in the Examples. FIG. 6 is a characteristic diagram showing the determination value calculated from the fluorescence intensity measured with the mutant probe when using the blocking nucleic acid (CD79B gene) designed in the Examples. 1 is a characteristic diagram showing the determination values ​​for each mutant probe calculated using v4-1 and v4-2 of the blocking nucleic acids (CD79B gene) designed in the Examples and using a wild-type model specimen or a 5% mutation model specimen. FIG. 2 is a characteristic diagram showing the fluorescence intensity values ​​for each mutant probe calculated using v4-1 and v4-2 of the blocking nucleic acids (CD79B gene) designed in the Examples and using a wild-type model specimen or a 5% mutation model specimen. FIG. 3 is a characteristic diagram showing the results of calculating the determination values ​​for the blocking nucleic acid (CD79B gene) v4-2 designed in the Examples by changing its concentration. FIG. 4 is a characteristic diagram showing the results of measuring the fluorescence intensity from each mutant probe when a 5% mutation model specimen is used and the concentration of the blocking nucleic acid (CD79B gene) v4-2 designed in the Examples is set to 500 nM, 750 nM, or 1000 nM. This is a characteristic diagram showing the results of calculating the judgment values ​​of each mutant probe when using a wild-type model sample or a 5% mutant model sample, when the concentration of the blocking nucleic acid (CD79B gene) v4-2 designed in the examples was 500 nM or 750 nM.

[0023] The gene mutation evaluation kit according to the present invention is described in detail below. The gene mutation evaluation kit simultaneously identifies at least gene mutations in the MYD88 gene and gene mutations in the CD79B gene. Therefore, the gene mutation evaluation kit can be used to diagnose diseases associated with gene mutations in the MYD88 gene and / or gene mutations in the CD79B gene. The MYD88 gene and CD79B gene encode proteins involved in the NF-κB signaling pathway, and gene mutations in these genes are thought to constitutively activate the NF-κB pathway, leading to the development of lymphoma and other diseases. For example, the MYD88 L265P mutation is frequently observed in lymphoplasmacytic lymphoma (LPL) / Waldenstrom macroglobulinemia (WM). Furthermore, 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 that amplifies a nucleic acid region containing a gene mutation and a probe set that detects the amplified nucleic acid fragment. That is, the gene mutation evaluation kit includes a primer set for MYD88 gene mutations that amplifies a nucleic acid region containing a gene mutation in the MYD88 gene and a primer set for CD79B gene mutations that amplify a nucleic acid region containing a gene mutation in the CD79B gene. The gene mutation evaluation kit also includes a probe set consisting of a MYD88 gene mutant probe and a MYD88 gene wild-type probe for a gene mutation in the MYD88 gene, and a probe set consisting of a CD79B gene mutant probe and a CD79B gene wild-type probe for a gene mutation in the CD79B gene.

[0025] MYD88 is a cytoplasmic adaptor protein that plays a central role in innate and adaptive immune responses and functions as an essential signal transduction pathway for interleukin-1 and Toll-like receptor signaling. MYD88 is also known as IMD68, MYD88D, and WM1. The genetic mutation in the MYD88 gene is a missense mutation resulting in a proline substitution at the leucine-encoding codon (CTG) at position 265 from the N-terminus. The missense mutation is a thymine-to-cytosine substitution at position 794 from the 5' end of the MYD88 coding region (794T>C).

[0026] On the other hand, CD79B exists as a heterodimer 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. Genetic mutations in the CD79B gene include three types of genetic mutations: a missense mutation resulting in a base substitution at the codon (TAC) encoding tyrosine at position 196 from the N-terminus, resulting in a different amino acid; a nonsense mutation resulting in a stop codon; and a silent mutation resulting in the encoded amino acid remaining as tyrosine.

[0027] Here, the missense mutations in the CD79B gene are a mutation in which thymine at position 589 from the 5' end of the CD79B coding region is replaced with cytosine (this mutation is referred to as 589T>C; hereinafter, the same description applies, with A: adenine, G: guanine, C: cytosine, and T: thymine), 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T. Furthermore, the nonsense mutations in the CD79B gene are 591C>G and 591C>A. Furthermore, a silent mutation in the CD79B gene is 591C>T.

[0028] These mutations at thymine 196 in the CD79B gene are summarized in Table 2. In Table 2, the wild-type sequence is marked with W in the type column, and M1 to M6 in the same column are missense mutations, M7 and M8 are nonsense mutations, and M9 is a silent mutation.

[0029]

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

[0031] As shown in the Examples below, the primer set for MYD88 gene mutation and the primer set for CD79B gene mutation are designed based on the following: the desired amount of nucleic acid fragments can be amplified with each primer set in the same reaction system; and nonspecific amplification is minimized. Specifically, a primer set is designed so that the amount (molar concentration) of nucleic acid fragments obtained when a nucleic acid amplification reaction is performed using a plasmid containing the nucleic acid region to be amplified as a template is similar to that obtained when a nucleic acid amplification reaction is performed using genomic DNA containing the nucleic acid region to be amplified as a template. Furthermore, a primer set with minimal nonspecific amplification can be designed by detecting the nucleic acid fragments contained in the reaction solution after the nucleic acid amplification reaction using techniques such as electrophoresis.

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

[0033] A primer set defined by these nucleotide sequences is designed so that the forward primer and reverse primer have similar Tm values. Therefore, the nucleotide sequences may have one or more nucleotides added to the 5'-end and / or 3'-end, or one or more nucleotides deleted from the 5'-end and / or 3'-end, as long as the difference in Tm between the forward primer and reverse primer is not too large.

[0034] The Tm value of the primers can be calculated using the nearest neighbor method or software that applies the nearest neighbor method. The difference in Tm value 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, even more preferably within 1.5°C, even more preferably within 1.0°C, and even more preferably within 0.5°C.

[0035] The base length of a 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 add one or more bases within this range. The one or more bases to be added can be determined based on the base sequence of the MYD88 gene (NCBI Gene ID: 4615) and the base sequence of the CD79B gene (NCBI Gene ID: 974) registered in publicly known databases.

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

[0037] Here, the term "labeling" refers to binding of a labeling substance, such as a radioisotope, a fluorescent dye, or an organic compound such as digoxigenin (DIG) or biotin, to the primer. The labeling substance can be appropriately selected from various conventionally known labeling substances. In particular, 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 are preferred, as they can be detected easily and with high sensitivity.

[0038] Furthermore, the ratio (concentration ratio) of the forward primer to the reverse primer contained in the primer set for MYD88 gene mutation is not particularly limited, but is preferably set so that the labeled primer is greater than the unlabeled primer. For example, when the reverse primer is labeled, 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. The ratio (reverse primer concentration: forward primer concentration) can also be 50:10 or 60:10. That is, when the reverse primer is labeled, the concentration of the reverse primer can be 1.5 to 6 times that of the forward primer, preferably 2 to 6 times, more preferably 3 to 5 times, and most preferably 4 times. The best fluorescence intensity values ​​were achieved when this ratio was used. By using such a ratio, the fluorescence intensity derived from the amplification product obtained with the primer set for MYD88 gene mutation can be increased.

[0039] Furthermore, the ratio (concentration ratio) of the forward primer to the reverse primer contained in the primer set for CD79B gene mutation is not particularly limited, but is preferably a ratio in which the labeled primer is greater than the unlabeled primer. For example, when the forward primer is labeled, the ratio of the forward primer to the reverse primer (forward primer concentration:reverse primer concentration) can be 15:10, 20:10, 30:10, 40:10, or 50:10. Furthermore, this ratio is preferably 60:10, and more preferably 70:10. Furthermore, this ratio can be 80:10, 90:10, or 100:10. That is, when the forward primer is labeled, the concentration of the forward primer can be 1.5 to 10 times that of the reverse primer, particularly 5 to 10 times, more preferably 6 to 7 times, and most preferably 7 times. By setting such a ratio, it is possible to increase the fluorescence intensity derived from the amplification product obtained with the primer set for CD79B gene mutation.

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

[0041] Each probe set will be described below. The mutant probes and wild-type probes contained in these probe sets are preferably nucleic acids, more preferably DNA. DNA includes both double-stranded and single-stranded DNA, but is preferably single-stranded DNA. The mutant probes and wild-type probes can be obtained by chemical synthesis using, for example, a nucleic acid synthesizer. As the nucleic acid synthesizer, a device called a DNA synthesizer, a fully automated nucleic acid synthesizer, an automatic nucleic acid synthesizer, etc. can be used.

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

[0043] The wild-type probe and the mutant-type probe may be designed so that only the base corresponding to the genetic mutation is different and the rest are identical, or they may be designed to have different base lengths or so that the 3' and / or 5' ends are different. The wild-type probe and the mutant-type probe are not particularly limited, but can be, for example, 10 to 30 bases long, preferably 15 to 25 bases long. Furthermore, in the wild-type probe and the mutant-type probe, the position of the base corresponding to the genetic mutation is preferably the center of the character string when the bases constituting the wild-type probe and the mutant-type probe are viewed as a character string. Note that the center of the character string includes cases where the wild-type probe and the mutant-type probe are shifted by one base toward the 5' or 3' end for wild-type probes and mutant probes consisting of an even number of bases.

[0044] More specifically, for nucleic acid fragments obtained by nucleic acid amplification using a set of the forward primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 (5'-CAGGTGCCCATCAGAAG-3') and the 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. These wild-type and mutant probes for the MYD88 gene produce high fluorescence intensity from the amplified nucleic acid fragments and have high resolution, allowing for highly accurate detection of genetic mutations to be tested. Here, resolution refers to the difference in the determination values ​​for each sample calculated according to the following formula 1, where nucleic acid fragments are amplified using samples with different mutation rates, and each nucleic acid fragment is detected with the wild-type and mutant probes: The samples with different mutation rates can be a 100% wild-type sample and a 5% mutant sample. Formula 1: [Signal intensity derived from mutant probe] / ([Signal intensity derived from wild-type probe]+[Signal intensity derived from mutant probe]).

[0045] Probe set for gene mutation in CD79B gene A probe set for gene mutation in CD79B gene can be designed based on the nucleotide sequence of a nucleic acid fragment amplified by a nucleic acid amplification reaction using the above-mentioned primer set for CD79B gene mutation. That is, the mutant probe and wild-type probe included in the probe set can be designed as nucleotide sequences complementary to a region containing the gene mutation site in the amplified nucleic acid fragment. As described above, the wild-type of a gene mutation in CD79B gene has a T at nucleotide 589, an A at nucleotide 590, and a C at nucleotide 591. Furthermore, as shown in Table 2 above, the mutation types in this gene mutation include missense mutations indicated as M1 to M6, nonsense mutations indicated as M7 and M8, and silent mutation indicated as M9.

[0046] The wild-type probe and the mutant-type probe may be designed so that only the base corresponding to the genetic mutation is different and the rest are identical, or they may be designed to have different base lengths or so that the 3' and / or 5' ends are different. The wild-type probe and the mutant-type probe are not particularly limited, but can be, for example, 10 to 30 bases long, preferably 15 to 25 bases long. Furthermore, in the wild-type probe and the mutant-type probe, the position of the base corresponding to the genetic mutation is preferably the center of the character string when the bases constituting the wild-type probe and the mutant-type probe are viewed as a character string. Note that the center of the character string includes cases where the wild-type probe and the mutant-type probe are shifted by one base toward the 5' or 3' end for wild-type probes and mutant probes consisting of an even number of bases.

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

[0048]

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

[0050] The wild-type and mutant probes designed for gene mutations in the CD79B gene shown in Table 3 have high fluorescence intensity derived from the amplified nucleic acid fragments and high resolution, enabling highly accurate detection of gene mutations to be tested. Here, resolution refers to the difference in the judgment values ​​for each sample calculated according to the following formula 1 after amplifying nucleic acid fragments using samples with different mutation rates and detecting each nucleic acid fragment with the wild-type and mutant probes. The samples with different mutation rates can be a 100% wild-type sample and a 5% mutant sample. Formula 1: [Signal intensity derived from mutant probe] / ([Signal intensity derived from wild-type probe]+[Signal intensity derived from mutant probe]).

[0051] DNA Chip (DNA Microarray) The wild-type probe and mutant-type probe designed as described above are preferably used in the form of a microarray (e.g., a DNA chip) by immobilizing their 5' ends on a carrier. In this case, the microarray has a first probe, a second probe, and a wild-type probe for the genetic mutation to be tested. The microarray may have a wild-type probe and a mutant-type probe for the genetic mutation of the MYD88 gene and a genetic mutation of the CD79B gene, respectively. Alternatively, a microarray having a wild-type probe and a mutant-type probe for the genetic mutation of the MYD88 gene and a microarray having a wild-type probe and a mutant-type probe for the genetic mutation of the CD79B gene may be prepared.

[0052] The carrier material may be any known material in the art and is not particularly limited. For example, conductive materials such as noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten, and compounds thereof, and carbon such as graphite and carbon fiber; silicon materials such as single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, and silicon nitride, and composite materials of these silicon materials such as SOI (silicon-on-insulator); inorganic materials such as glass, quartz glass, alumina, sapphire, ceramics, forsterite, and photosensitive glass; polyethylene, ethylene, and polypropylene. Examples of the carrier include organic materials such as cyclic polyolefins, polyisobutylene, polyethylene terephthalate, unsaturated polyesters, fluorine-containing resins, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resins, polyacrylonitrile, polystyrene, acetal resins, polycarbonate, polyamide, phenolic resins, urea resins, epoxy resins, melamine resins, styrene-acrylonitrile copolymers, acrylonitrile-butadiene styrene copolymers, polyphenylene oxide, and polysulfone. The shape of the carrier is not particularly limited, but is preferably flat.

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

[0054] In microarrays, carriers with a fine, flat structure are preferably used. Shapes include, but are not limited to, rectangular, square, and round. Typically, carriers measuring 1 to 75 mm square, preferably 1 to 10 mm square, and more preferably 3 to 5 mm square are used. Because carriers with a fine, flat structure are easily manufactured, substrates made of silicon or resin materials are preferred. Carriers with a carbon layer and chemically modified groups on the surface of a substrate made of single-crystal silicon are particularly preferred. Single-crystal silicon also includes those with slight variations in the orientation of the crystal axis in parts (sometimes called mosaic crystals) and those containing atomic-scale irregularities (lattice defects).

[0055] As the carbon layer that is formed on substrate, there is no particular limitation, but preferably use synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene, carbon nanotube), any of their mixtures, or their laminated material.In addition, can also use carbide 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.Here, soft diamond is a general term for the incomplete diamond structure that is the mixture of diamond and carbon, such as so-called diamond-like carbon (DLC: Diamond Like Carbon), and its mixture ratio is not particularly limited. Carbon layers are advantageous in that they have excellent chemical stability and can withstand subsequent introduction of chemical modification groups and reactions involving binding with the analyte; they bind to the analyte via electrostatic bonding, which allows for flexibility; they do not absorb UV light, making them transparent to the UV light of the detection system; and they can be electrified during electroblotting. Another advantage is that they exhibit minimal nonspecific adsorption during the binding reaction with the analyte. As mentioned above, a carrier in which the substrate itself is made of a carbon layer may also be used.

[0056] The carbon layer can be formed by a known method, such as microwave plasma CVD (chemical vapor deposition), ECRCVD (electric cyclotron resonance chemical vapor deposition), ICP (inductive coupled plasma), DC sputtering, ECR (electric cyclotron resonance) sputtering, ionization deposition, arc deposition, laser deposition, EB (electron beam) deposition, and resistance heating deposition.

[0057] In radio-frequency plasma CVD, the source gas (methane) is decomposed by glow discharge generated between electrodes using radio frequency waves, and a carbon layer is synthesized on the substrate. In ionized vapor deposition, the source gas (benzene) is decomposed and ionized using thermal electrons generated by a tungsten filament, and a carbon layer is formed on the substrate using a bias voltage. The carbon layer can also be formed by ionized vapor deposition in a mixed gas consisting of 1-99% by volume of hydrogen gas and the remaining 99-1% by volume of methane gas.

[0058] In the arc evaporation method, a DC voltage is applied between a solid graphite material (cathode evaporation source) and a vacuum chamber (anode), causing an arc discharge in a vacuum to generate a plasma of carbon atoms from the cathode, and a bias voltage more negative than that of the evaporation source is applied to the substrate, accelerating the carbon ions in the plasma toward the substrate and forming a carbon layer.

[0059] In the laser deposition method, for example, a graphite target plate is irradiated with light from an Nd:YAG laser (pulsed oscillation) to melt it, and carbon atoms are deposited on a glass substrate, thereby forming a carbon layer.

[0060] When a carbon layer is formed on the surface of a substrate, the thickness of the carbon layer is usually about a monolayer to 100 μm. If the carbon layer is too thin, the surface of the base substrate may be locally exposed, while if the carbon layer is too thick, productivity will decrease. Therefore, the thickness is preferably 2 nm to 1 μm, and more preferably 5 nm to 500 nm.

[0061] By introducing a chemical modifying group onto the surface of the substrate on which the carbon layer is formed, the probe can be firmly immobilized on the carrier. The chemical modifying group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, but examples thereof 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 amino groups can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiating it with ultraviolet light in chlorine gas, and then further irradiating it with ultraviolet light in ammonia gas. Alternatively, the introduction can be carried out by reacting the chlorinated carbon layer with a polyamine gas such as methylenediamine or ethylenediamine.

[0063] The introduction of a carboxyl group can be carried out, for example, by reacting an appropriate compound with the aminated carbon layer as described above. Examples of compounds that can be used to introduce 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, and 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, and phthalic acid; polyacrylic acid; and polymethacrylic 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 and malonic acid monochloride; and acid anhydrides such as phthalic anhydride, succinic acid anhydride, oxalic acid anhydride, maleic acid anhydride, and butanetetracarboxylic acid anhydride.

[0064] Epoxy groups can be introduced by, for example, reacting an appropriate polyepoxy compound with the aminated carbon layer as described above. Alternatively, they can be obtained by reacting an organic peracid with the carbon-carbon double bond contained in the carbon layer. Examples of organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

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

[0066] The introduction of a hydroxyl group can be carried out, for example, by reacting the chlorinated carbon layer with water.

[0067] The term "activated ester group" refers to an ester group that has a highly acidic electron-withdrawing group on the alcohol side of the ester group, activating nucleophilic reactions, i.e., a highly reactive ester group. The ester group has an electron-withdrawing group on the alcohol side of the ester group, making it more activated than alkyl esters. The activated ester group is reactive with groups such as amino groups, thiol groups, and hydroxyl groups. More specifically, phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, and esters of heterocyclic hydroxy compounds are known to be activated ester groups with much higher activity than alkyl esters. More specifically, examples of activated ester groups include p-nitrophenyl, N-hydroxysuccinimide, succinimide, phthalimide, and 5-norbornene-2,3-dicarboximide groups, with the N-hydroxysuccinimide group being particularly preferred.

[0068] The introduction of an active ester group can be carried out, for example, by converting the carboxyl group introduced as described above into an active ester using a dehydration condensation agent such as cyanamide or a carbodiimide (e.g., 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and a compound such as N-hydroxysuccinimide. This treatment allows the formation of a group in which an active ester group such as an N-hydroxysuccinimide group is bound to the end of a hydrocarbon group via an amide bond (JP 2001-139532 A).

[0069] A spotting solution is prepared by dissolving the wild-type probe or mutant-type probe in a spotting buffer, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted onto a carrier using a spotter device or the like to produce a microarray in which the wild-type probe and mutant-type probe are immobilized on the carrier. Alternatively, the spotting solution may be spotted manually using a micropipette.

[0070] After spotting, incubation is preferably performed to promote the reaction of binding of the wild-type probe or mutant probe to the carrier. Incubation is typically performed at temperatures between -20 and 100°C, preferably between 0 and 90°C, for typically 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is preferably performed in a high-humidity atmosphere, for example, at a humidity of 50 to 90%. Following incubation, washing with a washing solution (e.g., 50 mM TBS / 0.05% Tween 20, 2x SSC / 0.2% SDS solution, ultrapure water, etc.) is preferably performed to remove DNA not bound to the carrier.

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

[0072] The blocking nucleic acid can be mixed with a reaction solution containing a wild-type nucleic acid fragment and a mutant nucleic acid fragment, and the resulting mixture can be brought into contact with the above-mentioned DNA chip to allow the hybridization reaction between the mutant nucleic acid fragment and the mutant probe to proceed. Alternatively, the reaction solution containing the wild-type nucleic acid fragment and the mutant nucleic acid fragment can be mixed with a solution containing the blocking nucleic acid on the DNA chip to allow specific hybridization between the mutant nucleic acid fragment and the mutant probe to proceed simultaneously. In either case, the blocking nucleic acid specifically hybridizes with the wild-type nucleic acid fragment, thereby preventing the wild-type nucleic acid fragment from nonspecifically hybridizing with the mutant probe.

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

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

[0075] It should be noted that the use of a blocking nucleic acid is not necessary if the nonspecific hybridization between the wild-type nucleic acid fragment and the mutant probe is slight, but is unnecessary if the nonspecific hybridization between the wild-type nucleic acid fragment and the mutant probe is at a level that is not problematic. When a blocking nucleic acid is used, its concentration is not particularly limited, and can be appropriately set depending on, for example, the concentration of the non-target nucleic acid and / or the concentration of the target nucleic acid, or the concentration of the primer.

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

[0077] In particular, when using a set of the aforementioned forward primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 79 (5'-CAGGTGCCCATCAGAAG-3') and reverse primer for the MYD88 gene consisting of the nucleotide sequence of SEQ ID NO: 78 (5'-CGCAGACAGTGATGAACC-3'), nonspecific hybridization between the amplified wild-type nucleic acid fragment and the MYD88 gene mutant probe consisting of the nucleotide sequence of SEQ ID NO: 6 (5'-GAAGCGACCGATCCC-3') is slight, and it cannot be said that a blocking nucleic acid is necessarily required. In other words, the use of a blocking nucleic acid for MYD88 gene mutations is not necessary (blocking nucleic acid concentration is 0 nM). However, for more precise detection of mutant nucleic acid fragments, it is preferable to use a blocking nucleic acid for MYD88 gene mutations consisting of the nucleotide sequence of SEQ ID NO: 89. In this case, the concentration of the blocking nucleic acid for 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, and preferably 70 to 150 nM. As an example, the concentration of the blocking nucleic acid for MYD88 gene mutation can be set to 125 nM.

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

[0079] 4) Gene Mutation Testing Method As described above, the gene mutation assessment kit identifies gene mutations in the CD79B gene and the MYD88 gene and can be used to diagnose diseases associated with these gene mutations. Specifically, if these gene mutations are detected, the patient can be classified as having the MCD subtype of diffuse large B-cell lymphoma (DLBCL). Furthermore, Cancer Cell 39, 1643-1653, December 13, 2021, suggests that R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) in combination with ibrutinib, a BTK inhibitor, is effective in treating DLBCL classified as having the MCD subtype. Therefore, identifying the MCD type of DLBCL using the above-described gene mutation assessment kit can provide useful information for determining treatment strategies.

[0080] The following is a detailed description of the procedure for identifying genetic mutations in the MYD88 gene and the CD79B gene using a genetic mutation assessment kit. In the following description, a microarray having wild-type and mutant probes for genetic mutations in the MYD88 gene and wild-type and mutant probes for genetic mutations in the CD79B gene is used.

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

[0082] Specifically, testing for genetic mutations involves the steps of extracting DNA from a sample derived from a subject, using the extracted DNA as a template to amplify a region containing the genetic mutation to be tested, hybridizing the amplified nucleic acid fragment with wild-type and mutant probes using the microarray described above, and detecting signals from the wild-type and mutant probes. Note that in the hybridizing step, "wild-type and mutant probes" refers to both wild-type and mutant probes for genetic mutations in the MYD88 gene and CD79B gene. Similarly, "wild-type and mutant probes" in the signal detecting step refers to both wild-type and mutant probes for genetic mutations in the MYD88 gene and CD79B gene.

[0083] The subject of diagnosis is typically a human, and although there are no particular limitations on race, the subject is preferably a person of the Asian race, preferably an East Asian race, and particularly preferably a Japanese person. The subject of diagnosis may also be a patient suspected of having a disease associated with the genetic mutation being tested. The sample derived from the subject of diagnosis is not particularly limited. Examples include blood-related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ fragments, and extracts.

[0084] First, DNA is extracted from a sample collected from a subject. The extraction method 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 tested. Examples of applicable amplification reactions include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN). As described above, the amplified nucleic acid fragments can be labeled by labeling the primers used in the nucleic acid amplification reaction. Specifically, the amplified nucleic acid fragments can be labeled by adding a labeling substance to either the forward primer or the reverse primer contained in the primer set for CD79B gene mutations, or to either the forward primer or the reverse primer contained in the primer set for MYD88 gene mutations.

[0086] This reaction system also contains a buffer necessary for nucleic acid amplification and labeling, a heat-stable DNA polymerase, a primer set for MYD88 gene mutation and a primer set for CD79B gene mutation, labeled nucleotide triphosphates (specifically, nucleotide triphosphates labeled with a fluorescent label, etc.), nucleotide triphosphates, magnesium chloride, etc.

[0087] Furthermore, 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, but is preferably 1 kbp or less, more preferably 800 bp or less, even more preferably 500 bp or less, and particularly preferably 350 bp or less.

[0088] The amplified nucleic acid obtained as described above is subjected to a hybridization reaction with a wild-type probe and a mutant-type probe immobilized on a carrier, and signals from the wild-type probe and the mutant-type probe are detected based on the label of the hybridized nucleic acid fragment, allowing the above-mentioned genetic mutation in the subject to be examined.

[0089] For example, when a fluorescent label is used, the signal from the label can be detected using a fluorescent scanner and analyzed using image analysis software to quantify the signal intensity. The hybridization reaction is preferably performed under stringent conditions. Stringent conditions refer to conditions under which specific hybrids are formed and nonspecific hybrids are not formed, such as hybridization at 47-52°C (specifically, 51.5°C) for 15-90 minutes (specifically, 30 minutes), followed by 30 washes in 0.1xSSC / 0.1% SDS at 25°C, and then 80 washes in 1xSSC at 25°C. It should be noted that, if the probe chain length is short, it is more preferable to use a lower hybridization temperature, and, if the chain length is long, it is more preferable to use a higher hybridization temperature. It goes without saying that the hybridization temperature for specificity increases with increasing salt concentration, and conversely, the hybridization temperature for specificity decreases with decreasing salt concentration.

[0090] Furthermore, when a microarray equipped with a mutant probe and a wild-type probe is used for the above-mentioned genetic mutation, the genetic mutation can be tested using the signal intensities from these mutant probes and wild-type probes. Specifically, the signal intensities of the mutant probe and the wild-type probe are measured, respectively, and a judgment value for evaluating the signal intensity derived from the mutant probe is calculated. An example of calculating the judgment value is a method for evaluating the mutation corresponding to the mutant probe using Formula 1: [Signal intensity derived from the mutant probe] / ([Signal intensity derived from the wild-type probe]+[Signal intensity derived from the mutant probe]).

[0091] The judgment value calculated by the above formula 1 is then compared with a predetermined threshold value (cutoff value), and if the judgment value exceeds the threshold value, it is determined that the amplified nucleic acid contains a genetic mutation (mutation type) corresponding to the mutant probe, and if the judgment value is below the threshold value, it is determined that the amplified nucleic acid does not contain the genetic mutation (mutation type).

[0092] Here, the threshold is not particularly limited, but can be defined, for example, based on the judgment value calculated by the above formula 1 using a sample in which the genetic mutation has been confirmed to be wild-type. More specifically, multiple judgment values ​​can be calculated using multiple samples in which the genetic mutation has been confirmed to be wild-type, and the value of the average value + 3σ (σ: standard deviation) can be used as the threshold. Note that the value of the average value + 2σ or the average value + σ can also be used as the threshold.

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

[0094] In particular, when using a primer set for MYD88 gene mutations, which comprises 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'), and a primer set for CD79B gene mutations, which comprises 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'), the target nucleic acid fragments can be stably and highly amplified, and nonspecific amplification due to nonspecific hybridization of the forward primer and / or reverse primer can be suppressed. As a result, the use of these primer sets allows for identification of each gene mutation with greater sensitivity.

[0095] Furthermore, for nucleic acid fragments obtained using a set of the forward primer for the MYD88 gene consisting of the base sequence of SEQ ID NO: 79 (5'-CAGGTGCCCATCAGAAG-3') and the reverse primer for the MYD88 gene consisting of the base sequence of SEQ ID NO: 78 (5'-CGCAGACAGTGATGAACC-3'), genetic mutations in the MYD88 gene in subjects can be identified with high accuracy by using a wild-type probe for the MYD88 gene consisting of the base sequence of SEQ ID NO: 2 (5'-AGCGACTGATCCCCAT-3') and a mutant-type probe for the MYD88 gene consisting of the base sequence of SEQ ID NO: 6 (5'-GAAGCGACCGATCCC-3').

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

[0097] In this case, whether the genetic mutation in the CD79B gene is wild-type or mutant can be identified as follows: In particular, in the present invention, when the genetic mutation is detected as a silent mutation using the second probe, the genetic mutation can be determined to be wild-type.

[0098] Specifically, when a microarray comprising a first probe, a second probe, and a wild-type probe is used, genetic mutations in the CD79B gene can be tested using the signal intensities from these first probe, second probe, and wild-type probe. That is, the signal intensities of the first probe, second probe, and wild-type probe are each measured, and a judgment value for evaluating the signal intensity derived from the first probe or the second probe is calculated. Examples of calculation methods for the judgment value include, for example, using Formula 1: [signal intensity derived from the first probe] / ([signal intensity derived from the wild-type probe]+[signal intensity derived from the first probe]) to evaluate missense mutations and / or nonsense mutations corresponding to the first probe, and using Formula 2: [signal intensity derived from the second probe] / ([signal intensity derived from the wild-type probe]+[signal intensity derived from the second probe]) to evaluate silent mutations corresponding to the second probe.

[0099] The judgment value calculated by the above formula 1 is then compared with a predetermined threshold (cutoff value), and if the judgment value exceeds the threshold, it is determined that the amplified nucleic acid contains a missense mutation and / or a nonsense mutation corresponding to the first probe for the above genetic mutation, and if the judgment value is below the threshold, it is determined that the amplified nucleic acid does not contain the missense mutation and / or nonsense mutation.Furthermore, the judgment value calculated by the above formula 2 is compared with a predetermined threshold (cutoff value), and if the judgment value exceeds the threshold, it is determined that the amplified nucleic acid contains a silent mutation corresponding to the second probe for the above genetic mutation, and if the judgment value is below the threshold, it is determined that the amplified nucleic acid does not contain the silent mutation.

[0100] Then, if it is determined that a silent mutation is present based on the above formula 2, the genetic mutation in the CD79B gene being tested is determined to be wild-type. Furthermore, if it is determined that a missense mutation and / or a nonsense mutation is not present based on the above formula 1 and that a silent mutation is not present based on the above formula 2, the genetic mutation being tested is also determined to be wild-type. In this way, by using a microarray comprising a first probe, a second probe, and a wild-type probe for testing a genetic mutation in the CD79B gene, it is possible to identify with high accuracy whether the genetic mutation is a missense mutation and / or a nonsense mutation or a wild-type mutation.

[0101] As explained above, by identifying whether the genetic mutation in the MYD88 gene and the genetic mutation in the CD79B gene of a subject are wild-type or mutant, it is possible to provide information that is useful for diagnosing whether the subject has MCD type diffuse large B-cell lymphoma (DLBCL).

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

[0103] <Sequence design> In this example, for gene mutations in the MYD88 gene and gene mutations in the CD79B gene, a primer set was designed to amplify the region containing the gene mutation, a probe was designed to detect the wild-type or mutant type contained in the nucleic acid fragment amplified with the primer set, and blocking nucleic acids were designed to correspond to the wild-type gene mutations in the MYD88 gene and the CD79B gene.

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

[0105]

[0106] The probes designed in this example that correspond to gene mutations in the CD79B gene are shown in Tables 5 and 6. In the "Wild type (W) / Mutant type (M)" column in Tables 5 and 6, probes corresponding to the wild type are marked with W, and probes corresponding to the mutant types are marked with M1 to M9. The designations M1 to M9 are in accordance with Table 3.

[0107]

[0108]

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

[0110]

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

[0112]

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

[0114]

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

[0116]

[0117] <Investigation of optimal mutant probes> Fluorescence intensities were measured for the probes listed in Tables 4 to 6 using a 100% mutation model sample or a 5% mutation model sample. Specifically, DNA chips with the above-mentioned probes immobilized were placed in a BioShot HT-32 genetic analyzer (manufactured by Toyo Kohan Co., Ltd.) and hybridization reactions with the model samples were performed. The DNA chip needle with the immobilized DNA chip, along with a cleaning solution (0.1x SSC / 0.1% SDS solution, room temperature) for washing off excess reagent after the hybridization reaction of the DNA chip, a rinse solution (1x SSC solution, room temperature), and a detection solution for fluorescence detection (1x SSC solution, room temperature) were placed in the BioShot HT-32 and operation was started.

[0118] The reaction solution in the designated PCR tube was heated to 51.5°C by automatic operation, and 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 a washing solution bath and agitated 30 times so that the DNA chip went in and out of the washing solution surface. After washing, the DNA chip needle was immersed in a rinse solution bath and agitated 80 times so that the DNA chip went in and out of the rinse solution surface.

[0119] The rinsed DNA chip needle was slowly immersed in the detection solution tank, and a 640 nm single-wavelength laser was irradiated, capturing excitation light for 2 seconds on a CCD camera. The measured fluorescence intensity was then used to calculate the evaluation value according to the following formula: [Evaluation value] = [Fluorescence intensity of mutant probe] / ([Fluorescence intensity of mutant probe] + [Fluorescence intensity of wild-type probe]). The 100% mutation model sample refers to a sample in which the ratio of mutant to wild-type in the gene mutation to be detected is 100:0. The 5% mutation model sample refers to a sample in which the ratio of mutant to wild-type in the gene mutation to be detected is 5:95.

[0120] The results of measuring the fluorescence intensity values ​​when a 100% mutation model specimen was used are shown in Figure 1, the results of measuring the fluorescence intensity values ​​when a 5% mutation model specimen was used are shown in Figure 2, and the results of measuring the judged values ​​from the results shown in Figure 2 are shown in Figure 3. For comparison, Figures 1 to 3 also show the results of calculating the fluorescence intensity values ​​from each probe using a wild-type sample in which the ratio of mutant to wild-type in the gene mutation to be detected was 0:100.

[0121] The specificity of the mutant and wild-type probes was confirmed from the fluorescence intensity values ​​shown in Figure 1. The results showed that excellent fluorescence intensity values ​​were obtained for all mutant and wild-type probes tested. Furthermore, cross-reactivity was suppressed to approximately half of the specific fluorescence intensity value for all mutant and wild-type probes tested, indicating that it was possible to distinguish between the target gene mutations (mutant and wild-type).

[0122] Furthermore, as shown in Figure 1, the silent mutation 591C>T, designated M9, in the CD79B gene was detected with a strong fluorescence intensity value (47125) using the M8 probe (CD79B_M8_v3-2). Failure to detect the silent M9 mutation using a mutant probe could result in samples with the M9 silent mutation being erroneously identified as the M8 mutation. Therefore, using a mutant probe corresponding to the silent M9 mutation when identifying CD79B gene mutations can reliably identify the silent mutation and prevent erroneous identification. Furthermore, if a silent mutation is identified, the sample is classified as "wild type." This allows precise identification of whether the CD79B gene mutation is mutant or wild type, providing highly useful information about diseases associated with CD79B gene mutations.

[0123] On the other hand, Figure 2 shows the fluorescence intensity values ​​for the mutant and wild-type probes when a sample with a low mutation rate was used. Even with a sample with a low mutation rate, obtaining a fluorescence intensity value equal to or greater than the desired value allows for highly accurate detection of the target gene mutation. For example, assuming that a fluorescence intensity value of 10,000 or greater is desirable, the results shown in Figure 2 indicate that sufficient fluorescence intensity values ​​were not obtained for CD79B_M8_v3-3 and CD79B_M8_v4-3.

[0124] Figure 3 also shows the judgment values ​​calculated from the fluorescence intensity values ​​shown in Figure 2 according to the above formula. As can be seen from the formula for calculating the judgment value, a higher judgment value indicates better resolution between the mutant probe and the wild-type probe. It can also be said that a lower judgment value calculated for the mutant probe when a wild-type sample is used indicates better resolution. For example, if a judgment value of 0.05 or less calculated for the mutant probe when a wild-type sample is used is used as the standard, the results shown in Figure 3 indicate that CD79B_M4_v3-2, CD79B_M4_v4-4, and CD79B_M6-v4-3 do not have sufficient resolution.

[0125] Taking the above results into consideration, it was found that for genetic mutations in the MYD88 gene, it is preferable to use MYD88_M_v2-1 as the mutant probe, and for genetic mutations in the CD79B gene (nine types, M1 to M9), it is preferable to use 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] <Investigation of the optimal wild-type probe> First, we investigated a wild-type probe for detecting wild-type MYD88 gene mutations. Fluorescence intensity values ​​and judgement values ​​from the wild-type probe and mutant probe (v2-1) were calculated using the wild-type sample and a 5% mutant model specimen. Furthermore, fluorescence intensity values ​​and judgement values ​​were calculated in the same way using genomic DNA (gDNA-1) instead of the wild-type sample and the 5% mutant model specimen.

[0127] The results are shown in Figure 4. The wild-type probes were selected based on the following criteria: a higher fluorescence intensity value when using genomic DNA, a higher resolution (i.e., a larger difference between the mutant and wild-type scores), and a lower score calculated using the above formula from the fluorescence intensity value when using the wild-type sample. Specifically, the wild-type probes were evaluated comprehensively based on the following criteria: a fluorescence intensity value of 10,000 or greater when using genomic DNA, the highest resolution among the wild-type probes tested (the largest difference between the mutant and wild-type scores), and a score of 0.05 or less when using the wild-type sample. As a result, v2-1, which had the highest resolution, was selected as the optimal wild-type probe.

[0128] Next, we investigated wild-type probes for detecting wild-type CD79B gene mutations. The investigation method and evaluation criteria were similar to those used to select wild-type probes for MYD88 gene mutations described above. The mutant probes used were 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.

[0129] The results of measuring the fluorescence intensity values ​​are shown in Figure 5, and the results of calculating the judgment values ​​from the fluorescence intensity values ​​are shown in Figure 6. As can be seen from Figures 5 and 6, v3-1 was selected as the optimal wild-type probe, taking into consideration the high fluorescence intensity values ​​and high resolution when genomic DNA was used, and the low judgment value when a wild-type sample was used.

[0130] <Performance Evaluation of Selected Mutant and Wild-Type Probes> The mutant probe (MYD88_M_v2-1) and wild-type probe (MYD88_W_v2-1) were selected for MYD88 gene mutations, 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 probe (CD79B_W_v3-1) were selected for CD79B gene mutations to identify the same gene mutations in genomic DNA. Three types of genomic DNA were used: gDNA-1, gDNA-2, and gDNA-3. gDNA-1 is genomic DNA from a healthy individual, gDNA-2 is genomic DNA from a patient with non-Hodgkin's lymphoma, and gDNA-3 is genomic DNA from a patient with lymphoplasmacytic lymphoma.

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

[0132] To verify the results using the mutant and wild-type probes shown in Figure 7, we used direct sequencing to confirm the MYD88 and CD79B gene mutations for the three genomic DNAs tested (gDNA-1, gDNA-2, and gDNA-3). The results confirmed that both the MYD88 and CD79B mutations in gDNA-1 were wild-type. Furthermore, the MYD88 mutation in gDNA-1 was the L265P mutation, and the CD79B mutation was the Y196D mutation (M3). Furthermore, the MYD88 mutation in gDNA-3 was the L265P mutation, and the CD79B mutation was wild-type.

[0133] The results of identifying each gene mutation using the mutant and wild-type probes selected as described above were completely consistent with the results confirmed by direct sequencing, demonstrating that the mutant and wild-type probes selected as described above can accurately detect each gene mutation with high precision even in practical situations.

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

[0135] First, a reaction mixture was prepared using a wild-type model specimen or genomic DNA (gDNA) as a template. The PCR reaction mixture was then subjected to a thermal cycle consisting of 5 minutes at 95°C, 30 seconds at 95°C, 30 seconds at a predetermined annealing temperature (e.g., 59°C), and 45 seconds at 72°C. This was followed by 10 minutes at 72°C, with a final temperature of 4°C. The annealing temperatures were 56°C, 58°C, 60°C, 62°C, or 64°C.

[0136] The relationship between the amount of nucleic acid amplification and the annealing temperature is shown in Figure 9. As can be seen from Figure 9, the combinations [5], [6], and [7] were sufficient to amplify the regions containing the MYD88 gene mutation and the CD79B gene mutation in the wild-type model specimen and genomic DNA (multiplex amplification was possible).

[0137] Furthermore, the results of electrophoresis of the nucleic acid fragments contained in the reaction solution after PCR performed using combinations [5], [6], and [7] are shown in Figure 10. As shown in Figure 10, when combination [7] was used, multiple nucleic acid fragments due to non-specific amplification other than the target nucleic acid fragment were confirmed. This result shows that the combination of [5] and [6] is superior to combination [7].

[0138] Next, a reaction mixture was prepared using a 5% mutant model sample or genomic DNA (gDNA-1) as a template. PCR was performed using the reaction mixture in the same manner, and the fluorescence intensity of the amplified fragments was measured. The results are shown in Figure 11. As shown in Figure 11, when combination [5] was used, the fluorescence intensity from the probe corresponding to the mutation type in the MYD88 gene mutation was lower than that of the other combinations.

[0139] We then investigated the effect of varying the ratio of labeled primers to unlabeled primers on the fluorescence intensity of combinations [6] and [7]. The results are shown in Figure 12. As shown in Figure 12, for combination [6], the fluorescence intensity increased with increasing ratio of labeled primers, and the fluorescence intensity values ​​were proportional to the ratio of labeled primers. In contrast, for combination [7], the trend in the change in fluorescence intensity did not match the ratio of labeled primers. This was likely due to the nonspecific amplification observed when combination [7] was used. These results demonstrate that combination [6] is preferable for multiplex amplification of regions containing mutations in the CD79B gene and regions containing mutations in the MYD88 gene.

[0140] <Examination of Primer Ratio (Labeled vs. Unlabeled)> The ratio of labeled primers to unlabeled primers was varied for each primer set amplifying the region containing the CD79B gene mutation and the region containing the MYD88 gene mutation, and the changes in fluorescence intensity were examined to determine the optimal ratio of labeled primers to unlabeled primers. In this example, the primer set amplifying the region containing the CD79B gene mutation was a labeled primer (CD79B_Fw-4) as the forward primer and an unlabeled primer (CD79B_Rv-4) as the reverse primer. The primer set amplifying the region containing the MYD88 gene mutation was an unlabeled primer (MYD88_Fw-5-3) as the forward primer and a labeled primer (MYD88_Rv-5) as the reverse primer.

[0141] In this example, as shown in Figure 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 adjusted, and PCR was performed in the same manner. A 5% mutation model specimen and genomic DNA were used as templates. The results obtained using a primer set that amplifies the region containing the MYD88 gene mutation are shown in Figure 14, and the results obtained using a primer set that amplifies the region containing the CD79B gene mutation are shown in Figure 15.

[0142] As can be seen from Figure 14, excellent fluorescence intensity values ​​were achieved when the final concentration of the labeled primers amplifying the region containing the gene mutation in the MYD88 gene was between 2-fold (i.e., labeled primer:unlabeled primer = 20:10) and 6-fold higher than that of the unlabeled primers, and even better fluorescence intensity values ​​were achieved in the range of 3-fold to 5-fold, with the best fluorescence intensity value being achieved at 4-fold.

[0143] Furthermore, as can be seen from Figure 15, excellent fluorescence intensity values ​​were achieved when the final concentration of the labeled primers used to amplify the region containing the gene mutation in the CD79B gene was 5 to 10 times higher than that of the unlabeled primers, and even better fluorescence intensity values ​​were achieved when the final concentration was 6 to 7 times higher, with the best fluorescence intensity value being achieved when the final concentration was 7 times higher.

[0144] <Consideration of Optimal Blocking Nucleic Acids> Optimal blocking nucleic acids were selected based on their function as blocking nucleic acids for the MYD88 gene mutations shown in Table 9 and the CD79B gene mutations shown in Table 10. Blocking nucleic acids specifically hybridize to wild-type nucleic acid fragments among nucleic acid fragments obtained by amplifying a region containing a gene mutation, thereby preventing nonspecific hybridization of the wild-type nucleic acid fragment to a mutant probe. Therefore, when selecting an optimal blocking nucleic acid, nonspecific hybridization between a wild-type nucleic acid fragment and a mutant probe in the presence of the blocking nucleic acid can be determined based on the fluorescence intensity of the mutant probe. Furthermore, whether the blocking nucleic acid inhibits specific hybridization between a mutant nucleic acid fragment and a mutant probe in the presence of the blocking nucleic acid can be determined based on the fluorescence intensity of the mutant probe.

[0145] For the blocking nucleic acids related to the gene mutations of the MYD88 gene shown in Table 9, a wild-type model specimen was hybridized with a mutant probe (MYD88_M_v2-1) in the presence of the blocking nucleic acid (125 nM or 500 nM), and the fluorescence intensity of the mutant probe was measured. Also, a 100% mutant model specimen was hybridized with a mutant probe (MYD88_M_v2-1) 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, all of the blocking nucleic acids tested were able to suppress nonspecific hybridization between the wild-type model specimen and the mutant probe, but it was revealed that among the blocking nucleic acids, v1 and v2-1 inhibited specific hybridization between the 100% mutant model specimen and the mutant probe. Therefore, using the 5% mutant model specimen, we examined which of v2-2 and v2-3 functioned as a better blocking nucleic acid.

[0147] Specifically, a 5% mutant model sample was hybridized with a wild-type probe (MYD88_W_v2-1) and a mutant probe (MYD88_M_v2-1) in the presence of v2-2 or v2-3 (125 nM or 500 nM), and the fluorescence intensities of the wild-type and mutant probes were measured. Furthermore, a judgement value was calculated from the measured fluorescence intensity values ​​using the above formula. The results of the fluorescence intensity measurements are shown in Figure 17, and the results of the calculated judgement values ​​are shown in Figure 18.

[0148] As can be seen from Figures 17 and 18, the fluorescence intensity of the wild-type probe was lower when v2-2 was used compared to when v2-3 was used, demonstrating superior blocking nucleic acid functionality compared to v2-3. Based on these results, v2-2 was selected as the optimal blocking nucleic acid for MYD88 gene mutations among those listed in Table 9. Furthermore, these results indicated that even when no blocking nucleic acid for MYD88 gene mutations was used (i.e., at 0 nM), there was sufficient separation between the values ​​determined using the wild-type model sample and the 5% mutant model sample. These results suggest that a concentration of 0 to 500 nM is preferred for blocking nucleic acids for MYD88 gene mutations.

[0149] Next, for the blocking nucleic acids related to the gene mutations of the CD79B gene shown in Table 10, wild-type model samples or 5% mutant model samples were hybridized with the wild-type probe (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 the blocking nucleic acid, and the fluorescence intensity from the wild-type probe and mutant probe was measured. The judged value was calculated according to the above formula. The results are shown in Figure 19.

[0150] As shown in Figure 19, when a wild-type model sample was used, the only cases in which the judgment value for all mutant probes was 0.05 or less were those in which the v4-1 and v4-2 blocking nucleic acids were used. The judgment values ​​and fluorescence intensity values ​​for each mutant probe calculated using the wild-type model sample or the 5% mutant model sample for the v4-1 and v4-2 blocking nucleic acids are shown in Figures 20 and 21, respectively. As can be seen from Figures 20 and 21, when the judgment values ​​for v4-1 and v4-2 were compared, they were nearly equivalent, but when the fluorescence intensity values ​​for v4-1 and v4-2 were compared, it was found that v4-2 exhibited a higher value. Based on these results, v4-2 was selected as the optimal blocking nucleic acid for gene mutations in the CD79B gene from among those listed in Table 10.

[0151] <Investigation of Blocking Nucleic Acid Concentration> For v4-2, selected as a blocking nucleic acid for gene mutations in the CD79B gene, wild-type model samples or 5% mutant model samples were hybridized with the wild-type probe (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 various concentrations of blocking nucleic acid, and the fluorescence intensity from the wild-type probe and mutant probe was measured, and the judged value was calculated according to the above formula. The results are shown in Figure 22.

[0152] As shown in Figure 22, when the blocking nucleic acid concentration was 125 nM, there were several mutant probes whose judgement value exceeded 0.05 when using a wild-type model sample. These results indicated that a blocking nucleic acid concentration higher than 125 nM is preferable. Furthermore, Figure 23 shows the results of measuring the fluorescence intensity from each mutant probe when using a 5% mutant model sample with a blocking nucleic acid concentration of 500 nM, 750 nM, or 1000 nM. These results indicated that the fluorescence intensity value was lower when the blocking nucleic acid concentration was 1000 nM compared to when the concentration was 500 nM or 750 nM. These results suggest that a blocking nucleic acid concentration of 500 nM or 750 nM is preferable.

[0153] To confirm the above results, the judgment values ​​of each mutant probe when using a wild-type model sample or a 5% mutant model sample at a blocking nucleic acid concentration of 500 nM or 750 nM were calculated. The results are shown in Figure 24. As can be seen from Figure 24, when the blocking nucleic acid concentration was either 500 nM or 750 nM, the judgment values ​​when using the wild-type model sample and the judgment values ​​when using the 5% mutant model sample were sufficiently separated, indicating that the wild-type and each mutant could be accurately detected. In particular, when the blocking nucleic acid concentration was 750 nM, the judgment values ​​when using the wild-type model sample and the judgment values ​​when using the 5% mutant model sample were more widely separated than when the blocking nucleic acid 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, with 500 to 750 nM being preferred. As an example, it was found that the most preferred concentration of the blocking nucleic acid is 750 nM. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

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

2. The kit for evaluating gene mutations described in claim 1, characterized in that the gene mutation in the MYD88 gene is a substitution mutation of leucine at position 265 with proline, and the gene mutation in the CD79B gene is a missense mutation and / or nonsense mutation in the tyrosine residue at position 196 and a silent mutation in the tyrosine residue.

3. The gene mutation evaluation kit described in claim 1, characterized in that the primer set for MYD88 gene mutation is a set of a forward primer for MYD88 gene consisting of the base sequence of SEQ ID NO: 79 and a reverse primer for MYD88 gene consisting of the base sequence of SEQ ID NO: 78, and the primer set for CD79B gene mutation is a set of a forward primer for CD79B gene consisting of the base sequence of SEQ ID NO: 85 and a reverse primer for CD79B gene consisting of the base sequence of SEQ ID NO:

86.

4. The kit for evaluating gene mutations described in claim 3, characterized in that the reverse primer for the MYD88 gene is labeled and has a concentration 2 to 6 times that of the forward primer for the MYD88 gene, and the forward primer for the CD79B gene is labeled and has a concentration 5 to 10 times that of the reverse primer for the CD79B gene.

5. The kit for evaluating gene mutations described in claim 1, characterized in that 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 base sequences shown in Table 1.

6. The kit for evaluating a gene mutation described in claim 1, further comprising a blocking nucleic acid for MYD88 that hybridizes to a wild-type nucleic acid fragment among the nucleic acid fragments amplified with the primer set for MYD88 gene mutation, and / or a blocking nucleic acid for CD79B that hybridizes to a wild-type nucleic acid fragment among the nucleic acid fragments amplified with the primer set for CD79B gene mutation.

7. The kit for evaluating gene mutations according to claim 6, characterized in that the blocking nucleic acid for MYD88 has the base sequence of SEQ ID NO:89, and the blocking nucleic acid for CD79B has the base sequence of SEQ ID NO:

95.

8. The kit for evaluating gene mutations described in claim 6, characterized in that the blocking nucleic acid for MYD88 is prepared in the hybridization solution at a concentration in the range of 0 to 500 nM, and the blocking nucleic acid for CD79B is prepared in the hybridization solution at a concentration in the range of 125 to 1000 nM.

Images