Composition for identifying NUDT15 gene mutation and uses thereof

Abstract

The present invention relates to a composition for identifying NUDT15 gene mutations and uses thereof and, more specifically, to: probes and primers which can identify each of the rs746071566, rs116855232, rs147390019 and rs186364861 mutations of the NUDT15 gene; and a method and a kit for identifying NUDT15 gene mutations, using the probes and primers. The probes according to the present invention can not only identify NUDT15 gene mutations with high speed, high sensitivity, and high accuracy compared to conventional sequencing methods, but also facilitate the discrimination of false positives, and thus can be effectively used for predicting adverse effects, such as leukopenia or myelosuppression, induced by thiopurine-based drugs.

Description

Composition for detecting NUDT15 gene mutation and use thereof

[0001] The present invention relates to a composition for detecting a NUDT15 gene mutation and its use, and more specifically, to a probe and primer capable of detecting mutations of rs746071566, rs116855232, rs147390019, and rs186364861 of the NUDT15 gene, respectively, and a method and detection kit for detecting a NUDT15 gene mutation using the same.

[0002]

[0003] Thiopurine drugs, azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG), act as anti-metabolites in the purine metabolism process and are used to treat autoimmune diseases, inflammatory bowel disease (IBD) such as Crohn's disease and ulcerative colitis, acute lymphoblastic leukemia, and as immunosuppressants to reduce rejection of transplanted organs after solid organ transplantation. Thiopurine drugs undergo intracellular metabolic processes by various enzymes in the body, mimicking naturally occurring purines and active metabolites and binding to DNA or RNA. This process promotes cell death, demonstrating the drug's efficacy, but can also cause fatal toxicities such as liver toxicity or cytopenias (Moyer AM. Clin Biochem Vol.92, pp.1-8, 2021). It is well known that when thiopurine is administered to patients with reduced thiopurine S-methlytransferase (TPMT) enzyme activity, the concentration of the drug metabolite 6-thioguanine nucleotide (TGN) increases, increasing the risk of toxicity. Therefore, TPMT genotyping has been widely used to predict thiopurine toxicity and determine dosage, and has been presented as a representative pharmacogenetic test for clinical use in the domestic clinical test guideline 'Clinical Pharmacogenetic Testing and Application: Clinical Test Guidelines for Laboratory Medicine' published by the Korean Society for Laboratory Medicine (Kim S et al., Lab Med Online Vol.6, pp. 193-213 2016).However, because the frequency of TPMT gene mutations is low in Koreans, it has become necessary to consider factors other than the TPMT gene in the adverse effects of thiopurine drugs. In addition, as the association between the Nudix hydrolase15 (NUDT15) genotype, which has a high mutation frequency in Asians, and adverse drug effects has been discovered and verified, NUDT15 genotyping for appropriate thiopurine drug therapy has recently been included in major clinical pharmacogenetic testing guidelines (Yang SK, et al., Nat Genet Vol.46, pp. 1017-20, 2014).

[0004] The NUDT15 gene is located on chromosome 13q14.2 and has three exons. There are variants known to have functional or structural effects on the NUDT15 enzyme protein, and variants whose functional effects have not yet been clearly identified. When interpreting genotype results, it is necessary to check the type and characteristics of the observed variant, the relationship between allele determination, and the presence or absence of homozygosity or heterozygosity. The types of NUDT15 gene variants and the known frequencies of variants according to the CPIC guidelines nomenclature are shown in the table below (Yang JJ et al., Clin Pharmacol Ther Vol.105 pp. 1091-4, 2019). NUDT15 gene variants are most frequently found in East Asians, and the type and frequency of each allele vary by race. The most common alleles found in East Asians are NUDT15*2 and NUDT15*3, while NUDT15*9 is found only in Europeans and has not been reported in Asians.

[0005]

[0006]

[0007] In addition, if there is one non-functional allele, it is defined as an intermediate metabolizer, and if there are two, it is defined as a poor metabolizer. In addition, the alleles NUDT15 *4, *5, *6, which are unclear functional alleles, have also been reported at a low frequency in Koreans, and the genotypes containing these alleles are defined as possible intermediate metabolizers or indeterminates.

[0008]

[0009] The 2016 published guidelines for clinical pharmacogenetic testing in laboratory medicine recommended TPMT genotyping before starting thiopurine treatment to determine the appropriate initial dose for each individual. However, the frequency of TPMT gene mutations in Koreans was reported to be low at 0.8-2.5%, and as the importance of NUDT15 became known, the Asian Society for Inflammatory Bowel Disease and the Asia-Pacific Society of Gastroenterology recommended NUDT15 genotyping before starting treatment for patients with inflammatory bowel disease in 2020 (Sutiman N et al., Pharmacogenomics, Vol.19, pp.31-43, 2018). The US Food and Drug Administration and the European Medicines Agency have reflected pharmacogenetic information in the labels of thiopurine drugs, and the European Medicines Agency has suggested performing NUDT15 genetic testing before starting treatment, citing the risk of early leukopenia and alopecia in patients with NUDT15 genetic mutations in the label of 6-MP, which is used to treat childhood acute lymphoblastic leukemia. In Korea, according to the general principles of the coverage standards for hereditary genetic testing (Ministry of Health and Welfare Notice No. 2020-135), if the genetic test can meaningfully predict serious side effects of a specific drug, it is eligible for medical care benefits. Starting in 2020, TPMT genetic testing will be recognized as medical care benefits if it is performed to identify genetic mutations when drug side effects such as severe bone marrow suppression are suspected during thiopurine administration. For patients planning to take thiopurines and those who perform it in addition to the above, an 80% co-payment rate is applied. The NUDT15 genetic test has been covered by health insurance benefits since August 2020 in accordance with the general principles of genetic testing benefits.

[0010] However, the NUDT15 genetic test method developed to date determines the genotype based on Sanger sequencing, and has the disadvantage of being time-consuming and costly.

[0011] Korean Patent Registration No. 1704143 (December 30, 2014) describes a composition for predicting the risk of developing leukopenia, which comprises a polynucleotide-containing composition capable of detecting NUDT15 R139C or R139H mutation, and comprises the rs116855232 or rs147390019 marker itself or a nucleic acid complementary thereto, which is limited by the sequence number.

[0012] Korean Patent No. 2230252 (August 14, 2019) discloses a kit for genetic analysis to predict adverse drug reactions in multiple prescription drugs related to cancer and chronic diseases. The kit includes primers of sequence numbers 1 to 124 for determining mutations in multiple genes, including NUDT15 rs116855232. However, neither document describes a composition for detecting rs746071566, and has the disadvantage of using a complex method rather than real-time polymerase chain reaction for genotype detection.

[0013] Accordingly, the present inventors have made great efforts to solve the above problems and develop a method for detecting mutations in the NDUT15 gene. As a result, they have confirmed that mutations in the NUDT15 gene can be detected quickly and accurately when the NUDT15 gene is amplified with primers and then the melting curve is analyzed with a gene-specific probe, thereby completing the present invention.

[0014]

[0015] Summary of the invention

[0016] An object of the present invention is to provide a probe and primer pair capable of detecting a mutation in the NUDT15 gene.

[0017] Another object of the present invention is to provide a method and kit for detecting NUDT15 gene mutation using the probe and primer pair.

[0018] To achieve the above purpose, the present invention provides a probe for detecting a NUDT15 gene mutation comprising one or more sequences selected from the group consisting of SEQ ID NOs: 1 to 8.

[0019] The present invention also provides a primer pair for detecting a NUDT15 gene mutation, comprising: (i) a primer pair comprising a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10; (ii) a primer pair comprising a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12; or (iii) a primer pair comprising a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14.

[0020] The present invention also provides a composition for detecting a NUDT15 gene mutation comprising the probe and the primer pair.

[0021] The present invention also provides a NUDT15 gene mutation detection kit comprising the composition.

[0022] The present invention also provides a method for detecting a NUDT15 gene mutation, comprising the following steps:

[0023] (a) a step of amplifying nucleic acid extracted from a specimen using the primer pair and probe; and

[0024] (b) A step of analyzing the amplification product to determine the NUDT15 gene mutation.

[0025]

[0026] Figure 1 is a schematic diagram illustrating the concept of a method for detecting NUDT15 gene mutations using the probe and primer of the present invention.

[0027] Figure 2 is a graph showing the results of detecting the rs116855232 mutation of the NUDT15 gene using a probe and primer according to the present invention.

[0028] Figure 3 is a graph showing the results of detecting the rs147390019 mutation of the NUDT15 gene using a probe and primer according to the present invention.

[0029] Figure 4 is a graph showing the results of detecting the rs186364861 mutation of the NUDT15 gene using a probe and primer according to the present invention.

[0030] Figure 5 is a graph showing the results of detecting the rs746071566 mutation of the NUDT15 gene using a probe and primer according to the present invention.

[0031] Figure 6 is a graph confirming the sensitivity of detection of the rs116855232 mutation of the NUDT15 gene using a probe and primer according to the present invention.

[0032] Figure 7 is a graph confirming the sensitivity of detection of the rs147390019 mutation of the NUDT15 gene using a probe and primer according to the present invention.

[0033] Figure 8 is a graph confirming the sensitivity of detection of the rs186364861 mutation of the NUDT15 gene using a probe and primer according to the present invention.

[0034] Figure 9 is a graph confirming the sensitivity of detection of the rs746071566 mutation of the NUDT15 gene using a probe and primer according to the present invention.

[0035]

[0036] Detailed description of the invention and preferred embodiments

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0038]

[0039] In the present invention, it was attempted to confirm that when the NUDT15 gene is detected with a probe, a mutation in the NUDT15 gene can be identified simply by analyzing the melting curve without going through a complicated process.

[0040] That is, in one embodiment of the present invention, by designing a probe that specifically binds to the NUDT15 gene and analyzing a melting curve using the probe, it was confirmed that a mutation in the NUDT15 gene can be identified only by analyzing the melting curve without additional analysis work (Fig. 1).

[0041] Therefore, the present invention, from one point of view,

[0042] A probe for detecting a NUDT15 gene mutation comprising one or more sequences selected from the group consisting of sequence numbers 1 to 8.

[0043]

[0044] In the present invention, the probe may have a reporter and a quencher fluorescent material capable of quenching reporter fluorescence bound to both ends, and may include an intercalating fluorescent material.

[0045] In the present invention, the reporter may be characterized by being at least one fluorescent substance selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, ABY, VIC, JUN, cyanine series dyes, and thiadicarbocyanine dyes, but is not limited thereto.

[0046] The above intercalating fluorescent material is acridine homodimer and derivatives thereof, acridine orange and derivatives thereof, 7-aminoactinomycin D (7-AAD) and derivatives thereof, actinomycin D and derivatives thereof, ACMA (9-amino-6-chloro-2-methoxyacridine) and derivatives thereof, DAPI and derivatives thereof, dihydroethidium and derivatives thereof, ethidium bromide and derivatives thereof, ethidium homodimer-1 (EthD-1) and derivatives thereof, ethidium homodimer-2 (EthD-2) and derivatives thereof, ethidium monoazide and derivatives thereof. The derivatives may be selected from the group consisting of, but are not limited to, hexidium iodide and derivatives thereof, bisbenzimide (Hoechst 33258) and derivatives thereof, Hoechst 33342 and derivatives thereof, Hoechst 34580 and derivatives thereof, hydroxystilbamidine and derivatives thereof, LDS 751 and derivatives thereof, propidium iodide (PI) and derivatives thereof, and Cy-dyes derivatives.

[0047] In the present invention, the quencher may be characterized by being at least one selected from the group consisting of Dabcyl, TAMRA (6-carboxytetramethyl-rhodamine), Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa black RQ, and IRDye QC-1, but is not limited thereto.

[0048] In the present invention, a probe including a reporter and a quencher generates a fluorescent signal when a target nucleic acid is present, and as a PCR reaction progresses, the fluorescent signal increases according to the amount of amplification product generated by additional separation of the reporter from the quencher, and by analyzing this fluorescent signal, the presence or absence of the target nucleic acid can be detected.

[0049]

[0050] The term "probe" of the present invention refers to a nucleic acid fragment of a few bases to several hundred bases that can specifically bind to a gene or mRNA, and may be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, a PNA probe, an LNA probe, etc., and may be labeled for easier detection, but is not particularly limited thereto.

[0051] The term "hybridization" in the present invention refers to the formation of a double-stranded nucleic acid from complementary single-stranded nucleic acids. Hybridization can occur when the complementarity between the two nucleic acid strands is perfect (a perfect match), or even when some mismatched bases are present. The degree of complementarity required for hybridization can vary depending on the hybridization conditions, and can be particularly controlled by temperature.

[0052] In the present invention, the target nucleic acid refers to a nucleic acid sequence to be detected, and is annealed or hybridized with a primer or probe under hybridization, annealing, or amplification conditions.

[0053] In the present invention, the "probe" may be characterized by preferentially binding to a target nucleic acid when the target nucleic acid is present and increasing a fluorescence signal, and not exhibiting a fluorescence signal when the target nucleic acid is absent, and false positive and false negative signals can be distinguished using the increase value of the target nucleic acid fluorescence signal.

[0054]

[0055] In the present invention, the term "variant" is used interchangeably with "polymorphism." Polymorphism refers to the presence of two or more variants in a single genetic locus, and the term "polymorphic site" refers to the genetic locus where the above-mentioned allele exists. Among the polymorphic sites, those where only a single base differs from person to person are defined as "single nucleotide polymorphism," or SNP (single nucleotide polymorphism). In other words, the term "variant" in the present invention refers to SNP.

[0056]

[0057] From another aspect, the present invention provides (i) a primer pair comprising a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10;

[0058] (ii) a primer pair comprising a forward primer of sequence number 11 and a reverse primer of sequence number 12; or

[0059] (iii) a primer pair comprising a forward primer of sequence number 13 and a reverse primer of sequence number 14;

[0060] It relates to a primer pair for detecting NUDT15 gene mutations including .

[0061] In the present invention, the primer pair can specifically amplify the NUDT15 gene.

[0062]

[0063] In another aspect, the present invention relates to a composition for detecting a NUDT15 gene mutation comprising the probe and the primer pair.

[0064]

[0065] In another aspect, the present invention relates to a kit for detecting a NUDT15 gene mutation comprising the composition.

[0066] In the present invention, the kit may optionally include reagents necessary for performing a nucleic acid amplification reaction (e.g., polymerase chain reaction), such as a buffer, DNA polymerase, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate (dNTP). Optionally, the kit of the present invention may also include various oligonucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. Furthermore, the optimal amount of reagents to be used in a specific reaction of the kit can be readily determined by one skilled in the art having learned the teachings of the present specification. Typically, the device of the present invention may be manufactured as a separate package or compartment containing the aforementioned components.

[0067] In one embodiment, the kit may comprise a compartmented carrier means for containing a sample, a container containing a reagent, and a container containing a primer or probe.

[0068] The carrier means is suitable for containing one or more containers, such as bottles or tubes, each containing independent components used in the method of the present invention. Given the present disclosure, one skilled in the art can readily dispense the required formulations within the containers.

[0069]

[0070] In another aspect, the present invention relates to a method for detecting a NUDT15 gene mutation, comprising the following steps:

[0071] (a) a step of amplifying nucleic acid extracted from a specimen using the primer pair and probe; and

[0072] (c) A step of analyzing the amplified product to determine the NUDT15 gene mutation.

[0073]

[0074] In the present invention, the sample is derived from a specimen for which a NUDT15 gene mutation is to be detected, and comprises DNA or RNA molecules, which may be in a double-stranded or single-stranded form. If the nucleic acid as the starting material is double-stranded, it is preferable to convert the two strands into a single strand or a partially single-stranded form. Known methods for separating the strands include, but are not limited to, heat, alkali, formamide, urea, and glycoxal treatment, enzymatic methods (e.g., helicase action), and binding proteins. For example, strand separation can be achieved by heat treatment at a temperature of 80 to 105°C.

[0075] In the present invention, the sample means any material, biological fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, blood (including plasma and serum), sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate. The biological material may include, but is not limited to, blood, urine, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, semen, hair, saliva, urine, buccal cells, placental cells, cerebrospinal fluid, and mixtures thereof.

[0076] In the present invention, the amplification of step (a) may be characterized by being performed by a real-time polymerase chain reaction (real-time PCR) method, but is not limited thereto. The real-time polymerase chain reaction method of the present invention allows a fluorescent substance to bind (interchelate) to a double-stranded DNA chain during the PCR process, and, along with amplification of a PCR product, increases the temperature to unwind the DNA double strands, thereby analyzing the pattern of a melting curve in which the amount of the fluorescent substance existing between the DNA double strands decreases, particularly the temperature (Tm) at which DNA melts (denatures), thereby allowing analysis of the presence or absence of a variation in the base sequence between a normal control group and a mutant. The real-time polymerase chain reaction method of the present invention allows the presence or absence of a specific mutation to be analyzed by using the fact that a probe including a reporter and a quencher binds to a target nucleic acid during a PCR process, a primer is extended and is cleaved to amplify a fluorescent signal, an additional reporter is cleaved from the probe at each cycle, and the fluorescent intensity increases in proportion to the amount of the amplified product, and the fluorescent intensity does not increase when the target nucleic acid does not exist.

[0077]

[0078] Example

[0079] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0080]

[0081] Example 1. Preparation of primers and probes for detecting NUDT15 gene mutations.

[0082] Primers were prepared to detect mutations in the NUDT15 gene as shown in Table 3 below, and probes capable of hybridizing with each SNP were prepared (Table 4).

[0083]

[0084]

[0085]

[0086] Newly designed primers and NDUT15 SNP genes (*2, *3, *4, *5 and *6) were synthesized by Cosmogenetech (Korea), and all probes used in the present invention were synthesized by TQMN QSY PROBE 6K PMOLES (Thermo scientific, USA).

[0087]

[0088] Example 2. Analysis of NUDT15 gene mutation detection results

[0089] CFX96TM (Bio-Rad, USA) was used as the experimental equipment, and the experimental compositions as shown in Tables 6 to 9 below were used. Samples were prepared in each well, and the real-time polymerase chain reaction experimental conditions were performed at once as shown in Table 10. Here, the DNA samples contain specific SNP types in the NUDT15 gene, and each sample represents an independent SNP mutation.

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] As a result, as described in Fig. 2, it was confirmed that the ABY probe hybridizing to the wild type showed an amplification curve only in samples in which the SNP genotype included C (Fig. 2 A, B), the JUN probe hybridizing to NUDT15 *3 type showed an amplification curve only in samples in which the SNP genotype included T (Fig. 2 B, C), and as described in Fig. 3, it was confirmed that the FAM probe hybridizing to the wild type showed an amplification curve only in samples in which the SNP genotype included G (Fig. 3 A, B), and the VIC probe hybridizing to NUDT15 *4 type showed an amplification curve only in samples in which the SNP genotype included A (Fig. 3 B, C).

[0096] In addition, as described in Fig. 4, it was confirmed that the ABY probe hybridizing to the wild type showed an amplification curve only in samples in which the SNP genotype included G (Fig. 4 A, B), the JUN probe hybridizing to the NUDT15 *5 type showed an amplification curve only in samples in which the SNP genotype included A (Fig. 4 B, C), and as described in Fig. 5, it was confirmed that the FAM probe hybridizing to the wild type showed an amplification curve only in samples in which no mutation existed and heterozygous samples (Fig. 5 A, B), and the VIC probe hybridizing to the NUDT15 *6 type showed an amplification curve only in samples in which the mutation sequence was included (Fig. 5 B, C).

[0097] To verify the real-time polymerase chain reaction results as described above, they were compared with the Sanger sequencing results, and as a result, it was confirmed that the genotypes identified by real-time polymerase chain reaction and the genotypes identified by Sanger sequencing were consistent with each other (D of Figures 2 to 5).

[0098] As shown in Figures 2 to 5, it was confirmed that the wild type, mutant homozygous, and mutant heterozygous of NUDT15 *3 to *6 could be clearly distinguished using the probe and primer pairs of Tables 4 and 5.

[0099]

[0100] Example 3. Sensitivity analysis for detection of NUDT15 gene mutations

[0101] The sensitivity of primer and probe combinations for detecting each mutant type was evaluated under the same conditions as Example 2. Specifically, PCR reactions were performed using 1 ?L of each of standard DNA concentrations of 2.9×102 copies / ?L, 2.9×103 copies / ?L, 2.9×104 copies / ?L, and 5.8×104 copies / ?L.

[0102] As a result, as described in FIGS. 6 to 9, it was confirmed that all primer and probe combinations had a sensitivity capable of effectively detecting mutant DNA samples with a concentration of up to 2.9×102 copies / ?L.

[0103]

[0104] Example 4. Confirmation of NUDT15 Gene Variant Performance in Clinical Samples

[0105] Nucleic acids were extracted from 51 clinical blood samples using MagNA Pure 96 (Roche, Switzerland), and the genotype of the NUDT15 gene in the samples was confirmed using real-time polymerase chain reaction in the same manner as in Example 2.

[0106] As a result, as described in Table 11, it was confirmed that the primer and probe-based genotyping method of the present invention and the Sanger sequencing analysis method were consistent in all samples, confirming that the accuracy of the primer and probe set of the present invention was very excellent.

[0107]

[0108]

[0109]

[0110] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0111]

[0112] The probe according to the present invention can detect mutations in the NUDT15 gene with high speed, high sensitivity, and high accuracy compared to existing sequence analysis methods, and can also easily determine false positives, so that it can be usefully used in predicting side effects such as leukopenia or bone marrow suppression induced by thiopurine drugs.

[0113]

[0114] Electronic file attached.

Claims

1. A probe for detecting a NUDT15 gene mutation comprising one or more sequences selected from the group consisting of sequence numbers 1 to 8.

2. A probe for detecting a NUDT15 gene mutation, characterized in that in the first paragraph, the probe is combined with a reporter and a quencher.

3. A probe for detecting a NUDT15 gene mutation, characterized in that in the second paragraph, the reporter is at least one fluorescent substance selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, ABY, VIC, JUN, cyanine series dyes, and thiadicarbocyanine dyes.

4. A probe for detecting a NUDT15 gene mutation, characterized in that in the third paragraph, the quencher is at least one selected from the group consisting of Dabcyl, TAMRA (6-carboxytetramethyl-rhodamine), Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa black RQ, and IRDye QC-1. 5.(i) A primer pair comprising a forward primer of sequence number 9 and a reverse primer of sequence number 10; (ii) a primer pair comprising a forward primer of sequence number 11 and a reverse primer of sequence number 12; or (iii) a primer pair comprising a forward primer of sequence number 13 and a reverse primer of sequence number 14; Primer pairs for detection of NUDT15 gene mutations including .

6. A composition for detecting a NUDT15 gene mutation, comprising a probe of any one of claims 1 to 4 and a primer pair of claim 5.

7. A kit for detecting NUDT15 gene mutation comprising the composition of Article 6.

8. NUDT15 gene mutation detection method including the following steps: (a) a step of amplifying a nucleic acid extracted from a specimen using a primer pair of clause 5 and a probe of any one of clauses 1 to 4; and (b) A step of analyzing the amplification product to determine the NUDT15 gene mutation.

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