Probe set, kit for detecting genetic chronic granulomatous disease related gene mutation and application

Abstract

The application provides a probe set, a kit and application for detecting genetic chronic granulomatous disease related gene mutations, and relates to the technical field of molecular biology. The genetic chronic granulomatous disease related genes include CYBA genes, NCF1 genes, NCF2 genes, CYBB genes, WG genes, NCF4 genes, CYBC1 genes, ASAH1 genes, RAG2 genes and RAG1 genes. Through the probe customization of the application, the chronic granulomatous disease related main effect genes can be efficiently captured, the detection efficiency and the positive rate can be greatly improved, the cycle can be shortened, and the detection cost of the genetic CGD second-generation high-throughput sequencing can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a probe set, a kit and application for detecting genetic chronic granulomatous disease related gene mutation. BACKGROUND

[0002] Chronic granulomatous disease (CGD) is a rare genetic immunodeficiency disease. Due to the inability of phagocytes (such as neutrophils and macrophages) to effectively kill bacteria and fungi, repeated infections and chronic inflammation occur, resulting in characteristic granulomas. The core cause is NADPH oxidase deficiency, which leads to active oxygen generation disorder. Diagnosis is made by genetic testing and DHR test, and treatment is mainly anti-infection, immunomodulation and stem cell transplantation.

[0003] Genetic CGD is a disease caused by genetic factors. The immune system of patients is defective, which makes the body unable to effectively clear the invading pathogens, thereby triggering chronic inflammatory response and granuloma formation. Such diseases seriously affect the quality of life of patients, and most of them have poor prognosis. With the rapid development of molecular genetics technology, genetic testing plays an increasingly important role in the diagnosis, treatment and genetic counseling of genetic granuloma.

[0004] Traditional diagnostic methods mainly rely on clinical symptoms, signs and some laboratory tests, such as white blood cell count, C-reactive protein, erythrocyte sedimentation rate and other inflammatory markers, as well as tetrazolium nitroblue test (NBT), neutrophil membrane and cytoplasmic granule free cell oxidase activity test to detect phagocyte function. However, these methods often lack specificity and are difficult to accurately distinguish genetic granuloma from other causes of chronic inflammatory diseases. For patients with suspected genetic granuloma, genetic testing can provide key evidence for diagnosis. Genetic testing can directly detect mutations in related genes, determine the cause and achieve accurate diagnosis. SUMMARY

[0005] In order to solve the technical problems existing in the prior art, the embodiments of the present application provide a probe set, a kit and application for detecting chronic granulomatous disease related gene mutation. The technical solution is as follows:

[0006] A probe set for detecting genetic chronic granulomatous disease related gene mutation, the genetic chronic granulomatous disease related gene includes CYBA gene, NCF1 gene, NCF2 gene, CYBB gene, WG gene, NCF4 gene, CYBC1 gene, ASAH1 gene, RAG2 gene and RAG1 gene.

[0007] Optionally, the sequence of the probe set for detecting genetic chronic granulomatous disease related gene mutation is shown as SEQ ID No. 1 to SEQ ID No. 103.

[0008] Alternatively, the sequence of the probe set for detecting genetic CGD related gene mutation is the sequence synthesized after adding 5'-GACTACATGGGACAT-3' in front of each sequence shown as SEQ ID No. 1 to SEQ ID No. 103 and adding 5'-GGAACCTACGACGTA-3' behind each sequence.

[0009] A kit for detecting chronic granulomatous disease related gene mutation, the kit comprising: the probe set for detecting genetic chronic granulomatous disease related gene mutation.

[0010] Optionally, the kit further comprises at least one of hybrid enrichment buffer, hybridization buffer, binding buffer, rinse buffer 1, rinse buffer 2, PCR amplification enzyme and PCR primer mixture.

[0011] The hybrid enrichment buffer is composed of 3.5 parts by volume of human cot-1 DNA aqueous solution, 3.5 parts by volume of salmon sperm DNA aqueous solution and 3 parts of Universal Blockers;

[0012] The hybridization buffer is an aqueous solution containing 1.25M NaCl, 0.125M sodium citrate, 0.1g / 100mL BSA and 7% by volume of Tween 20;

[0013] The binding buffer is a Tris-HCl buffer with pH 7.5, 10mM containing 1M NaCl and 1mM EDTA;

[0014] The rinse buffer 1 is a sodium citrate buffer containing 0.1% (m / v) SDS; the sodium citrate buffer is an aqueous solution containing 175g / L NaCl and 88g / L trisodium citrate, with pH value of 7.4;

[0015] The rinse buffer 2: sodium citrate buffer containing 0.1% (m / v) SDS diluent; the sodium citrate buffer diluent is mixed by 1 part by volume of sodium citrate buffer and 9 parts by volume of water;

[0016] The PCR mixed enzyme: composed of KAPA HIFI Hotstart readymix;

[0017] The PCR primer mixture comprises: a primer sequence shown in SEQ ID No. 104 to SEQ ID No. 105, wherein the last nucleotide at the 5' end of the primer sequence is modified by thio, and the concentration of the primer sequence is 10 μM.

[0018] The probe set for detecting the genetic chronic granulomatous disease related gene mutation or the kit for detecting the genetic chronic granulomatous disease related gene mutation is applied to the preparation of a diagnostic kit for genetic chronic granulomatous disease.

[0019] Optionally, the detection sample of the diagnostic kit for genetic chronic granulomatous disease comprises blood.

[0020] A diagnostic kit for genetic chronic granulomatous disease comprises the probe set for detecting the genetic chronic granulomatous disease related gene mutation.

[0021] Optionally, the kit further comprises at least one of hybrid enrichment buffer, hybridization buffer, binding buffer, rinse buffer 1, rinse buffer 2, PCR amplification enzyme and PCR primer mixture.

[0022] The hybrid enrichment buffer is composed of 3.5 parts by volume of human cot-1 DNA aqueous solution, 3.5 parts by volume of salmon sperm DNA aqueous solution and 3 parts of Universal Blockers;

[0023] The hybridization buffer is an aqueous solution containing 1.25 M NaCl, 0.125 M sodium citrate, 0.1 g / 100 mL BSA and 7% by volume of Tween 20;

[0024] The binding buffer is a Tris-HCl buffer with a pH of 7.5, 10 mM, containing 1 M NaCl and 1 mM EDTA;

[0025] The rinse buffer 1 is a sodium citrate buffer containing 0.1% (m / v) SDS; the sodium citrate buffer is an aqueous solution containing 175 g / L NaCl and 88 g / L trisodium citrate, with a pH of 7.4;

[0026] The rinse buffer 2 is a sodium citrate buffer diluent containing 0.1% (m / v) SDS; the sodium citrate buffer diluent is a mixture of 1 part by volume of sodium citrate buffer and 9 parts by volume of water;

[0027] The PCR mixture enzyme is composed of KAPA HIFI Hotstart readymix;

[0028] The PCR primer mixture comprises: a primer sequence shown in SEQ ID No. 104 to SEQ ID No. 105, wherein the last nucleotide at the 5' end of the primer sequence is provided with a sulfur modification, and the concentration of the primer sequence is 10 muM.

[0029] The technical scheme provided by the embodiment of the application brings at least the following beneficial effects:

[0030] The detection technology on the market either detects individual hot spots of a single gene and does not cover all, or measures whole exome and other projects, covers a large number of non-target genes and sequences, and not only has a high cost, but also greatly reduces the data quality such as sequencing depth, so a CGD capture panel that covers comprehensively and has a high cost performance becomes an urgent need of the clinic. Taking genetic CGD as an example, by detecting CYBB, NCF1, NCF2, CYBA and other genes, if a specific pathogenic mutation is found, genetic CGD can be diagnosed. Even if the patient is in the early stage of the disease and the clinical symptoms are atypical, gene detection can play an important role in early diagnosis and timely intervention.

[0031] The inventor of the application has independently researched and developed and updated the capture probes related to genetic CGD, and through the targeted panel customized by the probes, the main genes related to chronic granuloma can be efficiently captured, the detection efficiency and the positive rate can be greatly improved, the cycle can be shortened, and the detection cost of genetic CGD next-generation high-throughput sequencing can be reduced.

[0032] The application can simultaneously detect the exon regions and part of the intron regions of CYBA, NCF1, NCF2, CYBB, WG, NCF4, CYBC1, ASAH1, RAG2 and RAG1 genes, and can simultaneously detect SNPs / Indels and CNV. Through detection of pathogenic mutations of CYBB, NCF1, NCF2, CYBA and other genes, early diagnosis and timely intervention can be achieved when the patient is in the early stage of the disease and the clinical symptoms are atypical, and the genetic counseling and family analysis can be provided, and the selection of a treatment plan and the evaluation of a disease prognosis can be guided. DETAILED DESCRIPTION

[0033] The technical scheme in the application will be described below in combination with the embodiments.

[0034] Embodiment 1: A method and a kit for detecting mutations of genes related to chronic granuloma

[0035] 1. Design and preparation of a probe for detecting mutations of genes related to chronic granulomatous disease

[0036] According to the guidelines such as "Diagnosis and Treatment Guidelines for Chronic Granulomatous Disease", "Pediatric Chronic Granulomatous Disease", "Multidisciplinary Expert Consensus on Diagnosis and Treatment of Eosinophilic Granulomatous Polyangiitis (2025 Edition)", and HGMD, OMIM, NCBI, HGNC, ENSEMBL databases, etc. Research and screening of genes related to granuloma (see Table 1), extract all gene sequences from the NCBI database, use RepeatMask software to analyze the repetitive regions of the extracted sequences and remove them, according to the shingled design, start from the first base and extract 120bp sequences, then move n bases back to extract 120bp sequences, until the last 120bp. Adjust the size of n according to the base distribution of the gene sequence to improve the uniformity of probe enrichment.

[0037] Table 1 Chronic granulomatous disease related genes

[0038]

[0039] The probe sequence is shown in Table 2. After adding 5'-GACTACATGGGACAT-3' at the front of each sequence in Table 2 and 5'-GGAACCTACGACGTA-3' at the back, synthesis is completed. The synthesis is completed by Beijing Maijinuo Gene Technology Co., Ltd. The probe of the present inventors is designed in a shingled manner, so there is not only one sequence for a gene. The number of probe strips covered by each gene will also differ according to the sequence base distribution, and the number of probe coverage strips in certain regions will be increased according to the base distribution.

[0040] Table 2 Probe sequence

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[0054]

[0055] The synthesized probe sequences were mixed in an equimolar ratio in 1.2 mL of enzyme-free water, and a portion was used for PCR primer pairs (consisting of primer 1: 5'-bio-GACTACATGGGACAT-3' (bio represents a biotin label at the 5' end) and primer 2: 5'-GGAACCTACGACGTA-3') for PCR amplification (which can be performed in three tubes), to obtain PCR amplification products. The PCR amplification system is as follows: probe sequence template 5 μL, primer 1 (25 μM) 2 μL, primer 2 (25 μM) 2 μL; MgCl2 (50 mM), 4 μL; 10x Platinum Taq buffer (thermo), 5 μL; dNTPs (10 mM), 4 μL; Platinum Taq (thermo), 1 μL; H2O, 27 μL; total volume 50 μL.

[0056] The PCR amplification conditions are as follows: 98°C, 30 s; (98°C, 30 s, 60°C, 25 s, 72°C, 45 s) 35 cycles; 72°C, 5 min.

[0057] After purification, the PCR products were combined with MyOne streptavidin magnetic beads (product of Invitrogen, product number 35602); then NaOH solution was added for treatment, the purpose of which was to denature and elute the complementary strand without a biotin label; then the entire magnetic beads were washed with 100°C formamide liquid to separate the probe from the magnetic beads; finally, ethanol precipitation was performed to obtain the biotin-labeled probe set.

[0058] The probe set is biotin-labeled; streptavidin magnetic beads are used in the subsequent process of preparing the target library. The biotin on the probe covalently binds to the streptavidin magnetic beads.

[0059] 2. Sample collection and genomic DNA extraction

[0060] 2 mL of peripheral blood was collected with the informed consent of the person to be tested, and genomic DNA was extracted using a nucleic acid extraction kit (magnetic bead method) (Chongqing Maijinuo, MGN-WZ-143). The specific process is as follows:

[0061] (1) Take 20 µL of proteinase K into a new centrifuge tube, add 250 µL of blood, 250 µL of lysis solution in order, vortex to mix, and place in a constant temperature instrument at 56°C, 1300 rpm for 15 min;

[0062] (2) Add 15 μL magnetic beads, 380 μL isopropyl alcohol to the above sample tube, shake and mix, then add to the rotating mixer and react for 15 min;

[0063] (3) Place the sample tube magnetic stand in the magnetic stand for 2 min, discard the supernatant, add 700 μL of rinse solution MW1, remove the sample tube and vortex for 30 s;

[0064] (4) Place the sample tube magnetic stand in the magnetic stand for 2 min, discard the supernatant, add 700 μL of rinse solution MW2, remove the sample tube and vortex for 30 s, repeat once;

[0065] (5) Place the sample tube magnetic stand in the magnetic stand for 2 min, discard the supernatant, and stand at room temperature for 5-10 min;

[0066] (6) Add 100 μL eluent to the sample, mix by pipetting, then react at 65°C and 1300 rpm on the thermostat for 5 min;

[0067] (7) Place the sample tube magnetic stand in the magnetic stand for 2 min, transfer the supernatant to a new centrifuge tube.

[0068] (8) Use Nanodrop to measure the concentration and perform quality inspection.

[0069] 3. Preparation of pre-library

[0070] Take 500 ng of genomic DNA, use Covaris M220 (Covaris) to fragment the DNA to about 150-250 bp according to the instructions, and prepare the pre-library according to the instructions of Rapid Max DNA LibPrep Kit (abclonal, PK20217) and Dual DNA Adapter 96 Kit (abclonal, RK20287).

[0071] 4. Preparation of target-enriched library

[0072] Target enrichment kit preparation:

[0073] Probes: The probes are composed of the probe set prepared in 1;

[0074] Hybridization enrichment buffer is composed of 3.5 parts of human cot-1 DNA aqueous solution (thermo, 15279011), 3.5 parts of salmon sperm DNA aqueous solution (thermo, 15634017), and 3 parts of Universal Blockers (abclonal, RK20269);

[0075] Hybridization buffer: 1.25 M NaCl, 0.125 M sodium citrate, 0.1 g / 100 mL BSA, and 7% (v / v) Tween 20 in water;

[0076] Binding buffer: 1 M NaCl and 1 mM EDTA in pH 7.5, 10 mM Tris-HCl buffer;

[0077] Rinse buffer 1: 0.1% (m / v) SDS in sodium citrate buffer. The sodium citrate buffer is a water solution containing 175 g / L NaCl and 88 g / L trisodium citrate; the pH value is 7.4;

[0078] Rinse buffer 2: 0.1% (m / v) SDS in sodium citrate buffer dilution. The sodium citrate buffer dilution is a mixture of 1 volume part of the sodium citrate buffer and 9 volume parts of water;

[0079] PCR mixed enzyme: composed of KAPA HIFI Hotstart readymix (KAPA, KK2604);

[0080] PCR primer mixture: the primer sequence is 5'-AATGATACGGCGACCACCGAG-3' (SEQ ID No. 104), 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID No. 105), wherein the last nucleotide at the 5' end of the primer sequence is thio-modified, and the concentration of the primer sequence is 10 μM.

[0081] Targeted enrichment library preparation

[0082] (1) Take 1 μg of pre-library, add 10 μL of enrichment buffer, 5 μL of probe, mix by pipetting, and place in a PCR instrument. The reaction conditions are 95℃ for 5 min, 65℃ for 5 min;

[0083] (2) Add 19 μL of hybridization buffer preheated at 65℃ to the system of (1), mix by pipetting, and hybridize at 65℃ for 12 h or more.

[0084] (3) Take 50 μL of streptavidin magnetic beads (thermo, 35002D) to a 1.5 mL centrifuge tube, place on a magnetic stand for 1 min, and discard the supernatant;

[0085] (4) Add 50 μL of binding buffer, shake after centrifugation, place on a magnetic stand for 1 min, discard the supernatant, and repeat three times.

[0086] (5) Add 50 μL binding buffer to the hybridized product, mix well, and rotate on a rotary mixer at room temperature for 30 min.

[0087] (6) After the product of the previous step is centrifuged briefly, it is placed on a magnetic stand for 1 min, the supernatant is discarded, 500 μL of rinse buffer 1 is added, and the mixture is vortexed and mixed on a rotary mixer at room temperature for 15 min.

[0088] (7) The product of the previous step is centrifuged briefly, placed on a magnetic stand for 1 min, the supernatant is discarded, and preheated rinse buffer 2 at 65°C is added. The mixture is vortexed and mixed, and then placed in a constant-temperature instrument at 65°C for 15 min, and the process is repeated 3 times.

[0089] (8) The product of the previous step is centrifuged briefly, placed on a magnetic stand for 1 min, the supernatant is discarded, and 1 mL of 80% ethanol is added and left to stand for 1 min. The mixture is placed on a magnetic stand for 1 min, and the supernatant is discarded.

[0090] (9) 23 μL of enzyme-free water is added to the product of the previous step, mixed well, and then 25 μL of PCR mixed enzyme and 5 μL of PCR primer mixture are added. After mixing well, PCR is performed, and the PCR reaction program is as follows: 98°C for 30 s, 1 cycle; 98°C for 25 s, 65°C for 30 s, 72°C for 30 s, 15 cycles; 72°C for 5 min, 1 cycle.

[0091] (10) 50 μL of purification magnetic beads is added to the PCR amplification product of the previous step, mixed well, and left to stand at room temperature for 5 min.

[0092] (11) The mixture is left to stand on a magnetic stand for 5 min, the supernatant is discarded, 200 μL of 80% ethanol is added, the mixture is left to stand on a magnetic stand for 1 min, the supernatant is discarded, and the process is repeated 2 times.

[0093] (12) 33 μL of enzyme-free water is added to the product of the previous step, mixed well, and left to stand at room temperature for 5 min.

[0094] (13) The mixture is left to stand on a magnetic stand for 5 min, and the supernatant is transferred to a new EP tube.

[0095] (14) The concentration of the product of the previous step is detected using qubit, and after passing the detection, the product is sequenced on Illumina, Huada, etc.

[0096] Bioinformatics analysis

[0097] (1) The raw data obtained by sequencing is cutadapt software to remove adapter sequences, low-quality bases (base quality less than Q15) and short sequences (length less than 40 bp), and then aligned to the corresponding position of the reference genome hg19 by bwa software.

[0098] (2) After step (1) is completed, use GATK software to remove redundant sequences generated by PCR amplification.

[0099] (3) After step (2) is completed, use GATK software to detect single nucleotide variations and insertion and deletion variations.

[0100] (4) After step (3) is completed, use ANNOVAR software to annotate all SNPs and INDELs in the relevant information obtained in step 3, and retain mutation sites that meet both (a) and (b):

[0101] (a) Mutation reads greater than 5 and mutation frequency not less than 30% mutation sites;

[0102] (b) Mutation sites with a frequency of less than 0.05 in normal human databases (such as 1000 Genomes, ESP6599, EXAC, and genomeAD).

[0103] (5) After step (4) is completed, delete synonymous mutations and mutation sites that have not been reported in the HGMD database (Human Gene Mutation Database).

[0104] (6) After step (5) is completed, count all retained mutation sites and determine the mutation site type. If the mutation frequency of the mutation site is between 30% and 70%, the mutation site is a heterozygous mutation.

[0105] Example 2 Detection results of mutations in genes related to chronic granuloma

[0106] Four blood DNA samples were tested using the method of Example 1, and the test statistics are shown in Table 3, which meet the conditions for gene mutation analysis.

[0107] Table 3 Test results

[0108]

[0109] The project is designed to detect several major genes related to genetic chronic granulomatous disease. The target region length is less than 100K, the capture efficiency is about 45%, and the average sequencing depth is as high as 400x. The target region size, capture efficiency, and average depth are statistics of sequencing data to reflect the capture effect of the kit. The target region size is calculated according to the base length covered by the probe sequence; the capture efficiency is calculated according to the data reads or bases of the probe coverage area, and then compared to the data reads or bases of the human genome; the average depth is the data of the probe coverage area / the size of the probe coverage area, which is high-throughput data. Compared with the large panel on the market, such as the commonly used gene whole exome detection, the target region length is more than 50M, the capture efficiency is about 40%, and the average sequencing depth is 100x. The data volume of the project is smaller, the capture efficiency is higher, and the average sequencing depth is higher. While improving the quality of sequencing data, smaller data volume not only greatly reduces the cost of detection reagents, but also reduces data redundancy, which is more conducive to subsequent rapid data screening, filtering and bioinformatics analysis, thereby greatly shortening the detection period. The detection cost of the project can be controlled to no more than 1000 yuan per case, and the detection period is 10-12 days. Compared with the whole exome cost of more than 4000 yuan per case and the detection period of 20-30 days, there has been a significant optimization and improvement.

[0110] After testing 50 cases of genetic CGD, it was found that due to the special design of the high-density probes in this panel, the positive detection rate was as high as 42%, which was significantly improved compared with the nearly 38% positive detection rate of the whole exome project.

[0111] Under the condition of informed consent of the person to be tested, peripheral blood was collected and detected using the method of Example 1. The results are shown in Table 4:

[0112] Table 4

[0113]

[0114] Subject 1: Sample No. 20C237134, clinical information: half-year-old boy, repeated fever, pneumonia, genetic detection found a c.573_587delTTCCTCCACCAAAAC mutation in exon 6 of CYBB gene. ACMG (American College of Medical Genetics and Genomics) guidelines evaluate that the site is suspiciously pathogenic and is related to X-linked chronic granulomatous disease. The clinical manifestations of the child are basically consistent. Through the detection of the kit, the child is diagnosed with CYBB-related chronic granulomatous disease, and early intervention treatment is achieved.

[0115] Subject 2: sample number is 20C237265, clinical information: 1-year-old boy, typical chronic granulomatous manifestations, gene detection in CYBB gene 7th exon detected a c.676C>T hotspot mutation, literature reports that this site is definitely pathogenic and is related to X-linked chronic granulomatous disease. Through the detection of the kit, not only the molecular diagnosis is completed, but also the molecular typing is realized. CYBB related chronic granuloma is usually severe, which provides a reference for subsequent treatment and prognosis evaluation such as transplantation;

[0116] Subject 3: sample number is 20C237329, 1-year-old boy, typical chronic granulomatous manifestations, gene detection in CYBB gene 7th exon detected a c.676C>T hotspot mutation, literature reports that this site is definitely pathogenic and is related to X-linked chronic granulomatous disease. Through the detection of the kit, not only the molecular diagnosis is completed, but also the molecular typing is realized. CYBB related chronic granuloma is usually severe, which provides a reference for subsequent treatment and prognosis evaluation such as transplantation;

[0117] Subject 4: sample number is 21C312226, clinical information: 4-year-old boy, 9-month-old boy, fungal pneumonia, disseminated BCG infection, primary immunodeficiency, gene detection in CYBB gene 9th exon detected a c.898-8_904delTTCTATAGGTGGTCA mutation, literature reports that this site is definitely pathogenic and is related to X-linked chronic granulomatous disease. Through the detection of the kit, CYBB related chronic granuloma is diagnosed, which provides a molecular basis for subsequent diagnosis and treatment such as transplantation.

[0118] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any skilled person in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A probe set for detecting gene mutations related to hereditary chronic granulomatous disease, characterized in that, The genes associated with hereditary chronic granulomatous disease include the CYBA gene, NCF1 gene, NCF2 gene, CYBB gene, WG gene, NCF4 gene, CYBC1 gene, ASAH1 gene, RAG2 gene, and RAG1 gene. The sequences of the probe set used to detect gene mutations related to hereditary chronic granulomatous disease are shown in SEQ ID No. 1 to SEQ ID No. 103; Alternatively, the probe set for detecting gene mutations related to hereditary chronic granulomatous disease may be a sequence synthesized by adding 5'-GACTACATGGGACAT-3' to the beginning of each sequence shown in SEQ ID No. 1 to SEQ ID No. 103 and adding 5'-GGAACCTACGACGTA-3' to the end of each sequence.

2. A kit for detecting gene mutations related to chronic granulomatous disease, characterized in that, The kit comprises: a probe set for detecting gene mutations associated with hereditary chronic granulomatous disease as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit further includes at least one of the following: hybridization enrichment buffer, hybridization buffer, binding buffer, wash buffer 1, wash buffer 2, PCR amplification enzyme, and PCR primer mixture; wherein... The hybridization enrichment buffer consists of 3.5 volumes of human cot-1 DNA aqueous solution, 3.5 volumes of salmon sperm DNA aqueous solution, and 3 volumes of Universal Blockers; The hybridization buffer is an aqueous solution containing 1.25M NaCl, 0.125M sodium citrate, 0.1g / 100mL BSA and 7% Tween 20 (volume). The binding buffer is a pH 7.5, 10mM Tris-HCl buffer containing 1M NaCl and 1mM EDTA. The rinsing buffer 1 is a sodium citrate buffer containing 0.1% m / v SDS; the sodium citrate buffer is an aqueous solution containing 175 g / L NaCl and 88 g / L trisodium citrate, with a pH of 7.4; The rinsing buffer 2 is a sodium citrate buffer dilution containing 0.1% m / v SDS; the sodium citrate buffer dilution is a mixture of 1 volume part sodium citrate buffer and 9 volumes water. The PCR amplification enzyme consists of KAPA HIFI Hotstart readymix; The PCR primer mixture comprises primer sequences as shown in SEQ ID No. 104 to SEQ ID No. 105, wherein the last nucleotide at the 5' end of the primer sequence is thiolated, and the concentration of the primer sequence is 10 μM.

4. The use of the probe set for detecting gene mutations related to hereditary chronic granulomatosis according to claim 1 or the kit for detecting gene mutations related to hereditary chronic granulomatosis according to any one of claims 2-3 in the preparation of a diagnostic kit for hereditary chronic granulomatosis.

5. The application according to claim 4, characterized in that, The test sample for the diagnostic kit for hereditary chronic granulomatous disease includes blood.

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