CN114317759A - A primer combination and method for detecting hematological malignancies IKZF family gene mutation - Google Patents

Abstract

The invention discloses a primer combination and a method for detecting gene mutation of an IKZF family of malignant hematological diseases, wherein the method comprises the steps of adopting multiple PCR library building and a high-throughput sequencing technology to carry out IKZF1-5 gene targeting exon sequencing screening on a patient with malignant hematological diseases. Discloses a primer sequence comprising 71 pairs and a combination for detecting an IKZF1-5 gene exon. The invention has the advantages of comprehensive detection range, high accuracy and sensitivity and strong specificity. The method has the advantages of low DNA amount of a patient required for detection, capability of meeting requirements by one-time blood collection or bone marrow puncture, strong clinical operability, capability of quickly and accurately detecting IKZF family gene mutation of a patient with malignant hemopathy, and application to clinical accurate typing diagnosis, early screening of high-risk subtypes, prognosis evaluation and accurate individualized targeted therapy guidance.

Description

A primer combination and method for detecting IKZF family gene mutation in hematological malignancies

technical field

The invention relates to the technical field of biomedicine, in particular to a primer combination and a method for detecting the mutation of IKZF family gene in malignant hematological diseases.

Background technique

The IKZF family is composed of several transcriptional regulatory proteins with zinc finger structures. The N-terminal zinc finger structure has DNA binding function, while the C-terminal zinc finger structure is used for mutual recognition and binding among IKZF family members to form homologous or heterologous two aggregates. The members of the IKZF family mainly include Ikaros, Helios, Aiolos, Eos and Pegasus, which are encoded by IKZF1-5 genes, respectively. IKZF1 and IKZF2 play important roles in the regulation of proliferation and differentiation of lymphoid cells. Meanwhile, studies have shown that the isoforms of IKZF1 and IKZF2 play an important role in the occurrence and development of leukemia. Ik-6 refers to the deletion of IKZF13-6 exon, which is the most important IKZF1 isoform discovered so far in the pathogenesis of leukemia, and is currently recognized as a high-risk prognostic marker. IKZF2 is a marker gene of regulatory T cells and plays an important role in the tumor microenvironment. Its gene mutation is closely related to tumor metastasis, treatment regimen selection and prognosis. The Aiolos protein encoded by IKZF3 plays a crucial role in the differentiation and development of lymphocytes. IKZF3 mutation plays a key role in the occurrence and development of B-cell malignancies and chronic lymphocytic leukemia. IKZF4 and IKZF5 are also important blood cell development genes and are highly expressed in blood tumor cells. Its gene mutation is not only related to the occurrence and development of tumors, but also closely related to the pathophysiology of thrombocytopenia, suggesting that its gene mutation can guide the prognosis stratification of patients and the choice of clinical treatment. At present, studies have reported that the mutation rate of IKZF family genes in patients with acute leukemia and myelodysplastic syndromes is higher than that of other hematological malignancies, and it is related to the prognosis of patients and is considered to be the driving factor of the disease. However, only mutations in the IKZF1 gene are currently used for clinical detection and typing of certain hematological malignancies. Therefore, the development of a new method to comprehensively detect the mutation characteristics and distribution of the five members of the IKZF family in hematological malignancies is of great significance for its clinical classification, prognosis judgment, and treatment plan selection.

The traditional morphology combined with flow immunophenotyping can no longer meet the needs of the diagnosis of hematological malignancies in the era of precision medicine. With the development of high-throughput sequencing technology, more and more molecular abnormalities associated with hematological malignancies have been discovered. Patients with these molecular abnormalities are often difficult to treat clinically, prone to recurrence, and have poor prognosis. Early identification of new targets for precise and individualized treatment will become the future direction of treatment. However, only IKZF1 mutation detection is currently included in some leukemia high-throughput sequencing detection panels, and there is a lack of high-throughput detection methods for mutations in the five members of the complete IKZF family. Therefore, there is an urgent need to develop a more comprehensive, efficient and simple detection method for IKZF family gene mutations. Early identification of molecular abnormalities in this family gene is expected to provide patients with more accurate individualized treatment, improve prognosis and survival, and has great clinical significance. value and application prospects.

Multiplex PCR technology can add multiple pairs of primers to a PCR reaction system for amplification. Combined with high-throughput sequencing technology, it can be used for sequencing after amplification of the locus to be detected. The addition of primers for multiple sites to be detected in a PCR reaction system can greatly reduce the workload and reagent costs, and at the same time, it can meet the clinical needs of comprehensive, accurate and rapid acquisition of test results, and truly achieve accurate detection of patients with hematological malignancies. , Efficient screening of high-risk subtypes. However, adding multiple pairs of primers to a PCR reaction system will inevitably increase the probability of primer-dimer formation, resulting in a decrease in detection efficiency. Therefore, the design and optimization of multiplex PCR primers and their combinations is the focus and difficulty of this technology. The more primers, the more difficult the design. In view of the above difficulties, the present invention will design primers and their combinations for IKZF1-5 gene and optimize them to adapt to the characteristics and needs of clinical detection.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a primer combination and method for detecting IKZF family gene mutation in hematological malignancies, so as to solve the problem that the current IKZF gene mutation detection in patients with hematological malignancies cannot fully cover the IKZF1-5 genes, and the detection is difficult, time-consuming, sensitive and accurate. The primer combination and method based on multiplex PCR combined with high-throughput sequencing technology are used to detect IKZF1-5 gene mutations in patients with hematological malignancies; the primers and combinations in the method have been optimized for many times, with specificity, High sensitivity and accuracy; this technology is applied to the detection of IKZF family gene mutations in patients with hematological malignancies, and plays an important role in guiding accurate clinical typing and diagnosis, early screening of high-risk subtypes, prognostic evaluation and individualized target therapy.

The object of the present invention can be realized through the following technical solutions:

A primer combination for detecting the gene mutation of IKZF family of malignant hematological diseases, including 71 pairs of primers for IKZF1-5 gene exon sequencing, divided into two primer pools, 36 pairs of primers constitute the first primer pool M1, 35 pairs of primers constitute Second primer pool M2.

Further, the M1 primer pool sequences include M1-1f to M1-36f and M1-1r to M1-36r, and the M2 primer pool sequences include M2-1f to M2-35f and M2-1r to M2-35r.

The method for detecting IKZF family gene mutation by primer combination, the steps are as follows:

1. Isolation of mononuclear cells from patients with hematological malignancies

1.1 Balance the blood collection tube and place it in a low-speed centrifuge, 2500rpm×5min. After centrifugation, the upper plasma was slowly aspirated and discarded, and the remaining blood cells were diluted to 5 mL with sterile normal saline, and then mixed well by pipetting.

1.2 Take a 15mL centrifuge tube, add 5mL of human lymphocyte separation solution after marking the patient's name, and then slowly add the bone marrow or peripheral blood dilution dropwise to the lymphocyte separation solution along the tube wall to form a layer to avoid mixing the two;

1.3 Using density gradient centrifugation, place the centrifuge tube in a low-speed centrifuge (pay attention to balance), 2000rpm × 20min; after centrifugation, the liquid in the tube is divided into three layers, the upper clear layer is the diluted plasma and platelets, and the middle buffy coat layer is Mononuclear cells, the lower red layer is granulocytes and red blood cells;

1.4 Slowly aspirate the middle mononuclear cell layer, place it in another clean centrifuge tube marked with the patient's name, add 10 mL of sterile normal saline, pipette and mix, and then centrifuge in a low-speed centrifuge, 2000 rpm × 5 min, and discard the supernatant;

1.5 Resuspend the washed cell pellet with 1 mL of sterile normal saline, count it with a cell counting plate under a high-power microscope, and calculate the total cell volume (the cell counting solution is 1% glacial acetic acid, and 20 μL of cell suspension is added to 380 μL of 1 % glacial acetic acid by pipetting and mixing, which is equivalent to a 20-fold dilution; add 20 μL to the cell counting plate, count the number of cells n in 16 grids under a microscope, and the total number of cells is n × 10000 × 20);

1.6 Dilute the cell suspension to 1×10 ⁷ mononuclear cells/mL; divide the cell suspension into 1.5mL EP tubes, 1mL per tube; centrifuge at high speed (8000rpm) after balancing, carefully aspirate the supernatant, do not touch it Cell pellet at the bottom of the tube. Mark the number and patient name on the tube wall.

2. Extraction of cellular genomic DNA

The reagents used for DNA extraction and purification were purchased from Qiagen (QIAamp DNA Blood Mini Kit) kit, and the method was a silicon membrane adsorption method, as follows:

2.1 Dissolve the isolated 5×10 ⁶ mononuclear cells from patients with hematological malignancies in 200 μL of PBS, and add 20 μL of proteinase K to the bottom of the tube (provided by the kit, prepared according to the instructions for use, and stored at 4°C);

2.2 Slowly add 200 μL of oily lysis solution AL, shake and mix, and then detach at low speed;

2.3 56 ℃ water bath for 20min, when the oily mixture becomes clear, wipe the liquid on the outer wall, and then instantaneously dissociate at a low speed to centrifuge the liquid adhering to the inner wall and the tube cover to the bottom of the tube;

2.4 Add 200 μL of absolute ethanol, invert and mix, and detach at low speed to remove the liquid on the tube wall;

2.5 Carefully transfer the mixture obtained in step 2.4 to the QIAamp Mini adsorption column (the adsorption column is placed on the 2mL collection tube provided by the kit), cover the tube cap, make a mark, centrifuge at high speed 12000rpm × 3min, discard the collection tube and filtrate;

2.6 Put the adsorption column into a new collection tube (provided in the kit), add 500 μL of buffer AW1 to the adsorption column (add anhydrous ethanol according to the product instructions and mix well), cover the tube and centrifuge, 12000rpm×3min, discard and collect Tube and filtrate;

2.7 Put the adsorption column into a new collection tube (provided by the kit), add 500 μL of buffer AW2 (add anhydrous ethanol according to the product instructions and mix well), cover the tube and centrifuge, 12000rpm×3min, discard the collection tube and filtrate;

2.8 Put the adsorption column in a clean and sterile 1.5mL EP tube prepared by yourself, open the cap of the adsorption column, centrifuge at 12000rpm for 3min to fully volatilize the ethanol, and discard the EP tube and filtrate;

2.9 Put the adsorption column in a clean and sterile 1.5mL EP tube prepared by yourself, add 100 μL of elution buffer AE dropwise, add AE dropwise to the middle part of the adsorption membrane of the adsorption column, and incubate at room temperature for 3 minutes to fully elute the adsorption membrane The DNA in the EP tube was centrifuged at 12000rpm×3min, and the filtrate in the EP tube was the genomic DNA solution;

2.10 In order to improve the DNA collection efficiency, repeat step 2.9, return the DNA solution collected in the EP tube to the adsorption column, carefully add it to the middle part of the adsorption membrane of the adsorption column, and incubate at room temperature for 3 minutes to fully dissolve the DNA in the adsorption membrane. Centrifuge at 12000rpm×3min, and collect the DNA solution again;

2.11 Use Nanodrop one micro UV-visible spectrophotometer to detect the concentration and purity of genomic DNA. The DNA quality with an OD260/280 ratio of 1.8-2.0 meets the requirements and can be used for the construction of the DNA library and high-throughput sequencing of the present invention.

3. DNA library construction and sequencing

3.1 IKZF1-5 gene exon PCR amplicon sequence

The information of the 71 amplicons detected is as follows:

Primers were designed according to the exon sequence of IKZF1-5 gene, and the primer synthesis was completed by Shanghai Sangong Company. Due to the large number of primers, in order to avoid the formation of primer-dimers and affect the amplification efficiency, the designed primers are divided into two primer pools (M1 and M2), where M1 is 36 pairs of primers, M2 is 35 pairs of primers, and the specific sequences of the primers The information is as follows:

3.2 Prepare and Quantitative PCR Amplification Template

Use Qubit dsDNA HS Assay Kit from Life Technologies, USA, product number Q32850, to detect the DNA template concentration in the PCR reaction, and ensure that 20-60ng of DNA is added to each reaction system.

3.2.1 Put the kit at room temperature;

3.2.2 Take the dsDNA HS Reagent (component A) in the kit, dilute it with Buffer (component B) at 1:200, make up the working solution, and use it now; 1 μL of component A+199 μL of component B;

3.2.3 Add 190 μL of the prepared working solution to the analysis tube;

3.2.4 Add dsDNA standard#1, standard#2 and 10-fold diluted dsDNA samples to the analysis tubes, 10 μL per tube, and label them correctly;

3.2.5 Incubate at room temperature for 2 minutes in the dark;

3.2.6 Detect the sample concentration.

3.3 Gene-specific PCR

The PCR amplification reaction system is (the reagents used are high-throughput sequencing library construction kits from Sangon):

The reaction systems of Panel A/B should be prepared separately; the purity and quality of the genome are very important. If the extracted DNA has impurities or is severely degraded, the amplification efficiency will drop sharply.

Mix well, place Panel A/B on the PCR machine respectively, and run the following reaction program:

3.4 Recovery of the first round of amplification products

3.4.1 Place the Agencourt AMPure XP magnetic beads at room temperature half an hour in advance, and mix thoroughly and gently to activate the magnetic beads;

3.4.2 Add 25 μL of magnetic beads (1.0 times the volume) and mix with 25 μL of reaction solution (PCR product in step 3.3), gently pipette 10 times to mix, and incubate at room temperature for 5 minutes. Prepare 80% ethanol (the volume ratio of ethanol to Nuclease-free water is 4:1);

3.4.3 Place the EP tube on the magnetic stand for 5 minutes until the liquid is clear, carefully aspirate the supernatant, and do not suck the magnetic beads on the tube wall;

3.4.4 Keep the EP tube on the magnetic stand, add 200 μL of 80% ethanol along the wall of the tube and leave it at room temperature for 30 seconds, discard the supernatant, and do not suck the magnetic beads on the tube wall;

3.4.5 Repeat the previous step to wash once with 80% ethanol;

3.4.6 Keep the EP tube on the magnetic frame to dry for 2-5 minutes, and observe that the surface of the magnetic beads is no longer reflective. Do not over-dry;

3.4.7 Remove the EP tube from the magnetic frame, add 30 μL of Nuclease-free water, gently pipette and mix, leave it at room temperature for 2 minutes, and then place the EP tube on the magnetic frame until the liquid is clear;

3.4.8 Carefully pipette 25 μL of supernatant into a new PCR tube, do not pipette the magnetic beads on the tube wall, and make a mark.

3.5Qubit 3.0 quantitative

Use Qubit dsDNA HS Assay Kit to quantify the recovered products, as in step 3.2.

3.6 Adapter ligation (reagents used are high-throughput sequencing kits from Sanggong)

Prepare the second round PCR reaction system (divided into Panel A/B):

reagent volume 2x Indexing PCR Master Mix2 10μL i5-primer adapter (10uM) 1μL i7-primer adapter (10uM) 1μL Purified PCR product 30ng Nuclease-free water Make up to 30 μL total capacity 30μL

The linker was synthesized by Shanghai Shenggong Company, and the specific sequence is:

serial number i5-primer linker i7-primer linker 1 AGCGCTAG CCGCGGTT 2 GATATCGA TTATAACC 3 CGCAGACG GGACTTGG 4 TATGAGTA AAGTCCAA 5 AGGTGCGT ATCCACTG 6 GAACATAC GCTTGTCA 7 ACATACGCG CAAGCTAG 8 GTGCGATA TGGATCGA 9 CCAACAGA AGTTCAGG 10 TTGGTGAG GACCTGAA

The reaction systems of Panel A/B should be prepared separately. There are two kinds of primers for this PCR adapter, namely i5 index and i7 index. Each primer has a number. For different samples, it needs to be used in combination, and Panel A and Panel B of the same sample need to use the same combination. The index combination of different samples in the same batch cannot be repeated, otherwise the data cannot be split.

3.7. After mixing, centrifuge quickly, place it on the PCR machine, and run the following reaction:

3.8 Library purification

3.8.1 Place the Agencourt AMPure XP magnetic beads at room temperature half an hour in advance, and mix thoroughly and gently to activate the magnetic beads;

3.8.2 Add 22.5μL of magnetic beads (0.9 times the volume) and mix with 25μL of reaction solution (PCR product in step 3.7), mix by gently pipetting 10 times, and incubate at room temperature for 5 minutes;

3.8.3 Place the EP tube on the magnetic stand for 5 minutes until the liquid is clear, carefully aspirate the supernatant, and do not suck the magnetic beads on the tube wall;

3.8.4 Keep the EP tube on the magnetic stand, add 200 μL of 80% ethanol along the wall of the tube and leave it at room temperature for 30 seconds, discard the supernatant, and do not suck the magnetic beads on the tube wall;

3.8.5 Repeat the previous step to wash once with 80% ethanol;

3.8.6 Keep the EP tube on the magnetic frame to dry for 2-5 minutes, and observe that the surface of the magnetic beads is no longer reflective. Do not over-dry;

3.8.7 Remove the EP tube from the magnetic frame, add 30 μL of Nuclease-free water, gently pipette and mix, leave it at room temperature for 2 minutes, and then place the EP tube on the magnetic frame until the liquid is clear;

3.8.8 Aspirate 25μL of supernatant into a new PCR tube, do not suck the magnetic beads on the tube wall.

3.9Qubit quantitative product concentration

Use Qubit dsDNA HS Assay Kit to quantify the recovered products, as in step 3.2.

3.10 Send for sequencing

After completing the above steps, the obtained product is the library for sequencing. After equimolar mixing of different samples, Illumina sequencing can be performed. The index combination of different samples in the same batch cannot be repeated, which will lead to data cannot be split.

Beneficial effects of the present invention:

The primer combination of the present invention effectively solves the problems of cumbersome, time-consuming and non-standard screening methods for IKZF family gene mutation in patients with hematological malignancies; clinical blood sampling can meet the detection requirements, and the present invention can be timely and accurately applied to clinical malignant hematological diseases The detection of IKZF family gene mutations in patients with hematological diseases is convenient for guiding the prognosis of clinical patients.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings.

Figure 1 is the quality control chart of high-throughput sequencing results;

Figure 2 is a quality control chart of high-throughput sequencing results;

Figure 3 is a quality control chart of high-throughput sequencing results;

Figure 4 is a quality control chart of high-throughput sequencing results;

Figure 5 is a quality control chart of high-throughput sequencing results;

Figure 6 is a quality control chart of high-throughput sequencing results;

Figure 7 is a quality control chart of high-throughput sequencing results;

Figure 8 is a quality control chart of high-throughput sequencing results;

Figure 9 is a quality control chart of high-throughput sequencing results.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

The specificity, sensitivity and precision of the method of the present invention are verified by testing a sample from a clinical patient. The patient was a patient with refractory acute myeloid leukemia M2, and the test specimen was the bone marrow of the patient before initial diagnosis and treatment.

The specific detection method is as follows:

1. Use density gradient centrifugation to separate the patient's leukemia cells, and use the human peripheral blood lymphocyte separation liquid from Tianjin Haoyang Company. The operation method is shown in the instruction manual.

2. Extraction of nucleic acid: It is recommended to use the QIAamp DNA Blood Mini Kit from Qiagen Company for DNA extraction. See the instruction manual for the operation method.

3. The operation steps of the gene mutation detection method are as follows:

The patient's DNA concentration was 10.5ng/μL using the Qubit kit. Add 3 μL of DNA template to Panel A/B respectively, prepare the first-step PCR reaction system according to technical scheme 3.3, run the first-step PCR reaction and carry out magnetic bead purification, using 1.0 times (25 μL) Agencourt AMPure XP magnetic beads. After purification, Qubit kit was performed again for quantification, and the concentrations of the first-step PCR reaction products were measured to be 54.6 ng/μL for Panel A and 50.2 ng/μL for Panel B, respectively, which were diluted to 30 ng/μL for the second-step PCR reaction. Select i5-primer connector 1 and i7-primer connector 1, and prepare the second-step PCR reaction system and operation program according to technical scheme 3.6. The obtained PCR product was purified with 0.9 times (22.5 μL) Agencourt AMPure XP magnetic beads, and Qubit quantified the PCR products of the second step, with the concentrations of Panel A 27.2 ng/μL and Panel B 26.6 ng/μL, respectively taking 3.68 μL and 3.76 μL. Mixing is performed. After the above steps are completed, high-throughput sequencing is performed. The quality control of the sequencing results is shown in Figure 1-3, indicating that the sequencing results are qualified. Analysis of sequencing data revealed that this patient had IKZF1:c.A476G, p.N159S mutations with a mutation frequency of 15.4%.

4. This example shows that the primer combination and method provided by the present invention can comprehensively and sensitively detect the IKZF family gene mutation in patients with hematological malignancies, and accurately detect the presence of IKZF1 p.N159S mutation in the acute myeloid leukemia patient in this case. The operation feasibility and results High reliability.

5. Clinical value: The patient failed to achieve remission after standard induction chemotherapy, and was treated with salvage chemotherapy again, but still did not achieve remission. He died 2 months after the diagnosis, and was a refractory high-risk patient. Using the primer combination and method provided by the present invention to detect the bone marrow specimen of the patient before treatment, it is found that there is IKZF1 p.N159S mutation, which is consistent with its poor clinical prognosis, indicating that the application of this method can detect high-risk subtypes early, which is helpful for early diagnosis of individual patients. chemical treatment.

Example 2

The specificity, sensitivity and precision of the method of the present invention are verified by testing a sample from a clinical patient. This patient is a case of de novo acute lymphoblastic leukemia. The test specimens were bone marrow before initial diagnosis and treatment.

The specific detection method is as follows:

1. The patient's leukemia cells are separated by density gradient centrifugation. It is recommended to use the human peripheral blood lymphocyte separation solution from Tianjin Haoyang Company. The operation method is described in the instructions.

2. Extraction of nucleic acid: It is recommended to use the QIAamp DNA Blood Mini Kit from Qiagen Company for DNA extraction. See the instruction manual for the operation method.

3. The detection method of gene mutation is operated according to the steps in the above technical scheme. The patient's DNA concentration was 10.2ng/μL using Qubit kit. Add 3 μL of DNA template to Panel A/B respectively, prepare the first-step PCR reaction system according to technical scheme 3.3, run the first-step PCR reaction and carry out magnetic bead purification, using 1.0 times (25 μL) Agencourt AMPureXP magnetic beads. After purification, Qubit kit was performed again for quantification, and the concentrations of the first-step PCR reaction products were measured as Panel A 59ng/μL and Panel B 56.2ng/μL, respectively, and were diluted to 30ng/μL for the second-step PCR reaction. Select i5-primer connector 1 and i7-primer connector 2, and prepare the second-step PCR reaction system and operating program according to technical scheme 3.6. The obtained PCR products were purified by 0.9 times (22.5 μL) Agencourt AMPure XP magnetic beads, and Qubit quantified the PCR products of the second step. Perform mixing, and perform high-throughput sequencing after the above steps are completed. The quality control of sequencing results is shown in Figure 4-6, indicating that the sequencing results are qualified. Analysis of sequencing data found that this patient had IKZF2:c.A278G, p.N93S mutations, with a mutation frequency of 52.6%.

4. It can be seen that the primer combination and method provided by the present invention can comprehensively and sensitively detect the IKZF family gene mutation in patients with hematological malignancies, and accurately detect the presence of IKZF2 p.N93S mutation in this patient with acute lymphoblastic leukemia, and the operation feasibility and results are acceptable. High reliability.

5. Clinical value: The patient was a Philadelphia chromosome-positive acute lymphoblastic leukemia, and the bone marrow specimen was found to have IKZF2 p.N93S mutation before the initial diagnosis and treatment. The treatment plan was targeted therapy combined with individualized induction chemotherapy, and a complete remission was obtained in one course of treatment, suggesting that the The invention has important clinical significance and value for guiding clinical individualized treatment.

Example 3

The specificity, sensitivity and precision of the method of the present invention are further verified by detecting a sample of a clinical patient. This patient is a case of primary Ph-negative acute lymphoblastic leukemia. The test specimens were bone marrow before initial diagnosis and treatment.

The specific detection method is as follows:

1. The patient's leukemia cells are separated by density gradient centrifugation. It is recommended to use the human peripheral blood lymphocyte separation solution from Tianjin Haoyang Company. The operation method is described in the instructions.

2. Extraction of nucleic acid: It is recommended to use the QIAamp DNA Blood Mini Kit from Qiagen Company for DNA extraction. See the instruction manual for the operation method.

3. The detection method of gene mutation is operated according to the steps in the above technical scheme. The patient's DNA concentration was 1.9ng/μL quantified using the Qubit kit. Add 7 μL of DNA template to Panel A/B respectively, prepare the first-step PCR reaction system according to technical scheme 3.3, run the first-step PCR reaction and then carry out magnetic bead purification, using 1.0 times (25 μL) Agencourt AMPureXP magnetic beads. After purification, Qubit kit was performed again for quantification, and the concentrations of the first-step PCR reaction products were measured to be 16.5 ng/μL for Panel A and 13 ng/μL for Panel B, respectively, and 1.8 μL and 2.3 μL were added for the second-step PCR reaction. Select the i5-primer linker 4 and the i7-primer linker 7, and prepare the second-step PCR reaction system and operation program according to technical scheme 3.6. The obtained PCR products were purified by 0.9 times (22.5 μL) Agencourt AMPure XP magnetic beads, and Qubit quantified the PCR products of the second step, with the concentrations of Panel A 13ng/μL and Panel B 25.4ng/μL, respectively taking 7.69 μL and 3.94 μL for Mixing, high-throughput sequencing is performed after the above steps are completed, and the quality control of sequencing results is shown in Figure 7-9, indicating that the sequencing results are qualified. Analysis of sequencing data found that the patient had IKZF1: c.A476C:p.N159T mutation, and the mutation frequency was 36.82%.

4. This example shows that the primer combination and method provided by the present invention can comprehensively and sensitively detect the IKZF family gene mutation in patients with hematological malignancies, and accurately detect the presence of IKZF1 p.N159T mutation in the acute lymphoblastic leukemia patient, and the operation feasibility and results are credible high degree.

5. Clinical value: The patient was Philadelphia chromosome-negative acute lymphoblastic leukemia. The patient relapsed in the stage of consolidation chemotherapy, and the patient died without remission after induction chemotherapy. The IKZF1 p.N159T mutation was found in the bone marrow specimen of the patient before treatment by applying the present invention, indicating that the present invention has clinical guiding value for prognosis evaluation and early detection of high-risk subtypes.

The above discussion and examples show that the method provided by the present invention can comprehensively, accurately and rapidly detect whether there are IKZF family gene (IKZF1-5) mutations, precise mutation sites and frequencies in patients with various hematological malignancies at different stages of the disease, which makes up for the existing Due to the limitations and incompleteness of IKZF gene mutation detection methods, it fills the blank of all IKZF family gene mutation detection methods. The clinical practical value and application prospect.

In the description of this specification, description with reference to the terms "one embodiment," "example," "specific example," etc. means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one aspect of the present invention. in one embodiment or example. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Various changes and modifications fall within the scope of the claimed invention.

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<212> DNA

<213> Artificial Sequence

<400> 19

atcaatttcc agttgtccaa tgaat 25

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 20

ctaaattgag ttgattctga gatgc 25

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 21

attataagat gctgaaaggc aggca 25

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 22

aggaagccta aaattcatcc ctgaa 25

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 23

ggtccaatac agaccatgcc gcaga 25

<210> 24

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 24

gagagtgcca ggaaactaaa ggtat 25

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 25

tacaagagaa gtcactgagc tctac 25

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 26

cccctacctg tcgatctccc taaag 25

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 27

ctgctttctt tccccaaacc gaatc 25

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 28

gcttggggtc ctctttggcg taggc 25

<210> 29

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 29

aaagcaaaac tccttgggaa atggt 25

<210> 30

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 30

gcaaccatga agaacgccag aatca 25

<210> 31

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 31

cgccactgct ttgatgtcaa ctata 25

<210> 32

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 32

ccagttttatt tccttttctc cccat 25

<210> 33

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 33

ggtaacctcc tccgccacat taaac 25

<210> 34

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 34

gagcctgaaa tcccttacag ctatt 25

<210> 35

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 35

ttgtgtgaag ctgaaagtta aatgg 25

<210> 36

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 36

ccggctcctg cccttttcag tctgc 25

<210> 37

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 37

gcagtatttc ttcatgtgca gttct 25

<210> 38

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 38

caaagctttg acatcctcct tcatg 25

<210> 39

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 39

agctccaagg taggtgattg cattg 25

<210> 40

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 40

catgttgaac tacaaaagca gtagg 25

<210> 41

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 41

cttttcttct caaggagttg gtgac 25

<210> 42

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 42

tcagtttacc attcggaagc cggat 25

<210> 43

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 43

caaaggtgaa attgtgaaca gagag 25

<210> 44

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 44

agaaaaaggt atatgcatcc cagca 25

<210> 45

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 45

gatgagtagc tgaacagtgg ttttg 25

<210> 46

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 46

tcactgttct attcgttaga cacct 25

<210> 47

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 47

cctatttgat tgtctttttg ctgct 25

<210> 48

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 48

gccccccgtgg gaaacaactt tctcg 25

<210> 49

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 49

agtcgcatga gttcttggta caatt 25

<210> 50

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 50

ttggacgcga ctgaaccctt taaac 25

<210> 51

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 51

tggtcccggt catcagcccg atgta 25

<210> 52

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 52

gctcttcctg gatcacgtca tgtac 25

<210> 53

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 53

cccatgacat cccatatgaa tagtg 25

<210> 54

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 54

gcagaaacta ctgcttgggt agagg 25

<210> 55

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 55

ggtaccattt tatgcttgcg ccttc 25

<210> 56

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 56

gctttttcca gctgtctttc catta 25

<210> 57

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 57

ctaactgatc cagaaatcat gttca 25

<210> 58

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 58

ctcaggcttt gacgggatag aatgg 25

<210> 59

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 59

cagcttcccc ctttgcctct ctcta 25

<210> 60

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 60

ctgtattgga cccaacgtgc tcatg 25

<210> 61

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 61

ctctgggatt tgttctttca cttgc 25

<210> 62

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 62

gcggtgccat aactacctac agagt 25

<210> 63

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 63

tacccctact caactgctac tactg 25

<210> 64

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 64

ggagcttcca ggatcccgag aagca 25

<210> 65

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 65

agcctgtgaa ggccttcaag tgtga 25

<210> 66

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 66

atgttacact cgaaagggtc acgga 25

<210> 67

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 67

gatgcttttc ttttccagct cttga 25

<210> 68

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 68

cttgtgtagc aagcatccta tgtta 25

<210> 69

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 69

taatggtcgt gtaattagcc ctaga 25

<210> 70

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 70

aagtaaccaa aagttccaaa ttcca 25

<210> 71

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 71

ttcttctcta cactaagcct aagca 25

<210> 72

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 72

agcaaaactg agattcagaa gaaac 25

<210> 73

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 73

cctgccttaa atcccaagag gaaac 25

<210> 74

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 74

ggaaaagctc atgcgattca gctac 25

<210> 75

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 75

agctttgtat catccaaaat gttga 25

<210> 76

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 76

tgagacacat aaagttacac tctgg 25

<210> 77

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 77

gggtagcagc ctagaagaac cccta 25

<210> 78

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 78

tatgcatgcc attgcgtgta taaag 25

<210> 79

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 79

ccagggaagc ttttctcatg aactt 25

<210> 80

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 80

tctttaatat gccagttgag ggaac 25

<210> 81

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 81

ggagtaatct caaagctgtc cagca 25

<210> 82

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 82

actacggaca caaactggaa taagg 25

<210> 83

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 83

tgagaagtcc tttacaaggc tgtac 25

<210> 84

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 84

ggtgcagtgg gaaaaggaga atatg 25

<210> 85

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 85

gaaactgatg ctattggcca aaaac 25

<210> 86

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 86

cggggtgccc tccgcgagcg gcttg 25

<210> 87

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 87

gcacatgttg cactcaaaag gatca 25

<210> 88

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 88

tgccagcact gtgatatgta ctttg 25

<210> 89

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 89

caagaactca tggttgataa ccctt 25

<210> 90

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 90

tgaattatgt tccttccgct gcagt 25

<210> 91

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 91

cctttgatgg gaagcttaag tgtcg 25

<210> 92

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 92

accagagcct ttggacttcg tgaaa 25

<210> 93

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 93

aggcttggga gtagttactg aattg 25

<210> 94

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 94

ggtgaattga aggccaaatg caaca 25

<210> 95

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 95

ccctctggct tacttaccag tgtga 25

<210> 96

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 96

tagttgcaga agggacattt aaagg 25

<210> 97

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 97

cccttttaac tccccagtag tacat 25

<210> 98

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 98

tccaggtcac gtatttcgtc accta 25

<210> 99

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 99

tgctttctgt gtctgtggag tcctg 25

<210> 100

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 100

atgtggatag tgaacatgac gtggt 25

<210> 101

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 101

ggtgatggat gtgtatcggt gtgac 25

<210> 102

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 102

cagttatcag cagcatgtat cccat 25

<210> 103

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 103

tattttaacc atccaaacac gttgc 25

<210> 104

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 104

ttctagtgaa cagttgtcac agagc 25

<210> 105

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 105

tctaatctgt gcatatgttt ctcca 25

<210> 106

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 106

tttgatagct tcaagtatag tcgtt 25

<210> 107

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 107

ttctgctgtt aagttttccg aaatg 25

<210> 108

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 108

tgaaaactca taacggtcct ggctt 25

<210> 109

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 109

ttttcatgac tatcagcagt ttccc 25

<210> 110

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 110

gggaacttgc cttttcctat taaca 25

<210> 111

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 111

tacatgcatc ctgcaagatt tatca 25

<210> 112

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 112

cacccctgaa taaaaaggaa agaga 25

<210> 113

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 113

ccccacagac ctaacaaatt aaagt 25

<210> 114

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 114

caaggtctgt gccagtctga tactc 25

<210> 115

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 115

gactcacact tcttctttct catca 25

<210> 116

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 116

aacaaaagca tgccttcatc tccta 25

<210> 117

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 117

tgcaaatgtg tccataaggt attgg 25

<210> 118

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 118

aaactaaagt gtgatatctg tggga 25

<210> 119

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 119

cttcaaatgc cacctctgca actac 25

<210> 120

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 120

cacaaatgtg gatattgtgg ccgaa 25

<210> 121

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 121

gccagacctg accggttccg gaggt 25

<210> 122

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 122

tcatctacct gaccaaccac atcgc 25

<210> 123

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 123

agcagacact ttattaggga tgacc 25

<210> 124

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 124

acctgccact ggactatagt tcctt 25

<210> 125

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 125

ttgccatact ttcataggag tcagt 25

<210> 126

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 126

cttctgacct gtttgtatgt tgtta 25

<210> 127

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 127

agaatgattc acattaatgc gcagt 25

<210> 128

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 128

cccctcactt ccaggaatcc acgct 25

<210> 129

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 129

aggcaatgga cagtagatat ctcca 25

<210> 130

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 130

catcaaggtg gagatgtaca gcgat 25

<210> 131

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 131

cataaatcag ggtttgtgtg cattg 25

<210> 132

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 132

cagtgtatac ttgctcttgg ctgaa 25

<210> 133

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 133

tctatatcct tggcctaatg ggaga 25

<210> 134

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 134

cgccttccac ccaccaattg catct 25

<210> 135

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 135

aaagcaacca cgaagatcgg gttgc 25

<210> 136

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 136

atgttttgag agcaatctgt taggc 25

<210> 137

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 137

acaggaccat gtgattttgc tgata 25

<210> 138

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 138

ctgtatgagc tcactctctt tctca 25

<210> 139

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 139

tactattgta tctcaccacc tagga 25

<210> 140

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 140

ttcctagaca aggaatgctc atttc 25

<210> 141

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 141

gatgcaggat aatccacaca catcg 25

<210> 142

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 142

cacacatttc catagcattt ttact 25

<210> 143

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 143

agcgctag 8

<210> 144

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 144

gatatcga 8

<210> 145

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 145

cgcagacg 8

<210> 146

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 146

tatgagta 8

<210> 147

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 147

aggtgcgt 8

<210> 148

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 148

gaacatac 8

<210> 149

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 149

acatagcg 8

<210> 150

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 150

gtgcgata 8

<210> 151

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 151

ccaacaga 8

<210> 152

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 152

ttggtgag 8

<210> 153

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 153

ccgcggtt 8

<210> 154

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 154

ttataacc 8

<210> 155

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 155

ggacttgg 8

<210> 156

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 156

aagtccaa 8

<210> 157

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 157

atccactg 8

<210> 158

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 158

gcttgtca 8

<210> 159

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 159

caagctag 8

<210> 160

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 160

tggatcga 8

<210> 161

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 161

agttcagg 8

<210> 162

<211> 8

<212> DNA

<213> Artificial Sequence

<400> 162

gacctgaa 8

Claims (8)

1. A primer combination for detecting gene mutation of an IKZF family of malignant blood diseases is characterized by comprising 71 pairs of primers for sequencing an exon of an IKZF1-5 gene, wherein the 71 pairs of primers are divided into two primer pools, 36 pairs of primers form a first primer pool M1, and 35 pairs of primers form a second primer pool M2.

2. The primer combination for detecting the IKZF family gene mutation of the hematological malignancy as claimed in claim 1, wherein the M1 primer pool sequence comprises M1-1 f-M1-36 f and M1-1 r-M1-36 r, and the M2 primer pool sequence comprises M2-1 f-M2-35 f and M2-1 r-M2-35 r.

3. The method for detecting IKZF family gene mutation by using the primer combination as claimed in claim 1, which comprises the following steps:

s1, separating mononuclear cells of the patient with the malignant blood disease;

s2, extracting cell genome DNA;

s3, constructing a DNA library and sequencing.

4. The method for detecting IKZF family gene mutation using the primer combination as claimed in claim 3, wherein the operation of S3 comprises:

s3.1, confirming an IKZF1-5 gene exon PCR amplicon sequence;

s3.2 preparation and quantification of PCR amplification templates

S3.2.1 the kit was left at room temperature;

s3.2.2 preparing working solution;

s3.2.3 Add 190 μ L of prepared working solution to the tube;

s3.2.4 adding dsDNA standard #1, standard #2 and diluted dsDNA samples in the analysis tube, respectively, and marking;

s3.2.5 incubating at room temperature in dark;

s3.2.6 detecting the sample concentration;

s3.3 Gene-specific PCR

Preparing a PCR amplification reaction system, mixing uniformly and placing on a PCR instrument for operation

S3.4 recovery of first round amplification products

S3.4.1 placing the magnetic beads at room temperature, and mixing thoroughly;

s3.4.2 adding magnetic beads, mixing with the reaction solution, gently blowing and sucking, mixing, and incubating at room temperature;

s3.4.3 placing the EP tube on a magnetic frame until the liquid is clear, and sucking and discarding the supernatant;

s3.4.4 holding the EP tube on a magnetic frame, adding 80% ethanol, standing at room temperature for 30 s, and discarding the supernatant;

s3.4.5 repeating the above step, and washing with 80% ethanol;

s3.4.6 keeping the EP tube on the magnetic rack to dry, observing that the surface of the magnetic bead is no longer reflecting;

s3.4.7 taking off the EP tube from the magnetic frame, adding clean-free water, gently blowing and beating, mixing, standing at room temperature for 2min, and placing the EP tube on the magnetic frame until the liquid is clear;

s3.4.8 sucking the supernatant to a new PCR tube and marking;

s3.5, quantifying the recovered product by using the Qubit 3.0;

s3.6 Joint connection

Preparing a second round of PCR reaction system, and synthesizing PCR joint primers;

s3.7, mixing uniformly, centrifuging, and placing on a PCR instrument for operation;

s3.8 library purification

3.8.1 placing the magnetic beads at room temperature, and mixing thoroughly;

3.8.2 adding magnetic beads, mixing with the reaction solution, gently blowing and sucking, mixing, and incubating at room temperature;

3.8.3 placing the EP tube on a magnetic frame until the liquid is clear, and sucking and discarding the supernatant;

3.8.4 holding the EP tube on a magnetic frame, adding 80% ethanol, standing at room temperature for 30 s, and discarding the supernatant;

3.8.5 repeat 3.8.4 steps with 80% ethanol wash;

3.8.6 keeping the EP tube on the magnetic rack to dry, observing that the surface of the magnetic bead is no longer reflecting;

3.8.7 taking off the EP tube from the magnetic frame, adding clean-free water, gently blowing and beating, mixing, standing at room temperature for 2min, and placing the EP tube on the magnetic frame until the liquid is clear;

3.8.8 sucking the supernatant into a new PCR tube;

3.9Qubit quantification of product concentration;

3.10 sequencing by hand

The obtained product is a library for sequencing; and mixing equimolar amounts of different samples, and carrying out Illumina sequencing.

5. The method for detecting IKZF family Gene mutation by using the primer combination as claimed in claim 4, wherein the PCR amplification reaction system reagent in S3.3 comprises Gene-specific PCR Master Mix1, PanelA/B, diluted DNA template and double distilled water.

6. The method for detecting IKZF family gene mutation by using the primer combination as claimed in claim 5, wherein the reaction system of the PanelA/B is separately prepared.

7. The method for detecting IKZF family gene mutation by using the primer combination as claimed in claim 4, wherein the PCR amplification reaction system reagent in S3.6 comprises 2x Indexing PCR MasterMix2, i5-primer linker, i7-primer linker, purified PCR product and Nuclear-free water.

8. The method for detecting IKZF family gene mutation by using the primer combination as claimed in claim 4, wherein the PCR adaptor primer comprises i5 index and i7 index.

Images