CN114317759A - Primer combination and method for detecting gene mutation of IKZF family of malignant blood disease - Google Patents
Primer combination and method for detecting gene mutation of IKZF family of malignant blood disease Download PDFInfo
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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
Technical Field
The invention relates to the technical field of biological medicines, in particular to a primer combination and a method for detecting gene mutation of an IKZF family of malignant blood diseases.
Background
The IKZF family consists of several transcriptional regulatory proteins with zinc fingers, the N-terminal zinc finger of which has DNA binding function, and the C-terminal zinc finger for mutual recognition and binding between IKZF family members to form homo-or heterodimers. IKZF family members mainly include Ikaros, Helios, Aiolos, Eos and Pegasus, each encoded by IKZF1-5 gene. IKZF1 and IKZF2 play important roles in the regulation of proliferation and differentiation of lymphoid lineage cells. Meanwhile, studies show that isomers of IKZF1 and IKZF2 play an important role in the development and progression of leukemia. Ik-6 refers to deletion of an IKZF13-6 exon, is the most important IKZF1 isomer discovered to date in leukemia pathogenesis, and is a high-risk prognosis marker recognized at present. IKZF2 is a marker gene of regulatory T cells, plays an important role in tumor microenvironment, and has close correlation with tumor metastasis, treatment scheme selection and prognosis. The Aiolos protein encoded by IKZF3 plays a crucial role in the differentiation and development of lymphocytes. The IKZF3 mutation plays a key role in the occurrence and development of B cell malignancy and chronic lymphocytic leukemia. IKZF4 and IKZF5 are also important blood cell developmental genes, and are expressed in high amount in blood tumor cells. The gene mutation is not only related to the occurrence and development of tumors, but also closely related to the pathophysiology of thrombocytopenia, and the gene mutation can guide the prognosis stratification and clinical treatment selection of patients. Research reports that the IKZF family gene has higher mutation rate in patients with acute leukemia and myelodysplastic syndrome than other malignant hematological diseases, is relevant to patient prognosis and is considered as a driving factor of diseases. However, only the IKZF1 gene mutation is currently used for clinical detection and typing of certain hematologic malignancies. Therefore, the new method for comprehensively detecting the mutation characteristics and distribution of the 5 IKZF family members in the hematological malignancy has very important significance on the aspects of clinical typing, prognosis judgment, treatment scheme selection and the like.
The traditional morphology combined with flow immunotyping can not meet the demand of the precise medical age for diagnosing patients with malignant hemopathy. With the development of high throughput sequencing technologies, more and more molecular abnormalities associated with hematological malignancies have been discovered. Patients with these molecular abnormalities often have difficulty in clinical treatment, relapse susceptibility, and poor prognosis. The early identification of new targets for accurate individual treatment becomes the treatment direction in the future. However, only IKZF1 mutation detection is currently included in certain leukemia high-throughput sequencing test suites, and there is a lack of a high-throughput method for detecting mutations in the 5 member genes of the complete IKZF family. Therefore, a new method for detecting IKZF family gene mutation more comprehensively, efficiently and simply is urgently needed to be developed, the molecular abnormality of the family gene is identified at an early stage, more accurate individual treatment is expected to be provided for patients, prognosis and survival are improved, and the method has great clinical value and application prospect.
The multiplex PCR technology can add a plurality of pairs of primers into a PCR reaction system for amplification, and can be used for sequencing after the amplification of the to-be-detected locus by combining a high-throughput sequencing technology. The addition of the primers with multiple sites to be detected in a PCR reaction system can greatly reduce the workload and the reagent cost, and simultaneously can meet the requirement of comprehensively, accurately and quickly acquiring a detection result in clinic hope, thereby truly realizing accurate and efficient high-risk subtype screening of patients with malignant hemopathy. However, the addition of multiple pairs of primers to a PCR reaction tends to 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 are the key and difficult points of the technology, and the more primers, the greater the design difficulty. In view of the difficulties, the invention designs primers and a combination thereof and optimizes the IKZF1-5 gene to adapt to the characteristics and the requirements of clinical detection.
Disclosure of Invention
The invention aims to provide a primer combination and a method for detecting IKZF family gene mutation of a malignant hematological disease, which solve the bottleneck technical problems that the IKZF gene mutation detection of the malignant hematological disease cannot completely cover the IKZF1-5 gene at present, and the detection is difficult, time-consuming, low in sensitivity and accuracy and the like, and are used for detecting the IKZF1-5 gene mutation of the malignant hematological disease patient based on the primer combination and the method combining multiple PCR with a high-throughput sequencing technology; the primers and the combination thereof in the method are optimized for many times, and the specificity, the sensitivity and the accuracy are high; the technology is applied to the detection of IKZF family gene mutation of a patient with malignant hemopathy, and plays an important role in guiding clinical accurate typing diagnosis, early screening high-risk subtypes, prognosis evaluation and individualized target treatment.
The purpose of the invention can be realized by the following technical scheme:
a primer combination for detecting IKZF family gene mutation of malignant blood disease comprises 71 pairs of primers for IKZF1-5 gene exon sequencing, which are divided into two primer pools, wherein 36 pairs of primers form a first primer pool M1, and 35 pairs of primers form a second primer pool M2.
Further, 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.
The method for detecting IKZF family gene mutation by using the primer combination comprises the following steps:
first, separation of mononuclear cells of patients with malignant blood disease
1.1 the blood collection tube is trimmed and placed in a low speed centrifuge at 2500rpm 5 min. Centrifuging, slowly absorbing upper plasma, diluting the remaining blood cells to 5mL with sterile normal saline, fully pumping and uniformly mixing;
1.2 taking a 15mL centrifuge tube, marking the name of a patient, adding 5mL human lymphocyte separation liquid, and slowly dripping the diluted liquid of bone marrow or peripheral blood on the lymphocyte separation liquid along the tube wall to form layering so as to avoid mixing the two;
1.3 adopting a density gradient centrifugation method, placing the centrifuge tube in a low-speed centrifuge (attention balancing), and rotating speed of 2000rpm is multiplied by 20 min; after centrifugation, the liquid in the tube is divided into three layers, wherein the upper clear layer is diluted plasma and platelets, the middle white membrane layer is mononuclear cells, and the lower red layer is granulocytes and red blood cells;
1.4 slowly absorbing the middle mononuclear cell layer, placing the middle mononuclear cell layer in another clean centrifuge tube for marking the name of a patient, adding 10mL of sterile physiological saline, blowing, uniformly mixing, placing the mixture in a low-speed centrifuge for centrifugation at 2000rpm multiplied by 5min, and discarding the supernatant;
1.5 resuspending the washed cell mass with 1mL of sterile physiological saline, counting the cell mass by using a cell counting plate under a high power microscope, and calculating the total cell amount (the cell counting solution is 1% of glacial acetic acid, 20 mu L of cell suspension is added into 380 mu L of 1% of glacial acetic acid, the mixture is blown and uniformly mixed, namely the cell mass is diluted by 20 times; 20 mu L of cell suspension is added into the cell counting plate, the number n of cells in 16 lattices is counted under the microscope, and then the total cell amount is n multiplied by 10000 multiplied by 20);
1.6 dilution of the cell suspension to 1X 107Single mononuclear cell/mL; 1.5mL of the cell suspension was dispensed into 1mL of EP tube; after trimming, the cells were centrifuged instantaneously at high speed (8000rpm) and the supernatant was carefully aspirated without touching the bottom cell pellet. The tube wall is marked with a number and a patient name.
Secondly, extracting cell genome DNA
The reagents for DNA extraction and purification were purchased from Qiagen (QIAamp DNA Blood Mini Kit) Kit, and the method was a silicon membrane adsorption method, which was as follows:
2.1 separation of the resulting 5X 106Mononuclear cells of a patient with malignant hemopathy are dissolved in 200 mu L PBS, and 20 mu L proteinase K (provided by a kit, prepared according to the instructions and stored at 4 ℃) is added at the bottom of a tube;
2.2 slowly adding 200 mul of oily lysate AL, shaking and uniformly mixing, and then quickly separating at a low speed;
performing water bath at 2.356 deg.C for 20min, clarifying the oily mixed solution, wiping off the outer wall liquid, and centrifuging the liquid adhered to the inner wall and the tube cover to the bottom of the tube;
2.4 adding 200 μ L of absolute ethyl alcohol, reversing, mixing uniformly, and carrying out low-speed instantaneous separation to remove liquid on the tube wall;
2.5 carefully transferring the mixed solution obtained in the step 2.4 to a QIAamp Mini adsorption column (the adsorption column is placed on a 2mL collection tube provided by the kit), covering a tube cover, marking, centrifuging at high speed of 12000rpm × 3min, and discarding the collection tube and the filtrate;
2.6 placing the adsorption column into a new collection tube (provided by kit), adding 500 μ L buffer AW1 (adding anhydrous ethanol according to product instruction, mixing well), covering tube cover, centrifuging, 12000rpm × 3min, discarding the collection tube and filtrate;
2.7 the adsorption column is put into a new collection tube (provided by the kit), 500. mu.L of buffer AW2 (adding absolute ethyl alcohol according to the product instruction for mixing uniformly) is added, the tube cover is covered and the centrifugation is carried out, 12000rpm is multiplied by 3min, and the collection tube and the filtrate are discarded;
2.8 placing the adsorption column in a self-prepared clean and sterile 1.5mL EP tube, opening the tube cover of the adsorption column, centrifuging at 12000rpm for 3min to fully volatilize ethanol, and discarding the EP tube and filtrate;
2.9 placing the adsorption column into a self-prepared clean sterile 1.5mL EP tube, suspending and dropping 100 μ L of elution buffer AE, dropping AE into the middle part of the adsorption membrane of the adsorption column, incubating at room temperature for 3min to fully elute DNA in the adsorption membrane, centrifuging at 12000rpm × 3min, and obtaining the filtrate in the EP tube as genome DNA solution;
2.10 to improve the collection efficiency of DNA, repeat step 2.9, add the DNA solution collected in the EP tube back to the adsorption column, carefully add to the middle part of the adsorption membrane of the adsorption column, incubate for 3min at room temperature to fully dissolve the DNA in the adsorption membrane, centrifuge 12000rpm x 3min, collect the DNA solution again;
2.11 using Nanodrop one trace ultraviolet visible spectrophotometer to detect the concentration and purity of genome DNA, the DNA quality with OD260/280 ratio of 1.8-2.0 meets the requirement, and can be used for the construction of DNA library and high-throughput sequencing.
Thirdly, DNA library construction and sequencing
3.1IKZF1-5 Gene exon PCR amplicon sequence
The information of the 71 amplicons tested is given in the following table:
primers are designed aiming at an IKZF1-5 gene exon sequence, and the primer synthesis is completed by Shanghai bio-chemical company. Because the number of the primers is large, in order to avoid forming primer dimers to influence the amplification efficiency, the designed primers are divided into 2 primer pools (M1 and M2), wherein M1 is 36 pairs of primers, M2 is 35 pairs of primers, and the specific sequence information of the primers is as follows:
3.2 preparation and quantification of PCR amplification templates
The DNA template concentration of the PCR reaction was determined using the Qubit dsDNA HS Assay Kit from Life Technologies, USA, cat # Q32850, ensuring that 20-60ng of DNA was added to each reaction system.
3.2.1 placing the kit at room temperature;
3.2.2 taking dsDNA HS Reagent (component A) in the kit, diluting the dsDNA HS Reagent (component A) with Buffer (component B) according to the ratio of 1:200 to prepare working solution which is used immediately; 1 μ L of component a +199 μ L of component B;
3.2.3 adding 190 μ L of the prepared working solution into the analysis tube;
3.2.4 Add dsDNA standard #1, standard #2 and 10-fold diluted dsDNA samples in the analysis tubes, 10. mu.L per tube, correctly labeled;
3.2.5 incubating for 2min at room temperature in the dark;
3.2.6 detecting the concentration of the sample.
3.3 Gene-specific PCR
The PCR amplification reaction system is (the used reagent is a high-throughput sequencing library construction kit of an engineering company):
the reaction system of Panel A/B needs to be prepared separately; the purity and quality of the genome are very important, and if the extracted DNA has impurities or is seriously degraded, the amplification efficiency is greatly reduced.
Mixing, respectively placing Panel A/B on a PCR instrument, and operating the following reaction programs:
3.4 recovery of first round amplification product
3.4.1 placing the Agencour AMPure XP magnetic beads in room temperature half an hour in advance, and fully and softly mixing the Agencour AMPure XP magnetic beads uniformly to activate the magnetic beads;
3.4.2 mu.L of magnetic beads (1.0 times volume) and 25. mu.L of the reaction solution (PCR product in step 3.3) were added, mixed by gentle pipetting 10 times, and incubated at room temperature for 5 minutes. Preparing 80% ethanol (the volume ratio of the ethanol to the clean-free water is 4: 1);
3.4.3 place the EP tube on the magnetic frame for 5min until the liquid is clear, carefully suck and discard the supernatant, do not suck the magnetic bead on the tube wall;
3.4.4 holding the EP tube on a magnetic stand, adding 200. mu.L of 80% ethanol (ready for preparation) along the tube wall, standing at room temperature for 30 seconds, discarding the supernatant, and cutting off the magnetic beads which are attracted to the tube wall;
3.4.5 repeating the above step and washing with 80% ethanol once;
3.4.6 keep the EP tube on the magnetic frame to dry for 2-5min, observe the surface of the magnetic bead not to reflect light any more, do not dry excessively;
3.4.7 taking down the EP tube from the magnetic frame, adding 30 μ L of clean-free water, gently blowing and beating, mixing, standing at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
3.4.8 carefully pipette 25. mu.L of the supernatant into a new PCR tube, and do not pipette the beads onto the tube wall and make the label.
3.5Qubit 3.0 quantitation
The recovery product was quantified using the Qubit dsDNA HS Assay Kit, as in step 3.2.
3.6 linker ligation (reagents used are high throughput sequencing kits for Industrial Co.)
Preparing a second round of PCR reaction system (divided into Panel A/B):
| reagent | Volume of |
| 2x Indexing PCR Master Mix2 | 10μL |
| i5-primer joint (10uM) | 1μL |
| i7-primer joint (10uM) | 1μL |
| Purification of PCR products | 30ng |
| Clean-free water | Make up to 30. mu.L |
| Total volume | 30μL |
The joint is synthesized by Shanghai chemical company, and has the specific sequence as follows:
| 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 | ACATAGCG | CAAGCTAG | |
| 8 | GTGCGATA | TGGATCGA | |
| | CCAACAGA | AGTTCAGG | |
| 10 | TTGGTGAG | GACCTGAA |
The Panel A/B reaction system is prepared separately. The PCR adaptor has two primers, i5 index and i7 index, each of which has a number, and different samples need to be combined, while the same combination needs to be used for Panel A and Panel B of the same sample. Index combinations for the same batch of different samples cannot be repeated, otherwise the data cannot be resolved.
3.7. After mixing, the mixture was centrifuged rapidly, placed on a PCR instrument, and the following reactions were run:
3.8 library purification
3.8.1 placing Agencour AMPure XP magnetic beads in room temperature half an hour in advance, and mixing the Agencour AMPure XP magnetic beads thoroughly and softly to activate the magnetic beads;
3.8.2 adding 22.5 μ L magnetic beads (0.9 times volume) and 25 μ L reaction solution (PCR product in step 3.7), mixing, gently blowing and sucking for 10 times, and incubating at room temperature for 5 min;
3.8.3 placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully aspirating the supernatant without aspirating the magnetic beads on the tube wall;
3.8.4 holding the EP tube on a magnetic frame, adding 200 μ L80% ethanol (now ready for use) along the tube wall, standing at room temperature for 30 s, discarding the supernatant, and cutting the magnetic beads which are not adsorbed onto the tube wall;
3.8.5 repeating the above step, and washing with 80% ethanol;
3.8.6 keeping the EP tube on a magnetic rack and drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and keeping from over-drying;
3.8.7 taking off the EP tube from the magnetic frame, adding 30 μ L of clean-free water, gently blowing and beating, standing at room temperature for 2min, and standing on the magnetic frame until the liquid is clear;
3.8.8 pipette 25. mu.L of supernatant into a new PCR tube, and do not pipette down the beads on the tube wall.
3.9Qubit quantification of product concentration
The recovery product was quantified using the Qubit dsDNA HS Assay Kit, as in step 3.2.
3.10 sequencing by hand
After the above steps are completed, the obtained product is the library for sequencing. After equimolar amounts of different samples are mixed, Illumina sequencing can be performed, and index combinations of the same batch of different samples cannot be repeated, so that data cannot be resolved.
The invention has the beneficial effects that:
the primer combination effectively solves the problems of complicated screening method, time consumption, non-standardization and the like of IKZF family gene mutation of the previous malignant hemopathy patient; the detection requirement can be met by one-time clinical blood collection, and the invention can be timely and accurately applied to the detection of IKZF family gene mutation of clinical malignant hemopathy patients, thereby being convenient for guiding the prognosis of clinical patients.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a diagram of quality control of high throughput sequencing results;
FIG. 2 is a quality control diagram of the results of high throughput sequencing;
FIG. 3 is a quality control diagram of the results of high throughput sequencing;
FIG. 4 is a quality control diagram of the results of high throughput sequencing;
FIG. 5 is a quality control diagram of the results of high throughput sequencing;
FIG. 6 is a quality control diagram of the results of high throughput sequencing;
FIG. 7 is a quality control diagram of the results of high throughput sequencing;
FIG. 8 is a quality control diagram of the results of high throughput sequencing;
FIG. 9 is a quality control diagram of the results of high throughput sequencing.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The specificity, sensitivity and accuracy of the method of the invention is verified by testing a sample of a clinical patient. This patient is an example of refractory acute myelogenous leukemia M2 patient, and the test specimen is the bone marrow before first diagnosis treatment.
The specific detection method comprises the following steps:
1. separating by density gradient centrifugation to obtain patient leukemia cells, and separating human peripheral blood lymphocyte separation solution from Tianjin N-P-ocean company by the method described in the specification.
2. Extracting nucleic acid: DNA extraction is suggested using QIAamp DNA Blood Mini Kit from Qiagen, see the manual for the procedure.
3. The operation steps of the detection method of the gene mutation are as follows:
the patient was quantified for DNA concentration of 10.5 ng/. mu.L using the Qubit kit. Adding 3 mu L of Panel A/B into a DNA template respectively, preparing a first-step PCR reaction system according to a technical scheme 3.3, performing magnetic bead purification after running the first-step PCR reaction, and selecting 1.0-time (25 mu L) Agencour AMPure XP magnetic beads. After purification, the Qubit kit is used for quantification again, the concentrations of the products of the first step PCR reaction are respectively 54.6 ng/muL of Panel A and 50.2 ng/muL of Panel B, and the products are respectively diluted to 30 ng/muL for carrying out the second step PCR reaction. Selecting i5-primer 1 and i7-primer 1, and preparing a second-step PCR reaction system and an operation program according to the technical scheme 3.6. And purifying the obtained PCR product by using 0.9-fold (22.5 mu L) Agencour AMPure XP magnetic beads, quantifying the PCR product in the second step by using Qubit, wherein the concentrations of the PCR product in the second step are respectively 27.2 ng/mu L of Panel A and 26.6 ng/mu L of Panel B, mixing 3.68 mu L and 3.76 mu L of the PCR product respectively, performing high-throughput sequencing after the steps are completed, and controlling the quality of the sequencing result as shown in FIGS. 1-3 to prompt that the sequencing result is qualified. Analysis of sequencing data revealed that the patient had IKZF1: c.A476G, p.N159S mutation with a mutation frequency of 15.4%.
4. The example shows that the primer combination and the method provided by the invention can comprehensively and sensitively detect IKZF family gene mutation of a patient with malignant hemopathy, accurately detect the IKZF1 p.N159S mutation of the patient with acute myelogenous leukemia, and have high operation feasibility and result reliability.
5. The clinical value is as follows: the patient in the case is not relieved after being treated by the standard induction chemotherapy scheme, and is still not relieved after being treated by the rescue treatment scheme again, and dies after 2 months of diagnosis, thereby being a patient with high risk of difficult treatment. The primer combination and the method provided by the invention are used for detecting the bone marrow specimen of the patient before treatment, and the IKZF1 p.N159S mutation is found to be in accordance with the clinical prognosis difference, so that the method can be used for early finding high-risk subtypes and is beneficial to individual treatment as soon as possible.
Example 2
The specificity, sensitivity and accuracy of the method of the invention is verified by testing a sample of a clinical patient. This patient is an example of a patient with incipient acute lymphoblastic leukemia. The detection specimen is bone marrow before initial diagnosis and treatment.
The specific detection method comprises the following steps:
1. separating leukemia cells of patient by density gradient centrifugation, and using human peripheral blood lymphocyte separation solution of Tianjin tertiary ocean company as suggested, the operation method is described in the specification.
2. Extracting nucleic acid: DNA extraction is suggested using QIAamp DNA Blood Mini Kit from Qiagen, see the manual for the procedure.
3. The detection method of gene mutation is operated according to the steps in the technical scheme. The patient was quantified for DNA concentration of 10.2 ng/. mu.L using the Qubit kit. Adding 3 mu L of Panel A/B into a DNA template respectively, preparing a first-step PCR reaction system according to a technical scheme 3.3, performing magnetic bead purification after running the first-step PCR reaction, and selecting 1.0-time (25 mu L) Agencour AMPure XP magnetic beads. After purification, the Qubit kit is used for quantification again, the concentrations of the products of the first step PCR reaction are measured to be 59 ng/mu L of Panel A and 56.2 ng/mu L of Panel B, and the products are respectively diluted to 30 ng/mu L for carrying out the second step PCR reaction. Selecting i5-primer 1 and i7-primer 2, and preparing a second-step PCR reaction system and an operation program according to the technical scheme 3.6. And (3) purifying the obtained PCR product by using 0.9-fold (22.5 mu L) Agencour AMPure XP magnetic beads, quantifying the PCR product in the second step by using Qubit, wherein the concentrations of the PCR product in the second step are respectively Panel A18.9 ng/mu L and Panel B23.6 ng/mu L, mixing 5.29 mu L and 4.24 mu L respectively, performing high-throughput sequencing after the steps are completed, and controlling the quality of a sequencing result as shown in FIGS. 4-6 to prompt that the sequencing result is qualified. Analysis of sequencing data revealed that the patient had IKZF2: c.A278G and p.N93S mutations with a mutation frequency of 52.6%.
4. Therefore, the primer combination and the method provided by the invention can comprehensively and sensitively detect IKZF family gene mutation of a patient with malignant hemopathy, accurately detect the IKZF2 p.N93S mutation of the patient with acute lymphoblastic leukemia, and have high operation feasibility and result reliability.
5. The clinical value is as follows: the patient is philadelphia chromosome positive acute lymphoblastic leukemia, the IKZF2 p.N93S mutation is found by bone marrow specimen detection before initial diagnosis treatment, a targeted treatment and individualized induction chemotherapy scheme is selected as a treatment scheme, and complete remission is obtained in one treatment course, so that the invention has important clinical significance and value for guiding clinical individualized treatment.
Example 3
The specificity, sensitivity and accuracy of the methods of the invention are further verified by testing a sample from a clinical patient. This patient is one with an initial Ph negative acute lymphoblastic leukemia. The detection specimen is bone marrow before initial diagnosis and treatment.
The specific detection method comprises the following steps:
1. separating leukemia cells of patient by density gradient centrifugation, and using human peripheral blood lymphocyte separation solution of Tianjin tertiary ocean company as suggested, the operation method is described in the specification.
2. Extracting nucleic acid: DNA extraction is suggested using QIAamp DNA Blood Mini Kit from Qiagen, see the manual for the procedure.
3. The detection method of gene mutation is operated according to the steps in the technical scheme. The patient was quantified for DNA concentration of 1.9 ng/. mu.L using the Qubit kit. Adding 7 mu L of Panel A/B into a DNA template respectively, preparing a first-step PCR reaction system according to a technical scheme 3.3, performing magnetic bead purification after running the first-step PCR reaction, and selecting 1.0-time (25 mu L) Agencour AMPure XP magnetic beads. After purification, the Qubit kit is used for quantification again, the concentrations of the products of the first step PCR reaction are measured to be respectively Panel A16.5 ng/muL and Panel B13 ng/muL, and 1.8 muL and 2.3 muL are respectively added for carrying out the second step PCR reaction. Selecting i5-primer 4 and i7-primer 7, and preparing a second-step PCR reaction system and an operation program according to the technical scheme 3.6. And (3) purifying the obtained PCR product by using 0.9-fold (22.5 mu L) Agencour AMPure XP magnetic beads, quantifying the PCR product in the second step by using Qubit, wherein the concentrations of the PCR product in the second step are respectively Panel A13 ng/mu L and Panel B25.4 ng/mu L, mixing 7.69 mu L and 3.94 mu L respectively, performing high-throughput sequencing after the steps are completed, and controlling the quality of the sequencing result as shown in FIGS. 7-9, thereby prompting that the sequencing result is qualified. Analysis of the sequencing data revealed that the patient had IKZF1: and c.A476C: p.N159T mutation, wherein the mutation frequency is 36.82%.
4. The example shows that the primer combination and the method provided by the invention can comprehensively and sensitively detect IKZF family gene mutation of a patient with malignant hemopathy, accurately detect the existence of IKZF1 p.N159T mutation in the patient with acute lymphoblastic leukemia, and have high operation feasibility and result reliability.
5. The clinical value is as follows: the patient is philadelphia chromosome negative acute lymphocytic leukemia, the relapse of the patient in the chemotherapy stage is consolidated, the re-induced chemotherapy is not relieved, and the patient dies. The IKZF1 p.N159T mutation is found in the bone marrow specimen of the patient before treatment by applying the invention, which shows that the invention has clinical guidance value for prognosis evaluation and early discovery of high-risk subtypes.
The above discussion and examples show that the method provided by the invention can comprehensively, accurately and rapidly detect whether IKZF family gene (IKZF1-5) mutation, accurate mutation site and frequency exist in different stages of diseases of various patients with malignant hemopathy, make up for the limitation and incompleteness of the existing IKZF gene mutation detection method, fill up the blank of the IKZF whole family gene mutation detection method, and have important clinical practical value and application prospect for guiding clinical typing diagnosis, early screening high-risk subtypes, prognosis evaluation and guiding individual treatment.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above 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 shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
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<213> Artificial Sequence (Artificial Sequence)
<400> 7
gcttggagct tcaaataagt taaga 25
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cacttaccca cgactctgtc actct 25
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ttgatgaagg aacatgcacg tacga 25
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tgaaaccatg ttcagttctt tgttg 25
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ccgttaggaa gtcgaatgcc tccaa 25
<210> 12
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atccagggga cctaccggag tgcgt 25
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgttcctct aaagagcttc gctgt 25
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
catttctact tgcatctaaa acacc 25
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ctcctccttg agcgacagcc cgttg 25
<210> 16
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aatccaaaca caacattgtc tggtc 25
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
agtttctgcc gtatcagcaa gtatt 25
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
accagactac cttaacgatt ttacc 25
<210> 19
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
atcaatttcc agttgtccaa tgaat 25
<210> 20
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ctaaattgag ttgattctga gatgc 25
<210> 21
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
attataagat gctgaaaggc aggca 25
<210> 22
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
aggaagccta aaattcatcc ctgaa 25
<210> 23
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ggtccaatac agaccatgcc gcaga 25
<210> 24
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gagagtgcca ggaaactaaa ggtat 25
<210> 25
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tacaagagaa gtcactgagc tctac 25
<210> 26
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
cccctacctg tcgatctccc taaag 25
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ctgctttctt tccccaaacc gaatc 25
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gcttggggtc ctctttggcg taggc 25
<210> 29
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aaagcaaaac tccttgggaa atggt 25
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gcaaccatga agaacgccag aatca 25
<210> 31
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cgccactgct ttgatgtcaa ctata 25
<210> 32
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ccagtttatt tccttttctc cccat 25
<210> 33
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ggtaacctcc tccgccacat taaac 25
<210> 34
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gagcctgaaa tcccttacag ctatt 25
<210> 35
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ttgtgtgaag ctgaaagtta aatgg 25
<210> 36
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ccggctcctg cccttttcag tctgc 25
<210> 37
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gcagtatttc ttcatgtgca gttct 25
<210> 38
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
caaagctttg acatcctcct tcatg 25
<210> 39
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
agctccaagg taggtgattg cattg 25
<210> 40
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
catgttgaac tacaaaagca gtagg 25
<210> 41
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cttttcttct caaggagttg gtgac 25
<210> 42
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tcagtttacc attcggaagc cggat 25
<210> 43
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
caaaggtgaa attgtgaaca gagag 25
<210> 44
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
agaaaaaggt atatgcatcc cagca 25
<210> 45
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gatgagtagc tgaacagtgg ttttg 25
<210> 46
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
tcactgttct attcgttaga cacct 25
<210> 47
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
cctatttgat tgtctttttg ctgct 25
<210> 48
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gcccccgtgg gaaacaactt tctcg 25
<210> 49
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
agtcgcatga gttcttggta caatt 25
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
ttggacgcga ctgaaccctt taaac 25
<210> 51
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tggtcccggt catcagcccg atgta 25
<210> 52
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gctcttcctg gatcacgtca tgtac 25
<210> 53
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
cccatgacat cccatatgaa tagtg 25
<210> 54
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gcagaaacta ctgcttgggt agagg 25
<210> 55
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
ggtaccattt tatgcttgcg ccttc 25
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gctttttcca gctgtctttc catta 25
<210> 57
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
ctaactgatc cagaaatcat gttca 25
<210> 58
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
ctcaggcttt gacgggatag aatgg 25
<210> 59
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
cagcttcccc ctttgcctct ctcta 25
<210> 60
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
ctgtattgga cccaacgtgc tcatg 25
<210> 61
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ctctgggatt tgttctttca cttgc 25
<210> 62
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gcggtgccat aactacctac agagt 25
<210> 63
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
tacccctact caactgctac tactg 25
<210> 64
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ggagcttcca ggatcccgag aagca 25
<210> 65
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
agcctgtgaa ggccttcaag tgtga 25
<210> 66
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
atgttacact cgaaagggtc acgga 25
<210> 67
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
gatgcttttc ttttccagct cttga 25
<210> 68
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
cttgtgtagc aagcatccta tgtta 25
<210> 69
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
taatggtcgt gtaattagcc ctaga 25
<210> 70
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
aagtaaccaa aagttccaaa ttcca 25
<210> 71
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ttcttctcta cactaagcct aagca 25
<210> 72
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
agcaaaactg agattcagaa gaaac 25
<210> 73
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cctgccttaa atcccaagag gaaac 25
<210> 74
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
ggaaaagctc atgcgattca gctac 25
<210> 75
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
agctttgtat catccaaaat gttga 25
<210> 76
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
tgagacacat aaagttacac tctgg 25
<210> 77
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gggtagcagc ctagaagaac cccta 25
<210> 78
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
tatgcatgcc attgcgtgta taaag 25
<210> 79
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ccagggaagc ttttctcatg aactt 25
<210> 80
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
tctttaatat gccagttgag ggaac 25
<210> 81
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
ggagtaatct caaagctgtc cagca 25
<210> 82
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
actacggaca caaactggaa taagg 25
<210> 83
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
tgagaagtcc tttacaaggc tgtac 25
<210> 84
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
ggtgcagtgg gaaaaggaga atatg 25
<210> 85
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
gaaactgatg ctattggcca aaaac 25
<210> 86
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
cggggtgccc tccgcgagcg gcttg 25
<210> 87
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gcacatgttg cactcaaaag gatca 25
<210> 88
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
tgccagcact gtgatatgta ctttg 25
<210> 89
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
caagaactca tggttgataa ccctt 25
<210> 90
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
tgaattatgt tccttccgct gcagt 25
<210> 91
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
cctttgatgg gaagcttaag tgtcg 25
<210> 92
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
accagagcct ttggacttcg tgaaa 25
<210> 93
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
aggcttggga gtagttactg aattg 25
<210> 94
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
ggtgaattga aggccaaatg caaca 25
<210> 95
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
ccctctggct tacttaccag tgtga 25
<210> 96
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
tagttgcaga agggacattt aaagg 25
<210> 97
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
cccttttaac tccccagtag tacat 25
<210> 98
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
tccaggtcac gtatttcgtc accta 25
<210> 99
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
tgctttctgt gtctgtggag tcctg 25
<210> 100
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
atgtggatag tgaacatgac gtggt 25
<210> 101
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
ggtgatggat gtgtatcggt gtgac 25
<210> 102
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
cagttatcag cagcatgtat cccat 25
<210> 103
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
tattttaacc atccaaacac gttgc 25
<210> 104
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ttctagtgaa cagttgtcac agagc 25
<210> 105
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
tctaatctgt gcatatgttt ctcca 25
<210> 106
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
tttgatagct tcaagtatag tcgtt 25
<210> 107
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ttctgctgtt aagttttccg aaatg 25
<210> 108
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
tgaaaactca taacggtcct ggctt 25
<210> 109
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
ttttcatgac tatcagcagt ttccc 25
<210> 110
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
gggaacttgc cttttcctat taaca 25
<210> 111
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
tacatgcatc ctgcaagatt tatca 25
<210> 112
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
cacccctgaa taaaaaggaa agaga 25
<210> 113
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
ccccacagac ctaacaaatt aaagt 25
<210> 114
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
caaggtctgt gccagtctga tactc 25
<210> 115
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
gactcacact tcttctttct catca 25
<210> 116
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
aacaaaagca tgccttcatc tccta 25
<210> 117
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
tgcaaatgtg tccataaggt attgg 25
<210> 118
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
aaactaaagt gtgatatctg tggga 25
<210> 119
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cttcaaatgc cacctctgca actac 25
<210> 120
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
cacaaatgtg gatattgtgg ccgaa 25
<210> 121
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gccagacctg accggttccg gaggt 25
<210> 122
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
tcatctacct gaccaaccac atcgc 25
<210> 123
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
agcagacact ttattaggga tgacc 25
<210> 124
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
acctgccact ggactatagt tcctt 25
<210> 125
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
ttgccatact ttcataggag tcagt 25
<210> 126
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
cttctgacct gtttgtatgt tgtta 25
<210> 127
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
agaatgattc acattaatgc gcagt 25
<210> 128
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
cccctcactt ccaggaatcc acgct 25
<210> 129
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
aggcaatgga cagtagatat ctcca 25
<210> 130
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
catcaaggtg gagatgtaca gcgat 25
<210> 131
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
cataaatcag ggtttgtgtg cattg 25
<210> 132
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
cagtgtatac ttgctcttgg ctgaa 25
<210> 133
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
tctatatcct tggcctaatg ggaga 25
<210> 134
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
cgccttccac ccaccaattg catct 25
<210> 135
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
aaagcaacca cgaagatcgg gttgc 25
<210> 136
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
atgttttgag agcaatctgt taggc 25
<210> 137
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
acaggaccat gtgattttgc tgata 25
<210> 138
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
ctgtatgagc tcactctctt tctca 25
<210> 139
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
tactattgta tctcaccacc tagga 25
<210> 140
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
ttcctagaca aggaatgctc atttc 25
<210> 141
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
gatgcaggat aatccacaca catcg 25
<210> 142
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
cacacatttc catagcattt ttact 25
<210> 143
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
agcgctag 8
<210> 144
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
gatatcga 8
<210> 145
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
cgcagacg 8
<210> 146
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
tatgagta 8
<210> 147
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
aggtgcgt 8
<210> 148
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
gaacatac 8
<210> 149
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
acatagcg 8
<210> 150
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
gtgcgata 8
<210> 151
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
ccaacaga 8
<210> 152
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
ttggtgag 8
<210> 153
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
ccgcggtt 8
<210> 154
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
ttataacc 8
<210> 155
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
ggacttgg 8
<210> 156
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
aagtccaa 8
<210> 157
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
atccactg 8
<210> 158
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
gcttgtca 8
<210> 159
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
caagctag 8
<210> 160
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
tggatcga 8
<210> 161
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
agttcagg 8
<210> 162
<211> 8
<212> DNA
<213> Artificial Sequence (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.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117604103A (en) * | 2023-12-22 | 2024-02-27 | 上海信诺佰世医学检验有限公司 | Primer and probe composition, kit and application thereof in detection of IKZF1 gene deletion |
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| CN107475398A (en) * | 2017-09-06 | 2017-12-15 | 杭州艾迪康医学检验中心有限公司 | Detect the primer and method of IKZF1 gene mutation typings |
| CN110511989A (en) * | 2019-08-01 | 2019-11-29 | 杭州和壹基因科技有限公司 | A kind of high-flux sequence method and its application of hypertension therapeutic pharmaceutical relevant gene |
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| CN107475398A (en) * | 2017-09-06 | 2017-12-15 | 杭州艾迪康医学检验中心有限公司 | Detect the primer and method of IKZF1 gene mutation typings |
| CN110511989A (en) * | 2019-08-01 | 2019-11-29 | 杭州和壹基因科技有限公司 | A kind of high-flux sequence method and its application of hypertension therapeutic pharmaceutical relevant gene |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117604103A (en) * | 2023-12-22 | 2024-02-27 | 上海信诺佰世医学检验有限公司 | Primer and probe composition, kit and application thereof in detection of IKZF1 gene deletion |
| CN117604103B (en) * | 2023-12-22 | 2024-05-28 | 上海信诺佰世医学检验有限公司 | Primer and probe composition, kit and application thereof in detection of IKZF1 gene deletion |
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