KMT2A fusion gene detection primer probe set, kit and detection method

Abstract

The invention discloses a KMT2A fusion gene detection primer probe group, a kit and a detection method, the primer probe group is specifically designed based on KMT2A gene fusion and 13 common partner genes MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3 and CBL fusion sites, the mutual interference is small, the primer probe group can be used for targeted amplification of a fusion gene specific region in a single-tube mixed solution, the detection sensitivity is high, the detection time is short, the detection time is short, and the detection efficiency is high. According to the present invention, the rapid detection of any one fusion gene type in 67 types can be achieved, and compared with the high-throughput sequencing, the sensitivity is high, the detection rate is high, the single sample detection cost is low, the method is suitable for the clinical auxiliary diagnosis of the large batch of samples, and the guidance is provided for the diagnosis, the treatment and the prognosis of the blood disease patients.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a primer probe set, a kit and a detection method for a KMT2A fusion gene. Background Art

[0002] The human KMT2A gene, whose full name is lysine methyltransferase 2A, also known as the MLL gene, is located at q23.3 on chromosome 11. This gene encodes a transcriptional coactivator that plays an important role in regulating gene expression during early development and hematopoiesis.

[0003] KMT2A gene rearrangement involves multiple partner genes. Its fusion with partner genes transcribes the fusion gene transcript and encodes the corresponding protein, which can cause abnormalities in hematopoietic progenitor cells, abnormal self-renewal and epigenetic regulation disorders, thereby inducing the occurrence of leukemia. It is one of the genes commonly seen in malignant blood diseases.

[0004] The fusion of KMT2A gene and partner gene has a positive rate of about 70% to 80% in acute lymphoblastic leukemia (ALL), about 50% to 60% in acute myeloid leukemia (AML), and about 5% to 10% in mixed phenotype acute leukemia (MPAL).

[0005] KMT2A gene rearrangement is an independent factor for poor prognosis in childhood acute leukemia (AL). Such patients have the characteristics of young onset, high white blood cell count, multi-organ infiltration, difficulty in remission with conventional chemotherapy, high relapse rate after remission, and short survival. The current combination chemotherapy and allogeneic hematopoietic stem cell transplantation have improved the survival rate of such patients, but the overall treatment effect is still not ideal. New targeted drugs and cell immunotherapy are expected to improve the prognosis of children with KMT2A gene rearrangement-positive leukemia.

[0006] At present, the prognosis of most children with positive KMT2A gene rearrangement is poor. In the past, more than 90 partner genes of KMT2A gene rearrangement were detected clinically, which can lead to more than 130 related transcript subtypes of KMT2A gene rearrangement.

[0007] Among the current clinical diagnostic methods for KMT2A gene rearrangement, chromosome karyotype analysis involves rearrangement in the 11q23 region, but it is easily affected by cell culture conditions and the interpretation experience of the experimenter, so there is a possibility of missed detection. Fluorescence in situ hybridization (FISH) technology can detect almost all KMT2A gene rearrangements, with a short cycle and high sensitivity, but due to the limitation of the probe, it cannot detect the partner gene of KMT2A. The commonly used fluorescent quantitative PCR method in clinical practice can detect common KMT2A gene rearrangements, but a single tube reaction solution can detect only a few KMT2A fusion types, and it is difficult to detect such a large number of fusion types using RT-PCR. RNA-based second-generation sequencing can obtain sequencing results at the whole transcriptome level, and can obtain the sequences and fusion breakpoints of all known and unknown partner genes, but it is difficult to promote on a large scale due to factors such as the long experimental process, the cumbersome bioinformatics analysis process, the high requirements for operators and analysts, the high requirements for instruments, the long detection cycle, and the high price of single sample detection. Summary of the invention

[0008] The present invention designs specific primers and probes for the KMT2A fusion gene, screens and obtains a primer and probe set for specific detection, which is used to configure into a single-tube mixed solution for detection, thereby achieving targeted amplification of the specific region of the fusion gene, and solving the defects of existing fusion gene type detection, such as high cost, long cycle, and low efficiency.

[0009] In view of this, the scheme of the present invention is: The first aspect of the present invention is to propose a primer-probe group for detecting a KMT2A fusion gene, comprising an upstream primer group and a probe group designed based on the break site of the KMT2A gene, and a downstream primer group designed based on the fusion exon of a partner gene; the partner genes include MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3, and CBL; the nucleotide sequence of the upstream primer group is shown in SEQ ID NOs: 1-6, the nucleotide sequence of the probe group is shown in SEQ ID NOs: 7-12, and the nucleotide sequence of the downstream primer group is shown in SEQ ID NOs: 13-42.

[0010] Furthermore, the KMT2A fusion gene detection primer probe group also includes an internal reference primer probe group; the internal reference primer probe group includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 43, a downstream primer as shown in SEQ ID NO: 44, and a probe as shown in SEQ ID NO: 45.

[0011] Furthermore, the 5' end of the probe group is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group.

[0012] The second aspect of the present invention is to provide a KMT2A fusion gene detection kit, comprising the primer probe set described in the first aspect above.

[0013] Furthermore, the kit also includes at least one of a positive quality control product, a negative quality control product, a PCR reaction mixture and purified water.

[0014] Preferably, the positive quality control product is a KMT2A fusion gene plasmid mixture, and the KMT2A fusion gene plasmid includes a plasmid formed by the fusion of the KMT2A gene and any one partner gene, and the partner gene is selected from MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3 or CBL.

[0015] Preferably, the negative control product is a KMT2A wild-type gene plasmid.

[0016] Furthermore, the kit also includes a nucleic acid extraction reagent and / or a cDNA synthesis reagent.

[0017] The third aspect of the present invention is to propose the use of the primer probe set described in the first aspect in the preparation of a KMT2A fusion gene detection product.

[0018] Furthermore, the steps of using the detection product for KMT2A fusion gene detection include: S1. Extract RNA from the sample to be tested and reverse transcribe it into cDNA; S2. Using cDNA as a template, PCR amplification is performed using the primer probe set described in the first aspect and the internal reference primer probe set to detect the fusion gene amplification signal and the internal reference gene amplification signal respectively; S3. Using the internal reference gene amplification signal value as a reference, determine the positive fusion gene type based on the fusion gene amplification signal value.

[0019] Furthermore, the fusion gene type is composed of any broken exon of the KMT2A gene fused with any broken exon of a partner gene; wherein: The KMT2A gene broken exons include exons 6-11; the partner gene broken exons are: MLLT3 gene exons 6, 9-10, AFF1 gene exons 5-7, 12, MLLT10 gene exons 3, 5, 8-9, 13-14, MLLT1 gene exons 2, 7, MLLT4 gene exon 2, ELL gene exon 2, EPS15 exons 2, 12, SEPT6 gene exon 2, MLLT11 gene exon 2, MLLT6 gene exons 7, 11, SEPT9 gene exons 2-3, AFF3 gene exons 7, 9, CBL gene exons 12, 16.

[0020] Furthermore, the PCR amplification reaction system is: 10×KMT2A Mix 2.5uL, 2×BR buffer mix 12.5uL, cDNA 10uL, total volume 25uL; Furthermore, the PCR amplification reaction program is: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 5 min, 95°C for 15 s, 60°C for 1 min, for a total of 40 cycles; and collecting amplification signals at 60°C.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The KMT2A fusion gene detection primer probe set provided by the present invention is based on KMT2A gene fusion, and 13 common partner genes MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3, CBL fusion site-specifically designed primer probe sets, which have little interference with each other and can be used for targeted amplification of fusion gene-specific regions in a single tube of mixed solution to meet the needs of efficiently detecting any one of the 67 types of fusion gene types.

[0022] The KMT2A fusion gene detection primer probe set provided by the present invention can be applied to the fluorescence quantitative PCR detection method to realize the rapid detection of any fusion gene type among 67 types. Compared with high-throughput sequencing, it has high sensitivity, high detection rate, low cost for single sample detection, short detection cycle, and low requirements for operating analysts; compared with conventional fluorescence quantitative PCR, it can detect more fusion types; compared with digital PCR, a single tube can detect more fusion gene types, and the operation is simple and low cost. It is suitable for clinical auxiliary diagnosis of large batches of samples, and provides guidance for the diagnosis, treatment and prognosis of patients with hematological diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the primer and probe design region of the present invention.

[0024] Figure 2 This is the positive amplification curve of KMT2A (E9) -MLLT3 (E6), KMT2A (E9) -AFF1 (E6) and KMT2A (E6) -MLLT10 (E13) fusion genes in Example 1 of the present invention.

[0025] Figure 3 This is the positive amplification curve of KMT2A(E9)-MLLT1(E2), KMT2A(E9)-MLLT4(E2) and KMT2A(E9)-ELL(E2) fusion genes in Example 1 of the present invention.

[0026] Figure 4 This is the positive amplification curve of KMT2A(E9)-EPS15(E2), KMT2A(E10)-SEPT6(E2) and KMT2A(E9)-ELL(E2) fusion genes in Example 1 of the present invention.

[0027] Figure 5 This is the positive amplification curve of KMT2A (E8) -MLLT6 (E7), KMT2A (E7) -SEPT9 (E3) and KMT2A (E10) -AFF3 (E7) fusion genes in Example 1 of the present invention.

[0028] Figure 6 This is the positive amplification curve of the KMT2A (E9) -CBL (E12) fusion gene in Example 1 of the present invention.

[0029] Figure 7 This is the negative amplification curve of the KMT2A target fusion gene in Example 1 of the present invention.

[0030] Figure 8 This is the negative amplification curve of the wild type KMT2A in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In one embodiment, a design process of a primer probe set for detecting a KMTA fusion gene and a method for detecting a KMTA fusion gene are proposed, as follows: 1. Selection process of target KMTA fusion gene 1) By searching the literature and finding the consensus of relevant experts in the field of leukemia, the latest KMT2A-related literature and reviews were summarized, the fusion types with the highest fusion probability were selected, and the target KMTA fusion gene list was determined.

[0033] Finally, 67 fusion gene types of 13 common partner genes are: KMT2A(E8)-MLLT3(E6), KMT2A(E8)-MLLT3(E9), KMT2A(E8)-MLLT3(E10), KMT2A(E9)-MLLT3(E6), KMT2A(E10)-MLLT 3(E6),KMT2A(E8)-AFF1(E5),KMT2A(E8)-AFF1(E6),KMT2A(E8)-AFF1(E7),KMT2A(E9)-AFF1(E5),KMT2A(E9)-AFF1(E6),KMT2A(E9)-AFF1(E7),KMT 2A(E9)-AFF1(E12),KMT2A(E10)-AFF1(E5),KMT2A(E10)-AFF1(E6),KMT2A(E10)-AFF1(E7),KMT2A(E10)-AFF1(E12),KMT2A(E11)-AFF1(E5),KMT2A (E11)-AFF1(E6),KMT2A(E11)-AFF1(E7),KMT2A(E6)-MLLT10(E13),KMT2A(E7)-MLLT10(E13),KMT2A(E8)-MLLT10(E3),KMT2A(E8)-MLLT10(E5),KM T2A(E8)-MLLT10(E8),KMT2A(E8)-MLLT10(E9),KMT2A(E8)-MLLT10(E14),KMT2A(E9)-MLLT10(E3),KMT2A(E9)-MLLT10(E5),KMT2A(E9)-MLLT10(E8) ), KMT2A(E9)-MLLT10(E9), KMT2A(E9)-MLLT10(E14), KMT2A(E10)-MLLT10(E8), KMT2A(E10)-MLLT10(E9), KMT2A(E10)-MLLT10(E14), KMT2A(E8)-M LLT1(E2),KMT2A(E8)-MLLT1(E7),KMT2A(E9)-MLLT1(E2),KMT2A(E9)-MLLT1(E7),KMT2A(E10)-MLLT1(E2),KMT2A(E10)-MLLT1(E7),KMT2A(E8)-ML LT4(E2),KMT2A(E9)-MLLT4(E2),KMT2A(E10)-MLLT4(E2),KMT2A(E8)-ELL(E2),KMT2A(E9)-ELL(E2),KMT2A(E10)-ELL(E2),KMT2A(E11)-ELL(E2),KMT2A(E8)-EPS15(E2), KMT2A(E8)-EPS15(E12), KMT2A(E9)-EPS15(E2), KMT2A(10)-EPS15(E2), KMT2A(E8)-SEPT6(E2), KMT2A(E9)-SEPT6(E2), KMT2A(E10)-SEPT6(E2), KMT2A(E8)-MLLT11(E2), KMT2A(E9)-MLLT11(E2), KMT2A(E6)-MLLT6(E1 1), KMT2A(E7)-MLLT6(E11), KMT2A(E8)-MLLT6(E7), KMT2A(E6)-SEPT9(E3), KMT2A(E7)-SEPT9(E2), KMT2A(E7)-SEPT9(E 3), KMT2A(E10)-AFF3(E7), KMT2A(E10)-AFF3(E9), KMT2A(E7)-CBL(E16), KMT2A(E8)-CBL(E12), KMT2A(E9)-CBL(E12);, 2) By summarizing the above fusion types, analyzing the KMT2A gene break exons and the 13 partner gene break exons, the target exons summarized in Table 1 were obtained.

[0034] Table 1:

[0035] 2. Design of specific primers, probes and kits 1) Search the relevant leukemia database, determine the gene sequences of 67 fusion gene types through software, design specific primer pairs and specific probes respectively, and try to unify the annealing temperature in the primer and probe design stage. The break sites of KMT2A gene often occur in exon 6, exon 7, exon 8, exon 9, exon 10, and exon 11, which can fuse with other genes. Select KMT2A gene to set a common forward primer and probe on different exons, and then set the exon-specific reverse primers of MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3, and CBL gene fusion sites respectively. Figure 1 ; 2) Select appropriate primers. During the primer design phase, use software to exclude non-specific amplification. For experimental screening, dilute the primers to a working solution concentration of 10uM, extract total RNA from the sample and reverse it into cDNA, amplify with primer pairs, perform agarose gel electrophoresis on the amplified product, verify the primers, remove primers that may cause primer dimers or have low amplification efficiency, and redesign and synthesize primers; 3) Confirmation of primer and probe specificity. Synthesize the corresponding plasmid for the KMTA fusion gene. Quantify the plasmid using digital PCR, prepare quality control products, and test the specificity of primers and probes; 4) Internal reference design. Use the ABL1 gene as the internal reference to design forward and reverse primers and probes; 5) Optimize PCR reaction conditions. Select the appropriate annealing temperature according to the characteristics of the primers, and test different annealing temperatures, annealing times, and PCR amplification cycle numbers; 6) The target detection primer probe set is obtained through screening through the above development process, as shown in Table 2. All primer and probe dry powders are dissolved into high-concentration primers and probes, and a primer probe pool is prepared to form a single-tube mixed solution.

[0036] Table 2:

[0037] 7) Set up quality control. The negative quality control is the KMT2A wild-type gene plasmid, and the positive quality control is the KMT2A fusion gene plasmid mixture.

[0038] 3. Detection of KMT2A fusion gene The KMT2A fusion gene test procedure is as follows: 1) Extract total RNA from the sample to be tested, with RNA concentration ≥ 50ng / uL; 2) Total RNA was reverse transcribed into cDNA. The RNA was reverse transcribed into cDNA using AG reverse transcription reagent. The reaction system was 5×RTase Reaction Buffer Mix II 4μl, EvoM-MLV RTase Enzyme Mix 1μl, OligodT (18T) Primer (50μM) 1μl, Random 6mers Primer (100μM) 1μl, Total RNA 2000ng, supplemented with RNase Free H 2 The reaction conditions of reverse transcription are: 37℃ 15min 1Cycle; 85℃ 5s 1Cycle, and the product is cooled to 12℃ after the reaction. 3) Fluorescence PCR detection, using cDNA as a template, using primer probe pool for PCR amplification, and detecting amplification signals. The reaction system for fluorescence PCR amplification is: 10×KMT2A Mix 2.5uL, 2×BR buffer mix 12.5uL, cDNA 10uL, total volume 25uL. The amplification reaction program of this kit is: 50℃ 2min 1 cycle, 95℃ 5min 1 cycle, 95℃ 15s, 60℃ 1min, a total of 40 cycles; collect fluorescence at 60℃; 4) Judgment result: When the internal reference ABL1 gene amplification is normal and the CT value is <30, the fusion gene CT value <35 is judged as positive; 5) If the test result of fluorescence quantitative PCR is judged to be positive, it is necessary to identify which of the 13 fusion genes, KMT2A-MLLT3, KMT2A-AFF1, KMT2A-MLLT10, KMT2A-MLLT1, KMT2A-MLLT4, KMT2A-ELL, KMT2A-EPS15, KMT2A-SEPT6, KMT2A-MLLT11, KMT2A-MLLT6, KMT2A-SEPT9, KMT2A-AFF3, and KMT2A-CBL, is positive. The corresponding gene mix can be used for detection.

[0039] The primer-probe set described in Table 2 can be used to accurately detect any one of the 67 types of 13 fusion genes in combination. The specific primer-probe combination is shown in Table 3.

[0040] Table 3: Example 1

[0041] Multiple samples were tested and 15 of them were selected for display.

[0042] Specimen information: Bone marrow specimens from patients with positive KMT2A(E9)-MLLT3(E6) fusion gene, bone marrow specimens from patients with positive KMT2A(E9)-AFF1(E6) fusion gene, bone marrow specimens from patients with positive KMT2A(E6)-MLLT10(E13) fusion gene, bone marrow specimens from patients with positive KMT2A(E9)-MLLT1(E2) fusion gene, bone marrow specimens from patients with positive KMT2A(E9)-MLLT4(E2) fusion gene, bone marrow specimens from patients with positive KMT2A(E9)-ELL(E2) fusion gene, bone marrow specimens from patients with positive KMT2A(E9)-EPS15(E2), and bone marrow specimens from patients with positive KMT2A(E10 )-SEPT6(E2) fusion gene positive patient bone marrow specimens, KMT2A(E9)-ELL(E2) fusion gene positive patient bone marrow specimens, KMT2A(E8)-MLLT6(E7) fusion gene positive patient bone marrow specimens, KMT2A(E7)-SEPT9(E3) fusion gene positive patient bone marrow specimens, KMT2A(E10)-AFF3(E7) fusion gene positive patient bone marrow specimens, KMT2A(E9)-CBL(E12) fusion gene positive patient bone marrow specimens, KMT2A-ACIN1 fusion gene positive patient bone marrow specimens. These samples are not within the detection range of this kit. There are a total of 15 KMT2A wild-type bone marrow specimens.

[0043] Experimental steps: 1. Sample nucleic acid extraction 1.1 Lysis: 1 mL sample + 5 mL red blood cell lysis buffer were added to a 15 mL centrifuge tube. The sample was mixed by inversion. Lysis was performed at 4°C for 10 min, and then centrifuged at 4°C, 450 g for 10 min. The supernatant was discarded.

[0044] 1.2 Add 1 mL Trizol to the leukocyte pellet, vortex to dissolve, transfer to a 1.5 mL centrifuge tube, and incubate at room temperature for 5 min to completely dissociate the nuclear protein.

[0045] 1.3 Add 200 μL of chloroform, vortex mix for 15 seconds, incubate at room temperature for 2-3 minutes. Centrifuge at 16000g for 15 minutes at 4°C, and the mixture is separated into a red phenol-chloroform phase (protein), an intermediate phase (DNA), and an upper colorless aqueous phase (RNA).

[0046] 1.4 Transfer the aqueous phase containing RNA to a new 1.5mL EP tube, tilt the tube 45° and remove the solution, avoiding aspirating any organic phase or other phase layers, and aspirate 400uL of supernatant.

[0047] 1.5 Add 500 μL of isopropanol to the sample, vortex briefly to mix, and incubate at -20°C for 10 min.

[0048] 1.6 Centrifuge at 16000g for 10 min at 4°C. Total RNA will form a gelatinous precipitate at the bottom.

[0049] 1.7 Carefully discard the supernatant and add 1 mL of 75% ethanol solution to resuspend the precipitate. Vortex the sample briefly, then centrifuge at 12,000 g for 5 min at 4°C and discard the supernatant.

[0050] 1.8 Dry the RNA precipitate for 5-10 minutes. Use 50 μL of RNase-free water to carefully pipette up and down to resuspend the precipitate and mix well. This is the extracted RNA.

[0051] 1.9 Use NanoDrop One to measure and record the concentration of RNA solution and A260 / A280, A260 / A230 purity. RNA concentration ≥ 50ng / uL.

[0052] 2. Nucleic acid reverse transcription Total RNA was reverse transcribed into cDNA. AG's reverse transcription reagent was used to reverse transcribe RNA into cDNA. The reaction system was 5×RTase Reaction Buffer Mix II 4μl, EvoM-MLV RTase Enzyme Mix 1μl, OligodT (18T) Primer (50μM) 1μl, Random 6mers Primer (100μM) 1μl, Total RNA 2000ng, and RNaseFree H2O was added to 20uL of the system. The reaction conditions for reverse transcription were: 37℃ 15min 1Cycle; 85℃ 5s 1Cycle, and the product was cooled to 12℃ after the reaction.

[0053] 3. Fluorescence PCR Detection Using cDNA as template, PCR amplification was performed with primer probe pool to detect amplification signals. The reaction system for fluorescence PCR amplification was: 10×KMT2A Mix 2.5uL, 2×BR buffer mix 12.5uL, cDNA 10uL, total volume 25uL. The amplification reaction program of the kit was: 1 cycle at 50℃ 2min, 1 cycle at 95℃ 5min, 1 cycle at 95℃ 15s, 60℃ 1min, for a total of 40 cycles; fluorescence was collected at 60℃.

[0054] 4. Judge the results When the internal reference ABL1 gene amplification is normal and the CT value is <30, the CT value of each fusion gene <35 is judged as positive. Figure 2-6 As shown in the figure, the internal reference ABL1 gene and the target detection gene of the sample produce the same amplification trend curve, and the CT value of the internal reference ABL1 gene is <30, and the result is judged to be positive, which is consistent with the specimen information. Figure 7-8 Only the internal reference gene ABL1 of the sample had a typical S-shaped amplification curve and a CT value <30. The target detection gene did not start to amplify, and the result was judged to be negative, which was consistent with the specimen information. Figure 7 The KMT2A-ACIN1 fusion is not within the detection range of this kit, and the result is negative, with good specificity. Example 2

[0055] In order to verify the accuracy of the sample test results provided by this kit, the above 15 samples were tested by NGS and fluorescent quantitative PCR at the same time, and the differences between the two results were compared. The results are shown in Table 4.

[0056] Table 4: Comparison of fluorescence PCR detection results and high-throughput sequencing (NGS) results

[0057] The results showed that the results of fluorescence PCR detection were consistent with the results of KMT2A fusion gene detected by high-throughput sequencing.

[0058] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A primer probe set for detecting KMT2A fusion gene, characterized in that: It includes an upstream primer set and a probe set designed based on the break site of the KMT2A gene, and a downstream primer set designed based on the fusion exon of the partner gene; the partner genes include MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3, and CBL; the nucleotide sequence of the upstream primer set is shown in SEQ ID NOs: 1-6, the nucleotide sequence of the probe set is shown in SEQ ID NOs: 7-12, and the nucleotide sequence of the downstream primer set is shown in SEQ ID NOs: 13-42.

2. The primer probe set according to claim 1, characterized in that: It also includes an internal reference primer probe set; the internal reference primer probe set includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 43, a downstream primer as shown in SEQ ID NO: 44, and a probe as shown in SEQ ID NO:

45.

3. The primer probe set according to claim 1, characterized in that: The 5' end of the probe group is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group.

4. A KMT2A fusion gene detection kit, characterized in that: A primer probe set comprising any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes at least one of a positive quality control product, a negative quality control product, a PCR reaction mixture and purified water.

6. The kit according to claim 5, characterized in that The positive quality control product is a KMT2A fusion gene plasmid mixture, wherein the KMT2A fusion gene plasmid includes a plasmid formed by fusion of the KMT2A gene with any one of the partner genes, wherein the partner gene is selected from MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3 or CBL; And / or, the negative control product is a KMT2A wild-type gene plasmid.

7. The kit according to claim 4, characterized in that The kit also includes a nucleic acid extraction reagent and / or a cDNA synthesis reagent.

8. Use of the primer probe set according to any one of claims 1 to 3 in preparing a KMT2A fusion gene detection product.

9. The use according to claim 8, characterized in that: The steps of using the detection product for KMT2A fusion gene detection include: S1. Extract RNA from the sample to be tested and reverse transcribe it into cDNA; S2. Using cDNA as a template, using the primer probe set and the internal reference primer probe set to perform PCR amplification, and detecting the fusion gene amplification signal and the internal reference gene amplification signal respectively; S3. Using the internal reference gene amplification signal value as a reference, determine the positive fusion gene type based on the fusion gene amplification signal value.

10. The use according to claim 9, characterized in that: The fusion gene type is composed of any broken exon of the KMT2A gene fused with any broken exon of a partner gene; wherein: The KMT2A gene broken exons include exons 6-11; the partner gene broken exons are: MLLT3 gene exons 6, 9-10, AFF1 gene exons 5-7, 12, MLLT10 gene exons 3, 5, 8-9, 13-14, MLLT1 gene exons 2, 7, MLLT4 gene exon 2, ELL gene exon 2, EPS15 exons 2, 12, SEPT6 gene exon 2, MLLT11 gene exon 2, MLLT6 gene exons 7, 11, SEPT9 gene exons 2-3, AFF3 gene exons 7, 9, CBL gene exons 12, 16; And / or, the PCR amplification reaction system is: 10×KMT2A Mix 2.5uL, 2×BR buffer mix 12.5uL, cDNA 10uL, total volume 25uL; And / or, the PCR amplification reaction program is: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 5 min, 95°C for 15 s, 60°C for 1 min, for a total of 40 cycles; and collecting amplification signals at 60°C.

Images