KMT2A Fusion Gene Detection Primer and Probe Set, Kit and Detection Method

Abstract

The present invention discloses a primer-probe set, a kit and a detection method for detecting KMT2A fusion genes. The primer-probe set is specifically designed based on the KMT2A gene fusion and the fusion sites of 13 common partner genes, namely MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3, and CBL. It has little interference with each other and can be used to target-amplify specific regions of fusion genes in a single-tube mixture. It can achieve rapid detection of any one of 67 types of fusion gene types. Compared with high-throughput sequencing, it has high sensitivity, high detection rate, and low single-sample detection cost. It is suitable for clinical auxiliary diagnosis of a large number of samples, and provides guidance for the diagnosis, treatment and prognosis of patients with hematological diseases.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic technologies, and specifically to a primer-probe set, a kit and a detection method for detecting KMT2A fusion genes. Background Art

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

[0003] The rearrangement of the KMT2A gene involves multiple partner genes. Its fusion with partner genes can transcribe fusion gene transcripts and encode corresponding proteins, which can cause abnormalities in hematopoietic progenitor cells, abnormal self-renewal and epigenetic regulation disorders, thus inducing the occurrence of leukemia. It is one of the genes commonly present in malignant blood diseases.

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

[0005] The rearrangement of the KMT2A gene is an independent factor for poor prognosis in children with acute leukemia (AL). Such patients have characteristics such as a younger onset age, high white blood cell count, multi-organ infiltration, difficulty in remission with conventional chemotherapy, easy relapse after remission, and short survival period. Current combined 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 cellular immunotherapy are expected to improve the prognosis of children with KMT2A gene rearrangement-positive leukemia.

[0006] Currently, the prognosis of the vast majority of children with KMT2A gene rearrangement-positive is poor. In the past, there were more than 90 partner genes detected for KMT2A gene rearrangement clinically, which could lead to more than 130 related transcript subtypes of KMT2A gene rearrangement.

[0007] Among the current clinical diagnostic methods for KMT2A gene rearrangement, chromosomal karyotype analysis detects rearrangements in the 11q23 region, but it is susceptible to cell culture conditions and the interpretation experience of laboratory personnel, 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. However, due to the limitation of probes, it cannot detect the partner genes of KMT2A. The commonly used fluorescence quantitative PCR method in clinics can detect common KMT2A gene rearrangements, but the number of KMT2A fusion types that can be detected in a single-tube reaction solution is small, and it is difficult to detect such a large number of fusion types by RT-PCR. RNA-based next-generation sequencing can obtain sequencing results at the whole-transcriptome level, and can obtain information on the sequences and fusion breakpoints of all known and unknown partner genes. However, due to factors such as a long experimental process, a cumbersome bioinformatics analysis process, high requirements for operators and analysts, high requirements for instruments, a long detection cycle, and high cost for single-sample detection, it is difficult to be widely promoted on a large scale. Summary of the Invention

[0008] Through the design of specific primers and probes for the KMT2A fusion gene, the present invention screens and obtains a primer-probe group for specific detection, which is used to configure a single-tube mixture for detection, so as to achieve targeted amplification of specific regions of the fusion gene, and solve the defects of high cost, long cycle, and low efficiency in the detection of existing fusion gene types.

[0009] In view of this, the solution of the present invention is as follows:

[0010] The first aspect of the present invention is to provide a primer-probe group for detecting KMT2A fusion gene, which includes an upstream primer group, a probe group designed based on the breakage site of the KMT2A gene, and a downstream primer group 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, CBL; the nucleotide sequences of the upstream primer group are shown in SEQ ID NO: 1-6, the nucleotide sequences of the probe group are shown in SEQ ID NO: 7-12, and the nucleotide sequences of the downstream primer group are shown in SEQ ID NO: 13-42.

[0011] Furthermore, the primer-probe group for detecting KMT2A fusion gene further includes an internal reference primer-probe group; the internal reference primer-probe group includes an upstream primer with a nucleotide sequence shown in SEQ ID NO: 43, a downstream primer with a nucleotide sequence shown in SEQ ID NO: 44, and a probe with a nucleotide sequence shown in SEQ ID NO: 45.

[0012] Furthermore, the 5'-ends of the probe sets are all labeled with fluorescent groups, and the 3'-ends are all labeled with fluorescence quenching groups.

[0013] The second aspect of the present invention lies in providing a kit for detecting KMT2A fusion genes, which comprises the primer-probe set described in the first aspect above.

[0014] Furthermore, the kit further comprises at least one of a positive control product, a negative control product, a PCR reaction mixture, and purified water.

[0015] Preferably, the positive control product is a plasmid mixture of KMT2A fusion genes, and the plasmid of KMT2A fusion genes comprises a plasmid after fusion of the KMT2A gene with any one of partner genes, and the partner genes are selected from MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3 or CBL.

[0016] Preferably, the negative control product is a plasmid of wild-type KMT2A gene.

[0017] Furthermore, the kit further comprises a nucleic acid extraction reagent and / or a cDNA synthesis reagent.

[0018] The third aspect of the present invention lies in providing the application of the primer-probe set described in the first aspect above in the preparation of a product for detecting KMT2A fusion genes.

[0019] Furthermore, the steps of the detection product for detecting KMT2A fusion genes include:

[0020] S1. Extract RNA from the sample to be detected and reverse transcribe it into cDNA;

[0021] S2. Using the cDNA as a template, perform PCR amplification with the primer-probe set described in the first aspect and an internal reference primer-probe set to detect the amplification signals of the fusion gene and the internal reference gene respectively;

[0022] S3. Taking the amplification signal value of the internal reference gene as a reference, determine the type of the fusion gene judged to be positive according to the amplification signal value of the fusion gene.

[0023] Furthermore, the fusion gene type is composed of the fusion of any one of the broken exons of the KMT2A gene and any one of the broken exons of the partner gene; wherein:

[0024] The KMT2A gene break exons include exons 6-11; the partner gene break 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.

[0025] Further, the PCR amplification reaction system is: 10×KMT2A Mix 2.5 uL, 2×BR buffer mix 12.5 uL, cDNA 10 uL, with a total volume of 25 uL;

[0026] Further, 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; the amplification signal is collected at 60°C.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The KMT2A fusion gene detection primer-probe set provided by the present invention is based on the KMT2A gene fusion and the fusion sites of 13 common partner genes, namely MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3, and CBL, to specifically design the primer-probe set. The interference between them is small, and it can be used to target-amplify the specific region of the fusion gene in a single-tube mixture to meet the need for efficient detection of any one of the 67 types of fusion gene types.

[0029] The KMT2A fusion gene detection primer-probe set provided by the present invention can be applied to the fluorescence quantitative PCR detection method to achieve rapid detection of any one of the 67 types of fusion gene types. Compared with high-throughput sequencing, it has high sensitivity, high detection rate, low single-sample detection cost, short detection cycle, and low requirements for operators; compared with conventional fluorescence quantitative PCR, it can detect more fusion types; compared with digital PCR, it can detect more fusion gene types in a single tube, with simple operation and low cost. It is suitable for clinical auxiliary diagnosis of a large number of samples and provides guidance for the diagnosis, treatment, and prognosis of patients with hematological diseases. Description of the Drawings

[0030] Figure 1 It is a schematic diagram of the primer and probe design regions of the present invention.

[0031] Figure 2 These are the positive amplification curves of the KMT2A(E9)-MLLT3(E6), KMT2A(E9)-AFF1(E6), and KMT2A(E6)-MLLT10(E13) fusion genes in Example 1 of the present invention.

[0032] Figure 3 These are the positive amplification curves of the KMT2A(E9)-MLLT1(E2), KMT2A(E9)-MLLT4(E2), and KMT2A(E9)-ELL(E2) fusion genes in Example 1 of the present invention.

[0033] Figure 4 These are the positive amplification curves of the KMT2A(E9)-EPS15(E2), KMT2A(E10)-SEPT6(E2), and KMT2A(E9)-ELL(E2) fusion genes in Example 1 of the present invention.

[0034] Figure 5 These are the positive amplification curves of the KMT2A(E8)-MLLT6(E7), KMT2A(E7)-SEPT9(E3), and KMT2A(E10)-AFF3(E7) fusion genes in Example 1 of the present invention.

[0035] Figure 6 These are the positive amplification curves of the KMT2A(E9)-CBL(E12) fusion gene in Example 1 of the present invention.

[0036] Figure 7 These are the negative amplification curves of the KMT2A target fusion gene in Example 1 of the present invention.

[0037] Figure 8 These are the negative amplification curves of the wild-type KMT2A in Example 1 of the present invention. Detailed implementation manners

[0038] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0039] In one embodiment, the design process of the primer-probe group for detecting the KMTA fusion gene and the method for detecting the KMTA fusion gene are proposed as follows:

[0040] 1 Selection process of the target KMTA fusion gene

[0041] 1) By searching the literature and relevant expert consensus in the field of leukemia, summarizing the latest KMT2A-related literature and reviews, selecting the fusion types with higher fusion probabilities, and determining the target KMTA fusion gene list.

[0042] A total of 67 fusion gene types of 13 common partner genes were finally determined, such as: KMT2A(E8)-MLLT3(E6), KMT2A(E8)-MLLT3(E9), KMT2A(E8)-MLLT3(E10), KMT2A(E9)-MLLT3(E6), KMT2A(E10)-MLLT3(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), KMT2A(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), KMT2A(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)-MLLT1(E2), KMT2A(E8)-MLLT1(E7), KMT2A(E9)-MLLT1(E2), KMT2A(E9)-MLLT1(E7), KMT2A(E10)-MLLT1(E2), KMT2A(E10)-MLLT1(E7), KMT2A(E8)-MLLT4(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(E11), KMT2A(E7)-MLLT6(E11), KMT2A(E8)-MLLT6(E7), KMT2A(E6)-SEPT9(E3), KMT2A(E7)-SEPT9(E2), KMT2A(E7)-SEPT9(E3), KMT2A(E10)-AFF3(E7), KMT2A(E10)-AFF3(E9), KMT2A(E7)-CBL(E16), KMT2A(E8)-CBL(E12), KMT2A(E9)-CBL(E12);,

[0043] 2) By summarizing the above fusion types, analyze the KMT2A gene break exons and the break exons of 13 partner genes to obtain the target exons summarized in Table 1.

[0044] Table 1:

[0045]

[0046] 2. Design of specific primers, probes and kits

[0047] 1) Retrieve relevant leukemia databases, determine the gene sequences of 67 fusion gene types through software, and design specific primer pairs and specific probes respectively. In the primer and probe design stage, try to unify the annealing temperature. The frequently occurring break sites of the KMT2A gene are in exons 6, 7, 8, 9, 10, and 11, which can fuse with other genes. Set a common forward primer and probe for the KMT2A gene on different exons, and then set specific reverse primers for the fusion sites of the MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3, and CBL genes as shown in Figure 1 ;

[0048] 2) Select appropriate primers. During the primer design stage, use software to exclude non-specific amplification. Through experimental screening, dilute the primers to the working solution concentration of 10 uM, reverse transcribe the total RNA extracted from the sample into cDNA, amplify with primer pairs, perform agarose gel electrophoresis on the amplification products, conduct primer verification, remove the primers that may cause primer dimers or have low amplification efficiency, and redesign and synthesize the primers;

[0049] 3) Confirm the specificity of primers and probes. Synthesize the corresponding plasmid for the KMTA fusion gene. Quantify the plasmid by digital PCR method, prepare the quality control product, and detect the specificity of primers and probes;

[0050] 4) Design internal reference. Design forward and reverse primers and probes using the ABL1 gene as the internal reference;

[0051] 5) Optimize the PCR reaction conditions. According to the primer characteristics, select an appropriate annealing temperature, and test different annealing temperatures, annealing times, and PCR amplification cycle numbers;

[0052] 6) Through the above development process, screen and obtain the target detection primer-probe set as shown in Table 2. Dissolve all the primer and probe dry powders into high-concentration primers and probes, and prepare the primer-probe pool, which is a single-tube mixture.

[0053] Table 2:

[0054]

[0055] 7) Set quality control. The negative quality control product is the KMT2A wild-type gene plasmid, and the positive quality control product is the KMT2A fusion gene plasmid mixture.

[0056] 3. Detection of KMT2A fusion gene

[0057] The operation method for detecting the KMT2A fusion gene is as follows:

[0058] 1) Extract the total RNA from the sample to be tested, with the RNA concentration ≥ 50 ng / μL;

[0059] 2) Reverse transcribe total RNA into cDNA. Use the reverse transcription reagent from AG to reverse transcribe RNA into cDNA. The reaction system is as follows: 4 μl of 5×RTase Reaction Buffer Mix II, 1 μl of EvoM-MLV RTase Enzyme Mix, 1 μl of OligodT(18T) Primer (50 μM), 1 μl of Random 6mers Primer (100 μM), 2000 ng of Total RNA, and supplement RNase Free H2O to a total volume of 20 μl. The reaction conditions for reverse transcription are: 37°C for 15 min for 1 cycle; 85°C for 5 s for 1 cycle. After the reaction, cool the product to 12°C;

[0060] 3) Fluorescent PCR detection. Using cDNA as a template, perform PCR amplification with the primer-probe pool and detect the amplification signal. The reaction system for fluorescent PCR amplification is: 2.5 μl of 10×KMT2A Mix, 12.5 μl of 2×BR buffer mix, 10 μl of cDNA, with a total volume of 25 μl. The amplification reaction program of this kit is: 50°C for 2 min for 1 cycle, 95°C for 5 min for 1 cycle, 95°C for 15 s, 60°C for 1 min, for a total of 40 cycles; collect fluorescence at 60°C;

[0061] 4) Result judgment: When the internal reference ABL1 gene is amplified normally and the CT value < 30, a CT value < 35 for each fusion gene is determined as positive;

[0062] 5) If the detection result in fluorescent quantitative PCR is determined as positive, it is necessary to identify which one of the 13 fusion genes, namely KMT2A-MLLT3, KMT2A-AFF1, KMT2A-MLLT10, KMT2A-MLLT1, KMT2A-MLLT4, KMT2A-ELL, KMT2A-EPS15, KMT2A-SEPT6, KMT2A-MLLT11, KMT2A-MLLT6, KMT2A-SEPT9, KMT2A-AFF3, KMT2A-CBL, is positive, and the corresponding gene mix can be used for detection.

[0063] The primer-probe sets 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 methods are shown in Table 3.

[0064] Table 3:

[0065] Example 1

[0066] Detect multiple samples and select 15 of them for display.

[0067] Specimen information: Bone marrow specimens from patients positive for the KMT2A(E9)-MLLT3(E6) fusion gene, bone marrow specimens from patients positive for the KMT2A(E9)-AFF1(E6) fusion gene, bone marrow specimens from patients positive for the KMT2A(E6)-MLLT10(E13) fusion gene, bone marrow specimens from patients positive for the KMT2A(E9)-MLLT1(E2) fusion gene, bone marrow specimens from patients positive for the KMT2A(E9)-MLLT4(E2) fusion gene, bone marrow specimens from patients positive for the KMT2A(E9)-ELL(E2) fusion gene, bone marrow specimens from patients positive for the KMT2A(E9)-EPS15(E2) fusion gene, bone marrow specimens from patients positive for the KMT2A(E10)-SEPT6(E2) fusion gene, bone marrow specimens from patients positive for the KMT2A(E9)-ELL(E2) fusion gene, bone marrow specimens from patients positive for the KMT2A(E8)-MLLT6(E7) fusion gene, bone marrow specimens from patients positive for the KMT2A(E7)-SEPT9(E3) fusion gene, bone marrow specimens from patients positive for the KMT2A(E10)-AFF3(E7) fusion gene, bone marrow specimens from patients positive for the KMT2A(E9)-CBL(E12) fusion gene, bone marrow specimens from patients positive for the KMT2A-ACIN1 fusion gene, which is not within the detection range of this kit, and 15 bone marrow specimens of wild-type KMT2A in total.

[0068] Experimental procedures:

[0069] 1. Nucleic acid extraction from samples

[0070] 1.1 Lysis: Add to a 15 mL centrifuge tube at a ratio of 1 mL of sample + 5 mL of red blood cell lysis buffer. Invert the sample to mix well, lyse at 4°C for 10 min, then centrifuge at 4°C and 450 g for 10 min, and pour off the supernatant.

[0071] 1.2 Add 1 mL of Trizol to the white blood cell pellet, vortex to dissolve, and transfer to a 1.5 mL centrifuge tube. Incubate at room temperature for 5 min to completely dissociate the nuclear proteins.

[0072] 1.3 Add 200 μL of chloroform, vortex for 15 s, and incubate at room temperature for 2 - 3 min. Centrifuge at 4°C and 16000 g for 15 min. The mixture separates into a lower red phenol-chloroform (protein), an intermediate phase (DNA), and an upper colorless aqueous phase (RNA).

[0073] 1.4 Transfer the aqueous phase containing RNA to a new 1.5 mL Eppendorf tube. Tilt the tube at a 45° angle and remove the solution, avoiding aspirating any organic phase or other phase layers, and aspirate 400 μL of the supernatant.

[0074] 1. Add 500 μL of isopropanol to the sample, vortex briefly to mix evenly, and incubate at -20 °C for 10 min.

[0075] 1.6 Centrifuge at 16000 g for 10 min at 4 °C. Total RNA will form a gelatinous precipitate at the bottom.

[0076] 1.7 Carefully aspirate and discard the supernatant, add 1 mL of 75% ethanol solution to resuspend the precipitate. Vortex the sample briefly, then centrifuge at 12000 g for 5 min at 4 °C, and discard the supernatant.

[0077] 1.8 Dry the RNA precipitate for 5 - 10 min. Resuspend the precipitate by carefully pipetting up and down with 50 μL of RNase-free water to obtain the extracted RNA.

[0078] 1.9 Use NanoDrop One to measure and record the concentration of the RNA solution, as well as the purity of A260 / A280 and A260 / A230. The RNA concentration ≥ 50 ng / μL.

[0079] 2. Nucleic acid reverse transcription

[0080] Reverse transcribe the total RNA into cDNA. Use the reverse transcription reagent from AG to reverse transcribe RNA into cDNA. The reaction system is 4 μL of 5×RTase Reaction Buffer Mix II, 1 μL of EvoM-MLV RTase Enzyme Mix, 1 μL of OligodT(18T)Primer(50 μM), 1 μL of Random 6mers Primer(100 μM), 2000 ng of Total RNA, and supplement RNase-Free H2O to a 20 μL system. The reaction conditions for reverse transcription are: 37 °C for 15 min for 1 cycle; 85 °C for 5 s for 1 cycle. After the reaction, cool the product to 12 °C.

[0081] 3. Fluorescent PCR detection

[0082] Use the cDNA as a template and perform PCR amplification with the primer-probe pool to detect the amplification signal. The reaction system for fluorescent PCR amplification is: 2.5 μL of 10×KMT2A Mix, 12.5 μL of 2×BR buffer mix, 10 μL of cDNA, with a total volume of 25 μL. The amplification reaction program of this kit is: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 5 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min; collect fluorescence at 60 °C.

[0083] 4. Judgment of results

[0084] When the internal reference ABL1 gene amplification is normal and the CT value < 30, a CT value < 35 for each fusion gene is determined as positive. If Figures 2 - 6 As shown, the internal reference ABL1 gene of the sample and the target detection gene produce the same amplification trend curve, and the CT value of the internal reference ABL1 gene < 30, and the judgment result is positive, which is consistent with the specimen information. If Figures 7 - 8 , only the internal reference gene ABL1 of the sample has a typical S-shaped amplification curve and the CT value < 30, and the target detection gene does not start to amplify, and the judgment result is negative, and the result is consistent with the specimen information. Figure 7 The KMT2A-ACIN1 fusion in is not within the detection range of this kit, and the result is negative, with good specificity. Example 2

[0085] To verify the accuracy of the sample detection results provided by this kit, the above 15 samples were subjected to NGS detection, and at the same time, fluorescence quantitative PCR detection was carried out to compare the differences in the results of the two. The results are shown in Table 4.

[0086] Table 4: Comparison of fluorescence PCR detection results and high-throughput sequencing (NGS) results

[0087]

[0088] The results show that the detection results of fluorescence PCR are consistent with the results of the KMT2A fusion gene detected by high-throughput sequencing.

[0089] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. 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, a probe set designed based on the breakpoint 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 sequences of the upstream primer set are as shown in SEQ ID NO: 1-6, the nucleotide sequences of the probe set are as shown in SEQ ID NO: 7-12, and the nucleotide sequences of the downstream primer set are as shown in SEQ ID NO: 13-42.

2. The primer-probe set according to claim 1, wherein It further 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 with a nucleotide sequence as shown in SEQ ID NO: 44, and a probe with a nucleotide sequence as shown in SEQ ID NO:

45.

3. The primer-probe set according to claim 1, wherein The 5' ends of the probe set are all labeled with a fluorescent group, and the 3' ends are all labeled with a fluorescence quenching group.

4. The KMT2A fusion gene detection kit, characterized in that, It includes the primer-probe set according to any one of claims 1-3.

5. The kit according to claim 4, wherein, The kit further includes at least one of a positive control product, a negative control product, a PCR reaction mixture, and purified water.

6. The kit according to claim 5, characterized in that, The positive control product is a mixture of KMT2A fusion gene plasmids, and the KMT2A fusion gene plasmids include plasmids after the fusion of the KMT2A gene and the partner gene, and the partner genes are MLLT3, AFF1, MLLT10, MLLT1, MLLT4, ELL, EPS15, SEPT6, MLLT11, MLLT6, SEPT9, AFF3, and CBL; and / or, the negative control product is a KMT2A wild-type gene plasmid.

7. The kit according to claim 4, wherein The kit further 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 1-3 in the preparation of a KMT2A fusion gene detection product.

9. The application according to claim 8, wherein The steps of 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 the cDNA as a template, perform PCR amplification using the primer-probe set and the internal reference primer-probe set to detect the fusion gene amplification signal and the internal reference gene amplification signal respectively; S3. Taking the internal reference gene amplification signal value as a reference, determine the positive fusion gene type according to the fusion gene amplification signal value.

10. The application according to claim 9, wherein The fusion gene type is composed of the fusion of any one of the broken exons of the KMT2A gene and any one of the broken exons of the partner gene; where: The KMT2A gene break exons include exons 6-11; the partner gene break exons are: exons 6, 9-10 of the MLLT3 gene, exons 5-7, 12 of the AFF1 gene, exons 3, 5, 8-9, 13-14 of the MLLT10 gene, exons 2, 7 of the MLLT1 gene, exon 2 of the MLLT4 gene, exon 2 of the ELL gene, exons 2, 12 of the EPS15, exon 2 of the SEPT6 gene, exon 2 of the MLLT11 gene, exons 7, 11 of the MLLT6 gene, exons 2-3 of the SEPT9 gene, exons 7, 9 of the AFF3 gene, exons 12, 16 of the CBL gene; And / or, the PCR amplification reaction program is: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 5 min, 15 s at 95°C, 1 min at 60°C, for a total of 40 cycles; the amplification signal is collected at 60°C.

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