Primer probe composition for detecting leukemia fusion gene and application thereof

Abstract

According to the invention, the primers are designed for 65 leukemia fusion genes through sufficient literature investigation in the early stage, the leukemia genotypes covering 90% or more are covered, and the primers are good in amplification performance and strong in specificity; based on a multiple digital PCR system of an innovative multicolor fluorescent channel, different fluorescently-labeled hydrolysis probes are designed, multi-gene multi-channel detection is combined, 65 leukemia fusion genes are detected at the same time, leukemia typing can be judged through one-time detection, and time is saved. Through high-throughput detection, the cost is reduced to 1 / 10 of that of foreign like products, and the sensitivity is high.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to a primer-probe combination for detecting leukemia fusion genes and an application thereof. Background Art

[0002] Hematological tumors are malignant tumors that originate from the hematopoietic system. Common clinical hematological tumors include three categories: leukemia, multiple myeloma, and malignant lymphoma. Leukemia originates from hematopoietic stem cells and hematopoietic progenitor cells. According to the degree of differentiation and the course of the disease, leukemia can be divided into two categories: acute and chronic. Acute leukemia can be divided into acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML); chronic leukemia can be divided into chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL).

[0003] Leukemia fusion gene detection can be used for auxiliary diagnosis, typing and medication guidance. In terms of auxiliary diagnosis, when the PML-RARα fusion gene is positive, it can be diagnosed as acute promyelocytic leukemia (AML M3 type). In terms of typing, the WHO guidelines clearly define the proportions of CBFβ-MYH11, PML-RARα, and AML1-ETO in different typing. For example, the positive rate of AML1-ETO in primary AML is about 6% to 8%, the positive rate in M2 type (partially differentiated acute myeloid leukemia) is 20% to 40%, and the positive rate in M2b (M2 is divided into a and b subtypes) is about 90%. In terms of guiding medication, Gleevec is approved for the treatment of BCR-ABL1-positive CML patients, and dasatinib is used to treat BCR-ABL1-positive and T315I mutation-resistant patients. AML1-ETO fusion gene positivity is an indicator of good prognosis.

[0004] At present, the commonly used methods for detecting blood tumors at home and abroad include FISH, qPCR, and NGS. The throughput of FISH technology is extremely low and can only include a few fusions; qPCR is mainly used for qualitative detection and cannot meet the needs of quantitative detection, and the sensitivity of qPCR is limited, which makes it difficult to meet the application scenario of minimal residual disease monitoring; NGS detection is too expensive, complicated to operate, and has low sensitivity. Therefore, the market urgently needs a low-cost, high-performance leukemia fusion detection product that can meet both qualitative and quantitative needs. Summary of the invention

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a primer-probe combination for detecting leukemia fusion gene, wherein the primer-probe combination comprises one or more of the following six groups;

[0006] The first group of primer-probe compositions includes PML-RARA-F, PML-RARA-LP, PML-RARA-VP, PML-RARA-SF, PML-RARA-SP, PML-RARA-R, AML1-F, AML1-P, AML1-MTG16-R1, AML1-MTG16-R2, MLL-F1, MLL-F2, MLL-ELL-R, MLL-ELL-P1, MLL-ELL-P2, MLL-AF4-P, MLL-AF4-R, MLL-AF6-P, MLL-AF6-R, ABL1-F, ABL1-R, and ABL-P, and their sequences are shown in SEQ ID NOs. 1 to 22, respectively; the probe sequences are modified with different fluorescent groups, and different fluorescent groups are excited to produce fluorescence of different wavelengths;

[0007] The second group of primer probe compositions includes MLL-F1, MLL-F2, dupMLL-P, dupMLL-R, 210-F1, 210-P1, 210-F2, 210-P2, BCR-R, 190-F, 190-P, BCR-R, 230-F, 230-P, BCR-R, TEL-PDGFRB-F, TEL-PDGFRB-P, TEL-PDGFRB-R, TEL-PDGFRA-F, TEL-PDGFRA-P, TEL-PDGFRA-R, TEL-JAK2-F, TEL-JAK2-P, TEL-JAK2-R1, TEL-JAK2-R2, TEL-JAK2-R3, TEL-JAK2-R4, TEL-JAK2-R5, AML1-F, AML1-EAP-R, and AML1-P, and their sequences are as shown in SEQ As shown in ID NO.23~53; the probe sequence is modified by different fluorescent groups, and different fluorescent groups are excited to produce fluorescence of different wavelengths;

[0008] The third group of primer probe combinations includes DEK-CAN-F, CAN-R, DEK-CAN-P, EVI1-F, EVI1-R, EVI1-P, E2A-HLF-F1, E2A-HLF-F2, E2A-HLF-R, E2A-HLF-P, AML1-ETO-F, AML1-ETO-R, AML1-ETO-P, CALM-AF10-F, CALM-AF10-P, CALM-AF10-R1, CALM-AF10-R2, CALM-AF10-R3, FIP1L1-PDGFR A-F1, FIP1L1-PDGFRA-F2, FIP1L1-PDGFRA-F3, FIP1L1-PDGFRA-F4, FIP1L1-PDGFRA-R1, FIP1L1-PDGFRA-R2, FIP1L1-PDGFRA-P1, FIP1L1-PDGFRA-P2, MLL-F1, MLL-P1, MLL-F2, MLL-P2, MLL-AF10-R1, MLL-AF10-R2, MLL-AF10-R3, MLL-AF10-R4, whose sequences are shown in SEQ ID NOs. 54 to 87, respectively; the probe sequences are modified with different fluorescent groups, and different fluorescent groups are excited to produce fluorescence of different wavelengths;

[0009] The fourth group of primer probe combinations includes TEL-ABL1-F1, TEL-ABL1-F2, BCR-R, TEL-ABL1-P, MLL-F1, MLL-P1, MLL-F2, MLL-P2, AF17-R, AF1q-R, AF1p-R, AFX1-R, SEPT6-R, AF5-R, PLZF-F, PRKAR1A-F, STAT5B-F, FIP1L1-F, NPM1-F, NUMA1-F, BCOR-F, NA BP1-F, RARA-R, RARA-P, E2A-PBX1-F, E2A-PBX1-R, E2A-PBX1-P, RPN1-EVI1-F, RPN1-EVI1-R, RPN1-EVI1-P, HLXB9-ETV6-F, HLXB9-ETV6-P, HLXB9-ETV6-R1, HLXB9-ETV6-R2, HOX11L2-F, HOX11L2-R, HOX11L2-P, whose sequences are shown in SEQ ID NOs. 88 to 124, respectively; the probe sequences are modified with different fluorescent groups, and different fluorescent groups are excited to produce fluorescence of different wavelengths;

[0010] The fifth set of primer probe compositions includes BCR-PDGFRA-F1, BCR-PDGFRA-F2, CDK5RAP2-PDGFRA-F, STRN-PDGFRA-F, KIF5B-PDGFRA-F, PDGFRA-R, PDGFRA-P, TEL-AML1-F, TEL-AML1-R, TEL-AML1-P, MLL-F1, MLL-P1, MLL-F2, MLL-P2, MLL-ENL-R1, MLL-ENL-R2, MLL-ENL-R3, MLL-ENL- R4, NUP98-F, NUP98-P, HOXA11-R, HOXA13-R, HOXA9-R, HOXC11-R, HOXD13-R, PMX1-R, NSD1-R, RARG-R, NPM1-ALK-F, NPM1-ALK-R, NPM1-ALK-P, NPM1-MLF1-F, NPM1-MLF1-R, NPM1-MLF1-P, CBFB-MF, CBFB-M-R1, CBFB-M-R2, CBFB-M-R3, CBFB-MP, whose sequences are shown in SEQ ID NOs. 125 to 163, respectively; the probe sequences are modified with different fluorescent groups, and different fluorescent groups are excited to produce fluorescence of different wavelengths;

[0011] The sixth set of primer probe combinations includes AML1-F, AML1-EVI1-F, AML1-EVI1-P1, AML1-EVI1-P2, AML1-EVI1-R1, AML1-EVI1-R2, BCR-ABL1 p190-F, BCR-ABL1 p210-F, BCR-ABL1p230-F, BCR-ABL1-P, BCR-R, MLL-F1, MLL-P1, MLL-F2, MLL-P2, AF9-R1, AF9-R2, AF9-R3, AF9-R4, SIL-TAL1-F, SIL-TAL1-R, SIL-TAL1-P, WT1-F, WT1-R, WT1-P, HOX11-F, HOX11-R, HOX11-P, SET-CAN-F, CAN-R, SET-CAN-P, TLS-ERG-F, TLS-ERG-R1, TLS-ERG-R2, TLS-ERG-P1, TLS-ERG-P2, whose sequences are shown in SEQ ID NOs. 164 to 199, respectively; the probe sequences are modified with different fluorescent groups, and different fluorescent groups are excited to produce fluorescence of different wavelengths.

[0012] The specific information of the six sets of primer-probe combinations is shown in Table 1. Among them, -F represents the forward primer, -R represents the reverse primer, and -P represents the fluorescent probe. The probe usually has 5' and 3' end modifications, and some have middle modifications. Different fusion genes can share the forward (-F) primer or the reverse primer (-R) or the fluorescent labeled probe (-P).

[0013] The above six sets of primer-probe combinations can cover 65 leukemia fusion genes. The corresponding information of the six sets of primer-probe combinations and leukemia fusion genes is shown in Table 2.

[0014] Table 1 Detailed information of six sets of primer-probe combinations

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Table 2 Corresponding information of six sets of primer-probe combinations and leukemia genes

[0026]

[0027]

[0028] In the second aspect of the present invention, a kit for detecting leukemia fusion genes is designed based on the primer-probe combination, the kit comprising an amplification reaction system solution, the amplification reaction system solution comprising one or more groups of primer-probe combinations from Group 1 to Group 6, and different groups of primer-probe combinations are separately arranged.

[0029] As a preferred embodiment, the amplification reaction system solution also includes cDNA reverse transcription buffer and PCR reaction solution.

[0030] As a preferred embodiment, the cDNA reverse transcription buffer comprises Evo M-MLV reverse transcriptase and water.

[0031] As a preferred embodiment, the PCR reaction solution includes DNA polymerase, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP.

[0032] As a preferred solution, the concentration of each primer in the amplification reaction system solution is 400 nmol / L, and the concentration of each probe in the amplification reaction system solution is 200 nmol / L.

[0033] The third aspect of the present invention provides the use of the primer-probe combination in a kit for detecting leukemia fusion genes, comprising the following application steps:

[0034] S1: Extract bone marrow blood or peripheral blood and extract RNA from it;

[0035] S2: reverse transcription of RNA into cDNA;

[0036] S3: According to the primer-probe combination group, the primer-probe combination is mixed with the PCR reaction solution and the reverse transcribed cDNA to prepare an amplification reaction system solution, and the amplification reaction system solution is added to different reaction wells, and then transferred to the chip sample row, droplet generation oil is added, and the solution is placed in a droplet generator to generate droplets;

[0037] S4: The chip with the generated droplets is transferred in parallel to a chip-based PCR instrument for PCR amplification and collection of fluorescence signals. The copy number of the fusion gene in the sample reaction well is calculated based on the fluorescence signals.

[0038] As a preferred solution, the concentration of each primer and probe in the amplification reaction system solution is 200-400 nmol / L.

[0039] As a preferred solution, the volume proportion of the reverse transcribed cDNA in the amplification reaction system solution is 30% to 40%.

[0040] As a preferred solution, the volume proportion of the PCR reaction solution in the amplification reaction system solution is 40% to 60%.

[0041] As a preferred embodiment, the volume ratio of the droplet generation oil to the amplification reaction system solution is 0.8:1 to 1.2:1.

[0042] Droplet digital PCR technology is a nucleic acid absolute quantification technology that has developed rapidly after the first and second generation PCR technologies. Droplet digital PCR technology is designed with microfluidic channels and reaction chamber chips, and thousands of tiny droplets are obtained in the reaction chamber through oil-water separation. After PCR amplification, the copy number of the target gene is calculated by counting the fluorescence signal of the droplets. This PCR reaction system based on a single oil-in-water droplet can achieve absolute quantification of "single molecule template amplification", greatly improves the detection sensitivity, and is easier to meet the needs of small volume and high throughput. In addition, the system is simple and low cost.

[0043] Through the above technical solution, the present invention produces the following beneficial effects:

[0044] (1) In the early stage of the present invention, through sufficient literature research, primers were designed for 65 leukemia fusion genes, covering more than 90% of leukemia gene typing, and the primers have good amplification performance and strong specificity.

[0045] (2) The present invention is based on an innovative multiplex digital PCR system with multicolor fluorescent channels, designs different fluorescent labeled hydrolysis probes, combines multi-gene multi-channel detection, and simultaneously detects 65 leukemia fusion genes. One test can determine the leukemia typing, saving time. Through high-throughput detection, the cost is reduced to 1 / 10 of similar foreign products.

[0046] (3) The use of a multiplex digital PCR system can reduce the amount of template used and improve sensitivity, reaching 0.1%.

[0047] (4) The multiplex digital PCR system has simple configuration, high degree of automation, easy operation, and short overall detection time for a single process. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The negative control and positive control effect diagrams of the amplification of the fusion gene by the first set of primer-probe combinations;

[0049] Figure 2 The negative control and positive control effect diagrams of the second set of primer-probe combination amplification of the fusion gene;

[0050] Figure 3 The negative control and positive control effect diagrams of the third set of primer-probe combination amplification of the fusion gene;

[0051] Figure 4 The negative control and positive control effect diagrams of the fusion gene amplification by the fourth set of primer-probe combination;

[0052] Figure 5 The negative control and positive control effect diagrams of the fifth set of primer-probe combination amplification of fusion gene;

[0053] Figure 6 The negative control and positive control effect diagrams of the 6th set of primer-probe combination amplification of fusion gene;

[0054] The PC column is the positive control, the NC column is the negative control, and the first column on the left is the gene name; the horizontal axis of the negative control and positive control effect diagram of the amplified fusion gene is the number of droplets; the vertical axis is the fluorescence signal intensity of each channel, where purple ATT0425 channel positive droplets, blue FAM channel positive droplets, green VIC channel positive droplets, orange ROX channel positive droplets, pink ATTO633 channel positive droplets, brown CY5.5 channel positive droplets, red CY7 channel positive droplets, gray Negative droplets. DETAILED DESCRIPTION

[0055] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.

[0056] This method has no special requirements for the RNA extraction method, as long as it complies with the conventional RNA extraction method in the art.

[0057] Example 1: Primer amplification performance evaluation

[0058] 1. Test methods

[0059] (1) Six sets of primer-probe combinations for detecting leukemia fusion genes in multiplex PCR were synthesized by Shanghai Bio-Tech Co., Ltd. The sequences are shown in Table 1.

[0060] (2) Select positive (PC) and negative (NC) plasmids as reference samples.

[0061] (3) Sample addition: According to the primer-probe combination group, the six sets of primer-probe combinations were mixed with the PCR reaction solution and 5 μL of the test sample (when the template was stored at -20°C, it was pre-thawed at room temperature and centrifuged at 10,000 rpm for 10 s) to prepare the amplification reaction system solution, and the amplification reaction system solution was added to different reaction wells respectively; 5 μL of double distilled water was used instead of the test sample in the blank control well; a total of 15 μL of the amplification reaction system solution was added to each well, vortexed, and centrifuged at 2,000 rpm for 1 min.

[0062] (4) Droplet generation: 15 μL of PCR amplification reaction system solution in the PCR eight-well tube (well) was transferred to the sample row of the chip, and then 14 μL of droplet generation oil was added and placed in a droplet generator to automatically generate droplets.

[0063] (5) PCR amplification: The chip with generated droplets was transferred in parallel to a chip-based qualitative PCR instrument and amplified according to the following reaction program: 95°C, 10 min; 40 cycles (98°C, 15 s; 62°C, 30 s); 28°C for 1 min; 28°C, hold; and removed when the temperature drops to 28°C after amplification.

[0064] 2. Results Analysis

[0065] The negative and positive control results of 6 sets of primer-probe combinations for amplifying 65 fusion genes are shown in the figure below. Figure 1 to Figure 6 As shown. Figure 1 to Figure 6 It can be seen that the six sets of primer-probe combinations have better amplification effects on the corresponding fusion genes and higher efficiency.

[0066] Example 2: Primer specificity assessment

[0067] 1. Test methods

[0068] (1) The primers and probes for detecting leukemia fusion multiplex PCR were synthesized by Shanghai Bio-Tech Co., Ltd. The sequences are shown in Table 1.

[0069] (2) RNA from 32 negative samples was selected and reverse transcribed into cDNA.

[0070] (3) Sample loading: According to the primer-probe combination group, 6 sets of primer-probe combinations (corresponding to 60 fusion genes, excluding the five expressed genes dupMLL, EVI1, HOX11, HOX11L2, and WT1) were mixed with PCR reaction solution and 5 μL of test sample (when the template was stored at -20°C, it was pre-thawed at room temperature and centrifuged at 10,000 rpm for 10 s) to prepare an amplification reaction system solution, and the amplification reaction system solution was added to different reaction wells respectively; 5 μL of double distilled water was used instead of the test sample in the blank control well; a total of 15 μL of amplification reaction system solution was added to each well, vortexed, and centrifuged at 2,000 rpm for 1 min.

[0071] (4) Droplet generation: 15 μL of PCR amplification reaction system solution in the PCR eight-well tube (well) was transferred to the sample row of the chip, and then 14 μL of droplet generation oil was added and placed in a droplet generator to automatically generate droplets.

[0072] (5) PCR amplification: The chip with generated droplets was transferred in parallel to a chip-based qualitative PCR instrument and amplified according to the following reaction program: 95°C, 10 min; 40 cycles (98°C, 15 s; 62°C, 30 s); 28°C for 1 min; 28°C, hold; and removed when the temperature drops to 28°C after amplification.

[0073] 2. Results Analysis

[0074] Six sets of primer-probe combinations (corresponding to 60 fusion genes, excluding the five expressed genes of dupMLL, EVI1, HOX11, HOX11L2, and WT1) amplified 32 negative samples, and the results were all negative (lower than the fusion positive judgment value ≤0.5 copies / μL), with a specificity of 100%. The six sets of primer-probe combinations have high amplification specificity.

[0075] Example 3: Sensitivity test

[0076] 1. Test methods

[0077] (1) The primers and probes for detecting leukemia fusion multiplex PCR were synthesized by Shanghai Bio-Tech Co., Ltd. The sequences are shown in Table 1.

[0078] (2) Artificially synthesize mutant plasmids of each fusion site. Take each tube of primer-probe premix as a unit to synthesize a long fragment plasmid containing all fusion sites of the premix in that tube, and use it after enzyme digestion; artificially synthesize the internal reference-ABL1 plasmid and use it after enzyme digestion.

[0079] (3) The fusion gene plasmid and ABL1 internal reference plasmid after enzyme digestion were diluted 1.0E+05 times, and the plasmid concentration was determined using the pilot digital PCR system. According to the plasmid concentration results, the theoretical concentrations of 5%, 1%, 0.5%, 0.1%, 0.05%, 0.00% and other different concentrations of standard products were prepared under the background of 2.0E+04 copies of ABL1 internal reference, and the corresponding theoretical copy numbers of each fusion site were 1.0E+03, 2.0E+02, 1.0E+02, 2.0E+01, and 1.0E+01 copies. Each concentration gradient was tested with 3 replicates.

[0080] (4) Sample addition: According to the primer-probe combination group, the six sets of primer-probe combinations were mixed with the PCR reaction solution and 5 μL of the test sample (when the template was stored at -20°C, it was pre-thawed at room temperature and centrifuged at 10,000 rpm for 10 s) to prepare the amplification reaction system solution, and the amplification reaction system solution was added to different reaction wells respectively; 5 μL of double distilled water was used instead of the test sample in the blank control well; a total of 15 μL of the amplification reaction system solution was added to each well, vortexed, and centrifuged at 2,000 rpm for 1 min.

[0081] (5) Droplet generation: 15 μL of PCR amplification reaction system solution in the PCR eight-well strip was transferred to the sample row of the chip, and then 14 μL of droplet generation oil was added and placed in a droplet generator to automatically generate droplets.

[0082] (6) PCR amplification: The chip with generated droplets was transferred in parallel to a chip-based qualitative PCR instrument and amplified according to the following reaction program: 95°C, 10 min; 40 cycles (98°C, 15 s; 62°C, 30 s); 28°C for 1 min; 28°C, hold; and removed when the temperature drops to 28°C after amplification.

[0083] 2. Results Analysis

[0084] From the sensitivity test data, it can be found that the positive quality control product with a theoretical concentration of 0.1% can be detected in all three wells, and the actual detection concentration is consistent with the theory; although the positive quality control product with a theoretical concentration of 0.05% can be detected in all three wells, the parallel data of the three wells are quite different (see Table 3 for specific results). In summary, the detection sensitivity of 65 fusion genes is 0.1%. At the same time, from the linear relationship between theoretical concentration and actual detection concentration, it can be found that all 65 fusion genes have a good linear relationship (R 2 ≥0.980).

[0085] Table 3 Sensitivity sample test results

[0086]

[0087]

[0088] Example 4: Accuracy Test

[0089] 1. Test methods

[0090] (1) The primers and probes for detecting leukemia fusion multiplex PCR were synthesized by Shanghai Bio-Tech Co., Ltd. The sequences are shown in Table 1.

[0091] (2) A total of 100 human peripheral blood / bone marrow RNA clinical samples were collected, including 33 negative samples and 67 positive samples. The positive samples covered 35 fusion genes and 5 expression genes within the scope of this project package. All positive samples were verified by leukemia fusion gene detection kit or second-generation sequencing. Clinical sample RNA was reverse transcribed into cDNA.

[0092] (3) Sample addition: According to the primer-probe combination group, the six sets of primer-probe combinations were mixed with the PCR reaction solution and 5 μL of the test sample (when the template was stored at -20°C, it was pre-thawed at room temperature and centrifuged at 10,000 rpm for 10 s) to prepare the amplification reaction system solution, and the amplification reaction system solution was added to different reaction wells respectively; 5 μL of double distilled water was used instead of the test sample in the blank control well; a total of 15 μL of the amplification reaction system solution was added to each well, vortexed, and centrifuged at 2,000 rpm for 1 min.

[0093] (4) Droplet generation: 15 μL of PCR amplification reaction system solution in the PCR eight-well tube (well) was transferred to the sample row of the chip, and then 14 μL of droplet generation oil was added and placed in a droplet generator to automatically generate droplets.

[0094] (5) PCR amplification: The chip with generated droplets was transferred in parallel to a chip-based qualitative PCR instrument and amplified according to the following reaction program: 95°C, 10 min; 40 cycles (98°C, 15 s; 62°C, 30 s); 28°C for 1 min; 28°C, hold; and removed when the temperature drops to 28°C after amplification.

[0095] 2. Results Analysis

[0096] The kit had a sensitivity of 98.5%, a specificity of 97.0%, and an overall compliance rate of 98%. The specific test results are shown in Table 4. The analysis of the 2 samples with inconsistent results is as follows:

[0097] (1) The peripheral blood sample of FG-50 patient was negative for the leukemia fusion gene detection reference kit, while the test result of this project was positive for BCR-ABL p190. Further analysis of the sample found that the number of fusion gene copies quantified by digital PCR was only 15 copies, and the abundance of fusion variants was only 0.11%, which was lower than the detection limit of the reference kit, so the reference kit had the risk of missing detection.

[0098] (2) The bone marrow sample of FG-97 patient was WT1 positive by next generation sequencing, but negative by this project. The WT1 expression abundance was 2.46%, which was lower than the positive judgment threshold (3%). We speculate that this may be related to the difference in target position design and the difference in positive judgment thresholds for expressed genes by different methodologies.

[0099] Table 4: Sample accuracy test results

[0100]

[0101]

[0102]

[0103]

[0104] This application uses a multiplex PCR method to simultaneously and comprehensively detect 65 leukemia fusion genes. By optimizing the combination of single-channel multi-gene and single-hole multiplex detection, a primer-probe combination with strong specificity and high sensitivity is screened out. It requires few samples, is low-cost, has high throughput, is simple and fast to operate and analyze, and has accurate results. Compared with existing FISH, qPCR, and NGS products, it has obvious advantages and improvements in performance, has a high application prospect, and can provide more accurate guidance for leukemia fusion gene typing.

[0105] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A primer-probe combination for detecting leukemia fusion gene, characterized in that: The primer-probe composition includes the first group of primer-probe compositions; the first group of primer-probe compositions includes PML-RARA-F, PML-RARA-LP, PML-RARA-VP, PML-RARA-SF, PML-RARA-SP, PML-RARA-R, AML1-F, AML1-P, AML1-MTG16-R1, AML1-MTG16-R2, MLL-F1, MLL-F2, MLL-ELL-R, MLL-ELL-P1, MLL-ELL-P2, MLL-AF4-P, MLL-AF4-R, MLL-AF6-P, MLL-AF6-R, ABL1-F, ABL1-R, and ABL-P, and their sequences are shown in SEQ ID NOs. 1 to 22, respectively; the probe sequences are modified by different fluorescent groups.

2. The primer-probe combination according to claim 1, characterized in that The second group of primer probe compositions is also included, and the second group of primer probe compositions includes MLL-F1, MLL-F2, dupMLL-P, dupMLL-R, 210-F1, 210-P1, 210-F2, 210-P2, BCR-R, 190-F, 190-P, BCR-R, 230-F, 230-P, BCR-R, TEL-PDGFRB-F, TEL-PDGFRB-P, TEL-PDG FRB-R, TEL-PDGFRA-F, TEL-PDGFRA-P, TEL-PDGFRA-R, TEL-JAK2-F, TEL-JAK2-P, TEL-JAK2-R1, TEL-JAK2-R2, TEL-JAK2-R3, TEL-JAK2-R4, TEL-JAK2-R5, AML1-F, AML1-EAP-R, AML1-P, whose sequences are shown in SEQ ID NOs. 23 to 53, respectively; the probe sequences are modified by different fluorescent groups.

3. The primer-probe composition according to claim 2, characterized in that: Also included is a third group of primer-probe combinations, which include DEK-CAN-F, CAN-R, DEK-CAN-P, EVI1-F, EVI1-R, EVI1-P, E2A-HLF-F1, E2A-HLF-F2, E2A-HLF-R, E2A-HLF-P, AML1-ETO-F, AML1-ETO-R, AML1-ETO-P, CALM-AF10-F, CALM-AF10-P, CALM-AF10-R1, CALM-AF10-R2, CALM-AF10-R3, and FIP1L 1-PDGFRA-F1, FIP1L1-PDGFRA-F2, FIP1L1-PDGFRA-F3, FIP1L1-PDGFRA-F4, FIP1L1-PDGFRA-R1, FIP1L1-PDGFRA-R2, FIP1L1-PDGFRA-P1, FIP1L1-PDGFRA-P2, MLL-F1, MLL-P1, MLL-F2, MLL-P2, MLL-AF10-R1, MLL-AF10-R2, MLL-AF10-R3, MLL-AF10-R4, whose sequences are shown in SEQ ID NOs.54 to 87, respectively; the probe sequences are modified by different fluorescent groups.

4. The primer-probe combination according to claim 3, characterized in that: Also included is a fourth group of primer probe combinations, which include TEL-ABL1-F1, TEL-ABL1-F2, BCR-R, TEL-ABL1-P, MLL-F1, MLL-P1, MLL-F2, MLL-P2, AF17-R, AF1q-R, AF1p-R, AFX1-R, SEPT6-R, AF5-R, PLZF-F, PRKAR1A-F, STAT5B-F, FIP1L1-F, NPM1-F, NUMA1-F, BC OR-F, NABP1-F, RARA-R, RARA-P, E2A-PBX1-F, E2A-PBX1-R, E2A-PBX1-P, RPN1-EVI1-F, RPN1-EVI1-R, RPN1-EVI1-P, HLXB9-ETV6-F, HLXB9-ETV6-P, HLXB9-ETV6-R1, HLXB9-ETV6-R2, HOX11L2-F, HOX11L2-R, HOX11L2-P, whose sequences are shown in SEQ ID NOs. 88 to 124, respectively; the probe sequences are modified by different fluorescent groups.

5. The primer-probe combination according to claim 4, characterized in that: Also included is a fifth group of primer-probe combinations, which include BCR-PDGFRA-F1, BCR-PDGFRA-F2, CDK5RAP2-PDGFRA-F, STRN-PDGFRA-F, KIF5B-PDGFRA-F, PDGFRA-R, PDGFRA-P, TEL-AML1-F, TEL-AML1-R, TEL-AML1-P, MLL-F1, MLL-P1, MLL-F2, MLL-P2, MLL-ENL-R1, MLL-ENL-R2, MLL-ENL-R3, MLL- LL-ENL-R4, NUP98-F, NUP98-P, HOXA11-R, HOXA13-R, HOXA9-R, HOXC11-R, HOXD13-R, PMX1-R, NSD1-R, RARG-R, NPM1-ALK-F, NPM1-ALK-R, NPM1-ALK-P, NPM1-MLF1-F, NPM1-MLF1-R, NPM1-MLF1-P, CBFB-MF, CBFB-M-R1, CBFB-M-R2, CBFB-M-R3, CBFB-MP, whose sequences are shown in SEQ ID NOs. 125 to 163, respectively; the probe sequences are modified by different fluorescent groups.

6. The primer-probe combination according to claim 5, characterized in that: The sixth group of primer probe compositions is also included, and the sixth group of primer probe compositions includes AML1-F, AML1-EVI1-F, AML1-EVI1-P1, AML1-EVI1-P2, AML1-EVI1-R1, AML1-EVI1-R2, BCR-ABL1 p190-F, BCR-ABL1 p210-F, BCR-ABL1 p230-F, BCR-ABL1-P, BCR-R, MLL-F1, MLL-P1, MLL-F2, MLL-P2, AF9-R1, AF9-R2, AF9-R3, AF9-R4, SIL-TAL1-F, SIL-TAL1-R, SIL-TAL1-P, WT1-F, WT1-R, WT1-P, HOX11-F, HOX11-R, HOX11-P, SET-CAN-F, CAN-R, SET-CAN-P, TLS-ERG-F, TLS-ERG-R1, TLS-ERG-R2, TLS-ERG-P1, TLS-ERG-P2, whose sequences are shown in SEQ ID NOs. 164 to 199, respectively; the probe sequences are modified by different fluorescent groups.

7. A kit for detecting leukemia fusion gene, characterized in that: The kit comprises an amplification reaction system solution, and the amplification reaction system solution comprises the primer-probe combination of claim 6, and different groups of primer-probe combinations are arranged separately.

8. The kit according to claim 7, characterized in that The amplification reaction system solution also includes cDNA reverse transcription buffer and PCR reaction solution.

9. Use of any one of the primer-probe combinations according to claim 1 to claim 6 in a kit for detecting leukemia fusion genes.

10. The use according to claim 9, characterized in that: The application consists of the following steps: S1: Extract bone marrow blood or peripheral blood and extract RNA from it; S2: reverse transcription of RNA into cDNA; S3: According to the primer-probe combination group, the primer-probe combination is mixed with the PCR reaction solution and the reverse transcribed cDNA to prepare an amplification reaction system solution, and the amplification reaction system solution is added to different reaction wells, and then transferred to the chip sample row, droplet generation oil is added, and the solution is placed in a droplet generator to generate droplets; S4: The chip with the generated droplets is transferred in parallel to a chip-based PCR instrument for PCR amplification and collection of fluorescence signals. The copy number of the fusion gene in the sample reaction well is calculated based on the fluorescence signals.

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