Kit for detecting common fusion genes of myelogenous leukemia based on multiple digital PCR (Polymerase Chain Reaction) method

Abstract

The invention discloses a kit for detecting common fusion genes of myelogenous leukemia based on a multiplex digital PCR (Polymerase Chain Reaction) method, which can be used for rapid and accurate molecular detection of 46 clinical common fusion genes of myelogenous leukemia by adopting a specific primer probe combination and an optimized seven-color multiplex digital PCR technical platform. According to the optimal primer probe combination and the detection system disclosed by the invention, the PCR enhancer is creatively added into the PCR reaction liquid, the ROX + CY5 and Atto 425 (A425) + VIC dual-channel detection system is creatively constructed, the PCR enhancer remarkably improves the efficiency and the specificity of a multiple amplification system, and the dual-channel system can realize that 46 fusion genes can be accurately detected by three detection holes. The kit has the advantages of multiple accurate quantitation, high sensitivity, strong specificity, strong anti-interference capability, good repeatability, simplicity, rapidness, high throughput, low cost and the like, has a good clinical application prospect, and can provide a reference basis for rapid and accurate diagnosis, treatment, medication selection, curative effect evaluation and prognosis judgment of clinical myelogenous leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological gene detection, and more specifically, relates to a kit for detecting common fusion genes of myeloid leukemia based on a multiplex digital PCR method. Background Art

[0002] Myeloid leukemia (ML) is a group of malignant tumors that affect the bone marrow and blood, primarily involving abnormal proliferation and maturation disorders of myeloid cells. Based on the rate of disease progression, ML is primarily divided into two categories: acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Treatment for AML typically includes intensive chemotherapy and hematopoietic stem cell transplantation, while treatment for CML relies on tyrosine kinase inhibitors that target the BCR-ABL fusion gene, such as imatinib. Fusion gene detection is crucial for the rapid and accurate diagnosis, risk and prognosis assessment, treatment selection, and efficacy monitoring of AML and CML.

[0003] In recent years, with the continuous advancement of molecular diagnostic technology, the detection techniques and methods for leukemia fusion genes have also been continuously developed and refined. Rapid and accurate detection of fusion genes, especially in the early stages of the disease, is crucial for achieving precise clinical diagnosis and treatment. Among these, RT-qPCR technology significantly shortens the length of target products and overcomes the problem of nucleic acid fragmentation that hinders detection. The closed system mitigates the problem of contamination of RT-PCR amplification products, offering high specificity, a wide quantitative range, and high sensitivity. Currently, a large number of fusion gene detection products based on this technology platform have been developed and dominate clinical applications. Several related products and technologies have been reported in the literature and patents. Two technical systems, one-step and two-step, are available based on this platform. However, the one-step RT-qPCR method can only detect a single fusion gene. This technique requires the use of a standard curve to determine the copy number of the fusion gene. A single target can only be detected in a single tube, requiring separate standard curves for the target gene and the internal standard gene. Furthermore, it is significantly affected by amplification efficiency and PCR inhibitors, resulting in limited relative quantitative accuracy, low throughput, complex operation, high cost, and a high RNA template requirement. Reagents for simultaneous detection of multiple fusion genes are generally two-step detection products. Due to the competitive inhibition of different targets, accurate quantification is impossible and they are all qualitative products. In addition, existing PCR reaction solutions have the following main problems: (1) low amplification efficiency. When primers or probes for multiple targets (such as BCR::ABL1, PML::RARA, RUNX1::RUNX1T1) are present at the same time, primer dimers or heterologous hybridization (such as BCR primers binding to RUNX1 probes) may form due to sequence similarity, consuming reaction reagents and reducing the effective concentration. At the same time, multiple pairs of primers compete for limited Taq DNA polymerase and dNTPs. Targets with low amplification efficiency (such as high GC content fusion genes) will be suppressed by targets with high efficiency (such as low GC content genes), resulting in amplification deviation; (2) poor specificity. Primers may not fully match the template and bind to it, resulting in mixed bands or false positive results; (3) limited sensitivity. Generally, there are trace inhibitors in the sample, and low-concentration templates may not be detected. The platform generally has 4 channels, and each well can detect 3 targets. Various fusion gene screening products generally require a large number of wells (8-16 wells), have high requirements for sample quantity and quality, low throughput, and high unit cost; multiple detection competition interference is relatively serious, resulting in low sensitivity (most detection limits are above 1000 copies / reaction), which cannot meet the clinical needs of high-sensitivity and precise diagnosis and treatment. Summary of the Invention

[0004] The present invention addresses the deficiencies in the prior art and provides a primer-probe combination and detection kit with high sensitivity, high accuracy, high specificity, simple and rapid operation, and the ability to simultaneously achieve absolute quantification of multiple fusion genes. Specifically, the present invention optimizes the composition of primers, probes, and fluorescent markers in the reaction units and innovatively adds PCR enhancers to the PCR reaction solution and innovatively constructs a dual-channel detection system of ROX+CY5 and Atto 425 (A425)+VIC. The PCR enhancer significantly improves the efficiency and specificity of the multiplex amplification system, and the dual-channel system can achieve accurate detection of 46 fusion genes using three detection wells. The present invention, while effectively avoiding interference between primers in each reaction unit, achieves rapid, accurate, high-throughput, and low-cost screening and detection of suspected myeloid leukemia patients through collaborative optimization between reaction units to make up for the deficiencies of existing detection methods and products.

[0005] To solve the above technical problems, the present invention adopts the following technical solution: a kit for detecting common fusion genes in myeloid leukemia based on multiplex digital PCR, the kit comprising at least a primer-probe mixture in well 1, a primer-probe mixture in well 2, a primer-probe mixture in well 3, and a PCR reaction solution:

[0006] Well 1 primer-probe mixture includes: nucleotides described in SEQ ID NO.001-SEQ ID NO.062; Well 2 primer-probe mixture includes: nucleotides described in SEQ ID NO.020, SEQ ID NO.022, SEQ ID NO.027-028, SEQ ID NO.035-036, SEQ ID NO.063-101; Well 3 primer-probe mixture includes: nucleotides described in SEQ ID NO.027-028, SEQ ID NO.035-036, SEQ ID NO.088, SEQ ID NO.090, SEQ ID NO.100-101, SEQ ID NO.102-136;

[0007] In addition, the PCR reaction solution of the kit contains a PCR enhancer, which is composed of betaine, DMSO, (NH4)2SO4, BSA and gelatin, and its concentration in the PCR reaction solution is 0.25M, 1.5wt%, 5mM, 0.05mg / mL and 0.25wt%, respectively.

[0008] Furthermore, in the primer-probe mixture solution in well 1 to the primer-probe mixture solution in well 3, the final concentration of the primers is 250 to 900 nM; and the final concentration of the probes is 250 to 500 nM.

[0009] Generally, the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group. The invention screened the fluorescent reporter group to achieve accurate detection, as follows:

[0010] Nucleotides 11-12, 65, and 106 are probes, which are linked to the fluorescent reporter group VIC;

[0011] Nucleotides 22-23, 70-71, and 90 are probes, which are linked to the fluorescent reporter group FAM;

[0012] Nucleotides 26, 74, and 112 are probes, which are linked to the fluorescent reporter group ROX;

[0013] Nucleotides 35-36 and 35-36 are probes, which are linked to the fluorescent reporter group A425;

[0014] Nucleotides 45-046, 22, and 121-122 are probes, which are linked to the fluorescent reporter group CY7;

[0015] Nucleotides 52-053, 90-091, and 125 are probes, which are linked to the fluorescent reporter group CY5;

[0016] Nucleotides 56, 94, and 130-131 are probes, which are linked to the fluorescent reporter group CY5.5;

[0017] Nucleotides 59, 97-098, and 135 are probes, which are linked to fluorescent reporter groups ROX and CY5;

[0018] Nucleotides 62 and 101 are probes, which are connected to the fluorescent reporter groups VIC and A425.

[0019] The fluorescence quenching group is selected from any one of BHQ1, BHQ2, and BHQ3.

[0020] Generally, the PCR reaction solution also contains DNA polymerase, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP, and positioning fluorescent dye.

[0021] In an embodiment of the present invention, the kit further comprises:

[0022] (1) Reverse transcription reagents: Contains RT enzyme, RNase Inhibitor, dNTP, Oligo dT(18T) Primer, Random 6mers Primer, and reaction buffer;

[0023] (2) Digital PCR microfluidic chip and droplet generation oil;

[0024] (3) Positive control No. 1: a mixed solution containing NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, KAT6A::CREBBP fusion gene variant plasmids and ABL1 internal reference gene plasmid;

[0025] (4) Positive control No. 2: a mixed solution containing FUS::ERG and NPM1::ALK fusion gene variant plasmids and ABL1 internal reference gene plasmid;

[0026] (5) Positive control No. 3: a mixed solution containing KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, PLZF::RARα, ETV6::PDGFRB fusion gene variant plasmids and ABL1 internal reference gene plasmid;

[0027] (6) Positive control No. 4: a mixed solution containing KMT2A::MLLT10 and SET::NUP214 fusion gene variant plasmids and ABL1 internal reference gene plasmid;

[0028] (7) Positive control No. 5: a mixed solution containing CBFB::MYH11, PML::RARA, NPM1::MLF1, KMT2A::ELL, ETV6::JAK2, RUNX1::RPL22, RUNX1::MECOM fusion gene variant plasmids and ABL1 internal reference gene plasmid;

[0029] (8) Positive control No. 6: a mixed solution containing RUNX1::CBFA2T3 and DEK::NUP214 fusion gene variant plasmids and ABL1 internal reference gene plasmid;

[0030] (9) NC: a mixed solution containing the ABL1 internal reference gene plasmid.

[0031] Furthermore, in the positive control, the concentration of each fusion gene variant plasmid was about 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was about 1500 copies / μL; in the NC, the concentration of the ABL1 internal reference plasmid was about 1500 copies / μL.

[0032] Based on the kit described in the present invention, its amplification system is as follows: 7.5 μL of PCR reaction solution, 2.5 μL of primer-probe mixture in well 1 or primer-probe mixture in well 2 or primer-probe mixture in well 3, 5 μL of test sample cDNA, and a total system of 15 μL; the PCR amplification conditions are: 95°C for 10 minutes, 40 cycles (98°C for 15 seconds, 62°C for 1 minute), 28°C for 5 minutes, and 28°C hold.

[0033] The beneficial effects of the present invention are: by optimizing the primers, probes and other components of the PCR reaction, innovatively adding a PCR enhancer to the PCR reaction solution, and innovatively constructing a ROX+CY5 and A425+VIC dual-channel detection system, rapid, accurate, high-throughput, and low-cost screening and detection of patients suspected of myeloid leukemia are achieved, specifically:

[0034] (1) Multiple detection targets and wide coverage. Currently, most blood disease fusion gene detection products are based on fluorescent quantitative PCR platforms, mainly targeting a single target or a small number of targets, such as the common BCR-ABL1 p190, BCR-ABL1 p210, TEL-AML1, AML1-ETO, E2A-PBX1, etc., covering a relatively small number of target types. The present invention constructs a ROX+CY5 and A425+VIC dual-channel detection system, which can achieve accurate detection of 46 common fusion genes related to myeloid leukemia with 3 detection wells;

[0035] (2) High amplification efficiency, high throughput, and low cost. The present invention specifically designs and develops specific and sensitive detection primer probes and detection systems, and configures PCR enhancers, which significantly improves the amplification efficiency and specificity of the multiplex amplification system. At the same time, combined with the preferred 7-color fluorescence (A425, VIC, FAM, ROX, CY5, CY5.5, CY7) digital PCR platform, the single-well of the kit can achieve 7-channel detection, the unit point cost can be reduced by more than 50%, the throughput is more than doubled, and half of the patient's precious blood sample can be saved.

[0036] (3) Fast speed and easy to use. Currently, some blood disease fusion gene detection products are based on high-channel sequencing platforms. Their advantages are that they can detect a wide range of targets and can detect unknown fusion forms. However, the platform equipment and reagents are expensive, the product detection process is time-consuming (3-5 days), the operation and result analysis are very complicated, and professional bioinformatics analysts are required. The sensitivity is low and it is easy to miss detections. The primer-probe combination and detection system described in the present invention are simple to operate, the process is short (3 hours), and the result analysis is intuitive and easy to understand, making it more suitable for application and promotion in hospitals at all levels.

[0037] (4) The performance is significantly improved compared to QPCR products, with multiple absolute quantification, high sensitivity, strong specificity, strong anti-interference and good reproducibility. Currently, the literature reports that a small number of blood disease fusion gene detection products are based on digital PCR platforms, but most of them are targeted at a single target, usually involving only two channels, FAM and VIC, with low detection throughput, few target coverage, and high cost. There are no screening products for multiple leukemia-related fusion genes (≥10 types) based on a multiple digital PCR platform with more than 5 colors, and no specific product involves the detection of multiple myeloid leukemia-related fusion genes. One reason is that the dye luminescence intensity of the three new fluorescent channels, A425, CY5.5 and CY7, is poor (A425, CY5.5) or the fluorescence background is very high (CY7), resulting in a poor signal-to-noise ratio. The requirements for probe development and design are very high, and it is necessary to adopt multiple methods and continuously try to optimize to meet the application requirements. It is very time-consuming and labor-intensive, requiring researchers to have rich R&D experience. The development investment required is huge and it is difficult to obtain satisfactory results. The present invention achieves the expansion of the number of targets for simultaneous ultra-sensitive quantitative detection in a single well through screening tests on multiple multi-color digital PCR platforms, optimized screening of multiple sets of primers and probes for each target, and attempts at different multiple combinations, as well as the effective introduction of PCR enhancers. Ultimately, a set of preferred three-well primer-probe combinations and detection kits for multiplex quantification of 46 fusion genes in myeloid leukemia has been successfully developed. Compared with existing reported similar technologies and products, the present invention has achieved very significant improvements in sensitivity (1 copy / reaction), accuracy and reproducibility (CV ≤ 10%), anti-interference, throughput, cost (1 / 5 of imported similar products), and ease of operation (3 hours), greatly enhancing its potential and practicality for clinical application.

[0038] (5) Strong scalability and platform optimization. The 7-color digital PCR system preferably used in the present invention has multiple channels, excellent performance, good scalability and flexibility. The existing system can be split and customized according to different clinical needs; or newly emerging clinical fusion genes can be added as needed. Compared with the QPCR system, the digital PCR system does not rely on amplification efficiency, and it can also complete testing and optimization in less time in terms of sensitivity and specificity optimization.

[0039] In summary, the preferred primer probe combination and supporting digital PCR kit for myeloid fusion gene detection described in the present invention have the following characteristics: high sensitivity, no need for a standard curve to achieve single-copy absolute quantification; high specificity, simultaneous quantitative detection of more than ten fusion genes in a single well, and no non-specific cross-reaction between different targets; independent of amplification efficiency, high accuracy, good repeatability, strong resistance to inhibitors and interference, and high tolerance; simple and fast operation, intuitive and easy-to-understand result interpretation, and the entire process from sample to result reporting is completed in 3 hours; the domestically produced 7-color digital PCR platform is preferred, and a single well can detect more than 10 targets, with low cost and sample saving; the system has strong scalability, and is convenient for flexible combination or addition or subtraction of targets as needed to match new clinical advances and new needs. The primer probe combination and kit described in the present invention can provide a scientific reference basis for the accurate and rapid screening, diagnosis, treatment, and prognosis evaluation of clinical myeloid leukemia-related fusion genes, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results for positive control No. 1 are shown. The vertical axis of the one-dimensional graph represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, respectively, corresponding to the detection of the NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, and KAT6A::CREBBP fusion genes and the ABL1 internal reference gene. The bottom of the one-dimensional graph represents negative droplets, while the top represents positive droplets in the VIC, FAM, ROX, A425, CY7, CY5, or CY5.5 channels. The software automatically converts the concentration to copy numbers.

[0041] Figure 2 This is the test result for positive control No. 2. The vertical axis of the one-dimensional graph represents the VIC, ROX, A425, CY5, and CY5.5 channels, respectively. The ROX+CY5 channel corresponds to the detection of FUS::ERG, the VIC+A425 channel corresponds to the detection of NPM1::ALK, and the CY5.5 channel corresponds to the detection of the ABL1 internal reference gene. The bottom of the one-dimensional graph represents negative droplets, while the top represents positive droplets in the VIC, ROX, A425, CY5, or CY5.5 channels. The software automatically converts this to copy concentration.

[0042] Figure 3The results for positive control No. 3 are shown. The vertical axis of the one-dimensional graph represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, respectively, corresponding to the detection of the KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, PLZF::RARα, and ETV6::PDGFRB fusion genes. The bottom of the one-dimensional graph represents negative droplets, while the top represents positive droplets in the VIC, FAM, ROX, A425, CY7, CY5, or CY5.5 channels. The software automatically converts the concentration to copy numbers.

[0043] Figure 4 The results for positive control No. 4 are shown on the vertical axis of the one-dimensional graph, representing the VIC, ROX, A425, and CY5 channels, respectively. The ROX+CY5 channel corresponds to the detection of KMT2A::MLLT10, and the VIC+A425 channel corresponds to the detection of SET::NUP214. The bottom of the one-dimensional graph represents negative droplets, while the top represents positive droplets in the VIC, ROX, A425, or CY5 channels. The software automatically converts this to copy concentration.

[0044] Figure 5 The results for positive control No. 5 are shown. The vertical axis of the one-dimensional graph represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, respectively, corresponding to the detection of the CBFB::MYH11, PML::RARA, NPM1::MLF1, KMT2A::ELL, ETV6::JAK2, RUNX1::RPL22, and RUNX1::MECOM fusion genes. The bottom of the one-dimensional graph represents negative droplets, while the top represents positive droplets in the VIC, FAM, ROX, A425, CY7, CY5, or CY5.5 channels. The software automatically converts the concentration to copy numbers.

[0045] Figure 6 The results for positive control No. 6 are shown on the vertical axis of the one-dimensional graph, representing the VIC, ROX, A425, and CY5 channels, respectively. The ROX+CY5 channel corresponds to the detection of RUNX1::CBFA2T3, and the VIC+A425 channel corresponds to the detection of DEK::NUP214. The bottom of the one-dimensional graph represents negative droplets, while the top represents positive droplets in the VIC, ROX, A425, or CY5 channels. The software automatically converts this to copy concentration.

[0046] Figure 7This is the detection result for the negative control NC in the primer-probe mix in well 1. The vertical axis of the one-dimensional graph represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, respectively, corresponding to the detection of the NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, and KAT6A::CREBBP fusion genes and the ABL1 internal reference gene. The bottom of the one-dimensional graph represents a negative droplet. The upper portion of the graph shows no positive droplets in the VIC, FAM, ROX, A425, CY7, or CY5 channels, and only a positive droplet in the CY5.5 channel. The software automatically converts this to copy concentration.

[0047] Figure 8 This is the detection result of the negative control NC in the primer-probe mixture in well 2. The vertical axis of the one-dimensional graph represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, respectively. The bottom of the one-dimensional graph represents the negative droplet, and the top does not contain the positive droplet.

[0048] Figure 9 This is the detection result of the negative control NC in the primer-probe mixture in well 3. The vertical axis of the one-dimensional graph represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, respectively. The bottom of the one-dimensional graph represents the negative droplet, and the top does not contain the positive droplet.

[0049] Figure 10 The results for a BCR::ABL1-positive patient are shown in Figure 1. The vertical axis of the one-dimensional graph represents the FAM and CY5.5 channels in the primer-probe mix in well 1, corresponding to the detection of the BCR::ABL1 fusion gene and the ABL1 internal reference gene, indicating a positive result. DETAILED DESCRIPTION

[0050] In order to better understand the technical means and effects of the present invention, the following examples are provided for further explanation. The contents of the examples are only used to illustrate the present invention and should not and will not limit the present invention.

[0051] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are all the same.

[0052] Example 1 Design, synthesis and screening of primer probes

[0053] Primer and probe design tools: The primer pairs and probes involved in the present invention were designed by Primer 5.0, PrimerExpress 3.0, NCBI Blast, and Clustalx software, and synthesized by Shanghai Bio-Tech Co., Ltd.

[0054] By studying extensive clinical data and relevant databases, we identified 46 fusion genes with high incidence rates associated with myeloid leukemia. We analyzed the mutation sites of each fusion gene, pinpointed the breakpoints between the partner gene and the driver gene, and designed multiple sets of primers and probes across the fusion breakpoint region.

[0055] For any fusion gene, three or more pairs of candidate target sequences were designed. Each set of candidate target sequences was tested individually in a single channel in the early stage. A PCR reaction solution without PCR enhancer was used. The copy concentration results of digital PCR and the one-dimensional or two-dimensional effect graph of the positive droplets were used as the judgment criteria until a set of excellent candidate target sequences was screened for each fusion gene, as shown in Table 1.

[0056] Well 1 primer-probe mix contains:

[0057] (1) Primers for amplifying NUP98 series fusion genes, whose nucleotide sequences are shown in SEQ ID NOs. 001-010, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 011-012, wherein the probes are linked to a fluorescent reporter group VIC.

[0058] (2) primers for amplifying the BCR::ABL1 fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 013-021, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 022-023, wherein the probes are linked to a fluorescent reporter group FAM;

[0059] (3) Primers for amplifying the RUNX1::RUNX1T1 fusion gene, whose nucleotide sequences are shown in SEQ ID NO.024-025, and a probe, whose nucleotide sequence is shown in SEQ ID NO.026, wherein the probe is connected to the fluorescent reporter group ROX.

[0060] (4) primers for amplifying MLL series fusion genes, whose nucleotide sequences are shown in SEQ ID NOs. 027-034, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 035-036, wherein the probes are linked to a fluorescent reporter group A425;

[0061] (5) primers for amplifying the FIP1L1::PDGFRA fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 037-044, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 046-046, wherein the probes are linked to a fluorescent reporter group CY7;

[0062] (6) primers for amplifying the KAT6A::CREBBP fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 047-051, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 052-053, wherein the probes are linked to a fluorescent reporter group CY5;

[0063] (7) primers for amplifying the ABL1 internal reference gene, whose nucleotide sequences are shown in SEQ ID NOs. 054-055, and a probe, whose nucleotide sequence is shown in SEQ ID NO. 056, wherein the probe is linked to a fluorescent reporter group CY5.5;

[0064] (8) primers for amplifying the FUS::ERG fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 057-058, and a probe, whose nucleotide sequence is shown in SEQ ID NO. 059, wherein the probe is linked to fluorescent reporter groups ROX and CY5;

[0065] (9) primers for amplifying the NPM1::ALK fusion gene, whose nucleotide sequences are shown in SEQ ID NO.060-061, and a probe, whose nucleotide sequence is shown in SEQ ID NO.062, wherein the probe is linked to fluorescent reporter groups VIC and A425;

[0066] Well 2 primer-probe mix contains:

[0067] (10) Primers for amplifying the KMT2A::AFDN fusion gene, whose nucleotide sequences are shown in SEQ ID NO.028 and SEQ ID NO.063-064, and a probe, whose nucleotide sequence is shown in SEQ ID NO.065, wherein the probe is connected to the fluorescent reporter group VIC.

[0068] (11) primers for amplifying the KMT2A::MLLT3 fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 066-069, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 070-071, wherein the probes are linked to a fluorescent reporter group FAM;

[0069] (12) Primers for amplifying the KMT2A::AFF1 fusion gene, whose nucleotide sequences are shown in SEQ ID NO.063, SEQ ID NO.028, and SEQ ID NO.072-073, and a probe, whose nucleotide sequence is shown in SEQ ID NO.074, wherein the probe is connected to the fluorescent reporter group ROX.

[0070] (13) primers for amplifying the KMT2A::MLLT1 fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 027-028 and 075-077, and a probe, whose nucleotide sequence is shown in SEQ ID NOs. 035-036, wherein the probe is linked to a fluorescent reporter group A425;

[0071] (14) primers for amplifying the ETV6::ABL1 fusion gene, whose nucleotide sequences are shown in SEQ ID NO.020 and SEQ ID NO.078-079, and a probe, whose nucleotide sequence is shown in SEQ ID NO.022, wherein the probe is linked to a fluorescent reporter group CY7;

[0072] (15) Primers for amplifying RARα series fusion genes, whose nucleotide sequences are shown in SEQ ID NOs. 080-089, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 090-091, wherein the probes are linked to a fluorescent reporter group CY5;

[0073] (16) primers for amplifying the ETV6::PDGFRB fusion gene, whose nucleotide sequences are shown in SEQ ID NO.092-093, and a probe, whose nucleotide sequence is shown in SEQ ID NO.094, wherein the probe is linked to a fluorescent reporter group CY5.5;

[0074] (17) primers for amplifying the KMT2A::MLLT10 fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 027-028 and SEQ ID NOs. 095-096, and a probe, whose nucleotide sequence is shown in SEQ ID NOs. 097-098, wherein the probe is linked to fluorescent reporter groups ROX and CY5;

[0075] (18) primers for amplifying the SET::NUP214 fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 099-100, and a probe, whose nucleotide sequence is shown in SEQ ID NO. 101, wherein the probe is linked to the fluorescent reporter groups VIC and A425;

[0076] Well 3 primer-probe mix contains:

[0077] (19) Primers for amplifying the CBFB::MYH11 fusion gene, whose nucleotide sequences are shown in SEQ ID NO.102-105, and a probe, whose nucleotide sequence is shown in SEQ ID NO.106, wherein the probe is connected to the fluorescent reporter group VIC.

[0078] (20) Primers for amplifying the PML::RARA fusion gene, whose nucleotide sequences are shown in SEQ ID NO.088 and SEQ ID NO.107-109, and a probe, whose nucleotide sequence is shown in SEQ ID NO.090, wherein the probe is linked to a fluorescent reporter group FAM;

[0079] (21) Primers for amplifying the NPM1::MLF1 fusion gene, whose nucleotide sequences are shown in SEQ ID NO.110-111, and a probe, whose nucleotide sequence is shown in SEQ ID NO.112, wherein the probe is connected to the fluorescent reporter group ROX.

[0080] (22) primers for amplifying the KMT2A::ELL fusion gene, whose nucleotide sequences are shown in SEQ ID NO.027-028 and SEQ ID NO.113, and a probe, whose nucleotide sequence is shown in SEQ ID NO.035-036, wherein the probe is linked to the fluorescent reporter group A425;

[0081] (23) primers for amplifying the ETV6::JAK2 fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 114-120, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 121-122, wherein the probes are linked to a fluorescent reporter group CY7;

[0082] (24) primers for amplifying the RUNX1::RPL22 fusion gene, whose nucleotide sequences are shown in SEQ ID NO.123-124, and a probe, whose nucleotide sequence is shown in SEQ ID NO.125, wherein the probe is linked to a fluorescent reporter group CY5;

[0083] (25) primers for amplifying the RUNX1::MECOM fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 126-129, and probes, whose nucleotide sequences are shown in SEQ ID NOs. 130-131, wherein the probes are linked to a fluorescent reporter group CY5.5;

[0084] (26) primers for amplifying the RUNX1::CBFA2T3 fusion gene, whose nucleotide sequences are shown in SEQ ID NOs. 132-134, and a probe, whose nucleotide sequence is shown in SEQ ID NO. 135, wherein the probe is linked to the fluorescent reporter groups ROX and CY5;

[0085] (27) primers for amplifying the DEK::NUP214 fusion gene, whose nucleotide sequences are shown in SEQ ID NO. 100 and SEQ ID NO. 136, and a probe, whose nucleotide sequence is shown in SEQ ID NO. 101, wherein the probe is linked to the fluorescent reporter groups VIC and A425;

[0086] Table 1 Sequence information of SEQ ID NO. 001-136

[0087]

[0088]

[0089] ·Here, + represents the following base-modified locked nucleic acid LNA.

[0090] Nucleotides 11-12, 65, and 106 are probes, which are linked to the fluorescent reporter group VIC;

[0091] Nucleotides 22-23, 70-71, and 90 are probes, which are linked to the fluorescent reporter group FAM;

[0092] Nucleotides 26, 74, and 112 are probes, which are linked to the fluorescent reporter group ROX;

[0093] Nucleotides 35-36 are probes, which are linked to the fluorescent reporter group A425;

[0094] Nucleotides 45-46, 22, and 121-122 are probes, which are linked to the fluorescent reporter group CY7;

[0095] Nucleotides 52-53, 90-91, and 125 are probes, which are linked to the fluorescent reporter group CY5;

[0096] Nucleotides 56, 94, and 130-131 are probes, which are linked to the fluorescent reporter group CY5.5;

[0097] Nucleotides 59, 97-98, and 135 are probes, which are linked to fluorescent reporter groups ROX and CY5;

[0098] Nucleotides 62 and 101 are probes, which are connected to the fluorescent reporter groups VIC and A425.

[0099] Example 2 Design and Synthesis of Plasmid

[0100] A positive plasmid is designed for any variant site of 46 myeloid leukemia-related fusion genes, that is, each positive plasmid has only one variant site, which is used to separately test the effect of any candidate target sequence of the variant site.

[0101] A plasmid targeting the ABL1 internal reference gene was synthesized and numbered NC. It was used to screen the primer and probe sequences of the ABL1 internal reference gene and was also used to test and optimize the single-well multiplex detection system.

[0102] A long fragment plasmid containing the fusion genes of NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, and KAT6A::CREBBP was synthesized and numbered PC-1 for testing and optimization of the single-well multiplex detection system.

[0103] A long plasmid containing the FUS::ERG and NPM1::ALK fusion genes was synthesized and numbered PC-2 for testing and optimization of the single-well multiplex detection system.

[0104] A long fragment plasmid containing the KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, PLZF::RARα, and ETV6::PDGFRB fusion genes was synthesized and numbered PC-3 for testing and optimization of the single-well multiplex detection system.

[0105] A long plasmid containing the KMT2A::MLLT10 and SET::NUP214 fusion genes was synthesized and numbered PC-4 for testing and optimizing the single-well multiplex detection system.

[0106] A long fragment plasmid containing the fusion genes of CBFB::MYH11, PML::RARA, NPM1::MLF1, KMT2A::ELL, ETV6::JAK2, RUNX1::RPL22, and RUNX1::MECOM was synthesized and numbered PC-5 for testing and optimization of the single-well multiplex detection system.

[0107] A long fragment plasmid containing the RUNX1::CBFA2T3 and DEK::NUP214 fusion genes was synthesized and numbered PC-6 for testing and optimization of the single-well multiplex detection system.

[0108] Example 3: Preparation and assembly of the kit

[0109] (1) Reverse transcription reagents: RT enzyme, RNase Inhibitor, dNTPs, Oligo dT(18T) Primer, Random6mers Primer, and reaction buffer;

[0110] (2) PCR reaction solution: containing DNA polymerase, Mg 2+, PCR reaction buffer, dATP, dCTP, dTTP and dGTP, localization fluorescent dye, betaine, DMSO, (NH4)2SO4, BSA and gelatin;

[0111] (3) Digital PCR microfluidic chip and microdroplet generation oil were purchased from Linghang Gene Technology (Hangzhou) Co., Ltd.

[0112] (4) Well 1 primer-probe mixture: SEQ ID NO. 001-062: Primer probe was dissolved in double-distilled water to a concentration of 100 μM and prepared into a tube of 6× primer-probe premix according to a specific ratio;

[0113] (5) Well 2 primer-probe mixture: SEQ ID NO.020, SEQ ID NO.022, SEQ ID NO.027-028, SEQ ID NO.035-036, SEQ ID NO.063-101: Primer probes were dissolved in double-distilled water to a concentration of 100 μM and prepared into a tube of 6× primer-probe premix according to a specific ratio;

[0114] (6) Well 3 primer-probe mixture: SEQ ID NO.027-028, SEQ ID NO.035-036, SEQ ID NO.088, SEQ ID NO.090, SEQ ID NO.100-101, SEQ ID NO.102-136: Primer probes were dissolved in double-distilled water to a concentration of 100 μM and prepared into a tube of 6× primer-probe premix according to a specific ratio;

[0115] (7) Positive control No. 1: a long fragment plasmid containing NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, and KAT6A::CREBBP fusion genes and an ABL1 internal reference gene plasmid were prepared in a specific ratio to form a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid was approximately 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was approximately 1500 copies / μL;

[0116] (8) Positive control No. 2: A long fragment plasmid containing the FUS::ERG and NPM1::ALK fusion genes and an ABL1 internal reference gene plasmid were prepared in a specific ratio to form a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid was approximately 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was approximately 1500 copies / μL;

[0117] (9) Positive control No. 3: A long fragment plasmid containing KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, PLZF::RARα, and ETV6::PDGFRB fusion genes and an ABL1 internal reference gene plasmid were prepared in a specific ratio to form a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid was approximately 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was approximately 1500 copies / μL;

[0118] (10) Positive control No. 4: a long fragment plasmid containing the KMT2A::MLLT10 and SET::NUP214 fusion genes and an ABL1 internal reference gene plasmid were prepared in a specific ratio to form a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid was approximately 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was approximately 1500 copies / μL;

[0119] (11) Positive control No. 5: a long fragment plasmid containing CBFB::MYH11, PML::RARA, NPM1::MLF1, KMT2A::ELL, ETV6::JAK2, RUNX1::RPL22, and RUNX1::MECOM fusion genes and an ABL1 internal reference gene plasmid were prepared in a specific ratio to form a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid was approximately 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was approximately 1500 copies / μL;

[0120] (12) Positive control No. 6: A long fragment plasmid containing the RUNX1::CBFA2T3 and DEK::NUP214 fusion genes and an ABL1 internal reference gene plasmid were prepared in a specific ratio to form a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid was approximately 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was approximately 1500 copies / μL;

[0121] (13) NC: ABL1 internal reference gene plasmid was diluted to the specified concentration, specifically, the concentration was about 1500 copies / μL.

[0122] Example 4 Detection Method for Myeloid Leukemia-Related Fusion Genes

[0123] Based on the raw materials of the kit prepared in Example 3, the detection method is as follows:

[0124] (1) Sample RNA template preparation: The test sample can be fresh bone marrow or peripheral blood. It is recommended to use a commercial kit to extract the sample RNA to be tested. The extraction process should strictly follow the instructions. It is recommended to immediately perform reverse transcription reaction on the extracted RNA sample. For short-term storage, please place it in a -20℃ refrigerator. For long-term storage, please place it in a -80℃ refrigerator.

[0125] (2) RNA reverse transcription: The extracted RNA was reverse transcribed into cDNA. The reaction system is shown in Table 2.

[0126] The reaction solution was vortexed for a few seconds and then briefly centrifuged. The reverse transcription reaction was performed according to the following steps: 37°C, 15 min; 85°C, 5 s; 4°C, hold.

[0127] (3) Preparation of digital PCR reaction solution:

[0128] The components contained in the PCR reaction buffer are shown in Table 3.

[0129] Take out the kit of the present invention from the -20°C refrigerator and equilibrate to room temperature. Vortex and shake for a few seconds to mix thoroughly. Prepare X μL of reaction premix according to the number of samples to be tested:

[0130] X = (7.5 μL PCR reaction solution + 2.5 μL primer-probe premix) × (n samples + 6 positive controls + 3 NCs + 1 loss).

[0131] Vortex the above reaction premix to mix thoroughly, then centrifuge briefly and pipette 10 μL / well of the reaction premix into a thin-walled PCR reaction tube or an eight-tube strip.

[0132] (4) Sample addition: Add 5 μL of the cDNA template to be tested, the positive control, and NC into the PCR tube or eight-tube strip, tightly cap the tube, vortex to mix, and centrifuge briefly.

[0133] (5) Droplet preparation: Add 15 μL / well of reaction solution to the injection hole of the digital PCR microfluidic chip, cover the injection hole and outlet with the four-cap, and place it in the droplet generator. Generally, droplets can be generated in about 20 minutes.

[0134] (6) PCR amplification: The microfluidic chip with generated droplets was slowly transferred to the PCR amplification instrument, and the PCR reaction parameters were set as follows: 95°C for 10 min, 40 cycles (98°C for 15 s, 62°C for 1 min), 28°C for 5 min, and 28°C hold.

[0135] (7) Result analysis: After the amplification is completed, place the microfluidic chip into the pilot chip reader, open the supporting software, simply set the information of each reaction well, and click on the photo scan after preheating. The instrument will automatically read and analyze the fluorescence, and calculate the copy number concentration and total copy number of each channel in each reaction well, and provide a one-dimensional map, a two-dimensional map and the original picture of each channel in each reaction well.

[0136] (8) The droplet count, positive control, NC, and blank control results must meet the following conditions:

[0137] 1) The number of droplets generated by the chip is greater than 20,000. If it is less than 20,000, the copy number result may be inaccurate;

[0138] 2) Positive control No. 1: All seven channels tested positive; Figure 1 shown.

[0139] 3) Positive control No. 2: A425, VIC, ROX, CY5 and CY5.5 channels all tested positive; Figure 2 shown.

[0140] 4) Positive control No. 3: All seven channels tested positive; Figure 3 shown.

[0141] 5) Positive control No. 4: A425, VIC, ROX, CY5 channels all tested positive; Figure 4 shown.

[0142] 6) Positive control No. 5: All seven channels tested positive; Figure 5 shown.

[0143] 7) Positive control No. 6: A425, VIC, ROX, CY5 channels all tested positive; Figure 6 shown.

[0144] 8) NC: The negative control NC in the primer-probe mixture in well 1 is positive in the CY5.5 channel and negative in the other channels. Figure 7 As shown; the detection results of the negative control NC in well 2 primer-probe mixture are negative in all seven channels. Figure 8 As shown; the detection results of the negative control NC in well 3 primer-probe mixture are negative in all seven channels. Figure 9 shown.

[0145] 9) Sample results were interpreted according to Table 4, and the variation frequency was calculated based on the test results.

[0146] 10) If the ABL1 internal standard gene detection value is less than 50 copies / μL, it indicates that the amount of cDNA added is insufficient. The sample should be increased, or the sample should be re-extracted and tested by PCR. The results should be determined according to the table above.

[0147] The expression level of each fusion gene is: fusion gene copy concentration / ABL1 internal standard gene copy concentration*100%.

[0148] Table 2 Reverse transcription system

[0149] Reagents 20μL system Reverse transcription reagents 4 μL RNA template Variable RNase / DNase-free water up to 20μL

[0150] Note: The amount of RNA can be increased as needed. In a 20 μL reverse transcription system, a maximum of 2 μg of total RNA is recommended.

[0151] Table 3 Components of PCR reaction buffer

[0152]

[0153] Note: * indicates enhancer component

[0154] Table 4 Rules for interpreting test kit results

[0155]

[0156] Example 5 Fluorescent labeling screening of the same target

[0157] The digital PCR platform used in this kit accommodates up to seven fluorescence channels per well: A425, VIC, FAM, ROX, CY5, CY5.5, and CY7. Compared to the four-channel combination commonly found in fluorescent quantitative PCR, this digital PCR platform can accommodate three more channels per well: A425, CY5.5, and CY7, increasing the potential for detecting more targets per well.

[0158] Furthermore, this kit cleverly incorporates dual-channel combinations of ROX+CY5 and A425+VIC. This means that when positive signals appear simultaneously in the ROX and CY5 channels, or the A425 and VIC channels, within a single well, these signals are interpreted as the presence of two additional detection targets. This effectively expands the number of channels per well from seven to nine, enabling the detection of more targets. Consequently, the requirements for fluorescent labeling of each target must be higher.

[0159] To this end, for the same target, we systematically screened the fluorescent markers VIC, FAM, ROX, A425, CY7, CY5, and CY5.5, as well as combinations of ROX+633 and VIC+A425. Nine tests were performed for each fusion gene (NUP98::HOXA11 was selected for the NUP98 series; MLL::AF17 was selected for the MLL series; and PLZF::RARα was selected for the RARα series), resulting in a total of 243 comparative tests. The optimal fluorescent marker was selected based on the signal-to-noise ratio. The results of the fluorescent marker screening are shown in Table 5, where the selected fluorescent channel is marked with a *.

[0160] According to the screening results in Table 5, the optimal fluorescent labeling channel (the one with the largest signal-to-noise ratio is the optimal) is selected for each fusion gene for the subsequent well position combination test.

[0161] Table 5 Fluorescent labeling screening results

[0162]

[0163]

[0164] Example 6 Optimization of the reaction system by PCR enhancer

[0165] Based on the screening results from Example 5, this kit primarily selected one combination for study to verify the effectiveness of the PCR enhancer in improving the performance of the myeloid leukemia-related fusion gene detection system. The PCR amplification system and its included enhancer components are shown in Table 3. Components marked with * in Table 3 are enhancer components.

[0166] The PCR amplification system without the addition of PCR enhancer was as follows: 5.5 μL of PCR reaction solution, 2.5 μL of primer-probe mixture in well 1 or primer-probe mixture in well 2 or primer-probe mixture in well 3, 5 μL of test sample cDNA, 2 μL of DEPC water, and a total system of 15 μL; the PCR amplification conditions were: 95°C for 10 min, 40 cycles (98°C for 15 s, 62°C for 1 min), 28°C for 5 min, and 28°C hold.

[0167] The PCR amplification system with the addition of PCR enhancer was as follows: 7.5 μL of PCR reaction solution, 2.5 μL of primer-probe mixture in well 1 or primer-probe mixture in well 2 or primer-probe mixture in well 3, 5 μL of test sample cDNA, and a total system of 15 μL; the PCR amplification conditions were: 95°C for 10 min, 40 cycles (98°C for 15 s, 62°C for 1 min), 28°C for 5 min, and 28°C hold.

[0168] In this embodiment, the probe combination scheme used is shown in Table 6, and the comparison of the effects of the enhancer under the same combination scheme is shown in Table 7.

[0169] When the above primer-probe combination scheme was used to permutate 46 fusion genes, the test results showed that the target concentration and signal-to-noise ratio were generally higher after adding PCR enhancers to the PCR reaction solution than without PCR enhancers. In addition, when the PCR enhancer was not added, nonspecific amplification occurred in wells 1 and 2. However, after adding the PCR enhancer to the PCR reaction solution, nonspecific amplification disappeared. In summary, PCR enhancers can optimize polymerase fidelity, reduce false extensions, and improve amplification efficiency; block background interference, improve signal-to-noise ratio, and inhibit primer dimers and nonspecific amplification.

[0170] Table 6: Probing combination scheme

[0171] aisle Kong 1 fusion gene name Kong 2 fusion gene name Name of the Kong 3 fusion gene VIC KMT2A::AFDN NUP98 Series CBFB::MYH11 FAM BCR::ABL1 KMT2A::MLLT3 PML::RARA ROX RUNX1::RUNX1T1 NPM1::MLF1 KMT2A::AFF1 A425 MLL Series KMT2A::MLLT1 KMT2A::ELL CY7 ETV6::ABL1 FIP1L1::PDGFRA ETV6::JAK2 CY5 KAT6A::CREBBP RARα series RUNX1::RPL22 CY5.5 ABL1 ETV6::PDGFRB RUNX1::MECOM ROX+633 FUS::ERG KMT2A::MLLT10 RUNX1::CBFA2T3 VIC+A425 DEK::NUP214 SET::NUP214 NPM1::ALK

[0172] Note: 1. The MLL series includes four fusion genes: MLL::AF17, MLL::AF1p, MLL::AF1q, and MLL::SEPT6;

[0173] 2. The RARα series includes seven fusion genes: PLZF::RARα, STAT5b::RARα, PRKAR1A::RARα, FIPIL1::RARα, NPM1::RARα, BCOR::RARA, and NABP1::RARA;

[0174] 3. The NUP98 series includes eight fusion genes: NUP98::HOXA11, NUP98::HOXA13, NUP98::HOXA9, NUP98::HOXC11, NUP98::HOXD13, NUP98::PMX1, NUP98::NSD1, and NUP98::RARG;

[0175] Table 7 Comparison of test results

[0176]

[0177]

[0178] Note: Template names starting with "Z" indicate that a PCR enhancer was added to the PCR reaction solution; template names without "Z" indicate that no PCR enhancer was added to the PCR reaction solution. For example, PC-1 indicates the test result when no PCR enhancer was added to the reaction solution; Z-PC-1 indicates the test result when a PCR enhancer was added to the reaction solution; and so on. The template names in wells 1, 2, and 3 have the same meanings as PC-1 and Z-PC-1.

[0179] Example 7 Kit Well Combination

[0180] While ensuring that the single-channel test effect of each candidate fusion gene target sequence is good enough, the more critical thing is that the primer probe sequence of any fusion gene does not interfere with the nucleotide sequence of other fusion genes in the well, and no dimers between primers or primers and probes will be formed. There will also be no non-specific amplification due to unreasonable primer-probe design or well arrangement.

[0181] To this end, based on the screening results of Example 5, this kit has tried a variety of well position combination schemes and selected a group of combinations in which the probes do not interfere with each other and the detection effect meets the requirements. Three of the combination schemes are mainly selected and shown in Tables 8, 10, and 12. The detection results of the three probe combination schemes are shown in Tables 9, 11, and 13 respectively.

[0182] When the first primer-probe combination (Table 8) was used to permutate the 46 fusion genes, as shown in Table 9, the six fusion genes and the ABL1 reference gene contained in the first well, PC-1, were detected normally. Since FUS::ERG in PC-2 was labeled with both ROX and CY5 probes, positive signals appeared simultaneously in both channels, and the detection concentrations were comparable. Furthermore, since NPM1::ALK was labeled with both A425 and VIC fluorescence signals, positive amplification also appeared simultaneously in both channels, and the detection concentrations were comparable. Overall, the detection results were normal. However, nonspecific amplification occurred in the A425 channel of NC. After investigation, it was found that the primer-probe conflict between RUNX1::RPL22 and the MLL series was present. The well position for RUNX1::RPL22 was subsequently adjusted accordingly.

[0183] In the second well, PC-3 detected 7 fusion genes normally, and PC-4 detected the KMT2A::MLLT10 fusion gene simultaneously labeled by ROX+CY5, and the SET::NUP214 fusion gene simultaneously labeled by A425+VIC normally.

[0184] In the third well, PC-5 detected 7 fusion genes normally, and PC-6 detected the RUNX1::CBFA2T3 fusion gene simultaneously labeled by ROX+CY5, and the DEK::NUP214 fusion gene simultaneously labeled by A425+VIC.

[0185] When the second primer combination scheme (Table 10) was used to arrange and combine the 46 fusion genes, PC-1 in the first well normally detected the six fusion genes and the ABL1 reference gene, and PC-2 normally detected the FUS::ERG fusion gene simultaneously labeled by ROX+CY5 and the NPM1::ALK fusion gene simultaneously labeled by A425+VIC.

[0186] The CY7 channel concentration of the second well PC-3 was low, only about 1 / 10 of the expected concentration. The reason may be that the competition effect was poor when the KMT2A::MLLT1 probe was fluorescently labeled with CY7 in the combination. Therefore, the fluorescent labeling of the KMT2A::MLLT1 fusion gene was adjusted accordingly.

[0187] In the third well, PC-5 detected 7 fusion genes normally, and PC-6 detected the RUNX1::CBFA2T3 fusion gene simultaneously labeled by ROX+CY5, and the DEK::NUP214 fusion gene simultaneously labeled by A425+VIC.

[0188] When the third primer combination scheme (Table 12) was used to arrange and combine the 46 fusion genes, PC-1 in the first well normally detected the six fusion genes and the ABL1 reference gene included, and PC-2 normally detected the FUS::ERG fusion gene simultaneously labeled by ROX+CY5 and the NPM1::ALK fusion gene simultaneously labeled by A425+VIC.

[0189] In the second well, PC-3 detected 7 fusion genes normally, and PC-4 detected the KMT2A::MLLT10 fusion gene simultaneously labeled by ROX+CY5, and the SET::NUP214 fusion gene simultaneously labeled by A425+VIC normally.

[0190] In the third well, PC-5 detected 7 fusion genes normally, and PC-6 detected the RUNX1::CBFA2T3 fusion gene simultaneously labeled by ROX+CY5, and the DEK::NUP214 fusion gene simultaneously labeled by A425+VIC.

[0191] In summary, all targets in the third primer combination were detected normally, the concentrations were consistent with the expected concentrations, and no nonspecific amplification occurred. Therefore, this combination was determined as the final fusion gene combination used in this kit.

[0192] Table 8 The first combination of induction and exploration scheme

[0193] aisle Kong 1 fusion gene name Kong 2 fusion gene name Name of the Kong 3 fusion gene VIC NUP98 Series KMT2A::AFDN CBFB::MYH11 FAM BCR::ABL1 KMT2A::MLLT3 PML::RARA ROX RUNX1::RUNX1T1 KMT2A::AFF1 NPM1::MLF1 A425 MLL Series KMT2A::MLLT1 KMT2A::ELL CY7 FIP1L1::PDGFRA ETV6::ABL1 ETV6::JAK2 CY5 RUNX1::RPL22 RARα series KAT6A::CREBBP CY5.5 ABL1 ETV6::PDGFRB RUNX1::MECOM ROX+CY5 FUS::ERG KMT2A::MLLT10 RUNX1::CBFA2T3 A425+VIC NPM1::ALK SET::NUP214 DEK::NUP214

[0194] Note: 1. The MLL series includes four fusion genes: MLL::AF17, MLL::AF1p, MLL::AF1q, and MLL::SEPT6;

[0195] 2. The RARα series includes seven fusion genes: PLZF::RARα, STAT5b::RARα, PRKAR1A::RARα, FIPIL1::RARα, NPM1::RARα, BCOR::RARA, and NABP1::RARA;

[0196] 3. The NUP98 series includes eight fusion genes: NUP98::HOXA11, NUP98::HOXA13, NUP98::HOXA9, NUP98::HOXC11, NUP98::HOXD13, NUP98::PMX1, NUP98::NSD1, and NUP98::RARG;

[0197] Table 9 Detection results of the first probe combination scheme

[0198]

[0199] Table 10 The second combination of induction and exploration scheme

[0200] aisle Kong 1 fusion gene name Kong 2 fusion gene name Name of the Kong 3 fusion gene VIC NUP98 Series KMT2A::AFDN CBFB::MYH11 FAM BCR::ABL1 KMT2A::MLLT3 PML::RARA ROX RUNX1::RUNX1T1 KMT2A::AFF1 NPM1::MLF1 A425 MLL Series ETV6::ABL1 KMT2A::ELL CY7 FIP1L1::PDGFRA KMT2A::MLLT1 ETV6::JAK2 CY5 KAT6A::CREBBP RARα series RUNX1::RPL22 CY5.5 ABL1 ETV6::PDGFRB RUNX1::MECOM ROX+CY5 FUS::ERG KMT2A::MLLT10 RUNX1::CBFA2T3 A425+VIC NPM1::ALK SET::NUP214 DEK::NUP214

[0201] Note: 1. The MLL series includes four fusion genes: MLL::AF17, MLL::AF1p, MLL::AF1q, and MLL::SEPT6;

[0202] 2. The RARα series includes seven fusion genes: PLZF::RARα, STAT5b::RARα, PRKAR1A::RARα, FIPIL1::RARα, NPM1::RARα, BCOR::RARA, and NABP1::RARA;

[0203] 3. The NUP98 series includes eight fusion genes: NUP98::HOXA11, NUP98::HOXA13, NUP98::HOXA9, NUP98::HOXC11, NUP98::HOXD13, NUP98::PMX1, NUP98::NSD1, and NUP98::RARG;

[0204] Table 11 Detection results of the second probe combination scheme

[0205]

[0206] Table 12 The third combination of induction and exploration

[0207]

[0208]

[0209] Note: 1. The MLL series includes four fusion genes: MLL::AF17, MLL::AF1p, MLL::AF1q, and MLL::SEPT6;

[0210] 2. The RARα series includes seven fusion genes: PLZF::RARα, STAT5b::RARα, PRKAR1A::RARα, FIPIL1::RARα, NPM1::RARα, BCOR::RARA, and NABP1::RARA;

[0211] 3. The NUP98 series includes eight fusion genes: NUP98::HOXA11, NUP98::HOXA13, NUP98::HOXA9, NUP98::HOXC11, NUP98::HOXD13, NUP98::PMX1, NUP98::NSD1, and NUP98::RARG;

[0212] Table 13 Detection results of the third probe combination scheme

[0213]

[0214] Example 8 Test Kit Blank Limit Verification

[0215] Peripheral blood or bone marrow samples were collected from 30 healthy individuals, and each clinical sample was tested twice using the detection method described in Example 4, i.e., a total of 60 tests. The experimental data were recorded and the results were analyzed, as shown in Tables 14-16.

[0216] According to Tables 14-16, the limit of blank (LOB) was calculated using a non-parametric method. The results are summarized in Table 17. It can be seen that the false positive rate of each fusion gene is less than 5%, which meets the specificity requirements of clinical detection.

[0217] Table 14 Blank limit verification results of primer-probe mixture in well 1

[0218]

[0219]

[0220]

[0221] Table 15 Blank limit verification results of primer-probe mixture in well 2

[0222]

[0223]

[0224]

[0225]

[0226] Table 16 Blank limit verification results of primer-probe mixture in well 3

[0227]

[0228]

[0229]

[0230] Table 17 Summary of test kit blank limit verification results

[0231] Fusion gene name Detection value range (copies / μL) 95th percentile (copies / μL) False positive rate (%) NUP98 Series 0.1-0.12 0 3.3％(2 / 60) BCR::ABL1 0 0 0％(0 / 60) RUNX1::RUNX1T1 0 0 0％(0 / 60) MLL Series 0.09-0.28 0.09 5％(3 / 60) FIP1L1::PDGFRA 0.09-0.18 0.09 3.3％(2 / 60) KAT6A::CREBBP 0.16-0.36 0.16 3.3％(2 / 60) FUS::ERG 0.11-0.34 0 3.3％(2 / 60) NPM1::ALK 0.21-0.37 0 3.3％(2 / 60) KMT2A::AFDN 0.19-0.25 0 3.3％(2 / 60) KMT2A::MLLT3 0.09-0.37 0.09 5％(3 / 60) KMT2A::AFF1 0.13-0.43 0.13 5％(3 / 60) KMT2A::MLLT1 0 0 0％ ETV6::ABL1 0.09-0.25 0.09 3.3％(2 / 60) RARα series 0.09-0.38 0.21 5％(3 / 60) ETV6::PDGFRB 0.09-0.18 0.09 3.3％(2 / 60) KMT2A::MLLT10 0.16-0.26 0 1.6％(1 / 60) SET::NUP214 0.24-0.27 0 1.6％(1 / 60) CBFB::MYH11 0 0 0％(0 / 60) PML::RARA 0 0 0％(0 / 60) NPM1::MLF1 0.09-0.21 0.09 5％(3 / 60) KMT2A::ELL 0.08-0.18 0.08 3.3％(2 / 60) ETV6::JAK2 0.15-0.25 0 3.3％(2 / 60) RUNX1::RPL22 0.07-0.27 0.07 3.3％(2 / 60) RUNX1::MECOM 0.06-0.25 0.08 3.3％(2 / 60) RUNX1::CBFA2T3 0.13-0.31 0 5％(3 / 60) DEK::NUP214 0.15-0.29 0 3.3％(2 / 60)

[0232] Example 9 Kit Detection Limit (LOD) Verification

[0233] Standards of different concentrations, such as 0.5%, 0.1%, 0.05%, 0.01%, 0.005% and 0%, were prepared based on the internal reference gene plasmid and the target gene long fragment plasmid. The concentration of the internal reference plasmid was controlled at around 15,000 copies / μL, and the concentrations of each target were gradiently diluted to 75 copies / μL, 15 copies / μL, 7.5 copies / μL, 1.5 copies / μL, 0.75 copies / μL and 0 copies / μL.

[0234] The detection method described in Example 4 was used for detection, and each sample was detected 5 times. The experimental data were recorded and the results were analyzed, as shown in Tables 18-20.

[0235] The results are summarized in Table 21. The detection limit of this kit was set as the lowest concentration with a detection rate of ≥95%, that is, the detection limit concentration of each fusion gene was 0.01%. Specifically, the detection limit of the fusion gene was 1.5 copies / μL.

[0236] Table 18 Detection limit verification results of primer-probe mixture in well 1

[0237]

[0238] Note: AH in the table correspond to:

[0239] A: NUP98 series detection value (copies / μL); B: BCR::ABL1 detection value (copies / μL); C: RUNX1::RUNX1T1 detection value (copies / μL); D: MLL series detection value (copies / μL); E: FIP1L1::PDGFRA detection value (copies / μL); F: KAT6A::CREBBP detection value (copies / μL); G: FUS::ERG detection value (copies / μL); H: NPM1::ALK detection value (copies / μL)

[0240] Table 19 Detection limit verification results of primer-probe mixture in well 2

[0241]

[0242]

[0243] Note: The AI in the table corresponds to:

[0244] A: KMT2A::AFDN detection value (copies / μL); B: KMT2A::MLLT3 detection value (copies / μL); C: KMT2A::AFF1 detection value (copies / μL); D: KMT2A::MLLT1 detection value (copies / μL); E: ETV6::ABL1 detection value (copies / μL); F: RARα series detection value (copies / μL)

[0245] G:ETV6::PDGFRB detection value (copies / μL); H:KMT2A::MLLT10 detection value (copies / μL); I:SET::NUP214 detection value (copies / μL)

[0246] Table 20 Detection limit verification results of primer-probe mixture in well 3

[0247]

[0248]

[0249] Note: The AI in the table corresponds to:

[0250] A: CBFB::MYH11 detection value (copies / μL); B: PML::RARA detection value (copies / μL); C: NPM1::MLF1 detection value (copies / μL);

[0251] D: KMT2A::ELL detection value (copies / μL); E: ETV6::JAK2 detection value (copies / μL); F: RUNX1::RPL22 detection value (copies / μL);

[0252] G: RUNX1::MECOM detection value (copies / μL); H: RUNX1::CBFA2T3 detection value (copies / μL); I: DEK::NUP214 detection value (copies / μL);

[0253] Table 21 Summary of kit detection limit verification results

[0254]

[0255] Example 10 Clinical Sample Results Verification

[0256] Positive blood or bone marrow samples from patients diagnosed with myeloid leukemia were collected. The samples had been tested using commercially available test kits, and the corresponding results were known. A total of 61 positive samples covering fusion genes within the detection range of this kit were selected. These 61 samples were tested using this kit and the detection method described in Example 4. The test results are shown in Table 22.

[0257] The kit provided by the present invention and a commercially available kit were tested on 61 clinical samples. The results showed that the kit provided by the present invention detected 44 positive results, while the commercially available kit detected only 43 positive results. Compared with the commercially available kit, the kit provided by the present invention detected one more BCR::ABL1 positive result (sample No. 46).

[0258] like Figure 10 As shown, further analysis of sample No. 46 revealed an internal standard gene detection concentration of 5537.32 copies / μL, a BCR::ABL1 fusion gene detection concentration of 0.92 copies / μL, and a fusion gene ratio of 0.02%, which is below the detection limit of commercially available kits. Given a detection limit of 0.01%, the present kit, according to the result interpretation method described in Example 4, was positive for BCR::ABL1, demonstrating the significant advantage of this kit in detecting low-abundance fusion genes.

[0259] Table 22 Summary of clinical sample comparison results

[0260]

[0261]

[0262] The primer-probe combination and the accompanying digital PCR kit described in the present invention have the advantages of high specificity, high sensitivity, high accuracy, high tolerance, convenient operation and low cost, and can provide a scientific reference for the diagnosis, treatment selection and prognosis evaluation of clinical myeloid leukemia.

Claims

1. A kit for detecting common fusion genes in myeloid leukemia based on multiplex digital PCR, characterized in that: The kit includes at least a primer-probe mixture in well 1, a primer-probe mixture in well 2, a primer-probe mixture in well 3, and a PCR reaction solution: Well 1 primer-probe mixture includes: nucleotides described in SEQ ID NO.001-SEQ ID NO.062; Well 2 primer-probe mixture includes: nucleotides described in SEQ ID NO.020, SEQ ID NO.022, SEQ ID NO.027-028, SEQ ID NO.035-036, SEQ ID NO.063-101; Well 3 primer-probe mixture includes: nucleotides described in SEQ ID NO.027-028, SEQ ID NO.035-036, SEQ ID NO.088, SEQ ID NO.090, SEQ ID NO.100-101, SEQ ID NO.102-136; Furthermore, the PCR reaction solution of the kit contains a PCR enhancer, which is composed of betaine, DMSO, (NH4)2SO4, BSA and gelatin, and the concentrations of the enhancers in the PCR reaction solution are 0.25M, 1.5wt%, 5mM, 0.05mg / mL and 0.25wt%, respectively; ·Here, + represents the following base-modified locked nucleic acid LNA.

2. The kit according to claim 1, wherein In the primer-probe mixture solution in well 1 to the primer-probe mixture solution in well 3, the final concentration of the primers is 250 to 900 nM; the final concentration of the probes is 250 to 500 nM.

3. The kit according to claim 1, wherein The 5' end of the probe is connected to a fluorescent reporter group, the 3' end is connected to a fluorescent quencher group, and, Nucleotides 11-12, 65, and 106 are probes, which are linked to the fluorescent reporter group VIC; Nucleotides 22-23, 70-71, and 90 are probes, which are linked to the fluorescent reporter group FAM; Nucleotides 26, 74, and 112 are probes, which are linked to the fluorescent reporter group ROX; Nucleotides 35-36 and 35-36 are probes, which are linked to the fluorescent reporter group A425; Nucleotides 45-046, 22, and 121-122 are probes, which are linked to the fluorescent reporter group CY7; Nucleotides 52-053, 90-091, and 125 are probes, which are linked to the fluorescent reporter group CY5; Nucleotides 56, 94, and 130-131 are probes, which are linked to the fluorescent reporter group CY5.5; Nucleotides 59, 97-098, and 135 are probes, which are linked to fluorescent reporter groups ROX and CY5; Nucleotides 62 and 101 are probes, which are connected to the fluorescent reporter groups VIC and A425.

4. The nucleotide sequence according to claim 3, characterized in that: The fluorescence quenching group is selected from any one of BHQ1, BHQ2, and BHQ3.

5. The kit according to claim 1, wherein The PCR reaction solution also contains DNA polymerase, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP, and positioning fluorescent dye.

6. The kit according to claim 1, wherein The kit also includes: (1) Reverse transcription reagents: Contains RT enzyme, RNase Inhibitor, dNTP, Oligo dT(18T) Primer, Random6mers Primer, and reaction buffer; (2) Digital PCR microfluidic chip and droplet generation oil; (3) Positive control No. 1: contains NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, A mixed solution of KAT6A::CREBBP fusion gene variant plasmid and ABL1 internal reference gene plasmid; (4) Positive control No. 2: a mixed solution containing FUS::ERG and NPM1::ALK fusion gene variant plasmids and ABL1 internal reference gene plasmid; (5) Positive control No. 3: a mixed solution containing KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, PLZF::RARα, ETV6::PDGFRB fusion gene variant plasmids and ABL1 internal reference gene plasmid; (6) Positive control No. 4: a mixed solution containing KMT2A::MLLT10 and SET::NUP214 fusion gene variant plasmids and ABL1 internal reference gene plasmid; (7) Positive control No. 5: a mixed solution containing CBFB::MYH11, PML::RARA, NPM1::MLF1, KMT2A::ELL, ETV6::JAK2, RUNX1::RPL22, RUNX1::MECOM fusion gene variant plasmids and ABL1 internal reference gene plasmid; (8) Positive control No. 6: a mixed solution containing RUNX1::CBFA2T3 and DEK::NUP214 fusion gene variant plasmids and ABL1 internal reference gene plasmid; (9) NC: a mixed solution containing the ABL1 internal reference gene plasmid.

7. The kit according to claim 6, characterized in that In the positive control, the concentration of each fusion gene variant plasmid was about 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was about 1500 copies / μL; in the NC, the concentration of the ABL1 internal reference plasmid was about 1500 copies / μL.

8. The kit according to claim 1, wherein The amplification system is as follows: 7.5 μL of PCR reaction solution, 2.5 μL of primer-probe mixture in well 1 or primer-probe mixture in well 2 or primer-probe mixture in well 3, 5 μL of test sample cDNA, and a total system of 15 μL; the PCR amplification conditions are: 95°C for 10 min, 40 cycles (98°C for 15 s, 62°C for 1 min), 28°C for 5 min, and 28°C hold.

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