A kit for detecting common fusion genes of myeloid leukemia based on multiplex digital PCR method

Abstract

The application discloses a kit for detecting common fusion genes of myeloid leukemia based on a multiple digital PCR method, wherein specific primer probe combinations are adopted, and through an optimized 7-color multiple digital PCR technology platform, the kit can be used for rapid and accurate molecular detection of 46 common fusion genes of myeloid leukemia in clinic. The preferred primer probe combinations and detection systems disclosed by the application innovatively add a PCR enhancer into a PCR reaction liquid and innovatively construct a ROX+CY5 and Atto 425 (A425)+VIC double-channel detection system, the PCR enhancer significantly improves the efficiency and specificity of the multiple amplification system, and the double-channel system can realize accurate detection of 46 fusion genes in three detection holes. The application has the advantages of multiple accurate quantification, high sensitivity, strong specificity, strong anti-interference ability, good repeatability, simplicity and rapidness, high throughput, low cost and the like, has a good clinical application prospect, and can provide reference bases for rapid and accurate diagnosis, treatment, drug selection, efficacy evaluation and prognosis judgment of myeloid leukemia in clinic.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology gene detection, and more specifically, relates to a kit for detecting common fusion genes in myeloid leukemia based on multiplex digital PCR. Background Technology

[0002] Myeloid leukemia (ML) is a group of malignant tumors affecting the bone marrow and blood, primarily involving abnormal proliferation and maturation disorders of myeloid cells. Based on the rate of disease progression, ML is mainly 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 targeting the BCR-ABL fusion gene, such as imatinib. Detection of the fusion gene is of significant value in the rapid and accurate diagnosis, risk and prognostic assessment, treatment selection, and efficacy monitoring of both 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 improved, enabling rapid and accurate detection of fusion genes, especially in the early stages of the disease, which is crucial for precision clinical diagnosis and treatment. Among these methods, RT-qPCR can significantly shorten the target product length and solve the problem of undetectable nucleic acid fragmentation. The closed system can solve the problem of RT-PCR amplification product contamination, and it features high specificity, a wide quantitative range, and high sensitivity. Currently, many fusion gene detection products based on this technology platform have been developed, occupying the mainstream of clinical applications, and some literature and patents have reported on related products and technologies. There are two technical systems based on this platform: one-step and two-step. However, the one-step RT-qPCR technology can only be used to detect a single fusion gene. This technology requires the use of a standard curve to determine the copy number of the fusion gene, and one tube can only detect a single target. Standard curves for the target gene and the internal control gene need to be prepared separately, and it is greatly affected by amplification efficiency and PCR inhibitors, resulting in limited quantitative accuracy, low throughput, complex operation, high cost, and high requirements for RNA templates. Reagents for simultaneous detection of multiple fusion genes are generally two-step detection products. Due to the competition and inhibition of different targets, accurate quantification is impossible. They are all qualitative products. The existing PCR reaction solutions have the following problems: (1) Low amplification efficiency. When primers or probes of multiple targets (such as BCR::ABL1, PML::RARA, RUNX1::RUNX1T1) are present at the same time, primer dimers or heterologous hybridization may be formed due to sequence similarity (such as the binding of BCR primers and RUNX1 probes), 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 be completely matched with the template, producing impurities or false positive results; (3) Limited sensitivity. There are usually trace amounts of inhibitors in the sample. Low concentrations of template may not be detected. The platform typically has 4 channels, with each well capable of detecting 3 targets. Multiple fusion gene screening products generally require a large number of wells (8-16 wells), which places high demands on the quantity and quality of samples, resulting in low throughput and high cost per unit point. The competition and interference from multiple detections are quite severe, leading to low sensitivity (the detection limit is mostly above 1000 copies / reaction), which cannot meet the needs of clinical high-sensitivity and precision diagnosis. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a primer-probe combination and detection kit that offers high sensitivity, high accuracy, high specificity, and is simple and rapid to operate, enabling simultaneous absolute quantification of multiple fusion genes. Specifically, this invention optimizes the composition of primers, probes, and fluorescent markers in the reaction units and innovatively adds PCR enhancers to the PCR reaction solution. It also 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 allows for accurate detection of 46 fusion genes using three detection wells. By effectively avoiding interference between primers within each reaction unit and through synergistic optimization between reaction units, this invention achieves rapid, accurate, high-throughput, and low-cost screening and detection of suspected myeloid leukemia patients, thus overcoming the deficiencies of existing detection methods and products.

[0005] To solve the above-mentioned 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 primer-probe mixture for well 1, primer-probe mixture for well 2, primer-probe mixture for well 3, and PCR reaction solution:

[0006] The primer-probe mixture in well 1 comprises the nucleotides described in SEQ ID NO. 001-SEQ ID NO. 062; the primer-probe mixture in well 2 comprises the nucleotides described in SEQ ID NO. 020, SEQ ID NO. 022, SEQ ID NO. 027-028, SEQ ID NO. 035-036, and SEQ ID NO. 063-101; the primer-probe mixture in well 3 comprises the 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, and SEQ ID NO. 102-136.

[0007] Furthermore, the PCR reaction solution of the kit contains PCR enhancers, which are composed of betaine, DMSO, (NH4)2SO4, BSA and gelatin, and their concentrations in the PCR reaction solution are 0.25M, 1.5wt%, 5mM, 0.05mg / mL and 0.25wt%, respectively.

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

[0009] Generally, the probe has a fluorescent reporter group attached to its 5' end and a fluorescent quencher group attached to its 3' end. The invention has screened for fluorescent reporter groups to achieve accurate detection, as detailed below:

[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 used as probes, which are linked to the fluorescent reporter group FAM.

[0012] Nucleotides 26, 74, and 112 are used as 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 used as probes, which are linked to the fluorescent reporter group CY5.5;

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

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

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

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

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

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

[0023] (2) Digital PCR microfluidic chip, droplet-generated 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 plasmid and ABL1 internal reference gene plasmid;

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

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

[0029] (8) Positive control No. 6: a mixed solution containing the RUNX1::CBFA2T3 and DEK::NUP214 fusion gene variant plasmid and the 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 is approximately 75 copies / μL, and the concentration of the ABL1 internal reference plasmid is approximately 1500 copies / μL; in the NC, the concentration of the ABL1 internal reference plasmid is approximately 1500 copies / μL.

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

[0033] The beneficial effects of this invention are as follows: By optimizing the primers, probes, and other components of the PCR reaction, an innovative PCR enhancer was added to the PCR reaction solution, and a dual-channel detection system of ROX+CY5 and A425+VIC was constructed. This enables rapid, accurate, high-throughput, and low-cost screening and detection of suspected myeloid leukemia patients, specifically manifested as follows:

[0034] (1) Numerous detection targets and broad coverage. Currently, most detection products for fusion genes in hematological diseases are based on the quantitative real-time PCR platform, mainly targeting a single target or a few 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. This invention constructs a dual-channel detection system of ROX+CY5 and A425+VIC, which can achieve accurate detection of 46 common fusion genes related to myeloid leukemia using 3 detection wells;

[0035] (2) High amplification efficiency, high throughput, and low cost. This invention specifically designs and develops specific and sensitive detection primers, 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 kit can achieve 7-channel detection per well, reducing the unit cost by more than 50%, increasing the throughput by more than double, and saving half of the amount of precious blood samples used by patients.

[0036] (3) Fast and easy to use. Currently, some detection products for fusion genes in hematological diseases are based on high-channel sequencing platforms. Their advantage lies in the wide range of targets that can be detected and the ability to detect unknown fusion forms. However, the equipment and reagents of this platform are expensive, the product detection process is time-consuming (3-5 days), and the operation and result analysis are very complex. It requires professional bioinformatics analysts, has low sensitivity, and is prone to missed detections. In contrast, the primer-probe combination and detection system described in this invention is easy to operate, has a short process (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 multiplex absolute quantification, high sensitivity, strong specificity, strong anti-interference, and good reproducibility. Currently, the few existing blood disease fusion gene detection products reported in the literature are based on digital PCR platforms, but most of them target a single target, usually only involving the FAM and VIC channels. The detection throughput is low, the number of targets covered is small, and the cost is high. There are no screening products based on 5-color or higher multiplex digital PCR platforms for multiple leukemia-related fusion genes (≥10 types), and there are no products specifically for 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. Multiple methods and continuous optimization are required to meet the application requirements. It is very time-consuming and labor-intensive. Researchers need to have rich research and development experience. The investment required for development is huge and it is difficult to obtain satisfactory results. This invention, through screening tests on multiple multicolor digital PCR platforms, optimization screening of multiple sets of primers and probes for each target, and attempts at different multiplex combinations, as well as the effective introduction of PCR enhancers, has achieved an expansion of the number of targets for simultaneous ultrasensitive quantitative detection in a single well. Ultimately, a set of optimized primer and probe combinations and detection kits for multiplex quantification of 46 fusion genes in myeloid leukemia has been successfully developed in 3 wells. Compared with existing reported similar technologies and products, it has achieved 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 (3h), greatly enhancing its potential and practicality in clinical applications.

[0038] (5) High scalability and optimal platform. The 7-color digital PCR system preferred in this invention has multiple channels, excellent performance, and good scalability and flexibility. Existing systems can be split and customized according to different clinical needs; or newly emerging fusion genes can be added as needed. Compared with the qPCR system, the digital PCR system does not depend on amplification efficiency, and can complete testing and optimization in less time in terms of sensitivity and specificity.

[0039] In summary, the preferred primer-probe combination and matching digital PCR kit for detecting myeloid fusion genes described in this invention offer the following advantages: high sensitivity, achieving absolute quantification of a single copy without the need for a standard curve; high specificity, enabling simultaneous quantification of more than ten fusion genes in a single well, with no non-specific cross-reactions between different targets; high accuracy and repeatability, independent of amplification efficiency, strong resistance to inhibitors and interference, and high tolerability; simple and rapid operation, intuitive and easy-to-understand result interpretation, with the entire process from sample to result reporting completed within 3 hours; a preferred domestic 7-color digital PCR platform, capable of detecting more than 10 targets per well, low cost, and sample saving; and strong scalability, allowing for flexible combination or addition / removal of targets as needed to match new clinical advances and requirements. The primer-probe combination and kit described in this invention can provide a scientific reference for the accurate and rapid screening, diagnosis, treatment, and prognostic evaluation of myeloid leukemia-related fusion genes in clinical practice, and have broad clinical application prospects. Attached Figure Description

[0040] Figure 1 This is the detection result for positive control 1. The vertical axis of the one-dimensional plot represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, corresponding to the detection of NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, KAT6A::CREBBP fusion genes and the ABL1 internal reference gene. The bottom of the one-dimensional plot shows negative droplets, and the top shows positive droplets for either the VIC, FAM, ROX, A425, CY7, CY5, or CY5.5 channels. The software automatically converts these values ​​to copy concentration.

[0041] Figure 2 This is the detection result for positive control No. 2. The vertical axis of the one-dimensional plot 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 plot shows negative droplets, and the top shows positive droplets for either the VIC, ROX, A425, CY5, or CY5.5 channels, automatically converted to copy concentration by the software.

[0042] Figure 3This is the detection result for positive control No. 3. The vertical axis of the one-dimensional graph represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, corresponding to the detection of KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, PLZF::RARα, and ETV6::PDGFRB fusion genes. The bottom of the one-dimensional graph shows negative droplets, and the top shows positive droplets for either the VIC, FAM, ROX, A425, CY7, CY5, or CY5.5 channels. The software automatically converts these values ​​to copy concentration.

[0043] Figure 4 This is the detection result for positive control 4. The vertical axis of the one-dimensional plot represents 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 plot shows negative droplets, and the top shows positive droplets from the VIC, ROX, A425, or CY5 channels, automatically converted to copy concentration by the software.

[0044] Figure 5 This is the detection result for positive control No. 5. The vertical axis of the one-dimensional plot represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, corresponding to the detection of CBFB::MYH11, PML::RARA, NPM1::MLF1, KMT2A::ELL, ETV6::JAK2, RUNX1::RPL22, and RUNX1::MECOM fusion genes. The bottom of the one-dimensional plot shows negative droplets, and the top shows positive droplets for either the VIC, FAM, ROX, A425, CY7, CY5, or CY5.5 channels, automatically converted to copy concentration by the software.

[0045] Figure 6 This is the detection result for positive control 6. The vertical axis of the one-dimensional plot represents 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 plot shows negative droplets, and the top shows positive droplets from the VIC, ROX, A425, or CY5 channels, automatically converted to copy concentration by the software.

[0046] Figure 7This is the detection result of the negative control NC in the primer-probe mixture of well 1. The vertical axis of the one-dimensional plot represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, corresponding to the detection of NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, KAT6A::CREBBP fusion genes and ABL1 internal reference gene. The bottom of the one-dimensional plot represents negative droplets. No positive droplets were found in the VIC, FAM, ROX, A425, CY7, or CY5 channels above. Only the CY5.5 channel showed positive droplets, which were automatically converted to copy concentration by the software.

[0047] Figure 8 This is the detection result of the negative control NC in the primer-probe mixture of well 2. The vertical axis of the one-dimensional plot represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, respectively. The bottom of the one-dimensional plot shows negative droplets, and no positive droplets appear at the top.

[0048] Figure 9 This is the detection result of the negative control NC in the primer-probe mixture of well 3. The vertical axis of the one-dimensional plot represents the VIC, FAM, ROX, A425, CY7, CY5, and CY5.5 channels, respectively. The bottom of the one-dimensional plot shows negative droplets, and no positive droplets appear at the top.

[0049] Figure 10 This is the test result for a BCR::ABL1 positive patient. The vertical axis of the one-dimensional graph represents the FAM channel and CY5.5 channel in the primer-probe mixture in well 1, corresponding to the detection of the BCR::ABL1 fusion gene and the ABL1 internal reference gene, and the result is positive. Detailed Implementation

[0050] To better understand the technical means and effects of this invention, further explanation is provided in conjunction with the following embodiments. The descriptions in these embodiments are for illustrative purposes only and should not, and will not, limit the scope of this invention.

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

[0052] Example 1: Design, Synthesis, and Screening of Primers and Probes

[0053] Primer and probe design tools: The primer pairs and probes involved in this invention were designed using Primer 5.0, PrimerExpress 3.0, NCBI Blast, and clustalx software, and synthesized by Shanghai Bailige Biotechnology Co., Ltd.

[0054] Extensive clinical data and relevant databases were studied to identify 46 fusion genes with high incidence rates associated with myeloid leukemia. The mutation sites of each fusion gene were analyzed to pinpoint the breakpoints in partner and driver genes, and multiple primer and probe sets were designed across the fusion breakpoint regions.

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

[0056] The primer-probe mixture in well 1 includes:

[0057] (1) Primers for amplifying the NUP98 series fusion genes, the nucleotide sequences of which are shown in SEQ ID NO.001-010, and probes, the nucleotide sequences of which are shown in SEQ ID NO.011-012, the probes being linked to the fluorescent reporter group VIC.

[0058] (2) Primers for amplifying the BCR::ABL1 fusion gene, the nucleotide sequence of which is shown in SEQ ID NO.013-021, and probe, the nucleotide sequence of which is shown in SEQ ID NO.022-023, the probe being linked to a fluorescent reporter group FAM;

[0059] (3) Primers for amplifying the RUNX1::RUNX1T1 fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.024-025, and probes, the nucleotide sequences of which are shown in SEQ ID NO.026, the probes being linked to the fluorescent reporter group ROX.

[0060] (4) Primers for amplifying MLL series fusion genes, the nucleotide sequences of which are shown in SEQ ID NO.027-034, and probes, the nucleotide sequences of which are shown in SEQ ID NO.035-036, the probes being linked to the fluorescent reporter group A425;

[0061] (5) Primers for amplifying the FIP1L1::PDGFRA fusion gene, the nucleotide sequence of which is shown in SEQ ID NO.037-044, and probe, the nucleotide sequence of which is shown in SEQ ID NO.045-046, the probe being linked to the fluorescent reporter group CY7;

[0062] (6) Primers for amplifying the KAT6A::CREBBP fusion gene, the nucleotide sequence of which is shown in SEQ ID NO.047-051, and probe, the nucleotide sequence of which is shown in SEQ ID NO.052-053, the probe being linked to the fluorescent reporter group CY5;

[0063] (7) Primers for amplifying the ABL1 internal reference gene, the nucleotide sequences of which are shown in SEQ ID NO.054-055, and probes, the nucleotide sequences of which are shown in SEQ ID NO.056, the probes being linked to the fluorescent reporter group CY5.5;

[0064] (8) Primers for amplifying the FUS::ERG fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.057-058, and probes, the nucleotide sequences of which are shown in SEQ ID NO.059, the probes being linked to the fluorescent reporter groups ROX and CY5;

[0065] (9) Primers for amplifying the NPM1::ALK fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.060-061, and probes, the nucleotide sequences of which are shown in SEQ ID NO.062, the probes being linked to the fluorescent reporter groups VIC and A425;

[0066] The primer-probe mixture in well 2 includes:

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

[0068] (11) Primers for amplifying the KMT2A::MLLT3 fusion gene, the nucleotide sequence of which is shown in SEQ ID NO.066-069, and probe, the nucleotide sequence of which is shown in SEQ ID NO.070-071, the probe being linked to a fluorescent reporter group FAM;

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

[0070] (13) Primers for amplifying the KMT2A::MLLT1 fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.027-028 and SEQ ID NO.075-077, and probe, the nucleotide sequence of which is shown in SEQ ID NO.035-036, the probe being linked to a fluorescent reporter group A425;

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

[0072] (15) Primers for amplifying the RARα series fusion genes, the nucleotide sequences of which are shown in SEQ ID NO.080-089, and probes, the nucleotide sequences of which are shown in SEQ ID NO.090-091, the probes being linked to the fluorescent reporter group CY5;

[0073] (16) Primers for amplifying the ETV6::PDGFRB fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.092-093, and probes, the nucleotide sequences of which are shown in SEQ ID NO.094, the probes being linked to the fluorescent reporter group CY5.5;

[0074] (17) Primers for amplifying the KMT2A::MLLT10 fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.027-028 and SEQ ID NO.095-096, and probe, the nucleotide sequences of which are shown in SEQ ID NO.097-098, the probe being linked to the fluorescent reporter groups ROX and CY5;

[0075] (18) Primers for amplifying the SET::NUP214 fusion gene, the nucleotide sequence of which is shown in SEQ ID NO.099-100, and probe, the nucleotide sequence of which is shown in SEQ ID NO.101, the probe being linked to the fluorescent reporter groups VIC and A425;

[0076] The primer-probe mixture in well 3 includes:

[0077] (19) Primers for amplifying the CBFB::MYH11 fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.102-105, and probes, the nucleotide sequences of which are shown in SEQ ID NO.106, the probes being linked to a fluorescent reporter group VIC.

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

[0079] (21) Primers for amplifying the NPM1::MLF1 fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.110-111, and probes, the nucleotide sequences of which are shown in SEQ ID NO.112, the probes being linked to the fluorescent reporter group ROX.

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

[0081] (23) Primers for amplifying the ETV6::JAK2 fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.114-120, and probes, the nucleotide sequences of which are shown in SEQ ID NO.121-122, the probes being linked to the fluorescent reporter group CY7;

[0082] (24) Primers for amplifying the RUNX1::RPL22 fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.123-124, and probe, the nucleotide sequences of which are shown in SEQ ID NO.125, the probe being linked to the fluorescent reporter group CY5;

[0083] (25) Primers for amplifying the RUNX1::MECOM fusion gene, the nucleotide sequences of which are shown in SEQ ID NO.126-129, and probes, the nucleotide sequences of which are shown in SEQ ID NO.130-131, the probes being linked to the fluorescent reporter group CY5.5;

[0084] (26) Primers for amplifying the RUNX1::CBFA2T3 fusion gene, the nucleotide sequences of which are shown in SEQ ID NO. 132-134, and probes, the nucleotide sequences of which are shown in SEQ ID NO. 135, the probes being linked to the fluorescent reporter groups ROX and CY5;

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

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

[0087]

[0088] The + signifies that the following base is modified into a locked nucleic acid (LNA).

[0089] Nucleotides 11-12, 65, and 106 are probes, which are linked to the fluorescent reporter group VIC.

[0090] Nucleotides 22-23, 70-71, and 90 are used as probes, which are linked to the fluorescent reporter group FAM.

[0091] Nucleotides 26, 74, and 112 are used as probes, which are linked to the fluorescent reporter group ROX.

[0092] Nucleotides 35-36 serve as probes, linked to a fluorescent reporter group A425;

[0093] Nucleotides 45-46, 22, and 121-122 are probes, which are linked to the fluorescent reporter group CY7;

[0094] Nucleotides 52-53, 90-91, and 125 are probes, which are linked to the fluorescent reporter group CY5;

[0095] Nucleotides 56, 94, and 130-131 are used as probes, which are linked to the fluorescent reporter group CY5.5;

[0096] Nucleotides 59, 97-98, and 135 are used as probes, which are linked to the fluorescent reporter groups ROX and CY5.

[0097] Nucleotides 62 and 101 are probes, which are linked to the fluorescent reporter groups VIC and A425.

[0098] Example 2: Design and Synthesis of Plasmids

[0099] Positive plasmids were designed for any variant site of any of the 46 myeloid leukemia-related fusion genes. Each positive plasmid has exactly one variant site and is used to test the effectiveness of candidate target sequences for any variant site individually.

[0100] A plasmid targeting the ABL1 internal reference gene was synthesized, numbered NC, to screen primer and probe sequences for the ABL1 internal reference gene, and also to test and optimize the single-well multiplex detection system.

[0101] A long plasmid containing the fusion genes NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, and KAT6A::CREBBP was synthesized and designated PC-1 for testing and optimizing single-well multiplex detection systems.

[0102] A long plasmid containing the fusion genes FUS::ERG and NPM1::ALK was synthesized, designated PC-2, for testing and optimization of single-well multiplex detection systems.

[0103] A long plasmid containing the fusion genes KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, PLZF::RARα, and ETV6::PDGFRB was synthesized and designated PC-3 for testing and optimization of single-well multiplex detection systems.

[0104] A long plasmid containing the KMT2A::MLLT10 and SET::NUP214 fusion genes was synthesized, designated PC-4, for testing and optimization of single-well multiplex detection systems.

[0105] A long plasmid containing the fusion genes CBFB::MYH11, PML::RARA, NPM1::MLF1, KMT2A::ELL, ETV6::JAK2, RUNX1::RPL22, and RUNX1::MECOM was synthesized and designated PC-5 for testing and optimizing single-well multiplex detection systems.

[0106] A long plasmid containing the fusion genes RUNX1::CBFA2T3 and DEK::NUP214 was synthesized, designated PC-6, for testing and optimizing single-well multiplex detection systems.

[0107] Example 3: Preparation and Assembly of the Reagent Kit

[0108] (1) Reverse transcription reagent: containing RT enzyme, RNase inhibitor, dNTP, Oligo dT (18T) Primer, Random 6 mers Primer, and reaction buffer;

[0109] (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;

[0110] (3) Digital PCR microfluidic chip and droplet generation oil were purchased from Leading Gene Technology (Hangzhou) Co., Ltd.

[0111] (4) Primer-probe mixture in well 1: SEQ ID NO.001-062: The primers and probes are 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;

[0112] (5) Primer-probe mixture in well 2: SEQ ID NO.020, SEQ ID NO.022, SEQ ID NO.027-028, SEQ ID NO.035-036, SEQ ID NO.063-101: The primers and probes are dissolved in double-distilled water to a concentration of 100 μM and prepared into a 6× primer-probe premix in a specific ratio;

[0113] (6) Primer-probe mixture in well 3: 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: The primers and probes are dissolved in double-distilled water to a concentration of 100 μM and prepared into a 6× primer-probe premix in a specific ratio;

[0114] (7) Positive control No. 1: a long fragment plasmid containing the fusion genes NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, KAT6A::CREBBP, and mixed with the ABL1 internal reference gene plasmid in a specific ratio to prepare a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid is about 75 copies / μL, and the concentration of the ABL1 internal reference plasmid is about 1500 copies / μL.

[0115] (8) Positive control No. 2: A long fragment plasmid containing the fusion genes FUS::ERG and NPM1::ALK, mixed with the ABL1 internal reference gene plasmid in a specific ratio to prepare a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid is about 75 copies / μL, and the concentration of the ABL1 internal reference plasmid is about 1500 copies / μL.

[0116] (9) Positive control No. 3: Long fragment plasmids containing the fusion genes KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, PLZF::RARα, and ETV6::PDGFRB are mixed with the ABL1 internal reference gene plasmid in a specific ratio to prepare a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid is about 75 copies / μL, and the concentration of the ABL1 internal reference plasmid is about 1500 copies / μL.

[0117] (10) Positive control No. 4: a long fragment plasmid containing the KMT2A::MLLT10 and SET::NUP214 fusion genes, mixed with the ABL1 internal reference gene plasmid in a specific ratio to prepare a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid is about 75 copies / μL, and the concentration of the ABL1 internal reference plasmid is about 1500 copies / μL.

[0118] (11) Positive control No. 5: Long fragment plasmids containing CBFB::MYH11, PML::RARA, NPM1::MLF1, KMT2A::ELL, ETV6::JAK2, RUNX1::RPL22, and RUNX1::MECOM fusion genes were mixed with the ABL1 internal reference gene plasmid in a specific ratio to prepare a 5% concentration mixture. Specifically, 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.

[0119] (12) Positive control No. 6: a long fragment plasmid containing the fusion genes RUNX1::CBFA2T3 and DEK::NUP214, mixed with the ABL1 internal reference gene plasmid in a specific ratio to prepare a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid is about 75 copies / μL, and the concentration of the ABL1 internal reference plasmid is about 1500 copies / μL.

[0120] (13) NC: ABL1 internal reference gene plasmid diluted to the specified concentration, specifically, the concentration is about 1500 copies / μL.

[0121] Example 4: Detection method for myeloid leukemia-related fusion genes

[0122] Based on the reagent kit materials prepared in Example 3, the detection method is as follows:

[0123] (1) Sample RNA template preparation: The test sample can be fresh bone marrow or peripheral blood. It is recommended to use a commercially available kit to extract RNA from the sample. During the extraction process, strictly follow the instructions. It is recommended to perform reverse transcription immediately after the RNA sample is extracted. For short-term storage, please store at -20℃; for long-term storage, please store at -80℃.

[0124] (2) RNA reverse transcription: The extracted RNA was reverse transcribed into cDNA. The reaction system is shown in Table 2.

[0125] After vortexing the reaction solution for a few seconds, briefly centrifuge and proceed with the reverse transcription reaction as follows: 37℃, 15 min; 85℃, 5 s; 4℃, hold.

[0126] (3) Preparation of digital PCR reaction solution:

[0127] The components contained in the PCR reaction buffer are shown in Table 3.

[0128] After removing the reagent kit from the -20°C freezer and equilibrating to room temperature, vortex for several seconds to thoroughly mix. Prepare X μL of reaction premix according to the number of samples to be tested:

[0129] X = (7.5 μL PCR reaction solution + 2.5 μL primer and probe premix) × (n samples + 6 positive controls + 3 NCs + 1 loss).

[0130] Vortex the above reaction premixed solution to mix well, then centrifuge briefly, and aspirate 10 μL / well of the reaction premixed solution into a thin-walled PCR reaction tube or an eight-tube strip.

[0131] (4) Sample addition: Add 5 μL of the cDNA template to be tested, positive control and NC to the PCR tube or octet tube, tighten the cap, vortex to mix and then centrifuge briefly.

[0132] (5) Droplet preparation: Add 15 μL of reaction solution to the injection well of the digital PCR microfluidic chip, tighten the four-cap on the injection well and the outlet well respectively, and place it in the droplet generator. Droplets can generally be generated in about 20 minutes.

[0133] (6) PCR amplification: The microfluidic chip with generated droplets is slowly transferred to the PCR amplification instrument. The PCR reaction parameters are set as follows: 95℃ for 10 min, 40 cycles (98℃ for 15 s, 62℃ for 1 min), 28℃ for 5 min, and 28℃ hold.

[0134] (7) Results analysis: After amplification, the microfluidic chip was placed in the Navigator chip reader, the supporting software was opened, the information of each reaction well was simply set, and after preheating, the camera was scanned. The instrument will automatically perform fluorescence reading and analysis, and calculate the copy number concentration and total copy number of each channel in each reaction well, providing one-dimensional map, two-dimensional map and original image of each channel in each reaction well.

[0135] (8) The results of droplet count, positive control, NC and blank control must simultaneously meet the following conditions:

[0136] 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.

[0137] 2) Positive control No. 1: All seven channels tested positive; such as Figure 1 As shown.

[0138] 3) Positive control No. 2: All five channels (A425, VIC, ROX, CY5, and CY5.5) tested positive; Figure 2 As shown.

[0139] 4) Positive control No. 3: All seven channels tested positive; if Figure 3 As shown.

[0140] 5) Positive control #4: All four channels (A425, VIC, ROX, and CY5) tested positive; Figure 4 As shown.

[0141] 6) Positive control No. 5: All seven channels tested positive; Figure 5 As shown.

[0142] 7) Positive control #6: All four channels (A425, VIC, ROX, CY5) tested positive; Figure 6 As shown.

[0143] 8) NC: The detection result of the primer-probe mixture negative control NC in Well 1 was positive in the CY5.5 channel and negative in the other channels. Figure 7 As shown; the detection results of the primer-probe mixture negative control NC in well 2 were all negative in all seven channels, as indicated. Figure 8 As shown; the detection results of the primer-probe mixture negative control NC in well 3 were all negative in all seven channels, as indicated. Figure 9 As shown.

[0144] 9) The sample results shall be interpreted according to Table 4, and the variation frequency shall be calculated based on the test results.

[0145] 10) If the ABL1 internal standard gene detection value of a sample is <50 copies / μL, it indicates that the amount of cDNA added is insufficient. The amount of cDNA added to the sample needs to be increased, or the sample needs to be re-extracted and then PCR detection should be performed. The result should be determined according to the table above.

[0146] The expression level of each fusion gene is calculated as: fusion gene copy concentration / ABL1 internal standard gene copy concentration * 100%.

[0147] Table 2 Reverse Transcription System

[0148]

[0149] Note: The amount of RNA can be added as needed. It is recommended to use a maximum of 2 μg of total RNA in a 20 μL reverse transcription system.

[0150] Table 3. Components of PCR reaction buffer

[0151]

[0152] Note: * indicates reinforcing agent components.

[0153] Table 4. Rules for interpreting reagent kit results

[0154]

[0155] Example 5: Screening of Fluorescent Labels on the Same Target

[0156] This kit utilizes a digital PCR platform that can accommodate up to seven fluorescence channels per well: A425, VIC, FAM, ROX, CY5, CY5.5, and CY7 channels. Compared to the common four-channel configuration in quantitative PCR, this digital PCR platform can accommodate three more channels (A425, CY5.5, and CY7) per well, increasing the possibility of detecting more targets per well.

[0157] Furthermore, this kit cleverly incorporates a dual-channel design with ROX+CY5 and A425+VIC channels. This means that when both the ROX and CY5 channels, or the A425 and VIC channels, show a positive signal in a single well, it is interpreted as detecting two other targets. This effectively expands the single-well range from 7 channels to 9 channels, allowing for the detection of more targets per well. Consequently, the requirements for the fluorescent labeling of each target are inevitably higher.

[0158] Therefore, for the same target, the system screened fluorescent markers VIC, FAM, ROX, A425, CY7, CY5, and CY5.5, as well as combinations of ROX+633 and VIC+A425 fluorescent markers. Nine tests were performed on each fusion gene (NUP98 series: NUP98::HOXA11 was used for testing; MLL series: MLL::AF17 was used for testing; RARα series: PLZF::RARα was used for testing), totaling 243 comparative tests. The optimal fluorescent marker was selected based on the signal-to-noise ratio. The results of the fluorescent marker selection are shown in Table 5, where the selected fluorescent channels are marked with ※.

[0159] Based on the screening results in Table 5, the optimal fluorescent labeling channel (with the highest signal-to-noise ratio) for each fusion gene was selected for subsequent well combination tests.

[0160] Table 5. Results of Fluorescent Label Screening

[0161]

[0162] Example 6: Optimization of the PCR Enhancer Reaction System

[0163] Based on the screening results of Example 5, this kit mainly focuses on one combination scheme to verify the performance enhancement effect of PCR enhancers on the detection system for myeloid leukemia-related fusion genes. The PCR amplification system and its included enhancer components are shown in Table 3. *In Table 3, the enhancer components are marked with an asterisk (*).

[0164] The PCR amplification system without added PCR enhancer is as follows: 5.5 μL PCR reaction solution, 2.5 μL primer-probe mixture for well 1, or primer-probe mixture for well 2, or primer-probe mixture for well 3, 5 μL cDNA from the detection sample, 2 μL DEPC water, and a total system volume of 15 μL; the PCR amplification conditions are: 95℃ for 10 min, 40 cycles (98℃ for 15 s, 62℃ for 1 min), 28℃ for 5 min, and 28℃ hold.

[0165] The PCR amplification system with added PCR enhancer is as follows: 7.5 μL PCR reaction solution, 2.5 μL primer-probe mixture for well 1, or primer-probe mixture for well 2, or primer-probe mixture for well 3, 5 μL cDNA from the detection sample, for a total system of 15 μL; the PCR amplification conditions are: 95℃ for 10 min, 40 cycles (98℃ for 15 s, 62℃ for 1 min), 28℃ for 5 min, and 28℃ hold.

[0166] In this embodiment, the probe combination scheme is shown in Table 6, and the comparison of the reinforcing agent effect under the same combination scheme is shown in Table 7.

[0167] When the above primer-probe combination scheme was used to arrange and combine 46 fusion genes, the detection results showed that the target concentration and signal-to-noise ratio of the PCR reaction solution with the addition of PCR enhancer were generally higher than those without PCR enhancer. Furthermore, without PCR enhancer, non-specific amplification occurred in the NC of well 1 and well 2; after adding PCR enhancer, the non-specific amplification disappeared. In summary, PCR enhancer can optimize polymerase fidelity, reduce erroneous extension, and improve amplification efficiency; it can block background interference and improve the signal-to-noise ratio; and it can inhibit primer dimerization and non-specific amplification.

[0168] Table 6 Probe Combination Scheme

[0169]

[0170] Note: 1. The MLL series includes four fusion genes: MLL::AF17, MLL::AF1p, MLL::AF1q, and MLL::SEPT6.

[0171] 2. The RARα series includes 7 fusion genes: PLZF::RARα, STAT5b::RARα, PRKAR1A::RARα, FIPIL1::RARα, NPM1::RARα, BCOR::RARA, and NABP1::RARA.

[0172] 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.

[0173] Table 7 Comparison of Detection Results

[0174]

[0175] Note: Templates starting with "Z" indicate that PCR enhancer was added to the PCR reaction solution, while those not starting with "Z" indicate that PCR enhancer was not added to the PCR reaction solution. For example, PC-1 indicates a test result without PCR enhancer; Z-PC-1 indicates a test result with PCR enhancer; and so on. The templates in wells 1, 2, and 3 have the same meaning as PC-1 and Z-PC-1.

[0176] Example 7: Reagent Kit Well Combination

[0177] While ensuring that the single-channel test results of each fusion gene candidate target sequence are good enough, the more crucial point is that the primer and probe sequences of any fusion gene do not interfere with the nucleotide sequences of other fusion genes in the same well, and will not form dimers between primers or between primers and probes. There will also be no non-specific amplification due to unreasonable primer and probe design or well arrangement.

[0178] Therefore, based on the screening results of Example 5, this kit has tried a variety of well combination schemes and selected a combination in which the probes do not interfere with each other and the detection effect meets the requirements. Three of these combination schemes are 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.

[0179] When the first primer-probe combination scheme (Table 8) was used to arrange and combine 46 fusion genes, as shown in Table 9, the six fusion genes contained in PC-1 of the first well, as well as the ABL1 internal reference gene, were detected normally. In PC-2, FUS::ERG, because it was labeled with both ROX and CY5 probes, showed positive signals in both channels simultaneously, and the detection concentrations were comparable. Similarly, NPM1::ALK, because it was labeled with both A425 and VIC fluorescence signals, also showed positive amplification in both channels simultaneously, and the detection concentrations were comparable. Overall, the detection results were normal. However, non-specific amplification occurred in the A425 channel of NC. After investigation, it was found that the primer-probe interaction between RUNX1::RPL22 and the MLL series was problematic. The well positions of RUNX1::RPL22 were then adjusted accordingly.

[0180] The second well, PC-3, normally detected 7 fusion genes, while PC-4 normally detected the KMT2A::MLLT10 fusion gene labeled with both ROX and CY5, and the SET::NUP214 fusion gene labeled with both A425 and VIC.

[0181] The third well, PC-5, normally detected 7 fusion genes, while PC-6 normally detected the RUNX1::CBFA2T3 fusion gene labeled with both ROX and CY5, and the DEK::NUP214 fusion gene labeled with both A425 and VIC.

[0182] When the second probe combination scheme (Table 10) was used to arrange and combine the 46 fusion genes, the first well PC-1 normally detected the 6 fusion genes and the ABL1 internal reference gene, and PC-2 normally detected the FUS::ERG fusion gene labeled with ROX+CY5 and the NPM1::ALK fusion gene labeled with A425+VIC.

[0183] The concentration of the CY7 channel in the second well, PC-3, was low, only about 1 / 10 of the expected concentration. This may be because the KMT2A::MLLT1 probe had poor competition when labeled with CY7 fluorescent markers in the combination. Therefore, the fluorescent labeling of the KMT2A::MLLT1 fusion gene was subsequently adjusted accordingly.

[0184] The third well, PC-5, normally detected 7 fusion genes, while PC-6 normally detected the RUNX1::CBFA2T3 fusion gene labeled with both ROX and CY5, and the DEK::NUP214 fusion gene labeled with both A425 and VIC.

[0185] When the third probe combination scheme (Table 12) was used to arrange and combine the 46 fusion genes, the first well PC-1 normally detected the 6 fusion genes and the ABL1 internal reference gene, and PC-2 normally detected the FUS::ERG fusion gene labeled with ROX+CY5 and the NPM1::ALK fusion gene labeled with A425+VIC.

[0186] The second well, PC-3, normally detected 7 fusion genes, while PC-4 normally detected the KMT2A::MLLT10 fusion gene labeled with both ROX and CY5, and the SET::NUP214 fusion gene labeled with both A425 and VIC.

[0187] The third well, PC-5, normally detected 7 fusion genes, while PC-6 normally detected the RUNX1::CBFA2T3 fusion gene labeled with both ROX and CY5, and the DEK::NUP214 fusion gene labeled with both A425 and VIC.

[0188] In summary, all targets in the third primer combination scheme were detected normally, the concentrations met the expectations, and no non-specific amplification occurred. Therefore, this combination scheme was selected as the final fusion gene combination scheme used in this kit.

[0189] Table 8 First Probe Combination Scheme

[0190]

[0191] Note: 1. The MLL series includes four fusion genes: MLL::AF17, MLL::AF1p, MLL::AF1q, and MLL::SEPT6.

[0192] 2. The RARα series includes 7 fusion genes: PLZF::RARα, STAT5b::RARα, PRKAR1A::RARα, FIPIL1::RARα, NPM1::RARα, BCOR::RARA, and NABP1::RARA.

[0193] 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.

[0194] Table 9. Detection results of the first probe combination scheme

[0195]

[0196] Table 10 Second Probe Combination Scheme

[0197]

[0198] Note: 1. The MLL series includes four fusion genes: MLL::AF17, MLL::AF1p, MLL::AF1q, and MLL::SEPT6.

[0199] 2. The RARα series includes 7 fusion genes: PLZF::RARα, STAT5b::RARα, PRKAR1A::RARα, FIPIL1::RARα, NPM1::RARα, BCOR::RARA, and NABP1::RARA.

[0200] 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.

[0201] Table 11 Detection results of the second probe combination scheme

[0202]

[0203] Table 12 Third Probe Combination Scheme

[0204]

[0205] Note: 1. The MLL series includes four fusion genes: MLL::AF17, MLL::AF1p, MLL::AF1q, and MLL::SEPT6.

[0206] 2. The RARα series includes 7 fusion genes: PLZF::RARα, STAT5b::RARα, PRKAR1A::RARα, FIPIL1::RARα, NPM1::RARα, BCOR::RARA, and NABP1::RARA.

[0207] 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.

[0208] Table 13 Detection results of the third probe combination scheme

[0209]

[0210] Example 8: Blank Limit Validation of the Reagent Kit

[0211] Peripheral blood or bone marrow samples were collected from 30 healthy individuals. Each clinical sample was tested twice using the detection method described in Example 4, for a total of 60 tests. Experimental data were recorded and the results were analyzed, as shown in Tables 14-16.

[0212] According to Tables 14-16, the blank limit (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 for clinical testing.

[0213] Table 14. Blank limit verification results for primer-probe mixture in Well 1

[0214]

[0215] Table 15. Blank limit verification results of primer-probe mixture in well 2

[0216]

[0217] Table 16. Blank limit verification results for the primer-probe mixture in well 3.

[0218]

[0219] Table 17 Summary of Blank Limit Validation Results for Reagent Kits

[0220]

[0221] Example 9: Validation of the Limit of Detection (LOD) of the Reagent Kit

[0222] Standards at different concentrations of 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, and 0% were prepared based on the internal control gene plasmid and the target gene long fragment plasmid. The concentration of the internal control plasmid was controlled at around 15,000 copies / μL, and the concentrations of each target gene were serially diluted to 75 copies / μL, 15 copies / μL, 7.5 copies / μL, 1.5 copies / μL, 0.75 copies / μL, and 0 copies / μL.

[0223] The detection was performed according to the detection method described in Example 4. Each sample was tested 5 times. The experimental data were recorded and the results were analyzed, as shown in Tables 18-20.

[0224] The results are summarized in Table 21. The lowest concentration with a detection rate ≥95% is taken as the limit of detection of this kit, that is, the limit of detection concentration of each fusion gene is 0.01%, specifically, the limit of detection of the fusion gene is 1.5 copies / μL.

[0225] Table 1. Validation results of the detection limit of the primer-probe mixture in 18 wells.

[0226]

[0227] Note: AH in the table corresponds to:

[0228] A: NUP98 series detection value (copies / μL); B: BCR::ABL1 detection value (copies / μL); C: RUNX1::RUNX1T1 detection value (copies / μL)

[0229] D: MLL series detection value (copies / μL); E: FIP1L1::PDGFRA detection value (copies / μL); F: KAT6A::CREBBP detection value (copies / μL)

[0230] G: FUS::ERG detection value (copies / μL); H: NPM1::ALK detection value (copies / μL)

[0231] Table 19 Detection limit verification results of the primer-probe mixture in well 2

[0232]

[0233] Note: AI in the table corresponds to:

[0234] A: KMT2A::AFDN detection value (copies / μL); B: KMT2A::MLLT3 detection value (copies / μL); C: KMT2A::AFF1 detection value (copies / μL)

[0235] D: KMT2A::MLLT1 detection value (copies / μL); E: ETV6::ABL1 detection value (copies / μL); F: RARα series detection value (copies / μL)

[0236] G: ETV6::PDGFRB detection value (copies / μL); H: KMT2A::MLLT10 detection value (copies / μL); I: SET::NUP214 detection value (copies / μL)

[0237] Table 20 Detection limit validation results for the primer-probe mixture in well 3

[0238]

[0239] Note: AI in the table corresponds to:

[0240] A:CBFB::MYH11 detection value (copies / μL); B:PML::RARA detection value (copies / μL); C:NPM1::MLF1 detection value (copies / μL);

[0241] D:KMT2A::ELL detection value (copies / μL); E:ETV6::JAK2 detection value (copies / μL); F:RUNX1::RPL22 detection value (copies / μL);

[0242] G: RUNX1::MECOM detection value (copies / μL); H: RUNX1::CBFA2T3 detection value (copies / μL); I:DEK::NUP214 detection value (copies / μL);

[0243] Table 21 Summary of Detection Limit Validation Results for Reagent Kits

[0244]

[0245] Example 10: Validation of Clinical Sample Results

[0246] Positive blood or bone marrow samples were collected from patients diagnosed with myeloid leukemia. The samples had been tested with commercially available kits and the corresponding results were known. A total of 61 positive samples that covered the fusion gene detection range of this kit were selected. The kit was used to test the 61 samples according to the detection method described in Example 4. The test results are shown in Table 22.

[0247] The kit provided by this invention was compared with a commercially available kit to test 61 clinical samples. The results showed that the kit provided by this invention detected 44 positive cases, while the commercially available kit detected only 43 positive cases. Compared with the commercially available kit, the kit described by this invention detected one more BCR::ABL1 positive case (sample No. 46).

[0248] like Figure 10 As shown, further analysis of sample NO.46 revealed that the concentration of the internal standard gene was 5537.32 copies / μL, and the concentration of the BCR::ABL1 fusion gene was 0.92 copies / μL, with a fusion gene ratio of 0.02%, which is lower than the detection limit of commercially available kits. Since the detection limit of this kit is 0.01%, and according to the result interpretation method described in Example 4, it is considered positive for BCR::ABL1, verifying that this kit has a significant advantage in detecting low-abundance fusion genes.

[0249] Table 22 Summary of Clinical Sample Comparison Results

[0250]

[0251] The primer-probe combination and matching digital PCR kit described in this invention have the advantages of high specificity, high sensitivity, high accuracy, high tolerability, convenient operation, and low cost, and can provide a scientific reference for the diagnosis, treatment selection, and prognostic evaluation of myeloid leukemia in clinical practice.

Claims

1. A kit for detecting common fusion genes in myeloid leukemia based on multiplex digital PCR, characterized in that, This kit includes at least primer-probe mixture for well 1, primer-probe mixture for well 2, primer-probe mixture for well 3, and PCR reaction solution: The primer-probe mixture in well 1 includes the nucleotides shown in SEQ ID NO.001-SEQ ID NO.062; the primer-probe mixture in well 2 includes the nucleotides shown in SEQ ID NO.020, SEQ ID NO.022, SEQ ID NO.027-028, SEQ ID NO.035-036, and SEQ ID NO.063-101; the primer-probe mixture in well 3 includes the nucleotides shown in SEQ ID NO.027-028, SEQ ID NO.035-036, SEQ ID NO.088, SEQ ID NO.090, SEQ ID NO.100-101, and 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, with concentrations of 0.25M, 1.5wt%, 5mM, 0.05 mg / mL, and 0.25wt% respectively in the PCR reaction solution. • In this context, + indicates that the following base is modified to lock nucleic acid (LNA).

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

3. The reagent kit according to claim 1, characterized in that, The probe has a fluorescent reporter group attached to its 5' end and a fluorescent quencher group attached to its 3' end. Specifically, the attached fluorescent reporter group is: In the primer-probe mixture of well 1, The probes shown in SEQ ID NO.11-12 are linked to the fluorescent reporter group VIC; The probes shown in SEQ ID NO.22-23 are linked to the fluorescent reporter group FAM; The probe shown in SEQ ID NO.26 is linked to the fluorescent reporter group ROX; The probe shown in SEQ ID NO.35-36 is linked to the fluorescent reporter group A425; The probes shown in SEQ ID NO.45-46 are linked to the fluorescent reporter group CY7; The probes shown in SEQ ID NO.52-53 are linked to the fluorescent reporter group CY5; The probe shown in SEQ ID NO. 56 is linked to the fluorescent reporter group CY5.5; The probe shown in SEQ ID NO.59 is linked to the fluorescent reporter groups ROX and CY5; The probe shown in SEQ ID NO.62 is linked to the fluorescent reporter groups VIC and A425; In the primer-probe mixture of well 2: The probe shown in SEQ ID NO. 65 is linked to the fluorescent reporter group VIC; The probe shown in SEQ ID NO.70-71 is linked to the fluorescent reporter group FAM; The probe shown in SEQ ID NO.74 is linked to the fluorescent reporter group ROX; The probe shown in SEQ ID NO.35-36 is linked to the fluorescent reporter group A425; The probe shown in SEQ ID NO.22 is linked to the fluorescent reporter group CY7; The probe shown in SEQ ID NO. 90-91 is linked to the fluorescent reporter group CY5; The probe shown in SEQ ID NO.94 is linked to the fluorescent reporter group CY5.5; The probes shown in SEQ ID NO. 97-98 are linked to the fluorescent reporter groups ROX and CY5; The probe shown in SEQ ID NO. 101 is linked to the fluorescent reporter groups VIC and A425; In the primer-probe mixture of well 3: The probe shown in SEQ ID NO. 106 is linked to the fluorescent reporter group VIC; The probe shown in SEQ ID NO. 90 is linked to the fluorescent reporter group FAM; The probe shown in SEQ ID NO. 112 is linked to the fluorescent reporter group ROX; The probe shown in SEQ ID NO.35-36 is linked to the fluorescent reporter group A425; The probes shown in SEQ ID NO.121-122 are linked to the fluorescent reporter group CY7; The probe shown in SEQ ID NO.125 is linked to the fluorescent reporter group CY5; The probe shown in SEQ ID NO. 130-131 is linked to the fluorescent reporter group CY5.5; The probe shown in SEQ ID NO.135 is linked to the fluorescent reporter groups ROX and CY5; The probe shown in SEQ ID NO.101 is linked to the fluorescent reporter groups VIC and A425.

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

5. The reagent kit according to claim 1, characterized in that, The PCR reaction solution also contains DNA polymerase, Mg 2+ PCR reaction buffer, dATP, dCTP, dTTP and dGTP, and localization fluorescent dye.

6. The reagent kit according to claim 1, characterized in that, The kit also includes: (1) Reverse transcription reagent: containing RT enzyme, RNase inhibitor, dNTP, Oligo (dT)18 Primer, Random 6mers Primer, and reaction buffer; (2) Digital PCR microfluidic chip, droplet-generated oil; (3) Positive control No. 1: a mixed solution containing NUP98::HOXA11, BCR::ABL1, RUNX1::RUNX1T1, MLL::AF17, FIP1L1::PDGFRA, KAT6A::CREBBP fusion gene variant plasmid and ABL1 internal reference gene plasmid; (4) Positive control No. 2: a mixed solution containing the FUS::ERG and NPM1::ALK fusion gene variant plasmid and the 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 plasmid and ABL1 internal reference gene plasmid; (6) Positive control No. 4: a mixed solution containing the KMT2A::MLLT10 and SET::NUP214 fusion gene variant plasmid and the 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 plasmid and ABL1 internal reference gene plasmid; (8) Positive control No. 6: a mixed solution containing the RUNX1::CBFA2T3 and DEK::NUP214 fusion gene variant plasmid and the ABL1 internal reference gene plasmid; (9) NC: A mixed solution containing the ABL1 internal reference gene plasmid.

7. The reagent kit according to claim 6, characterized in that, In the positive controls, 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.

8. The reagent kit according to claim 1, characterized in that, The amplification system is as follows: 7.5 μL PCR reaction solution, 2.5 μL primer-probe mixture for well 1, or primer-probe mixture for well 2, or primer-probe mixture for well 3, 5 μL cDNA from the detection sample, and a total system of 15 μL. The PCR amplification conditions were: 95℃ for 10 min, 98℃ for 15 s, 62℃ for 1 min, 40 cycles; 28℃ for 5 min, 28℃ hold.

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