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

Abstract

The application provides a kit and method for detecting common fusion genes of lymphoid leukemia based on a multiple digital PCR method. By constructing a synergistic mechanism of primers and fluorescent probes, optimizing the collocation of primers, probes and fluorescent markers, and effectively avoiding the non-specific binding between amplification products. The innovative introduction of a composite reaction aid with a specific formula, namely a PCR enhancer, significantly improves the amplification efficiency and specificity of the multiple detection system. The kit provided by the application realizes the integrated detection capability of seven fluorescent channels, Atto 425, VIC, FAM, ROX, CY5, CY5.5 and CY7 in a single hole design. By innovatively constructing a ROX+CY5 and Atto 425+VIC dual-channel combined detection system, when the ROX and CY5 channels or the Atto 425 and VIC channels in the same detection hole simultaneously present positive signals, two additional detection targets can be specifically interpreted. This innovative detection strategy based on channel combination enables the kit to break through and achieve accurate detection of 18 fusion genes with only 2 detection holes.

Description

Technical Field

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

[0002] Lymphoid leukemia is a malignant hematologic disorder caused by the abnormal proliferation of lymphocytes. Its occurrence is closely related to fusion genes resulting from chromosomal translocations such as BCR::ABL1, ETV6::RUNX1, and KMT2A::AFF1. These fusion genes are not only core molecular markers for disease diagnosis and subtyping, but also have important guiding value for treatment planning and prognostic assessment. Therefore, rapid and accurate detection technologies are urgently needed in clinical practice. Currently, the mainstream detection methods include fluorescence in situ hybridization (FISH), quantitative real-time PCR (qPCR), and next-generation sequencing (NGS). FISH technology can visually display chromosomal translocations, but its sensitivity is low (usually >5%) and it cannot quantify the copy number of fusion genes. qPCR has high sensitivity (up to 0.1%), but it depends on a standard curve, the number of targets detected per well is limited (usually ≤4), and multiplex detection efficiency is low. Although NGS can comprehensively screen for gene variations, it has disadvantages such as high cost and long cycle (3-5 days), and the detection rate of low-abundance fusion genes (such as minimal residual disease, MRD) is unstable. The above methods generally suffer from insufficient multiplex detection capability and difficulty in balancing sensitivity and specificity, especially in complex samples (such as whole blood or bone marrow) where they are susceptible to interference from non-specific amplification. Existing PCR reaction solutions face the following main problems in multiplex digital PCR applications: (1) It is difficult to simultaneously meet the high-efficiency amplification of multiple primer / probe pairs, which easily leads to uneven amplification efficiency and loss of some target signals; (2) When detecting low-abundance fusion genes, non-specific amplification will significantly reduce the signal-to-noise ratio and affect the detection accuracy; (3) Conventional PCR enhancers have limited promoting effect on highly complex templates and multiplex detection systems. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a kit and method for detecting common fusion genes in lymphoblastic leukemia using multiplex digital PCR. The kit described in this invention offers high sensitivity, high specificity, high accuracy, convenient operation, and cost-effectiveness in detecting lymphoblastic leukemia and its subtypes, aiming to assist in the clinical diagnosis, subtyping, clinical treatment selection, and prognostic evaluation of leukemia.

[0004] By constructing a synergistic mechanism between primers and fluorescent probes and optimizing the combination of primers, probes, and fluorescent labels, non-specific binding between amplification products is effectively avoided. A novel composite reaction aid, the PCR enhancer, is introduced. In a multiplex detection system, the PCR enhancer significantly improves the amplification efficiency of each target gene, effectively suppresses non-specific amplification, increases the throughput of single-well multiplex detection, and improves the signal-to-noise ratio of target gene detection, thereby meeting the sensitivity and specificity requirements of detection. The kit provided by this invention achieves integrated detection capabilities for seven fluorescent channels—Atto 425, VIC, FAM, ROX, CY5, CY5.5, and CY7—in a single-well design. Through the innovative construction of a dual-channel joint detection system of ROX+CY5 and Atto 425+VIC, when the ROX and CY5 channels or the Atto 425 and VIC channels simultaneously show positive signals in the same detection well, two additional detection targets can be specifically identified. This innovative detection strategy based on channel combination enables this kit to achieve the breakthrough of accurately detecting 18 fusion genes with only two detection wells.

[0005] This invention provides a kit for detecting common fusion genes in lymphoblastic leukemia based on multiplex digital PCR, the kit comprising at least: (1) Primer-probe mixture in well 1: containing the primer-probe sequences shown in SEQ ID NO. 001-033; wherein SEQ ID NO. 001~002, 004, 006, 008~015, 018~019, 021~023, 025~026, 028~029, and 031~032 are primer nucleotide sequences, and the rest are probe nucleotide sequences; the final concentration of primers and probes is 100~1000 nM; (2) Primer-probe mixture in well 2: contains the primer-probe sequences shown in SEQ ID NO.034-064; of which 034~035, 037~040, 043~044, 046~048, 049~051, 053~054, 056~057, 059~060, and 062~063 are primer nucleotide sequences, and the rest are probe nucleotide sequences; the final concentration of primers and probes is 100~1000 nM.

[0006] Furthermore, the PCR reaction solution in the kit contains PCR enhancers, the components and final concentrations of which are betaine (0.25M), DMSO (1.5wt%), (NH4)2SO4 (5mM), BSA (0.05 mg / mL) and gelatin (0.25wt%).

[0007] Furthermore, the probe is a Taqman probe, with a fluorescent reporter group attached to the 5' end and a fluorescent quencher group attached to the 3' end; the fluorescent reporter group is one or more of Atto 425, VIC, FAM, ROX, CY5, CY5.5, and CY7, and the fluorescent quencher group is one of BHQ1, BHQ2, and BHQ3.

[0008] Furthermore, the common fusion genes in lymphoid leukemia include: BCR::ABL1 e1a2, BCR::ABL1e13a2 / e14a2, BCR::ABL1 e19a2, KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, KMT2A::AFF4, ETV6::RUNX1, TCF3::HLF, FUS::ERG, NPM1::ALK, KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, ETV6::PDGFRB, STIL::TAL1, SET::NUP214.

[0009] (1) The primer nucleotide sequences for amplifying the BCR::ABL1 e1a2 fusion gene are shown in SEQ ID NO: 001~002, and the probe nucleotide sequences are shown in SEQ ID NO: 003; (2) The primer nucleotide sequences for amplifying the BCR::ABL1 e13a2 / e14a2 fusion gene are shown in SEQ ID NO: 002 and SEQ ID NO: 004, and the probe nucleotide sequence is shown in SEQ ID NO: 005; (3) The primer nucleotide sequences for amplifying the BCR::ABL1 e19a2 fusion gene are shown in SEQ ID NO: 002 and SEQ ID NO: 006, and the probe nucleotide sequence is shown in SEQ ID NO: 007; (4) The primer nucleotide sequences for amplifying the MLL series fusion genes are shown in SEQ ID NO: 008~015, and the probe nucleotide sequences are shown in SEQ ID NO: 016~017; the MLL series fusion genes include KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4; (5) The primer nucleotide sequences for amplifying the ETV6::RUNX1 fusion gene are shown in SEQ ID NO: 018~019, and the probe nucleotide sequences are shown in SEQ ID NO: 020; (6) The primer nucleotide sequences for amplifying the TCF3::HLF fusion gene are shown in SEQ ID NO: 021~023, and the probe nucleotide sequences are shown in SEQ ID NO: 024; (7) The primer nucleotide sequences for amplifying the ABL1 internal control gene are shown in SEQ ID NO: 025~026, and the probe nucleotide sequences are shown in SEQ ID NO: 027; (8) The primer nucleotide sequences for amplifying the FUS::ERG fusion gene are shown in SEQ ID NO: 028~029, and the probe nucleotide sequences are shown in SEQ ID NO: 030; (9) The primer nucleotide sequences for amplifying the NPM1::ALK fusion gene are shown in SEQ ID NO: 031~032, and the probe nucleotide sequences are shown in SEQ ID NO: 033; (10) The primer nucleotide sequences for amplifying the KMT2A::AFDN fusion gene are shown in SEQ ID NO: 009 and SEQ ID NO: 034~035, and the probe nucleotide sequences are shown in SEQ ID NO: 036; (11) The primer nucleotide sequences for amplifying the KMT2A::MLLT3 fusion gene are shown in SEQ ID NO: 037~040, and the probe nucleotide sequences are shown in SEQ ID NO: 041~042; (12) The primer nucleotide sequences for amplifying the KMT2A::AFF1 fusion gene are shown in SEQ ID NO: 009, SEQ ID NO: 034 and SEQ ID NO: 043~044, and the probe nucleotide sequence is shown in SEQ ID NO: 045; (13) The primer nucleotide sequences for amplifying the KMT2A::MLLT1 fusion gene are shown in SEQ ID NO: 008~009 and SEQ ID NO: 046~048, and the probe nucleotide sequences are shown in SEQ ID NO: 016~017; (14) The primer nucleotide sequences for amplifying the ETV6::ABL1 fusion gene are shown in SEQ ID NO: 049~051, and the probe nucleotide sequences are shown in SEQ ID NO: 052; (15) The primer nucleotide sequences for amplifying the TCF3::PBX1 fusion gene are shown in SEQ ID NO: 053~054, and the probe nucleotide sequences are shown in SEQ ID NO: 055; (16) The primer nucleotide sequences for amplifying the ETV6::PDGFRB fusion gene are shown in SEQ ID NO: 056~057, and the probe nucleotide sequences are shown in SEQ ID NO: 058; (17) The primer nucleotide sequences for amplifying the STIL::TAL1 fusion gene are shown in SEQ ID NO: 059~060, and the probe nucleotide sequences are shown in SEQ ID NO: 061; (18) The primer nucleotide sequences for amplifying the SET::NUP214 fusion gene are shown in SEQ ID NO: 062~063, and the probe nucleotide sequences are shown in SEQ ID NO: 064; Furthermore, the probe for the BCR::ABL1 e1a2 fusion gene is SEQ ID NO.3, and its 5' end is attached with a fluorescent reporter group VIC; The probe for the BCR::ABL1 e13a2 / e14a2 fusion gene is SEQ ID NO: 005, and its 5' end is attached with a fluorescent reporter group FAM. The probe for the BCR::ABL1 e19a2 fusion gene is SEQ ID NO: 007, and its 5' end is attached with a fluorescent reporter group ROX. The probes for the MLL series fusion genes are SEQ ID NO: 016~017, and their 5' ends are connected to the fluorescent reporter group Atto425; the MLL series fusion genes include KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4; The probe for the ETV6::RUNX1 fusion gene is SEQ ID NO: 020, and its 5' end is attached with a fluorescent reporter group CY7; The probe for the TCF3::HLF fusion gene is SEQ ID NO: 024, and its 5' end is attached with a fluorescent reporter group CY5; ABL1 is an internal control gene, and the probe is SEQ ID NO: 027, with a fluorescent reporter group CY5.5 attached to its 5' end; The probe for the FUS::ERG fusion gene is SEQ ID NO: 030, and its 5' end is connected to the fluorescent reporter groups ROX and CY5; The probe for the NPM1::ALK fusion gene is SEQ ID NO: 033, and its 5' end is connected to the fluorescent reporter groups VIC and Atto 425; The probe for the KMT2A::AFDN fusion gene is SEQ ID NO: 036, and its 5' end is attached with a fluorescent reporter group VIC. The probe for the KMT2A::MLLT3 fusion gene is SEQ ID NO: 041~042, and its 5' end is attached with a fluorescent reporter group FAM; The probe for the KMT2A::AFF1 fusion gene is SEQ ID NO: 045, and its 5' end is attached with a fluorescent reporter group ROX; The probe for the KMT2A::MLLT1 fusion gene is SEQ ID NO: 016~017, and its 5' end is attached with a fluorescent reporter group Atto 425; The probe for the ETV6::ABL1 fusion gene is SEQ ID NO: 052, and its 5' end is attached with a fluorescent reporter group CY7; The probe for the TCF3::PBX1 fusion gene is SEQ ID NO: 055, and its 5' end is attached with a fluorescent reporter group CY5. The probe for the ETV6::PDGFRB fusion gene is SEQ ID NO: 058, and its 5' end is attached with a fluorescent reporter group CY5.5; The probe for the STIL::TAL1 fusion gene is SEQ ID NO: 061, and its 5' end is connected to the fluorescent reporter groups ROX and CY5; The probe for the SET::NUP214 fusion gene is SEQ ID NO: 064, and its 5' end is attached with fluorescent reporter groups VIC and Atto 425.

[0010] Furthermore, the PCR reaction solution also contains DNA polymerase, Mg2+, PCR reaction buffer, dATP, dCTP, dTTP and dGTP, and a localization fluorescent dye.

[0011] Furthermore, the kit also includes: (1) reverse transcription reagents: containing RT enzyme, RNase inhibitor, dNTP, Oligo dT (18T) Primer, Random 6 mers Primer, and reaction buffer; (2) Digital PCR microfluidic chip, droplet-generated oil; (3) Positive control No. 1: a mixed solution containing BCR::ABL1 e1a2, BCR::ABL1 e13a2 / e14a2, BCR::ABL1e19a2, KMT2A::EPS15, ETV6::RUNX1, TCF3::HLF 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, TCF3::PBX1, ETV6::PDGFRB fusion gene variant plasmid and ABL1 internal reference gene plasmid; (6) Positive control No. 4: a mixed solution containing the STIL::TAL1 and SET::NUP214 fusion gene variant plasmid and the ABL1 internal reference gene plasmid; (7) Negative control: A mixed solution containing the ABL1 internal reference gene plasmid; The positive control had a concentration of approximately 75 copies / μL for each fusion gene variant plasmid and a concentration of approximately 1500 copies / μL for the ABL1 internal reference plasmid; the negative control had a concentration of approximately 1500 copies / μL for the ABL1 internal reference plasmid.

[0012] Furthermore, the primer-probe mixture in Well 1 also contains an internal standard system; the internal standard probe has a fluorescent reporter group CY5.5 attached to its 5' end and a fluorescent quencher group BHQ3 attached to its 3' end.

[0013] The present invention also provides a method of using the above-mentioned reagent kit, comprising the following steps: (1) RNA extraction from the sample to be tested, wherein the sample to be tested includes bone marrow and peripheral blood; (2) Perform cDNA reverse transcription; (3) Using the kit described in this invention, perform multiplex digital PCR on the cDNA template; (4) Collect fluorescence signals using a digital PCR reader, and analyze whether the test sample contains the 18 common fusion genes of lymphoid leukemia in the kit described in this invention based on the negative / positive control results, and give the judgment result; The PCR amplification system is as follows: 7.5 μL PCR reaction solution, 2.5 μL primer-probe mixture for well 1 or well 2, 5 μL cDNA from 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.

[0014] The advantages of this invention are: (1) This invention innovatively achieves efficient detection of 18 lymphoblastic leukemia fusion genes by systematically screening the optimal well combination and deeply optimizing the PCR reaction system, requiring only 2 reaction wells.

[0015] 1) Significantly increased detection throughput: Under the same detection cost conditions, the number of targets detected is significantly increased; 2) Cost-effectiveness optimization: Under the same target detection requirements, it not only significantly reduces reagent consumption costs and the amount of bone marrow, whole blood and other samples required for testing, effectively reducing the burden on patients, but also simplifies the experimental operation process and reduces human resource input; 3) Excellent detection performance: While maintaining high throughput and cost-effectiveness, it achieves absolute quantification of target fusion genes, ensuring high sensitivity, high specificity and high accuracy of the detection system; 4) Technological integration and innovation: Through the optimization of the reaction system, technical problems such as uneven amplification efficiency and non-specific interference in multiplex digital PCR have been solved, ensuring the reliability and consistency of the detection results of 18 fusion genes.

[0016] (2) The kit described in this invention can accommodate seven fluorescent channels of Atto 425, VIC, FAM, ROX, CY5, CY5.5 and CY7 in a single well. Furthermore, this invention ingeniously designs a dual-channel combination of ROX+CY5 and Atto 425+VIC. This can be interpreted as the simultaneous appearance of positive signals in the ROX and CY5 channels or the Atto 425 and VIC channels in a single well, indicating two other detection targets. This expands the possibility of detecting more targets and meets the requirement of detecting 18 fusion genes in just 2 wells.

[0017] (3) The kit described in this invention has the basic advantages of PCR, is easy to operate and low in cost, and can meet the requirement of detecting 18 fusion genes in 2 wells through multiple combinations of the system.

[0018] 1) Breaking through the limitation of conventional digital PCR with only 2 channels per well, by using a combined signal interpretation strategy, the target capacity per well is increased to 30-40 nucleotide sequences; 2) Compared to the drawback of requiring multiple wells for detection in a single tube of quantitative real-time PCR with 3-4 channels, this technology effectively increases throughput through a 7-channel + dual-combination design; Attached Figure Description

[0019] Figure 1 This is the detection result for positive control 1. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents droplet index; these represent the VIC, FAM, ROX, Atto 425, CY7, CY5, and CY5.5 channels, respectively, corresponding to the detection of BCR::ABL1 e1a2, BCR::ABL1 e13a2 / e14a2, BCR::ABL1 e19a2, KMT2A::EPS15, ETV6::RUNX1, TCF3::HLF fusion genes, and the ABL1 internal reference gene. The bottom of the one-dimensional graph shows negative droplets, and the top shows positive droplets for either the VIC, FAM, ROX, Atto 425, CY7, CY5, or CY5.5 channels, automatically converted to copy concentration by the software.

[0020] Figure 2 This is the detection result for positive control No. 2. The vertical axis of the one-dimensional plot represents fluorescence intensity, and the horizontal axis represents the droplet index; these represent the VIC, ROX, Atto 425, CY5, and CY5.5 channels, respectively. The ROX+CY5 channel corresponds to the detection of FUS::ERG, and the VIC+Atto 425 channel corresponds to the detection of NPM1::ALK. The bottom of the one-dimensional plot shows negative droplets, and the top shows positive droplets from either the VIC, ROX, Atto 425, CY5, or CY5.5 channels, automatically converted to copy concentration by the software.

[0021] Figure 3 This is the detection result for positive control No. 3. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents the droplet index; these represent the VIC, FAM, ROX, Atto 425, CY7, CY5, and CY5.5 channels, corresponding to the detection of KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, and ETV6::PDGFRB fusion genes, respectively. The bottom of the one-dimensional graph shows negative droplets, and the top shows positive droplets for either the VIC, FAM, ROX, Atto 425, CY7, CY5, or CY5.5 channels, automatically converted to copy concentration by the software.

[0022] Figure 4 This is the detection result for positive control 4. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents the droplet index; these represent the VIC, ROX, Atto 425, and CY5 channels, respectively. The ROX+CY5 channel corresponds to the detection of STIL::TAL1, and the VIC+Atto 425 channel corresponds to the detection of SET::NUP214. The bottom of the one-dimensional graph shows negative droplets, and the top shows positive droplets from the VIC, ROX, Atto 425, or CY5 channels, automatically converted to copy concentration by the software.

[0023] Figure 5 This is the detection result for the negative control. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents the droplet index; these represent the VIC, FAM, ROX, Atto 425, CY7, CY5, and CY5.5 channels, respectively, and the corresponding targets detected in wells 1 and 2. The bottom of the one-dimensional graph shows negative droplets, and the top shows positive droplets from either the VIC, FAM, ROX, Atto 425, CY7, CY5, or CY5.5 channels, which are automatically converted to copy concentration by the software.

[0024] Figure 6This is the test result for a patient who tested positive for BCR::ABL1 e1a2. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents the droplet index; it represents the VIC channel in well 1, corresponding to the detection of BCR::ABL1 e1a2 fusion gene mutations.

[0025] Figure 7 This is the detection result for a SET::NUP214 positive patient. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents the droplet index; they represent the VIC and Atto 425 channels in well 2, respectively, both corresponding to the detection of SET::NUP214 fusion gene mutations. Detailed Implementation

[0026] The following examples are provided to further illustrate the present invention and are intended to explain the invention, not to limit its scope. Unless otherwise specified, all figures are expressed in parts by weight and weight percentages.

[0027] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0028] The embodiments of the present invention will be further described below with reference to several examples.

[0029] It should be understood that the described embodiments are merely some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0030] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0031] Example 1: Design, synthesis, and screening of primers and probes 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.

[0032] Extensive clinical data and relevant databases were studied to identify 18 fusion genes with high incidence rates associated with lymphoblastic leukemia. The mutation sites of each fusion gene were analyzed to determine the breakpoints of the partner and driver genes, and multiple sets of primers and probes were designed across the fusion breakpoint regions.

[0033] For each fusion gene, three or more candidate target sequences were designed. In the early stage, commercially available and universal PCR reaction solutions were used to test each set of candidate target sequences independently in a single channel. 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 target sequences with excellent effect could be screened for each fusion gene.

[0034] Locked nucleotides (LNAs) are introduced into certain sequences, such as SEQ ID NO.004 and SEQ ID NO.005, to reduce interference between primers / probes in multiplex systems, effectively suppress non-target amplification, and improve the specificity of primers and probes. The advantages of LNA sequences are briefly summarized in the table below, focusing on two main genes: Table 1 Comparison of LNA efficacy with and without primer / probe sequences Some sequences use specially designed MGB (Minor Groove Binder) probes, such as SEQ ID NO.016 and SEQ ID NO.017, which have advantages such as high specificity and high sensitivity compared to traditional TaqMan probes.

[0035] Table 2 Primer and probe sequence information Some sequences have a "+" sign in front of the base, indicating that the base is replaced by an LNA-modified nucleotide to improve sequence specificity.

[0036] Example 2 PCR reaction solution optimization Positive plasmids were designed for any mutation site in any of the 18 lymphoblastic leukemia-related fusion genes. Each positive plasmid has exactly one mutation site and is used to test the effectiveness of candidate target sequences for any mutation site individually.

[0037] 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.

[0038] A long plasmid containing the fusion genes BCR::ABL1 e1a2, BCR::ABL1 e13a2 / e14a2, BCR::ABL1 e19a2, KMT2A::EPS15, ETV6::RUNX1, and TCF3::HLF was synthesized and designated PC-1 for testing and optimizing single-well multiplex detection systems.

[0039] 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.

[0040] A long plasmid containing the fusion genes KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, and ETV6::PDGFRB was synthesized and designated PC-3 for testing and optimization of single-well multiplex detection systems.

[0041] A long plasmid containing the STIL::TAL1 and SET::NUP214 fusion genes was synthesized, designated PC-4, for testing and optimization of single-well multiplex detection systems.

[0042] For any of the above plasmids, HindIII restriction sites are introduced at both ends of the target nucleotide sequence to improve the plasmid's reaction efficiency.

[0043] This kit innovatively constructs a dual-channel combined detection capability of ROX+CY5 and Atto 425+VIC based on 7 fluorescence detection channels. The increased detection throughput expands the possibility of detecting more targets in a single well and increases the potential of the system to accommodate more primer-probe pairs. Consequently, the performance requirements of the PCR reaction solution are also increased.

[0044] This kit optimizes the PCR reaction system, screening and validating various reaction solution formulations to ultimately determine the PCR enhancer with specific components added to the PCR reaction solution. In a multiplex detection system, this PCR enhancer significantly improves the amplification efficiency of each target gene, effectively inhibits non-specific amplification, increases the throughput of single-well multiplex detection, and improves the signal-to-noise ratio of target gene detection, thereby meeting the sensitivity and specificity requirements of the detection.

[0045] Table 3 PCR Enhancer Formulation Table 4 Detection results of Well 1 Table 5 Detection results of Well 2 Add the PCR enhancer prepared according to the above formula to the PCR reaction solution. The first well, PC-1, normally detected all the included fusion genes and the ABL1 internal reference gene, with a good signal-to-noise ratio. PC-2 normally detected all fusion genes, with a good signal-to-noise ratio.

[0046] In the second well, PC-3 detected all targets normally except for channel CY7. PC-4 detected all targets normally with a good signal-to-noise ratio.

[0047] The aforementioned PCR enhancer is an essential component of the multiplex detection system of this invention. Experimental verification has shown that by adding the PCR enhancer and optimizing its concentration, the ultimately determined optimal concentration can significantly improve the overall performance of the multiplex detection system, including increasing the signal-to-noise ratio and reducing non-specific amplification.

[0048] Comparative Example 1 Table 6 Detection results of Well 1 Table 7 Detection results of Well 2 When no PCR enhancer is added to the PCR reaction solution, the detection concentrations of PC-1 and PC-2 in the first well are reduced, resulting in a poor signal-to-noise ratio.

[0049] No positive signal was detected in channel CY7 of the second well PC-3, and the detection concentrations in the other channels were all reduced, resulting in a poor signal-to-noise ratio. The detection concentrations of all targets in PC-4 were also reduced, failing to meet the detection requirements.

[0050] Comparative Example 2 Table 8 PCR Enhancer Formulation Table 9 Detection results of Well 1 Table 10 Detection results of Well 2 Adding the PCR enhancer prepared according to the above formula to the PCR reaction solution resulted in normal detection of all targets in PC-1 and PC-2 wells in the first well, with a good signal-to-noise ratio. Non-specific amplification occurred in the VIC and Atto 425 channels of NC, possibly due to excessively high amplification efficiency.

[0051] In the second well, all channels of PC-3 except CY7 were detected normally with a good signal-to-noise ratio. PC-4 detected all targets normally with a good signal-to-noise ratio. Non-specific amplification was observed in FAM, CY7, and CY5.5 of NC, which did not meet the detection requirements.

[0052] Example 3: Optimization of pore site combinations and screening of probe dye modification groups This kit utilizes a digital PCR platform that can accommodate up to seven fluorescence channels per well: Atto 425, VIC, FAM, ROX, CY5, CY5.5, and CY7. Compared to the common four-channel configuration in quantitative PCR, this digital PCR platform can accommodate three additional channels (Atto 425, CY5.5, and CY7) per well, increasing the possibility of detecting more targets per well.

[0053] Table 11 Probe Combination Scheme Note: The MLL series includes four fusion genes: KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4. The PCR reaction solution used in the reaction contains DNA polymerase and Mg. 2+ PCR reaction buffer, dATP, dCTP, dTTP and dGTP, localization fluorescent dye, PCR enhancer; Table 12 Test results of the probe combination scheme When the probe combination scheme was used to arrange and combine 18 fusion genes, the first well PC-1 normally detected all the included 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 Atto 425+VIC.

[0054] The second well, PC-3, normally detected 7 fusion genes, while PC-4 normally detected the STIL::TAL1 fusion gene co-labeled with ROX+CY5 and the SET::NUP214 fusion gene co-labeled with Atto 425+VIC. While ensuring sufficient single-channel testing results for each fusion gene candidate target sequence, the crucial point is that the primer-probe sequences of any fusion gene do not interfere with the nucleotide sequences of other fusion genes in the same well, preventing the formation of primer-to-primer or primer-to-probe dimers, and avoiding non-specific amplification due to improper primer-probe design or well placement.

[0055] Comparative Example 3 Table 13 Probe Combination Scheme Note: The MLL series includes four fusion genes: KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4.

[0056] Table 14 Test results of the probe combination scheme When this primer-probe combination scheme was used to arrange and combine 18 fusion genes, the fusion gene contained in PC-1 of the first well and 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 Atto425 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, nonspecific amplification occurred in the FAM channel of NC. After investigation, it was found that the primer-probe interaction between BCR::ABL1 e13a2 / e14a2 and the MLL series was problematic.

[0057] Comparative Example 4 Table 15 Probe Combination Scheme Note: The MLL series includes four fusion genes: KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4.

[0058] Table 16 Test Results of the Probe Combination Scheme When the probe combination scheme was used to arrange and combine 18 fusion genes, the first well PC-1 normally detected all the included 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 Atto 425+VIC.

[0059] The concentration of the CY7 channel in the second well, PC-3, was low, only about 1 / 10 of the expected concentration. This is because the KMT2A::MLLT1 probe did not perform well when labeled with CY7 fluorescence.

[0060] Comparative Example 5 The TCF3::HLF and TCF3::PBX1 genes were selected, and probes labeled with CY5 and Atto 633 dye modification groups were synthesized respectively. The concentration, signal intensity and signal-to-noise ratio of the detection targets were compared in a multiplex digital PCR system to screen for the optimal fluorescent dye modification group.

[0061] The ABL1 and ETV6::PDGFRB genes were selected, and probes labeled with CY5.5 and Quasar705 dye modification groups were synthesized respectively. The concentration, signal intensity and signal-to-noise ratio of the detection targets were compared in a multiplex digital PCR system to screen for the optimal fluorescent dye modification group.

[0062] The ETV6::RUNX1 and ETV6::ABL1 genes were selected, and probes labeled with CY7 and DYlight 755 dye modification groups were synthesized respectively. The concentration, signal intensity and signal-to-noise ratio of the detection targets were compared in a multiplex digital PCR system to screen for the optimal fluorescent dye modification group.

[0063] Table 17 Screening of dye modification groups for probe 1 well Table 18 Screening of dye modification groups for well 2 probes The detection concentrations of the two probes labeled with CY5 dye-modified groups were not significantly different from those labeled with Atto 633 dye-modified groups, but the former had a better signal-to-noise ratio than the latter.

[0064] Two sets of probes labeled with CY5.5 dye modification groups were selected. The detection concentration of one set of probes was significantly higher than that of the probes labeled with Quasar705 dye modification groups, and the signal-to-noise ratio of the former was higher than that of the latter.

[0065] The detection concentrations of the two probes labeled with CY7 dye modification groups were not significantly different from those labeled with DYlight 755 dye modification groups, but the former had a better signal-to-noise ratio than the latter.

[0066] Example 4: Preparation and Assembly of the Reagent Kit (1) Reverse transcription reagent: containing RT enzyme, RNase inhibitor, dNTP, Oligo dT (18T) Primer, Random 6 mers Primer, and reaction buffer; (2) PCR reaction solution: containing DNA polymerase, Mg 2+ PCR reaction buffer, dATP, dCTP, dTTP and dGTP, localization fluorescent dye, PCR enhancer; (3) Digital PCR microfluidic chip and droplet generation oil were purchased from Leading Gene Technology (Hangzhou) Co., Ltd. (4) Primer-probe mixture in well 1: 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 the following ratio; Table 19 Preparation scheme for primer-probe mixture in well 1 Well 2 primer-probe mixture: 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; Table 20 Preparation scheme for primer-probe mixture in well 2 Positive control 1: Long fragment plasmids containing BCR::ABL1 e1a2, BCR::ABL1 e13a2 / e14a2, BCR::ABL1e19a2, KMT2A::EPS15, ETV6::RUNX1, and TCF3::HLF fusion genes are mixed with the ABL1 internal control gene plasmid in a specific ratio to prepare a 5% concentration mixture. Specifically, the concentration of each fusion gene variant plasmid is approximately 75 copies / μL, and the concentration of the ABL1 internal control plasmid is approximately 1500 copies / μL. Positive control No. 2: A long fragment plasmid containing the FUS::ERG and NPM1::ALK fusion genes, mixed with the ABL1 internal reference gene plasmid in a specific ratio to prepare a 5% concentration solution. 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. Positive control No. 3: Long fragment plasmids containing the KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, and ETV6::PDGFRB fusion genes were mixed with the ABL1 internal control gene plasmid in a specific ratio to prepare 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 control plasmid was approximately 1500 copies / μL. Positive control No. 4: A long fragment plasmid containing the STIL::TAL1 and SET::NUP214 fusion genes, mixed with the ABL1 internal reference gene plasmid in a specific ratio to prepare a 5% concentration solution. Specifically, 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. Negative control: ABL1 internal reference gene plasmid diluted to the specified concentration, specifically, approximately 1500 copies / μL.

[0067] Example 5: Detection method for lymphoblastic leukemia-related fusion genes (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℃.

[0068] (2) RNA reverse transcription: The extracted RNA was reverse transcribed into cDNA. The specific reaction system is as follows. Table 21 Reverse Transcription System 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.

[0069] After vortexing the above 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.

[0070] (3) Preparation of digital PCR reaction solution: After removing the kit described in this invention from the -20℃ freezer, bring it to room temperature and vortex for a few seconds to mix thoroughly. Prepare X μL of reaction premix according to the number of samples to be tested: X = (7.5 μL PCR reaction solution + 2.5 μL primer and probe premix) × (n samples + 4 positive controls + 2 negative controls + 1 loss).

[0071] 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.

[0072] (4) Sample addition: Add 5 μL of the cDNA template to be tested, positive control and negative control to the PCR tube or octet tube, tighten the cap, vortex to mix and then centrifuge briefly.

[0073] (5) Droplet preparation: Add 15 μL / well of reaction solution to the sample inlet end of the digital PCR microfluidic chip described in this invention, and tighten the four-cap provided to the sample inlet end and the outlet end respectively. Place it in the droplet generator, and droplets can generally be generated in about 20 minutes.

[0074] (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.

[0075] (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.

[0076] (8) The results of droplet count, positive control, NC and blank control must simultaneously meet the following conditions: 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. 2) Positive control No. 1: All seven channels tested positive; 3) Positive control No. 2: All five channels (Atto 425, VIC, ROX, CY5, and CY5.5) tested positive; 4) Positive control No. 3: All seven channels tested positive; 5) Positive control No. 4: All four channels (Atto 425, VIC, ROX, and CY5) tested positive; 6) Negative control: The CY5.5 channel test was positive, while the other channels were negative.

[0077] (9) The sample results shall be interpreted according to the table below. The variation frequency shall be calculated based on the test results, as follows: Table 22 Rules for Interpreting Kit Results 1) 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.

[0078] 2) The expression level of each fusion gene is: fusion gene copy concentration / ABL1 internal standard gene copy concentration * 100%.

[0079] Example 6: Validation of the Blank Limit of the Reagent Kit 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 6, for a total of 60 tests. Experimental data were recorded and the results analyzed, as follows: Table 23 Blank Limit Verification Results for Well 1 Table 24. Blank Limit Verification Results for Well 2 The blank limit (LOB) was calculated using the nonparametric method, and the results are summarized below: Table 25 Summary of reagent blank limit validation results The blank limits for this kit in healthy human samples are shown in the table above (95th percentile). The false positive rate for each fusion gene is no higher than 5%, meeting the specificity requirements for clinical testing.

[0080] Example 7: Validation of the Limit of Detection (LOD) of the Reagent Kit 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.

[0081] The detection was performed according to the detection method described in Example 6. Each sample was tested five times. The experimental data were recorded and the results were analyzed, as follows: Table 26 Detection limit verification results for Well 1 Table 27 Detection limit verification results for Well 2 Further analysis of the data is as follows: Table 28 Summary of reagent kit detection limit validation results The lowest concentration with a detection rate of ≥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.

[0082] Example 8: Validation of Clinical Sample Results Blood or bone marrow samples were collected from patients diagnosed with lymphoblastic leukemia. These samples had been tested using commercially available kits, and the corresponding results were known. Fifty positive samples were selected, all of which contained fusion genes within the detection range of this kit. These 50 samples were then tested using this kit and the detection method described in Example 6. The results are as follows: Table 29 Summary of Clinical Sample Comparison Results The kit provided by this invention was compared with a commercially available kit to test 50 clinical samples. The results showed that the kit provided by this invention detected 33 positive cases, while the commercially available kit detected only 32 positive cases. Compared with the commercially available kit, the kit described by this invention detected one more positive case of BCR::ABL1 e1a2 (sample NO.35).

[0083] Further analysis of sample NO.35 revealed that the concentration of the internal standard gene was 5537.32 copies / μL, and the concentration of the BCR::ABL1 e1a2 fusion gene was 0.92 copies / μL, with a fusion gene ratio of 0.02%, which was below the detection limit of commercially available kits and therefore undetectable. Since this kit has a detection limit of 0.01%, and according to the result interpretation method described in Example 6, it was considered positive for BCR::ABL1 e1a2, verifying that this kit has a significant advantage in detecting low-abundance fusion genes.

[0084] Figure 6 This is the test result for a patient who tested positive for BCR::ABL1 e1a2. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents the droplet index. The VIC channel in well 1 corresponds to the detection of BCR::ABL1 e1a2 fusion gene mutations, and the result was positive, with a copy concentration of 483.5 copies / μL, representing 67.32%. No positive droplets were found in the remaining channels in well 1 (excluding the internal control) and in all channels in well 2, indicating that the remaining 17 fusion genes included in this kit were negative.

[0085] Figure 7 This is the test result for a SET::NUP214 positive patient. The vertical axis of the one-dimensional plot represents fluorescence intensity, and the horizontal axis represents droplet index; the VIC and Atto 425 channels in well 2 represent the detection of SET::NUP214 fusion gene variants, respectively. The result was positive, with a copy concentration of 37.65 copies / μL and a proportion of 9.24%. No positive droplets were found in the remaining channels of well 1 (excluding the internal control) and the remaining channels of well 2, indicating that the other 17 fusion genes included in this kit were negative.

[0086] This invention employs a digital PCR platform based on a combination of chip-based and water-in-oil technology. It can divide each sample's 15 μL reaction solution into at least 20,000 independent reaction units, with each unit independently performing single-molecule-level PCR amplification and fluorescence signal acquisition. Compared to qPCR, digital PCR enables absolute quantification of the target without the need for a standard curve, making it particularly suitable for monitoring disease progression. Furthermore, thanks to the principle of extreme dilution, background sequences or PCR inhibitors in the system are also distributed to each reaction unit, significantly reducing interference from background sequences or PCR inhibitors in the unit containing the target sequence, thus improving the tolerance and accuracy of PCR.

[0087] 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 clinical lymphocytic leukemia.

[0088] The above embodiments describe in detail the structure, features, and effects of the present invention. The above description is only a preferred embodiment of the present invention. Any changes made in accordance with the concept of the present invention, or equivalent embodiments modified to have equivalent changes, shall still fall within the scope of protection of the present invention if they do not exceed the scope covered by the specification.

Claims

1. A kit for detecting common fusion genes in lymphoblastic leukemia based on multiplex digital PCR, characterized in that, The kit includes at least: (1) Primer-probe mixture in well 1: containing the primer-probe sequences shown in SEQ ID NO. 001-033; wherein SEQ ID NO. 001~002, 004, 006, 008~015, 018~019, 021~023, 025~026, 028~029, and 031~032 are primer nucleotide sequences, and the rest are probe nucleotide sequences; the final concentration of primers and probes is 100~1000 nM; (2) Primer-probe mixture in well 2: containing the primer-probe sequences shown in SEQ ID NO. 008-009, 016-017, and 034-064; of which SEQ ID NO. 008-009, 034~035, 037~040, 043~044, 046~048, 049~051, 053~054, 056~057, 059~060, and 062~063 are primer nucleotide sequences, and the rest are probe nucleotide sequences; the final concentration of primers and probes is 100~1000 nM; Furthermore, the PCR reaction solution in the kit contains PCR enhancers, the components and final concentrations of which are betaine (0.25M), DMSO (1.5wt%), (NH4)2SO4 (5mM), BSA (0.05 mg / mL) and gelatin (0.25wt%). The primer and probe sequences are as follows: A "+" sign preceding a base in the sequence indicates that the base has been replaced with an LNA-modified nucleotide instead of a regular nucleotide.

2. The reagent kit according to claim 1, characterized in that, The probe is a Taqman probe, with a fluorescent reporter group attached to the 5' end and a fluorescent quencher group attached to the 3' end; the fluorescent reporter group is one or more of Atto 425, VIC, FAM, ROX, CY5, CY5.5, and CY7, and the fluorescent quencher group is one of BHQ1, BHQ2, and BHQ3.

3. The reagent kit according to claim 1, characterized in that, The fusion genes include BCR::ABL1 e1a2, BCR::ABL1 e13a2 / e14a2, BCR::ABL1 e19a2, KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, KMT2A::AFF4, ETV6::RUNX1, TCF3::HLF, FUS::ERG, NPM1::ALK, KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, ETV6::PDGFRB, STIL::TAL1, SET::NUP214; The probe for the BCR::ABL1 e1a2 fusion gene is SEQ ID NO.003, and its 5' end is attached with a fluorescent reporter group VIC; The probe for the BCR::ABL1 e13a2 / e14a2 fusion gene is SEQ ID NO: 005, and its 5' end is attached with a fluorescent reporter group FAM. The probe for the BCR::ABL1 e19a2 fusion gene is SEQ ID NO: 007, and its 5' end is attached with a fluorescent reporter group ROX. The probes for the MLL series fusion genes are SEQ ID NO: 016~017, and their 5' ends are connected to the fluorescent reporter group Atto425; the MLL series fusion genes include KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4; The probe for the ETV6::RUNX1 fusion gene is SEQ ID NO: 020, and its 5' end is attached with a fluorescent reporter group CY7; The probe for the TCF3::HLF fusion gene is SEQ ID NO: 024, and its 5' end is attached with a fluorescent reporter group CY5; ABL1 is an internal control gene, and the probe is SEQ ID NO: 027, with a fluorescent reporter group CY5.5 attached to its 5' end; The probe for the FUS::ERG fusion gene is SEQ ID NO: 030, and its 5' end is connected to the fluorescent reporter groups ROX and CY5; The probe for the NPM1::ALK fusion gene is SEQ ID NO: 033, and its 5' end is connected to the fluorescent reporter groups VIC and Atto425; The probe for the KMT2A::AFDN fusion gene is SEQ ID NO: 036, and its 5' end is attached with a fluorescent reporter group VIC. The probe for the KMT2A::MLLT3 fusion gene is SEQ ID NO: 041~042, and its 5' end is attached with a fluorescent reporter group FAM; The probe for the KMT2A::AFF1 fusion gene is SEQ ID NO: 045, and its 5' end is attached with a fluorescent reporter group ROX; The probe for the KMT2A::MLLT1 fusion gene is SEQ ID NO: 016~017, and its 5' end is attached with a fluorescent reporter group Atto 425; The probe for the ETV6::ABL1 fusion gene is SEQ ID NO: 052, and its 5' end is attached with a fluorescent reporter group CY7; The probe for the TCF3::PBX1 fusion gene is SEQ ID NO: 055, and its 5' end is attached with a fluorescent reporter group CY5; The probe for the ETV6::PDGFRB fusion gene is SEQ ID NO: 058, and its 5' end is attached with a fluorescent reporter group CY5.5; The probe for the STIL::TAL1 fusion gene is SEQ ID NO: 061, and its 5' end is connected to the fluorescent reporter groups ROX and CY5; The probe for the SET::NUP214 fusion gene is SEQ ID NO: 064, and its 5' end is attached with fluorescent reporter groups VIC and Atto 425.

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

5. The reagent kit according to claim 1, characterized in that, The kit also includes: (1) Reverse transcription reagents: containing RT enzyme, RNase inhibitor, dNTP, Oligo dT (18T) Primer, Random 6 mers Primer, and reaction buffer; (2) Digital PCR microfluidic chip, droplet-generated oil; (3) Positive control No. 1: a mixed solution containing BCR::ABL1 e1a2, BCR::ABL1 e13a2 / e14a2, BCR::ABL1e19a2, KMT2A::EPS15, ETV6::RUNX1, TCF3::HLF 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, TCF3::PBX1, ETV6::PDGFRB fusion gene variant plasmid and ABL1 internal reference gene plasmid; (6) Positive control No. 4: a mixed solution containing the STIL::TAL1 and SET::NUP214 fusion gene variant plasmid and the ABL1 internal reference gene plasmid; (7) Negative control: A mixed solution containing the ABL1 internal reference gene plasmid; The positive control consisted of 75 copies / μL for each fusion gene variant plasmid and 1500 copies / μL for the ABL1 internal reference plasmid; the negative control consisted of 1500 copies / μL for the ABL1 internal reference plasmid.

6. The reagent kit according to claim 1, characterized in that, The primer-probe mixture in Well 1 also includes an internal standard system; the internal standard probe in the internal standard system has a fluorescent reporter group CY5.5 attached to its 5' end and a fluorescent quencher group BHQ3 attached to its 3' end.

7. A method of using the reagent kit as described in claim 1, characterized in that, The steps include: (1) RNA extraction from the sample to be tested, wherein the sample to be tested includes bone marrow and peripheral blood; (2) Perform cDNA reverse transcription; (3) Using the kit described in this invention, perform multiplex digital PCR on the cDNA template; (4) Collect fluorescence signals using a digital PCR reader, and analyze whether the test sample contains the 18 common fusion genes of lymphoid leukemia in the kit described in this invention based on the negative / positive control results, and give the judgment result; The PCR amplification system is as follows: 7.5 μL PCR reaction solution, 2.5 μL primer-probe mixture for well 1 or well 2, 5 μL cDNA from 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.

Images