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

Abstract

The invention provides a kit and a method for detecting common fusion genes of lymphoid leukemia based on a multiple digital PCR (Polymerase Chain Reaction) method. By constructing a synergistic effect mechanism of the primer and the fluorescent probe and optimizing matching of the primer, the probe and a fluorescent marker, non-specific binding among amplification products is effectively avoided. A composite reaction aid with a specific formula, namely a PCR enhancer, is innovatively introduced, so that the amplification efficiency and specificity of a multiple detection system are remarkably improved. According to the kit provided by the invention, the integrated detection capability on seven fluorescent channels, namely Atto 425, VIC, FAM, ROX, CY5, CY5.5 and CY7, is realized in a single-hole design. By innovatively constructing an ROX + CY5 and Atto425 + VIC dual-channel joint detection system, when the ROX and CY5 channels or Atto425 and VIC channels in the same detection hole simultaneously present positive signals, two additional detection targets can be specifically interpreted. The innovative detection strategy based on channel combination enables the kit to realize precise detection of 18 fusion genes only through two detection holes in a breakthrough manner.

Description

Technical Field

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

[0002] Lymphoid leukemia is a hematologic malignancy caused by abnormal lymphocyte proliferation. Its development is closely associated with chromosomal translocations, such as BCR::ABL1, ETV6::RUNX1, and KMT2A::AFF1, leading to fusion genes. These fusion genes are not only core molecular markers for disease diagnosis and typing, but also provide valuable guidance for treatment planning and prognosis assessment. Therefore, rapid and accurate detection technologies are urgently needed in clinical practice. Currently, mainstream detection methods include fluorescence in situ hybridization (FISH), real-time quantitative 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 number of fusion gene copies; qPCR has a high sensitivity (up to 0.1%), but it relies on a standard curve, the number of targets detected in a single well is limited (usually ≤4), and the efficiency of multiplex detection is low; although NGS can comprehensively screen gene variations, it has the disadvantages of 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 have the problems of insufficient multiplex detection capabilities and difficulty in balancing sensitivity and specificity, especially in complex samples (such as whole blood or bone marrow), which are easily interfered by nonspecific amplification. Existing PCR reaction solutions mainly face the following problems in multiplex digital PCR applications: (1) It is difficult to simultaneously meet the efficient amplification of multiple primer / probe pairs, and uneven amplification efficiency is prone to occur, resulting in the loss of some target signals; (2) When detecting low-abundance fusion genes, nonspecific amplification will significantly reduce the signal-to-noise ratio, affecting the accuracy of detection; (3) Conventional PCR enhancers have limited promoting effects on highly complex templates and multiplex detection systems. Summary of the Invention

[0003] To address these issues, the present invention provides a kit and method for detecting common fusion genes in lymphoid leukemia using multiplex digital PCR. The kit is capable of detecting lymphoid leukemia and its typing with high sensitivity, specificity, accuracy, ease of use, and cost-effectiveness, aiming to facilitate the clinical diagnosis, typing, treatment selection, and prognosis and efficacy evaluation of leukemia.

[0004] By constructing a synergistic mechanism between primers and fluorescent probes, the combination of primers, probes and fluorescent markers is optimized, effectively avoiding nonspecific binding between amplified products. The innovative introduction of a composite reaction aid, a PCR enhancer, can significantly improve the amplification efficiency of each target gene in the multiple detection system, effectively inhibit nonspecific amplification, increase the single-well multiple detection throughput, and improve the signal-to-noise ratio of target gene detection, thereby meeting the sensitivity and specificity requirements of the detection. The kit provided by the present invention realizes the integrated detection capability of seven fluorescent channels: Atto 425, VIC, FAM, ROX, CY5, CY5.5 and CY7 in a single-well design. By innovatively constructing a dual-channel combined detection system of ROX+CY5 and Atto 425+VIC, when the ROX and CY5 channels or the Atto 425 and VIC channels in the same detection well simultaneously present positive signals, two additional detection targets can be specifically read out. This detection strategy based on innovative channel combination enables this kit to achieve a breakthrough in the accurate detection of 18 fusion genes with only two detection wells.

[0005] The present invention provides a kit for detecting common fusion genes in lymphoid leukemia based on a multiplex digital PCR method, the kit comprising at least:

[0006] (1) Well 1 primer-probe mixture: contains the primer-probe sequences shown in SEQ ID NOs. 001-033; wherein SEQ ID NOs. 001-002, 004, 006, 008-015, 018-019, 021-023, 025-026, 028-029, 031-032 are primer nucleotide sequences, and the rest are probe nucleotide sequences; the final concentration of primers and probes is 100-1000 nM;

[0007] (2) Well 2 primer-probe mixture: contains the primer-probe sequences shown in SEQ ID NO. 034-064; wherein 034-035, 037-040, 043-044, 046-048, 049-051, 053-054, 056-057, 059-060, 062-063 are primer nucleotide sequences, and the rest are probe nucleotide sequences; the final concentration of primers and probes is 100-1000 nM.

[0008] In addition, the PCR reaction solution of the kit contains a PCR enhancer, and the components and final concentrations of the PCR enhancer are betaine (0.25M), DMSO (1.5wt%), (NH4)2SO4 (5mM), BSA (0.05mg / mL) and gelatin (0.25wt%).

[0009] Furthermore, the probe is a Taqman probe, the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group; the 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.

[0010] Furthermore, the common fusion genes of 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.

[0011] (1) The nucleotide sequences of the primers for amplifying the BCR::ABL1 e1a2 fusion gene are shown in SEQ ID NOs: 001 to 002, and the nucleotide sequence of the probe is shown in SEQ ID NO: 003;

[0012] (2) The nucleotide sequences of the primers for amplifying the BCR::ABL1 e13a2 / e14a2 fusion gene are shown in SEQ ID NO: 002 and SEQ ID NO: 004, and the nucleotide sequence of the probe is shown in SEQ ID NO: 005;

[0013] (3) The nucleotide sequences of the primers for amplifying the BCR::ABL1 e19a2 fusion gene are shown in SEQ ID NO: 002 and SEQ ID NO: 006, and the nucleotide sequence of the probe is shown in SEQ ID NO: 007;

[0014] (4) The primer nucleotide sequences for amplifying the MLL series fusion genes are shown in SEQ ID NOs: 008 to 015, and the probe nucleotide sequences are shown in SEQ ID NOs: 016 to 017; the MLL series fusion genes include KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4;

[0015] (5) The nucleotide sequences of the primers for amplifying the ETV6::RUNX1 fusion gene are shown in SEQ ID NOs: 018-019, and the nucleotide sequence of the probe is shown in SEQ ID NO: 020;

[0016] (6) The nucleotide sequences of the primers for amplifying the TCF3::HLF fusion gene are shown in SEQ ID NOs: 021 to 023, and the nucleotide sequence of the probe is shown in SEQ ID NO: 024;

[0017] (7) The nucleotide sequences of the primers for amplifying the ABL1 internal standard gene are shown in SEQ ID NOs: 025-026, and the nucleotide sequence of the probe is shown in SEQ ID NO: 027;

[0018] (8) The nucleotide sequences of the primers for amplifying the FUS::ERG fusion gene are shown in SEQ ID NOs: 028-029, and the nucleotide sequence of the probe is shown in SEQ ID NO: 030;

[0019] (9) The nucleotide sequences of the primers for amplifying the NPM1::ALK fusion gene are shown in SEQ ID NOs: 031-032, and the nucleotide sequence of the probe is shown in SEQ ID NO: 033;

[0020] (10) The nucleotide sequences of the primers for amplifying the KMT2A::AFDN fusion gene are shown in SEQ ID NO: 009 and SEQ ID NO: 034-035, and the nucleotide sequence of the probe is shown in SEQ ID NO: 036;

[0021] (11) The nucleotide sequences of the primers for amplifying the KMT2A::MLLT3 fusion gene are shown in SEQ ID NOs: 037-040, and the nucleotide sequences of the probes are shown in SEQ ID NOs: 041-042;

[0022] (12) The nucleotide sequences of the primers 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 nucleotide sequence of the probe is shown in SEQ ID NO: 045;

[0023] (13) The nucleotide sequences of the primers for amplifying the KMT2A::MLLT1 fusion gene are shown in SEQ ID NOs: 008-009 and SEQ ID NOs: 046-048, and the nucleotide sequence of the probe is shown in SEQ ID NOs: 016-017;

[0024] (14) The nucleotide sequences of the primers for amplifying the ETV6::ABL1 fusion gene are shown in SEQ ID NOs: 049 to 051, and the nucleotide sequence of the probe is shown in SEQ ID NO: 052;

[0025] (15) The nucleotide sequences of the primers for amplifying the TCF3::PBX1 fusion gene are shown in SEQ ID NOs: 053-054, and the nucleotide sequence of the probe is shown in SEQ ID NO: 055;

[0026] (16) The nucleotide sequences of the primers for amplifying the ETV6::PDGFRB fusion gene are shown in SEQ ID NOs: 056-057, and the nucleotide sequence of the probe is shown in SEQ ID NO: 058;

[0027] (17) The nucleotide sequences of the primers for amplifying the STIL::TAL1 fusion gene are shown in SEQ ID NOs: 059-060, and the nucleotide sequence of the probe is shown in SEQ ID NO: 061;

[0028] (18) The nucleotide sequences of the primers for amplifying the SET::NUP214 fusion gene are shown in SEQ ID NOs: 062-063, and the nucleotide sequence of the probe is shown in SEQ ID NO: 064;

[0029] Furthermore, the probe for the BCR::ABL1 e1a2 fusion gene is SEQ ID NO. 3, and the 5′ end thereof is connected to a fluorescent reporter group VIC;

[0030] The probe for the BCR::ABL1 e13a2 / e14a2 fusion gene is SEQ ID NO: 005, with a fluorescent reporter group FAM attached to its 5′ end;

[0031] The probe for the BCR::ABL1 e19a2 fusion gene is SEQ ID NO: 007, with a fluorescent reporter group ROX linked to its 5′ end;

[0032] The probes for the MLL series fusion genes are SEQ ID NOs: 016-017, with the fluorescent reporter group Atto 425 attached to their 5' ends; the MLL series fusion genes include KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4;

[0033] The probe for the ETV6::RUNX1 fusion gene is SEQ ID NO: 020, with a fluorescent reporter group CY7 attached to its 5′ end;

[0034] The probe for the TCF3::HLF fusion gene is SEQ ID NO: 024, with a fluorescent reporter group CY5 attached to its 5′ end;

[0035] ABL1 was the internal standard gene, and the probe was SEQ ID NO: 027, with the fluorescent reporter group CY5.5 linked to its 5′ end;

[0036] The probe for the FUS::ERG fusion gene is SEQ ID NO: 030, with fluorescent reporter groups ROX and CY5 linked to its 5′ end;

[0037] The probe for the NPM1::ALK fusion gene is SEQ ID NO: 033, with the fluorescent reporter groups VIC and Atto 425 linked to its 5′ end;

[0038] The probe for the KMT2A::AFDN fusion gene is SEQ ID NO: 036, with a fluorescent reporter group VIC attached to its 5′ end;

[0039] The probe for the KMT2A::MLLT3 fusion gene is SEQ ID NO: 041-042, and the 5' end thereof is connected to a fluorescent reporter group FAM;

[0040] The probe for the KMT2A::AFF1 fusion gene is SEQ ID NO: 045, with a fluorescent reporter group ROX linked to its 5′ end;

[0041] The probe for the KMT2A::MLLT1 fusion gene is SEQ ID NO: 016-017, with the fluorescent reporter group Atto 425 linked to its 5' end;

[0042] The probe for the ETV6::ABL1 fusion gene is SEQ ID NO: 052, with a fluorescent reporter group CY7 attached to its 5′ end;

[0043] The probe for the TCF3::PBX1 fusion gene is SEQ ID NO: 055, with a fluorescent reporter group CY5 attached to its 5′ end;

[0044] The probe for the ETV6::PDGFRB fusion gene is SEQ ID NO: 058, with a fluorescent reporter group CY5.5 attached to its 5′ end;

[0045] The probe for the STIL::TAL1 fusion gene is SEQ ID NO: 061, with fluorescent reporter groups ROX and CY5 linked to its 5′ end;

[0046] The probe of the SET::NUP214 fusion gene is SEQ ID NO: 064, and the fluorescent reporter groups VIC and Atto 425 are connected to the 5' end of the probe.

[0047] Furthermore, the PCR reaction solution also contains DNA polymerase, Mg2+, PCR reaction buffer, dATP, dCTP, dTTP and dGTP, and positioning fluorescent dye.

[0048] Furthermore, the kit also includes: (1) reverse transcription reagent: containing RT enzyme, RNase Inhibitor, dNTP, Oligo dT (18T) Primer, Random6mers Primer, reaction buffer;

[0049] (2) Digital PCR microfluidic chip and droplet generation oil;

[0050] (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 plasmids and ABL1 internal reference gene plasmid;

[0051] (4) Positive control No. 2: a mixed solution containing FUS::ERG and NPM1::ALK fusion gene variant plasmids and ABL1 internal reference gene plasmid;

[0052] (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 plasmids and ABL1 internal reference gene plasmid;

[0053] (6) Positive control No. 4: a mixed solution containing STIL::TAL1 and SET::NUP214 fusion gene variant plasmids and ABL1 internal reference gene plasmid;

[0054] (7) Negative control: a mixed solution containing the ABL1 internal reference gene plasmid;

[0055] For the positive control, the concentration of each fusion gene variant plasmid was about 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was about 1500 copies / μL; for the negative control, the concentration of the ABL1 internal reference plasmid was about 1500 copies / μL.

[0056] Furthermore, the primer-probe mixture in well 1 also contains an internal standard system; the 5' end of the internal standard probe is connected to a fluorescent reporter group CY5.5, and the 3' end is connected to a fluorescent quencher group BHQ3.

[0057] The present invention also provides a method for using the above-mentioned kit, comprising the following steps: (1) extracting RNA from a sample to be tested, wherein the sample to be tested includes bone marrow and peripheral blood;

[0058] (2) Perform cDNA reverse transcription reaction;

[0059] (3) using the kit of the present invention to perform a multiplex digital PCR reaction on the cDNA template;

[0060] (4) Collecting fluorescence signals using a digital PCR reader, analyzing the sample based on the negative / positive control results to determine whether it contains the 18 common fusion genes of lymphoid leukemia in the kit of the present invention, and providing a determination result;

[0061] The amplification system of the PCR reaction is as follows: 7.5 μL of PCR reaction solution, 2.5 μL of primer-probe mixture in well 1 or well 2, 5 μL of test sample cDNA, and a total system of 15 μL; the PCR amplification conditions are: 95°C for 10 min, 40 cycles (98°C for 15 s, 62°C for 1 min), 28°C for 5 min, and 28°C hold.

[0062] The present invention is beneficial in that:

[0063] (1) The present invention systematically screened the optimal well position combination and deeply optimized the PCR reaction system, innovatively achieving efficient detection of 18 lymphoid leukemia fusion genes with only two reaction wells.

[0064] 1) Significant improvement in detection throughput: Under the same detection cost conditions, the number of target detections has been significantly increased;

[0065] 2) Optimized cost-effectiveness: Under the same target detection requirements, this 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 experimental operation procedures and reduces human resource investment;

[0066] 3) Excellent detection performance: While maintaining high throughput and cost-effectiveness, it achieves absolute quantification of the target fusion gene, ensuring high sensitivity, specificity, and accuracy of the detection system;

[0067] 4) Technological integration innovation: Through reaction system optimization, technical difficulties such as uneven amplification efficiency and nonspecific interference in multiplex digital PCR were resolved, ensuring the reliability and consistency of the detection results of 18 fusion genes.

[0068] (2) A single well of the kit described in the present invention can accommodate seven fluorescent channels, namely, Atto 425, VIC, FAM, ROX, CY5, CY5.5, and CY7. In addition, the present invention has cleverly designed dual-channel combinations of ROX+CY5 and Atto 425+VIC. This means that when positive signals appear simultaneously in the ROX and CY5 channels, or in the Atto 425 and VIC channels in a single well, they are interpreted as two other detection targets, thus expanding the possibility of detecting more targets and realizing the need to detect 18 fusion genes in just two wells.

[0069] (3) The kit described in the present invention has the basic advantages of PCR, is easy to operate and low-cost, and can detect 18 fusion genes in 2 wells through multiple combinations of the system.

[0070] 1) Breaking through the limitation of conventional digital PCR with only two channels per well, by utilizing a combined signal interpretation strategy, the target capacity of a single well is increased to 30-40 nucleotide sequences;

[0071] 2) Compared to the drawback of fluorescence quantitative PCR single-tube 3-4 channels requiring multiple wells for detection, this technology effectively increases throughput through a 7-channel + dual combination design; BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The results for positive control No. 1 are shown. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents droplet index. The VIC, FAM, ROX, Atto 425, CY7, CY5, and CY5.5 channels are represented, respectively, for 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 represents negative droplets, while the top represents positive droplets in the VIC, FAM, ROX, Atto 425, CY7, CY5, or CY5.5 channels. The software automatically converts this to copy concentration.

[0073] Figure 2 This is the test result for positive control No. 2. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents droplet index; these represent the VIC, ROX, Atto425, 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 graph represents negative droplets, while the top represents positive droplets in the VIC, ROX, Atto425, CY5, or CY5.5 channels. The software automatically converts this to copy concentration.

[0074] Figure 3 The results for positive control No. 3 are shown. 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 the KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, and ETV6::PDGFRB fusion genes. The bottom of the one-dimensional graph represents negative droplets, while the top represents positive droplets in the VIC, FAM, ROX, Atto 425, CY7, CY5, or CY5.5 channels. The software automatically converts this to copy concentration.

[0075] Figure 4 The results for positive control No. 4 are shown. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents droplet index; these represent the VIC, ROX, Atto425, 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 represents negative droplets, while the top represents positive droplets in the VIC, ROX, Atto425, or CY5 channels. The software automatically converts these to copy concentrations.

[0076] Figure 5 The results for the negative control are shown. 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, detecting the corresponding targets in wells 1 and 2. The bottom of the one-dimensional graph represents negative droplets, while the top represents positive droplets in the VIC, FAM, ROX, Atto 425, CY7, CY5, or CY5.5 channels. The software automatically converts this to copy concentration.

[0077] Figure 6 The results for a BCR::ABL1 e1a2-positive patient are shown in Figure 1. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents droplet index. This represents the VIC channel in well 1, corresponding to the detection of BCR::ABL1 e1a2 fusion gene mutations.

[0078] Figure 7 The results for a SET::NUP214-positive patient are shown in Figure 1. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents droplet index; they represent the VIC and Atto 425 channels in well 2, respectively, and both correspond to detecting SET::NUP214 fusion gene mutations. DETAILED DESCRIPTION

[0079] The following examples are used to further illustrate the present invention. Their purpose is to illustrate the present invention and should not be construed as limiting the scope of the present invention. Unless otherwise specified, all references are by weight and weight percentage.

[0080] Unless otherwise specified, the raw materials used in the present invention are conventional commercial products; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0081] The embodiments of the present invention are further described below with reference to a number of embodiments.

[0082] It should be clear that the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0083] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0084] Example 1 Design, synthesis and screening of primer probes

[0085] Primer and probe design tools: The primer pairs and probes involved in the present invention were designed by Primer 5.0, PrimerExpress 3.0, NCBI Blast, and Clustalx software, and synthesized by Shanghai Bio-Tech Co., Ltd.

[0086] By studying extensive clinical data and relevant databases, we identified 18 fusion genes with high incidence associated with lymphoid leukemia. We analyzed the mutation sites of each fusion gene, clarified the breakpoints between the partner gene and the driver gene, and designed multiple sets of primers and probes across the fusion breakpoint region.

[0087] For any fusion gene, three or more pairs of candidate target sequences are designed. In the early stage, commercial universal PCR reaction solution is used to conduct independent single-channel testing on each group of candidate target sequences. The copy concentration results of digital PCR and the one-dimensional or two-dimensional effect diagram of the positive droplets are used as the judgment criteria until one group of target sequences with excellent effects can be screened for each fusion gene.

[0088] Locked Nucleic Acid (LNA) sequences are introduced into some sequences, such as SEQ ID NO.004 and SEQ ID NO.005, to reduce mutual interference between primers and probes in multiplex systems, effectively inhibit non-target amplification, and improve the specificity of primers and probes. The advantages of LNA sequences are briefly summarized in the following table, which mainly selects two genes:

[0089] Table 1 Comparison of primer and probe sequences with and without LNA effects

[0090]

[0091] 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.

[0092] Table 2 Primer and probe sequence information

[0093]

[0094]

[0095] Some sequence bases are preceded by a "+" sign, indicating that the base is replaced by an LNA-modified nucleotide instead of an ordinary nucleotide to improve sequence specificity.

[0096] Example 2 Optimization of PCR reaction solution

[0097] Positive plasmids were designed for any variant site of 18 lymphoid leukemia-related fusion genes, that is, each positive plasmid had only one variant site, which was used to test the effect of any candidate target sequence of the variant site alone.

[0098] A plasmid targeting the ABL1 internal reference gene was synthesized and numbered NC. It was used to screen the primer and probe sequences of the ABL1 internal reference gene and was also used to test and optimize the single-well multiplex detection system.

[0099] A long plasmid containing the BCR::ABL1 e1a2, BCR::ABL1 e13a2 / e14a2, BCR::ABL1 e19a2, KMT2A::EPS15, ETV6::RUNX1, and TCF3::HLF fusion genes was synthesized and numbered PC-1 for testing and optimization of the single-well multiplex detection system.

[0100] A long plasmid containing the FUS::ERG and NPM1::ALK fusion genes was synthesized and numbered PC-2 for testing and optimization of the single-well multiplex detection system.

[0101] A long fragment plasmid containing the KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, and ETV6::PDGFRB fusion genes was synthesized and numbered PC-3 for testing and optimization of the single-well multiplex detection system.

[0102] A long plasmid containing the STIL::TAL1 and SET::NUP214 fusion genes was synthesized and numbered PC-4 for testing and optimization of the single-well multiplex detection system.

[0103] For any of the above plasmids, HindIII restriction sites were introduced at both ends of the target nucleotide sequence to improve the reaction efficiency of the plasmid.

[0104] This kit innovatively builds the dual-channel combined detection capability of ROX+CY5 and Atto 425+VIC based on 7 fluorescence detection channels. The increase in detection throughput expands the possibility of detecting more targets in a single well and increases the potential to accommodate more primer-probe pairs in the system, which in turn increases the performance requirements of the PCR reaction solution.

[0105] This kit optimizes the PCR reaction system, screens and validates multiple reaction solution formulations, and ultimately identifies a specific PCR enhancer added to the PCR reaction solution. This PCR enhancer significantly improves the amplification efficiency of each target gene in a multiplex detection system, effectively inhibits nonspecific amplification, increases single-well multiplex detection throughput, and improves the signal-to-noise ratio of target gene detection, thereby meeting the sensitivity and specificity requirements of the assay.

[0106] Table 3 PCR enhancer formula

[0107] Components Betaine DMSO <![CDATA[(NH4)2SO4]]> BSA gelatin Final concentration 0.25M 1.50wt% 5mM 0.05mg / mL 0.25wt%

[0108] Table 4 Test results of hole 1

[0109]

[0110]

[0111] Table 5 Test results of hole 2

[0112] aisle PC-3 Signal-to-noise ratio PC-4 Signal-to-noise ratio NC VIC 34.9 2.5 32.834 2.6 0 FAM 29.382 2.5 0 0 0 ROX 33.943 2.4 30.63 2.6 0 Atto425 30.32 2.1 31.65 2.4 0 CY7 3.92 2.2 0 0 0 CY5 30.38 2.8 31.273 2.5 0 CY5.5 30.372 2.8 0 0 0

[0113] The PCR enhancer prepared using the above formula was added to the PCR reaction solution. PC-1 in the first well detected all fusion genes and the ABL1 internal reference gene normally, with a good signal-to-noise ratio. PC-2 also detected all fusion genes normally, with a good signal-to-noise ratio.

[0114] In the second well, PC-3 detected all channels except CY7 normally. PC-4 detected all targets normally with a good signal-to-noise ratio.

[0115] The aforementioned PCR enhancer is an essential component of the multiplex detection system of the present invention. Experimental validation has shown that by adding the PCR enhancer and optimizing its concentration, the optimal concentration determined can significantly improve the overall performance of the multiplex detection system, including increasing the signal-to-noise ratio and reducing nonspecific amplification.

[0116] Comparative Example 1

[0117] Table 6 Test results of well 1

[0118] aisle PC-1 Signal-to-noise ratio PC-2 Signal-to-noise ratio NC VIC 18.64 1.8 20.372 1.3 0 FAM 20.521 1.5 0 0 0 ROX 21.832 1.4 21.34 1.4 0 Atto425 19.372 1.2 19.32 1.3 0 CY7 20.42 1.4 0 0 0 CY5 21.37 1.5 18.283 1.4 0 CY5.5 300.45 2.4 300.52 2.4 338.402

[0119] Table 7 Test results of hole 2

[0120] aisle PC-3 Signal-to-noise ratio PC-4 Signal-to-noise ratio NC VIC 23.621 1.4 19.372 1.5 0 FAM 25.34 1.3 0 0 0 ROX 19.372 1.4 16.372 1.3 0 Atto425 15.372 1.3 18.31 1.4 0 CY7 0 1.2 0 0 0 CY5 20.271 1.5 18.54 1.4 0 CY5.5 15.982 2.0 0 0 0

[0121] When no PCR enhancer was added to the PCR reaction solution, the detection concentrations of PC-1 and PC-2 in the first well were reduced, and the signal-to-noise ratio was poor.

[0122] In the second well, PC-3, the CY7 channel did not detect any positive signals, and the detection concentrations of the other channels were reduced, resulting in a poor signal-to-noise ratio. The detection concentrations of all targets in PC-4 were also reduced, not meeting the detection requirements.

[0123] Comparative Example 2

[0124] Table 8 PCR enhancer formula

[0125] Components Betaine DMSO Final concentration 0.5M 1.50wt%

[0126] Table 9 Test results of hole 1

[0127] aisle PC-1 Signal-to-noise ratio PC-2 Signal-to-noise ratio NC VIC 34.271 3.4 33.281 2.5 2.54 FAM 32.63 2.6 0 0 0 ROX 30.261 2.8 33.291 2.5 0 Atto425 32.63 2.4 32.98 2.6 3.02 CY7 32.609 2.5 0 0 0 CY5 33.61 2.4 33.281 3.4 0 CY5.5 352.51 4.0 365.291 4.0 349.29

[0128] Table 10 Test results of hole 2

[0129] aisle PC-3 Signal-to-noise ratio PC-4 Signal-to-noise ratio NC VIC 32.172 2.6 31.821 2.8 0 FAM 34.921 2.6 0 0 2.8 ROX 31.84 2.5 32.743 2.6 0 Atto425 31.83 2.2 33.28 2.8 0 CY7 10.93 2.1 0 0 0.98 CY5 33.281 2.6 33.76 2.8 0 CY5.5 32.63 3.0 0 0 0.72

[0130] When the PCR enhancer prepared using the above formula was added to the PCR reaction solution, PC-1 and PC-2 in the first well detected all targets normally, with a good signal-to-noise ratio. Nonspecific amplification was observed in the VIC and Atto 425 channels of the NC, possibly due to excessive amplification efficiency.

[0131] In the second well, PC-3 detected all channels except CY7 normally, with a good signal-to-noise ratio. PC-4 detected all targets normally with a good signal-to-noise ratio. However, nonspecific amplification was observed for FAM, CY7, and CY5.5 in NC, which did not meet the detection requirements.

[0132] Example 3: Optimization of pore position combination and screening of probe dye modification groups

[0133] The digital PCR platform used in this kit accommodates up to seven fluorescence channels per well: Atto 425, VIC, FAM, ROX, CY5, CY5.5, and CY7. Compared to the four-channel combination commonly found in fluorescent quantitative PCR, this digital PCR platform can accommodate three more channels per well: Atto 425, CY5.5, and CY7, increasing the potential for detecting more targets per well.

[0134] Table 11: Probing combination scheme

[0135] aisle Kong 1 fusion gene name Kong 2 fusion gene name VIC BCR::ABL1e1a2 KMT2A::AFDN FAM BCR::ABL1e13a2 / e14a2 KMT2A::MLLT3 ROX BCR::ABL1e19a2 KMT2A::AFF1 Atto425 MLL Series KMT2A::MLLT1 CY7 ETV6::RUNX1 ETV6::ABL1 CY5 TCF3::HLF TCF3::PBX1 CY5.5 ABL1 ETV6::PDGFRB ROX+CY5 FUS::ERG STIL::TAL1 Atto425+VIC NPM1::ALK SET::NUP214

[0136] 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, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP, localization fluorescent dye, PCR enhancer;

[0137] Table 12 Detection results of the combined lead-in and probe scheme

[0138]

[0139] When this primer combination scheme was used to arrange and combine 18 fusion genes, PC-1 in the first well normally detected all the fusion genes included and the ABL1 reference gene, and PC-2 normally detected the FUS::ERG fusion gene simultaneously labeled by ROX+CY5 and the NPM1::ALK fusion gene simultaneously labeled by Atto 425+VIC.

[0140] In the second well, PC-3 detected seven fusion genes, while PC-4 detected the STIL::TAL1 fusion gene, simultaneously labeled with ROX and CY5, and the SET::NUP214 fusion gene, simultaneously labeled with Atto425 and VIC. While ensuring sufficient single-channel testing performance for each candidate fusion gene target sequence, the key was ensuring that the primer and probe sequences of any fusion gene did 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 preventing nonspecific amplification due to improper primer-probe design or well placement.

[0141] Comparative Example 3

[0142] Table 13: Probing combination scheme

[0143] aisle Kong 1 fusion gene name Kong 2 fusion gene name VIC BCR::ABL1e1a2 KMT2A::AFDN FAM BCR::ABL1e13a2 / e14a2 KMT2A::MLLT3 ROX BCR::ABL1e19a2 KMT2A::AFF1 Atto425 MLL Series KMT2A::MLLT1 CY7 ETV6::RUNX1 ETV6::ABL1 CY5 TCF3::PBX1 TCF3::HLF CY5.5 ABL1 ETV6::PDGFRB ROX+CY5 FUS::ERG STIL::TAL1 Atto425+VIC NPM1::ALK SET::NUP214

[0144] Note: The MLL series includes four fusion genes: KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4.

[0145] Table 14 Detection results of the combined lead-in and probe scheme

[0146]

[0147] When this primer-probe combination was used to sequence 18 fusion genes, the fusion gene contained in PC-1 in the first well and the ABL1 reference gene were detected normally. FUS::ERG in PC-2, labeled with both ROX and CY5 probes, showed positive signals simultaneously in both channels, with comparable detection concentrations. Furthermore, NPM1::ALK, labeled with both Atto425 and VIC fluorescence signals, also showed positive amplification simultaneously in both channels, with comparable detection concentrations. However, nonspecific amplification was observed in the Atto 425 channel of the NC well. Investigation revealed that this was due to a conflict between the primers and probes for TCF3::PBX1 and the MLL series.

[0148] Comparative Example 4

[0149] Table 15: Probing combination scheme

[0150] aisle Kong 1 fusion gene name Kong 2 fusion gene name VIC BCR::ABL1e1a2 KMT2A::AFDN FAM BCR::ABL1e13a2 / e14a2 KMT2A::MLLT3 ROX BCR::ABL1e19a2 KMT2A::AFF1 Atto425 MLL Series ETV6::ABL1 CY7 ETV6::RUNX1 KMT2A::MLLT1 CY5 TCF3::HLF TCF3::PBX1 CY5.5 ABL1 ETV6::PDGFRB ROX+CY5 FUS::ERG STIL::TAL1 Atto425+VIC NPM1::ALK SET::NUP214

[0151] Note: The MLL series includes four fusion genes: KMT2A::EPS15, KMT2A::MLLT11, KMT2A::FOXO4, and KMT2A::AFF4.

[0152] Table 16 Detection results of the combined lead-in and probe scheme

[0153]

[0154] When this primer combination scheme was used to arrange and combine 18 fusion genes, PC-1 in the first well normally detected all the fusion genes included and the ABL1 reference gene, and PC-2 normally detected the FUS::ERG fusion gene simultaneously labeled by ROX+CY5 and the NPM1::ALK fusion gene simultaneously labeled by Atto 425+VIC.

[0155] The CY7 channel concentration of the second well PC-3 was low, only about 1 / 10 of the expected concentration. This was because the KMT2A::MLLT1 probe had a poor effect when labeled with CY7 fluorescence.

[0156] Comparative Example 5

[0157] TCF3::HLF and TCF3::PBX1 genes were selected, and probes labeled with CY5 and Atto 633 dye modifiers were synthesized, respectively. The concentration, signal intensity, and signal-to-noise ratio of the detected targets were compared in a multiplex digital PCR system to screen the optimal fluorescent dye modifier.

[0158] The ABL1 and ETV6::PDGFRB genes were selected, and probes labeled with CY5.5 and Quasar705 dye modifiers were synthesized, respectively. The concentration, signal intensity, and signal-to-noise ratio of the detected targets were compared in a multiplex digital PCR system to screen the optimal fluorescent dye modifier.

[0159] The ETV6::RUNX1 and ETV6::ABL1 genes were selected, and probes labeled with CY7 and DYlight 755 dye modifiers were synthesized, respectively. The concentration, signal intensity, and signal-to-noise ratio of the detected targets were compared in a multiplex digital PCR system to screen the optimal fluorescent dye modifier.

[0160] Table 17 Screening of dye-modified groups for probes in well 1

[0161]

[0162]

[0163] Table 18 Screening of dye modification groups for probes in well 2

[0164] Gene aisle PC-3 Signal-to-noise ratio aisle PC-3 Signal-to-noise ratio KMT2A::AFDN VIC 29.485 2.0 VIC 28.482 2.0 KMT2A::MLLT3 FAM 28.054 2.0 FAM 29.473 2.0 KMT2A::AFF1 ROX 29.43 2.0 ROX 30.482 2.0 KMT2A::MLLT1 Atto425 27.483 2.2 Atto425 29.83 2.2 ETV6::ABL1 CY7 28.59 4.5 DYlight755 28.473 3.2 TCF3::PBX1 CY5 28.831 1.6 Atto633 29.483 1.2 ETV6::PDGFRB CY5.5 30.592 3.2 Quasar705 20.481 1.8

[0165] There was no significant difference in the detection concentrations of the two groups of probes labeled with CY5 dye modifiers and those labeled with Atto 633 dye modifiers, but the signal-to-noise ratio of the former was better than that of the latter.

[0166] Two groups of probes labeled with CY5.5 dye-modifying groups were selected. The detection concentration of one group of probes was significantly higher than that of the probe labeled with Quasar705 dye-modifying groups, and the signal-to-noise ratio of the former was higher than that of the latter.

[0167] There was no significant difference in the detection concentrations of the two groups of probes labeled with the CY7 dye modifier group and the DYlight 755 dye modifier group, but the signal-to-noise ratio of the former was better than that of the latter.

[0168] Example 4 Preparation and Assembly of Kit

[0169] (1) Reverse transcription reagents: RT enzyme, RNase Inhibitor, dNTPs, Oligo dT(18T) Primer, Random6mers Primer, and reaction buffer;

[0170] (2) PCR reaction solution: containing DNA polymerase, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP, localization fluorescent dye, PCR enhancer;

[0171] (3) Digital PCR microfluidic chip and microdroplet generation oil were purchased from Linghang Gene Technology (Hangzhou) Co., Ltd.

[0172] (4) Primer-probe mixture for well 1: Dissolve the primer-probe in double-distilled water to a concentration of 100 μM and prepare a 6× primer-probe premix according to the following ratios;

[0173] Table 19: Preparation of primer-probe mixture in well 1

[0174]

[0175]

[0176] Well 2 primer-probe mixture: Dissolve the primer-probe in double-distilled water to a concentration of 100 μM and prepare a 6× primer-probe premix according to the specified ratio.

[0177] Table 20: Preparation of primer-probe mixture for well 2

[0178] Serial number Concentration (μM) 1 box (50 tests / box) Serial number Concentration (μM) 1 box (50 tests / box) SEQ ID NO.008 100 6 SEQ ID NO.048 100 3 SEQ ID NO.009 100 6 SEQ ID NO.049 100 4.5 SEQ ID NO.016 100 1.5 SEQ ID NO.050 100 4.5 SEQ ID NO.017 100 1.5 SEQ ID NO.051 100 4.5 SEQ ID NO.034 100 4.5 SEQ ID NO.052 100 1 SEQ ID NO.035 100 4.5 SEQ ID NO.053 100 3 SEQ ID NO.036 100 1 SEQ ID NO.054 100 3 SEQ ID NO.037 100 6 SEQ ID NO.055 100 3 SEQ ID NO.038 100 6 SEQ ID NO.056 100 4.5 SEQ ID NO.039 100 6 SEQ ID NO.057 100 4.5 SEQ ID NO.040 100 6 SEQ ID NO.058 100 1 SEQ ID NO.041 100 1.5 SEQ ID NO.059 100 3 SEQ ID NO.042 100 1.5 SEQ ID NO.060 100 3 SEQ ID NO.043 100 3 SEQ ID NO.061 100 1.5 SEQ ID NO.044 100 3 SEQ ID NO.062 100 4.5 SEQ ID NO.045 100 1 SEQ ID NO.063 100 4.5 SEQ ID NO.046 100 3 SEQ ID NO.064 100 3 SEQ ID NO.047 100 3 Ultrapure water / 4

[0179] Positive Control No. 1: A 5% mixture containing long fragment plasmids of the BCR::ABL1 e1a2, BCR::ABL1 e13a2 / e14a2, BCR::ABL1e19a2, KMT2A::EPS15, ETV6::RUNX1, and TCF3::HLF fusion genes and an ABL1 reference gene plasmid at a specific ratio. Specifically, the concentration of each fusion gene variant plasmid is approximately 75 copies / μL, and the concentration of the ABL1 reference plasmid is approximately 1500 copies / μL.

[0180] Positive Control No. 2: A 5% mixture of a long fragment plasmid containing the FUS::ERG and NPM1::ALK fusion genes and an ABL1 reference gene plasmid at a specific ratio. Specifically, the concentration of each fusion gene variant plasmid is approximately 75 copies / μL, and the concentration of the ABL1 reference plasmid is approximately 1500 copies / μL.

[0181] Positive Control No. 3: A 5% mixture containing a long plasmid containing the KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, and ETV6::PDGFRB fusion genes and an ABL1 reference gene plasmid at a specific ratio. Specifically, the concentration of each fusion gene variant plasmid is approximately 75 copies / μL, and the concentration of the ABL1 reference plasmid is approximately 1500 copies / μL.

[0182] Positive Control No. 4: A 5% mixture of a long plasmid containing the STIL::TAL1 and SET::NUP214 fusion genes and an ABL1 reference gene plasmid at a specific ratio. Specifically, the concentration of each fusion gene variant plasmid is approximately 75 copies / μL, and the concentration of the ABL1 reference plasmid is approximately 1500 copies / μL.

[0183] Negative control: ABL1 internal reference gene plasmid was diluted to a specified concentration, specifically, a concentration of approximately 1500 copies / μL.

[0184] Example 5 Detection Method for Lymphoid Leukemia-Related Fusion Genes

[0185] (1) Sample RNA template preparation: The test sample can be fresh bone marrow or peripheral blood. It is recommended to use a commercial kit to extract the sample RNA to be tested. The extraction process should strictly follow the instructions. It is recommended to immediately perform reverse transcription reaction on the extracted RNA sample. For short-term storage, please place it in a -20℃ refrigerator. For long-term storage, please place it in a -80℃ refrigerator.

[0186] (2) RNA reverse transcription: The extracted RNA is reverse transcribed into cDNA. The reaction system is as follows:

[0187] Table 21 Reverse transcription system

[0188]

[0189]

[0190] Note: The amount of RNA can be increased as needed. In a 20 μL reverse transcription system, a maximum of 2 μg of total RNA is recommended.

[0191] The reaction solution was vortexed for a few seconds and then briefly centrifuged. The reverse transcription reaction was performed according to the following steps: 37°C, 15 min; 85°C, 5 s; 4°C, hold.

[0192] (3) Preparation of digital PCR reaction solution: Take out the reagent kit of the present invention from the -20°C refrigerator and equilibrate to room temperature. Vortex and shake for a few seconds to mix thoroughly. Prepare X μL of reaction premix according to the number of samples to be tested:

[0193] X = (7.5 μL PCR reaction solution + 2.5 μL primer-probe premix) × (n samples + 4 positive controls + 2 negative controls + 1 loss).

[0194] Vortex the above reaction premix to mix thoroughly, then centrifuge briefly and pipette 10 μL / well of the reaction premix into a thin-walled PCR reaction tube or an eight-tube strip.

[0195] (4) Sample addition: Add 5 μL of the cDNA template to be tested, the positive control, and the negative control into the PCR tube or eight-tube strip, tightly cap the tube, vortex to mix, and then centrifuge briefly.

[0196] (5) Droplet preparation: Add 15 μL / well of reaction solution to the injection hole of the digital PCR microfluidic chip described in the present invention, cover the injection hole and outlet with the four-caps provided, and place the chip in a droplet generator. Generally, droplets can be generated in about 20 minutes.

[0197] (6) PCR amplification: The microfluidic chip with generated droplets was slowly transferred to the PCR amplification instrument, and the PCR reaction parameters were set as follows: 95°C for 10 min, 40 cycles (98°C for 15 s, 62°C for 1 min), 28°C for 5 min, and 28°C hold.

[0198] (7) Result analysis: After the amplification is completed, place the microfluidic chip into the pilot chip reader, open the supporting software, simply set the information of each reaction well, and click on the photo scan after preheating. The instrument will automatically read and analyze the fluorescence, and calculate the copy number concentration and total copy number of each channel in each reaction well, and provide a one-dimensional map, a two-dimensional map and the original picture of each channel in each reaction well.

[0199] (8) The droplet count, positive control, NC, and blank control results must meet the following conditions:

[0200] 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;

[0201] 2) Positive control No. 1: All seven channels tested positive;

[0202] 3) Positive control No. 2: Atto 425, VIC, ROX, CY5, and CY5.5 channels all tested positive;

[0203] 4) Positive control No. 3: All seven channels tested positive;

[0204] 5) Positive control No. 4: Atto 425, VIC, ROX, and CY5 all tested positive;

[0205] 6) Negative control: CY5.5 channel detection is positive, and the other channels are negative.

[0206] (9) The sample results are interpreted as shown in the table below. The mutation frequency is calculated based on the test results, as follows:

[0207] Table 22 Rules for interpreting test kit results

[0208]

[0209] 1) If the ABL1 internal standard gene detection value is less than 50 copies / μL, it indicates that the amount of cDNA added is insufficient. The sample should be increased or re-extracted and then tested by PCR. The results should be determined according to the table above.

[0210] 2) The expression level of each fusion gene is: fusion gene copy concentration / ABL1 internal standard gene copy concentration*100%.

[0211] Example 6 Test Kit Blank Limit Verification

[0212] 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. The experimental data were recorded and the results were analyzed as follows:

[0213] Table 23 Blank limit verification results for well 1

[0214]

[0215]

[0216] Table 24 Blank limit verification results for well 2

[0217]

[0218]

[0219]

[0220] The limit of blank (LOB) was calculated according to the non-parametric method and the results are summarized as follows:

[0221] Table 25 Summary of the blank limit verification results of the test kit

[0222]

[0223] The blank limit of this kit in healthy human samples is specifically shown in the above table (95th percentile). The false positive rate of each fusion gene is less than 5%, meeting the specific requirements of clinical detection.

[0224] Example 7 Kit Detection Limit (LOD) Verification

[0225] Standards of different concentrations, such as 0.5%, 0.1%, 0.05%, 0.01%, 0.005% and 0%, were prepared based on the internal reference gene plasmid and the target gene long fragment plasmid. The concentration of the internal reference plasmid was controlled at around 15,000 copies / μL, and the concentrations of each target were gradiently diluted to 75 copies / μL, 15 copies / μL, 7.5 copies / μL, 1.5 copies / μL, 0.75 copies / μL and 0 copies / μL.

[0226] The detection method described in Example 6 was used for detection, and each sample was tested 5 times. The experimental data were recorded and the results were analyzed as follows:

[0227] Table 26 Detection limit verification results for well 1

[0228]

[0229]

[0230] Table 27 Detection limit verification results for well 2

[0231]

[0232]

[0233] The data were further analyzed as follows:

[0234] Table 28 Summary of kit detection limit verification results

[0235]

[0236]

[0237] The lowest concentration with a detection rate of ≥95% was taken as the detection limit of this kit, that is, the detection limit concentration of each fusion gene was 0.01%. Specifically, the detection limit of the fusion gene was 1.5 copies / μL.

[0238] Example 8 Clinical Sample Results Verification

[0239] Blood or bone marrow samples were collected from patients diagnosed with lymphoid leukemia. The samples had been tested with commercially available kits, and the corresponding results were known. 50 positive samples covering fusion genes within the detection range of this kit were selected and tested using this kit according to the detection method described in Example 6. The test results are as follows:

[0240] Table 29 Summary of clinical sample comparison results

[0241]

[0242]

[0243] The kit provided by the present invention and a commercially available kit were tested on 50 clinical samples. The results showed that the kit provided by the present invention detected 33 positive results, while the commercially available kit detected only 32 positive results. Compared with the commercially available kit, the kit provided by the present invention detected one more BCR::ABL1 e1a2-positive sample (sample No. 35).

[0244] Further analysis of sample No. 35 revealed an internal standard gene concentration of 5537.32 copies / μL and a BCR::ABL1 e1a2 fusion gene concentration of 0.92 copies / μL, resulting in a fusion gene ratio of 0.02%, which falls below the detection limit of commercially available kits and is therefore undetectable. Given the 0.01% detection limit of this kit, the result was positive for BCR::ABL1 e1a2 according to the interpretation method described in Example 6, demonstrating the significant advantages of this kit for detecting low-abundance fusion genes.

[0245] Figure 6 The results for a patient with BCR::ABL1 e1a2 positive results are shown in the 1D graph. The vertical axis represents fluorescence intensity, and the horizontal axis represents droplet index. The VIC channel in well 1 detects the BCR::ABL1 e1a2 fusion gene mutation, showing a positive result with a copy concentration of 483.5 copies / μL and a ratio of 67.32%. The remaining channels in well 1, excluding the internal control, and all channels in well 2 show no positive droplets, indicating that the remaining 17 fusion genes included in this kit are negative.

[0246] Figure 7The results for a SET::NUP214-positive patient are shown in Figure 1. The vertical axis of the one-dimensional graph represents fluorescence intensity, and the horizontal axis represents droplet index. These indicators represent the VIC and Atto 425 channels in well 2, respectively, both corresponding to detection of the SET::NUP214 fusion gene variant. The results showed a positive result with a copy concentration of 37.65 copies / μL and a ratio of 9.24%. The remaining channels in well 1, excluding the internal control, and the remaining channels in well 2 showed no positive droplets, indicating that the remaining 17 fusion genes included in this kit were negative.

[0247] The present invention adopts a digital PCR platform based on a combination of chip-type and oil-in-water technology, which can divide the 15μL reaction solution of each sample into at least more than 20,000 independent reaction units, and independently perform single-molecule level PCR amplification and fluorescence signal acquisition in each reaction unit. Compared with QPCR, digital PCR can achieve absolute quantification of the target without the help of a standard curve, and is particularly suitable for monitoring the progression of the disease. In addition, thanks to the principle of limiting dilution, the background sequence or PCR reaction inhibitor in the system is also distributed to each reaction unit, which significantly reduces the interference of the background sequence or PCR inhibitor in the reaction unit where the target sequence is located, and improves the tolerance and accuracy of PCR.

[0248] The primer-probe combination and the accompanying digital PCR kit described in the present invention have the advantages of high specificity, high sensitivity, high accuracy, high tolerance, convenient operation and low cost, and can provide a scientific reference for the diagnosis, treatment selection and prognosis evaluation of clinical lymphoid leukemia.

[0249] The above embodiments describe in detail the structure, features and effects of the present invention. The above are only preferred embodiments of the present invention. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which do not exceed the scope covered by the specification, should be within the scope of protection of the present invention.

Claims

1. A kit for detecting common fusion genes in lymphoid leukemia based on multiplex digital PCR, characterized in that: The kit at least comprises: (1) Well 1 primer-probe mixture: contains the primer-probe sequences shown in SEQ ID NOs. 001-033; wherein SEQ ID NOs. 001-002, 004, 006, 008-015, 018-019, 021-023, 025-026, 028-029, 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) Well 2 primer-probe mixture: contains the primer-probe sequences shown in SEQ ID NOs. 008-009, 016-017, and 034-064; wherein SEQ ID NOs. 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 of the kit contains a PCR enhancer, the components and final concentrations of which are betaine (0.25 M), DMSO (1.5 wt %), (NH 4 ) 2 SO 4 (5 mM), BSA (0.05 mg / mL) and gelatin (0.25 wt %); The primer probe sequences are as follows: The "+" sign in front of the sequence base indicates that the base is replaced by an LNA-modified nucleotide instead of an ordinary nucleotide.

2. The kit according to claim 1, wherein The probe is a Taqman probe, the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group; the 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 kit according to claim 1, wherein 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. 3, with a fluorescent reporter group VIC attached to its 5' end; The probe for the BCR::ABL1 e13a2 / e14a2 fusion gene is SEQ ID NO: 005, with a fluorescent reporter group FAM attached to its 5′ end; The probe for the BCR::ABL1 e19a2 fusion gene is SEQ ID NO: 007, with a fluorescent reporter group ROX linked to its 5′ end; The probes for the MLL series fusion genes are SEQ ID NOs: 016-017, with the fluorescent reporter group Atto425 linked to their 5' ends; 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, with a fluorescent reporter group CY7 attached to its 5′ end; The probe for the TCF3::HLF fusion gene is SEQ ID NO: 024, with a fluorescent reporter group CY5 attached to its 5′ end; ABL1 was the internal standard gene, and the probe was SEQ ID NO: 027, with the fluorescent reporter group CY5.5 linked to its 5′ end; The probe for the FUS::ERG fusion gene is SEQ ID NO: 030, with fluorescent reporter groups ROX and CY5 linked to its 5′ end; The probe for the NPM1::ALK fusion gene is SEQ ID NO: 033, with the fluorescent reporter groups VIC and Atto425 linked to its 5′ end; The probe for the KMT2A::AFDN fusion gene is SEQ ID NO: 036, with a fluorescent reporter group VIC attached to its 5′ end; The probe for the KMT2A::MLLT3 fusion gene is SEQ ID NO: 041-042, and the 5' end thereof is connected to a fluorescent reporter group FAM; The probe for the KMT2A::AFF1 fusion gene is SEQ ID NO: 045, with a fluorescent reporter group ROX linked to its 5′ end; The probe for the KMT2A::MLLT1 fusion gene is SEQ ID NO: 016-017, with the fluorescent reporter group Atto 425 linked to its 5' end; The probe for the ETV6::ABL1 fusion gene is SEQ ID NO: 052, with a fluorescent reporter group CY7 attached to its 5′ end; The probe for the TCF3::PBX1 fusion gene is SEQ ID NO: 055, with a fluorescent reporter group CY5 attached to its 5′ end; The probe for the ETV6::PDGFRB fusion gene is SEQ ID NO: 058, with a fluorescent reporter group CY5.5 attached to its 5′ end; The probe for the STIL::TAL1 fusion gene is SEQ ID NO: 061, with fluorescent reporter groups ROX and CY5 linked to its 5′ end; The probe of the SET::NUP214 fusion gene is SEQ ID NO: 064, and the fluorescent reporter groups VIC and Atto 425 are connected to the 5' end of the probe.

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

5. The kit according to claim 1, wherein The kit also includes: (1) reverse transcription reagents: containing RT enzyme, RNase Inhibitor, dNTP, Oligo dT (18T) Primer, Random 6mers Primer, and reaction buffer; (2) Digital PCR microfluidic chip and droplet generation 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 plasmids and ABL1 internal reference gene plasmid; (4) Positive control No. 2: a mixed solution containing FUS::ERG and NPM1::ALK fusion gene variant plasmids and ABL1 internal reference gene plasmid; (5) Positive control No. 3: a mixed solution containing KMT2A::AFDN, KMT2A::MLLT3, KMT2A::AFF1, KMT2A::MLLT1, ETV6::ABL1, TCF3::PBX1, ETV6::PDGFRB fusion gene variant plasmids and ABL1 internal reference gene plasmid; (6) Positive control No. 4: a mixed solution containing STIL::TAL1 and SET::NUP214 fusion gene variant plasmids and ABL1 internal reference gene plasmid; (7) Negative control: a mixed solution containing the ABL1 internal reference gene plasmid; For the positive control, the concentration of each fusion gene variant plasmid was 75 copies / μL, and the concentration of the ABL1 internal reference plasmid was 1500 copies / μL; for the negative control, the concentration of the ABL1 internal reference plasmid was 1500 copies / μL.

6. The kit according to claim 1, wherein The primer-probe mixture in well 1 also contains an internal standard system; the internal standard probe in the internal standard system has a fluorescent reporter group CY5.5 connected to its 5' end and a fluorescent quencher group BHQ3 connected to its 3' end.

7. A method for using the kit according to claim 1, characterized in that: The method comprises the following steps: (1) extracting RNA from a sample to be tested, wherein the sample to be tested includes bone marrow and peripheral blood; (2) Perform cDNA reverse transcription reaction; (3) using the kit of the present invention to perform a multiplex digital PCR reaction on the cDNA template; (4) Collecting fluorescence signals using a digital PCR reader, analyzing the sample based on the negative / positive control results to determine whether it contains the 18 common fusion genes of lymphoid leukemia in the kit of the present invention, and providing a determination result; The amplification system of the PCR reaction is as follows: 7.5 μL of PCR reaction solution, 2.5 μL of primer-probe mixture in well 1 or well 2, 5 μL of test sample cDNA, and a total system of 15 μL; the PCR amplification conditions are: 95°C for 10 min, 40 cycles (98°C for 15 s, 62°C for 1 min), 28°C for 5 min, and 28°C hold.

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