Primer combinations, kits, and methods for quantitative analysis of the BCR-ABL1 fusion gene and simultaneous detection of hotspot mutations in the ABL1 kinase domain.

Abstract

This invention discloses primer combinations, kits, and methods for the simultaneous detection of BCR-ABL1 fusion gene quantification and hotspot mutations in the ABL1 kinase domain, belonging to the field of molecular diagnostics and gene detection technology. Based on an integrated detection strategy of "RT-PCR + capillary electrophoresis," this invention solves the problems of low throughput, cumbersome operation, and high cost of existing technologies by optimizing primer design and detection procedures. It can simultaneously identify 13 fusion subtypes of BCR-ABL1 and cover more than 90% of clinically relevant drug resistance mutation sites in the ABL1 kinase domain. It has multiple technical advantages such as an integrated detection process, high specificity, high detection sensitivity, and rapid and efficient operation, making it particularly suitable for clinical application.

Description

Technical Field

[0001] This invention relates to primer combinations, kits, and methods for the simultaneous detection of BCR-ABL1 fusion gene quantification and hotspot mutations in the ABL1 kinase region. Specifically, it involves specific primer combinations, matching kits, and detection methods designed for the simultaneous detection of BCR-ABL1 fusion gene expression and drug resistance mutations in the ABL1 kinase region based on RT-PCR-capillary electrophoresis technology, belonging to the field of molecular diagnostics and gene detection technology. Background Technology

[0002] Chronic myeloid leukemia (CML) and a portion (20-30%) of adult acute lymphoblastic leukemia (ALL) involve a translocation of chromosomes 9 and 22, forming the BCR-ABL1 fusion gene. The fusion protein encoded by this gene possesses persistently activated tyrosine kinase activity, leading to uncontrolled cell proliferation and inhibited apoptosis. Tyrosine kinase inhibitors (TKIs, such as imatinib and nilotinib) target and inhibit BCR-ABL1 kinase activity, and have become first-line treatments for CML and Ph. + BCR-ABL1 is a crucial component of ALL treatment regimens and can significantly prolong patient survival. However, approximately 20-30% of patients develop resistance to first- and / or second-generation TKIs due to acquired mutations in the ABL1 kinase domain (such as p.T315I, p.G250E, p.Y253H), leading to treatment failure or disease progression. Therefore, simultaneously detecting BCR-ABL1 fusion gene expression and ABL1 kinase domain mutation status is a key step in guiding TKI drug selection, optimizing treatment regimens, and predicting prognosis. Clinically, there is an urgent need to simultaneously detect BCR-ABL1 fusion gene expression levels and ABL1 kinase domain mutation status to guide precision medicine.

[0003] Analysis of existing technologies reveals the following main limitations of current detection methods:

[0004] (1) Detection separation: Existing technologies (such as Chinese patents with publication numbers CN117568455B, CN116536402A, and CN116970704B) cannot simultaneously detect the expression level of the BCR-ABL1 fusion gene and hotspot mutations in the ABL1 kinase domain in the same reaction system, resulting in cumbersome clinical testing procedures and increased sample consumption;

[0005] (2) Limited detection throughput: In the detection of mutations in the ABL1 kinase domain, existing methods (such as ARMS-PCR, multiplex fluorescent PCR, etc.) rely on multi-tube amplification or stepwise detection, which makes it difficult to achieve high-throughput parallel screening of multiple mutations and affects detection efficiency.

[0006] (3) The contradiction between sensitivity and cost: Although digital PCR, NGS and other technologies can improve the sensitivity of mutation detection, they have problems such as expensive equipment and complex data analysis, making it difficult to promote them in primary medical institutions.

[0007] (4) Primer design challenges: Since some mutation sites in the ABL1 kinase region (such as p.Y253H and p.E255K) are spatially adjacent, traditional PCR methods are prone to false positives or missed detections due to primer cross-interference, which affects the accuracy of detection.

[0008] In summary, current technologies lack a highly sensitive (≤1%), low-cost, integrated detection solution that can simultaneously quantify the BCR-ABL1 fusion gene and screen for multiple mutations in the ABL1 kinase domain, in order to meet the urgent needs of precise clinical diagnosis and treatment and drug resistance monitoring. Summary of the Invention

[0009] The purpose of this invention is to provide primer combinations, kits, and methods for the simultaneous detection of BCR-ABL1 fusion gene quantification and hotspot mutations in the ABL1 kinase domain. This technical solution is based on an integrated detection strategy of "RT-PCR + capillary electrophoresis". By optimizing primer design and detection process, it solves the problems of low detection throughput, cumbersome operation, and high cost of existing technologies. It can simultaneously identify 13 fusion subtypes of BCR-ABL1 and cover more than 90% of clinically relevant drug resistance mutation sites in the ABL1 kinase domain. It has many technical advantages such as integrated detection process, high specificity, high detection sensitivity, and rapid and efficient operation, and is particularly suitable for clinical application.

[0010] This invention is achieved through the following technical solution:

[0011] One technical solution: Primer combinations for simultaneous detection of BCR-ABL1 fusion gene quantification and ABL1 kinase region hotspot mutations, including the following two major primer groups:

[0012] The first set of primers was designed to target 13 mutation sites in the ABL1 kinase region, and its primer sequences are shown in SEQ ID NO.1 to SEQ ID NO.29.

[0013] Thirteen mutation sites: M244V, G250E, Q252H, E255V, D276G, V299L, T315I, M351T, F359C, L387M, H396R, E459K, and F486S;

[0014] The second set of primers was designed targeting 13 fusion forms of the BCR-ABL1 fusion gene and 7 hotspot mutations in the ABL1 kinase region. The primer sequences are shown in SEQ ID NO.30 to SEQ ID NO.46, SEQ ID NO.16 to SEQ ID NO.17, SEQ ID NO.20, and SEQ ID NO.25.

[0015] Thirteen fusion forms: BCR-ABL1-E13A2, BCR-ABL1-E14A2, BCR-ABL1-E19A2, BCR-ABL1-E1A2, BCR-ABL1-E13A3, BCR-ABL1-E14A3, BCR-ABL1-E19A3, BCR-ABL1-E6A3, BCR-ABL1-E12A3, BCR-ABL1-E12A2, BCR-ABL1-E6A2, BCR-ABL1-E1A3, BCR-ABL-E8A2.

[0016] Seven hotspot mutations: F359V, E355G, F317L, E279K, E255K, Y253H, and L248V.

[0017] The primer sequences SEQ ID NO.16 and SEQ ID NO.17 are quality control primers common to both the first and second primer groups.

[0018] The first and second primer sets also contain blocker probes that specifically block wild-type templates.

[0019] The blocker probes of the first primer group are formed by labeling the primers of the sequences shown in SEQ ID NO.18 to SEQ ID NO.29 with a repression modification group; the blocker probes of the second primer group are formed by labeling the primers of the sequences shown in SEQ ID NO.20, SEQ ID NO.25, SEQ ID NO.43 to SEQ ID NO.46 with a repression modification group.

[0020] The labeling of the repressor modifying group is selected from any one of 3'-Spacer C3, 3'-Phosphat, 3'-ddC, 3'-Inverted End, or 3'-MGB.

[0021] The primer sequences in the first primer group and the second primer group are labeled with different fluorescent markers, and the fluorescent markers are selected from any one of the fluorescent genes FAM, HEX, VIC, TAMRA, ROX or TEXRED.

[0022] The fluorescent gene is labeled on the universal primers for amplification corresponding to the mutation detection site.

[0023] Technical Solution Two: A method for quantifying the BCR-ABL1 fusion gene and simultaneously detecting hotspot mutations in the ABL1 kinase region, comprising the primer combination mentioned above.

[0024] The kit also includes an amplification enzyme mixture, a positive control, a negative control, and a capillary electrophoresis internal standard reagent.

[0025] Technical Solution 3: A method for simultaneously detecting hotspot mutations in the BCR-ABL1 fusion gene and the ABL1 kinase region based on RT-PCR-capillary electrophoresis, comprising the following steps:

[0026] S1. Prepare the cDNA template for the sample to be tested;

[0027] S2. Perform multiplex PCR amplification using the primer combination described above;

[0028] S3. Perform capillary electrophoresis analysis on the amplification products;

[0029] S4. Result determination: The peak position of each detection site is determined by the positive control. When the peak signal of the sample detection is >100RFU, it is determined to be positive.

[0030] The method of using the primer combination includes: mixing all primer sequences of the first primer group to form the first detection primer group, mixing all primer sequences of the second primer group to form the second detection primer group, and performing independent multiplex PCR amplification using the first detection primer group and the second detection primer group respectively.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] 1. This invention enables integrated detection, simultaneously completing the typing of 13 fusion forms of the BCR-ABL1 fusion gene and screening for 20 hotspot mutations in the ABL1 kinase region in a single experiment. Its advantages are:

[0033] Comprehensive coverage, avoiding missed detections, and providing more comprehensive and precise guidance for clinical treatment plans: The BCR-ABL1 fusion gene has multiple subtypes (such as p190, p210, p230, etc.), and different subtypes are closely related to disease types (such as chronic myeloid leukemia CML, acute lymphoblastic leukemia ALL) and prognosis. Meanwhile, ABL1 kinase domain mutations are one of the main causes of resistance to tyrosine kinase inhibitors (TKIs). A single experiment simultaneously detects 13 fusion forms and 20 hotspot mutations, obtaining complete molecular biological information in one go, avoiding the risk of missed detections caused by stepwise testing, and enabling comprehensive diagnosis and precise treatment guidance for newly diagnosed patients.

[0034] Reduced sample loss and experimental error: Traditional stepwise detection requires multiple samplings or batch experiments, which may affect the results due to insufficient sample size, storage conditions, or operational errors. Simultaneous detection requires only one experiment, reducing sample processing steps, minimizing result deviations caused by sample loss or experimental fluctuations, and improving data reliability.

[0035] Shortening the diagnostic cycle and improving patient prognosis: For leukemia patients, early diagnosis and treatment are key to improving prognosis. Traditional testing methods may take several days or even more than a week, while simultaneous testing can complete typing and mutation screening in a single experiment, significantly shortening the diagnostic cycle (reports can be issued within 1 to 2 days), enabling patients to receive targeted treatment more quickly, which is especially significant for critically ill patients;

[0036] Significantly reduces operational intensity and testing costs: Traditional step-by-step testing requires multiple tubes to detect different fusion types of the BCR-ABL1 fusion gene. After confirming a positive BCR-ABL1 fusion, multiple tubes are then used to detect mutations in the ABL1 kinase region, either through PCR-Sanger sequencing, or by using RT-PCR or quantitative real-time RT-PCR in 20 tubes. This process is cumbersome and demanding. This new method, however, requires only two tubes of multiplex PCR amplification reactions to simultaneously detect 13 fusion forms of the BCR-ABL1 fusion gene and 20 hotspot mutations in the ABL1 kinase region. The operational intensity is reduced tenfold per test, and the number of sites detected per tube is increased several times.

[0037] 2. The mutation site is amplified using AS-QF-PCR, and the amplification products are then detected by capillary electrophoresis using a genetic analyzer to obtain the detection results. The main advantages are:

[0038] High detection sensitivity: By optimizing primer design and shielding wild-type templates, as well as amplifying fluorescence signals, the target gene amplification product can be detected sensitively. It only needs to focus on the presence or absence of mutation characteristic signals, eliminating the interference of wild-type background peaks. The resolution is 100 to 200 times higher than that of Sange sequencing, and the mutation detection limit is 0.1%.

[0039] The test results can provide preliminary quantification: the test results will show specific values ​​for the height of specific peaks, and the peak height and the initial amount of template have a linear correspondence within a certain range. It can be compared with the internal reference (quality control gene) to a certain extent to achieve preliminary quantification of the mutation content of the mutant gene.

[0040] The system is expandable and has a wide detection range: the capillary electrophoresis detection platform can effectively distinguish fragments of 1 bp in size within the detection range of 60-600 bp, and the genetic analysis instrument can simultaneously receive 6 kinds of fluorescence signals with minimal interference between them. One channel is used for molecular internal standard detection, and the other 5 channels can detect different target gene loci in the sample. Target fragments within the same fluorescence channel can be distinguished by the difference in fragment length. With a distinguishable length of over 500 bp, theoretically, more than 500 fragment peaks can be distinguished. The 5 detectable channels can distinguish more than 2000 fragment peaks. In practical applications, we control the position of the same fragment peak to be at least 4-5 bp apart. Therefore, ideally, one tube of detection can amplify 400-500 target gene loci. This invention only shows the use of 2 channels for sample detection, with each channel detecting only 3-20 target fragments. In practice, it can be expanded to detect more genes and more loci.

[0041] Fast detection speed: Automated batch detection is possible: The capillary electrophoresis process can complete the detection in 40 minutes. Depending on the sample volume, it can detect 16 wells, 32 wells, or 96 wells at a time.

[0042] The test results can be automatically analyzed and interpreted using analysis software.

[0043] 3. This detection methodology enables simultaneous quality control: internal control targets are added simultaneously in the same detection tube to control the experimental process and sample quality throughout the entire process, ensuring the controllability of the experimental results. Attached Figure Description

[0044] Figure 1 The amplification map of the positive control sample in Example 4 is shown, where each vertical axis is displayed from bottom to top as 30, 130, 230, 330, 430, 530 (bp), and each horizontal axis is displayed from left to right as (RFU), 2400, 1600, 800, 0.

[0045] Figure 2 The amplification map of the wild-type sample in Example 4 is shown, where each vertical axis is displayed from bottom to top as 30, 130, 230, 330, 430, 530 (bp), and each horizontal axis is displayed from left to right as (RFU), 12000, 8000, 4000, 0.

[0046] Figure 3The detection spectrum of the 50% mutation rate of the T315I strong positive reference in Example 4 is shown. Each vertical axis is displayed from bottom to top as 0, 100, 200, 300, 400, 500, 600, 700 (bp), and each horizontal axis is displayed from left to right as (RFU), 12000, 8000, 4000, 0.

[0047] Figure 4 The image shows the detection spectrum of the 10% mutation rate of the strong positive reference material T315I in Example 4. The vertical axis from bottom to top shows 0, 100, 200, 300, 400, 500, 600 (bp), and the horizontal axis from left to right shows (RFU), 12000, 8000, 4000, 0.

[0048] Figure 5 The detection spectrum of the 1% mutation rate of the T315I weak positive reference material in Example 4 is shown. Each vertical axis is displayed from bottom to top as 0, 100, 200, 300, 400, 500, 600 (bp), and each horizontal axis is displayed from left to right as (RFU), 12000, 8000, 4000, 0.

[0049] Figure 6 The detection spectrum of the T315I detection limit positive reference material with a mutation rate of 0.1% in Example 4 is shown. Each vertical axis displays 0, 100, 200, 300, 400, 500, 600 (bp) from bottom to top, and each horizontal axis displays (RFU), 12000, 8000, 4000, 0 from left to right.

[0050] Figure 7 The detection spectrum of BCR-ABL1 P210 positive with M351T mutation in Example 5 is shown. Each vertical axis is displayed from bottom to top as 0, 100, 200, 300, 400, 500, 600 (bp), and each horizontal axis is displayed from left to right as (RFU), 12000, 8000, 4000, 0.

[0051] Figure 8 The amplification map of the wild-type sample in Comparative Example 1 is shown, where each vertical axis from bottom to top shows 50, 90, 130, 170, 210, 250, 290, 330, 370, 410, 450, 490, 530 (bp), and each horizontal axis from left to right shows (RFU), 12000, 8000, 4000, 0. Detailed Implementation

[0052] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0053] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0054] This invention addresses the clinical need for monitoring drug resistance in leukemia treatment by innovatively developing a high-efficiency detection system based on the combined use of multiplex fluorescent AS-RT-PCR and capillary electrophoresis. The technical solution includes: a specific primer combination capable of simultaneously detecting the expression level of the BCR-ABL1 fusion gene and key drug resistance mutation sites in the ABL1 kinase region; a matching detection kit containing this specific primer combination; and a standardized detection method based on a capillary electrophoresis platform. This solution effectively overcomes the limitations of traditional detection methods in terms of sensitivity, throughput, and cost, providing a reliable molecular diagnostic tool for personalized leukemia treatment.

[0055] The following is a further summary of the technical solution of the present invention:

[0056] (a) Specific primer combinations

[0057] Specific primer sets mainly consist of two primer groups: the first primer group and the second primer group.

[0058] The primer sequences of the first primer group are shown in SEQ ID NO.1 to SEQ ID NO.29, and they detect 13 mutation sites in the ABL1 kinase region: M244V, G250E, Q252H, E255V, D276G, V299L, T315I, M351T, F359C, L387M, H396R, E459K, and F486S.

[0059] The primer sequences of the second primer set are as follows: SEQ ID NO.30 to SEQ ID NO.46, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.20 and SEQ ID NO. As shown in NO.25, the detection included 13 fusion forms of the BCR-ABL1 fusion gene in leukemia: BCR-ABL1-E13A2, BCR-ABL1-E14A2, BCR-ABL1-E19A2, BCR-ABL1-E1A2, BCR-ABL1-E13A3, BCR-ABL1-E14A3, BCR-ABL1-E19A3, BCR-ABL1-E6A3, BCR-ABL1-E12A3, BCR-ABL1-E12A2, BCR-ABL1-E6A2, BCR-ABL1-E1A3, and BCR-ABL-E8A2, and 7 hotspot mutations in the ABL1 kinase domain: F359V, E355G, F317L, E279K, E255K, Y253H, and L248V.

[0060] In the first and second primer groups, SEQ ID NO.16 and SEQ ID NO.17 are universal quality control primers.

[0061] The first and second primer groups also contain blocker probes that specifically block wild-type templates, or use different fluorescent labels.

[0062] In some embodiments of the present invention, regarding the design of the blocker probes, optionally, the blocker probes of the first primer group are formed by labeling the primers of the sequences shown in SEQ ID NO.18 to SEQ ID NO.29 with repression modifying groups, and the blocker probes of the second primer group are formed by labeling the primers of the sequences shown in SEQ ID NO.20, SEQ ID NO.25, and SEQ ID NO.43 to SEQ ID NO.46 with repression modifying groups. That is, in the first primer group, the blocker probes of SEQ ID NO.18 to SEQ ID NO.29 can specifically bind to the wild-type template, and their 3' ends are labeled with different repression modifying groups; in the second primer group, the blocker probes of SEQ ID NO.20, SEQ ID NO.25, and SEQ ID NO.43 to SEQ ID NO.46 can specifically bind to the wild-type template. Among them, SEQ ID NO.20 and SEQ ID NO.25 are the blocker repression probes used in the first set of primers. Optionally, the repression modification group can be selected from any one of 3'-Spacer C3, 3'-Phosphat, 3'-ddC, 3'-Inverted End or 3'-MGB, depending on the labeling of different sites.

[0063] In some embodiments of the present invention, for fluorescent labeling, any one of the fluorescent groups such as FAM, HEX, VIC, TAMRA, ROX, or TEXRED can be selected. Preferably, the fluorescent group is labeled on the universal primers for amplification of each mutation detection site as much as possible to reduce primer synthesis costs and reduce fluorescence interference caused by too many fluorescent primers in amplification. For example, in a specific embodiment of the present invention, the 5' ends of the primer sequences shown in SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.16 in the first primer group are fluorescently labeled, wherein SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.16 respectively label different fluorescent genes; the 5' ends of the primer sequences shown in SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.16 in the second primer group are fluorescently labeled, wherein SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.16 respectively label different fluorescent tags.

[0064] It should be noted that the specific primer combination described in this invention employs an innovative design strategy, and has the following key design points in primer design:

[0065] (1) Design basis: Referencing the primer design principles for the BCR-ABL1 fusion gene in patent CN117568455B, the high efficiency detection capability for leukemia-related fusion genes is retained;

[0066] (2) Innovative design: Targeting 13 high-frequency mutation sites in the ABL1 kinase domain, 7 key clinical drug resistance mutation hotspots were specially optimized, and a brand-new primer design rule system was established;

[0067] (3) Technological breakthrough: Overcoming the limitations of traditional single-site detection, achieving simultaneous detection of fusion gene expression and drug resistance mutations, ensuring the specificity and sensitivity of multiplex PCR reactions;

[0068] (4) Design advantages: Optimize primer Tm value matching, avoid cross-reaction between primers, and improve the consistency of amplification efficiency.

[0069] This primer design strategy, through innovative rule optimization, successfully solves the technical challenge of balancing primer compatibility and reaction efficiency in multiplex detection, providing a more comprehensive gene testing solution for clinical use.

[0070] (II) Supporting test kits

[0071] The accompanying test kit contains at least the above-mentioned specific primer combination, as well as amplification enzyme mixture, positive control, negative control, capillary electrophoresis internal standard reagent, etc.

[0072] In some embodiments of the present invention, the amplification enzyme mixture is a PCR amplification buffer, Mg + The mixture of hot-start enzyme and the positive control consisted of 10 positive plasmids prepared from 13 fusion forms of the BCR-ABL1 fusion gene and 20 hotspot mutations in the ABL1 kinase region, along with wild-type plasmids at a copy number ratio of 1:10. 4 A 10% mutation-positive plasmid mixture was used as the copy number; the negative control was cDNA obtained by reverse transcription of RNA from normal human blood.

[0073] (III) Standardized Testing Methods

[0074] The steps of the detection method are as follows:

[0075] S1. Prepare the cDNA template for the sample to be tested;

[0076] S2. Multiplex PCR amplification is performed using the primer combination described above. Specifically, all primer sequences from the first primer group are mixed to form the first detection primer group, and all primer sequences from the second primer group are mixed to form the second detection primer group. Then, multiplex PCR amplification is performed independently using the first detection primer group and the second detection primer group respectively.

[0077] S3. Perform capillary electrophoresis analysis on the amplification products;

[0078] S4. Result determination: The target bin is accurately positioned by using the positive peak positions of all detection sites obtained from positive control amplification; when a target peak higher than 100 RFU appears on the target bin of the sample, the sample is determined to be positive for gene mutation or fusion gene in the corresponding bin.

[0079] In some embodiments of the present invention, the amplification system for the two PCR amplification sets is as follows: 2 μL of 5×PCR master mix, 1 μL of detection primer mix, 2 μL of cDNA to be tested, and 2 μL of deionized purified water, for a total system volume of 10 μL. Specifically, all primer sequences shown in SEQ ID NO.1 to SEQ ID NO.29 are mixed to form the first detection primer mix; and all primer sequences shown in SEQ ID NO.30 to SEQ ID NO.46, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.20, and SEQ ID NO.25 are mixed to form the second detection primer mix.

[0080] In some specific embodiments of the present invention, in order to minimize the generation of nonspecific fragments during multiplex PCR amplification, a touchdown PCR amplification program is used for PCR amplification. The amplification program is as follows: pre-denaturation at 95°C for 10 min for 1 cycle; denaturation to extension for 5 cycles: denaturation at 95°C for 10 s, annealing at 62°C for 30 s, decreasing by 1°C per cycle, extension at 70°C for 30 s; denaturation to extension for 26 cycles: denaturation at 95°C for 10 s, annealing at 58°C for 30 s, extension at 70°C for 30 s; post-amplification extension at 60°C for 30 min for 1 cycle; the amplification product is stored at 4°C or 16°C.

[0081] In some embodiments of the present invention, the primer combination in the amplification system has the following concentrations: SEQ ID NO.1 to SEQ ID NO.17 and SEQ ID NO.30 to SEQ ID NO.42, except for the shared fluorescent primers SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.34 and SEQ ID NO.35, all of which are 1 pmol / μL; the concentration of primer SEQ ID NO.12 is 8 pmol / μL; the concentration of primer SEQ ID NO.15 is 2 pmol / μL; and the concentrations of the blocking probes SEQ ID NO.18 to SEQ ID NO.29 and SEQ ID NO.43 to SEQ ID NO.46 are all 1.2 pmol / μL.

[0082] In some specific embodiments of the present invention, the capillary electrophoresis detection method for PCR amplification products is as follows:

[0083] Prepare a loading mixture containing a molecular weight internal standard and formamide (the ratio of molecular weight internal standard to formamide is 1:500). Calculate the number of tubes based on the number of PCR amplification products per sample (each sample contains 2 tubes of negative control and 2 tubes of positive control PCR products). Use a pipette to dispense 9 μL of the molecular weight internal standard and formamide mixture into each well. Add 1 μL of PCR amplification product to the mixture, cover with a sealing film, and centrifuge briefly to remove air bubbles from the sample loading tubes. Denature the loading plate at 95°C for 5 min, then immediately incubate on ice for 2 min to ensure complete denaturation of the PCR products into single-stranded nucleic acids. Perform capillary electrophoresis detection according to the steps in the genetic analyzer user manual. Parameters are set as follows: injection time 15 s, injection voltage 3 kV, and run time 2000 s.

[0084] In some specific embodiments of the present invention, the result interpretation scheme is as follows:

[0085] (1) Interpretation of experimental validity: In the negative control test results, the amplification results of the first and second groups showed the target peak only at the quality control position, and the peak height was greater than 300 RFU. There were no obvious non-specific peaks (peak height < 100 RFU) at other bins. In the positive control test results, the amplification results of the first and second groups showed specific peaks greater than 200 at the target peak positions of all detection sites.

[0086] (2) Interpretation of sample validity: In the test results of the sample, for the two groups of amplification, the target peak must appear at the quality control bin position, and the peak height must not be less than 300 RFU. This indicates that the sample cDNA template is normal.

[0087] (3) Result interpretation: Under the premise of experimental operation and sample validity, the amplification results of sample 2 groups are analyzed. If a target peak with a value higher than 100 RFU appears in the target bin except for the quality control peak, the fusion gene or mutation site in the corresponding bin of the sample is determined to be positive.

[0088] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0089] Example 1:

[0090] Primer and blocker probe design and screening

[0091] Based on the latest data from the COSMIC database (Human Cancer-Related Somatic Mutations Database) and literature reports, we compiled the possible fusion forms of the BCR-ABL1 fusion gene and the top 20 mutation sites with the most reported mutations in the ABL1 kinase domain. Primers were designed to detect all currently collected fusion forms and 20 hotspot mutations of the BCR-ABL1 fusion gene. The specific details of the detected fusion forms and mutation sites are shown in Table 1: List of detected BCR-ABL1 fusion gene fusion forms and ABL1 kinase domain mutation sites.

[0092] Table 1: List of BCR-ABL1 fusion gene fusion modes and ABL1 kinase domain mutation sites detected

[0093]

[0094] The primers for detecting different fusion forms of the above-mentioned fusion genes are designed according to the patent CN117568455B, and the primer sequences are shown in SEQ ID NO.30 to SEQ ID NO.35.

[0095] Furthermore, the design of specific primers for point mutations at 20 sites should follow these principles:

[0096] (1) For mutation site detection specific primers, the last base must fall on the mutation site and be consistent with the mutant base (if there are two mutation types at the mutation site, use degenerate primers instead).

[0097] (2) For closely spaced mutation sites, in order to prevent compound mutations from affecting primer specificity, degenerate primers are used to replace wild-type bases at positions in the middle of the primers where mutations may occur. The degenerate primer codes are M=A / C, R=A / G, W=A / T, S=G / C, Y=C / T, K=G / T, and B=G / C / T.

[0098] (3) In order to enhance primer specificity, a mismatch of one base will be made in the last 2-5 bases of the primer: In the first round of design, the mismatch is mainly set in the mismatch of the -2 and -3 bases (the -2 position originally has a mutation form, and there is a degenerate base, so the mismatch is made in the -3 and -4 bases). Each specific primer has 3 mismatch forms in the -2 and -3 positions, plus one form without mismatch, for a total of 7 specific primer design forms. Given that the primer synthesis cost is low at present, in the first round of design, we will design 7 primers for each site. There is a mismatch of one base in the -2 or -3 position between each primer, and then compare and screen the most suitable primer.

[0099] (4) The Tm values ​​of the primers designed in the primer combination should be controlled as close as possible to the temperature, around 58℃, so that when paired with other primers, they can be amplified efficiently in one amplification system, avoid the generation of primer dimers, and improve the efficiency and specificity of multiplex amplification.

[0100] (5) In order to enhance primer balance and stability, for specific primers with excessively high GC content at individual sites, 1-3 A bases are introduced at the 5' end of the primer while meeting the primer TM value, thereby increasing primer balance and stability while ensuring primer specificity.

[0101] (6) To ensure that the amplification of all sites is within the range of 75-600 bp for the length of the target sequence, capillary electrophoresis can clearly and effectively distinguish a 1 bp difference within this length range.

[0102] (7) The corresponding products generated by the amplification of primers with the same fluorescent label in each group of primers should be at least 3 bp apart, so as to distinguish the specific amplified target fragment.

[0103] In this specific embodiment, since the target mutation sites are concentrated in distance, and some sites are even adjacent, the following principles are further introduced into the primer design to take into account the characteristics of this detection target:

[0104] (8) For mutation sites that are close to each other (fragment distance within 500 bp), the other primer paired with the specific primer should be a universal primer. For example, the same downstream primer was used for 11 sites, such as M244V, G250E, Q252H, E255V, D276G, V299L, T315I, M351T, F359C, L387M, and H396R. The number of primers should be minimized to avoid cross-reaction between primers. The fluorescent group should be labeled on the universal primer.

[0105] (9) For adjacent (within 1-10 bp) mutation sites, primers are designed to amplify from different template strands and the reaction tubes are separated. For example, for the two sites Q252H and Y253H, which are only 1 bp apart, primers are designed from the reverse complementary strands and amplified in two separate tubes to minimize the impact of different mutations on each other.

[0106] Based on the principles in (8) and (9) above, the primer group is further divided into two large primer groups to detect the target separately:

[0107] The first channel of the first primer group amplifies 11 sites: M244V, G250E, Q252H, E255V, D276G, V299L, T315I, M351T, F359C, L387M, and H396R, using the same universal fluorescent primers. The second channel amplifies two sites, E459K and F486S, which are more than 600 bp away from the M244V site, the most prominent site in the genome among the sites detected in the first channel. At the same time, the quality control primers for ABL1 are also placed in the second channel for amplification. The first channel of the second primer group amplifies the BCR-ABL1 fusion gene; the second channel amplifies the remaining 7 mutation sites in the ABL1 kinase region by designing primers with the reverse complementary strands from the primers designed for the mutation sites in the first group, namely L248V, Y253H, E255K, E279K, F317L, E355G, and F359V. At the same time, the quality control primers for ABL1 are also placed in the second channel for amplification.

[0108] Based on the above primer design principles (1) to (9), seven specific primers with different mutated bases were designed for each detection site. Through comparative screening experiments, primers with strong specificity and high amplification efficiency were selected.

[0109] For this comparative screening experiment, since seven specific primers were designed for each locus initially, seven groups of experiments were conducted to screen and verify the specific primers for each locus. For example, the first specific primer was used for each locus, and the first screening primer group was configured. Each screening primer group was used to perform QF-PCR amplification on templates with three different mutation ratios for each locus, followed by capillary electrophoresis detection. Based on the difference between the specific peak height and the non-specific peak height, the most suitable specific amplification primer for each mutation locus was selected. The three different mutation ratio templates were prepared by configuring the corresponding site mutant plasmid and wild-type plasmid in a certain ratio: each containing 3 × 10 4 The total template amount (equivalent to 100 ng of human genome copies) was set to 50%, 10%, 1% and 0% (wild-type template), respectively.

[0110] In the comparative screening experiment, the screening criteria for primers with optimal specificity include:

[0111] (1) For templates with 50%, 10% and 1% mutations, the amplification-specific peak heights can be gradients according to the multiples of the template concentration;

[0112] (2) It can stably detect 1% of mutant templates;

[0113] (3) No amplification or the amplification peak height is as low as possible for pure wild-type template (0%), so that there is the most significant peak height difference with the peak of 1% mutation.

[0114] By setting up 7 sets of primer combinations, each mutation site was verified and screened one by one. Finally, the optimal specific primers for each mutation site were selected.

[0115] To improve the specificity of point mutation detection at 20 sites and avoid non-specific peaks due to the presence of high concentrations of wild-type template, this invention also incorporates a Blocker probe that can block the wild-type template. The design principles of the Blocker probe are as follows:

[0116] (1) In order to achieve the best blocking effect, in principle, we will design a separate blocking blocker probe for each detected mutation site, and try to keep the blocking probes of different mutation sites from having overlapping sequences. However, some mutation sites are less than 5 bp away from other mutation sites before and after, and the staggered design cannot avoid this. We will choose to share one blocker probe, but the number of shared blockers should be as small as possible.

[0117] (2) Since the target detection sites are very close to each other, in order to reduce the sequence sharing conflict of the blocker, the length of the blocker will be controlled at 15-20 bases.

[0118] (3) Modify the 3' end of the blocker to prevent the blocker from extending. The modified group should preferably increase the Tm value of the probe to promote the preferential binding of the probe primer to the wild-type probe. Therefore, the probe needs to be modified to increase the probe Tm value, such as MGB, locked nucleic acid modification, etc.

[0119] (4) The sequence is completely complementary to the wild-type template, and the blocker's Tm value is 4-7°C higher than the primer's Tm value.

[0120] In summary, based on the above principles, this embodiment designed and screened the optimal specific primers and suitable blocker probes for each mutation site, as shown in Table 2: Fusion and point mutation primer and blocker probe sequences and grouping table.

[0121] Table 2: Fusion and point mutation primers and Blocker probe sequences and grouping table

[0122]

[0123] Note: (1) The primer name is marked with " The line indicates that the primer is fluorescently labeled at the 5' end; the fluorescent labeling fluorescent group is selected from any one of FAM, HEX, VIC, TAMRA, ROX, TEXRED, etc. Different fluorescent groups are selected for primers of different channels in the same group. In this invention, FAM and HEX are used to fluorescently label primers of different channels.

[0124] (2) The primer (probe) name is marked with " The "-line" indicates that the primer is labeled with a repressor group at the 3' end, such as 3'-Spacer C3, 3'-Phosphat, 3'-ddC, 3'-Inverted End, 3'-MGB, and / or has undergone locked nucleic acid modification. In this invention, most repressor probes are labeled with 3'-MGB, which can inhibit probe amplification while increasing the primer annealing temperature; a small number of probes with high GC content and TM values ​​that are 4-7°C higher than the primer TM values ​​are labeled with 3'-Spacer C3.

[0125] (3) The bases in the primer sequence are marked with " The "" symbol indicates that the base uses a degenerate base.

[0126] (4) Use “” after the bases in the primer sequence The "" symbol indicates that the base has been mismatched.

[0127] (5) Use “” after the bases in the primer sequence The "" symbol indicates that polyA was introduced at the 5' end to balance and stabilize high GC primers.

[0128] Example 2:

[0129] Simultaneous detection kit for BCR-ABL1 fusion gene and ABL1 kinase domain hotspot mutations

[0130] The detection kit in this embodiment contains the primer combination from Example 1, as well as PCRmaster MIX, positive control, negative control, and capillary electrophoresis internal standard reagent. Using this kit, 13 fusion forms of the BCR-ABL1 fusion gene and 20 mutations in the ABL1 kinase region can be detected through two sets of RT-PCR amplification systems.

[0131] The amplification enzyme mixture in the kit consists of PCR amplification buffer, Mg+ It is a mixture of heat-starting enzymes.

[0132] The positive control was prepared by mixing positive plasmids of 13 fusion forms of the BCR-ABL1 fusion gene and positive plasmids of 20 hotspot mutations in the ABL1 kinase region with wild-type plasmids at a copy number ratio of 1:10, with a total copy number of 3 × 10⁻⁶. 4 A 10% copy of a mutant-positive plasmid mixture.

[0133] The negative control was prepared by reverse transcription of total RNA extracted from cells cultured in the H293T cell line to obtain cDNA.

[0134] Two primer mixtures, consisting of a first primer group and a second primer group respectively, were prepared and combined in the kit at a final concentration of 10×. Among them:

[0135] The first primer group consists of amplification primers SEQ ID NO.1 to SEQ ID NO.17 and amplification repression probes SEQ ID NO.18 to SEQ ID NO.29. In the first primer group, except for the common fluorescent primer SEQ ID NO.12 with a primer concentration of 80 pmol / μL and the primer concentration of SEQ ID NO.15 with a primer concentration of 20 pmol / μL, the concentration of the other amplification primers is 10 pmol / μL, while the primer concentration of the amplification repression probes SEQ ID NO.18 to SEQ ID NO.29 is 12 pmol / μL.

[0136] The second primer group consists of amplification primers SEQ ID NO.30 to SEQ ID NO.42, SEQ ID NO.16, and SEQ ID NO.17, and amplification repression probes SEQ ID NO.43 to SEQ ID NO.46, SEQ ID NO.20, and SEQ ID NO.25. Except for the primers that share SEQ ID NO.34 and SEQ ID NO.35, which have a concentration of 50 pmol / μL, the concentration of the other amplification primers is 10 pmol / μL, and the concentration of the amplification repression probes SEQ ID NO.43 to SEQ ID NO.46, SEQ ID NO.20, and SEQ ID NO.25 is 12 pmol / μL.

[0137] Example 3:

[0138] A multiplex RT-PCR method for simultaneous detection of 20 hotspot mutations in the BCR-ABL1 fusion gene and ABL1 kinase domain

[0139] Includes the following steps:

[0140] 1. Extract RNA from the sample to be tested.

[0141] 2. Obtain the cDNA template of the sample to be tested through reverse transcription;

[0142] 3. Amplification system configuration: Using the two primer mixtures from Example 2 and the amplification enzyme mixture from the kit in Example 2, PCR amplification was performed in two tubes using the cDNA obtained in step 2 as a template.

[0143] The total PCR amplification volume for each tube is 10 μL. See Table 3 for the amplification system.

[0144] Table 3 Amplification System

[0145] Element Sample volume 5×PCR master MIX 2μL Primer mix 1μL cDNA to be tested 2μL Deionized purified water 2μL Total volume 10μL

[0146] 4. PCR amplification: In order to minimize the generation of non-specific fragments during multiplex PCR amplification, this invention uses a touch-down PCR amplification program for PCR amplification. The amplification program is shown in Table 4: Amplification Program.

[0147] Table 4 Amplification Procedure

[0148]

[0149] 5. Capillary electrophoresis detection of PCR amplification products:

[0150] Prepare a loading mixture containing a molecular weight internal standard and formamide (mixing ratio of molecular weight internal standard to formamide: 1:500). Calculate the number of tubes based on the number of PCR amplification products per sample (each sample includes two tubes of PCR products from negative and positive controls). Use a pipette to dispense 9 μL of the mixture into each well. Add 1 μL of PCR amplification product to the mixture, cover with a sealing film, and centrifuge briefly to remove air bubbles. Denature the sample tube at 95°C for 5 minutes, then immediately incubate on ice for 2 minutes to ensure complete denaturation of the PCR products into single-stranded nucleic acids. Perform capillary electrophoresis detection according to the user manual for the genetic analyzer. Parameters are set as follows: injection time 15 s, injection voltage 3 kV, and run time 2000 s.

[0151] 6. Data Analysis

[0152] Import relevant files into GeneMapper software, including Panel, Bin, corresponding Analysis Method, and Orange600 internal standard. Input the source sample data (.fsa file), select the previously imported file in the relevant parameter selection bar, and analyze the data. The creation of the Bin file requires accurate positioning using the peak positions of the positive control amplification.

[0153] 7. Result Interpretation

[0154] (1) Interpretation of experimental validity: In the negative control test results, the amplification results of the first and second groups showed the target peak only at the quality control position, and the peak height was greater than 300 RFU. There were no obvious non-specific peaks (peak height < 100 RFU) at other bins. In the positive control test results, the amplification results of the first and second groups showed a specific peak with a value of greater than or equal to 200 RFU at the target peak position of all detection sites.

[0155] (2) Interpretation of sample validity: In the test results of the sample, for the two groups of amplification, the target peak must appear at the quality control bin position, and the peak height must not be less than 300 RFU. This indicates that the sample cDNA template is normal.

[0156] (3) Result interpretation: Under the premise of experimental operation and sample validity, the amplification results of sample 2 groups are analyzed. If a target peak of greater than or equal to 100 RFU appears in the target bin except for the quality control peak, the fusion gene or mutation site in the corresponding bin of the sample is determined to be positive.

[0157] Example 4:

[0158] Primer combinations and kits for specificity and sensitivity detection

[0159] RNA extracted from routinely cultured, stable H293T cells that were confirmed to be negative for mutations in the BCR-ABL1 fusion gene and ABL1 kinase region was reverse transcribed to obtain cDNA, which served as the wild-type template. Positive plasmids were synthesized using cDNA sequence fragments generated from 13 different fusion forms of the BCR-ABL1 fusion gene, and plasmids synthesized using mutant sequence fragments at 20 sites were used as positive templates for each site. Positive reference samples with different mutation frequencies were prepared by mixing wild-type and mutant templates to test the specificity and sensitivity of the kit.

[0160] The specific testing methods are as follows:

[0161] (1) RNA extraction from wild-type reference sample

[0162] RNA was extracted from H293T cell line using the Tiangen Biotech RNA Easy Fast Animal Tissue Cell Total RNA Extraction Kit, following the kit instructions. RNA concentration was measured; the extracted RNA concentration needed to be above 100 ng / μL, with an OD260 / OD280 ratio between 1.9 and 2.1.

[0163] (2) Wild-type reference reverse transcription

[0164] The Novizan HiScript III RT SuperMix for qPCR (+gDNA wiper) kit was used, with a total RNA input of 1 μg, to obtain cDNA according to the kit instructions.

[0165] (3) Quantitative analysis of the copy number of wild-type reference specimens

[0166] The copy number of the ABL1 gene in the wild-type reference sample was absolutely quantified using a real-time PCR platform following standard laboratory procedures for absolute quantification of target genes. With 1 μg of total RNA input, the copy number of the ABL1 gene cDNA obtained through reverse transcription was 2.5 × 10⁻⁶. 5 Copy / μL.

[0167] (4) Preparation of positive reference samples with different mutation ratios

[0168] Positive plasmid templates synthesized from different mutation types were concentrated using nanodrop and brought to a final volume of 100 ng / μL. The corresponding copy number concentration was then calculated based on the molecular weight of each plasmid. Serial dilutions were performed to obtain positive plasmid templates at different concentrations for each mutation site. Positive plasmid templates were mixed with wild-type templates in specific ratios to prepare four gradient mutation positive reference standards for each of the 20 mutation sites: 50% mutation rate 3 × 10⁻⁶. 4 A strong positive reference sample of copies / μL, with a 10% mutation rate of 3×10⁻⁶. 4 Medium-concentration positive reference material at 1% concentration (copies / μL), mutation rate 3×10⁻⁶. 4 A weak positive reference sample of copies / μL; 0.1% mutation rate 3×10⁻⁶. 4 Sensitivity reference at copies / μL. A high concentration of 2 × 10⁻⁶ is used for the wild-type reference. 5 Copy / μL.

[0169] (5) Multiplex PCR amplification

[0170] Using the two primer mixtures described in Example 1, the reaction solution was prepared according to the amplification system of Example 3; for each of the four positive gradient references for each mutation site, one tube of wild-type reference was added (all sites were wild-type), along with the positive control template and negative (blank) control template of the positive control plasmid mixture included in all tests of the kit, for a total of 83 templates, two tubes of PCR amplification were prepared for each.

[0171] After the system was configured, PCR amplification was performed, and the amplification procedure was the same as in Example 3.

[0172] (6) Capillary electrophoresis detection

[0173] Prepare a loading mixture containing a molecular weight internal standard and formamide (mixing ratio of molecular weight internal standard and formamide 1:500). Calculate the number of tubes based on the number of PCR amplification products in each sample. Use a pipette to dispense 9 μL of the mixture into each well. Add 1 μL of PCR amplification product to the mixture, cover with a sealing film, and centrifuge briefly to remove air bubbles. Denature the sample tube at 95°C for 5 min, then immediately incubate on ice for 2 min to ensure complete denaturation of the PCR products into single-stranded nucleic acids. Perform capillary electrophoresis detection according to the user manual for the genetic analyzer. Parameters are set as follows: injection time 15 s, injection voltage 3 kV, and run time 2000 s.

[0174] (7) Data Analysis

[0175] Import the relevant files into the GeneMapper software, including Panel, Bin, corresponding Analysis Method, and Orange600 internal standard. Input the source sample data (.fsa file), select the previously imported file in the relevant parameter selection bar, and analyze the data.

[0176] Amplification chromatogram of positive control sample as follows Figure 1 As shown; the amplification map of the wild-type sample is as follows. Figure 2 As shown; the detection spectra of four T315I mutation reference samples, from 50% mutation, 10% mutation, 1% mutation, and 0.1% mutation are as follows. Figures 3 to 6 As shown.

[0177] from Figure 1 As can be seen, peaks of ≥300 RUF appeared at the target positions of the first group of 13 detected mutation sites, and specific peaks of ≥300 RUF appeared at the target peak positions of the second group of 13 fusion forms of BCR-ABL1 fusion gene and 7 mutation detection sites.

[0178] from Figure 2 As can be seen from the data, in the first and second groups, except for the quality control peak position which has a main peak of ≥300 RFU, there are no obvious amplification peaks at other sites.

[0179] from Figures 3 to 6 As can be seen, both the first and second groups have obvious quality control peaks, and the peak heights of the quality control peaks are not significantly different. The first group shows obvious amplification at the T315I position, and the peak height increases from... Figures 3 to 6 Decreasing sequentially.

[0180] Example 5:

[0181] Application of the kit in detecting BCR-ABL1 fusion gene and ABL1 kinase domain mutations in leukemia

[0182] Using the primer combination and kit for detecting hotspot mutations in the BCR-ABL1 fusion gene and ABL1 kinase region described in Example 1, we validated and tested 10 positive RNA samples with mutations at different sites collected routinely from the Department of Hematology at the First Affiliated Hospital of Ningbo University (the mutation types in the ABL1 kinase region have been determined by PCR-Sanger sequencing).

[0183] The specific testing methods are as follows:

[0184] 1. Reverse transcription

[0185] The Novizan HiScript III RT SuperMix for qPCR (+gDNA wiper) kit was used, with a total RNA input of 1 μg, to obtain cDNA according to the kit instructions.

[0186] 2. Multiplex RT-PCR amplification

[0187] Using the two primer mixtures described in Example 1, the reaction solution was prepared according to the same amplification system as in Example 3. Two tubes of total PCR amplification were prepared for each of the reverse-transcribed cDNA from 10 confirmed genotyped samples, one negative control sample, and a positive control.

[0188] After the system was configured, PCR amplification was performed, and the amplification procedure was the same as in Example 3.

[0189] 3. Capillary electrophoresis detection

[0190] Prepare a loading mixture containing a molecular weight internal standard and formamide (the ratio of molecular weight internal standard to formamide is 1:500). Calculate the number of tubes based on the number of PCR amplification products in each sample. Use a pipette to dispense 9 μL of the mixture into each well. Add 1 μL of PCR amplification product or 1 μL of positive genotyping reference standard to the mixture. Cover with a sealing film, centrifuge briefly to remove air bubbles from the sample loading tubes. Denature the loading plate at 95°C for 5 min, then immediately incubate on ice for 2 min to ensure complete denaturation of the PCR products into single-stranded nucleic acids. Perform capillary electrophoresis detection according to the user manual for the genetic analyzer. Parameters are set as follows: injection time 15 s, injection voltage 3 kV, and run time 2000 s.

[0191] 4. Data Analysis

[0192] Import the relevant files into the GeneMapper software, including Panel, Bin, corresponding Analysis Method, and Orange600 internal standard. Input the source sample data (.fsa file), select the previously imported file in the relevant parameter selection bar, and analyze the data.

[0193] 5. Result Interpretation

[0194] Based on the data analysis atlas interpretation criteria, 10 clinical samples were analyzed. The results are shown in Table 5: Interpretation of Test Results for 10 Clinical Samples.

[0195] Table 5: Interpretation of test results from 10 clinical samples

[0196]

[0197] Figure 7 This is the amplification pattern of one case of BCR-ABL1 P210 positivity with M351T mutation. Figure 7 It can be seen that in the first set of amplification patterns, in addition to the quality control peak at the quality control peak position, a specific peak also appeared at the corresponding position of M351T; the second set of amplification patterns also showed specific peaks at both the quality control peak position and the BCR-ABL1 fusion gene position.

[0198] The clinical samples were all tested for BCR-ABL1 fusion gene using nested or real-time quantitative PCR, and then the ABL1 kinase domain mutation status was checked using first-generation sequencing. The method used in this embodiment simultaneously detected both BCR-ABL1 fusion status and ABL1 kinase domain mutation status, and the test results were consistent with the clinical results, with a consistency rate of 100%.

[0199] Therefore, the primer combinations, kits, and methods for detecting hotspot mutations in the BCR-ABL1 fusion gene and ABL1 kinase region of leukemia, as described in this invention, are consistent with clinical diagnosis. This demonstrates that the primer combinations, kits, and detection methods for simultaneously detecting hotspot mutations in the BCR-ABL1 fusion gene and ABL1 kinase region of leukemia based on capillary electrophoresis analysis of this invention have high specificity and sensitivity, and can be fully applied clinically for one-time detection of BCR-ABL1 fusion gene and ABL1 kinase region mutations in leukemia. The detection method of this invention is simple to operate, highly specific, highly sensitive, rapid, efficient, high-throughput, low-cost, and requires a small sample size, thus possessing extremely high clinical application value.

[0200] Comparative Example 1: Design of Control Primer Set

[0201] Primers were designed using Primer 5 software targeting 13 mutation sites (M244V, G250E, Q252H, E255V, D276G, V299L, T315I, M351T, F359C, L387M, H396R, E459K, and F486S) and 7 hotspot mutations (F359V, E355G, F317L, E279K, E255K, Y253H, and L248V) in the ABL1 kinase domain. The sequences of these primers are shown in SEQ ID NO.47 to SEQ ID NO.59. This primer set was mixed with the same common reverse primers SEQ ID NO.12 ABL-E7-UR1 and SEQ ID NO.15 ABL-E10-UR1, as well as the quality control primers SEQ ID NO.16 and SEQ ID NO.17 to form control primer set 1. See Table 6: Compare the sequences of primer set 1.

[0202] Table 6: Sequences of Comparison Primer Set 1

[0203]

[0204] Primer sets were designed for 13 fusion forms of the BCR-ABL1 fusion gene in leukemia (BCR-ABL1-E13A2, BCR-ABL1-E14A2, BCR-ABL1-E19A2, BCR-ABL1-E1A2, BCR-ABL1-E13A3, BCR-ABL1-E14A3, BCR-ABL1-E19A3, BCR-ABL1-E6A3, BCR-ABL1-E12A3, BCR-ABL1-E12A2, BCR-ABL1-E6A2, BCR-ABL1-E1A3, and BCR-ABL-E8A2) using the primer design principles in patent CN117568455B. The primer sequences are shown in SEQ ID NO. 30 to SEQ ID NO. 35.

[0205] Primer sets were designed using Primer5 software for seven hotspot mutations in the ABL1 kinase domain (F359V, E355G, F317L, E279K, E255K, Y253H, and L248V), and their sequences are shown in SEQ ID NO. 60 to SEQ ID NO. 66.

[0206] Primer sequences SEQ ID NO.30 to SEQ ID NO.35, SEQ ID NO.61 to SEQ ID NO.66, and quality control primers SEQ ID NO.16 and SEQ ID NO.17 were mixed to form control primer set 2. See Table 7: Sequences of control primer set 2.

[0207] Table 7: Sequences of Comparison Primer Set 2

[0208]

[0209] The detection kit was prepared using the same method as described in Example 2, employing primer sets 1 and 2. The wild-type samples were then specifically tested using this detection kit as described in Example 4, and the results are shown below. Figure 8 .Depend on Figure 8 As shown, in addition to the quality control peak, non-characteristic peaks of varying heights appeared at the ABL1 kinase domain mutation detection sites, affecting the interpretation.

[0210] By comparing the specificity detection results of Comparative Example 1 with those of Example 4, it can be seen that: the primers for detecting mutations were designed entirely according to the conventional AS primer design method, and the detection was performed according to the primer mixing method and detection method of the invention. As a result, the detection specificity was poor, and most detection sites would have false positive amplification peaks of varying degrees, making it impossible to interpret the results effectively.

[0211] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Primer combination for simultaneous detection of BCR-ABL1 fusion gene quantification and hotspot mutations in the ABL1 kinase region, characterized in that: This includes the following two major groups of primers: The first set of primers was designed to target 13 mutation sites in the ABL1 kinase region, and its primer sequences are shown in SEQ ID NO.1 to SEQ ID NO.

29. Thirteen mutation sites: M244V, G250E, Q252H, E255V, D276G, V299L, T315I, M351T, F359C, L387M, H396R, E459K, and F486S; The second set of primers was designed to target 13 fusion forms of the BCR-ABL1 fusion gene and 7 hotspot mutations in the ABL1 kinase region. The primer sequences are shown in SEQ ID NO.30 to SEQ ID NO.46, SEQ ID NO.16 to SEQ ID NO.17, SEQ ID NO.20, and SEQ ID NO.

25. Thirteen fusion forms: BCR-ABL1-E13A2, BCR-ABL1-E14A2, BCR-ABL1-E19A2, BCR-ABL1-E1A2, BCR-ABL1-E13A3, BCR-ABL1-E14A3, BCR-ABL1-E19A3, BCR-ABL1-E6A3, BCR-ABL1-E12A3, BCR-ABL1-E12A2, BCR-ABL1-E6A2, BCR-ABL1-E1A3, BCR-ABL-E8A2. Seven hotspot mutations: F359V, E355G, F317L, E279K, E255K, Y253H, and L248V; The primer sequences SEQ ID NO.16 and SEQ ID NO.17 are quality control primers common to both the first and second primer groups; The first and second primer sets also contain blocker probes that specifically block wild-type templates. The blocker probes of the first primer group are formed by labeling the primers of the sequences shown in SEQ ID NO.18 to SEQ ID NO.29 with a repression modification group; the blocker probes of the second primer group are formed by labeling the primers of the sequences shown in SEQ ID NO.20, SEQ ID NO.25, SEQ ID NO.43 to SEQ ID NO.46 with a repression modification group.

2. The primer combination according to claim 1, characterized in that: The labeling of the repressor modifying group is selected from any one of 3'-Spacer C3, 3'-Phosphat, 3'-ddC, 3'-Inverted End, or 3'-MGB.

3. The primer combination according to claim 1, characterized in that: The primer sequences in the first primer group and the second primer group are labeled with different fluorescent markers, and the fluorescent markers are selected from any one of the fluorescent genes FAM, HEX, VIC, TAMRA, ROX or TEXRED.

4. The primer combination according to claim 3, characterized in that: The fluorescent gene is labeled on the universal primers for amplification corresponding to the mutation detection site.

5. A kit for the simultaneous detection of BCR-ABL1 fusion gene quantification and hotspot mutations in the ABL1 kinase domain, characterized in that: It comprises the primer combination as described in any one of claims 1 to 4.

6. The reagent kit according to claim 5, characterized in that: The kit also includes an amplification enzyme mixture, a positive control, a negative control, and a capillary electrophoresis internal standard reagent.

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