Primer combination, kit and application for detecting bcr::abl1 fusion gene transcript and tyrosine kinase domain mutation based on targeted sequencing

Abstract

This application discloses a primer combination, kit, and application for detecting mutations in the transcript and tyrosine kinase region of the BCR::ABL1 fusion gene based on targeted sequencing, belonging to the field of gene detection technology. The primer combination or kit of this invention can cover all known types of BCR::ABL1 fusion genes, effectively avoiding the problem of missed detection of fusion subtypes. Furthermore, it provides more comprehensive coverage of the ABL1 kinase region, enabling the complete capture of known and potential drug resistance mutations within this region, further improving the comprehensiveness and accuracy of detection, and providing a more reliable basis for clinical treatment.

Description

Technical Field

[0001] This application relates to the field of gene detection technology, and in particular to a primer combination, kit, and application for detecting mutations in the transcript and tyrosine kinase region of the BCR::ABL1 fusion gene based on targeted sequencing. Background Technology

[0002] Chronic myeloid leukemia (CML) and some types of acute lymphoblastic leukemia (ALL) are characterized by a cytogenetic abnormality: the Philadelphia chromosome (Ph chromosome) formed by the t(9;22)(q34;q11) translocation. At the molecular level, this manifests as a BCR::ABL1 fusion gene, leading to persistent activation of the ABL1 tyrosine kinase. ABL1 tyrosine kinase inhibitors (TKIs), represented by imatinib, have become core drugs for first-line treatment of CML and for the treatment of Ph+ALL, significantly improving patient prognosis. However, TKI resistance remains a key problem for most patients in clinical treatment, and mutations in the BCR::ABL1 tyrosine kinase domain (TK domain) are an important molecular mechanism mediating resistance.

[0003] Direct sequencing (Sanger sequencing) is currently the standard clinical method for detecting mutations in the BCR::ABL1 tyrosine kinase region, and its results can provide a basis for the clinical selection of TKI drugs. However, direct sequencing has low sensitivity and is prone to missing low-abundance mutations. In the early stages of TKI treatment in CML patients, resistance mutations often exist in a low-abundance form (e.g., the proportion of mutated cells <5%), and direct sequencing is prone to missing them, leading to clinical misjudgment as "no mutation," thus delaying the timing of resistance intervention. In addition, multiple mutations can coexist in the BCR::ABL1 tyrosine kinase region (i.e., compound mutations), and the signals of different mutations overlap during direct sequencing, making it difficult to accurately identify the type and abundance of each mutation.

[0004] Real-time quantitative PCR (qPCR) is also a commonly used technique for detecting mutations in the BCR::ABL1 tyrosine kinase region. Allele-specific qPCR (AS-qPCR) has higher sensitivity than direct sequencing (typically reaching 0.1%–1%) and is widely used clinically for screening known drug resistance mutations. However, qPCR technology (especially AS-qPCR) relies on designing specific primers or probes (such as T315I, E255K, etc.) based on known mutation sites, and can only detect preset mutation types, failing to detect unknown mutations. Furthermore, the specificity of AS-qPCR is highly dependent on the precise binding of primers / probes to the mutation sequence, making it prone to cross-reactivity and resulting in a high risk of false positives.

[0005] In addition, direct sequencing and qPCR methods have the following drawbacks: First, it is impossible to confirm whether the detected mutations originate from the BCR::ABL1 fusion gene transcript. Other methods are needed to confirm the positivity of the BCR::ABL1 fusion before the detected variants can be identified as drug-resistant mutations. The process is complex and the traceability of the results is insufficient.

[0006] Secondly, the detection of fusion types is incomplete, posing a risk of missed detection. The BCR::ABL1 fusion gene has multiple subtypes (such as p210, p190, p230, etc.), and current technology can only detect a few common fusion subtypes (such as only detecting p210 and p190), which cannot cover all known fusion types. This can easily lead to missed detection of rare fusion subtypes, thereby affecting the comprehensiveness of clinical diagnosis and the accuracy of treatment planning.

[0007] Finally, the coverage of the ABL1 kinase domain is incomplete, easily missing key mutation sites. Current detection methods have limited coverage of the ABL1 tyrosine kinase domain, failing to cover all key functional regions of the kinase domain. Some drug resistance mutations located in the uncovered regions cannot be detected, leading to the failure to discover drug resistance mechanisms in a timely manner, delaying the timing of treatment adjustment, and affecting patient prognosis. Summary of the Invention

[0008] To solve at least one of the above-mentioned technical problems, the technical solution adopted in this application is as follows.

[0009] The first aspect of this application provides a primer combination for detecting mutations in the transcript and tyrosine kinase region of the BCR::ABL1 fusion gene based on targeted sequencing, comprising: The first-round amplification primer combination consists of the primers shown in SEQ ID No. 1 to SEQ ID No. 5, which are used to amplify the BCR::ABL1 fusion gene transcript using cDNA obtained by reverse transcription of RNA as a template, to obtain the first-round amplification product; The second-round amplification primer combination 1, consisting of primers shown in SEQ ID No. 6 to SEQ ID No. 18, is used to amplify the breakpoint sequence of the BCR::ABL1 fusion gene and part of the ABL1 tyrosine kinase region using the first-round amplification product as a template. The second-round amplification primer combination 2 consists of primers shown in SEQ ID No. 19 to SEQ ID No. 26, and is used to amplify the ABL1 tyrosine kinase region using the first-round amplification product as a template.

[0010] ABL1 is a proto-oncogene located on chromosome 9, q34 (long arm, region 3, band 4). Its gene product is a non-receptor tyrosine protein kinase. The BCR gene is located on chromosome 22, q11 (long arm, region 1, band 1). The BCR::ABL1 fusion gene is an oncogene formed by the translocation of the ABL1 and BCR genes. Its product has abnormal tyrosine kinase activity and anti-apoptotic function, leading to uncontrolled cell proliferation and abnormal cell survival. This fusion gene is carried on the Philadelphia chromosome, generated by t(9;22)(q34;q11), and is present in more than 90% of chronic myeloid leukemia (CML), 30% of adult acute lymphoblastic leukemia, and some other hematologic malignancies. Based on the different breakpoints in the BCR gene, the BCR::ABL1 fusion gene can be mainly divided into three types: M-BCR (p210), m-BCR (p190), and µ-BCR (p230). Over 95% of CML patients have the BCR::ABL1 fusion gene of type M, primarily type b2a2 (40%) and type b3a2 (55%). In this application, the first-round amplification primer combination is capable of amplifying all types of BCR::ABL1 fusion genes, including but not limited to p210, p190, p230, p195, p200, and p225.

[0011] In some embodiments of this application, each primer has a non-human characteristic sequence attached to its 5' end.

[0012] In some specific embodiments of this application, the nucleotide sequence of the non-human characteristic sequence is shown in SEQ ID No. 27. Therefore, the first round of amplification primer combination consists of primers shown in SEQ ID No. 28 to SEQ ID No. 31, the second round of amplification primer combination 1 consists of primers shown in SEQ ID No. 33 to SEQ ID No. 45, and the second round of amplification primer combination 2 consists of primers shown in SEQ ID No. 46 to SEQ ID No. 53.

[0013] The second aspect of this application provides a kit for detecting mutations in the transcript and tyrosine kinase region of the BCR::ABL1 fusion gene based on targeted sequencing, including any of the primer combinations described in the first aspect of this application.

[0014] In some embodiments of this application, the kit further includes RNA extraction reagents, reverse transcription reagents, PCR amplification buffers, and / or DNA polymerase.

[0015] In some embodiments of this application, the kit further includes sequencing library preparation reagents, including but not limited to sequencing adapter primers.

[0016] The third aspect of this application provides the use of any of the primer combinations described in the first aspect of this application in the preparation of a kit for detecting mutations in the BCR::ABL1 fusion gene transcript and tyrosine kinase region based on targeted sequencing.

[0017] In some embodiments of this application, the targeted sequencing includes the following steps: Obtain the RNA sample of the sample to be tested, and reverse transcribe it to obtain the cDNA sample; Using the cDNA sample as a template, the first round of PCR amplification was performed using the first round of amplification primer combination to obtain the first amplification product; Using the first amplification product as a template, a second round of PCR amplification was performed using the second round amplification primer combination 1 and the second round amplification primer combination 2, respectively, to obtain the second round PCR amplification product; The sequencing library was constructed using the second round of PCR amplification products and then sequenced. Analysis of the sequencing data yielded transcript information for the BCR::ABL1 fusion gene and mutation information for the tyrosine kinase region.

[0018] In some embodiments of this application, the first round of PCR amplification program is as follows: 95℃ for 3 min 30 sec; 98℃ for 20 sec, 55℃ for 30 sec, 60℃ for 30 sec, 65℃ for 1 min, 12 cycles; 72℃ for 5 min; and 4℃ hold.

[0019] In some embodiments of this application, the second round of PCR amplification program is as follows: 95℃ for 3 min 30 sec; 98℃ for 20 sec, 58℃ for 1 min, 72℃ for 30 sec, 9 cycles; 72℃ for 5 min; hold at 4℃.

[0020] In some embodiments of this application, after constructing the sequencing library, quality control of the library is performed, requiring the library concentration to be greater than 0.5 ng / μL and the library fragments to be 250 bp ≤ 450 bp.

[0021] Compared with the prior art, this application has the following advantages: Using the primer combination and kit of this application, the BCR::ABL1 fusion gene transcript can be identified simultaneously, and the detected mutation can be directly confirmed to originate from the BCR::ABL1 fusion gene transcript. The detection results can be directly used to guide the individualized selection of clinical TKI drugs and the adjustment of treatment plans.

[0022] The primer combination or kit in this solution covers all known types of BCR::ABL1 fusion genes, including p210, p190, p230, p195, p200, and p225, effectively avoiding missed detections of fusion subtypes. Furthermore, the primer combination and kit provide more comprehensive coverage of the ABL1 kinase region, precisely covering the 109-516 amino acid range, enabling comprehensive capture of known and potential drug resistance mutations within this region. This further enhances the comprehensiveness and accuracy of the detection, providing a more reliable basis for clinical treatment.

[0023] The first-round amplification primer combination of this application uses cDNA reverse transcribed from an RNA sample as a template for amplification, thereby obtaining the amplification product of the BCR::ABL1 fusion gene transcript. Further, using the first-round amplification product as a template, the second-round amplification primer combination 1 and the second-round amplification primer combination 2 are used to specifically amplify the ABL1 tyrosine kinase region and the breakpoint region of the BCR::ABL1 fusion gene, respectively, to obtain the second-round amplification product. Library construction and sequencing are performed using the second-round amplification product, and analysis of the sequencing data allows for the simultaneous detection of BCR::ABL1 fusion gene transcript information and ABL1 tyrosine kinase region mutation information, which has significant clinical application value.

[0024] The primer combination and kit of this application have a detection sensitivity of up to 0.1% for BCR::ABL1 fusion gene transcripts and a detection sensitivity of 1% for drug resistance mutations in the ABL1 tyrosine kinase region. They can also accurately identify multiple fusion gene transcripts and complex mutations containing one or more mutation sites.

[0025] Because this invention has a wide detection coverage and involves a complex number and combination of primers, system optimization is quite challenging. Before optimization, problems such as primer interference and non-specific amplification can lead to uneven amplification efficiency, resulting in decreased detection capability for known fusion transcripts and weaker signals at mutation sites, thus affecting the accuracy and reliability of the detection results. After targeted system optimization, cross-competition and non-specific binding between multiple primers can be effectively eliminated, significantly improving the uniformity of amplification at each target site and inhibiting the formation of non-specific amplification products. While ensuring simultaneous amplification of multiple targets, it still maintains high detection sensitivity for fusion transcripts and drug-resistant mutations, ensuring that various fusion subtypes and complex mutation sites can be effectively detected, greatly improving the accuracy, repeatability, and reliability of the detection results.

[0026] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0027] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which: Figure 1 This document illustrates a flowchart of targeted sequencing and analysis using the primer combination from Example 1 in Embodiment 2 of this application. Figure 2 This illustrates the effect of the number of first-round multiplex PCR amplification cycles on the sequencing depth of the kinase region amplicon in Example 2 of this application; Figure 3 The effect of different parameter combinations on the sequencing depth of the kinase region amplicon is shown in Example 2 of this application. Detailed Implementation

[0028] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0029] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0030] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0031] Unless otherwise defined, 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 application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0032] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0034] Example 1: Primer Design and Screening for Multiplex PCR Detection This invention provides a multiplex primer set that can simultaneously identify the BCR::ABL1 fusion gene transcript and detect drug resistance mutations in the tyrosine kinase region. The primer design and screening process is as follows.

[0035] 1) Primer design ideas This primer set targets the known fusion breakpoint of BCR::ABL1 and the full-length sequence of the ABL1 tyrosine kinase region. It adopts a shingled design to achieve full coverage of the target region without any detection blind spots. At the same time, the primers adopt a cross-exon design to effectively avoid interference from wild-type fusion sequences / genomic DNA. Combined with the regular amplicon arrangement and spacing design, it is suitable for multiplex PCR amplification systems, taking into account the specificity, integrity and uniformity of detection.

[0036] 2) Primer design method Wrap-in amplification design: A continuous wavy amplicon arrangement is used to cover all drug resistance hotspots and rare mutation sites in the ABL1 kinase region; custom amplicon is designed for known BCR::ABL1 fusion subtypes to achieve specific capture of fusion transcripts.

[0037] Precise control of amplicon spacing: The amplicon fragment length is uniform, and a small overlap and gapless arrangement is adopted to eliminate the blank area of ​​missed detection of sites and avoid primer interference and non-specific amplification caused by excessive overlap of amplicon, thus ensuring the integrity of regional coverage.

[0038] Cross-exon specific design: ABL1 kinase domain detection primers are designed to cross exons, specifically amplifying only the cDNA sequence without binding to genomic DNA, thus reducing background interference from the source.

[0039] Multiple system adaptation and optimization: unify the physicochemical parameters such as Tm value, GC content, and length of all primers, avoid primer dimer, hairpin structure and cross complementarity issues, and adapt to the conditions for simultaneous amplification of multiplex PCR.

[0040] 3) Primer screening criteria Physicochemical parameters are qualified: that is, primer length, GC content, and Tm value differences all meet the requirements for multiplex amplification, and there are no undesirable secondary structures; Good specificity: no wild-type or non-specific amplification; Excellent amplification uniformity: No obvious amplification bias after multiplex mixed amplification; It provides complete coverage, fully covering target fusion breakpoints and drug resistance mutation sites, with no detection blind spots.

[0041] 4) Primer screening process First, candidate primers were designed in batches based on reference sequences, and primers with abnormal parameters or structural defects were initially screened out. Then, single PCR was used to verify and screen qualified primers with single amplification bands and stable efficiency. Next, multiple system compatibility tests were conducted to eliminate primers that interfered with each other or had abnormal amplification. Finally, after multiple rounds of optimization and fine-tuning of the amplicon arrangement, the optimal primer combination was finally obtained, as shown in Table 1.

[0042] Table 1: Primer set for BCR::ABL1 fusion gene transcript identification and tyrosine kinase domain drug resistance mutation detection .

[0043] To effectively avoid environmental and reagent contamination and reduce interference with other detection items, a non-human characteristic sequence, GTCAGGATCTGACG (SEQ ID No. 27), was introduced at the 5' end of the primer sequence. The primer sequences after adding the above non-human characteristic sequence are shown in Table 2.

[0044] Table 2: Primer set for BCR::ABL1 fusion gene transcript identification and tyrosine kinase region drug resistance mutation detection (with added non-human characteristic sequences) .

[0045] Example 2: Targeted sequencing and analysis using the primer combination from Example 1. refer to Figure 1 This embodiment provides the use of primer combinations from Example 1 for targeted sequencing and data analysis to obtain transcript information of the BCR::ABL1 fusion gene and mutation information of the tyrosine kinase region.

[0046] 1. RNA reverse transcription Thaw the Novizan PR111 reagent components 10× RT Mix, PureScript Enzyme Mix, Oligo (dT)20VN, and Random hexamers on ice.

[0047] RNA template denaturation: Incubate 14 μL of RNA template (100 or 500 ng) at 65°C for 5 min, with the heating cap set to 75°C. After the reaction, incubate on ice for 2 min.

[0048] Prepare the cDNA synthesis reaction solution according to Table 3.

[0049] Table 3: cDNA synthesis reaction system .

[0050] Place the PCR tubes on the PCR instrument and set the heating cap to 95℃, 25℃ for 5 min; 37℃ for 45 min; 85℃ for 5 sec.

[0051] 2. First round of multiplex PCR amplification: amplification of the BCR::ABL1 fusion gene Remove the TOYOBO KFX-201 reagent components: 2× PCR Buffer for KODFX Neo, 2 mM dNTPs, KODFX Neo (1 U / μL), and Primer pool A, and thaw them on ice. Primer pool A consists of primers shown in SEQ ID No. 28 to SEQ ID No. 32, with each primer at a concentration of 10 μM. Prepare the first-round multiplex PCR amplification reaction system in the PCR tubes according to Table 4.

[0052] Table 4: First-round multiplex PCR amplification reaction system .

[0053] Add 6 μL of reverse transcription product to the 19 μL amplification reaction mixture. Gently pipette to mix and briefly centrifuge, keeping the entire process on ice.

[0054] Place the PCR tubes on the PCR instrument and set the heat cap to 105℃. The procedure for the first round of multiplex PCR amplification is shown in Table 5 below.

[0055] Table 5: First-round multiplex PCR amplification procedure .

[0056] 3. Second round of multiplex PCR amplification: amplification of the ABL1 tyrosine kinase region and the breakpoint sequence of the BCR::ABL1 fusion gene. Thaw the MultipSeq® Library Prep Kit M61022 reagent components—Enhancer buffer NB(1N), IGT-EM808 polymerase mixture, Enhancer buffer M, YF buffer B, and Primer pools B1 / B2—on ice. Primer pool B1 consists of primers shown in SEQ ID No. 33–SEQ ID No. 45, each with a concentration of 10 μM; Primer pool B2 consists of primers shown in SEQ ID No. 46–SEQ ID No. 53, each with a concentration of 10 μM. Prepare the second-round multiplex PCR amplification reaction system in PCR tubes according to Table 6.

[0057] Table 6: Second-round multiplex PCR amplification reaction system .

[0058] Two 21 μL reaction systems (two PCR tubes) were prepared using Primer pool B1 and Primer pool B2. 9 μL of the amplification product from the first round of multiplex PCR was added to each tube. The mixture was gently pipetted and briefly centrifuged, with the entire process performed on ice.

[0059] Place the PCR tubes on the PCR instrument and set the heat cap to 105°C. The procedure for the second round of multiplex PCR amplification is shown in Table 7 below.

[0060] Table 7: Second Round Multiplex PCR Amplification Procedure .

[0061] 4. Purification of PCR products (1) Preparation before purification: Remove VAHTS® DNA Clean Beads from the 4°C freezer, vortex to mix thoroughly, and allow to equilibrate at room temperature for at least 30 minutes before use. Vortex to mix thoroughly again before each subsequent use. Prepare 80% ethanol using anhydrous ethanol and nuclease-free water for cleaning the magnetic beads.

[0062] (2) Add 27 μL (0.9X) VAHTS® DNA Clean Beads to the new PCR tube, and then add 15 μL of the product obtained by Primer pool B1 and 15 μL of the product obtained by Primer pool B2 to the corresponding PCR tubes with magnetic beads. Vortex to mix thoroughly and let stand at room temperature for 5 min.

[0063] (3) After the PCR tube is centrifuged briefly, it is placed on a magnetic rack and left to stand for 2 minutes. After the solution is clear, the supernatant is removed.

[0064] (4) Remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B into the tube, mix well, and let stand at room temperature for 5 mins.

[0065] (5) Centrifuge briefly, place the PCR tube on a magnetic rack and let it stand for 2 minutes. After the solution is clear, remove the supernatant.

[0066] (6) Add 180 μL of 80% ethanol to the PCR tube to rinse the magnetic beads, incubate for 30 seconds and then remove the supernatant.

[0067] (7) Repeat step (6) and rinse the magnetic beads once more with 80% ethanol.

[0068] (8) After taking out the PCR tube and centrifuging briefly, place it on a magnetic rack, use a 20μL pipette to remove the residual ethanol at the bottom of the tube, and open the tube cap to dry until there is no ethanol residue.

[0069] (9) Add 21 μL of nuclease-free water to each tube and gently pipette to resuspend the magnetic beads. Let stand at room temperature for 5 minutes to allow the DNA on the magnetic beads to be fully released.

[0070] (10) After the PCR tube is detached, place it on a magnetic rack and let it stand for 2 minutes. Then transfer 20 μL of supernatant to a new PCR tube.

[0071] 5. Add connector sequence Thaw the IGT-EM808 polymerase mixture, Enhancer buffer M, and VAHTS UDI Primer (catalog number: N351) on ice. Prepare the reaction system according to Table 8 below.

[0072] Table 8: Adding a Connector Reaction System .

[0073] Add the above reaction components to a new PCR tube, along with 12.5 μL of the purified product from the previous round, for a total volume of 30 μL. Vortex to mix thoroughly and then centrifuge briefly.

[0074] Place the PCR tubes on the PCR instrument and set the heat cap to 105°C. Add the adapter reaction procedure as shown in Table 9.

[0075] Table 9: Adding Connector Reaction Procedure .

[0076] After the reaction was completed, the product with added adapter was purified according to the aforementioned PCR product purification method.

[0077] 6. Document Quality Inspection The library was vortexed to mix thoroughly, then briefly centrifuged. 1 μL of the sample was taken for concentration analysis, and fragment analysis was performed using an Agilent 4200 instrument. The fragment size and adapter ratio were recorded. The library concentration was required to be greater than 0.5 ng / μL, and the fragment size should be between 250 bp and 450 bp.

[0078] 7. Sequencing The sequencing platform and sequencing mode were Illumina NovaSeq-6000. Sequencing reagents with a sequencing read length of 300 cycles (paired-end sequencing, 2 × 150 cycles) were used for sequencing, with each library containing at least 50 Mb of data. Dilutes were prepared according to the following method for sequencing.

[0079]

[0080] 8. Data Analysis The following steps are involved in analyzing raw data from the Illumina platform: Sequencing data format conversion: Illumina's bcl2fastq v2.20.0.422 software was used to convert the BCL files generated by the sequencer to generate FastQ files for each sample.

[0081] Data preprocessing and quality control: The sequencing quality of each sample data was analyzed based on the Q30 base percentage. If the Q30 base percentage was ≥90%, the quality control passed; otherwise, it failed. Simultaneously, adapter sequences and low-quality base fragments introduced during library construction were removed.

[0082] Sequence alignment: The base sequences in the FastQ file are aligned to the hg19 (GRCh37) human reference genome to generate a BAM file. The BAM file is then sorted according to the genome coordinates, and sequence re-alignment and correction are performed on complex regions of the genome.

[0083] Comparison result quality control: Calculate statistical indicators such as the average sequencing depth (raw depth) and the total reads (depth) of the target region for each sample. If all key indicators are higher than the threshold, that is, if the sample simultaneously meets the requirements of a total number of reads in the target region ≥ 100,000 and an average sequencing depth ≥ 10,000X, then the quality control is passed and the sample data is considered qualified. Otherwise, if any of the key indicators is unqualified, the sample data is considered unqualified and the experiment is repeated.

[0084] Mutation analysis: The processed BAM files were analyzed to identify point mutations (SNVs) and insertion / deletion mutations (InDels). Specifically, firstly, rearranged reads were assembled in local regions using De Bruijn diagrams to generate candidate haplotypes; then, supporting evidence for each candidate allele was calculated based on sequencing data, and the posterior probability of the genotype was calculated using Bayes' theorem. Filtering thresholds included: sequencing depth of a single variant site ≥10,000, variant support number ≥70, variant allele frequency (VAF) ≥0.7% (SNV / InDel), and no significant strand bias.

[0085] Mutation annotation: Identified variant coordinates are cross-referenced with various public databases (such as dbSNP, gnomAD, COSMIC, and ClinVar) to obtain information such as population frequency, clinical significance, and tumor association. Using the ANNOVAR algorithm, the impact of variants on gene function (such as missense, nonsense, and splice site alterations) is predicted and calculated according to preset rules (such as "clinical pathogenicity > ...). Functional prediction harmful > "High-frequency polymorphism" prioritizes and formats the output of annotation information.

[0086] In this embodiment, the inventors optimized the detection system using one clinical sample. The optimizations mainly included the amount of RNA template loaded (100-500 ng), the amount of reverse transcription product (cDNA) loaded (1-14 μL), the number of cycles for the first round of multiplex PCR amplification (C1) (30-35), the amount of the first round of multiplex PCR amplification product used (1-9 μL), the number of cycles for the second round of multiplex PCR amplification (C2) (12-20), and the number of cycles for adding adapter sequences (C3) (7-9).

[0087] The above conditions were combined and tested, and the results were comprehensively evaluated to select the optimal combination of parameters for the entire reaction system: RNA template loading amount of 500ng, reverse transcription product (cDNA) loading amount of 14μL, C1 of 30, first round multiplex PCR amplification product volume of 9μL, C2 of 12, and C3 of 9.

[0088] The effects of different cycle numbers on the sequencing depth of the kinase region in one sample are shown in Table 10 and... Figure 2 As shown.

[0089] Table 10: Effect of the number of first-round multiplex PCR amplification cycles on the sequencing depth of kinase region amplicon (only some combinations are shown) .

[0090] From Table 10 and Figure 2 It can be seen that when the number of cycles for the first round of multiplex PCR amplification is 30, the sequencing depth of each amplicon is more uniform.

[0091] Assuming a first-round multiplex PCR amplification cycle count of 30, the amount of reverse transcription initiation, the number of second-round multiplex PCR amplification cycles, and the number of cycles for adding adapter sequences will also affect sequencing depth, as shown in Tables 11 and 12. Figure 3 As shown.

[0092] Table 11: Partial parameter combinations consisting of reverse transcription initiation amount, first-round multiplex PCR amplification cycle number, second-round multiplex PCR amplification cycle number, and adapter sequence addition cycle number. .

[0093] Table 12: Effects of different parameter combinations on sequencing depth .

[0094] From Table 12 and Figure 3It can be seen that when parameter combination 5 is selected (i.e., reverse transcription initiation amount is 500ng, C1 is 30, C2 is 12, and C3 is 9), the average sequencing depth of each amplicon is higher and the CV value is smaller, indicating that the sequencing depth of each amplicon is less different.

[0095] In this embodiment, the system has been optimized, significantly reducing the differences in sequencing depth among the amplicones and significantly improving amplification uniformity.

[0096] Example 3 Clinical Sample Testing Thirteen clinical bone marrow or whole blood samples that were verified to be positive for ABL1 kinase domain mutations by direct sequencing and 15 whole blood samples from healthy volunteers were selected. The BCR::ABL1 fusion gene transcripts of 28 samples were all unknown.

[0097] The BCR::ABL1 kinase domain mutation was detected using the method described in Example 2. Specifically, RNA was first extracted, followed by RNA reverse transcription, a first round of multiplex PCR amplification, a second round of multiplex PCR amplification, PCR product purification, addition of adapter sequences and purification, library quality control, and sequencing. Data analysis was then performed to obtain the mutation results.

[0098] The detection results of 13 positive clinical samples are shown in Table 13.

[0099] Table 13: Clinical Sample Test Results .

[0100] As shown in Table 13, multiplex PCR amplification sequencing can effectively detect known kinase domain mutations. Compared with direct sequencing, it can identify low-abundance drug resistance mutations in 46.2% of samples (samples 3, 4, 7, 8, 11, and 12). The detection sensitivity of 13 clinical samples reached 100%.

[0101] The test results of the healthy volunteer samples are shown in Table 14.

[0102] Table 14: Detection Results of Samples from Healthy Volunteers .

[0103] As shown in Table 14, the fusion detection results of all 15 samples were negative, and the kinase domain mutation detection results were all negative, indicating that the multiplex PCR amplification sequencing of this application has a detection specificity of 100% for 15 healthy volunteer samples.

[0104] Furthermore, it should be understood that after reading the foregoing contents of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A primer combination for detecting mutations in the transcript and tyrosine kinase region of the BCR::ABL1 fusion gene based on targeted sequencing, characterized in that, include: The first-round amplification primer combination consists of the primers shown in SEQ ID No. 1 to SEQ ID No. 5, which are used to amplify the BCR::ABL1 fusion gene transcript using cDNA obtained from RNA transcription as a template to obtain the first-round amplification product; The second-round amplification primer combination 1, consisting of primers shown in SEQ ID No. 6 to SEQ ID No. 18, is used to amplify the breakpoint sequence of the BCR::ABL1 fusion gene using the first-round amplification product as a template. The second-round amplification primer combination 2 consists of primers shown in SEQ ID No. 19 to SEQ ID No. 26, and is used to amplify the ABL1 tyrosine kinase region using the first-round amplification product as a template.

2. The primer combination according to claim 1, characterized in that, Each primer has a non-human characteristic sequence attached to its 5' end.

3. The primer combination according to claim 1, characterized in that, The nucleotide sequence of the non-human characteristic sequence is shown in SEQ ID No.

27.

4. A kit for detecting mutations in the transcript and tyrosine kinase region of the BCR::ABL1 fusion gene based on targeted sequencing, characterized in that, Includes the primer combination described in any one of claims 1 to 3.

5. The use of the primer combination according to any one of claims 1 to 3 in the preparation of a kit for detecting mutations in the BCR::ABL1 fusion gene transcript and tyrosine kinase region based on targeted sequencing.

6. The application according to claim 5, characterized in that, The targeted sequencing includes the following steps: Obtain the RNA sample of the sample to be tested, and reverse transcribe it to obtain the cDNA sample; Using the cDNA sample as a template, the first round of PCR amplification was performed using the first round of amplification primer combination to obtain the first amplification product; Using the first amplification product as a template, a second round of PCR amplification was performed using the second round amplification primer combination 1 and the second round amplification primer combination 2, respectively, to obtain the second round PCR amplification product; The sequencing library was constructed using the second round of PCR amplification products and then sequenced. Analysis of the sequencing data yielded transcript information for the BCR::ABL1 fusion gene and mutation information for the tyrosine kinase region.

7. The application according to claim 6, characterized in that, The first round of PCR amplification program was as follows: 95℃ for 3 min 30 sec; 98℃ for 20 sec, 55℃ for 30 sec, 60℃ for 30 sec, 65℃ for 1 min, 12 cycles; 72℃ for 5 min; and 4℃ for holding.

8. The application according to claim 6, characterized in that, The second round of PCR amplification program was as follows: 95℃ for 3 min 30 sec; 98℃ for 20 sec, 58℃ for 1 min, 72℃ for 30 sec, 9 cycles; 72℃ for 5 min; hold at 4℃.

9. The application according to any one of claims 6 to 8, characterized in that, After constructing the sequencing library, quality control of the library is required, which requires the library concentration to be greater than 0.5 ng / μL and the library fragments to be 250 bp ≤ 450 bp.

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