Hematologic tumor diagnosis and treatment primer group, kit and application

Abstract

The invention discloses a hematologic tumor diagnosis and treatment primer group, a kit and application. The primer group comprises a first primer group and / or a second primer group, the first primer group is used for amplifying IGH, IGDH, IGK and IGL; the first primer group is used for amplifying TRB V-J, TRB D-J, TRG and TRD, the second primer group is used for amplifying TRB V-J, TRB D-J, TRG and TRD, after the primer groups amplify and rearrange sequences, the positive detection of clinical samples can be remarkably improved, the obtained CDR3 sequence can be used for clinical monitoring and can independently evaluate an immune group library of each patient, the immune state of an individual can be accurately reflected, the change condition of tumor cells of the patient can be dynamically monitored, and the clinical detection efficiency can be improved. And the method has good reliability and repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of immune repertoire sequencing, and specifically to a primer set, a kit and an application for the diagnosis and treatment of hematological malignancies. Background Art

[0002] Lymphocytes can differentiate into B cells, T cells and natural killer cells. The normal functions of B cells and T cells are crucial for protecting the body from foreign invasions by viruses or bacteria. This proper function depends on the structures of B cells and T cells. The antigen receptor on the surface of T cells is called the T cell receptor (TCR). Approximately 95% of TCRs are composed of an α chain and a β chain connected by a disulfide bond, and about 5% of TCR chains are composed of a γ chain and a δ chain. The antigen receptor on the surface of B cells is called the B cell receptor (BCR), which is composed of two identical heavy chains and two identical light chains. The diversity of TCRs and BCRs determines the diversity of antigen recognition by T / B cells.

[0003] The amino acids near the N-terminus in TCRs and BCRs vary greatly and are the variable regions (V regions), while the amino acids at the C-terminus are relatively constant and are called the constant regions (C regions). Among the complementarity determining regions (CDRs) in the variable regions, CDR3 has the highest diversity, and the part that directly binds to the antigen during the antigen recognition process has the highest diversity. The gene clusters encoding TCRs and BCRs exist in a fragmented gene segment form in clusters on chromosomes at the germline stage before the development of T / B cells. The 5'-end and 3'-end of the gene cluster contain V gene segments and J gene segments respectively, and the D gene segment is in the middle. The TCR / BCR encoding genes of each T / B cell are different, and this diversity is caused by the rearrangement of V, D, and J genes during the development of T / B cells. During the rearrangement process, when different gene segments are ligated, the homologous recombination repair system will randomly delete some nucleotides at the ends of germline genes during the repair process. At the same time, terminal deoxynucleotidyl transferase will randomly add other non-template nucleotides to the ligation site, and these randomly added nucleotides will generate new sequences that do not exist in the original sequence. During this V(D)J rearrangement, the randomness of V, D, and J gene selection and the imprecision of rearrangement ligation result in each T cell or B cell having a unique TCR or BCR encoding gene, forming an immune repertoire with a large capacity.

[0004] Minimal residual disease (MRD) is defined as residual tumor cells that cannot be detected by conventional methodologies. This result is an important prognostic factor, and treatment strategies adjusted based on MRD results can greatly improve the prognosis of patients with hematological malignancies. Among the conventional methods for monitoring MRD, multi-parameter flow cytometry (MFC) and PCR methods are the main methodologies. Among them, the MRD measurement by MFC depends on the immunophenotype on the surface of tumor cells, but its sensitivity is usually limited, only 10 -3 -10 -4 , and this result highly depends on the labeled antibodies and may not comprehensively cover all possible case subtypes. The PCR method detects specific fusion genes or related mutant genes in tumor cells and highly depends on known rearrangements and mutations, making it impossible to conduct comprehensive monitoring. Therefore, antigen changes may occur in some clonal tumor cells after treatment, and tumor heterogeneity can lead to false negatives, thus limiting the application of traditional methodologies. Multiple recent studies have shown that in hematological malignancies, the sensitivity and precision of using NGS methods for monitoring are higher than those of MFC or PCR methods. For example, in leukemia patients, using sensitive and robust NGS methods for MRD monitoring can enhance the identification of the recurrence risk after hematopoietic stem cell transplantation or chimeric antigen receptor-modified T cell therapy, and new emerging clones can be identified at each time point of monitoring. In addition, for the MRD quantitative results, the immunoassay repertoire detection technology based on NGS methods can complete quantification at the molecular level even at 10 6 or even more cell numbers, accurately evaluating the MRD burden. Moreover, the CDR3 sequences obtained by NGS sequencing will serve as unique identification sequences to distinguish different subpopulations in the same type of clonal population, and this more detailed differentiation can provide a more refined and accurate assessment when monitoring the treatment effect of MRD later.

[0005] Currently, domestic immunoassay repertoire-related products for MRD detection are still in the initial R & D stage. Although certain progress has been made, in terms of both market demand and technical maturity, existing products still cannot fully meet the needs of clinical patients and scientific researchers. At the same time, in clinical detection, there is a lack of effective technologies that can comprehensively reflect the overall state of the patient's immune system. With the combination of immunoassay repertoire and NGS technologies, this innovative approach has brought unprecedented opportunities to the fields of clinical treatment and related scientific research, providing strong technical support for future precision medicine and personalized treatment. Summary of the Invention

[0006] The present invention provides a primer set suitable for multiplex PCR of the human immune repertoire, which is used for multiplex amplification of completely rearranged and incompletely rearranged CDR3 sequences in human immune cells. The rearranged sequences include IGH, IGDH, IGK, IGL, TRB V-J, TRB D-J, TRG, and TRD. Clinical data results show that after the multiplex PCR primer set amplifies the rearranged sequences, the positive detection rate of clinical samples can be significantly improved. The obtained CDR3 sequences can be used for clinical monitoring and can independently evaluate the immune repertoire of each patient, accurately reflecting the immune status of an individual.

[0007] In view of this, the solution of the present invention is as follows:

[0008] The first aspect of the present invention is to provide a primer set for the diagnosis and treatment of hematological malignancies, including a first primer set and / or a second primer set; the first primer set is used for amplifying IGH, IGDH, IGK, and IGL; the second primer set is used for amplifying TRB V-J, TRB D-J, TRG, and TRD;

[0009] The nucleotide sequences of the upstream and downstream primers for amplifying IGH are respectively shown as SEQ ID NO: 1-25 and 26-32;

[0010] The nucleotide sequences of the upstream and downstream primers for amplifying IGDH are respectively shown as SEQ ID NO: 33-39 and 40-44;

[0011] The nucleotide sequences of the upstream and downstream primers for amplifying IGK are respectively shown as SEQ ID NO: 45-54 and 55-63;

[0012] The nucleotide sequences of the upstream and downstream primers for amplifying IGL are respectively shown as SEQ ID NO: 64-73 and 74-80;

[0013] The nucleotide primer sequences of the upstream and downstream primers for amplifying TRB V-J are respectively shown as SEQ ID NO: 81-119 and 120-129;

[0014] The nucleotide sequences of the upstream and downstream primers for amplifying TRB D-J are respectively shown as SEQ ID NO: 130-145 and 146-156;

[0015] The nucleotide sequences of the upstream and downstream primers for amplifying TRD are respectively shown as SEQ IDNO: 157-169 and 170-177;

[0016] The nucleotide sequences of the upstream and downstream primers for amplifying TRG are respectively shown as SEQ ID NO: 178-196 and 197-205.

[0017] Furthermore, the primer set further includes at least one of an artificial internal reference sequence set that mimics natural CDR3, a housekeeping gene sequence, and a sequencing primer.

[0018] Furthermore, the first primer set includes the artificial internal reference sequence set shown in SEQ ID NO: 206 - 281, and the second primer set includes the artificial internal reference sequence set shown in SEQ ID NO: 282 - 352;

[0019] The housekeeping gene is GAPDH, and the sequence is as shown in SEQ ID NO: 353 - 354;

[0020] The sequencing primer sequence is as shown in SEQ ID NO: 355 - 356.

[0021] The second aspect of the present invention is to provide a blood tumor diagnosis and treatment kit, including the primer set described in the first aspect.

[0022] Furthermore, the kit has two tubes, which contain the first primer set and the second primer set respectively.

[0023] Furthermore, the final concentrations of the primers for amplifying IGH, IGDH, IGK, and IGL are 1 μM, 0.8 μM, 0.5 μM, and 0.35 μM respectively; and / or, the final concentrations of the primers for amplifying TRB V-J, TRB D-J, TRG, and TRD are 1.2 μM, 1.0 μM, 0.85 μM, and 0.5 μM respectively.

[0024] Furthermore, the kit further includes at least one of a PCR amplification reaction system, a nucleic acid extraction reagent, an RNA reverse transcription reagent, and a PCR product sequencing reagent. The nucleic acid extraction reagent is used to extract sample DNA and / or RNA.

[0025] The third aspect of the present invention is to provide the application of the primer set described in the first aspect, or the kit described in the second aspect, in the preparation of blood tumor diagnosis and treatment products.

[0026] Furthermore, the blood tumor diagnosis and treatment products are used for at least one of the following purposes:

[0027] 1) Immune repertoire analysis;

[0028] 2) Minimal residual disease tracking and detection;

[0029] 3) Dynamic monitoring of immune status.

[0030] Furthermore, the application includes the following steps:

[0031] Obtain nuclear cell DNA, cDNA or cfDNA samples, add artificial internal reference sets, perform multiplex PCR amplification using the amplification primer sets described in the first aspect, perform secondary amplification on the PCR products to add sequencing adapters, construct a library and perform sequencing;

[0032] The immune repertoire files obtained by sequencing need to be quality controlled, preprocessed and spliced, and then compared with the sequences in the IMGT database. Clone clustering is performed based on the CDR3 sequences or incomplete rearrangement sequences, and a file sorted according to clone abundance is generated; The MRD formula is calculated as follows:

[0033] MRD = [(Corrected_Clones * C_int * η_GC * η_total) / (Corrected_R_int * N_cells)] * F_batch * Q, where:

[0034] Corrected_Clones is the number of reads of tumor-related sequences obtained by corrected sequencing; C_int is the copy number of each internal reference set added; η_GC: GC% content difference correction coefficient; η_total is the DNA extraction, amplification, ligation, and sequencing correction coefficient; Corrected_R_int is the number of reads of the internal reference sequence obtained by sequencing; F_batch is the technical deviation correction coefficient; Q is the fragment ratio and genomic DNA ratio weight coefficient; N_cells is the total amount of nuclear cells in the sample;

[0035] Calculate the total amount of nuclear cells according to the following formula: N_cells = (M_DNA * η_DV / A_DNA) * [(1 + 0.1 * (Batch_effect_score)], where:

[0036] M_DNA is the total amount of DNA extracted from the experimental sample (ng); η_DV is the fragment ratio weight coefficient; A_DNA is the average DNA amount of a single nuclear cell; Batch_effect_score is the deviation coefficient.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] In the primer set design process provided by the present invention, the CDR3 core region is clustered, and the most complete and comprehensive subtypes are amplified in the form of the fewest primer combinations, reducing the complexity of the primer combinations and avoiding adverse factors such as poor normalization and many dimers caused by excessive primer combinations. After the multiplex PCR primer set amplifies the rearrangement sequences, the positive detection rate of clinical samples can be significantly improved. The obtained CDR3 sequences can be used for clinical monitoring and can independently evaluate the immune repertoire of each patient, accurately reflecting the individual's immune status and dynamically monitoring the changes of the patient's tumor cells, with good reliability and repeatability.

[0039] In the application of the diagnosis and treatment detection of hematological tumors of the present invention, by reasonably introducing a correction coefficient, the true residual level of tumor cells can be accurately restored, providing strong technical support for future precision medicine and personalized treatment. Brief Description of the Drawings

[0040] Figure 1 Schematic diagram of the hematological tumor detection and analysis process in the embodiment of the present invention.

[0041] Figure 2 It is the analysis result of the myeloid library fragment before treatment in the embodiment of the present invention.

[0042] Figure 3 It is the schematic diagram of the bioinformatics analysis process in the embodiment of the present invention. Detailed Embodiments

[0043] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0044] Example 1 Design of Primer Sets for Hematological Tumor Detection

[0045] In this embodiment, a complete set of primer combinations for the detection of hematological tumors is provided through a large number of tests and analyses. The primers cluster the CDR3 core region and amplify the most complete and comprehensive subtypes in the form of the least primer combinations, reducing the complexity of the primer combinations, avoiding adverse factors such as poor normalization and many dimers caused by excessive primer combinations, improving the accuracy of the analysis of sequence changes in the CDR3 region, dynamically monitoring the changes of tumor cells in patients, and indicating the disease process. The primer combination includes at least one tube of primers for amplifying IGH, IGDH, IGK (including KDE), IGL, and one tube of primer sets for amplifying TRB V-J, TRB D-J, TRG, and TRD.

[0046] Specifically, the upstream primer sequences of the IGH amplification primers are as shown in SEQ ID NO: 1-25, and the downstream primer sequences are as shown in SEQ ID NO: 26-32; the upstream primer sequences of the IGDH amplification primers are as shown in SEQ ID NO: 33-39, and the downstream primer sequences are as shown in SEQ ID NO: 40-44; the upstream primer sequences of the IGK amplification primers are as shown in SEQ ID NO: 45-54, and the downstream primer sequences are as shown in SEQ ID NO: 55-63; the upstream primer sequences of the IGL amplification primers are as shown in SEQ ID NO: 64-73, and the downstream primer sequences are as shown in SEQ ID NO: 74-80; the upstream primer sequences of the TRB V-J amplification primers are as shown in SEQ ID NO: 81-119, and the downstream primer sequences are as shown in SEQ ID NO: 120-129; the upstream primer sequences of the TRB D-J amplification primers are as shown in SEQ ID NO: 130-145, and the downstream primer sequences are as shown in SEQ ID NO: 146-156; the upstream primer sequences of the TRD amplification primers are as shown in SEQ ID NO: 157-169, and the downstream primer sequences are as shown in SEQ ID NO: 170-177; the upstream primer sequences of the TRG amplification primers are as shown in SEQ ID NO: 178-196, and the downstream primer sequences are as shown in SEQ ID NO: 197-205.

[0047] In the above embodiments, the primer set further includes an artificial internal reference set synthesized to simulate the natural CDR3 sequence, and preferably the artificial internal reference set sequences are as shown in SEQ ID NO: 206-352.

[0048] In the above embodiments, the primer set further includes the housekeeping gene GAPDH sequence; preferably the housekeeping gene GAPDH sequences are as shown in SEQ ID NO: 353-354.

[0049] In the above embodiments, the primer set further includes sequencing primers, wherein the upstream sequencing primer sequence is as shown in SEQ ID NO: 355, and the downstream sequencing primer sequence is as shown in SEQ ID NO: 356.

[0050] The above primer set is shown in Table 1 (wherein, F represents the upstream primer and R represents the downstream primer).

[0051] Table 1:

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] In the above primer sets, artificial internal references corresponding to the respective genes are added when amplifying IGH, IGDH, IGK, IGL, TRB V-J, TRB D-J, TRG, and TRD. For example, when amplifying IGH, IGH-Internal control is added to facilitate accurate calculation of copy numbers.

[0066] For each gene, its forward primer and reverse primer are randomly combined for amplification to meet the detection of different individual samples and samples at different stages.

[0067] For the sequencing primers shown in SEQ ID NO: 355-356, the underlined part is the independent number corresponding to each library, N is a degenerate base, which can be A, T, G, or C; the other parts are the same sequences.

[0068] Example 2 Diagnostic and Therapeutic Kit and Sample Detection and Analysis

[0069] This example provides a sample detection and analysis method. By extracting sample DNA and RNA, performing multiplex PCR amplification based on the primer sets provided in Example 1, and performing a second round of PCR amplification by adding sequencing adapters, high-throughput sequencing of BCR and TCR is carried out, and then immunoglobulin repertoire information analysis and main clone sequence marking are performed. The process is as Figure 1 shown. Specifically as follows:

[0070] 1. Sample acquisition:

[0071] Bone marrow samples are collected in EDTA anticoagulant tubes;

[0072] Peripheral blood samples are collected in EDTA anticoagulant tubes.

[0073] 2. Multiplex PCR Primer Mixing:

[0074] Mix the primers used in the first-step PCR reaction in proportion to obtain a primer pool. The final concentration of the IGH primer is 1 μM, the final concentration of the primer for amplifying IGDH is 0.8 μM, the final concentration of the primer for amplifying IGK is 0.5 μM, and the final concentration of the primer for amplifying IGL is 0.35 μM; the final concentration of the primer for amplifying TRB V-J is 1.2 μM, the final concentration of the primer for amplifying TRB D-J is 1.0 μM, the final concentration of the primer for amplifying TRG is 0.85 μM, and the final concentration of the primer for amplifying TRD is 0.5 μM.

[0075] 3. Library Construction with DNA as Template:

[0076] Use Spin gDNA Extraction Kit to extract genomic DNA from nucleated cells.

[0077] According to the primer pool concentrations of different strands, the internal reference set or the internal reference set and the primer pool of the housekeeping gene GAPDH are specifically amplified with the samples. Different calculation methods are used according to different amplification strategies. The number of strands used in the internal reference set is as follows: 54 for IGH, 20 for IGDH, 18 for IGK, 23 for IGL, 62 for TRB V-J, 12 for TRB D-J, 16 for TRG, and 24 for TRD.

[0078] Use Multiplex PCR Assay Kit Ver.2 to configure the reagents for the first-step multiplex PCR amplification according to the reaction system in the following table. Invert and mix the prepared reagents, briefly centrifuge, and place them in a PCR instrument. Perform the PCR amplification reaction according to the following PCR amplification reaction conditions:

[0079] Reaction System:

[0080] Reagent Usage amount <![CDATA[2×Multiplex PCR Buffer(Mg 2+ , dNTP plus)]]> 25 μL Primer Mix*1 Appropriate amount Multiplex PCR Enzyme Mix 0.25 μL DNA template 2 μL Internal control 5 μL <![CDATA[dH2O (Sterile water)]]> up to 50 μL

[0081] First-step PCR Amplification Reaction Conditions:

[0082]

[0083] 4. Library Amplification with RNA Reverse Transcription Product as Template

[0084] Use RevertSAid RT Reverse Transcription Kit for efficient and rapid RNA reverse transcription, and then perform library construction according to step 3 with the DNA obtained by reverse transcription. The reverse transcription system is as follows:

[0085] Reagent Usage amount total RNA 1 μg oligo(dT)primer(0.5 μg / μL) 1 μL DEPC-treated water to 12 μL

[0086] Gently mix and place in a centrifuge for 3 - 5 s.

[0087] Incubate the mixture at 70 °C for 5 min, cool it on ice and centrifuge briefly.

[0088] Place the centrifuge tube on ice and add the following components:

[0089] Reagent Usage amount 5x reaction buffer 4 μL RiboLock Ribonuclease Inhibitor(20 u / μL) 1 μL 10 mM dNTP mix 2 μL

[0090] Incubate at 37 °C for 5 minutes;

[0091] Add RevertAid TMM-MuLV Reverse Transcriptase (200 u / μL), 1 μL, and make up to 20 μL with DEPC water;

[0092] Incubate the mixture at 42 °C for 60 min;

[0093] Terminate the reaction by incubating at 70 °C for 10 min and quickly place on ice.

[0094] 5. PCR amplification of the target product Add sequencing primers

[0095] Component Volume (μL) 2×KAPA HiFi Premix 12.5 10 μM Forward primer 0.75 10 μM Reverse primer 0.75 Template DNA 1 PCR-grade water up to 25

[0096] PCR running conditions:

[0097]

[0098]

[0099] Transfer the PCR reaction mixture to a 1.5 mL centrifuge tube and purify the amplified product 1.0-fold with the AMPure XP DNA Purification kit (Thermo) as follows:

[0100] a) Take out the Ampure XP Beads at 4 °C and equilibrate at room temperature for 30 min;

[0101] b) Vortex well before use, add magnetic beads at a ratio of 1:1 by sample volume and mix well, let stand for 5 min;

[0102] c) Transfer the 1.5 mL centrifuge tube to a magnetic stand and let stand for 3 - 5 min until clear;

[0103] d) Keep the centrifuge tube on the magnetic stand and carefully aspirate the supernatant without touching the magnetic beads;

[0104] e) Add 500 μL of 75% ethanol, gently pipette the magnetic beads 2 - 3 times, wait for 30 s, and discard the supernatant (when adding ethanol, add it slowly and try not to let the liquid add towards the magnetic beads, otherwise the magnetic beads will detach from the tube and be lost);

[0105] f) Repeat step e to remove the supernatant as much as possible (without pipetting the magnetic beads);

[0106] g) Place it in a thermostatic mixer and dry at 37°C for 3 - 5 min until there is no moisture on the surface of the magnetic beads;

[0107] h) Add 31 μL of nuclease - free water to a 1.5 mL centrifuge tube, mix well, let stand for 5

[0108] min, and then place it on a magnetic stand for about 5 min until it becomes clear;

[0109] i) Transfer 30 μL of the liquid to a new pre - prepared 1.5 mL centrifuge tube.

[0110] The concentration of the purified DNA is measured using Qbit BR, and the library concentration should be greater than 5 μg / μL.

[0111] Using a pre - treatment bone marrow sample, library fragments are obtained by the above method, and the lengths of the library fragments are analyzed. The results are as Figure 2 shown, indicating that the lengths of the constructed library fragments meet the expectations.

[0112] 6. Sequencing and bioinformatics analysis

[0113] The DNA library is sequenced on the Illumina NovaseqTM6000 platform, PE150, the denatured concentration of the library is 2 nM, and the loading concentration is 20 nM. After the sequenced data is quality - controlled and assembled, it is compared with the IMGT (http: / / www.imgt.org / ) database. According to the CDR3 sequence, clones are clustered, and result files are generated based on the clone abundances. The process is as Figure 3 shown, and the specific steps are as follows:

[0114] 1) Construction of reference sequences

[0115] The complete sequences of V, D, and J genes were downloaded from the International Immunogenetics Information System (IMGT). The specific primers for each strand were aligned with the V, D, and J sequences, and the matching results with no more than 2 base mismatches between the primer and the template sequence were retained. According to the start and end positions of the primers, the part that could generate the PCR product was selected as the reference sequence. For those with mismatches between the primer and the template sequence, they were recorded as two reference sequences, and the reference sequence library was merged. Duplicate reference sequences were removed, and finally, only unique sequences were retained. To improve the accuracy of sequence alignment, the construction of the reference sequence further clarified the start and end position information of the CDR3 region. This step is helpful for subsequent immune repertoire analysis, especially in-depth analysis of the antibody and receptor recombination processes of B cells and T cells, and can more accurately analyze the key sequences related to antibody production and immune response. In addition, by combining algorithm improvement and machine learning technology, the generation and screening process of the reference sequence was automatically optimized, which can greatly improve the efficiency and accuracy in large-scale data processing.

[0116] 2) Sequencing data processing

[0117] The sequencing data needs to remove adapters, low-quality sequences, and sequences with low alignment rates.

[0118] 3) Paired-end data merging

[0119] The sequences obtained by sequencing were spliced and merged, that is, there should be at least a 5bp overlapping region.

[0120] 4) Alignment of merged data with reference sequences

[0121] When aligning the spliced sequences with the constructed reference sequences, first, the overlapping regions were separately aligned with the V, D, and J sequences. To improve the alignment accuracy, a distributed alignment strategy was adopted: the V region was extended towards the 3' end, the D region was extended bidirectionally, and the J region was extended towards the 5' end. After the initial alignment, the BLAST results were used to optimize the alignment positions. By re-aligning, calculating scores and identities, the V, D, and J regions with high scores were finally selected as the best results. A specific mismatch tolerance range was set for the V, D, and J regions, and the number of allowed mismatches for each strand differed according to the sequence characteristics. Combining machine learning algorithms, the historical alignment results and sequence features were used to automatically adjust the mismatch tolerance. Especially when dealing with the highly variable CDR3 region, this dynamic adjustment helps to improve the alignment accuracy. Using efficient parallel computing and alignment algorithms to accelerate large-scale data processing further ensures the accuracy and reliability of the analysis results.

[0122] 5) Screening of alignment results

[0123] Sequences with a low frequency (<3%) will be excluded. If the alignment results of the V gene and the J gene are inconsistent with the positive and negative strands of the genome, they will be excluded. The alignment results of pseudogenes will be excluded. When the identified CDR3 sequence is translated into amino acids, if a terminator appears and the complete sequence cannot be normally translated, it will be excluded.

[0124] 6) Data statistics

[0125] Based on the CDR3 region position, translate the DNA sequence into an amino acid sequence and count the polypeptide frequency. After filtering out the sequences that do not meet the standards through the above steps, the usage frequencies and variations (insertions, deletions, mutations, etc.) of the V, D, and J genes can be counted to deeply understand the relationship between gene mutation patterns and immune responses, sequence lengths, and base compositions. In addition, evaluate the diversity of the immune repertoire, and visualize the expression patterns of the V and J genes through bar charts, heat maps, etc. to comprehensively evaluate the diversity characteristics of the immune repertoire.

[0126] Perform clone clustering based on the CDR3 sequence or incomplete rearrangement sequence, and generate a file sorted by clone abundance; calculate the MRD formula as follows:

[0127] MRD = [(Corrected_Clones * C_int * η_GC * η_total) / (Corrected_R_int * N_cells)] * F_batch * Q, where:

[0128] Corrected_Clones is the number of reads of tumor-related sequences obtained by corrected sequencing; C_int is the copy number of each internal reference set added; η_GC: GC% content difference correction coefficient; η_total is the DNA extraction, amplification, ligation, and sequencing correction coefficient; Corrected_R_int is the number of reads of the internal reference sequence obtained by sequencing; F_batch is the technical bias correction coefficient; Q is the fragment ratio and genomic DNA ratio weight coefficient; N_cells is the total number of nucleated cells in the sample;

[0129] Calculate the total number of nucleated cells according to the following formula: N_cells = (M_DNA * η_DV / A_DNA) * [(1 + 0.1 * (Batch_effect_score)], where:

[0130] M_DNA is the total amount of DNA extracted from the experimental sample (ng); η_DV is the fragment ratio weight coefficient; A_DNA is the average DNA amount of a single nucleated cell; Batch_effect_score is the bias coefficient.

[0131] After verification with 50 samples, after introducing the correction coefficient, the coefficient of variation of the proportion of tumor cells monitored by flow cytometry and the calculated result value is very low (CV < 20%), while the CV reaches 30% - 115% without correction. The results show that this correction coefficient can accurately restore the true level of residual tumor cells.

[0132] Analysis of the working results of primer pairs in Example 3

[0133] 100 peripheral blood samples of healthy people were collected and completed according to the steps in Example 2. The detection results of 2 tubes for bioinformatics analysis (1 tube detected IGH, IGDH, IGK, IGL, and the other tube detected TRB V-J, TRB D-J, TRD, TRG) were used to evaluate whether the primer pairs within the same gene could work properly and whether there was cross-amplification between primers of different genes. The results show that in each of the 2 tubes, the primers in each tube can only amplify their respective gene fragments, and different upstream and downstream primers will be used according to the template differences in the samples. NGS sequencing results show that the upstream and downstream primers of each gene will randomly combine to amplify the target sequences in the samples, and each primer can detect the amplification of different target sequences in the same sample. The amplification results of the primer sets of IGH and TRB V-J are shown in Table 2 (in Table 2, the primer names F_IGH 1 to 25 are the upstream primers with sequence numbers 1 to 25 in Table 1, and R_IGH 1 to 7 correspond to the downstream primers with numbers 26 - 32 in Table 1 respectively; the naming of TRB V-J primers is similar), and the primer amplification results of other genes (IGDH, IGK, IGL, TRB D-J, TRD, TRG) are similar to those of these two genes.

[0134] Table 2:

[0135]

[0136]

[0137]

[0138] Example 4 Clinical sample analysis

[0139] For 110 samples of patients clinically diagnosed with ALL and positive samples monitored by flow cytometry, detection and analysis were carried out according to the method provided in Example 2. The results are shown in the following table.

[0140]

[0141]

[0142] The results are as follows:

[0143] Disease type Initial diagnosis concordance rate MRD concordance rate B-ALL 95.4％ 92.3％ T-ALL 96.3％ 95％

[0144] The results showed that the positive rates of the composition in the clinical diagnosis and treatment of hematological malignancies were both higher than 95% for B-ALL and T-ALL patients, and the monitoring positive rates were both higher than 90%, with good reliability and repeatability.

[0145] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A primer set for the diagnosis and treatment of hematological malignancies, characterized in that, Comprising a first primer set and / or a second primer set; the first primer set is used for amplifying IGH, IGDH, IGK and IGL; the second primer set is used for amplifying TRB V-J, TRB D-J, TRG and TRD; The nucleotide sequences of the upstream and downstream primers for amplifying IGH are respectively shown as SEQ ID NO: 1-25 and 26-32; The nucleotide sequences of the upstream and downstream primers for amplifying IGDH are respectively shown as SEQ ID NO: 33-39 and 40-44; The nucleotide sequences of the upstream and downstream primers for amplifying IGK are respectively shown as SEQ ID NO: 45-54 and 55-63; The nucleotide sequences of the upstream and downstream primers for amplifying IGL are respectively shown as SEQ ID NO: 64-73 and 74-80; The nucleotide primer sequences of the upstream and downstream primers for amplifying TRB V-J are respectively shown as SEQ ID NO: 81-119 and 120-129; The nucleotide sequences of the upstream and downstream primers for amplifying TRB D-J are respectively shown as SEQ ID NO: 130-145 and 146-156; The nucleotide sequences of the upstream and downstream primers for amplifying TRD are respectively shown as SEQ ID NO: 157-169 and 170-177; The nucleotide sequences of the upstream and downstream primers for amplifying TRG are respectively shown as SEQ ID NO: 178-196 and 197-205.

2. The primer set according to claim 1, wherein It further comprises at least one of an artificial internal reference sequence set simulating natural CDR3, a housekeeping gene sequence and a sequencing primer.

3. The primer set according to claim 2, wherein The first primer set comprises the artificial internal reference sequence set shown as SEQ ID NO: 206-281, and the second primer set comprises the artificial internal reference sequence set shown as SEQ ID NO: 282-352; And / or, the housekeeping gene is GAPDH, and the sequence is shown as SEQ ID NO: 353-354; And / or, the sequencing primer sequence is shown as SEQ ID NO: 355-356.

4. Blood tumor diagnosis and treatment kit, characterized in that Comprising the primer set according to any one of claims 1-3.

5. The kit according to claim 4, wherein The kit is in two tubes, respectively containing the first primer set and the second primer set.

6. The kit according to claim 4, wherein The final concentrations of the primers for amplifying IGH, IGDH, IGK and IGL are 1 μM, 0.8 μM, 0.5 μM and 0.35 μM respectively; and / or, the final concentrations of the primers for amplifying TRB V-J, TRB D-J, TRG and TRD are 1.2 μM, 1.0 μM, 0.85 μM and 0.5 μM respectively.

7. The kit according to claim 4, wherein The kit further comprises at least one of a PCR amplification reaction system, a nucleic acid extraction reagent, an RNA reverse transcription reagent and a PCR product sequencing reagent.

8. Use of the primer set according to any one of claims 1-3, or the kit according to any one of claims 4-7 in the preparation of a blood tumor diagnosis and treatment product.

9. The application according to claim 8, wherein The blood tumor diagnosis and treatment product is used for at least one of the following purposes: 1) Immunoglobulin repertoire analysis; 2) Minimal residual disease tracking detection; 3) Dynamic monitoring of immune status.

10. The application according to claim 8, wherein The application includes the following steps: Obtain a sample of nucleated cell DNA, cDNA or cfDNA, add an artificial internal reference set, perform multiplex PCR amplification using a primer set, perform secondary amplification on the PCR product to add sequencing adapters, construct a library and perform sequencing; The immune repertoire library file obtained by sequencing needs to undergo quality control, preprocessing and splicing, and then be compared with the sequences in the IMGT database. Clone clustering is performed based on the CDR3 sequence or incomplete rearrangement sequence, and a file sorted according to clone abundance is generated; The MRD formula is calculated as follows: MRD = [(Corrected_Clones * C_int * η_GC * η_total) / (Corrected_R_int * N_cells)] * F_batch * Q, where: Corrected_Clones is the number of reads of tumor-related sequences obtained by corrected sequencing; C_int is the copy number of each group of internal reference sets added; η_GC: GC% content difference correction coefficient; η_total is the DNA extraction, amplification, ligation, sequencing correction coefficient; Corrected_R_int is the number of reads of internal reference sequences obtained by sequencing; F_batch is the technical deviation correction coefficient; Q is the fragment ratio, genomic DNA ratio weight coefficient; N_cells is the total amount of nucleated cells in the sample; The total amount of nucleated cells is calculated according to the following formula: N_cells = (M_DNA * η_DV / A_DNA) * [(1 + 0.1 * (Batch_effect_score)], where: M_DNA is the total amount of DNA extracted from the experimental sample (ng); η_DV is the fragment ratio weight coefficient; A_DNA is the average DNA amount of a single nucleated cell; Batch_effect_score is the deviation coefficient.

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