Method for detecting circulating tumor DNA (deoxyribonucleic acid) of B lymphocyte tumor and application

Abstract

The invention discloses a method for detecting circulating tumor DNA of B lymphocyte tumor. The method comprises the following steps: S1, preparing a DNA sample; s2, preparing a pre-hybridization library; s3, hybridizing and capturing; and S4, sequencing on a machine. Probe design is carried out on a target area genome based on a multi-factor algorithm, an effective specific capture probe is synthesized, the probe can be partially or completely complementary with a section for capturing a target gene, a captured fragment can be subjected to next-generation sequencing, and after sequencing data is filtered and analyzed, the detection accuracy is improved. Auxiliary reference basis can be provided for personalized treatment schemes of patients, the cure rate and survival time of the patients are improved, and the prognosis condition of the patients is improved.

Description

Technical Field

[0001] The present invention relates to the field of cancer monitoring, involves C12Q, and specifically relates to a method and application for detecting circulating tumor DNA of B-lymphocyte tumors. Background Art

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common pathological subtype among B-lymphocyte tumors. Different subtypes have different clinical characteristics, genetic manifestations, and treatment responses. R-CHOP (rituximab + cyclophosphamide + doxorubicin + vincristine / vindesine + prednisone) is currently the most common treatment regimen for DLBCL. However, a large number of patients have problems of drug resistance and recurrence. Therefore, in clinical treatment, it is necessary to make early judgments on these patients and adopt targeted intensive treatment to improve the treatment effect. The mutation sites in ctDNA (circulating tumor DNA) can be used as markers for diffuse large B-lymphocyte circulating tumor cells. The existing tumor informed method is a common method for detecting ctDNA in this field. However, the early detection rate of low-risk diffuse large B-cell lymphoma is relatively low. Therefore, it is crucial to develop a detection method with high detection limits in the early, middle, and late stages of diffuse large B-cell lymphoma.

[0003] Chinese invention patent CN116479122A discloses a digital PCR detection kit for detecting mutations at the Y646 site of the human EZH2 gene. Using the digital PCR method, it is used for in vitro quantitative detection of the mutation situation at the Y646 site of the EZH2 gene in bone marrow DNA samples or free DNA samples of patients with germinal center diffuse large B-cell lymphoma (GCB-DLBCL) and follicular B-cell lymphoma (FL). It has high detection sensitivity and strong specificity. However, it cannot achieve long-term tracking and detection of changes in the detection object. Chinese invention patent CN111662983B discloses a kit for detecting lymphoma gene mutations and its application. Using a set of DNA probes to perform cell origin typing on patients with diffuse large B-cell lymphoma for auxiliary diagnosis, prognosis judgment, and / or targeted drug prediction. However, the detection process of multiple groups of probes is complex, and it is difficult to perform long-term monitoring. Summary of the Invention

[0004] In order to develop a detection method with high detection limits in the early, middle, and late stages of B-lymphocyte tumors, the first aspect of the present invention provides a method for detecting circulating tumor DNA of B-lymphocyte tumors, including the following steps:

[0005] S1 Prepare a DNA sample: Take tumor tissue, oral mucosa, and plasma samples to extract the samples;

[0006] S2 Prepare pre-hybridization libraries: Prepare pre-hybridization library one and pre-hybridization library two from the gDNA samples obtained from tumor tissues and oral mucosa respectively; prepare pre-hybridization library three from the cfDNA sample obtained from plasma.

[0007] S3 Hybridization capture: Combine the pre-hybridization libraries with capture probes to perform a hybridization reaction to capture the target regions and obtain the final hybridization libraries.

[0008] S4 Sequence the final hybridization libraries on a sequencer, and that's it.

[0009] In the present invention, by continuously detecting the gDNA mutations in tumor tissues and oral swabs and the cfDNA in blood samples, and analyzing the decline amplitude of ctDNA after a treatment stage, a certain change threshold needs to be reached, and targeted enhanced treatment can improve the treatment effect. And it has a certain predictive effect on both low-risk and high-risk diffuse large B-cell lymphomas. Especially after targeted intensive treatment, the cure rate can be significantly improved, and the impact on other parts caused by large-scale treatment can be prevented.

[0010] As a preferred embodiment, the hybridization reaction in step S3 includes the following steps:

[0011] 1) Place the probe solution in a thermal cycler and heat it to 95 - 98 °C for 1 - 2 min, set the heated lid to 105 °C, and then immediately cool it on ice for 5 - 10 min.

[0012] 2) When cooling the probe solution on ice, place the tube containing the resuspended tag mixed library in a thermal cycler, heat it at 95 - 98 °C for 5 - 10 min, set the heated lid to 105 °C, and then place both the probe solution and the resuspended tag mixed library on the laboratory bench and let them equilibrate at room temperature for 5 - 10 min.

[0013] 3) Vortex and slowly rotate the probe solution, and then transfer all of it to the resuspended tag mixed library. Mix well by vortexing.

[0014] 4) Centrifuge the tube transiently to ensure that all the solution is at the bottom of the tube.

[0015] 5) Add 15 - 20 μL of hybridization accelerator to the entire capture reaction solution.

[0016] 6) Centrifuge the tube transiently to ensure there are no air bubbles. (Note: Seal the tube tightly to prevent excessive evaporation during incubation)

[0017] 7) Place the hybridization reaction solution in a thermal cycler and incubate it at 70 - 73 °C for 16 - 18 h, set the heated lid to 85 - 90 °C.

[0018] As a preferred embodiment, after the hybridization reaction, binding to streptavidin magnetic beads includes the following steps:

[0019] 1) Vortex the pre-equilibrated streptavidin-binding magnetic beads until well mixed.

[0020] 2) Add 50 - 55 μL of streptavidin-binding magnetic beads to a 1.5 mL microcentrifuge tube. Prepare one tube for each hybridization reaction.

[0021] 3) Add 100 μL of binding buffer to the tube and pipette to mix well.

[0022] 4) Let the tube stand on the magnetic rack for 1 - 1.5 min, then aspirate and discard the supernatant. Do not disrupt the magnetic bead pellet. Remove the tube from the magnetic rack.

[0023] 5) Repeat the washing twice (steps 3 and 4), for a total of three washes.

[0024] 6) After the third wash, remove the supernatant, then add 100 μL of binding buffer and vortex to resuspend the magnetic beads until well mixed.

[0025] 7) Heat the resuspended magnetic beads at 68 - 70 °C for 10 - 15 min.

[0026] 8) After completing the hybridization (step 3.3), open the lid of the thermal cycler and directly transfer each hybridization reaction solution to the corresponding tube containing the pre-heated (step 7) streptavidin-binding magnetic beads. Mix the reagents by pipetting and flicking.

[0027] 9) Incubate the tubes containing the hybridization reaction solution and streptavidin-binding magnetic beads at 68 - 70 °C for 5 - 8 min without stirring. (Do not vortex, no vigorous mixing is required)

[0028] 10) Remove the tubes containing the hybridization reaction solution and streptavidin-binding magnetic beads from the mixer and perform a brief centrifugation to ensure that all the solution is at the bottom of the tube.

[0029] 11) Let the tube stand on the magnetic rack for 1 - 1.5 min.

[0030] 12) Aspirate and discard the supernatant, including the hybridization accelerator. Do not disrupt the magnetic bead pellet.

[0031] (Note: Some hybridization accelerator can be seen after aspirating the supernatant and can also be seen in each washing step. It does not affect the final captured product.)

[0032] 13) Remove the tube from the magnetic rack, add 100 μL of standard wash buffer 1 at 68 - 70 °C. Pipette to mix well

[0033] 14) Incubate the test tube at 68 - 70 °C for 5 - 8 min.

[0034] 15) Perform a transient centrifugation to ensure that all the solution is at the bottom of the test tube.

[0035] 16) Transfer all the solution in step 15) to a new 1.5 mL microcentrifuge tube, and transfer each hybridization reaction to one test tube. Let the test tube stand on the magnetic rack for 1 - 1.5 min.

[0036] 17) Aspirate and discard the supernatant. Do not disrupt the magnetic bead precipitate. Remove the test tube from the magnetic rack and add 100 μL of Wash Buffer 2 at 48 - 50 °C. Mix well by pipetting, and then perform a transient centrifugation to ensure that all the solution is at the bottom of the test tube.

[0037] 18) Incubate the test tube at 48 - 50 °C for 5 - 8 min.

[0038] 19) Let the test tube stand on the magnetic rack for 1 - 1.5 min.

[0039] 20) Aspirate and discard the supernatant. Do not disrupt the magnetic bead precipitate.

[0040] 21) Repeat the washing twice (repeat steps 17) - 20)), for a total of three washes.

[0041] 22) After completing the last wash, aspirate all the supernatant using a 10 μL pipette. Proceed to the next step immediately. Do not allow the magnetic beads to dry.

[0042] 23) Remove the test tube from the magnetic rack and add 22.5 - 25 μL of water. Mix well by pipetting, and then incubate this solution on ice (hereinafter referred to as the streptavidin - bound magnetic bead suspension).

[0043] The applicant found during the experiment that when binding the target region captured by the reaction solution after hybridization to streptavidin magnetic beads, it is necessary to maintain the temperature above 70 °C, and transfer the reaction quickly, let it stand for the reaction, and the obtained target binding rate is relatively high. It is speculated that the reason may be that when the temperature cools below 70 °C, the binding activity of the captured target region decreases, and the binding rate to streptavidin magnetic beads decreases, which can cause a 10 - 20% off - target rate in a short time, thus reducing the detection accuracy.

[0044] The applicant further found that when the hybridization reaction solution is incubated with streptavidin magnetic beads, stirring or vortexing will also cause a certain off - target rate. The reason may be that under the condition of the presence of external force, the binding resistance between the target region strand and streptavidin magnetic beads is large, so it is easy to be off - target.

[0045] During the experiment, the applicant found that transferring the target magnetic beads after washing with washing buffer to a new test tube for reaction can reduce the background interference of detection. The possible reason is speculated that when the target area is incubated with streptavidin magnetic beads, the hybridization reaction liquid in the target area will undergo non-specific binding on the surface of the test tube wall, causing detection errors. By replacing the new test tube, the probability of non-specific binding can be reduced, thereby reducing background interference.

[0046] As a preferred implementation, after sequencing on the machine in step S4, filtering conditions are set to generate a somatic mutation list.

[0047] The present invention uses the BGI sequencer DNBSEQ-T7RS for sequencing, and the target depths of cancer tissue samples, oral mucosal control samples and plasma samples are 1000X, 500X and 1000X, respectively. For the original fastq (nucleic acid sequence) data of sequencing, fastp and fastQC (quality control) are used for quality assessment and removal of joints, and BWA-mem software is used to align to the reference genome hg19, and samtools software is used to convert the alignment result BAM file, and GATK software is used to sort, remove duplication and base quality correction of the data, and the processed BAM file and quality control results are obtained. Lianti software is used to identify the point mutation SNV and insertion and deletion INDEL results, and Annovar software is used for gene annotation to generate mutation tables.

[0048] As a preferred embodiment, the filtering conditions include filtering background noise and germline variation, and the filtering conditions include: filtering mutation sites with low base quality, filtering mutation sites with a population mutation rate ≥ 0.001 in any database, filtering mutation frequency <1%, filtering sites with a mutation site depth <100X, filtering sites with a mutation frequency less than 2% and no pathogenicity records in cosmic and clinvar, filtering sites with qualified base quality and mutation frequency > 1% in germline control samples, filtering mutation results that are not within the range of panel reportable genes, and filtering at least one of false positive mutation results caused by sequencing errors.

[0049] As a preferred embodiment, after sequencing on the machine in step S4, the equivalent of ctDNA and cfDNA in the free DNA is calculated.

[0050] As a preferred embodiment, the equivalent calculation formula of the ctDNA is: Among them, J: a total of J individual cell mutations were detected in the test subject, the mutation read at the jth site is Mj, and the sequencing depth is Nj.

[0051] As a preferred embodiment, the ctDNA concentration calculation formula is: Among them, the plasma volume is V i , and the total amount of cfDNA extracted from the plasma is m i .

[0052] As a preferred embodiment, after sequencing on the machine in step S4, a prognostic risk prediction model is established, and the change rate calculation formula of ctDNA is as follows:

[0053] Among them, c 0 : The detected value of the ctDNA concentration of the sample collected from the detection object at time point t 0 ; c 1 : The detected value of the ctDNA concentration of the sample collected from the detection object at time point t 1 .

[0054] As a preferred embodiment, in the magnetic bead suspension in step S3, the volume ratio of the amplification primer to the prehybridization library is (7-11):1:(8-12).

[0055] As a preferred embodiment, in the magnetic bead suspension in step S3, the volume ratio of the amplification primer to the prehybridization library is 22.5 μL:2.5 μL:25 μL.

[0056] As a preferred embodiment, the amplification reaction in step S3 is PCR amplification, and the conditions of the PCR amplification are: pre-denaturation at 95-99 °C for 43-48 s; 95-99 °C for 13-33 s, 58-62 °C for 28-32 s, 70-75 °C for 28-32 s, cycling 12 times; extension at 70-75 °C for 55-65 s; constant temperature at 3-6 °C.

[0057] As a preferred embodiment, the amplification reaction in step S3 is PCR amplification, and the conditions of the PCR amplification are: pre-denaturation at 98 °C for 45 s; 98 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, cycling 12 times; extension at 72 °C for 60 s; constant temperature at 4 °C.

[0058] The second aspect of the present invention provides an application of a method for detecting circulating tumor DNA in B-lymphocyte tumors, which is applied to the detection of lesions in B-lymphocyte tumors.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] (1) The detection method for circulating tumor DNA in B-lymphocyte tumors of the present invention uses multi-sample detection of tumor tissues, oral swabs, and blood samples, forms a sequencing library, and performs DNA mutation detection, and has a high detection rate for B-lymphocyte tumors.

[0061] (2) The detection method for circulating tumor DNA of B-lymphocyte tumors according to the present invention can continuously detect the ctDNA equivalent value before, during, and after treatment, and can be used to assist in predicting the efficacy of the standard treatment plan for patients in the middle and late stages.

[0062] (3) The detection method for circulating tumor DNA of B-lymphocyte tumors according to the present invention analyzes the decrease amplitude of the ctDNA value after a course of treatment by continuously monitoring the ctDNA equivalent in a blood sample. When the decrease amplitude does not reach a threshold, targeted enhanced treatment will bring an improvement in the treatment effect for the patient.

[0063] (4) When the captured target region binds to streptavidin magnetic beads in the detection method for circulating tumor DNA of B-lymphocyte tumors according to the present invention, the temperature needs to be maintained above 70 °C, and the reaction should be transferred quickly and allowed to stand still to improve the detection accuracy and avoid off-target errors.

[0064] (5) In the detection method for circulating tumor DNA of B-lymphocyte tumors according to the present invention, the target magnetic beads after being washed with a washing buffer are transferred to a new test tube for reaction, which can reduce the background interference of the detection.

[0065] (6) In the detection method for circulating tumor DNA of B-lymphocyte tumors according to the present invention, probe design is carried out on the target region genome based on a multi-factor algorithm, and effective specific capture probes are synthesized. These probes can be complementary to part or all of the section capturing the target gene, and the captured fragments can be subjected to next-generation sequencing. After filtering and analyzing the sequencing data, the detection accuracy is improved.

[0066] (7) The detection method for circulating tumor DNA of B-lymphocyte tumors according to the present invention can provide an auxiliary reference basis for the personalized treatment plan of patients by evaluating the treatment efficacy of B-cell lymphoma patients according to the ctDNA equivalent value and the change in the equivalent value multiple, improve the cure rate and survival time of patients, and improve the prognosis of patients. At the same time, it provides an auxiliary reference basis for the efficacy evaluation of patients. The method of the present invention can predict the treatment effect and recurrence risk of patients only by monitoring the blood samples of patients, so as to more conveniently manage the prognosis of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Detection rate of ctDNA in peripheral blood plasma before treatment for diffuse large B-cell lymphoma with medium-high risk;

[0068] Figure 2 Detection rate of ctDNA in peripheral blood plasma before treatment for diffuse large B-cell lymphoma with low risk;

[0069] Figure 3To predict the mid-term efficacy based on the ctDNA equivalent before treatment;

[0070] Figure 4 To predict the end-term efficacy based on the ctDNA equivalent before treatment;

[0071] Figure 5 To be the change in the equivalent multiple of mid-term CR after one treatment course based on ctDNA detection;

[0072] Figure 6 To be the change in the equivalent multiple of end-term CR after one treatment course based on ctDNA detection. Specific implementation mode

[0073] The reagents and materials in the specific implementation mode of the present invention can be obtained through commercial channels; the room temperature in the specific implementation mode of the present invention is 25°C; the solvent of the solution in the specific implementation mode of the present invention is water.

[0074] The blocker solution, universal blocker, amplification primer, and binding buffer are from a universal hybridization capture kit, purchased from: Aegitekang, Twist, Diwin, Novizan, etc.

[0075] The washing buffers 1 and 2 are the buffers supporting a universal library construction kit, purchased from: Aegitekang, Novizan, Diwin, Twist Biosciences, etc.

[0076] The hybridization promoter is from the hybridization capture kit of Twist Biosciences.

[0077] Example 1

[0078] A method for detecting DNA of diffuse large B lymphoma circulation tumors, comprising the following steps:

[0079] S1 Prepare a DNA sample: Take tumor tissue, oral mucosa, and plasma samples to extract samples;

[0080] S2 Prepare a pre-hybridization library: Prepare a pre-hybridization library one from the ctDNA samples obtained from tumor tissue and oral mucosa respectively; prepare a pre-hybridization library two from the cfDNA samples obtained from plasma;

[0081] S3 Hybridization capture: Combine the pre-hybridization library with the capture probe to carry out a hybridization reaction, capture the target region, and obtain the final hybridization library;

[0082] S4 Sequence the final hybridization library on a machine, and that's it.

[0083] The method for preparing a DNA sample from the tumor tissue is as follows: Obtain a tumor sample by puncture, remove the normal tissue around the tumor to obtain a fresh tumor tissue sample, and use a general genomic DNA extraction method to prepare the DNA of the tumor tissue sample.

[0084] The method for preparing a DNA sample from oral mucosa is as follows: rinse the mouth 5 times with physiological saline, use an oral mucosa collection brush to scrape the oral mucosa samples of the patient 10 times forcefully on the left and right side walls of the oral cavity, and place them in the preservation solution. The oral mucosa samples are prepared for DNA of oral mucosa samples using a general genomic DNA extraction method.

[0085] The method for preparing a DNA sample from plasma samples is as follows: collect 10 mL of venous blood using a dedicated preservation tube (streck tube) for cell-free DNA, centrifuge at 2000 rpm for 10 min within 5 hours, separate the upper plasma for cell-free DNA extraction (if subsequent operations are not carried out immediately, the plasma is stored temporarily at 4 °C or frozen at -80 °C). Using a cell-free DNA extraction kit (supplier: Kangwei Century), the plasma samples are used to extract plasma cfDNA with reference to the "Kangwei Cell-free Nucleic Acid DNA Extraction Kit". Finally, use 60 μL of ddH 2 O eluent to elute cfDNA and then store it, which is used as a template for subsequent experiments.

[0086] The operation of step S2 is specifically as follows:

[0087] 1. Preparation of pre-hybridization library (for genomic DNA samples)

[0088] In this section, Twist Library Preparation EF Kit 2.0 is used.

[0089] 1.1 Fragmentation, end repair, and dA tailing of Input DNA

[0090] 1.1.1. Take out the Frag / AT buffer and Frag / AT enzyme in the kit, thaw and mix well thoroughly, briefly centrifuge to collect at the bottom of the tube, and place it on ice for later use. All the following steps are carried out on ice.

[0091] 1.1.2. Prepare the following reaction in a sterilized PCR tube:

[0092] Reagent Volume (μL) Input DNA 10 Frag / AT Buffer 4 Frag / AT Enzyme 6 <![CDATA[ddH 2 O]]> 30

[0093] The Input DNA is 100 ng. The Input DNA is tumor tissue, DNA extracted from oral mucosa.

[0094] 1.1.3. Use a pipette to blow and oscillate to mix well, and briefly centrifuge for 1 min to collect the reaction solution at the bottom of the tube.

[0095] 1.1.4. Place the PCR tube in a PCR instrument and carry out the following reaction:

[0096] Temperature Time Hot lid 105°C On 37℃ 18 min 65℃ 30 min 4℃ Hold

[0097] Note: When the thermal cycler program ends and the sample module returns to 4°C, remove the samples from the template and place them on ice. Proceed to the next step immediately.

[0098] 1.2 Ligate with universal adapter and purify

[0099] This step ligates an adapter to the ends of the fragmented and end-repaired products from the previous step.

[0100] 1.2.1. Take out the universal adapter from -20°C, thaw it, mix well thoroughly, briefly centrifuge to collect it at the bottom of the tube, and place it on ice for standby.

[0101] 1.2.2. Prepare the reaction system according to the following table:

[0102] Reagent Volume (μL) Product of previous step 50 Ligation Master Mix 20 DNA Adapter X 5 Total 75

[0103] 1.2.3. Gently pipette to mix well (do not mix by shaking), and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0104] 1.2.4. Place the PCR tube in a PCR instrument and perform the following reaction:

[0105] Temperature Time Hot lid 105°C On 20℃ 15 min 4℃ Hold

[0106] 1.2.5. Purify the reaction product using magnetic beads:

[0107] 1) After equilibrating the magnetic beads to room temperature (30 min), vortex to mix the magnetic beads well.

[0108] 2) Pipette 60 μL (0.8x) of magnetic beads into 75 μL of Adapter Ligation product, vortex or gently pipette 10 times to mix well thoroughly.

[0109] 3) Incubate at room temperature for 5 min.

[0110] 4) Briefly centrifuge the PCR tube and place it in a magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (5 min), carefully remove the supernatant.

[0111] 5) Keep the PCR tube in the magnetic stand all the time, add 180 μL of freshly prepared 80 vol% ethanol aqueous solution to wash the magnetic beads, incubate at room temperature for 30 s, and carefully remove the supernatant.

[0112] 6) Repeat step 5) for a total of two washes.

[0113] 7) Keep the PCR tube in the magnetic stand all the time, open the lid and air-dry the magnetic beads for 5 - 10 min until there is no ethanol residue.

[0114] 8) Remove the PCR tube from the magnetic stand, add 17 μL of ddH 2 O for elution, vortex or gently pipette to mix well, let it stand at room temperature for 2 min, briefly centrifuge the PCR tube and place it on the magnetic stand. After the solution becomes clear (5 min), carefully transfer 15 μL of the supernatant to a new EP tube, taking care not to touch the magnetic beads.

[0115] 1.3 Perform PCR amplification, purification, and quality control using index adapter primers

[0116] 1.3.1. Thaw the index adapter primers and Equinox library amplification mix (2x) in the kit and invert to mix well. Prepare the following reaction:

[0117] Reagent Volume (μL) Purified Adapter Ligation product 15 Index adapter primer 10 Equinox Library Amplification Mix (2x) 25 Total 50

[0118] The purified Adapter Ligation product is the product after step 1.3.

[0119] 1.3.2. Gently pipette to mix well (do not vortex to mix), and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0120] 1.3.3. Place the PCR tube in a PCR instrument and start the PCR program as follows: hot lid temperature 105 °C,

[0121]

[0122] 1.3.4. Purify the reaction product:

[0123] 1) After the magnetic beads are equilibrated to room temperature, vortex to mix the magnetic beads well.

[0124] 2) Pipette 50 μL (1x) of magnetic beads into 50 μL of the Library Amplification product and gently pipette 10 times to mix well.

[0125] 3) Incubate at room temperature for 5 min.

[0126] 4) Briefly centrifuge the PCR tube and place it on the magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (5 min), carefully remove the supernatant.

[0127] 5) Keep the PCR tube on the magnetic stand at all times, add 180 μL of freshly prepared 80 vol% ethanol aqueous solution to wash the magnetic beads, incubate at room temperature for 30 s, and carefully remove the supernatant.

[0128] 6) Repeat step 5) for a total of two washes.

[0129] 7) Keep the PCR tube in the magnetic stand all the time, open the lid and air-dry the magnetic beads for 5 - 10 min until there is no ethanol residue.

[0130] 8) Take out the PCR tube from the magnetic stand, add 65 μL of ddH 2 O for elution, vortex or gently pipette to mix well, let it stand at room temperature for 2 min, centrifuge the PCR tube briefly and place it in the magnetic stand to stand still. After the solution becomes clear (5 min), carefully transfer 65 μL of the supernatant to a new EP tube. The supernatant in the tube is the prepared pre-library.

[0131] 1.4 Library Quantification

[0132] Take 1 μL of the library and use a Qubit 2.0 Fluorometer (Qubit dsDNA HS Assay Kit) to measure the library concentration and record the library concentration. The concentration range of a normal library is above 50 ng / μL, and the library concentration is mainly related to the quality of the template.

[0133] 1.5 Library Quality Detection

[0134] Use a Qsep100 fully automatic nucleic acid and protein analysis system to measure the library fragment length and purity. The target fragment distribution range of a normal library is between 300 bp and 400 bp.

[0135] 2. Preparation of Pre-Hybridization Library (for cfDNA Samples)

[0136] This section uses Fast Library Prep Kit v2.0.

[0137] 2.1 End Repair and dA-Tailing of Input DNA

[0138] 2.1.1. Take out the End Repair&A-Tailing Buffer in the kit from the -20°C refrigerator in advance, place it on the ice box to melt, vortex briefly and centrifuge instantaneously after melting, and place it on the ice box for standby. Take out the End Repair&A-Tailing Enzyme Mix from the -20°C refrigerator, invert to mix well and centrifuge instantaneously, and place it on the ice box for standby. Prepare the reaction system according to the following table. This operation needs to be carried out on the ice box;

[0139] Reagent Volume (μL) cfDNA X Nuclease-free water 5 End Repair&A-Tailing Buffer 7 End Repair&A-Tailing Enzyme Mix 3 Total volume 60

[0140] The Input cfDNA is 10 ng. cfDNA is the DNA extracted from plasma.

[0141] 2.1.2. Use a pipette to mix well on ice (avoid violent shaking).

[0142] 2.1.3. Set the parameters of the PCR instrument according to the following table, and place the reaction system on the PCR instrument to run the program:

[0143]

[0144] Note: After the program ends, immediately proceed to the next step of adapter ligation reaction. This step cannot be paused!

[0145] 2.2 IGT UMI Adapter Ligation and Purification

[0146] In this step, the UMI adapter will be ligated to the ends of the fragmented and end-repaired products from the previous step.

[0147] 2.2.1. Take out the Adapter from the -20°C refrigerator in advance, place it on an ice box to melt. After melting, briefly vortex and centrifuge it instantaneously, then place it on the ice box for standby.

[0148] 2.2.2. Dilute the IGT UMI Adapter (15 μM) to an appropriate concentration in advance according to the amount of DNA input for library construction:

[0149] DNA input amount Adapter concentration Dilution factor 50 ng - 1 μg 15 μM Not diluted 25 ng 7.5 μM Diluted 2-fold 10 ng 3 μM Diluted 5-fold 5 ng 1.5 μM Diluted 10-fold 2.5 ng 750 nM Diluted 20-fold 1 ng 300 nM Diluted 50-fold

[0150] Note: Excessive input of the Adaper may cause self-ligation of the Adapter; insufficient input will affect the ligation efficiency and result in a decrease in library output.

[0151] 2.2.3. In a PCR tube, prepare the reaction system on an ice box according to the following table:

[0152]

[0153]

[0154] Note: If the sample size for one operation is relatively large, the reaction reagents need to be pre-mixed. Do not pre-mix the Adapter. The best operation method is to first mix the Adapter and the sample after the completion of STEP 1, and then add the pre-mixed reaction reagents, which can effectively reduce adapter self-ligation.

[0155] 2.2.4. Gently pipette and mix 6 times to avoid generating bubbles, then centrifuge briefly.

[0156] 2.2.5. Set the parameters of the PCR instrument according to the following table, close the heated lid. Put the PCR tube into the PCR instrument and run the program.

[0157]

[0158] Note: After the program ends, immediately proceed with the next step of magnetic bead purification. This step cannot be paused!

[0159] 2.3 Purification after Ligation

[0160] 2.3.1. Place the Agencourt AM Pure XP magnetic beads at room temperature 30 min in advance, shake well and set aside for use.

[0161] 2.3.2. After the program runs to completion, add 0.8 times the volume of magnetic beads (88 μL) to the 110 μL reaction system after 2.2.5, pipette or vortex to mix well.

[0162] 2.3.3. Let stand at room temperature for 5 min, place the centrifuge tube on the magnetic stand for 3 min until the solution is clear.

[0163] 2.3.4. Keep the centrifuge tube on the magnetic stand, remove the supernatant, add 200 μL of 80 vol% ethanol aqueous solution to the centrifuge tube, and let stand for 30 s.

[0164] 2.3.5. Remove the supernatant, add another 200 μL of 80 vol% ethanol aqueous solution to the centrifuge tube, let stand for 30 s and then completely remove the supernatant, and use a 10 μL pipette to remove the residual ethanol solution at the bottom.

[0165] 2.3.6. Let stand at room temperature for 5 min until the magnetic beads are dry and the residual ethanol has completely evaporated.

[0166] 2.3.7. Add 22 μL of nuclease-free water, remove the centrifuge tube from the magnetic stand, gently pipette to resuspend the magnetic beads, avoid generating bubbles, briefly centrifuge, and let stand at room temperature for 2 min.

[0167] 2.3.8. Place the centrifuge tube on the magnetic stand for 2 min until the solution is clear.

[0168] 2.3.9. Use a pipette to aspirate 20 μL of the supernatant and transfer it to a new PCR tube to prepare for the next reaction.

[0169] Note: This step is a pausable step and can be stored in a -20 °C refrigerator for 1 month.

[0170] 2.4 Pre-PCR Reaction

[0171] 2.4.1. Take out the pre-PCR amplification reaction solution, I5-end adapter primer, and I7-end adapter primer from the kit stored at -20 °C in advance, place them on an ice box to melt, mix well and place on ice for later use.

[0172] 2.4.2. Prepare the reaction system according to the following table (this operation is carried out on an ice box):

[0173]

[0174] 2.4.3. Use a pipette to gently pipette up and down to mix evenly, and then centrifuge briefly.

[0175] 2.4.4. Set the parameters of the PCR instrument according to the following table. The hot lid is at 105 °C. Place the above reaction system on the PCR instrument to run the program:

[0176]

[0177] Note: Immediately perform magnetic bead purification after the program runs to completion.

[0178] 2.4.5 Magnetic bead purification after PCR amplification

[0179] 1) Place the Agencourt AMPure XP magnetic beads at room temperature 30 min in advance, shake and mix evenly for standby;

[0180] 2) After the program runs to completion, transfer the 50 μL product in the PCR tube to a new 1.5 mL centrifuge tube, add 1 volume of magnetic beads (50 μL), pipette up and down to mix evenly, and centrifuge briefly.

[0181] 3) Let it stand at room temperature for 5 min, place the centrifuge tube on the magnetic rack for 3 min until the solution becomes clear.

[0182] 4) Keep the centrifuge tube on the magnetic rack, remove the supernatant, add 200 μL of 80% ethanol solution to the centrifuge tube, and let it stand for 30 s.

[0183] 5) Remove the supernatant, then add 200 μL of 80 vol% ethanol aqueous solution to the centrifuge tube. After standing for 30 s, completely remove the supernatant, and use a 10 μL pipette to remove the residual ethanol solution at the bottom.

[0184] 6) Let it stand at room temperature for 5 min until the magnetic beads are dry and the residual ethanol completely evaporates.

[0185] 7) Add 50 μL of nuclease-free water, remove the centrifuge tube from the magnetic rack, gently pipette up and down to resuspend the magnetic beads, avoid generating bubbles, centrifuge briefly, and let it stand at room temperature for 2 min.

[0186] 8) Place the centrifuge tube on the magnetic rack for 2 min until the solution becomes clear.

[0187] 9) Use a pipette to aspirate 48 μL of the supernatant and transfer it to a new centrifuge tube, and make a mark.

[0188] 10) Perform the subsequent experimental steps, or store the pre-hybridization library at -20 °C.

[0189] 2.4.6 Pre-library quality control

[0190] 1) Take 1 μL of the pre-hybridization library and measure the library concentration using the Qubit dsDNA HS Assay Kit. Record the concentration of the pre-library, and the pre-library concentration should be > 25 ng / μL.

[0191] 2) Take 1 μL of the pre-library and perform quality inspection on the library fragment distribution using nucleic acid analyzers such as the Agilent 2100 Bioanalyzer System and Qsep100 (Bioptic). The average length of the pre-library is between 250 - 350 bp.

[0192] The operation of step S3 is specifically as follows:

[0193] 3.1.1 The amount of the pre-hybridization library depends on the number of samples in each pooled library.

[0194] Use the concentration of each tagged library to calculate the volume (μL) of each library required for hybridization:

[0195] Determine the amount of each individual library in each pooled library from the following table.

[0196]

[0197] Note: If the amount of the library is insufficient, a smaller amount can be selected, but this may reduce the complexity of the library. The total DNA amount is 1.5 μg - 4 μg. Otherwise, it will lead to a decrease in enrichment performance. Tagged libraries: UDI primer, the I5 adapter primer and the I7 adapter primer.

[0198] 3.1.2. Transfer each amplified tagged library to a tagged pooled library reaction tube according to the calculated volume for hybridization.

[0199] Note: Check whether the tube is properly sealed because the liquid may evaporate, resulting in a decrease in performance.

[0200] 3.1.3. Centrifuge the tagged pooled library tube briefly to reduce bubbles.

[0201] 3.1.4. Dry the tagged pooled library using a centrifugal concentrator at low temperature or without heating.

[0202] Note: If the next step is not carried out immediately, the dried tagged pooled library can be stored at -20 °C for a maximum of 24 hours.

[0203] 3.2 Combine the capture probe (see Patent 202310828992.6) with the pooled library (prepared in 3.1.1) to prepare the probe solution.

[0204] 3.2.1. Place the hybridization reaction premix (the mixed library solution prepared in 3.1.1) into a heater and heat it at 65 °C for 10 min until all the precipitates dissolve. Then place it on the laboratory bench for 5 min to allow it to cool to room temperature.

[0205] 3.2.2. Prepare the probe solution as shown in the following table. Mix by flicking the test tube with your finger.

[0206] Reagent Volume (μL) Hybridization reaction premix 10 Custom 152-panel capture probe 1 Water 3 Total volume 14

[0207] Note: The hybridization reaction premix is a viscous reagent. Please operate slowly when pipetting to ensure accurate volume. There may be small white particles in the customized probe combination test tube. This will not affect the final capture product.

[0208] 3.2.3. Add the reagents as shown in the following table and resuspend the vacuum centrifuged dried label mixed library (prepared in step 3.1.1). Mix by flicking the test tube with your finger.

[0209] Reagent Volume Blocking agent solution 2.5 μL Universal blocker 3.5 μL Total volume 6

[0210] 3.3 Conduct the hybridization reaction

[0211] 1) Place the probe solution into a thermal cycler and heat it to 95 °C for 2 min. Set the heated lid to 105 °C, and then immediately cool it on ice for 5 min.

[0212] 2) While the probe solution is cooling on ice, place the test tube containing the resuspended label mixed library into the thermal cycler and heat it at 95 °C for 5 min. Set the heated lid to 105 °C, and then place both the probe solution and the resuspended label mixed library on the laboratory bench to allow them to equilibrate at room temperature for 5 min.

[0213] 3) Vortex and slowly rotate the probe solution, and then transfer all of it into the resuspended label mixed library. Mix well by vortexing.

[0214] 4) Centrifuge the test tube briefly to ensure that all the solution is at the bottom of the test tube.

[0215] 5) Add 15 μL of hybridization promoter to the entire capture reaction solution.

[0216] 6) Centrifuge the test tube briefly to ensure there are no air bubbles. (Note: Seal the test tube tightly to prevent excessive evaporation during the 16-hour incubation)

[0217] 7) Place the hybridization reaction solution into the thermal cycler and incubate it at 70 °C for 16 h. Set the heated lid to 85 °C. (Note: Stopping the hybridization during the 15 - 17 h period will not affect the downstream capture quality)

[0218] 2.4 Bind streptavidin magnetic beads

[0219] 1) The vortex passed through the pre-equilibrated streptavidin-coated magnetic beads until they were mixed evenly.

[0220] 2) Add 50 μL of streptavidin-coated magnetic beads to a 1.5 mL microcentrifuge tube. Prepare one tube for each hybridization reaction.

[0221] 3) Add 100 μL of binding buffer to the tube and pipette to mix evenly.

[0222] 4) Let the tube stand on the magnetic rack for 1 minute, then aspirate and discard the supernatant. Do not disrupt the magnetic bead pellet. Remove the tube from the magnetic rack.

[0223] 5) Repeat the washing twice (steps 3 and 4), for a total of three washes.

[0224] 6) After the third wash, remove the supernatant and add 100 μL of binding buffer. Resuspend the magnetic beads by vortexing until evenly mixed.

[0225] 7) Heat the resuspended magnetic beads at 68 °C for 10 minutes.

[0226] 8) After the hybridization (step 3.3) is completed, open the lid of the thermal cycler and directly transfer each hybridization reaction solution to the corresponding tube containing the pre-heated (step 7) streptavidin-coated magnetic beads. Mix the reagents by pipetting and flicking.

[0227] 9) Incubate the tubes containing the hybridization reaction solution and streptavidin-coated magnetic beads at 68 °C for 5 minutes without stirring. (Do not vortex, no vigorous mixing is required)

[0228] 10) Remove the tubes containing the hybridization reaction solution and streptavidin-coated magnetic beads from the mixer and perform a brief centrifugation to ensure that all the solution is at the bottom of the tube.

[0229] 11) Let the tube stand on the magnetic rack for 1 minute.

[0230] 12) Aspirate and discard the supernatant, including the hybridization enhancer. Do not disrupt the magnetic bead pellet.

[0231] (Note: Some hybridization enhancer can be seen after aspirating the supernatant and can also be seen in each washing step. It does not affect the final captured product.)

[0232] 13) Remove the tube from the magnetic rack and add 100 μL of standard wash buffer 1 at 68 °C. Pipette to mix evenly

[0233] 14) Incubate the tube at 68 °C for 5 minutes.

[0234] 15) Perform instantaneous centrifugation to ensure that all solutions are at the bottom of the test tube.

[0235] 16) Transfer all the solutions from step 15) to new 1.5 mL microcentrifuge tubes, with each hybridization reaction transferred to one tube. Let the tubes stand on the magnetic rack for 1 minute.

[0236] 17) Aspirate and discard the supernatant. Do not disrupt the magnetic bead pellet. Remove the tube from the magnetic rack and add 100 μL of Wash Buffer 2 at 48 °C. Mix well by pipetting up and down, then perform instantaneous centrifugation to ensure that all solutions are at the bottom of the tube.

[0237] 18) Incubate the tube at 48 °C for 5 min.

[0238] 19) Let the tube stand on the magnetic rack for 1 min.

[0239] 20) Aspirate and discard the supernatant. Do not disrupt the magnetic bead pellet.

[0240] 21) Repeat the washing twice (repeat steps 17) - 20)), for a total of three washes.

[0241] 22) After the last wash, use a 10 μL pipette to aspirate all the supernatant. Proceed to the next step immediately. Do not allow the magnetic beads to dry.

[0242] 23) Remove the tube from the magnetic rack and add 22.5 μL of water. Mix well by pipetting up and down, then incubate this solution on ice (hereinafter referred to as the streptavidin - bound magnetic bead suspension).

[0243] 3.4 Post - capture PCR amplification, purification, and quality control

[0244] 1) Enter the program in the thermal cycler according to the following conditions. Set the heated lid to 105 °C.

[0245]

[0246] 2) If the streptavidin - bound magnetic bead suspension shows sedimentation, mix well by pipetting up and down.

[0247] 3) Transfer 22.5 μL of the streptavidin - bound magnetic bead suspension to a 0.2 mL thin - walled PCR strip tube. Place it on ice until used in the next step.

[0248] (Note: Store the remaining 22.5 μL of the streptavidin - bound magnetic bead suspension at - 20 °C for future use.)

[0249] 4) Add the following reagents to the tube containing the streptavidin - bound magnetic bead suspension to prepare the PCR mixture. Mix well by pipetting up and down.

[0250] Reagent Volume (μL) Streptavidin-binding magnetic bead suspension 22.5 Amplification primer, ILMN 2.5 Equinox Library Amplification Mix (2x) 25 Total volume 50

[0251] 5) Centrifuge the test tube transiently, transfer it to a thermal cycler and start the cycling program.

[0252] 6) After the thermal cycler program ends, remove the test tube and immediately perform purification.

[0253] 7) Vortex the pre-equilibrated DNA purification magnetic beads until thoroughly mixed.

[0254] 8) Add 90 μL (1.8x) of the mixed DNA purification magnetic beads to the test tube in step 6). Mix by vortexing.

[0255] 9) Incubate at room temperature for 5 min.

[0256] 10) Let the test tube stand on the magnetic rack for 1 min until the supernatant becomes clear.

[0257] 11) The DNA purification magnetic beads form a precipitate. Keep the test tube on the magnetic rack, aspirate and discard the supernatant.

[0258] 12) Wash the magnetic bead precipitate with 200 μL of freshly prepared 80 vol% aqueous ethanol solution (operate gently without blowing away the precipitate). Incubate for 1 min, then aspirate and discard the ethanol.

[0259] 13) Repeat the washing twice, keeping the test tube on the magnetic rack during the process.

[0260] 14) Carefully aspirate all the remaining ethanol using a 10 μL pipette without disturbing the magnetic bead precipitate.

[0261] (Note: Before aspiration, briefly centrifuge the magnetic bead precipitate to aspirate the ethanol at the bottom of the plate or test tube, then place it back on the magnetic rack)

[0262] 15) Air-dry the magnetic bead precipitate on the magnetic rack (for 5 min until the magnetic bead precipitate is dry). Do not over-dry the magnetic bead precipitate.

[0263] 16) Remove the test tube from the magnetic rack and add 32 μL of water to each captured magnetic bead precipitate. Mix by pipetting up and down.

[0264] 17) Incubate at room temperature for 2 min.

[0265] 18) Place the test tube on the magnetic rack and let it stand for 3 minutes or until the magnetic beads completely form a precipitate.

[0266] 19) Transfer 30 μL of the supernatant (containing the enriched library: the DNA enriched by the method of this patent) to a clean 0.2 mL thin-walled PCR tube or a 96-well plate without disturbing the magnetic bead precipitate.

[0267] 20) Take 1 μL of the final hybridization library and quantify it using the Qubit dsDNA HS Assay Kit. Record the concentration of the final library, which is 1 ng / μL.

[0268] 21) Take 1 μL of the final library and perform quality inspection on the library fragment distribution using a nucleic acid analyzer. The average length of the cfDNA final library is between 270 - 390 bp, and the average length of the gDNA final library is between 330 - 450 bp.

[0269] 3.5 Library pooling and sequencing on the machine

[0270] Dilute each final library to 4 ng / μL, and the data volume is between 1 - 5 G.

[0271] After sequencing on the machine in step S4, set filtering conditions to generate a list of somatic mutations.

[0272] The filtering conditions include filtering background noise and germline variations, and the filtering conditions are as follows: filter mutation sites with low base quality, filter mutation sites with a population mutation rate ≥ 0.001 in any one database, filter mutation frequencies < 1%, filter sites with a mutation site depth < 100X, filter sites with a mutation frequency less than 2% and no pathogenicity records in cosmic and clinvar, filter sites with qualified base quality and a mutation frequency > 1% in the germline control sample, filter mutation results outside the range of Panel reportable genes, and filter false positive mutation results caused by sequencing errors.

[0273] After sequencing on the machine in step S4, calculate the equivalents of ctDNA and cfDNA in the cell-free DNA.

[0274] The equivalent calculation formula of ctDNA is: Where, J: A total of J somatic mutations are detected in the test subject. The mutation reads at the j-th site are Mj, and the sequencing depth is Nj.

[0275] The concentration calculation formula of ctDNA is: Where the plasma volume is V i , the total amount of cfDNA extracted from the plasma is m i , pg / haploid copy means pg per haploid genome.

[0276] The ctDNA concentration represents the number of ctDNA molecules contained in a unit volume of plasma. We define a sample with a ctDNA concentration greater than a certain threshold (e.g., 1.0 hGE / mL) as a ctDNA-positive sample, otherwise as a ctDNA-negative sample.

[0277] After the above-mentioned step S4, a prognostic risk prediction model is established after next-generation sequencing. The calculation formula for the change rate of ctDNA is as follows:

[0278] where c 0 : the detected value of the ctDNA concentration in the sample collected from the subject at time point t 0 ; c 1 : the detected value of the ctDNA concentration in the sample collected from the subject at time point t 1 .

[0279] Samples with a ctDNA change rate less than a certain threshold (e.g., 2) are defined as patients with better treatment efficacy, otherwise as patients with poorer treatment efficacy.

[0280] Apply this detection method to DLBCL patients, detect the punctured tumor tissue samples, oral swabs and blood samples of the patients, and calculate the ctDNA equivalent in the samples using the corresponding algorithm, so as to predict the prognosis of the patients.

[0281] Patient group 1 is composed of patients with intermediate-high risk diffuse large B-cell lymphoma, and patient group 2 is composed of patients with low risk diffuse large B-cell lymphoma.

[0282] The IPI (a scoring system used to evaluate the prognosis of lymphoma patients) score of the intermediate-high risk diffuse large B-cell lymphoma in patient group 1 is 2-5, and the detection rate of ctDNA in peripheral blood plasma before treatment is 96.73% (148 / 153), see Figure 1 . Different colors represent patient samples with different IPI scores.

[0283] The ctDNA equivalent before treatment predicts the intermediate-term efficacy (PD, SD, PR, CR); the higher the ctDNA equivalent before treatment, the fewer patients can achieve complete remission CR in the intermediate term. See Figure 3 . In the figure: a. disease progression; b. stable condition; c. partial response; d. complete response.

[0284] The IPI score of the low risk diffuse large B-cell lymphoma in patient group 2 is 0-1, and the detection rate of ctDNA in peripheral blood plasma before treatment is 89.5% (102 / 114), see Figure 2 . The ctDNA equivalent before treatment predicts the terminal efficacy (PD, PR, CR); the higher the ctDNA equivalent before treatment, the fewer patients can achieve complete remission CR at the terminal stage. See Figure 4 . In the figure: a. disease progression; c. partial response; d. complete response.

[0285] Based on the change in the equivalent multiple after 1 cycle of ctDNA detection, for DLBCL patients (IPI ≥ 2) in the group with a decline amplitude < 2LFC (poor efficacy, positive group), the CR rate increased significantly after intensive treatment. The mid-term CR rate increased from 40% to 58% as shown in Figure 5 , and the late-stage CR rate increased from 57% to 85.7% as shown in Figure 6 . In the figure: 1: Intensive treatment; 2. Standard treatment.

Claims

1. A method for detecting circulating tumor DNA in B lymphocyte tumors, characterized in that: The following steps are involved: S1 Preparation of DNA samples: Extract samples from tumor tissue, oral mucosa, and plasma; S2 Preparation of pre-hybridization library: Pre-hybridization library 1 and pre-hybridization library 2 were prepared using gDNA samples obtained from tumor tissue and oral mucosa, respectively; pre-hybridization library 3 was prepared using cfDNA samples obtained from plasma; S3 hybridization capture: combine the pre-hybridization library with the capture probe, perform hybridization reaction, capture the target area, and obtain the final hybridization library; S4 performs sequencing on the final hybridization library.

2. The method for detecting circulating tumor DNA in B lymphocyte tumors according to claim 1, characterized in that: After sequencing in the step S4, filtering conditions are set to generate a somatic mutation list.

3. The method for detecting circulating tumor DNA in B lymphocyte tumors according to claim 2, characterized in that: The filtering conditions include filtering background noise and germline variation, and the filtering conditions include: filtering mutation sites with low base quality, filtering mutation sites with a population mutation rate ≥0.1% in any database, filtering mutation frequency <1%, filtering sites with a mutation site depth <100X, filtering sites with a mutation frequency less than 2% and no pathogenicity records in cosmic and clinvar, filtering sites with qualified base quality and mutation frequency >1% in germline control samples, filtering mutation results that are not within the range of panel reportable genes, and filtering at least one of false positive mutation results caused by sequencing errors.

4. The method for detecting circulating tumor DNA in B lymphocyte tumors according to claim 1, characterized in that: After sequencing in step S4, the equivalent of ctDNA in free DNA is calculated.

5. The method for detecting circulating tumor DNA in B lymphocyte tumors according to claim 4, characterized in that: The calculation formula for the proportion of ctDNA is: Among them, J: a total of J individual cell mutations were detected in the test subject, the mutation read at the jth site is Mj, and the sequencing depth is Nj.

6. The method for detecting circulating tumor DNA in B lymphocyte tumors according to claim 5, characterized in that: The concentration calculation formula of the ctDNA is: The plasma volume is V i The total amount of cfDNA extracted from plasma is m i .

7. The method for detecting circulating tumor DNA in B lymphocyte tumors according to claim 6, characterized in that: After sequencing in step S4, a prognostic risk prediction model is established, and the ctDNA change rate calculation formula is as follows: Wherein, c0: the detection value of ctDNA concentration of the sample collected from the detection object at time point t0; c1: the detection value of ctDNA concentration of the sample collected from the detection object at time point t1.

8. The method for detecting circulating tumor DNA in B lymphocyte tumors according to claim 1, characterized in that: The hybridization reaction in step S3 is a PCR amplification reaction.

9. The method for detecting circulating tumor DNA in B lymphocyte tumors according to claim 1, characterized in that: The conditions for the PCR amplification are: pre-denaturation at 95-99°C for 43-48s; 12 cycles of 95-99°C for 13-18s, 58-62°C for 28-32s, and 70-75°C for 28-32s; extension at 70-75°C for 55-65s; constant temperature at 3-6°C.

10. An application of the method for detecting circulating tumor DNA in B lymphocyte tumors according to any one of claims 1 to 9, characterized in that: Applied to the detection of lesions of diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

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