Hematopoietic stem cell transplantation matching and prognosis evaluation kit and use method thereof

Abstract

The invention relates to a hematopoietic stem cell transplantation matching and prognosis evaluation kit and a use method thereof. The kit contains a probe for capturing target genes, and the target genes comprise an HLA gene, a KIR gene, an MICA gene, an MICB gene and an SUFU rs17114808 site. By selecting a specific exon region and limiting the Tm value, GC content, Hairpin free energy, sequence complexity and genome internal homology of probe design, all necessary gene loci can be detected at a time, and a scientific basis is provided for a personalized treatment scheme of hematopoietic stem cell transplantation; the method is beneficial to predicting and preventing graft versus host diseases after transplantation, and also can optimize the treatment scheme of transplantation pretreatment and perioperative period, so that better prognosis is brought to patients; meanwhile, a plurality of important genes and sites related to hematopoietic stem cell transplantation are covered, so that comprehensive assessment of transplantation risks is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection kits, and in particular to a hematopoietic stem cell transplantation matching and prognosis assessment kit and a method for using the kit. Background Art

[0002] With advances in haploidentical hematopoietic stem cell transplantation, differences in HLA typing between donors and recipients have a significant impact on treatment outcomes. While existing PCR-SBT methods can perform two types of HLA typing, this approach cannot provide more precise typing to identify the optimal donor for multiple haploidentical siblings. Furthermore, in addition to HLA genes, other immune-related genes, such as KIR, MICA, and SUFU, are closely associated with a patient's immune status and post-transplantation risks. Post-transplant immune reconstitution is a complex process, with innate immunity being particularly crucial, directly impacting defenses against tumors and pathogens. Natural killer (NK) cell function depends on its education, and the presence of KIR receptors mediates the generation of alloreactive NK cell activity, which in turn influences the development of significant post-transplant complications such as infection and GVHD. To improve transplant success rates and reduce the risk of rejection, detailed genotyping analysis of both donors and recipients of hematopoietic stem cell transplantation is crucial. These included HLA genotyping, KIR genotyping, MICA genotyping and its mutation analysis, and SUFU (suppressor of fusion) rs17114808 locus genotyping.

[0003] Chinese invention patent CN102877136B discloses a method and kit for constructing a DNA library based on genome simplification and second-generation sequencing in the field of biotechnology. This method and kit address the shortcomings of existing library construction methods and can be used for whole-genome SNP detection and genotyping of species with incomplete reference genomes, unclear research population pedigrees, and no haplotype maps. However, it focuses on whole-genome SNP detection and its application in species with incomplete reference genomes, and is less targeted to clinical transplantation scenarios. Summary of the Invention

[0004] The first aspect of the present invention provides a hematopoietic stem cell transplantation matching and prognosis assessment kit, which contains probes for capturing target genes, including: HLA gene, KIR gene, MICA gene, MICB gene and SUFU rs17114808 site.

[0005] The probe information corresponding to the HLA gene includes: located on chromosome 6, the exon regions are 29942531-29945870, 31268748-31272092, 31353874-31357179, 32578768-32589836, 32637405-32654846, 32659466-32666657, 33064568-33089696 and 32637405-32654846.

[0006] The probe information corresponding to the KIR gene includes: located on chromosome 19, exon region: 54724175-54724616, 54725174-54726394, 54726433-54728353, 54728473-54728833, 54728872-54728992, 54729197-54730277, 54730877-54730997, 54731357-54732258, 54732557-54733277, 54733517-54734237, 54735072-54735357, 54735393-5473551 3. 54735820-54735872, 54735971-54736633, 54738245-54738724, 54738864-54739016, 54739136-54739256, 54739357-54739613, 54739700-5474 0576、54740577-54741038、54741380-54742472、54742585-54742857、54742858-54743000、54743060-54743180、54743569-54743689、54743795-54 744088、54745419-54745539、54747127-54747385、54747549-54747667、54747936-54748087、54748148-54748268、54748497-54748617、54748960 -54749446、54749922-54750042、54750052-54750172、54750313-54750635、54750727-54750847、54751378-54751497、54751649-54751753、54751 916-54752165、54752216-54752268、54752367-54753052、54756308-54756764、54757034-54757274、54757280-54757514、54757993-54758113、54 758373-54758729、54758833-54759102、54759130-54759335、54759369-54759489、54759490-54759812、54760165-54760325、54760662-54760782、54760926-54761142、54761622-54761742、54763796-54764096、54764353-54764473、54764752-54764872、54764982-54765342、54765555-54765795、54766768-54766888、54766927-54767084、54767418-54767538、54769582-54769942、54770595-54770715、54770782-54770955、54771021-54771262、54771320-54771502、54771622-54772462、54772602-54772822、54772914-54773662、54773940-54774060、54774142-54774537、54774819-54774939、54775165-54775458、54775539-54775779、54775899-54776038、54776642-54776762、54777749-54777869、54778612-54778662、54778705-54778944、54779213-54779333、54779425-54779894、54780237-54780754、54781199-54781563、54781755-54782055、54782065-54782415、54782922-54783023、54783315-54783614、54783637-54784322、54785807-54786167、54786327-54786561、54786567-54787134、54787254-54787374、54787558-54788094、54788168-54788692、54789174-54789400、54789414-54789635、54789691-54789921、54790014-54790327、54803384-54803984、54803985-54806470、54806590-54806710、54807032-54807426、54808144-54808265、54808505-54810160、54810423-54811143、54811383-54812343、54812463-54813781、54813786-54814518、54816022-54816143、54816352-54816467、54816468-54816534、54816814-54816934、54817313-54818753、54818873-54819233、54819353-54820257、54820299-54820419、54820433-54820913、54821110-54821470、54821565-54822233、54822593-54822713、54823816-54823936、54824873-54825353、54825628-54825779、54825867-54825987、54826427-54826548、54826597-54826717、54826827-54826982、54827336-54827456、54827590-54828070、54828093-54828297、54828299-54828493、54828540-54828681、54829053-54829173、54829189-54829309、54829361-54829465、54829652-54829772、54829811-54829980、54830099-54830779、54832707-54832798、54832958-54833097、54833279-54833399、54833510-54833630、54833741-54833861、54833971-54834091、54834177-54834417、54834546-54834666、54834729-54834897、54834950-54835070、54835079-54835114、54835137-54835617、54835840-54836006、54836049-54836401、54836457-54836697、54836937-54837057、54837114-54837417、54837576-54837893、54837950-54838069、54838090-54838617、54839097-54839333、54839410-54839911、54840035-54840155、54842773-54842918、54842954-54843200、54843207-54843327、54843708-54844038、54844268-54844389、54844438-54844633、54844932-54845152、54845410-54845967、54846013- 54846133、54846154-54846540、54847047-54847341、54847451-54847595、54847634-54848575、54850207-54850327、54850447-54850567、54851047-54851508、54851566-54851686、 54851806-54852526、54852828-54852948、54853327-54853447、54853586-54853706、54853747-54854047、54854066-54854186、54854407-54854887、54858924-54859284、54859447- 54859567, 54860025-54860385, 54860625-54860855, 54861074-54861194, 54861222-54861342, 54861704-54861960, 54862064-54862519, 54865548-54866508, and 54866522-54867216.

[0007] The probe information corresponding to the MICA gene and the MICB gene includes: located on chromosome 6, the exon regions are 31400710-31415315 and 31494880-31511124.

[0008] The probe information corresponding to the SUFU rs17114808 site includes: located on chromosome 10, and the exon region is 102631527-102631528.

[0009] The probe is designed using the following method:

[0010] S1: Obtain the exon region of the target gene according to the database;

[0011] S2: Cut off 1 to 2 unit bp of an exon region as the first probe unit, cut off (n-1) unit to (n+1) unit bp of the exon region as the nth probe unit, and cut off in sequence, Unit = 60 bp, to obtain the probe of the exon region;

[0012] S3: The nucleotide sequence of the probes was evaluated using parameters including Tm of 65 ± 2°C, GC content of 45% to 55%, Hairpin free energy, sequence complexity, and intragenomic homology to ensure specific capture of the target sequence and stable binding of the probes under fixed temperature conditions during hybridization.

[0013] S4: Adjust the probes with scores below the threshold: the unit value of the probe sequence is 60±10bp when it is truncated; repeat steps S2 and S3 to optimize the probe sequence.

[0014] The second aspect of the present invention provides a method for using a hematopoietic stem cell transplantation matching and prognosis assessment kit, comprising the following steps: step 1, DNA sample preparation: genomic DNA extraction is performed using gDNA of target cells as a sample; step 2, pre-library preparation; step 3, hybridization capture; step 4, library mixing and sequencing; step 5, bioinformatics process analysis.

[0015] The target cells include bone marrow blood cells or peripheral blood leukocytes.

[0016] The prelibrary preparation includes: sequentially performing genomic DNA fragmentation, linker ligation and purification, and PCR amplification and purification.

[0017] The hybridization capture comprises: labeling the probe with biotin and hybridizing it with the prelibrary, and then using streptavidin magnetic beads to bind the probe to capture the target area.

[0018] The PCR amplification and purification also include library quantification, library quality detection and pre-library quality control.

[0019] Beneficial effects

[0020] 1. The present invention selects specific exon regions and defines the Tm value, GC content, Hairpin free energy, sequence complexity, and intragenomic homology of probe design, enabling the detection of all necessary gene loci at one time, providing a scientific basis for personalized treatment plans for hematopoietic stem cell transplantation.

[0021] 2. The kit of the present invention can simultaneously detect the HLA gene, KIR gene, MICA gene, MICB gene and SUFU rs17114808 locus of hematopoietic stem cell transplantation donors and recipients, covering multiple important genes and loci related to hematopoietic stem cell transplantation, which helps to comprehensively assess the risk of transplantation.

[0022] 3. The kit of the present invention can obtain all exon sequences of HLA, KIR, MICA, and MICB genes, making typing more accurate and more precise.

[0023] 4. The kit of the present invention not only helps to predict and prevent graft-versus-host disease after transplantation, but also optimizes transplantation pretreatment and perioperative treatment plans, thereby bringing better prognosis to patients.

[0024] 5. The kit of the present invention is easy to use and has low cost. Compared with the tedious operation of detecting multiple genes separately in the past, the kit of the present invention only requires one test, which simplifies the detection process and improves the detection efficiency.

[0025] 6. During the use of the kit of the present invention, the required sample volume is smaller. The donor does not need to draw blood, but only needs to provide an oral swab to obtain the DNA sample volume required for the test. DETAILED DESCRIPTION

[0026] Example

[0027] A hematopoietic stem cell transplantation matching and prognosis assessment kit contains probes for capturing target genes, wherein the target genes include: HLA gene, KIR gene, MICA gene, MICB gene and SUFU rs17114808 site.

[0028] The probe information corresponding to the HLA gene is: located on chromosome 6, the exon regions are 29942531-29945870, 31268748-31272092, 31353874-31357179, 32578768-32589836, 32637405-32654846, 32659466-32666657, 33064568-33089696 and 32637405-32654846.

[0029] The probe information corresponding to the KIR gene is: located on chromosome 19, exon region: 54724175-54724616, 54725174-54726394, 54726433-54728353, 54728473-54728833, 54728872-54728992, 54729197-54730277, 54730877-54730997, 54731357-54732258, 54732557-54733277, 54733517-54734237, 54735072-54735357, 54735393-54735513, 54735820-54735872、54735971-54736633、54738245-54738724、54738864-54739016、54739136-54739256、54739357-54739613、54739700-547405 76、54740577-54741038、54741380-54742472、54742585-54742857、54742858-54743000、54743060-54743180、54743569-54743689、54743795-547 44088、54745419-54745539、54747127-54747385、54747549-54747667、54747936-54748087、54748148-54748268、54748497-54748617、54748960- 54749446、54749922-54750042、54750052-54750172、54750313-54750635、54750727-54750847、54751378-54751497、54751649-54751753、547519 16-54752165、54752216-54752268、54752367-54753052、54756308-54756764、54757034-54757274、54757280-54757514、54757993-54758113、547 58373-54758729、54758833-54759102、54759130-54759335、54759369-54759489、54759490-54759812、54760165-54760325、54760662-54760782、54760926-54761142、54761622-54761742、54763796-54764096、54764353-54764473、54764752-54764872、54764982-54765342、54765555-54765795、54766768-54766888、54766927-54767084、54767418-54767538、54769582-54769942、54770595-54770715、54770782-54770955、54771021-54771262、54771320-54771502、54771622-54772462、54772602-54772822、54772914-54773662、54773940-54774060、54774142-54774537、54774819-54774939、54775165-54775458、54775539-54775779、54775899-54776038、54776642-54776762、54777749-54777869、54778612-54778662、54778705-54778944、54779213-54779333、54779425-54779894、54780237-54780754、54781199-54781563、54781755-54782055、54782065-54782415、54782922-54783023、54783315-54783614、54783637-54784322、54785807-54786167、54786327-54786561、54786567-54787134、54787254-54787374、54787558-54788094、54788168-54788692、54789174-54789400、54789414-54789635、54789691-54789921、54790014-54790327、54803384-54803984、54803985-54806470、54806590-54806710、54807032-54807426、54808144-54808265、54808505-54810160、54810423-54811143、54811383-54812343、54812463-54813781、54813786-54814518、54816022-54816143、54816352-54816467、54816468-54816534、54816814-54816934、54817313-54818753、54818873-54819233、54819353-54820257、54820299-54820419、54820433-54820913、54821110-54821470、54821565-54822233、54822593-54822713、54823816-54823936、54824873-54825353、54825628-54825779、54825867-54825987、54826427-54826548、54826597-54826717、54826827-54826982、54827336-54827456、54827590-54828070、54828093-54828297、54828299-54828493、54828540-54828681、54829053-54829173、54829189-54829309、54829361-54829465、54829652-54829772、54829811-54829980、54830099-54830779、54832707-54832798、54832958-54833097、54833279-54833399、54833510-54833630、54833741-54833861、54833971-54834091、54834177-54834417、54834546-54834666、54834729-54834897、54834950-54835070、54835079-54835114、54835137-54835617、54835840-54836006、54836049-54836401、54836457-54836697、54836937-54837057、54837114-54837417、54837576-54837893、54837950-54838069、54838090-54838617、54839097-54839333、54839410-54839911、54840035-54840155、54842773-54842918、54842954-54843200、54843207-54843327、54843708-54844038、54844268-54844389、54844438-54844633、54844932-54845152、54845410-54845967、54846013- 54846133、54846154-54846540、54847047-54847341、54847451-54847595、54847634-54848575、54850207-54850327、54850447-54850567、54851047-54851508、54851566-54851686、 54851806-54852526、54852828-54852948、54853327-54853447、54853586-54853706、54853747-54854047、54854066-54854186、54854407-54854887、54858924-54859284、54859447- 54859567, 54860025-54860385, 54860625-54860855, 54861074-54861194, 54861222-54861342, 54861704-54861960, 54862064-54862519, 54865548-54866508, and 54866522-54867216.

[0030] The probe information corresponding to the MICA gene and MICB gene is: located on chromosome 6, the exon regions are 31400710-31415315 and 31494880-31511124.

[0031] The probe information corresponding to the SUFU rs17114808 site is located on chromosome 10, and the exon region is 102631527-102631528.

[0032] The probe is designed using the following method:

[0033] S1: Obtain the exon region of the target gene according to the database;

[0034] S2: Cut off 1 to 2 unit bp of an exon region as the first probe unit, cut off (n-1) unit to (n+1) unit bp of the exon region as the nth probe unit, and cut off in sequence, Unit = 60 bp, to obtain the probe of the exon region;

[0035] S3: The nucleotide sequence of the probe was evaluated. The evaluation parameters included: Tm value of 65±2℃, GC content of 45% to 55%, Hairpin free energy, sequence complexity and intragenomic homology;

[0036] S4: Adjust the probes with scores below the threshold: the unit value of the probe sequence is 60±10bp when it is truncated; repeat steps S2 and S3 to optimize the probe sequence.

[0037] A method for using a hematopoietic stem cell transplantation matching and prognosis assessment kit comprises the following steps:

[0038] 1. DNA sample preparation: Genomic DNA was extracted from peripheral blood leukocyte gDNA samples according to the "Standard Operating Procedure for Genomic DNA Extraction from Blood". The DNA was eluted with 100 μL of ddH2O (double deionized water) and stored after elution. This DNA was used as a template for subsequent experiments.

[0039] 2. The pre-library preparation: using QuarPrep enzyme digestion DNA library construction kit.

[0040] 2.1 Genomic DNA fragmentation

[0041] Remove the Frag / AT buffer and Frag / AT enzyme from the kit, thaw and mix thoroughly, centrifuge briefly to collect the residue at the bottom of the tube, and place on ice for later use. All the following steps are performed on ice. Prepare the reaction as shown in Table 1 in a sterile PCR tube:

[0042] Table 1

[0043] Reagents content Input DNA* (template obtained in step 1) 100ng Frag / AT buffer 4 μL Frag / AT enzyme 6μL <![CDATA[ddH2O]]> Dissolve Input DNA* and adjust the volume to 40 μL

[0044] Use a pipette to pipette or shake to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Immediately place the tube in a PCR instrument (Agielnt 8800) to perform the following reactions in Table 2:

[0045] Table 2

[0046] temperature time Heated cover 105℃ 0 37℃ 18min 65℃ 30min 4℃ /

[0047] When the thermal cycler program is finished and the sample block returns to 4°C, remove the sample from the template, place it on ice, and proceed immediately to the next step.

[0048] 2.2. Adapter ligation and purification: Attach adapters to the ends of the fragmented end-repair products from the previous step.

[0049] Remove the universal adapter from -20°C, thaw, mix thoroughly, centrifuge briefly to collect the residue at the bottom of the tube, and place on ice for later use; prepare the reaction system according to Table 3 below:

[0050] Table 3

[0051] Reagents Volume (μL) Product of the previous step (step 2.1) 50 LigationMasterMix 20 DNAAdapterX 5 total 75

[0052] Use a pipette to gently pipette to mix (do not oscillate), and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a PCR instrument and perform the following reactions in Table 4:

[0053] Table 4

[0054] temperature time Heated cover 105℃ 0 20℃ 15min 4℃ /

[0055] Purify the reaction product using magnetic beads: After the magnetic beads are equilibrated to room temperature (30 minutes), vortex and mix the beads. Pipette 60μL of magnetic beads into 75μL of Adapter Ligation product and vortex or use a pipette to gently pipette 10 times to mix thoroughly. Incubate at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear (about 5 minutes), remove the supernatant. Keep the PCR tube in the magnetic rack at all times, add 180μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and remove the supernatant. Repeat step 5 for a total of two rinses. Keep the PCR tube in the magnetic rack at all times and air-dry the magnetic beads with the lid open for 5-10 minutes until no ethanol remains. Remove the PCR tube from the magnetic rack, add 17 μL ddH2O for elution, vortex or use a pipette to mix thoroughly, and place at room temperature for 2 minutes. Centrifuge the PCR tube briefly and place it on the magnetic rack to stand. After the solution is clear (about 5 minutes), transfer 15 μL of the supernatant to a new EP tube. Do not touch the magnetic beads.

[0056] 2.3 PCR amplification and purification

[0057] Thaw the UDI primers and Equinox library amplification mixture (2x) in the kit and mix thoroughly by inversion. Prepare the following reaction in a sterile PCR tube as shown in Table 5:

[0058] Table 5

[0059] Use a pipette to gently pipette to mix (do not oscillate to mix), and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a PCR instrument and start the PCR program as follows: hot cover temperature 105℃; 98℃, 45s; 98℃, 15s; 60℃, 30s; 72℃, 30s; 72℃, 1min; cycle 8 times, and store at 4℃

[0060] Purify the reaction product: After the magnetic beads are equilibrated to room temperature, vortex and oscillate to mix them. Pipette 50ul (1x) magnetic beads into 50μL LibraryAmplification product, vortex or use a pipette to gently blow 10 times to mix thoroughly. Incubate at room temperature for 5min. Centrifuge the PCR tube briefly and place it on a magnetic stand to separate the magnetic beads and liquid. After the solution is clear (about 5min), carefully remove the supernatant. Keep the PCR tube in the magnetic stand at all times, add 180μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30s, and carefully remove the supernatant. Repeat step 5 for a total of two rinses. Keep the PCR tube in the magnetic stand at all times, open the lid and air-dry the magnetic beads for 5-10min until no ethanol remains. Remove the PCR tube from the magnetic stand, add 65 μL ddH2O to elute, vortex or use a pipette to mix thoroughly, and place at room temperature for 2 minutes. Centrifuge the PCR tube briefly and place it on the magnetic stand to stand. After the solution is clear (about 5 minutes), carefully transfer 65 μL of the supernatant to a new EP tube. The supernatant in the tube is the prepared pre-library.

[0061] 2.4 Library quantification: Take 1 μL of library and use Qubit 2.0 Fluorometer (Qubit dsDNA HS Assay Kit) to measure the library concentration and record the library concentration.

[0062] 2.5. Library quality detection: The Qsep100 fully automatic nucleic acid protein analysis system was used to measure the length and purity of the library fragments. The target fragment distribution range of a normal library is between 300bp and 400bp.

[0063] 2.6. Prelibrary quality control: Take 1 μL of prelibrary and use the Qubit dsDNA HS Assay Kit to measure the library concentration and record the prelibrary concentration. The prelibrary concentration should be >25 ng / μL. Take 1 μL of prelibrary and use a nucleic acid analyzer such as Agilent 2100 Bioanalyzer System or Qsep100 (Bioptic) to perform library fragment distribution quality control. The average length of the prelibrary should be between 250-350 bp.

[0064] 3. The hybrid capture method uses Aijitaikang TargetSeq Kit.

[0065] 3.1. Take out the Hyb Human Block, Adapter Blocker, RNase Block, and Target Probe from the refrigerator in advance and place them on an ice box to melt. After melting, mix well and place on ice or at 4°C until use.

[0066] 3.2. For hybridization of multiple libraries, add equal amounts of each library to a PCR tube, totaling approximately 1200 ng, and label. Place the PCR tube in a vacuum concentrator centrifuge, open the PCR tube cap, start the centrifuge, turn on the vacuum pump, and begin concentrating to dryness. After library concentration is complete, add the hybridization reaction system listed in Table 6 below:

[0067] Table 6

[0068] Reagent Volume (μL) TargetSeqOneHybBuffer 13 HybHumanBlock 5 AdapterBlocker (select according to library type) 2 RnaseBlock 5 NucleaseFreeWater 3 TargetProbe 1 Total volume 30

[0069] Gently pipette to mix and centrifuge briefly. Place the PCR tube in a PCR instrument and program the following: hot cover temperature 85°C; 80°C for 5 minutes; incubate at 50°C overnight.

[0070] 3.3. Prepare capture magnetic beads: Cap beads are required for capture.

[0071] Remove the capture magnetic beads (T1) from 4°C, equilibrate at room temperature for 30 minutes, and then vortex to resuspend; for one hybridization library, take 50 μL of magnetic beads and add them to a new PCR tube, place it on the magnetic stand for 1 minute to allow the solution to clarify, and remove the supernatant; remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, gently pipette several times to mix, and resuspend the magnetic beads; place it on the magnetic stand for 1 minute, and remove the supernatant; repeat steps 3-4 twice, and wash the magnetic beads 3 times in total; remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, gently pipette to resuspend the magnetic beads and set aside.

[0072] 3.4. Capture target region DNA library

[0073] When capturing the target area, add the resuspended capture magnetic beads to the PCR tube and keep the PCR tube on the PCR instrument during the process.

[0074] The probe and magnetic beads were combined and incubated at room temperature for 30 minutes. A homogenizer was used to prevent the magnetic beads from settling. The rotation speed should not be too high, and it is recommended not to exceed 10 rpm.

[0075] After the capture is completed, be careful not to discard the magnetic beads. The next step is the PCR reaction with the magnetic beads in the system.

[0076] If any precipitate forms in Wash Buffer 1 stored at room temperature, heat it in a 37°C water bath and wait until the reagent is completely dissolved before use.

[0077] TargetSeq Preheat the Wash Buffer to 50°C in advance: 1) Keep the hybridization product on the PCR instrument and add 180 μL of Cap beads resuspended in step 6 of 6.3.2 to the hybridization product. Pipette and mix well. Place on a rotating mixer at room temperature for 30 minutes. 2) Place the PCR tube on a magnetic rack for 2 minutes to clarify the solution and remove the supernatant. 3) Add 150 μL of Wash Buffer 1 to the hybridization product and gently pipette and mix well. Place on a rotating mixer for 15 minutes, then centrifuge briefly. Place the PCR tube on a magnetic rack for 2 minutes to clarify the solution and remove the supernatant. 4) Add 150 μL of TargetSeq preheated at 50°C. Wash Buffer, gently pipette to mix, then briefly centrifuge and incubate on a thermostatic shaker at 50°C for 10 minutes. 5) Briefly centrifuge and place the PCR tube on a magnetic rack for 2 minutes to clarify the solution. Remove the supernatant. 6) Repeat steps 4-5 twice, washing the beads three times in total. 7) Keeping the sample on the magnetic rack, add 150μL of 80% ethanol to the PCR tube. Let it stand for 30 seconds, then completely remove the ethanol solution (a 10μL pipette can be used to remove any residue), and let it dry at room temperature. 8) Add 24μL of Nuclease-free Water to the PCR tube, remove the PCR tube from the magnetic rack, and gently pipette to resuspend the magnetic beads for later use.

[0078] 3.5. Post-capture PCR amplification

[0079] Take out the Post PCR Master Mix and Post PCR Primer from the -20°C freezer, place them on an ice box to thaw, mix well, and place on ice or at 4°C for later use. After capture, the DNA library needs to be amplified by PCR. Prepare the reaction system according to Table 7 below:

[0080] Table 7

[0081] Reagent Volume (μL) Sample after the previous step of reaction 24 PostPCRMasterMix 25 PostPCR Primer 1 Total volume 50

[0082] Adjust the pipette to 40 μL, gently pipette and mix 6 times, and then immediately place it on the PCR instrument; place the PCR tube in the PCR instrument, and the program is: 95°C, 1 min; 98°C, 20 s; 60°C, 30 s; 72°C, 30 s; 72°C, 5 min; after cycling, store at 4°C.

[0083] After PCR, add 55 μL of Agencourt AMPure XP magnetic beads to the sample, vortex and mix by pipetting, and let it stand at room temperature for 5 minutes; centrifuge briefly, and place the PCR tube on a magnetic rack for 3 minutes to wait for the solution to clarify; keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds; keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and then completely remove the supernatant; let it stand at room temperature for 3-5 minutes to allow the residual ethanol to completely evaporate; add 25 μL of Nuclease-free water, remove the PCR tube from the magnetic rack, vortex and mix by pipetting 10 times, and let it stand at room temperature for 2 minutes; centrifuge briefly, and place the PCR tube on a magnetic rack for 2 minutes to wait for the solution to clarify; use a pipette to transfer 23 μL of supernatant to a 1.5 mL centrifuge tube and mark the sample information.

[0084] 3.6 Library Quantification

[0085] 1 μL of library was taken and the library concentration was measured using a Qubit 2.0 Fluorometer (Qubit dsDNA HS Assay Kit) and the library concentration was recorded. The library fragment length and purity were measured using the Qsep100 fully automated nucleic acid protein analysis system. The main peak length of a normal library was approximately between 330 bp and 450 bp.

[0086] 4. Library mixing and sequencing

[0087] Dilute each final library to 4 ng / μL. Based on the data volume for each project, add the corresponding volume of final library to a new 1.5 mL EP tube. Vortex thoroughly to mix, and then measure the library concentration. The sequencing data volume is 1 GB. Select the appropriate instrument for sequencing based on the final expected data volume.

[0088] 5 Bioinformatics Process Analysis

[0089] First, the raw sequencing data was processed using Fastp software to remove sequencing adapters and low-quality reads. The filtered reads were aligned to the human reference genome (hg38) using BWA-mem software. Next, the aligned bam files were sorted, deduplicated, and quality-corrected using GATK software. Finally, bioinformatics genotyping software was used to align the data with the IPD-IMGT / HLA database to obtain high-resolution HLA genotyping results.

[0090] Performance testing methods

[0091] 1. Validation of HLA and KIR genotyping: 29 samples (from Shanghai Sitai Medical Testing Laboratory) with known HLA and KIR genotyping results were tested using the methods of the examples. For HLA genotyping, as shown in Tables 8-10, the results for all 29 samples were consistent with the binary typing results obtained by PCR-SBT, and all samples had all HLA loci typed at once. For KIR genotyping, as shown in Tables 11 and 12, where "1" indicates that the sample contains the gene and "0" indicates that the sample does not contain the gene, the results for all 29 samples were consistent with the control results obtained by PCR-SSP. This demonstrates that the kit accurately performs typing.

[0092] 2. Quality Control Testing: 29 samples were subjected to quality control testing using the method described in the Example. As shown in Table 13, the quality control results all met the quality control requirements of average sequencing depth ≥1000, on-target rate ≥40%, sequencing alignment rate >80%, and uniformity >60%, indicating good performance of the kit. Simultaneously, sequencing results for specific loci of MICA, MICB, and SUFU rs171148083 from 29 samples were analyzed using IGV software. The sequencing depth of the relevant loci all met the analysis requirements (≥1000), and the results were reliable and without abnormalities.

[0093] 3. Detection injection volume (detection limit) test: Two detection limit samples (samples with known content) were used, with injection volumes of 5 ng, 10 ng, 20 ng, 50 ng, and 100 ng, respectively, for library construction. As shown in Table 14, according to the example method, the four samples of 5 ng and 10 ng failed to be constructed. The samples of 20 ng, 50 ng, and 100 ng for library construction were tested and all met the quality control standards. It can be seen that the detection limit of the present invention can reach 20 ng.

[0094] Performance test data

[0095] Table 8

[0096]

[0097]

[0098] Table 9

[0099]

[0100]

[0101]

[0102] Table 10

[0103]

[0104]

[0105] Table 11

[0106]

[0107]

[0108] Table 12

[0109] KIR2DL5 KIR3DS1 KIR3DL1 KIR3DL2 KIR3DL3 KIR2DP1 KIR3DP1 KIR2DL23 Sample 1 1 1 0 1 1 1 1 1 Sample 2 0 0 1 1 1 1 1 1 Sample 3 0 0 1 1 1 1 1 1 Sample 4 0 0 1 1 1 1 1 1 Sample 5 1 1 1 1 1 1 1 1 Sample 6 0 0 1 1 1 1 1 1 Sample 7 0 0 1 1 1 1 1 1 Sample 8 0 0 1 1 1 1 1 1 Sample 9 0 0 1 1 1 1 1 1 Sample 10 0 0 1 1 1 1 1 1 Sample 11 0 0 1 1 1 1 1 1 Sample 12 1 1 1 1 1 1 1 1 Sample 13 0 0 1 1 1 1 1 1 Sample 14 0 0 1 1 1 1 1 1 Sample 15 1 0 1 1 1 1 1 1 Sample 16 0 0 1 1 1 1 1 1 Sample 17 0 0 1 1 1 1 1 1 Sample 18 0 0 1 1 1 1 1 1 Sample 19 1 0 1 1 1 1 1 1 Sample 20 1 1 1 1 1 1 1 1 Sample 21 1 1 1 1 1 1 1 1 Sample 22 0 0 1 1 1 1 1 1 Sample 23 1 1 1 1 1 1 1 1 Sample 24 1 1 1 1 1 1 1 1 Sample 25 0 0 1 1 1 1 1 1 Sample 26 0 0 1 1 1 1 1 1 Sample 27 0 0 1 1 1 1 1 1 Sample 28 0 0 1 1 1 1 1 1 Sample 29 0 0 1 1 1 1 1 1

[0110] Table 13

[0111] Average sequencing depth On-target rate Sequencing alignment rate Homogeneity Sample 1 3213.54 52.60％ 89.14％ 68.72％ Sample 2 2723.08 53.54％ 88.86％ 68.40％ Sample 3 2905.77 56.07％ 86.66％ 66.39％ Sample 4 2989.04 54.27％ 88.16％ 68.37％ Sample 5 3081.23 55.53％ 87.97％ 67.26％ Sample 6 3229.6 50.77％ 85.18％ 67.24％ Sample 7 2355.58 56.02％ 87.08％ 66.97％ Sample 8 3745.88 57.87％ 87.07％ 68.30％ Sample 9 3048.69 54.45％ 87.88％ 67.03％ Sample 10 3172.85 55.33％ 85.73％ 64.94％ Sample 11 3147.88 54.44％ 86.29％ 65.76％ Sample 12 2941.01 53.86％ 88.54％ 67.47％ Sample 13 1713.45 57.31％ 86.42％ 68.27％ Sample 14 3296.87 57.09％ 87.92％ 68.20％ Sample 15 2860.73 54.21％ 86.28％ 63.68％ Sample 16 2611.13 53.55％ 86.69％ 67.62％ Sample 17 3621.18 54.46％ 85.35％ 65.03％ Sample 18 2810.36 51.09％ 85.12％ 64.21％ Sample 19 1002.86 42.49％ 87.67％ 68.85％ Sample 20 1825.13 48.23％ 87.78％ 68.71％ Sample 21 1808.06 49.21％ 89.93％ 67.64％ Sample 22 2705.3 53.72％ 86.79％ 67.50％ Sample 23 2339.27 51.92％ 87.92％ 67.17％ Sample 24 2864.67 54.79％ 87.27％ 68.77％ Sample 25 3004.89 53.43％ 86.56％ 66.49％ Sample 26 2323.5 52.08％ 85.72％ 67.17％ Sample 27 2169.27 52.64％ 86.75％ 67.02％ Sample 28 3388.61 58.22％ 87.21％ 67.26％ Sample 29 2666.09 55.75％ 84.57％ 65.12％

[0112] Table 14

[0113]

Claims

1. A hematopoietic stem cell transplantation matching and prognosis assessment kit, characterized in that: The kit contains probes for capturing target genes, including HLA gene, KIR gene, MICA gene, MICB gene and SUFUrs17114808 site.

2. The hematopoietic stem cell transplantation matching and prognosis assessment kit according to claim 1, characterized in that: The probe information corresponding to the HLA gene includes: located on chromosome 6, the exon regions are 29942531-29945870, 31268748-31272092, 31353874-31357179, 32578768-32589836, 32637405-32654846, 32659466-32666657, 33064568-33089696 and 32637405-32654846.

3. The hematopoietic stem cell transplantation matching and prognosis assessment kit according to claim 1, characterized in that: The probe information corresponding to the KIR gene includes: located on chromosome 19, exon region: 54724175-54724616, 54725174-54726394, 54726433-54728353, 54728473-54728833, 54728872-54728992, 54729197-54730277, 54730877-54730997, 54731357-54732258, 54732557-54733277, 54733517-54734237, 54735072-54735357, 54735393-5473551 3. 54735820-54735872, 54735971-54736633, 54738245-54738724, 54738864-54739016, 54739136-54739256, 54739357-54739613, 54739700-5474 0576、54740577-54741038、54741380-54742472、54742585-54742857、54742858-54743000、54743060-54743180、54743569-54743689、54743795-54 744088、54745419-54745539、54747127-54747385、54747549-54747667、54747936-54748087、54748148-54748268、54748497-54748617、54748960 -54749446、54749922-54750042、54750052-54750172、54750313-54750635、54750727-54750847、54751378-54751497、54751649-54751753、54751 916-54752165、54752216-54752268、54752367-54753052、54756308-54756764、54757034-54757274、54757280-54757514、54757993-54758113、54 758373-54758729、54758833-54759102、54759130-54759335、54759369-54759489、54759490-54759812、54760165-54760325、54760662-54760782、54760926-54761142、54761622-54761742、54763796-54764096、54764353-54764473、54764752-54764872、54764982-54765342、54765555-54765795、54766768-54766888、54766927-54767084、54767418-54767538、54769582-54769942、54770595-54770715、54770782-54770955、54771021-54771262、54771320-54771502、54771622-54772462、54772602-54772822、54772914-54773662、54773940-54774060、54774142-54774537、54774819-54774939、54775165-54775458、54775539-54775779、54775899-54776038、54776642-54776762、54777749-54777869、54778612-54778662、54778705-54778944、54779213-54779333、54779425-54779894、54780237-54780754、54781199-54781563、54781755-54782055、54782065-54782415、54782922-54783023、54783315-54783614、54783637-54784322、54785807-54786167、54786327-54786561、54786567-54787134、54787254-54787374、54787558-54788094、54788168-54788692、54789174-54789400、54789414-54789635、54789691-54789921、54790014-54790327、54803384-54803984、54803985-54806470、54806590-54806710、54807032-54807426、54808144-54808265、54808505-54810160、54810423-54811143、54811383-54812343、54812463-54813781、54813786-54814518、54816022-54816143、54816352-54816467、54816468-54816534、54816814-54816934、54817313-54818753、54818873-54819233、54819353-54820257、54820299-54820419、54820433-54820913、54821110-54821470、54821565-54822233、54822593-54822713、54823816-54823936、54824873-54825353、54825628-54825779、54825867-54825987、54826427-54826548、54826597-54826717、54826827-54826982、54827336-54827456、54827590-54828070、54828093-54828297、54828299-54828493、54828540-54828681、54829053-54829173、54829189-54829309、54829361-54829465、54829652-54829772、54829811-54829980、54830099-54830779、54832707-54832798、54832958-54833097、54833279-54833399、54833510-54833630、54833741-54833861、54833971-54834091、54834177-54834417、54834546-54834666、54834729-54834897、54834950-54835070、54835079-54835114、54835137-54835617、54835840-54836006、54836049-54836401、54836457-54836697、54836937-54837057、54837114-54837417、54837576-54837893、54837950-54838069、54838090-54838617、54839097-54839333、54839410-54839911、54840035-54840155、54842773-54842918、54842954-54843200、54843207-54843327、54843708-54844038、54844268-54844389、54844438-54844633、54844932-54845152、54845410-54845967、54846013- 54846133、54846154-54846540、54847047-54847341、54847451-54847595、54847634-54848575、54850207-54850327、54850447-54850567、54851047-54851508、54851566-54851686、 54851806-54852526、54852828-54852948、54853327-54853447、54853586-54853706、54853747-54854047、54854066-54854186、54854407-54854887、54858924-54859284、54859447- 54859567, 54860025-54860385, 54860625-54860855, 54861074-54861194, 54861222-54861342, 54861704-54861960, 54862064-54862519, 54865548-54866508, and 54866522-54867216.

4. The hematopoietic stem cell transplantation matching and prognosis assessment kit according to claim 1, characterized in that: The probe information corresponding to the MICA gene and the MICB gene includes: located on chromosome 6, the exon regions are 31400710-31415315 and 31494880-31511124.

5. The hematopoietic stem cell transplantation matching and prognosis assessment kit according to claim 1, characterized in that: The probe information corresponding to the SUFU rs17114808 site includes: located on chromosome 10, and the exon region is 102631527-102631528.

6. The hematopoietic stem cell transplantation matching and prognosis assessment kit according to any one of claims 2 to 5, characterized in that: The probe is designed using the following method: S1: Obtain the exon region of the target gene according to the database; S2: Cut off 1 to 2 unit bp of an exon region as the first probe unit, cut off (n-1) unit to (n+1) unit bp of the exon region as the nth probe unit, and cut off in sequence, Unit = 60 bp, to obtain the probe of the exon region; S3: Evaluate the nucleotide sequence of the probe. The evaluation parameters include: Tm value, GC content, Hairpin free energy, sequence complexity and intragenomic homology; S4: Adjust the probes with scores below the threshold: the unit value of the probe sequence is 60±10bp when it is truncated; repeat steps S2 and S3 to optimize the probe sequence.

7. A method for using the hematopoietic stem cell transplantation matching and prognosis assessment kit according to claim 1, characterized in that: The following steps are involved: Step 1, DNA sample preparation: genomic DNA extraction using gDNA from target cells; Step 2, pre-library preparation; Step 3, hybridization capture; Step 4, library mixing and sequencing; Step 5, bioinformatics process analysis.

8. The method for using the hematopoietic stem cell transplantation matching and prognosis assessment kit according to claim 7, characterized in that: The target cells include bone marrow blood cells or peripheral blood leukocytes.

9. The method for using the hematopoietic stem cell transplantation matching and prognosis assessment kit according to claim 7, characterized in that: The prelibrary preparation includes: sequentially performing genomic DNA fragmentation, linker ligation and purification, and PCR amplification and purification.

10. The method for using the hematopoietic stem cell transplantation matching and prognosis assessment kit according to claim 7, characterized in that: The hybridization capture comprises: labeling the probe with biotin and hybridizing it with the prelibrary, and then using streptavidin magnetic beads to bind the probe to capture the target area.

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