A gene set and kit for evaluating the curative effect after allogeneic hematopoietic stem cell transplantation of hematopathy

Abstract

The application provides a gene set and kit for evaluating the curative effect after allogeneic hematopoietic stem cell transplantation of hematopathy, and belongs to the field of gene detection. The application develops a new post-transplantation curative effect evaluation panel detection design method. The panel comprises: 1. single nucleotide polymorphism (SNP) sites characteristic of individual genetic characteristics, which can be used for graft implantation evaluation; 2. high-frequency mutation sites of blood tumor hotspot genes, which can be used for MRD detection and disease state evaluation; and 3. HLA gene characteristic SNP sites on the short arm of human chromosome 6, which can be used for HLA gene typing, combined with the state of graft implantation, to analyze the occurrence of HLA-loss. The technical scheme of the application can solve the above-mentioned multiple contents of post-transplantation curative effect evaluation at one time, is more accurate and sensitive than the previous detection method, and can greatly reduce the detection cost and shorten the clinical report time.

Description

Technical Field

[0001] This invention belongs to the field of gene detection, and in particular relates to a gene set and kit for evaluating the efficacy of allogeneic hematopoietic stem cell transplantation in hematological diseases. Background Technology

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a treatment that involves intravenously infusing hematopoietic stem cells from a healthy donor into a patient to replace their abnormal or damaged hematopoietic system. This technique, by rebuilding the patient's immune and hematopoietic function, is used to treat hematologic disorders such as leukemia, lymphoma, and aplastic anemia. In the treatment of malignant hematologic diseases: ① Acute leukemia: For high-risk or relapsed acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), allo-HSCT can significantly improve long-term survival rates; ② Chronic myeloid leukemia (CML): Despite the widespread use of tyrosine kinase inhibitors (TKIs), allo-HSCT remains an effective option for patients who are TKI-resistant or intolerant; ③ Myelodysplastic syndromes (MDS): For high-risk MDS patients, allo-HSCT is the only potentially curative treatment. In the treatment of non-malignant hematological diseases, ① severe aplastic anemia: allo-HSCT is the first-line treatment for young patients with suitable donors; ② hereditary hematological diseases: such as thalassemia and sickle cell anemia, allo-HSCT can provide a chance of cure; ③ treatment of immunodeficiency diseases: primary immunodeficiency diseases: such as severe combined immunodeficiency, allo-HSCT can rebuild immune function.

[0003] Post-transplant efficacy assessment is crucial for ensuring treatment success and long-term patient survival. Currently, efficacy assessment primarily involves the following: 1. Graft implantation assessment: using capillary electrophoresis to analyze the donor cell ratio based on the donor and patient's gene sequence characteristics—short tandem repeats (STRs)—to assess graft implantation during the transplantation process; 2. Disease status assessment: based on the patient's pre-transplant disease proteins or gene markers, using methods such as flow cytometry and PCR to detect minimal residual disease (MRD) and assess whether disease relapse has occurred; 3. Post-transplant immune escape analysis: in the event of disease relapse, analyzing whether the human leukocyte antigen (HLA) gene is lost to assess the relationship between relapse and HLA-loss, providing a basis for salvage therapies such as donor lymphocyte infusion (DLI) and secondary transplantation. Therefore, accurate and comprehensive post-transplant efficacy assessment, addressing all of the above aspects, is of significant clinical value in ensuring patients achieve optimal treatment outcomes and long-term survival after allogeneic hematopoietic stem cell transplantation.

[0004] However, current assessment methods for post-transplant efficacy evaluation have the following drawbacks: ① Multiple assessment components require multiple testing methods, making data integration and analysis difficult. For example, graft implantation assessment uses capillary electrophoresis, MRD testing uses flow cytometry or PCR, and HLA-loss testing uses PCR or high-throughput sequencing. Different testing technologies result in different analytical standards between data, making it difficult to comprehensively judge post-transplant efficacy and disease status. ② Graft implantation assessment methods have poor sensitivity. For example, analyzing graft implantation status using donor and patient STR data, although widely used clinically, has poor sensitivity (5%–10%) due to the use of multiplex PCR to amplify STR sequences combined with capillary electrophoresis. It also cannot detect patients with missing pre-transplant information and presents challenges in multi-faceted graft implantation assessment. ③ Multiple tests are costly in total. Analyzing different aspects of graft implantation, MRD, and HLA-loss requires multiple tests, resulting in high total costs. Therefore, how to achieve a single test that can solve the analysis problems of different contents after transplantation, while ensuring the sensitivity and accuracy of the test, reducing the test cost, and enabling data integration and analysis, is an urgent problem that needs to be solved in the evaluation of the efficacy of transplantation. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a gene set and kit for evaluating the efficacy of allogeneic hematopoietic stem cell transplantation in hematological diseases.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a gene set for evaluating the efficacy of allogeneic hematopoietic stem cell transplantation in hematological diseases, the gene set including SNP sites of Panel A, genes of Panel B, and HLA gene SNP sites of Panel C.

[0008] The SNP sites of Panel A are shown in Table 1:

[0009] Table 1. SNP sites of Panel A

[0010]

[0011]

[0012]

[0013] The genes of Panel B are shown in Table 2:

[0014] Table 2. Genes of Panel B

[0015] EP300 KRAS SETBP1 GATA2 BCORL1 MYD88 KMT2D BCL2 KLHL6 PHF6 CSF3R ERBB3 EPOR IMPDH2 STAT3 MPL SH2B3 CALR KIT BRAF JAK1 PTPN11 JAK3 NFKB1 EZH2 GFI1 FLT3 CEBPA TET2 ETV6 NRAS DIS3 IKZF1 FBXW7 JAK2 NOTCH2 ABCC4 XRCC1 FAT1 CDKN2A ABCC2 PML DNMT3A ABCG2 PAX5 EGR2 IDH2 TTN TERT ABL1 PTEN CREBBP SF3B1 IL7R NOTCH1 CBL CYLD IDH1 APC PIGA STAT5A NQO1 ATIC CSF1R ZRSR2 WT1 PRPF8 UGT1A8 NPM1 BCOR GSTP1 TP53 ASXL1 DDX41 KDM6A BIRC3 NF1 RUNX1 STAG2 ATRX KMT2A STAT5B U2AF1 TNFAIP3

[0016] The HLA gene SNP sites in Panel C are shown in Table 3:

[0017] Table 3 HLA gene SNP sites in Panel C

[0018]

[0019]

[0020] The present invention provides a primer set for amplifying the gene set, wherein the primer set comprises primers for amplifying the chromosomal region of Panel A, primers for detecting genes of Panel B, and primers for detecting HLA gene SNP sites of Panel C;

[0021] The primer sequences for amplifying the chromosomal region of Panel A are shown in SEQ ID NO.1-SEQ ID NO.44;

[0022] The primer sequences for detecting the gene in Panel B are shown in SEQ ID NO.45-SEQ ID NO.588;

[0023] The primer sequences for detecting HLA gene SNP sites in Panel C are shown in SEQ ID NO.589-SEQ ID NO.600.

[0024] This invention provides a kit for evaluating the efficacy of allogeneic hematopoietic stem cell transplantation in hematological diseases, the kit comprising the aforementioned primer set.

[0025] Preferably, the method of use includes the following steps:

[0026] 1) Extract DNA from the sample to be tested;

[0027] 2) Perform PCR amplification using the primer set described above to obtain the amplification product;

[0028] 3) Perform high-throughput sequencing on the amplified products, analyze them after quality control, and obtain the efficacy evaluation results;

[0029] The analysis involved performing chimerism analysis based on the sequencing results of the Panel A chromosome region.

[0030] Gene mutation analysis was performed based on the MRD status of Panel B gene mutations;

[0031] HLA-loss analysis was performed based on the sequencing results of HLA gene SNP sites in Panel C.

[0032] Preferably, the sample to be tested in step 1) includes a bone marrow or peripheral blood sample.

[0033] Preferably, the quality control in step 3) includes aligning the amplification product with the human reference genome hg19 and removing multiple alignments, PCR duplicates, and unaligned sequences.

[0034] Preferably, the chimera analysis includes SNV and Indel extraction, differential site screening, and mutation frequency correction.

[0035] Preferably, the gene mutation analysis includes local re-alignment, base mass fraction re-correction, variant detection, and detection of insertions, deletions, inversions, tandem duplications, and chromosomal translocations.

[0036] Preferably, the HLA-loss analysis includes SNV extraction, Indel extraction, differential site screening, and mutation frequency correction.

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

[0038] This invention provides a gene set and kit for evaluating the efficacy of allogeneic hematopoietic stem cell transplantation in hematological diseases. Based on high-throughput targeted amplification sequencing technology, a novel panel design method for post-transplant efficacy evaluation has been developed. This panel includes: 1. Individually characteristic single nucleotide polymorphism (SNP) sites for graft implantation assessment; 2. High-frequency mutation sites of hotspot genes in hematological malignancies for MRD detection and disease status assessment; 3. Characteristic HLA gene SNP sites on the short arm of human chromosome 6 for HLA genotyping and, combined with graft implantation status, analysis of HLA-loss. This invention innovatively utilizes individually characteristic SNP sites and disease-characteristic hotspot mutations to address multiple aspects of post-transplant efficacy evaluation with a single detection technology. It is more accurate and sensitive than previous methods, and significantly reduces testing costs and shortens clinical reporting time. This method is highly portable and applicable to current sequencing instruments or newly developed testing platforms in the domestic market. Attached Figure Description

[0039] Figure 1 Comparative analysis of SNPseq and STR methods for mixed cell samples with different proportions (the next-generation sequencing is the evaluation method of this invention);

[0040] Figure 2 The pre-transplant gene mutation status of 98 transplant patients;

[0041] Figure 3 Correlation analysis of the proportion of HLA-loss detection specific to 100 transplant patients and the graft implantation rate;

[0042] Figure 4This is a technical flowchart. Detailed Implementation

[0043] This invention provides a gene set for evaluating the efficacy of allogeneic hematopoietic stem cell transplantation in hematological diseases, the gene set including SNP sites of Panel A, genes of Panel B, and HLA gene SNP sites of Panel C.

[0044] The SNP sites of Panel A are shown in Table 1;

[0045] The genes of Panel B are shown in Table 2;

[0046] The HLA gene SNP sites in Panel C are shown in Table 3.

[0047] The present invention provides a primer set for amplifying the gene set, wherein the primer set comprises primers for amplifying the chromosomal region of Panel A, primers for detecting genes of Panel B, and primers for detecting HLA gene SNP sites of Panel C;

[0048] The primer sequences for amplifying the chromosomal region of Panel A are shown in SEQ ID NO.1-SEQ ID NO.44;

[0049] The primer sequences for detecting the gene in Panel B are shown in SEQ ID NO.45-SEQ ID NO.588;

[0050] The primer sequences for detecting HLA gene SNP sites in Panel C are shown in SEQ ID NO. 589-SEQ ID NO. 600. In this invention, the specific sequences of SEQ ID NO. 1-SEQ ID NO. 600 are detailed in Table 4.

[0051] Table 4 shows the specific sequences of SEQ ID NO.1-SEQ ID NO.600.

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] This invention provides a kit for evaluating the efficacy of allogeneic hematopoietic stem cell transplantation in hematological diseases, the kit comprising the aforementioned primer set.

[0066] In this invention, the method of use includes the following steps:

[0067] 1) Extract DNA from the sample to be tested;

[0068] 2) Perform PCR amplification using the primer set described above to obtain the amplification product;

[0069] 3) Perform high-throughput sequencing on the amplified products, analyze them after quality control, and obtain the efficacy evaluation results;

[0070] The analysis involved performing chimerism analysis based on the sequencing results of the Panel A chromosome region.

[0071] Gene mutation analysis was performed based on the MRD status of Panel B gene mutations;

[0072] HLA-loss analysis was performed based on the sequencing results of HLA gene SNP sites in Panel C.

[0073] In this invention, the sample to be tested in step 1) preferably includes a bone marrow or peripheral blood sample.

[0074] In this invention, the PCR amplification is as follows:

[0075] Amplification system: 5X IonAmpliSeq TM HiFi Mix 2μL, Water 3.3μL, DNA 1.7μL, Primers 3μL

[0076] PCR amplification reaction system: 99℃ for 2 min → (99℃ for 15 s → 60℃ for 4 min) × 18 cycles → store at 10℃

[0077] The FUPA system consists of: amplified PCR product + 1 μL Fupa reagent.

[0078] FUPAPCR reaction system: 50℃ for 10 min → 55℃ for 10 min → 60℃ for 20 min → 10℃ Hold (≤1 h)

[0079] Connector connection system: Switch Solution 2μL, Water 0.5μL, DAN Ligase 1μL, Ion P1 Adapter 0.25μL, Bcacode X 0.25μL

[0080] Adapter ligation PCR reaction system: 22℃ 30min → 72℃ 10min → 10℃ hold.

[0081] In this invention, the quality control in step 3) includes aligning the amplification product with the human reference genome hg19 and removing multiple alignments, PCR duplicates, and unaligned sequences.

[0082] In this invention, the chimera analysis includes SNV extraction, Indel extraction, differential site screening, and mutation frequency correction.

[0083] In this invention, the gene mutation analysis includes local re-alignment, base mass fraction re-correction, variant detection, and detection of insertions, deletions, inversions, tandem duplications, and chromosomal translocations.

[0084] In this invention, the HLA-loss analysis includes SNV extraction, Indel extraction, differential site screening, and mutation frequency correction.

[0085] Significance of gene mutation analysis: Tumor cells contain characteristic gene mutations compared to normal cells, and gene mutation detection can serve as a marker to detect the presence of tumor cells. If gene mutations reappear after treatment, it indicates tumor recurrence.

[0086] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0087] A. The nucleic acid extraction method is as follows:

[0088] DNA / RNA were extracted using the Lab-Aid 824s / 808 nucleic acid extractor.

[0089] B. Library preparation steps

[0090] DNA purification (XP magnetic beads incubated at room temperature for 30 min, 85% ethanol prepared fresh before use).

[0091] After DNA extraction, use nonadrope to measure the concentration. If the sample concentration exceeds 100 ng / μL, dilute the DNA to about 100 ng / μL.

[0092] Aliquot 45 μL (1.5 × DNA volume) of magnetic beads into an eight-bead array, add 30 μL of DNA, vortex to mix, incubate at room temperature for 5 min, centrifuge, magnetically attach for 3 min, and discard the supernatant.

[0093] Add 200 μL of 85% alcohol and move the eight-row magnetic beads back and forth about 10 times to clean them. Discard the alcohol. Repeat once.

[0094] Use a small gun to remove the remaining liquid, cap and centrifuge, then remove the liquid again. If there are any droplets in the tube, use the small gun to separate them and let it dry.

[0095] Add 30 μL of water to wash the magnetic beads (if the initial DNA concentration is low, you can add less water), vortex to mix, let stand at room temperature for 5 min, centrifuge, and magnetic rack adsorption for 3 min.

[0096] Aspirate 28 μL of supernatant into a new eight-tube strip to obtain purified DNA, and measure its concentration.

[0097] PCR amplification:

[0098] After the DNA extraction is complete, use Nanodrop to measure the concentration. If the concentration is below 120 ng / μL, it can be used directly for subsequent applications. If the concentration is above 120 ng / μL, it needs to be diluted to below 120 ng / μL (the highest concentration that Qubit can measure is 120 ng / μL).

[0099] Re-measure the DNA concentration using Qubit, and then dilute the DNA to 10 ng / μL according to the concentration measured by Qubit (i.e., according to the formula: (concentration / 10)). -1 Calculate the required volume of water by multiplying the volume by 2, and then add 2 μL of the sample whose concentration has been measured using Qubit to the corresponding tube.

[0100] The amplification system was: 5X IonAmpliSeq TM HiFi Mix 2μL, Water 3.3μL, DNA 1.7μL, Primers (1μmol / L) 3μL.

[0101] The PCR reaction conditions were: 99℃ for 2 min → (99℃ for 15 s → 60℃ for 4 min) × 18 cycles → stored at 10℃.

[0102] Add 1 μL FUPA reagent to the PCR product (replace the cap of the eight-tube after adding FUPA), and run the PCR machine according to the following procedure: 50℃ for 10 min → 55℃ for 10 min → 60℃ for 20 min → 10℃ Hold (≤1 h).

[0103] Connector connection:

[0104] The required number of adapter ligation reagents should be prepared according to the number of specimens used in the library (the barcode numbers of specimens from the same batch cannot be repeated). The ligation system is as follows: Switch Solution 2μL, Water 0.5μL, DAN Ligase 1μL, Ion P1 Adapter 0.25μL, Bcacode X 0.25μL.

[0105] Transfer all PCR amplification products to the Switch system prepared above, run PCR on the machine, and follow the PCR program as follows: 22℃ 30min → 72℃ 10min → 10℃ hold.

[0106] Library expansion:

[0107] Add 22.5 μL (1.5x of the previous amplification system) of XP magnetic beads to each well, vortex to mix, let stand at room temperature for 5 min, centrifuge, magnetically attach for 3 min, and discard the supernatant.

[0108] Add 200 μL of 70% alcohol and move the eight-row magnetic beads back and forth about 10 times to clean them. Discard the alcohol. Repeat this step once.

[0109] Use a small pipette to aspirate the liquid, cap and centrifuge, then aspirate again. If there are droplets in the PCR tube, use a small pipette to separate them and let it air dry.

[0110] Add 25 μL of LibraryAmp Mix and 1 μL of LibraryAmp Primers to each well, shake to mix, let stand at room temperature for 5 min, centrifuge, and magnetic rack adsorption for 3 min.

[0111] Aspirate 25 μL of supernatant into a new 8-tube PCR instrument and run the PCR program as follows: 98℃ for 2 min → (98℃ for 15 s → 64℃ for 1 min) × 5 cycles → store at 10℃.

[0112] Segment sorting:

[0113] After PCR, add 12.5 μL (0.5x of the previous amplification system) of XP magnetic beads to each well, vortex to mix, let stand at room temperature for 5 min, centrifuge, and magnetic rack adsorption for 3 min.

[0114] Pre-allocate 30 μL of XP magnetic beads into a new tube, aspirate the supernatant from step 1 into 37.5 μL (all supernatant) of magnetic beads, vortex to mix, let stand at room temperature for 5 min, centrifuge, magnetically attach for 3 min, and discard the supernatant.

[0115] Add 200 μL of 70% alcohol and move the eight-row magnetic beads back and forth about 10 times to clean them. Discard the alcohol. Repeat this step once.

[0116] Then, use a pipette to aspirate the liquid, cap and centrifuge, then aspirate again. If there are droplets in the PCR tube, use a pipette to separate them and let it air dry.

[0117] Add 30 μL of water, shake to mix, let stand at room temperature for 5 min, centrifuge, and magnetic rack adsorption for 3 min.

[0118] Aspirate 28 μL of supernatant into a new tube to obtain a library. Measure the concentration; this library can then be used for subsequent library mixing.

[0119] The library can be used directly for sequencing or stored at -20°C.

[0120] C. Sequencing steps

[0121] Document preparation

[0122] Dilute the library sample to a final loading amount of 110 pM.

[0123] Sequencing was performed using a sequencer; the sequencing method was described in the sequencer's instruction manual. The instrument was an Ion Chemie PCR machine. TM .

[0124] Example 1

[0125] Panel Design: Primarily designed for screening relevant SNP sites and gene mutations in the efficacy evaluation of allogeneic hematopoietic stem cell transplantation. Based on the evaluation content, it is divided into Panel A: SNP sites with individual genetic characteristics (for graft implantation evaluation), Panel B: Hotspot gene mutations in hematologic malignancies (for MRD detection), and Panel C: HLA gene-related SNP sites (for HLA-loss identification).

[0126] Gene screening: Genes relevant to post-transplant efficacy assessment were selected from publicly available databases and literature resources, including: SNP loci in the dbSNP database, high-frequency mutation genes in hematologic malignancies, hematologic malignancy treatment-related genes from applied standards, treatment guidelines, and general databases, genes related to molecular subtyping, treatment, prognosis, and induction of hematologic malignancies, and HLA-encoding genes. The assessment was divided into Panel A SNP loci (for graft implantation assessment), Panel B gene mutations (for MRD detection), and Panel C HLA gene SNP loci (for HLA-loss identification). See Tables 1-3 for details.

[0127] The above SNP sites and gene mutations were identified using the HGNC database (HUGO Gene Nomenclature Committee) to determine the standard gene name, gene transcript number, and the chromosomal location of the relevant gene exons, which were then used for primer design and synthesis.

[0128] Primer evaluation: Assessing the amplification ability of relevant primers based on specificity and coverage.

[0129] PanelA selects 22 fragments (average length approximately 200 bp) related to SNP sites with individual genetic characteristics from the dbSNP database as detection targets for primer design. PanelA contains a total of 22 primer pairs, covering 100% of the target region. The primers are quality controlled by electrospray ionization mass spectrometry (ESI-MS) to ensure the accuracy of their synthesis.

[0130] Panel B selected 510 important mutation sites of 84 target genes related to hematological malignancies as detection targets for primer design. Panel B contains a total of 272 primer pairs, covering 100% of the target region. The primers were quality controlled by electrospray mass spectrometry (ESI-MS) to ensure the accuracy of their synthesis.

[0131] Panel C selects HLA sequences as the detection target and designs primers. Panel C includes 6 pairs of primers, covering 99.98% of the target region. The primers are quality controlled by electrospray mass spectrometry (ESI-MS) to ensure the accuracy of their synthesis.

[0132] Primer synthesis: A total of 300 primer pairs were synthesized by combining the amplification primers of Panel A (SEQ ID NO.1-SEQ ID NO.44), Panel B (SEQ ID NO.45-SEQ ID NO.588), and Panel C (SEQ ID NO.589-SEQ ID NO.600).

[0133] Example 2

[0134] The performance of the synthesized panel was verified in accordance with relevant quality control requirements.

[0135] (2.1) Preparation of performance verification samples

[0136] (2.1.1) Panel A performance verification sample preparation

[0137] (2.1.1.1) Mixed chimeric samples: There are two types. One type is prepared from bone marrow samples from patients who show incomplete chimerism according to STR testing. DNA is extracted and then prepared from these samples. The resulting DNA is subjected to concentration and purity quality control using NanoDrop. The nucleic acid concentration is required to be ≥10 ng / μL, and the purity is required to be between 1.8 and 2.0. This is called a clinical mixed chimeric sample. The other type is a mixed sample of blood cell proportions from healthy individuals A and B (where the proportions of A blood cells in the mixed sample are: 0.01%, 0.1%, 0.5%, 1%, 6.25%, 12.5%, 25%, 50%). This is called an artificial mixed chimeric sample. The chimeric samples used are first processed using Applied... TrueScience TM The STR method of the Aneuploidy STR Kits was validated. The amplification system, amplification parameters, and analytical methods were performed according to the instructions of the commercial kit to ensure the accuracy of the mixed samples. Nucleic acid was extracted from all samples, and the concentration and purity of the extracted nucleic acid were tested. After meeting the conditions (nucleic acid concentration ≥10 ng / ul, OD260 / 280 between 1.8 and 2.0), the samples were stored.

[0138] (2.1.1.1) Complete chimerism: This refers to samples from patients who have received allogeneic hematopoietic stem cell transplantation but whose STR test results confirm complete chimerism (or below the detection limit).

[0139] (2.1.1.1) Each prepared sample should be aliquoted into EP tubes and stored at -70°C or below. Ensure sufficient sample volume is prepared.

[0140] (2.1.2) Panel B Performance Verification Sample Preparation

[0141] (2.1.2.1) Positive Sample: One type is prepared from bone marrow samples collected from patients who were found to have three gene mutation sites, including CYP2C19rs4244285, ABCB1rs1045642, and CYP3A5rs776746, as determined by first-generation sequencing. DNA was extracted and the obtained DNA was used for concentration and purity quality control using NanoDrop. The nucleic acid concentration was required to be ≥10 ng / μL, and the purity was required to be between 1.8 and 2.0 OD260 / 280. This is hereinafter referred to as a positive sample. In addition, the positive samples were verified using digital PCR to ensure their accuracy.

[0142] Positive sample: Nucleic acid is extracted from previously confirmed positive clinical samples, and the concentration and purity of the obtained nucleic acid are tested. After meeting the quality control requirements (≥10ng), the positive samples are proportionally diluted using nucleic acid confirmed as negative (concentrations of 1%, 5%, 10%, 25%, and 50% for positive samples, with the dilutions using nucleic acid from negative samples). Positive quality control samples provided by qualified parties may also be used.

[0143] (2.1.2.2) Negative samples: Specimens that are confirmed to be negative by digital PCR (below the detection limit or zero concentration).

[0144] (2.1.3) Panel C performance verification sample preparation

[0145] (2.1.3.1) HLA-loss positive samples: These are HLA-loss positive samples detected by HLA-KMR. The detected HLA-loss percentages were 3%, 13%, 21%, 31%, and 38%, hereinafter referred to as positive samples. All positive samples were verified by real-time PCR (HLA-KMR, GenDx, Netherlands). The amplification system, amplification parameters, and analytical methods were performed according to the instructions of the commercially available kit to ensure the accuracy of the positive samples.

[0146] (2.1.3.2) HLA-loss pooled sample: This is prepared by mixing HLA-loss samples with corresponding donor nucleic acids (dilution ratios of 1%, 5%, 10%, 20%, and 50%), and is called an HLA-loss pooled sample. Nucleic acid must be extracted from all samples, and the concentration and purity of the extracted nucleic acids must be tested. Once the conditions are met, the samples are stored.

[0147] (2.1.3.3) Negative samples: Specimens that are confirmed to be negative by quantitative real-time PCR.

[0148] (2.2) Quality control of amplification primer efficiency

[0149] Primers for Panel A, Panel B, and Panel C were combined and synthesized. The performance verification samples in (2.1) were sequenced. After sequencing, the performance verification samples in (2.1) were quality controlled. The primer amplification specificity reached about 70%, and the total coverage of the amplification products was about 99%.

[0150] Table 4100 Sample Detection Results

[0151]

[0152] (2.3) Precision verification

[0153] (2.3.1) Panel A Precision Validation Scheme

[0154] (2.3.1.1) Operation Procedure

[0155] a) Sample preparation:

[0156] According to the precision testing requirements, precision testing was performed in the complete chimerism group and the mixed chimerism group respectively. The complete chimerism group selected complete chimeric samples, and the mixed chimerism group selected artificial mixed chimeric samples. The ratio of mixed cells between healthy person A and healthy person B was 1:1 (i.e., the chimerism rate was 50%).

[0157] b) Sequencing detection:

[0158] The complete chimeric group and the mixed chimeric group were divided into 5 batches, with 20 samples in each batch. The individual genetic characteristic single nucleotide polymorphism (SNP) sites were detected by NGS, and the corresponding intra-batch and inter-batch SD values ​​and coefficient of variation (CV) values ​​were calculated.

[0159] (2.3.1.2) Result Judgment

[0160] The complete chimerism group had an intra-assay mean of 100% and an intra-assay coefficient of variation of 0.74%, an inter-assay mean of 99.89% and an inter-assay coefficient of variation of 1.18%, and the mixed chimerism group had an intra-assay mean of 50.56% and an intra-assay coefficient of variation of 1.72%, an inter-assay mean of 48.05% and an inter-assay coefficient of variation of 6.56%.

[0161] (2.3.2) PanelB Precision Validation Scheme

[0162] (2.3.2.1) Operation Procedure

[0163] a) Sample preparation:

[0164] Panel B: According to the precision detection requirements, precision detection was performed in the high mutation rate group and the critical mutation rate group respectively. The high mutation rate group selected SNP sites with a mutation frequency of 50% (including mutations in the three genes CYP2C19, ABCB1 and CYP3A5), and the low mutation rate group selected mixed cells with a mutation frequency of 1% (a mixture of CYP2C19 positive cells and cells without this mutation at a ratio of 1:99).

[0165] b) Sequencing detection:

[0166] Panel B assay: 100 samples from the high mutation rate group were selected and divided into 5 batches. 20 samples from each batch were randomly selected for Panel B sequencing and analysis. The intra-batch and inter-batch SD values ​​and coefficient of variation (CV) values ​​of the high mutation rate group were calculated. 50 samples from the low mutation rate group, with a CYP2C19 mutation frequency of 1%, were selected and divided into 5 batches. 10 samples from each batch were tested. The intra-batch and inter-batch SD values ​​and coefficient of variation (CV) values ​​of the low mutation rate group were calculated.

[0167] (2.3.2.2) Result Judgment

[0168] The high mutation rate group had an intra-batch mean of 48.05% and an intra-batch coefficient of variation of 6.56%, while the inter-batch mean was 48.01% and the intra-batch coefficient of variation was 7.44%. The low mutation rate group had an intra-batch mean of 1.36% and an intra-batch coefficient of variation of 13.42%, while the inter-batch mean was 1.13% and the intra-batch coefficient of variation was 23.23%.

[0169] (2.3.3) Panel C Precision Verification Scheme

[0170] (2.3.3.1) Operation Procedure

[0171] a) Sample preparation:

[0172] According to the precision testing requirements, precision testing was performed on the HLA-loss sample group, which consisted of 5 samples (loss percentages of 3%, 13%, 21%, 31%, and 38%, respectively).

[0173] b) Sequencing detection:

[0174] Five samples from the HLA-loss group were subjected to Panel C sequencing analysis. Each sample was repeated five times within a batch, and five batches were repeated. The intra-batch and inter-batch SD values ​​and coefficient of variation (CV) values ​​were calculated.

[0175] (2.3.3.2) Result Judgment

[0176] The intra-assay mean HLA-loss positive samples were 3.12%, 13.81%, 20.05%, 32.17%, and 40.21%, with intra-assay coefficients of variation of 5.21%, 8.54%, 3.48%, 5.25%, and 6.81%. The inter-assay mean HLA-loss positive samples were 3.21%, 13.92%, 20.21%, 32.25%, and 37.81%, with inter-assay coefficients of variation of 8.37%, 9.21%, 2.79%, 6.31%, and 2.17%.

[0177] (2.4) Accuracy Verification

[0178] (2.4.1) Methods and requirements for evaluating the accuracy of qualitative determinations

[0179] The study primarily employed comparative methodologies. The STR method is currently the most widely used method for detecting graft implantation status. Clinically, the short tandem repeat (STR) method, based on PCR and capillary electrophoresis, is widely used for graft implantation detection. However, due to variations in the luminescence efficiency of different fluorescent labels leading to deviations in chimerism detection, shadow peaks during capillary electrophoresis causing misinterpretation of results, and low sensitivity (5%–10%), coupled with a limited number of detection sites resulting in poor result stability, the STR method is increasingly unable to meet the needs of clinical chimerism detection. Panel A selected the STR method for accuracy verification. Digital PCR is currently the most sensitive method for detecting mutation sites; Panel B selected this method to verify its accuracy against the current method. Panel C selected quantitative real-time PCR (HLA-KMR, GenDx, Netherlands) for accuracy verification.

[0180] Operators must be fully familiar with the equipment's operation, maintenance procedures, and evaluation schemes. During the comparison process, both the method to be evaluated and the reference method must have appropriate quality control, and sufficient data must be available to ensure that the results are representative.

[0181] (2.4.2) Panel A Accuracy Verification

[0182] (2.4.2.1) Test Scheme

[0183] a) Reference method: Multiplex PCR amplification of short tandem repeat (STR) sequences.

[0184] b) Prepare: (1) 50 fully chimeric samples for STR testing; (2) 50 artificially mixed chimeric samples.

[0185] c) The selected samples were simultaneously subjected to SNP genotyping and STR methods for chimerism detection.

[0186] d) For each experiment, all raw test data should be recorded and verified immediately to identify sources of systemic and human error early. If any results are found to be caused by explainable errors, they should be recorded and not used for data analysis. If the cause of the error cannot be determined, the raw results should be retained.

[0187] (2.4.2.2) Result Analysis and Judgment:

[0188] a) The results are presented in a four-cell table:

[0189] Table 5. STR test results

[0190]

[0191] b) Result interpretation: Comparison of SNP and STR results, the accuracy of SNP typing of chimeras is 100%, and the specificity of incomplete chimeras is improved by 26%.

[0192] (2.4.3) Panel B Accuracy Verification

[0193] (2.4.3.1) Test Scheme

[0194] a) Reference method: Digital PCR.

[0195] b) Sample preparation: 50 gene mutation positive samples; 50 gene mutation negative samples.

[0196] c) The selected samples were tested using both NGS and digital PCR methods.

[0197] d) For each experiment, all raw test data should be recorded and verified immediately to identify sources of systemic and human error early. If any results are found to be caused by explainable errors, they should be recorded and not used for data analysis. If the cause of the error cannot be determined, the raw results should be retained.

[0198] 2.4.3.2 Result Analysis and Judgment:

[0199] a) The results are presented in a four-cell table:

[0200] Table 6. Digital PCR Sequencing Results

[0201]

[0202] b) Result interpretation: After comparison with the results of digital PCR, the gene mutation detection sensitivity of the present invention is 100% and the specificity is 100%.

[0203] (2.4.4) Panel C Accuracy Verification

[0204] (2.4.4.1) Test Scheme

[0205] a) Reference method: Real-time PCR.

[0206] b) Sample preparation: (1) 50 HLA-loss positive samples (5 HLA-loss positive samples + 45 HLA-loss mixed samples); (2) 50 negative samples.

[0207] c) The selected samples were tested using two methods simultaneously: the evaluation method of this invention and quantitative real-time PCR. Quantitative real-time PCR was performed using a commercially available kit (HLA-KMR, GenDx, Netherlands); please refer to the kit instructions for specific instructions.

[0208] d) For each experiment, all raw test data should be recorded and verified immediately to identify sources of systemic and human error early. If any results are found to be caused by explainable errors, they should be recorded and not used for data analysis. If the cause of the error cannot be determined, the raw results should be retained.

[0209] (2.4.4.2) Result Analysis and Judgment:

[0210] a) The results show:

[0211] Table 7 Results of Real-Time PCR Detection

[0212]

[0213] b) Result interpretation: After comparison with the results of quantitative real-time PCR, the sensitivity and specificity of next-generation sequencing (NGS) for detecting gene mutations were 100%.

[0214] (4.5) Analytical sensitivity verification

[0215] (4.5.1) The sensitivity of the analysis is generally verified by a series of dilutions of samples known to contain known mutations. That is, under the same experimental conditions and with the same batch of reagents, the high-dilution samples are repeatedly measured, and the results are statistically analyzed.

[0216] (4.5.2) Experimental Scheme

[0217] Panel A: The artificially mixed chimeric samples were diluted proportionally to chimerism rates of 0.01%, 0.1%, 0.5%, 1%, 6.25%, 12.5%, 25%, and 50%. After dilution, each concentration of the sample was tested 10 times in accordance with the standard sample testing procedure, and analyzed according to the standard analytical steps.

[0218] Panel B: Dilute known positive samples with known negative samples to mutation frequencies of 0.5%, 1%, and 5%. After dilution, each concentration of the sample is tested 10 times according to the routine sample testing procedure and analyzed according to standard analytical steps.

[0219] Panel C: HLA-loss mixed samples were diluted to 0.5%, 1%, 5%, and 10% according to the standard procedure. Each concentration of the diluted sample was tested 10 times repeatedly according to the standard analytical procedure.

[0220] (4.5.3) Result Interpretation:

[0221] The detection limit (LOD) for Panel A is 0.01%, the detection sensitivity (LOD) for Panel B is 1%, and the detection sensitivity (LOD) for Panel C is 1%.

[0222] The results showed that PanleA improved the ability to detect graft implantation rates, ① increasing sensitivity by approximately 100 times; ② enabling graft implantation analysis even when pre-transplantation information is lacking; and ③ allowing analysis of implantation rates for ≥2 types of grafts.

[0223] PanleA aims to differentiate graft implantation status through linkage analysis of multiple SNP loci. This project utilizes NGS technology to establish a novel SNP-seq method for graft analysis. This method achieves a sensitivity of 0.01%, approximately 100 times higher than the STR method (STR sensitivity is 1%–5%), and its accuracy is also superior to the STR method (R...). 2 SNPseq = 0.9998 > R 2 (STR = 0.9968), and can solve clinical challenges such as the inability to detect grafts due to lack of pre-transplant information, and the analysis of the implantation ratio of ≥2 grafts based on SNP information of multiple grafts. For a detailed comparison of SNPseq and STR methods for mixed cell samples with different proportions, please refer to [link to relevant documentation]. Figure 1 .

[0224] Panle B can supplement the detection capabilities of gene mutations. Approximately 20% of patients still have detectable gene mutations before transplantation. Based on changes in gene mutations before and after transplantation, it can ① predict disease relapse and prognostic risk after transplantation; ② identify the source of disease relapse, such as: relapse of the original disease (the new gene mutation is consistent with the patient's original gene mutation), clonal evolution of the disease (the new gene mutation differs from the patient's original gene mutation), or relapse from the donor (the new gene mutation is consistent with the donor's gene mutation). For details on the pre-transplantation gene mutation status of 98 transplant patients, see [link to relevant documentation]. Figure 2 .

[0225] Panle C can supplement HLA-loss analysis capabilities, providing a basis for salvage therapies such as donor lymphocyte infusion (DLI) and secondary transplantation. Through linkage analysis of multiple SNP sites in six HLA genes, it aims to identify the deletion status of transplant patient-specific HLA genotypes. This project utilizes NGS technology to establish a new method for SNPseq analysis of HLA-loss. Through a simulated validation experiment with a mixed sample of 100 cases with a graft implantation rate >1.0%, Panle C achieved 100% accuracy in detecting HLA-loss, with a sensitivity of 1%. The correlation between the HLA-loss detection rate and the graft implantation rate was [value missing]. 2 =0.9948. For a detailed correlation analysis of the proportion of transplant-specific HLA gene HLA-loss detection and the graft implantation rate in 100 transplant patients, please refer to [link to relevant documentation]. Figure 3 .

[0226] For the complete design and technical flowchart of this invention, please refer to [link / reference]. Figure 4 .

[0227] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A gene set for efficacy evaluation after allogeneic hematopoietic stem cell transplantation for hematological diseases, characterized in that, The gene set comprises SNP sites of Panel A, genes of Panel B and HLA gene SNP sites of Panel C; The SNP sites of Panel A are shown in Table 1: Table 1 SNP sites of Panel A The genes of Panel B are shown in Table 2: Table 2 Genes of Panel B The HLA gene SNP sites of Panel C are shown in Table 3: Table 3 HLA gene SNP sites of Panel C 。 2. A primer set for amplifying the gene set of claim 1, characterized in that, The primer set is a primer for amplifying the chromosome region of Panel A, a primer for detecting the genes of Panel B and a primer for detecting the HLA gene SNP sites of Panel C; The primer sequence for amplifying the chromosome region of Panel A is shown in SEQ ID NO. 1-SEQ ID NO. 44; The primer sequence for detecting the genes of Panel B is shown in SEQ ID NO. 45-SEQ ID NO. 588; The primer sequence for detecting the HLA gene SNP sites of Panel C is shown in SEQ ID NO. 589-SEQ ID NO.

600.

3. A kit for efficacy evaluation after allogeneic hematopoietic stem cell transplantation for hematological diseases, characterized in that, The kit comprises the primer set of claim 2.

4. The kit of claim 3, wherein The use method comprises the following steps: 1) extracting DNA of the sample to be tested; 2) performing PCR amplification using the primer set of claim 2 to obtain an amplification product; 3) performing high-throughput sequencing on the amplification product, analyzing after quality control to obtain a therapeutic effect evaluation result; The analysis is: according to the sequencing result of the chromosome region of Panel A, performing chimera analysis; According to the MRD state of the mutation of the genes of Panel B, performing gene mutation analysis; According to the sequencing result of the HLA gene SNP sites of Panel C, performing HLA-loss analysis.

5. The kit of claim 4, wherein The sample to be tested of step 1) comprises a bone marrow or peripheral blood sample.

6. The kit of claim 4, wherein The quality control of step 3) comprises aligning the amplification product with the human reference genome hg19, removing multiple alignments, PCR repeats and unaligned sequences.

7. The kit of claim 4, wherein The chimera analysis comprises SNV, Indel extraction, differential site screening, mutation frequency correction.

8. The kit of claim 4, wherein The gene mutation analysis comprises local realignment, base quality score re-calling, variant detection, insertion, deletion, inversion, tandem repeat and chromosome translocation detection.

9. The kit of claim 4, wherein The HLA-loss analysis comprises SNV, Indel extraction, differential site screening, mutation frequency correction.

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