Preparation method of molecular residual lesion reference product and application thereof

Abstract

The application provides a preparation method of a molecular residual lesion reference product and application thereof, and the MRD reference product is prepared by screening cells containing commonly used mutation sites in clinical MRD detection, combining high-precision digital PCR value determination technology and gradient dilution strategy, and taking the mutation frequency as 0%, 0.5%, 0.05% and 0.005%. The MRD reference product is subjected to concentration value determination, and is prepared into the molecular residual lesion reference product after being divided and assembled. The MRD reference product prepared by the application is comprehensive in covered sites, stable and uniform in product, and can be produced on a large scale, and provides a reliable quality control material for tumor MRD detection.

Description

Technical Field

[0001] This invention belongs to the field of micro-lesion residue detection technology, specifically relating to a method for preparing a molecular residual lesion reference and its application. Background Technology

[0002] Molecular residual disease (MRD), also known as minimal residual disease or measurable residual disease, is a detection technology that has seen significant progress in recent years. Numerous clinical studies both domestically and internationally have demonstrated a correlation between MRD status and the risk of recurrence in cancer patients, making it useful for prognostic assessment and guiding individualized treatment decisions.

[0003] The concept of minimal residual disease (MRD) originated from hematologic malignancies. Initially, it was defined as the presence of a small number of leukemia cells remaining in the body after complete remission following induction chemotherapy or bone marrow transplantation, used to predict relapse risk and guide subsequent treatment. Since 10%–40% of newly diagnosed leukemia patients do not achieve complete remission after induction therapy, researchers later proposed the concept of measurable residual disease, referring to the presence of leukemia cells remaining in the body of newly diagnosed, refractory, or relapsed leukemia patients after treatment, regardless of whether complete remission is achieved. Relatively speaking, minimal residual disease is a more widely used concept now, referring to the detection of trace amounts of tumor cells or tumor-derived molecules in bone marrow or peripheral blood samples after disease remission following treatment for hematologic malignancies (leukemia / lymphoma / myeloma, etc.) using highly sensitive detection methods such as flow cytometry, quantitative polymerase chain reaction (PCR), and next-generation sequencing (NGS). MRD status has become one of the criteria for evaluating the efficacy of hematologic malignancy treatment and is significantly correlated with patient disease relapse and prognostic stratification. In solid tumors, MRD typically refers to molecular residual disease, which is a potential lesion that cannot be detected by imaging (including positron emission tomography) or traditional laboratory methods, indicating the persistence of the tumor and the potential for clinical progression, through molecular abnormalities of tumor-derived molecules such as circulating tumor DNA (ctDNA). MRD testing can help clinicians identify high-risk patients for recurrence earlier, providing a basis for subsequent clinical decisions. In the practical application of MRD testing technology, simultaneous comparison with MRD-related reference materials can significantly improve the accuracy of test results. Currently, MRD testing technologies or test kits on the market show a promising future, but research and commercialization of related MRD reference materials are still relatively scarce. This invention focuses on the research, development, and production process optimization of MRD reference materials, aiming to develop a reference product that can cover most mutation hotspot genes and is stable and can be systematically and scalably produced. Summary of the Invention

[0004] The technical problem to be solved: Existing technologies suffer from the scarcity of commercially available MRD reference materials, limited coverage of mutation sites, and a lack of systematic large-scale production processes. The purpose of this invention is to provide a method for preparing a molecular residual lesion reference material and its application. This invention utilizes genomic DNA (gDNA) from multiple cell lines, combined with high-precision digital PCR quantification technology and a gradient dilution strategy, to prepare an MRD reference material with high coverage, accurate abundance, stability, uniformity, and scalable production, providing a reliable quality control material for tumor MRD detection.

[0005] Technical Solution: This invention provides a molecular residual lesion reference material, which comprises the following 24 raw material cell lines: SW48, NCI-H1975, Hs-746T, HCC827, Lc-2 / ad, DLD-1, HT-1080, 5637, NCI-H2228, T-47D, HCC1395, Hs-766T, NCI-H2122, MOLT-4, 293T Lenti-M3, HEY, 293T Lenti-M7, IHH4, 293T Lenti-M21, 293T Lenti-M34, 293T Lenti-M36, GM12878, OCI-AML3, and HEL; the raw material cell lines contain the following 29 gene loci: EGFR G719S, EGFR L858R, ... T790M, TP53R273H, MET exon 14 Skipping c.3028+1G>T, EGFR E746-A750DEL, CCDC6-RET, BRCA2C3598-3599DELTG, KRAS G13D, PICK3CA E545K, NRAS Q61K, ERBB2 S310F, EML4(E6)-ALK(20), PICK3CA H1047R, BRCA1 R1772, KRAS Q61H, KRAS G12C, POLE L698, ERBB2 A775-G776insYVMA, KRAS G12D, EGFR V769-D770INS, AKT1 E17K, BRAF V600E, KIT D816V, IDH1R132H, IDH2 R172K, RPPH1, NPM1_W288fs, and TP53_M133K.

[0006] This invention provides a method for preparing the above-mentioned molecular residual lesion reference material, comprising the following steps:

[0007] S1. Input the cell lines from the cell line raw material library into the Cell Model Passports database to obtain all mutation site information of the candidate cell lines, and filter the required mutation sites and raw material cell lines;

[0008] S2. Digital PCR was used to detect the wild-type copy number, mutant copy number, and mutation ratio at the mutation sites in the above-mentioned raw material cell lines;

[0009] S3. Mix the gDNA of 22 raw material cell lines other than OCI-AML3 and HEL, so that the mutation frequency of each site in the mixture is 0.5%, and prepare a 0.5% MRD reference. Use the primers and fluorescent probes of digital PCR described in S2, as well as the primers and fluorescent probes corresponding to the 29 sites, to determine the mutation ratio of the 0.5% MRD reference.

[0010] S4. The gDNA of the OCI-AML3 cell line was diluted 10-fold by volume. 100 μL of 0.5% MRD reference and 900 μL of OCI-AML3 were mixed to prepare 0.05% MRD reference. Digital PCR was performed on the 0.05% MRD reference to determine the NPM1_W288fs site.

[0011] S5. HEL cell line gDNA was diluted 10-fold by volume. 100 μL of 0.05% MRD reference standard and 900 μL of HEL were mixed to prepare 0.005% MRD reference standard. Digital PCR was performed on the 0.005% MRD reference standard to determine the NPM1_W288fs and TP53_M133K sites.

[0012] S6. Wild-type MRD reference material was prepared using GM12878 cell line. Wild-type MRD reference material, 0.5% MRD reference material, 0.05% MRD reference material and 0.005% MRD reference material were sonicated and fragment length was detected.

[0013] S7. Concentration determination of wild-type MRD reference material, 0.5% MRD reference material, 0.05% MRD reference material and 0.005% MRD reference material, and dilution of MRD reference material to a final concentration of 20.0 ng / μL;

[0014] S8. Detection of the KRAS G12D site in wild-type MRD reference, 0.5% MRD reference, 0.05% MRD reference and 0.005% MRD reference with determined final concentrations;

[0015] S9. Wild-type MRD reference material, 0.5% MRD reference material, 0.05% MRD reference material and 0.005% MRD reference material were separately packaged and assembled to obtain molecular residual lesion reference material.

[0016] Preferably, the nucleotide sequences of the primers and probes for digital PCR in steps S2 and S3 are as shown in SEQ ID NO.1 to SEQ ID NO.117.

[0017] Preferably, the preparation system of the 22 raw material cell lines gDNA in step S3 is shown in the table below:

[0018]

[0019] Preferably, the nucleotide sequences of the primers and fluorescent probes for the NPM1_W288fs site in step S4 are shown in SEQ ID NO.105~SEQ ID NO.107.

[0020] Preferably, the nucleotide sequences of the primer and fluorescent probe for the TP53_M133K site in step S5 are shown in SEQ ID NO.100~SEQ ID NO.103.

[0021] Preferably, the conditions for ultrasonic treatment in step S6 are: peak power 65~85W, duty cycle 20~25%, pulse train cycle number 190~210, and duration 380~410 s.

[0022] Preferably, the nucleotide sequences of the primers and fluorescent probes for the KRAS G12D site in step S5 are shown in SEQ ID NO. 69 to SEQ ID NO. 72.

[0023] This invention also provides the application of the above-mentioned molecular residual lesion reference material in the quality control of the preparation of molecular residual lesion kits.

[0024] Beneficial effects:

[0025] 1. Existing customized MRD reference materials have limited loci and lack a universal approach. The MRD reference material prepared in this invention integrates 29 gene loci, all of which are mutation types with clear auxiliary diagnostic value and wide clinical application in relevant tumor hotspot genes. This invention's MRD reference material integrates multiple loci, simultaneously meeting the needs of various MRD panel detection methods. Furthermore, it has been validated and compared with relevant NGS platforms, demonstrating applicable scenarios and requirements, and the validation results are satisfactory.

[0026] 2. Multi-site combination MRD reference materials can optimize the production process and reduce preparation costs. Compared with the traditional mode of single-batch customized production for a few sites, this invention significantly shortens the preparation cycle and reduces the unit production cost under a stable and controllable production process, while effectively improving batch-to-batch consistency and product stability.

[0027] 3. Using cell line gDNA with a clear genetic background as raw material, with standardized sources and controllable batch differences, combined with a complete formulation and value determination process, large-scale industrial production can be achieved, providing a long-term stable and quality-controllable quality control material for MRD detection. Attached Figure Description

[0028] Figure 1 For MRD reference product manufacturing process flow chart;

[0029] Figure 2 Digital PCR amplification patterns of 0.5% MRD reference material at AKT1 E17K, BRAF V600E, BRCA1 R1772, BRCA2 C3598-3599DELTG, CCDC6-RET and EGFR E746-A750DEL sites;

[0030] Figure 3 Digital PCR amplification patterns of 0.5% MRD reference material at EGFR G719S, EGFR L858R, EGFR T790M, EGFR V769-D770INS, EML4(E6)-ALK(E20) and ERBB2 A775-G776insYVMA sites;

[0031] Figure 4 Digital PCR amplification patterns of 0.5% MRD reference at ERBB2 S310F, IDH1 R132H, IDH2 R172K, KIT D816V, KRASG12C and KRAS G12D sites;

[0032] Figure 5 Digital PCR amplification patterns of 0.5% MRD reference material at KRAS G13D, KRAS Q61H, MET exon 14 Skipping c.3028+1G>T, NRAS Q61K, PIK3CA E545K and PIK3CA H1047R sites;

[0033] Figure 6 Digital PCR amplification patterns of 0.5% MRD reference material at POLE L698 and TP53 R273H sites;

[0034] Figure 7 Digital PCR amplification patterns of OCI-AML3, 0.5% MRD reference, 0.05% MRD reference and 0.005% MRD reference at the NPM1_W288fs site;

[0035] Figure 8 Digital PCR amplification patterns of HEL, 0.05% MRD reference, and 0.005% MRD reference at the TP53_M133K site;

[0036] Figure 9The image shows the fragment size detection results for 0.5% MRD reference, 0.05% MRD reference, 0.005% MRD reference and wild-type MRD reference.

[0037] Figure 10 Digital PCR amplification patterns of 0.5% MRD reference, 0.05% MRD reference, 0.005% MRD reference and wild-type MRD reference at the KRAS G12D site. Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0039] Example 1: Mutation site search of raw material cell lines

[0040] The applicant's cell line library currently contains 24 candidate cell lines: SW48, NCI-H1975, Hs-746T, HCC827, Lc-2 / ad, DLD-1, HT-1080, 5637, NCI-H2228, T-47D, HCC1395, Hs-766T, NCI-H2122, MOLT-4, 293T Lenti-M3, HEY, 293T Lenti-M7, IHH4, 293T Lenti-M21, 293T Lenti-M34, 293T Lenti-M36, GM12878, OCI-AML3, and HEL.

[0041] The candidate cell lines were input into the Cell Model Passports database to obtain information on all mutation sites of the candidate cell lines, and the mutation sites required for this invention were screened. A total of 29 gene loci were obtained, namely EGFR G719S, EGFR L858R, EGFR T790M, TP53 R273H, MET exon 14 Skipping c.3028+1G>T, EGFR E746-A750DEL, CCDC6-RET, BRCA2 C3598-3599DELTG, KRAS G13D, PICK3CA E545KNRAS Q61K, ERBB2 S310F, EML4(E6)-ALK(20), PICK3CA H1047R, BRCA1 R1772, KRAS Q61H, KRAS G12C, POLE L698, ERBB2 A775-G776insYVMA, KRAS G12D, EGFR V769-D770INS, AKT1 E17K, BRAFV600E, KIT D816V, IDH1 R132H, IDH2 R172K, RPPH1, NPM1_W288fs, and TP53_M133K. Among these, 26 are gene mutation hotspots, 2 are quality control sites (NPM1_W288fs and TP53_M133K), which are also tumor gene mutation hotspots, and 1 is a housekeeping gene site (RPPH1).

[0042] Of the 24 candidate cell lines mentioned above, one is a normal human lymphocyte line (used as a background cell line for reference), and 23 are human immortalized tumor cell lines. Among them, the raw materials for five human immortalized tumor cell lines were obtained from gene editing screening, and their corresponding numbers are as follows: 293T Lenti-M3, 293T Lenti-M7, 293T Lenti-M21, 293T Lenti-M34, and 293T Lenti-M36. They were obtained through commercial paid customization by technical service companies.

[0043] Example 2: Digital PCR Validation of Related Mutation Sites in Raw Material Cell Lines

[0044] The BIO-RAD QX200 Droplet Reader digital PCR platform was used for detection and verification. The PCR primers and probes are shown in Table 1, the digital PCR reaction system is shown in Table 2, and the reaction program is shown in Table 3.

[0045] Table 1 PCR Primers

[0046]

[0047]

[0048] Table 2 Digital PCR Reaction System

[0049]

[0050] Table 3 Reaction Procedure

[0051]

[0052] Based on the analysis and calculation of the digital PCR analysis software (QuantaSoft), the wild-type copy number, mutant copy number, and mutation ratio of the candidate raw material cell lines at the corresponding mutation sites, as well as the MRD raw material cell line names and mutation site copy number determination results are shown in Table 4.

[0053] Table 4. Results of digital PCR assays

[0054]

[0055] Example 3: Preparation and determination of 0.5% MRD reference material

[0056] The MRD reference samples provided by this invention have mutation ratios of 0.5%, 0.05%, 0.005%, and 0% for each gene locus, totaling four groups. Each mutation ratio gradient is separated by dilution using raw material gDNA containing specific gene loci. This allows different gene loci in the reference sample mixture to be simultaneously diluted to the next gradient. In subsequent reference samples with lower mutation ratios, only the proportion of specific gene loci contained in the diluted background DNA needs to be detected. Simultaneously, during quality control verification, a few gene loci contained in the reference sample itself are detected as verification of the accuracy of the mutation ratio values.

[0057] The dilution gradient strategy described above enables successful detection of reference materials with ultra-low mutation frequencies, overcoming the limitations of detection platform technology. Furthermore, this method consistently achieves stable detection results in actual production processes and comparisons with different platforms. For specific production process details, please refer to [reference needed]. Figure 1 .

[0058] The 22 single-component raw materials gDNA (except OCI-AML3 and HEL) in the raw material determination step of Example 1 were mixed according to the single-component volumes in the table below, so that the mutation frequency of each site in the mixture was 0.5%.

[0059] Table 5. Preparation of 0.5% MRD Reference Sample

[0060]

[0061] After the 0.5% MRD reference was mixed, the mutation ratio of the 0.5% MRD reference was determined using the same digital PCR amplification detection system as the raw material and the specific primers and probes corresponding to each site, as in Example 1. The detection results are shown in Table 6.

[0062] Table 6. Results of Digital PCR Assay

[0063]

[0064] The mutation rate at each mutation site must be within the range of 0.35% to 0.85%. The tested mutation rates for the above 26 mutation sites are within the range of 0.46% to 0.84%, which is within the acceptable range for the product design. The digital PCR amplification pattern for each site is shown below. Figures 2-6 As shown.

[0065] Example 4: Preparation and determination of 0.05% MRD reference material

[0066] Digital PCR values ​​of the NPM1_W288fs site were determined for the OCI-AML3 cell line and 0.5% MRD reference material, as shown in Table 7. The M-0.5% mixture did not contain the NPM1_W288fs site mutation, and its mutation rate was 0%.

[0067] Table 7. Digital PCR values ​​of the NPM1_W288fs locus.

[0068]

[0069] Preparation of 0.05% MRD reference: Using OCI-AML3 cell line gDNA (NPM1_W288fs 49.44%), dilute 10-fold by volume and prepare the system according to Table 8. Mix 1 part MRD-0.5% and 9 parts OCI-AML3 to obtain 0.05% MRD reference.

[0070] Table 8 Formulation System

[0071]

[0072] After the 0.05% MRD reference sample was mixed, the specific primers and probes corresponding to the NPM1_W288fs site were used to determine the mutation ratio at this site in the 0.05% MRD reference sample. The specific detection results are shown in Table 9.

[0073] Table 9. Proportion of mutations at the NPM1_W288fs site in the 0.05% MRD reference standard.

[0074]

[0075] The mutation rate at the NPM1_W288fs mutation site was 42.03%, which is within the acceptable range for this site in the product design. For the quality control site NPM1_W288fs, repeated testing was performed, and the average value of the measured mutation frequency was calculated. The value X should be within the range of 31% ≤ X ≤ 58%. The digital PCR amplification patterns of OCI-AML3, 0.5% MRD reference, 0.05% MRD reference, and 0.005% MRD reference at the quality control site NPM1_W288fs are shown below. Figure 7 As shown.

[0076] Example 5: Preparation and determination of 0.005% MRD reference material

[0077] Digital PCR values ​​of the TP53_M133K site were determined for the HEL cell line and 0.05% MRD reference material, as shown in Table 10. The M-0.05% mixture did not contain the TP53 M133K site mutation, and its mutation rate was 0%.

[0078] Table 10. Digital PCR values ​​of the TP53_M133K locus

[0079]

[0080] Preparation of 0.005% MRD reference: Using HEL cell line gDNA (TP53 p.M133K 100%), dilute 10-fold by volume and prepare the system according to Table 11. Mix 1 part MRD-0.05% and 9 parts HEL to obtain 0.005% MRD reference.

[0081] Table 11 Formulation System

[0082]

[0083] After the 0.005% MRD reference was mixed, specific primers and probes corresponding to the two sites NPM1_W288fs and TP53_M133K were used to determine the mutation ratio of the 0.005% MRD reference. The specific detection results are shown in Table 12.

[0084] Table 12 Digital PCR values ​​of NPM1_W288fs and TP53_M133K loci

[0085]

[0086] The mutation rate at the NPM1_W288fs mutation site was 3.42%, and the mutation rate at the TP53_M133K mutation site was 88.22%, both within the acceptable range for mutation rates at these sites as designed in the product. For the quality control site NPM1_W288fs, repeated testing was performed, and the average measured mutation frequency (X) should be within the range of 3.1% ≤ X ≤ 5.8%. For the quality control site TP53_M133K, repeated testing was performed, and the average measured mutation frequency (Y) should be within the range of 85% ≤ Y ≤ 95%. The digital PCR amplification pattern for the quality control site TP53_M133K is shown below. Figure 8 As shown.

[0087] Example 6: Preparation of Wild-Type MRD Reference Material

[0088] In the early stages of this invention, it was verified that GM12878 gDNA is the wild-type MRD gDNA. After assigning values ​​to each site of the wild-type MRD gDNA and performing digital PCR detection, the mutation frequency was calculated to be less than 0.0035%.

[0089] Example 7: Fragmentation and fragment size detection of 0.5% MRD reference, 0.05% MRD reference, 0.005% MRD reference and wild-type MRD reference.

[0090] The four groups of MRD reference samples (Examples 2-4) were ultrasonically fragmented using a Covaris M220 non-contact ultrasonic disruptor. The ultrasonic fragmentation parameters were: peak power: 65-85W, duty cycle: 20%-25%, pulse train cycles: 190-210, and duration: 380-410 s. The fragment lengths of the four samples fragmented into ctDNA were then measured using an Agilent 4150 TapeStation system. If the Agilent 4150 result was within the range of 162-198 bp, the fragmented reference samples were confirmed to meet the technical requirements of this invention. The fragment length detection results are as follows: Figure 9 As shown.

[0091] Example 8: Concentration determination and dilution determination of 0.5% MRD reference, 0.05% MRD reference, 0.005% MRD reference and wild-type MRD reference.

[0092] The four reference standards were measured in triplicate using Qubit, and the average value of the three replicates was calculated. Based on the concentration adjustment formula C1×V1=C2×V2, the four reference standards were diluted with TE Buffer to a final concentration of 20.0 ng / μL. The final concentrations are shown in Table 13.

[0093] Table 13 Final Concentrations of MRD Reference Materials

[0094]

[0095] Example 9 MRD Reference Validation

[0096] The KRAS G12D site was detected in the four sets of reference samples with confirmed concentrations to verify whether the mutation ratio after dilution was within an acceptable range when using a gradient dilution process that included the detection of specific mutation sites. The acceptable range for the mutation ratio at this site is shown in Table 14.

[0097] Table 14. Range of mutation rates at the KRAS G12D site

[0098]

[0099] Using a digital PCR detection system, the detection results were analyzed. The average value of the measured data for the four groups of MRD reference samples and the result determination are as follows. The KRAS G12D site was verified to be feasible and meets the technical requirements set forth in this invention in the production process of this invention. The digital PCR amplification patterns of the four groups of MRD reference samples at the KRAS G12D site are as follows. Figure 10 As shown.

[0100] Table 15 Average values ​​of measured data for MRD reference materials

[0101]

[0102] Example 9: Comparative Validation of Next-Generation Sequencing

[0103] The 0.5% and 0.05% fractions from the above MRD reference samples were sent to Aijitech for next-generation sequencing verification. The results are summarized below, with sequencing verification performed using two different probe panel combinations.

[0104] Table 16 Next-generation sequencing results

[0105]

[0106] Both of the aforementioned panels effectively detected 26 target mutation sites in the reference sample, with trends of 0.5% and 0.05%, respectively.

[0107] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A molecular residual lesion reference material, characterized in that: The molecular residual lesion reference material includes the following 24 raw material cell lines: SW48, NCI-H1975, Hs-746T, HCC827, Lc-2 / ad, DLD-1, HT-1080, 5637, NCI-H2228, T-47D, HCC1395, Hs-766T, NCI-H2122, MOLT-4, 293T Lenti-M3, HEY, 293T Lenti-M7, IHH4, 293T Lenti-M21, 293T Lenti-M34, 293T Lenti-M36, GM12878, OCI-AML3, and HEL; the raw material cell lines contain the following 29 gene loci: EGFR G719S, EGFR L858R, EGFR T790M, TP53 R273H, MET exon 14Skipping. c.3028+1G>T, EGFR E746-A750DEL, CCDC6-RET, BRCA2 C3598-3599DELTG, KRASG13D, PICK3CA E545K, NRAS Q61K, ERBB2 S310F, EML4(E6)-ALK(20), PICK3CA H1047R, BRCA1 R1772, KRAS Q61H, KRAS G12C, POLE L698, ERBB2 A775-G776insYVMA, KRAS G12D, EGFR V769-D770INS, AKT1 E17K, BRAF V600E, KIT D816V, IDH1 R132H, IDH2 R172K, RPPH1, NPM1_W288fs and TP53_M133K.

2. The method for preparing a molecular residual lesion reference article according to claim 1, characterized in that, Includes the following steps: S1. Input the cell lines from the cell line raw material library into the Cell Model Passports database to obtain all mutation site information of the candidate cell lines, and filter the required mutation sites and raw material cell lines; S2. Digital PCR was used to detect the wild-type copy number, mutant copy number, and mutation ratio at the mutation sites in the above-mentioned raw material cell lines; S3. Mix the gDNA of 22 raw material cell lines other than OCI-AML3 and HEL, so that the mutation frequency of each site in the mixture is 0.5%, and prepare a 0.5% MRD reference. Use digital PCR to determine the mutation ratio of the 0.5% MRD reference. S4. The gDNA of the OCI-AML3 cell line was diluted 10-fold by volume. 100 μL of 0.5% MRD reference and 900 μL of OCI-AML3 were mixed to prepare 0.05% MRD reference. Digital PCR was performed on the 0.05% MRD reference to determine the NPM1_W288fs site. S5. HEL cell line gDNA was diluted 10-fold by volume. 100 μL of 0.05% MRD reference standard and 900 μL of HEL were mixed to prepare 0.005% MRD reference standard. Digital PCR was performed on the 0.005% MRD reference standard to determine the NPM1_W288fs and TP53_M133K sites. S6. Wild-type MRD reference material was prepared using GM12878 cell line. Wild-type MRD reference material, 0.5% MRD reference material, 0.05% MRD reference material and 0.005% MRD reference material were sonicated and fragment length was detected. S7. Concentration determination of wild-type MRD reference material, 0.5% MRD reference material, 0.05% MRD reference material and 0.005% MRD reference material, and dilution of MRD reference material to a final concentration of 20.0 ng / μL; S8. Detection of the KRAS G12D site in wild-type MRD reference, 0.5% MRD reference, 0.05% MRD reference and 0.005% MRD reference with determined final concentrations; S9. Wild-type MRD reference material, 0.5% MRD reference material, 0.05% MRD reference material and 0.005% MRD reference material were separately packaged and assembled to obtain molecular residual lesion reference material.

3. The preparation method according to claim 2, characterized in that, The nucleotide sequences of the primers and probes for digital PCR in steps S2 and S3 are shown in SEQ ID NO.1~SEQ ID NO.

117.

4. The preparation method according to claim 2, characterized in that, The preparation systems for the 22 raw material cell lines of gDNA in step S3 are shown in the table below:

5. The preparation method according to claim 2, characterized in that, The nucleotide sequences of the primers and fluorescent probes for the NPM1_W288fs site in step S4 are shown in SEQ ID NO.105~SEQ ID NO.

107.

6. The preparation method according to claim 2, characterized in that, The nucleotide sequences of the primers and fluorescent probes for the TP53_M133K site in step S5 are shown in SEQ ID NO.100~SEQ ID NO.

103.

7. The preparation method according to claim 2, characterized in that, The conditions for ultrasonic treatment in step S6 are: peak power 65~85W, duty cycle 20~25%, pulse train cycle number 190~210, duration 380~410 s.

8. The preparation method according to claim 2, characterized in that, The nucleotide sequences of the primers and fluorescent probes for the KRAS G12D site in step S5 are shown in SEQ ID NO.69~SEQ ID NO.

72.

9. The use of the molecular residual lesion reference material according to claim 1 in the quality control material for the preparation of the molecular residual lesion kit.

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