An analytical method for identifying biological relationships

Abstract

The present invention provides an analytical method for identifying biological kinship, which is characterized in that a magnetic bead suspension coupled with a nano-antibody against H2B antigen on the surface of a cfDNA / nucleosome complex as shown in SEQ ID NO: 9 is introduced into a size exclusion chromatography column, and then the size exclusion chromatography column is placed on a magnetic stand with a strong magnetic field, and the movement rate of the magnetic bead complex in the size exclusion chromatography column is changed by the strength of the magnetic field to obtain high-concentration and high-purity cfDNA, and then high-throughput sequencing analysis is performed to calculate the CFSI and CLR kinship indexes. The method has high application efficiency in kinship identification.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an analytical method for identifying biological kinship. Background Art

[0002] Kinship identification traditionally relies on collecting blood or other tissue samples from individuals to determine kinship by analyzing short tandem repeat (STR) or single nucleotide polymorphism (SNP) markers. However, this method may be limited by sample acquisition in certain circumstances. As a non-invasive sample, cfDNA overcomes this limitation. Kinship identification can be effectively performed by analyzing cfDNA fragments in plasma, such as analyzing the genetic information of the fetus through cfDNA in maternal blood before birth to identify the father-son relationship. In addition, in legal or forensic investigations, cfDNA also provides a possible way to identify kinship for relatives involved in missing persons or where it is difficult to collect samples. The high sensitivity and stability of cfDNA make it a tool with great potential for accurate kinship analysis without interfering with the normal physiological state of the individual.

[0003] In recent years, research on cfDNA-nucleosome complexes has gradually revealed its unique value in liquid biopsy. For example, in the blood of cancer patients, the distribution characteristics of cfDNA-nucleosome complexes can reflect tumor-specific genomic changes. These complexes not only carry sequence information, but also retain epigenetic information of chromatin state, such as methylation status. Therefore, analyzing cfDNA-nucleosome complexes can provide more comprehensive genomic and epigenetic data, which helps to improve the sensitivity and specificity of liquid biopsy. In addition, the potential application of cfDNA-nucleosome complexes in inflammation, cardiovascular disease, and kinship identification has also received increasing attention and has become an important research direction in the field of precision medicine.

[0004] The methods reported in the prior art for separating cfDNA fragments from nucleosomes include: 1. High-Salt Buffer. The high-salt buffer can destroy the ionic bonds between histones and DNA in nucleosomes, thereby releasing cfDNA from nucleosomes. This buffer destroys the interaction between histones and DNA through high salt concentration, thereby separating cfDNA from nucleosomes; 2. Phenol-Chloroform-Isoamyl Alcohol Extraction: This is a classic organic solvent extraction method that can effectively separate cfDNA from nucleosomes. This method can release cfDNA bound to nucleosomes through deproteinization; 3. Proteinase K treatment (Proteinase K Digestion): Proteinase K can degrade histones in nucleosomes, thereby releasing cfDNA; 4. Nucleic acid extraction kit: Some commercial nucleic acid extraction kits, such as QIAGEN's QIAamp series, contain lysis buffers that can effectively lyse nucleosomes and release cfDNA. These kits are usually designed to process plasma or serum samples, from which high-purity cfDNA can be extracted, and nucleosome complexes can also be lysed.

[0005] Therefore, based on the characteristics that cfDNA and nucleosomes bind to each other to form a complex, this application proposes for the first time the use of magnetic beads coupled with nano-antibodies against the H2B antigen on the surface of nucleosomes to specifically enrich and separate cfDNA-nucleosome complexes and combine them with size exclusion chromatography columns to extract high-concentration, high-purity cfDNA, and further provide a more efficient and accurate method for kinship identification through sequencing analysis and data interpretation. Summary of the invention

[0006] In order to solve the above problems, the present invention first provides an analytical method and a kit for identifying biological relatedness.

[0007] The present invention adopts the following technical solution:

[0008] An analytical method for identifying biological relatedness, characterized in that it comprises the following steps:

[0009] S1. Adding lysis binding solution, proteinase K and magnetic bead suspension coupled with NH-7 nanoantibody as shown in SEQ ID NO: 9 to the plasma sample of the relative to be identified, inverting and mixing, and after the lysis at room temperature is completed, transferring the mixed lysis solution containing magnetic beads to a size exclusion chromatography column, and then placing the size exclusion chromatography column on a magnetic stand with a strong magnetic field, changing the movement rate of the cfDNA / nucleosome-nanoantibody-magnetic bead complex in the size exclusion chromatography column by the strength of the magnetic field, thereby forming a rate difference with other impurities, and finally collecting the mixed solution containing the cfDNA / nucleosome-nanoantibody-magnetic bead complex after repeated elution with PBS eluent;

[0010] S2: Add washing solution to the centrifuge tube containing cfDNA / nucleosome-nanobody-magnetic bead complex, vortex to mix, place the centrifuge tube on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant; repeat step S2 three times and place at room temperature for 2-10 minutes;

[0011] S3. Add the eluent into the centrifuge tube of step S2, vortex to mix, and incubate at 37-60°C for 5-15 minutes;

[0012] S4: Place the centrifuge tube in step S3 on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant;

[0013] S5: add the lysis binding solution, proteinase K and carboxyl-modified superparamagnetic nanosphere suspension to the supernatant of step S4 again, invert and mix, and after the room temperature lysis is completed, place the reaction tube on a magnetic rack, let it stand, discard the residual liquid, repeat steps S2-S4, and further separate and purify to obtain the cfDNA extract.

[0014] S6 performs high-throughput sequencing on the cfDNA extracted in step S5. The detection genes related to CFSI and secondary kinship CLR are selected for sequencing, namely, STR marker genes selected from D3S1358, D18S51, D8S1179, D5S818, D7S820 and CSF1PO, and SNP marker genes selected from rs1805007, rs1042522 and rs1799782. The sequencing results are analyzed for different kinship types using Familias software, and the kinship index of CFSI and secondary kinship CLR is calculated.

[0015] The present invention also provides a kit for identifying biological affinity, characterized in that the kit comprises a lysis binding solution, an elution solution, a washing solution, a proteinase K, a size exclusion chromatography column, a magnetic frame with a strong magnetic field, a magnetic bead suspension coupled with the NH-7 nano-antibody shown in SEQ ID NO: 9, and a carboxyl-modified superparamagnetic nano-microsphere suspension.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] (1) The present application proposes for the first time a method for specifically enriching, separating and purifying cfDNA by using a nanobody-coupled magnetic bead suspension and a size exclusion chromatography column based on the anti-cfDNA-nucleosome complex surface H2B antigen. This method can achieve high-concentration and high-purity extraction of cfDNA, and successfully prepare a nanobody against the cfDNA-nucleosome complex surface H2B antigen as shown in SEQ ID NO: 9.

[0018] (2) A nano-antibody coupled to a magnetic bead suspension against the H2B antigen on the nucleosome surface was used to specifically enrich, separate and purify cfDNA for the construction of a sequencing library. At the same time, detection genes related to CFSI and secondary kinship CLR, namely STR marker genes (D3S1358, D18S51, D8S1179, D5S818, D7S820, CSF1PO) and SNP marker genes (rs1805007, rs1042522, rs1799782), were selected for sequencing analysis for kinship identification. The results showed that the kinship index effect of CFSI and secondary kinship CLR was much higher than that of the existing technology, that is, this method has a high application efficiency in kinship identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 ELISA screening of phages displaying nanobodies;

[0020] Figure 2 NH-7 nanobody inhibition curve;

[0021] Figure 3 cfDNA concentration detection based on separation and extraction of NH-7 nanoantibody-coupled magnetic beads;

[0022] Figure 4 cfDNA purity detection based on NH-7 nanoantibody-coupled magnetic beads separation and extraction. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1 Preparation of Nanobodies Against NCP-H2B Antigen Fragments

[0025] Preparation of NCP-H2B antigen fragment: Using the H2B (Chain H, Histone H2B type 1-J, PDB: 8YBJ_H) nucleosome gene of the nucleosome core particle recorded in NCBI as a template, a PCR reaction was performed to obtain the NCP-H2B antigen fragment shown in SEQ ID NO: 1, and a recombinant expression plasmid was constructed. The expression plasmid was transferred into BL21 (DE 3) competent cells, and cultured until OD600 was 0.6-0.8, and then induced to express overnight at 25°C with a final concentration of 1 mmol / L PTG, and purified by Ni-Agarose Resin column treatment to prepare the NCP-H2B antigen fragment for camel immunization: APAPKKGSK KAVTKAQKKDGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKR (SEQ ID NO: 1);

[0026] Immunization with NCP-H2B antigen fragment: The purified NCP-H2B antigen fragment was mixed with an equal volume of Freund's complete adjuvant / Freund's incomplete adjuvant at a final concentration of 300ug / mL to prepare an emulsified vaccine for immunization of 24-month-old healthy male alpacas. The vaccine prepared with Freund's complete adjuvant was used for the first immunization, and was injected intramuscularly at 4 points on both sides of the spine and in the axilla, with 0.2 mL inoculated at each point. The second, third and fourth immunizations were carried out on the 14th, 28th and 42nd days after the first immunization, respectively. The vaccine prepared with Freund's incomplete adjuvant was used for the booster immunization, and the immunization method and dosage were the same as those of the first immunization. Peripheral whole blood of the immunized alpacas was collected two weeks after the third and fourth immunizations, and the serum was separated by centrifugation. The titer of NCP-H2B antibody was detected by ELISA. 30 mL of peripheral blood of the immunized alpacas was collected on the 14th day after the fourth immunization, and lymphocytes were separated according to the instructions of the lymphocyte separation kit. Trizol was used to extract RNA and reverse transcribed it into cDNA. Two-round PCR primers were designed according to the principle of nested PCR, and Sfil restriction sites were introduced into the second-round primers. The amplified products were identified and recovered by 1% agarose gel electrophoresis.

[0027] Construction of phage display nanoantibody library: The recovered second-round PCR products and pComb3x vector were digested with Sfil enzyme, and the digested products were recovered after agarose gel identification. The VHH gene fragments after digestion were ligated with the pComb3x vector using T4 DNA ligase, and the ligated products were introduced into TG1 competent cells by electroporation. All colonies grown on the plate were collected into 5 mL LB medium, which was the initial antibody library of anti-NCP-H2B nanoantibodies. Take 100uL of the initial library solution, add it to 100ml LB medium, and culture at 37°C for 6h. After adding the helper phage M13K07 and standing at 37°C for 30 minutes, the cells were shaken and cultured at 37°C and 180r / min for 30 minutes. This is the anti-NCP-H2B specific recombinant phage library. The purified NCP-H2B was used as the target antigen to coat the ELISA plate. The anti-NCP-H2B specific recombinant phage library was subjected to three rounds of panning to screen out the anti-NCP-H2B specific recombinant phage with higher affinity. The specific method is as follows: the monoclonal clone of the phage was used as the primary antibody for ELISA detection, the purified NCP-H2B was diluted to 1ug / mL with the coating buffer to coat the ELISA plate, and the secondary antibody was 100mL of 1000-fold diluted HRP-labeled mouse anti-M13 antibody. After washing, TMB was added for color development, 100uL / well, incubated for 15min, and finally 50uLH2SO4 was added to terminate the reaction. The OD450nm value was read. The results are shown in the table. Figure 1 .

[0028] Figure 1 The results showed that the NH-7 phage-displayed nanoantibody with the strongest binding ability to the NCP-H2B antigen fragment was the positive phage.

[0029] Identification of the sequence of the nanoantibody against the NCP-H2B antigen fragment: Based on the sequence of the selected NH-7 phage, universal primers with XmaI and XhoI restriction sites were designed and synthesized, and a HIS tag sequence was added to the N-terminus of the antibody sequence. The target gene was amplified in the NH-7 bacteria, and the VHH target gene and pcDNA3.1 vector were successively digested, connected, transformed, PCR identified and sequenced. The sequencing results were analyzed using Bioedit software, and the IMGT website (www.http: / / www.imgt.org / ) was logged in to analyze the antibody gene sequence to determine the framework region and complementary determining region of the VHH antibody sequence.

[0030] Table 1: NH-7 Nanobody Sequences

[0031]

[0032] NH-7 nano antibody inhibition curve detection:

[0033] NCP-H2B was prepared into coating antigen solutions with different gradient concentrations using PBS buffer, 100 μl / well, incubated overnight at 4°C; 5% skim milk was used for blocking at room temperature for 2 hours. Then, 50 μl of 1.5 μg / ml NH-7 nanoantibody was added to each well and incubated at room temperature for 1 hour. After washing with PBST 3 times, Anti-His tagAntibody (HRP) secondary antibody was added and incubated at room temperature for 1 hour, and washed with PBST 7 times; 100 μl / well of TMB substrate color development solution was added for color development, and OD450 was measured after the reaction was terminated. The inhibition curve was drawn, and the results are shown in the figure. Figure 2 .

[0034] Figure 2 The results showed that the IC50 value of NH-7 was 0.954 ng / mL.

[0035] Example 2 Detection of cfDNA in pregnant women based on separation and extraction of NH-7 nanoantibody coupled magnetic beads

[0036] Lysis and binding solution composition: 10mM Tris-HCl (pH 7.5), 500mM NaCl, 1mM EDTA, 0.5% TritonX-100, 70mmol / L SDS and 1% dodecyl dimethyl betaine;

[0037] Washing solution composition: 0.1% Triton X-100, 150 mM NaCl and 50 mM Tris-HCl, pH 7.5;

[0038] Desorption solution composition: 1M NaCl and 0.1% Triton X-100.

[0039] Group A extraction method:

[0040] S1: Add 1000 μL of lysis binding solution, 25 μL of proteinase K and 10 μL of magnetic bead suspension to 500 μL of maternal plasma sample to be processed, invert and mix, and lyse at room temperature for 15-60 minutes. After lysis, place the reaction tube on a magnetic rack and let it stand for 1-10 minutes, and discard the residual liquid. The magnetic bead suspension is a superparamagnetic nanosphere suspension with silanol modified on the surface, which can bind to free cfDNA;

[0041] S2: Add 500 μL of washing solution to the centrifuge tube, vortex to mix, place the centrifuge tube on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant; repeat step S2 three times and place at room temperature for 2-10 minutes;

[0042] S3. Add 30-50 μL of the eluent into the centrifuge tube of step S2, vortex to mix, and incubate at 37-60°C for 5-15 min.

[0043] S4 places the centrifuge tube in step S3 on the magnetic rack, and after the magnetic beads are completely aggregated, aspirates the supernatant to separate and obtain the cfDNA extract.

[0044] Group B extraction method:

[0045] S1: Add 1000 μL of lysis binding solution, 25 μL of proteinase K and 10 μL of NH-7 nanoantibody-coupled magnetic bead suspension to 500 μL of maternal plasma sample to be processed, invert and mix, and lyse at room temperature for 15-60 minutes. After lysis, place the reaction tube on a magnetic stand, let it stand for 1-10 minutes, and discard the residual liquid;

[0046] S2: Add 500 μL of washing solution to the centrifuge tube, vortex to mix, place the centrifuge tube on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant; repeat step S2 three times and place at room temperature for 2-10 minutes;

[0047] S3. Add 30-50 μL of the eluent into the centrifuge tube of step S2, vortex to mix, and incubate at 37-60°C for 5-15 min.

[0048] S4: Place the centrifuge tube in step S3 on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant;

[0049] S5: Add 1000 μL of lysis binding solution, 25 μL of proteinase K and 10 μL of carboxyl-modified superparamagnetic nanosphere suspension to the supernatant of step S4 again, invert to mix, and lyse at room temperature for 15-60 minutes. After lysis, place the reaction tube on a magnetic rack and let it stand for 1-10 minutes. Discard the residual liquid and repeat steps S2-S4 to further separate and purify to obtain cfDNA extract.

[0050] Group C extraction method:

[0051] S1: add 1000 μL of lysis binding solution, 25 μL of proteinase K and 10 μL of NH-7 nanoantibody-coupled magnetic bead suspension to 500 μL of maternal plasma sample to be processed, invert and mix, and lyse at room temperature for 15-60 minutes. After the lysis, transfer the mixed lysate containing magnetic beads to a Tosoh TSKgel G3000SWxl size exclusion chromatography column, and then place the Tosoh TS KgelG3000SWxl size exclusion chromatography column on a magnetic stand with a strong magnetic field. The movement rate of the cfDNA / nucleosome-nanoantibody-magnetic bead complex in the Tosoh TSKgel G3000SWxl size exclusion chromatography column is changed by the strength of the magnetic field, thereby forming a rate difference with other impurities. Finally, after repeated elution with PBS eluent, a mixed solution containing the cfDNA / nucleosome-nanoantibody-magnetic bead complex is collected;

[0052] S2: Add 500 μL of washing solution to the centrifuge tube containing the cfDNA / nucleosome-nanobody-magnetic bead complex, vortex to mix, place the centrifuge tube on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant; repeat step S2 three times and place at room temperature for 2-10 minutes;

[0053] S3. Add 30-50 μL of the eluent into the centrifuge tube of step S2, vortex to mix, and incubate at 37-60°C for 5-15 min.

[0054] S4: Place the centrifuge tube in step S3 on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant;

[0055] S5: Add 1000 μL of lysis binding solution, 25 μL of proteinase K and 10 μL of carboxyl-modified superparamagnetic nanosphere suspension to the supernatant of step S4 again, invert to mix, and lyse at room temperature for 15-60 minutes. After lysis, place the reaction tube on a magnetic rack and let it stand for 1-10 minutes. Discard the residual liquid and repeat steps S2-S4 to further separate and purify to obtain cfDNA extract.

[0056] Detection and analysis of cfDNA concentration and purity after extraction: 15 plasma samples of pregnant women were taken and cfDNA was extracted using the methods of group A, group B and group C respectively. The concentration and purity of cfDNA were detected using an ultra-micro UV spectrophotometer. Each sample was tested three times. The specific operation steps were carried out in accordance with the standard operating procedures of the ultra-micro UV spectrophotometer. The concentration and purity of the measured cfDNA after extraction were statistically analyzed to evaluate the extraction of cfDNA concentration by the methods of group A, group B and group C. The results are shown in Figure 3 ; and the purity of the advantages and disadvantages, the results are shown in Figure 4 .

[0057] Figure 3 The results showed that the concentration of cfDNA extracted from pregnant women by group C method was significantly better than that of cfDNA extracted by group A and group B methods (P<0.01), which was statistically significant.

[0058] Figure 4 The results showed that the purity of cfDNA extracted from pregnant women by the methods of group B and group C was significantly better than that of cfDNA extracted by the method of group A (P<0.01), which was statistically significant.

[0059] Analysis of the amplification efficiency Ct value of cfDNA after extraction: Primers designed with the DNA sequence of human ACTG1 gene (F: 5'-ATGGCCCTGAGGCTATCC-3'; R: 5'-TG GGCACCTCCATAGAAG-3') and Taqman probe (5'-FAM-TCCATCAA GTCTGACCGTGATGATG-TAMRA-3') were used to quantitatively amplify ACTG1 using the standard curve. Three plasma samples of pregnant women (named as sample 1, sample 2, and sample 3) were selected, and each person's plasma was divided into two parts, one for extracting cfDNA using the method of group A, one for extracting cfDNA using the method of group B, and the other for extracting cfDNA using the method of group C. The two extracted cfDNAs were used as templates to amplify ACTG1, and three replicates were made for each sample. The extraction effects of the methods of group A, group B, and group C were judged according to the average Ct value.

[0060] Table 2 Ct values ​​of cfDNA extracted by the methods of group A, group B and group C as template amplification

[0061]

[0062] The results in Table 2 show that for samples 1, 2, and 3, the Ct values ​​of cfDNA amplification extracted by the C group method were significantly advanced compared with the Ct values ​​of cfDNA amplification extracted by the A group and the B group methods, at least by about 2 cycles, indicating that the extraction effect of cfDNA in group C is better than that in group A and group B.

[0063] Example 3 Analysis of the application efficacy of cfDNA extracted from magnetic beads coupled to NH-7 nanoantibodies for kinship identification

[0064] Plasma samples were collected from 113 related individuals from five families, and all volunteers signed informed consent. The related samples included 43 triplets, 107 pairs of dyads, 38 pairs of full siblings, and 148 pairs of second-degree related pairs (61 pairs of grandparents and grandchildren, 4 pairs of half siblings, and 83 pairs of uncles and nephews / aunts and nephews / uncles and nephews / aunts and nephews (daughters)). cfDNA was extracted using the methods of group A, group B, and group C, respectively, and then used Fluorometer instrument (Thermo Fisher Scientific, USA) and QubitTM dsDNA HS Assay kit (Thermo Fisher Scientific) were used to quantify DNA, and the quantified DNA was diluted to 1 ng / μL for amplification. The amplified product was detected by 3500xL Genetic Analyzer (Thermo Fisher Scientific), and the detection system was 10 μL, containing 8.7 μL deionized formamide (Thermo Fisher Scientific), 0.3 μL GeneScanTM 600LIZTM size standard v2.0 (Thermo Fisher Scientific) and 1 μL amplified product.

[0065] The library was constructed according to the user manual of the Hybrid DNA Identification Kit (Identification Center of the Ministry of Public Security, China), and the prepared library was quantified using the KAPA Library Quantification Kit (Roche, Switzerland) on a 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific). All libraries were then diluted to 6 nM and mixed in equal volumes. The library was diluted to 12.5 pM and 10% PhiX Control V3 (Illumina) was added. Reagent Nano v2 kit (Illumina) and Reagent v2 kit (Illumina) was used to sequence the mixture containing denatured library and PhiX on MiSeq FGx sequencer (Verogen, USA). The measured data were analyzed using MHTyper data analysis software. The detection genes related to CFSI were selected, including STR marker genes (D3S1358, D18S51, D8S1179) and SNP marker genes (rs1805007, rs1042522, rs1799782), and the detection genes related to secondary kinship CLR, including STR marker genes (D5S818, D7S820, CSF1PO), for sequencing analysis. Then, Familias software was used to simulate different kinship types. The sample size was set to 10,000 pairs of kinship and 10,000 pairs of unrelated individuals for each type. In the simulation of triplets and dyads, 10 -9The mutation rate of other relatedness was not set. The ITO method was used to calculate the cumulative full sibling index (CFSI) and the combined likelihood ratio (CLR) of the second-level relatedness. The CFSI and CLR indices were used to evaluate the application efficiency of cfDNA extracted from group A, group B, and group C in relatedness identification.

[0066] Table 3 Comparison of cfDNA extracted by group A, group B and group C for kinship index

[0067]

[0068] The cfDNA extracted by the methods of Group A, Group B and Group C was used to calculate the values ​​of the full sibling index CFSI and the secondary kinship CLR. The results showed that the numerical range and kinship support probability calculated by Group C were higher than those of Group A and Group B, that is, the application efficiency of cfDNA extracted by Group C in kinship identification was stronger than that of Group A and Group B.

[0069] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An analytical method for identifying biological relatedness, characterized in that The steps include: S1. Adding a lysis binding solution, proteinase K and a magnetic bead suspension coupled with a nanobody based on an anti-cfDNA-nucleosome complex surface H2B antigen to a human plasma sample to be identified for kinship, wherein the nanobody against the cfDNA-nucleosome complex surface H2B antigen is as shown in SEQ ID NO: 9, inverting the magnetic bead suspension to mix evenly, and after the room temperature lysis is completed, transferring the mixed lysate containing the magnetic beads to a size exclusion chromatography column, and then placing the size exclusion chromatography column on a magnetic stand with a strong magnetic field, changing the movement rate of the cfDNA-nucleosome-nanobody-magnetic bead complex in the size exclusion chromatography column by the strength of the magnetic field, thereby forming a rate difference with other impurities, and finally collecting the mixed solution containing the cfDNA-nucleosome-nanobody-magnetic bead complex after repeated cyclic elution with a PBS eluent; S2: Add washing solution to the centrifuge tube containing the cfDNA-nucleosome-nanobody-magnetic bead complex, vortex to mix, place the centrifuge tube on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant; repeat step S2 three times and place at room temperature for 2-10 minutes; S3. Add the eluent into the centrifuge tube of step S2, vortex to mix, and incubate at 37-60°C for 5-15 minutes; S4: Place the centrifuge tube in step S3 on the magnetic rack, and after the magnetic beads are completely aggregated, aspirate the supernatant; S5: add the lysis binding solution, proteinase K and carboxyl-modified superparamagnetic nanosphere suspension to the supernatant of step S4 again, invert and mix, and place the reaction tube on a magnetic rack after the room temperature lysis is completed, let it stand, discard the residual liquid, repeat steps S2-S4, and further separate and purify to obtain the cfDNA extract; S6 performs high-throughput sequencing on the cfDNA extracted in step S5, and the sequencing selects detection genes related to CFSI and secondary kinship CLR; the CFSI detection genes include STR marker genes of D3S1358, D18S51 and D8S1179 and SNP marker genes of rs1805007, rs1042522 and rs1799782; the detection genes related to the secondary kinship CLR include STR marker genes of D5S818, D7S820 and CSF1PO, and sequencing analysis is performed. The sequencing results are analyzed for different kinship types using Familias software, and the kinship index of CFSI and secondary kinship CLR is calculated.

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