DPCR quantitative kit for detecting donor proportion in recipient and application of dPCR quantitative kit

Abstract

The invention discloses a dPCR quantitative kit for detecting the proportion of donors in recipients and application of the dPCR quantitative kit, and belongs to the technical field of gene detection. The dPCR quantitative kit provided by the invention can rapidly and efficiently detect 29 InDel sites of a recipient and / or a donor, so that the content of the donor in the recipient can be rapidly and accurately obtained, and the dPCR quantitative kit has the advantages of good accuracy, high sensitivity and the like; besides, the dPCR quantitative kit can be simultaneously used for detecting the content of donors (micro-chimeric rate and dd-cfDNA) in transplanted samples with donors and without donors, the whole test process is simple and convenient, and the test result is reliable.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a dPCR quantitative kit for detecting the ratio of donors in recipients and an application thereof. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is the main treatment for many malignant blood diseases and some non-malignant diseases. The chimerism of hematopoietic stem cells after transplantation is mainly detected based on polymorphic genetic markers in the population, such as red blood cell antigens, human leukocyte antigen typing, short tandem repeat analysis (STR-PCR), etc. At present, the method commonly used for chimerism detection is SNP combined with RT-PCR technology, which uses the genotyping of donors and recipients at SNP sites to evaluate the chimerism.

[0003] Cell-free DNA (cfDNA) is a fragmented and degraded DNA that can be detected in body fluids such as blood and urine. It is generally believed that cfDNA originates from cell necrosis and apoptosis. Necrotic cells are engulfed by macrophages and release digested DNA into the blood to form cfDNA. cfDNA mainly comes from blood cells, tissue cells, fat cells, etc., and under certain conditions, it may also come from tumors, transplants or fetuses. The plasma cfDNA of organ transplant patients contains transplant-derived cfDNA (donor-derived cell-free DNA, dd-cfDNA). Transplant rejection will release dd-cfDNA. Related studies on liver transplantation, lung transplantation, and pancreas-kidney combined transplantation have found that its content is significantly increased when patients have graft damage or rejection. Therefore, dd-cfDNA levels may be used as a biomarker for organ transplant prognosis and for the diagnosis and monitoring of graft damage and transplant rejection. It has great potential as a non-invasive, early diagnosis of graft damage and real-time monitoring of transplant patient rejection biomarker, and has practical clinical application value.

[0004] Currently, it is of great significance to provide a method to accurately and sensitively detect the donor ratio in the recipient. Summary of the invention

[0005] The object of the present invention is to provide a dPCR quantitative kit for detecting the ratio of donors in a recipient and its application. The dPCR quantitative kit for detecting the ratio of donors in a recipient and the method for detecting the ratio of donors in a recipient provided in the present invention have the advantages of high accuracy and high sensitivity, and therefore have good application prospects.

[0006] In a first aspect, the present invention provides a dPCR quantitative kit for detecting the donor ratio in a recipient, comprising a primer set and a probe set; wherein the primer set comprises a first primer set having a nucleotide sequence as shown in SEQ ID NO.1-3 and amplifying a sequence of the rs59841142 site, a second primer set having a nucleotide sequence as shown in SEQ ID NO.5-7 and amplifying a sequence of the rs34076006 site, a third primer set having a nucleotide sequence as shown in SEQ ID NO.9-11 and amplifying a sequence of the rs3049448 site, a fourth primer set having a nucleotide sequence as shown in SEQ ID NO.13-15 and amplifying a sequence of the rs3029189 site, a fifth primer set having a nucleotide sequence as shown in SEQ ID NO.17-19 and amplifying a sequence of the rs35309403 site, a sixth primer set having a nucleotide sequence as shown in SEQ ID NO.21-23 and amplifying a sequence of the rs2307696 site, and a sixth primer set having a nucleotide sequence as shown in SEQ ID NO. The seventh primer set as shown in SEQ ID NOs.25-27 and amplifies the sequence of rs1610937 site, the eighth primer set as shown in SEQ ID NOs.29-31 and amplifies the sequence of rs2308010 site, the ninth primer set as shown in SEQ ID NOs.33-35 and amplifies the sequence of rs16663 site, the tenth primer set as shown in SEQ ID NOs.37-39 and amplifies the sequence of rs34209360 site, the eleventh primer set as shown in SEQ ID NOs.41-43 and amplifies the sequence of rs1610932 site, the twelfth primer set as shown in SEQ ID NOs.45-47 and amplifies the sequence of rs35453727 site, the thirteenth primer set as shown in SEQ ID NOs.49-51 and amplifies the sequence of rs5787309 site, the nucleotide sequence as shown in SEQ ID NOs. NO.53-55 and amplify the rs60564093 site sequence, the 14th primer set with nucleotide sequences as shown in SEQ ID NO.57-59 and amplify the rs3038530 site sequence, the 16th primer set with nucleotide sequences as shown in SEQ ID NO.61-63 and amplify the rs60867863 site sequence, the 17th primer set with nucleotide sequences as shown in SEQ ID NO.65-67 and amplify the rs56024302 site sequence, the 18th primer set with nucleotide sequences as shown in SEQ ID NO.69-71 and amplify the rs140790 site sequence, the 19th primer set with nucleotide sequences as shown in SEQ ID NO.73-75 and amplify the rs769299 site sequence, the 20th primer set with nucleotide sequences as shown in SEQ ID NO.77-79 and amplify the rs67405073 site sequence, the 21st primer set with nucleotide sequences as shown in SEQ ID NO.81-83 and amplify the rs16458 site sequence, the 21st primer set with nucleotide sequences as shown in SEQ ID NOs.85-87 and amplify the rs4646006 site sequence, the 23rd primer set with nucleotide sequences as shown in SEQ ID NOs.89-91 and amplify the rs771783753 site sequence, the 24th primer set with nucleotide sequences as shown in SEQ ID NOs.93-95 and amplify the rs34419736 site sequence, the 25th primer set with nucleotide sequences as shown in SEQ ID NOs.97-99 and amplify the rs2308112 site sequence, the 26th primer set with nucleotide sequences as shown in SEQ ID NOs.101-103 and amplify the rs2067204 site sequence, the 27th primer set with nucleotide sequences as shown in SEQ ID NOs.105-107 and amplify the rs34855933 site sequence, the 28th primer set with nucleotide sequences as shown in SEQ ID NOs. The 28th primer set shown in SEQ ID NO.109-111 and amplifying the sequence of rs34076006 site, and the 29th primer set with nucleotide sequences shown in SEQ ID NO.113-115 and amplifying the sequence of rs72031009 site.

[0007] The dPCR quantitative kit provided by the present invention can quickly and efficiently detect 29 InDel sites (wide coverage, located on 16 autosomes and the Y chromosome) of the recipient and / or donor, thereby quickly and accurately obtaining the content of the donor in the recipient, and the dPCR quantitative kit has the advantages of good accuracy and high sensitivity.

[0008] In some embodiments, the probe group includes a first probe having a nucleotide sequence as shown in SEQ ID NO.4 and detecting a sequence of rs59841142, a second probe having a nucleotide sequence as shown in SEQ ID NO.8 and detecting a sequence of rs34076006, a third probe having a nucleotide sequence as shown in SEQ ID NO.12 and detecting a sequence of rs3049448, a fourth probe having a nucleotide sequence as shown in SEQ ID NO.16 and detecting a sequence of rs3029189, a fifth probe having a nucleotide sequence as shown in SEQ ID NO.20 and detecting a sequence of rs35309403, a sixth probe having a nucleotide sequence as shown in SEQ ID NO.24 and detecting a sequence of rs2307696, a seventh probe having a nucleotide sequence as shown in SEQ ID NO.28 and detecting a sequence of rs1610937, an eighth probe having a nucleotide sequence as shown in SEQ ID NO.32 and detecting a sequence of rs2308010, a ninth probe having a nucleotide sequence as shown in SEQ ID NO.36 and detecting a sequence of rs16663, and a nucleotide sequence as shown in SEQ ID NO. NO.40 and detecting the rs34209360 site sequence, the eleventh probe whose nucleotide sequence is shown in SEQ ID NO.44 and detecting the rs1610932 site sequence, the twelfth probe whose nucleotide sequence is shown in SEQ ID NO.48 and detecting the rs35453727 site sequence, the thirteenth probe whose nucleotide sequence is shown in SEQ ID NO.52 and detecting the rs5787309 site sequence, the fourteenth probe whose nucleotide sequence is shown in SEQ ID NO.56 and detecting the rs60564093 site sequence, the fifteenth probe whose nucleotide sequence is shown in SEQ ID NO.60 and detecting the rs3038530 site sequence, the sixteenth probe whose nucleotide sequence is shown in SEQ ID NO.64 and detecting the rs60867863 site sequence, the seventeenth probe whose nucleotide sequence is shown in SEQ ID NO.68 and detecting the rs56024302 site sequence, the eighteenth probe whose nucleotide sequence is shown in SEQ ID NO.72 and detecting the rs140790 site sequence, the The nucleotide sequence of the nineteenth probe shown in SEQ ID NO.76 and detecting the sequence of the rs769299 site, the nucleotide sequence of the twentieth probe shown in SEQ ID NO.80 and detecting the sequence of the rs67405073 site, the nucleotide sequence of the twenty-first probe shown in SEQ ID NO.84 and detecting the sequence of the rs16458 site, the nucleotide sequence of the twenty-second probe shown in SEQ ID NO.88 and detecting the sequence of the rs4646006 site, the nucleotide sequence of the twenty-second probe shown in SEQ ID NO.92 and detecting the 23rd probe of the rs771783753 site sequence, the 24th probe of the nucleotide sequence as shown in SEQ ID NO.96 and detecting the rs34419736 site sequence, the 25th probe of the nucleotide sequence as shown in SEQ ID NO.100 and detecting the rs2308112 site sequence, the 26th probe of the nucleotide sequence as shown in SEQ ID NO.104 and detecting the rs2067204 site sequence, the 27th probe of the nucleotide sequence as shown in SEQ ID NO.108 and detecting the rs34855933 site sequence, the 28th probe of the nucleotide sequence as shown in SEQ ID NO.112 and detecting the rs34076006 site sequence, and the 29th probe of the nucleotide sequence as shown in SEQ ID NO.116 and detecting the rs72031009 site sequence.

[0009] In some embodiments, each probe in the probe set contains a fluorescent group at the 5' end and a quencher group at the 3' end.

[0010] In some embodiments, the fluorescent group includes at least one of FAM, 6-FAM, HEX, VIC, Cy5, and ROX; and the quenching group includes at least one of BHQ1, BHQ2, and MGB.

[0011] In some embodiments, the dPCR quantification kit further comprises at least one of a DNA extraction reagent and a PCR amplification reagent.

[0012] In a second aspect, the present invention provides a use of any of the above-mentioned dPCR quantitative kits in detecting the donor ratio in a recipient.

[0013] It can be understood that the dPCR quantitative kit of the present invention can be used in allogeneic hematopoietic stem cell transplantation, organ transplantation dd-cfDNA, allogeneic cell immunotherapy car-T, car-NK, to detect the proportion of donors after transplantation in recipients, and can also be used to detect the proportion of donors after transplantation in recipients after any two human cells from different sources are transplanted.

[0014] In a third aspect, the present invention provides a method for detecting the content of a donor in a post-transplantation sample with a donor, comprising the following steps: S1, using a primer sequence such as a nucleotide sequence shown in SEQ ID NO.117-176 to detect the InDel site combination of the transplanted donor and the recipient to obtain the differentiated sites of the donor and the recipient; S2, using any of the above-mentioned dPCR quantitative kits to detect the differentiated sites to obtain the content of the donor in the post-transplantation sample with a donor.

[0015] In the present invention, the inventors have found that first, specific primer sequences are designed to perform typing detection on the InDel site combinations of the transplanted donor and recipient, so as to quickly, simply and efficiently obtain the differentiated sites of the donor and recipient, and then a dPCR quantitative kit is used to detect the differentiated sites to obtain the content of the donor in the post-transplant sample with the donor. The entire testing process is simple and the test results are reliable.

[0016] In some embodiments, in step S2, the method for calculating the content of the donor in the transplanted sample with the donor is as follows: when the recipient's InDel site genotype is homozygous and the donor's differential InDel site genotype is homozygous, the donor's content = Q 供 / (Q 供 +Q 受 ); When the recipient's InDel site genotype is homozygous and the donor's differential InDel site genotype is heterozygous, the donor's content = 2×Q 供 / (Q 供 +Q 受 ), where Q 供 is the gene content of the donor InDel site, Q 受 is the gene content of the recipient InDel site.

[0017] In a fourth aspect, the present invention provides a method for detecting the content of donors in a post-transplantation sample without a donor, comprising the following steps: 1) using a primer sequence such as a nucleotide sequence shown in SEQ ID NO.120-179 to detect the InDel site combination of the transplanted recipient to obtain an InDel site with a homozygous genotype; 2) using any of the above-mentioned dPCR quantitative kits to detect the homozygous InDel site to obtain the content of donors in the post-transplantation sample without a donor.

[0018] In the present invention, the inventors further studied and found that first, a specific primer sequence was designed to perform typing detection on the InDel site combination of the transplanted recipient, so as to quickly, simply and efficiently obtain the InDel site with a homozygous genotype, and then a dPCR quantitative kit was used to detect the homozygous InDel site to obtain the content of the donor in the donor-free post-transplant sample. The entire testing process is simple and the test results are reliable.

[0019] In some embodiments, in step 2), the method for calculating the content of the donor in the post-transplantation sample without a donor is as follows: when the number of homozygous InDel sites is N, and the genotype of the donor at the homozygous InDel site is heterozygous or homozygous, the proportion of the donor InDel site is set to X = Q 供 / (Q 供 +Q 受), if the donor's genotypes at the homozygous InDel sites are all heterozygous, the donor InDel site ratio is 2X, if the donor's genotypes at the homozygous InDel sites are all homozygous, the donor InDel site ratio is X, and the final donor content in the transplanted sample without a donor is 1.5X; when the number of homozygous InDel sites is N', and the donor's genotypes at the homozygous InDel sites are heterozygous and homozygous, the donor InDel site ratio is set to X = Q 供 / (Q 供 +Q 受 ), if the genotypes of the donor at the homozygous InDel loci are all heterozygous, the proportion of the donor InDel loci is 2X, if the genotypes of the donor at the homozygous InDel loci are all homozygous, the proportion of the donor InDel loci is X, therefore, the proportion of the donor InDel loci conforms to the probability distribution of X and 2X, the donor InDel loci proportion values ​​are arranged from small to large, 1.6-2.4 times the minimum value is determined as homozygous, and the rest are heterozygous, and the average value of the proportion of each donor InDel loci is calculated, which is the content of the donor in the transplanted sample without a donor; wherein N and N' are positive integers greater than 3, Q 供 is the gene content of the donor InDel site, Q 受 is the gene content of the recipient InDel site.

[0020] In some embodiments, N and N' are preferably 3-10, more preferably 5-10.

[0021] In some embodiments, the present invention also provides a method for continuously monitoring the content of donors in post-transplant samples, comprising the following steps: 1) using N or N' homozygous InDel sites in the above-mentioned method for detecting the content of donors in post-transplant samples without donors to obtain preliminary detection results of the content of donors in post-transplant samples without donors; 2) selecting 1-2 InDel sites in the above-mentioned method for detecting the content of donors in post-transplant samples without donors that have a smaller deviation from the actual results, and continuing to use the dPCR quantitative kit for detection to obtain re-test results of the content of donors in post-transplant samples without donors.

[0022] In some embodiments, the present invention also provides a method for continuously monitoring the content of donors in post-transplant samples, comprising the following steps: 1) performing NGS sequencing on the above-mentioned post-transplant samples without donors to obtain 901 SNP sites and 29 InDel site information, fitting a probability density map based on the 901 SNP sites, analyzing the InDel sites according to the probability density map, obtaining the preliminary test results of the content of donors in the post-transplant samples without donors, and analyzing the deviation of the InDel sites at the same time; 2) selecting 1-2 InDel sites with small deviation in the above step 1), and continuing to use the dPCR quantitative kit for detection to obtain the re-test results of the content of donors in the post-transplant samples without donors.

[0023] The beneficial effects of the present invention are as follows: Different from the prior art, the dPCR quantitative kit provided by the present invention can quickly and efficiently detect 29 InDel sites (wide coverage, located on 16 autosomes and the Y chromosome) of the recipient and / or donor, thereby quickly and accurately obtaining the content of the donor in the recipient, and the dPCR quantitative kit has the advantages of good accuracy and high sensitivity; in addition, the dPCR quantitative kit can be used to simultaneously detect the content of the donor (microchimerism rate and dd-cfDNA) in post-transplantation samples with and without donors, and the entire testing process is simple and the test results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph of the typing results of random samples in Example 1 of the invention, where "+" represents insertion type, "-" represents deletion type, del represents "-", and in represents "+";

[0025] Figure 2 This is a diagram showing the results of dPCR detection of the specificity of the A2 site using the primer probe set in Example 2 of the present invention;

[0026] Figure 3 This is a diagram showing the results of dPCR detection of the specificity of the B5 site using the primer probe set in Example 2 of the present invention;

[0027] Figure 4 This is a diagram showing the results of dPCR detection of the B7 site using the primer probe set in Example 2 of the present invention;

[0028] Figure 5 This is a diagram showing the results of dPCR detection of the specificity of the C4 site using the primer probe set in Example 2 of the present invention;

[0029] Figure 6 This is a diagram showing the results of dPCR detection of the specificity of the D5 site using the primer probe set in Example 2 of the present invention;

[0030] Figure 7This is a graph showing the results of dPCR detection of the amplification efficiency of the A2 site using the primer probe set in Example 2 of the present invention;

[0031] Figure 8 This is a graph showing the results of dPCR testing of the amplification efficiency of the B5 site using the primer probe set in Example 2 of the present invention;

[0032] Fig. 9 This is a graph showing the results of dPCR detection of the amplification efficiency of the B7 site using the primer probe set in Example 2 of the present invention;

[0033] Fig.10 This is a graph showing the results of dPCR detection of the amplification efficiency of the C4 site using the primer probe set in Example 2 of the present invention;

[0034] Fig.11 This is a graph showing the result of dPCR detection of the amplification efficiency of the D5 site using the primer probe set in Example 2 of the present invention;

[0035] Fig.12 This is a graph showing the results of dPCR detection of the proportion of sample 1 in a mixed sample using a primer probe set in Example 2 of the present invention;

[0036] Fig.13 This is a graph showing the results of dPCR detection of the proportion of sample 3 in a mixed sample using a primer probe set in Example 2 of the present invention;

[0037] Fig.14 This is a diagram of the donor typing results in Example 3 of the present invention, wherein "+" represents insertion type, "-" represents deletion type, del represents "-", and in represents "+";

[0038] Fig.15 This is a diagram of the recipient typing results in Example 3 of the present invention, wherein "+" represents an insertion type, "-" represents a deletion type, del represents "-", and in represents "+";

[0039] Fig.16 This is a graph showing the results of dPCR detection of sites A6 and B2 using a single channel and a combined channel using a primer probe set in Example 3 of the present invention;

[0040] Fig.17 The probability distribution diagram of the donor InDel site proportions conforming to X and 2X in Example 4 of the present invention;

[0041] Fig.18 This is a graph showing the dPCR test results of the recipient sample containing homozygous sites A5, B2, C6, and D3 in Example 4 of the present invention;

[0042] Fig.19This is a graph showing the dPCR test results of the recipient sample containing homozygous sites A3, B5, and C6 in Example 4 of the present invention;

[0043] Fig. 20 This is a graph showing the dPCR test results of the recipient sample containing homozygous sites A3, B4, and C2 in Example 4 of the present invention;

[0044] Fig.21 This is a probability density diagram of NGS sequencing in Example 5 of the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0047] Example 1 Site design and fragment analysis

[0048] Screening suitable InDel sites from population frequency databases such as gnomad, HapMap, Exac, 1000G 1000 Genomes, and UCSC literature requires the following screening conditions to be met:

[0049] 1. Minimum allele frequency (MAF) is between 0.30-0.50;

[0050] 2. The insertion / deletion bases are between 3-10bp;

[0051] 3. The selected loci should be located on different chromosomes as much as possible;

[0052] 4. The selected loci are located on the same chromosome, ensuring that the distance is greater than 10Mb and does not form linkage, and conform to Hardy-Weinberg equilibrium in the population;

[0053] 5. The number of selected sites combined with the gene frequency ensures that the feasibility of donor-free sample detection is greater than 99.99%.

[0054] Based on the above principles, 29 InDel sites were finally selected, located on 16 different autosomes and the Y chromosome, and divided into four groups: A, B, C, and D, as shown in Table 1 below:

[0055] Table 1 InDel site information table

[0056]

[0057]

[0058] According to the above grouping, A, B, C, and D were assigned to 4 fluorescent channels (spectral calibrants were independently developed, and the 4 channels were fluorescently labeled with FAM, HEX, TAMRA, and ROX, respectively), and amelogenin gender determination sites were added, a total of 30 sites, and fragment analysis amplification primers were designed (as shown in Table 5 below, "*" represents fluorescently labeled primers). The product size was between 100-300 bp, and the size spacing of adjacent site fragments was >10 bp. The site layout and the final concentration of the primers for single-well reactions are shown in Tables 3 and 4 below, respectively:

[0059] Table 3 Site layout and final concentration of site primers

[0060] FAM A1 A2 A3 A4 A5 A6 A7 A8 HEX B1 B2 B3 B4 B5 B6 B7 B8 TAMRA C0 C1 CY C2 C3 C4 C5 C6 ROX D1 D2 D3 D4 D5 D6

[0061] Table 4 Final concentration of primers in single well (μM)

[0062] Location A1 A2 A3 A4 A5 A6 A7 A8 Primers 45 50 50 75 60 40 100 275 Location B1 B2 B3 B4 B5 B6 B7 B8 Primers 80 45 300 50 60 40 50 120 Location C0 C1 CY C2 C3 C4 C5 C6 Primers 125 60 225 200 50 150 200 200 Location D1 D2 D3 D4 D5 D6 Primers 50 50 50 50 75 300

[0063] Table 5 Fragment analysis amplification primers

[0064]

[0065]

[0066] First, a DNA extraction kit is used to obtain template DNA of a random sample (peripheral blood gDNA or plasma cfDNA), and then a PCR amplification reaction is performed as follows:

[0067] 10μl amplification system: 5μl 2×PCR buffer; 0.3μl ARMS PCR Taq; 1.0μl Primers Mix; 2ng DNA; RNase-free Water to complete the reaction system.

[0068] PCR program: 95°C pre-denaturation for 30 s, 95°C denaturation for 15 s, 60°C annealing for 20 s, 72°C extension for 40 s, for a total of 32 cycles, and finally 72°C extension for 14 min.

[0069] The PCR products obtained above were directly loaded into the ABI 3500 capillary electrophoresis instrument for analysis, and the typing results of all loci of the random sample could be obtained at one time. Figure 1 shown.

[0070] from Figure 1 It can be seen that 12 of the InDel sites are homozygous and the other sites are heterozygous. The results show that the fragment analysis method of the present invention can quickly type the sites.

[0071] Further, according to the above-mentioned InDel site, a primer probe set for microchimerism and dd-cfDNA detection was designed (wherein, F(+) and R or F and R(+) are used to specifically amplify the inserted fragment; F(-) and R or F and R(-) are used to specifically amplify the deleted fragment; P is the probe sequence; the underlined part in F(+) is the inserted specific base), as shown in Table 6 below:

[0072] Table 6 Primer and probe sequence list

[0073]

[0074]

[0075]

[0076] Example 2 Performance test of dPCR quantitative kit

[0077] The dPCR quantitative kit provided by the present invention comprises the primer probe set used for detecting the donor ratio in the recipient in Example 1, reagents required for DNA extraction, and reagents required for PCR amplification.

[0078] Among them, the dPCR amplification reaction system and procedure are as follows:

[0079] 20μl amplification system: 2μl 10×Digital AMP PCR buffer; 1.0μl Primers Mix; DNA 20-100ng; RNase-free Water to supplement the reaction system.

[0080] The PCR instrument used in the present invention is a micro-well chip digital PCR (dPCR). The "solid phase partitioning" chip used generally has 20,000 to 30,000 reaction units. After "system partitioning", the nucleic acid molecules in the reaction system conform to the Poisson distribution. The sensitivity can be determined according to the amount of DNA input, generally not exceeding 5 times the upper limit of the reaction unit. Usually, 20-100ng of gDNA is input according to demand to meet the needs of the detection system.

[0081] dPCR program: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 60°C extension for 1 min, for a total of 40 cycles.

[0082] 2.1 Specificity detection of primer probe sets in dPCR quantitative kit

[0083] The samples typed in Example 1 are used as recipients and donors, respectively, and any InDel site in the above samples is selected, such as B5, which has two sets of primer probes, B5(+) and B5(-) (specific sequences are shown in Table 6), and dPCR is used to detect the "+ / +" type specimens (recipients) and "- / -" type specimens (donors) of the B5 site, respectively. If B5(+) amplifies "+ / +" with a signal and "- / -" without a signal, it can be regarded that the B5(+) primer probe group has good specificity; if B5(-) amplifies "- / -" with a signal and "+ / +" without a signal, it can be regarded that the B5(-) primer probe group has good specificity.

[0084] The concentration of the primer probe set in each InDel site is shown in Table 7 below:

[0085] Table 7 Concentration of primer probe sets in InDel sites

[0086]

[0087]

[0088]

[0089] Taking A2, B5, B7, C4, and D5 as examples, dPCR detection was performed, and the results were as follows: Figure 2-6 shown.

[0090] from Figure 2-6 It can be seen that the primer probe set in the dPCR quantitative kit provided by the present invention has good specificity.

[0091] 2.2 Test of the efficiency of primer probe set in dPCR quantitative kit to amplify alleles

[0092] Select the heterozygous sample typed in Example 1, select any InDel site in the above sample, such as B5, which has two sets of primer probes B5(+) and B5(-) (specific sequences are shown in Table 6), and use dPCR to amplify the "+ / -" type specimen (heterozygous sample) of the B5 site. If the copy number ratios of "+" and "-" are both within the range of 50%±10%, it can be considered that the amplification efficiency of B5(+) and B5(-) is close, and the copy number "distortion" will not occur when detecting alleles.

[0093] Among them, the copy number ratio of "+" = the copy number of "+" / (the copy number of "+" + the copy number of "-") × 100%; the copy number ratio of "-" = the copy number of "-" / (the copy number of "+" + the copy number of "-") × 100%.

[0094] Taking the A2, B5, B7, C4, and D5 sites in the heterozygous sample as an example, dPCR amplification was performed, and the results were as follows: Figure 7-11 shown.

[0095] from Figure 7-11 It can be seen that when the primer probe set in the dPCR quantitative kit provided by the present invention amplifies the "+ / -" type specimens of each site, for the A2 site, the copy number ratio of "+" is 48.5%, and the copy number ratio of "-" is 51.5%, for the B5 site, the copy number ratio of "+" is 52.9%, and the copy number ratio of "-" is 47.1%, for the B7 site, the copy number ratio of "+" is 49.6%, and the copy number ratio of "-" is 50.4%, for the C4 site, the copy number ratio of "+" is 55.1%, and the copy number ratio of "-" is 44.9%, for the D5 site, the copy number ratio of "+" is 49.7%, and the copy number ratio of "-" is 50.3%. In the above-mentioned sites, the copy number ratios of "+" and "-" are all in the range of 50%±10%. The results show that the primer probe set of the present invention has a good amplification efficiency for the allele.

[0096] 2. Accuracy and sensitivity test of primer probe set in 3dPCR quantitative kit

[0097] Sample 1 (B5 site is "- / -") and sample 2 (B5 site is "+ / +") typed in Example 1 were mixed in different proportions (sample 1 / sample 2: 10%, 2%, 0.05%, 0.25%) to obtain standards with different concentrations of 20 ng / μL. The prepared standards were used as templates for quantitative analysis. 2 O is the template as a blank control. The dPCR reaction system and conditions are the same as above.

[0098] The copy numbers of the B5(+) primer probe group and the B5(-) primer probe group obtained by dPCR detection of different proportions of standards were obtained, and the proportion of sample 1 in the mixed sample was calculated, which was the copy number of the B5(-) primer probe group / (the copy number of the B5(-) primer probe group+the copy number of the B5(+) primer probe group)×100%. The results are as follows: Fig.12 shown.

[0099] from Fig.12 It can be seen that in each proportion standard, the proportion of sample 1 in the mixed sample is 11.58%, 2.05%, 0.056% and 0.028% respectively. The test results are close to the theoretical values. The results show that the primer probe set has good accuracy and high sensitivity.

[0100] Similarly, sample 3 (A5 site is "- / -") and sample 4 (A5 site is "+ / +") typed in Example 1 were mixed in different proportions (sample 3 / sample 4: 2%, 0.2%, 0.1%) to obtain standard products with a concentration of 20 ng / μL in different proportions. The above method was used for testing, and the results were as follows: Fig.13 shown.

[0101] from Fig.13 It can be seen that in each proportion standard, the proportion of sample 3 in the mixed sample is 2.46%, 0.19%, and 0.094%, respectively. The test results are close to the theoretical values. The results show that the primer probe set has good accuracy and high sensitivity.

[0102] In summary, the primer probe set in the dPCR quantitative kit provided by the present invention has good specificity, good amplification efficiency for alleles, good accuracy, and high sensitivity.

[0103] Example 3 Detection of microchimerism rate and dd-cfDNA content in post-transplant samples with donors using dPCR quantitative kit

[0104] First, the fragment analysis method in Example 1 was used to detect the InDel site combinations of the transplant donor and recipient. The typing results of the donor and recipient were as follows: Fig.14 and 15 shown.

[0105] from Fig.14 and 15In the method, the differential sites of the donor and the recipient are selected, wherein the selection of the differential sites meets the following conditions: 1) the recipient genotype is homozygous (the phenotype in the figure is a single peak); 2) there are differences between the donor and the recipient, and the donor is preferably homozygous and the second choice is heterozygous; 3) 1-4 differential sites can be selected (the probes of the four groups of sites A, B, C, and D in this system can be marked with fluorescent markers such as FAM, HEX, CY5, and CY5.5 on the dPCR platform, and primer probes of different channels can be combined on two chips to complete the detection of "+" and "-", that is, 1-4 sites can be determined on two chips).

[0106] Then, the dPCR quantitative kit in Example 2 was used to detect the above-mentioned differential sites to obtain the microchimerism rate and dd-cfDNA content in the donor's post-transplantation samples.

[0107] Among them, the calculation method of microchimerism rate and dd-cfDNA content in post-transplant samples with donors is as follows:

[0108] When the recipient's InDel site genotype is homozygous and the donor's InDel site genotype is homozygous, the microchimerism rate and dd-cfDNA content = Q 供 / (Q 供 +Q 受 );

[0109] When the recipient's InDel site genotype is homozygous and the donor's InDel site genotype is heterozygous Aa, the microchimerism rate and dd-cfDNA content = 2×Q 供 / (Q 供 +Q 受 );

[0110] Among them, Q 供 is the gene content of the donor InDel site, Q 受 is the gene content of the recipient InDel site, Q 供 , Q 受 dPCR was used for detection.

[0111] Taking the differential sites A6 (recipient is "+ / +", donor is "- / -") and B2 (recipient is "+ / +", donor is "+ / -") as examples, the above method was used for single channel and combined channel determination, and the above method was used to calculate the donor microchimerism rate and dd-cfDNA content. The results are as follows Fig.16 shown.

[0112] from Fig.16 It can be seen that in both the single channel and the merged channel, the microchimerism rate and dd-cfDNA content of the differential sites A6 and B2 are both 0.

[0113] Example 4 Detection of microchimerism rate and dd-cfDNA content in post-transplant samples without donors using dPCR quantitative kit

[0114] First, the InDel sites of the recipients were analyzed using a commercial STR typing method to exclude the InDel sites that appeared heterozygous in the STR typing map.

[0115] Then, the fragment analysis method in Example 1 was used to detect the InDel site combination of the above recipient to obtain the InDel site with a homozygous genotype.

[0116] The number of InDel sites with homozygous genotypes required for detecting microchimerism rate and dd-cfDNA content in post-transplantation samples without donors is selected as follows:

[0117] The InDel loci in the present invention conform to the Hardy-Weinberg law. Assuming that the minimum allele frequency (MAF) of the "+" genotype is m and the probability of the "-" genotype is 1-m, the probability of a random sample being a "+ / +" type is m. 2 、The probability of "- / -" type is (1-m) 2 、“+ / -” type probability is 2m-2m 2 ,

[0118] There are three situations for mixed samples of donor D and recipient R:

[0119] (1)P1=P(R=“+ / +”,D=“- / -” or R=“- / -”,D=“+ / +”)=2×m 2 ×(1-m) 2 ;

[0120] (2)P2=P(R=“+ / +”,D=“+ / -”or R=“- / -”,D=“+ / -”)=(2m-2m 2 )×m 2 +(2m-2m 2 )×(1-m) 2 =2m-6m 2 +8m 3 -4m 4 ;

[0121] (3) P3 = 1 - P1 - P2;

[0122] Assume that the expected number of InDels that (1) occurs i times and (2) occurs j times is N i,j,0 , then N i,j,0 =P1×(N i-1,j,0 +1)+P2×(Ni,j-1,0 +1)+P3×(N i,j,0 +1); further get the following formula:

[0123]

[0124] For the detection of microchimerism rate and dd-cfDNA content in post-transplant samples without donors, N 1,1,0 The InDel sites can be expected to meet the needs of detecting microchimerism or ddcfDNA in unrelated donors and recipients using dPCR.

[0125] If the recipient homozygous ("+ / +" or "- / -") loci are screened in advance before dPCR testing, then N 1,1,0 '≈N 1,1,0 / 2(0.3 <MAF<0.5)。

[0126] For example, when m = 0.4, N 1,1,0 =10, N 1,1,0 '≈5, so for the detection of microchimerism rate and dd-cfDNA content in post-transplant samples without donors, after fragment analysis screening, the detection scheme can select 5-10 InDel homozygous sites for combination (in the entire detection system, m can be the geometric mean of MAF of all InDel sites screened out).

[0127] Theoretically, 5-10 InDel homozygous sites are selected for combination and the dPCR quantitative kit of the present invention is used for detection, and the microchimerism rate and dd-cfDNA content in the transplanted sample without a donor can be obtained. However, when the dPCR quantitative kit is actually used, it is not necessary to select 5-10 InDel homozygous sites for combined detection. For example, only 2, 3, or 4 InDel homozygous sites are selected for combination detection, and the microchimerism rate and dd-cfDNA content in the transplanted sample without a donor can also be obtained.

[0128] Finally, the homozygous InDel loci were detected using a dPCR quantitative kit to obtain the microchimerism rate and dd-cfDNA content in the donor-free post-transplantation samples. The calculation method is as follows:

[0129] 1) When there are N homozygous InDel sites, and the donor's genotype at the homozygous InDel site is heterozygous or homozygous (at least N 1,0,0 or N 0,1,0 ), set the donor InDel site ratio X = Q 供 / (Q 供 +Q 受), if the donor's genotypes at the homozygous InDel sites are all heterozygous, the proportion of donor InDel sites is 2X; if the donor's genotypes at the homozygous InDel sites are all homozygous, the proportion of donor InDel sites is X, and the final microchimerism rate and dd-cfDNA content in the post-transplantation samples without donors are 1.5X;

[0130] Specifically, the recipient samples containing homozygous sites A5, B2, C6, and D3 in the fragment analysis in Example 1 were selected and tested using dPCR. The results are as follows: Fig.18 shown.

[0131] from Fig.18 It can be seen that the X contents in sites A5, B2, C6, and D3 are 3.26%, 4.15%, 0, and 0, respectively. According to the calculation method in 1), the microchimerism rate and dd-cfDNA content in the recipient sample can be obtained as (3.26%+4.15%) / 2×1.5=5.56%.

[0132] Similarly, the recipient samples containing homozygous sites A3, B5, and C6 in the fragment analysis in Example 1 were selected and tested using dPCR. The results are as follows: Fig.19 shown.

[0133] from Fig.19 It can be seen that the X contents in sites A3, B5, and C6 are 3.14%, 3.22%, and 3.28%, respectively. According to the calculation method in 1), the microchimerism rate and dd-cfDNA content in the recipient sample can be obtained as (3.14%+3.22%2+3.28%) / 3×1.5=4.82%.

[0134] 2) When there are N' homozygous InDel sites, and the donor's genotype at the homozygous InDel site is heterozygous and homozygous (at least N 1,1,0 ), set the donor InDel site ratio X = Q 供 / (Q 供 +Q 受 ), if the donor's genotype at the homozygous InDel site is all heterozygous, then the donor's InDel site ratio is 2X; if the donor's genotype at the homozygous InDel site is all homozygous, then the donor's InDel site ratio is X. Therefore, the donor's InDel site ratio conforms to the probability distribution of X and 2X (such as Fig.17 As shown in the figure, the donor InDel site ratio values ​​were arranged from small to large, and the values ​​1.6-2.4 times the minimum value were determined as homozygous, and the rest were heterozygous. The average value of the InDel site ratio of each donor was calculated, which was the microchimerism rate and dd-cfDNA content in the transplanted samples without donors;

[0135] 1) and 2), N and N' are positive integers greater than 3, Q 供 is the gene content of the donor InDel site, Q 受 is the gene content of the recipient InDel site.

[0136] Specifically, the recipient samples containing homozygous sites A3, B4, and C2 in the fragment analysis in Example 1 were selected and tested using dPCR. The results are as follows: Fig. 20 shown.

[0137] from Fig. 20 It can be seen that the X contents in sites A3, B4, and C2 are 12.62%, 5.75%, and 9.99%, respectively. According to the calculation method in 2), the values ​​are arranged from small to large: 5.75%, 9.99%, and 12.62%, and the values ​​between 9.2% and 13.8% are taken as homozygotes. It can be obtained that the microchimerism rate and dd-cfDNA content in the recipient sample are (5.75%×2+9.99%+12.62%) / 3=11.37%.

[0138] In summary, the dPCR quantitative kit of the present invention can be used to simultaneously detect the microchimerism rate and dd-cfDNA content in post-transplantation samples with and without donors. The entire testing process is simple and the test results are reliable.

[0139] Example 5 Method for Continuously Monitoring Donor Content in Post-Transplantation Samples

[0140] This embodiment provides a method for continuously monitoring the content of donor in a sample after transplantation, comprising the following steps:

[0141] 1) using the N or N' homozygous InDel sites in the method for detecting the content of donors in post-transplantation samples without donors in the above Example 4 to obtain preliminary detection results of the content of donors in post-transplantation samples without donors;

[0142] 2) Select 1-2 InDel sites with smaller deviation from the actual result in the detection method of the content of donors in the post-transplantation sample without donors in the above step 1), and continue to use the dPCR quantitative kit for detection to obtain the re-test results of the content of donors in the post-transplantation sample without donors.

[0143] In the actual application of continuous monitoring of the donor content in the post-transplantation sample, NGS can also be used for initial testing, and then the dPCR quantitative kit can be used for retesting. The specific method is as follows:

[0144] S1) The above-mentioned post-transplantation samples without donors were sequenced by NGS to obtain 901 SNP sites and 29 InDel site information. The probability density map (such as Fig.21As shown, wherein the x-axis is the minor allele frequency (minor allele frequency), the y-axis is the density (Density), Peak1 is the first peak, Peak2 is the second peak, the first peak (Peak1) corresponds to the P2 event (i.e., R = "+ / +", D = "+ / -" or R = "- / -", D = "+ / -"), and the second peak (Peak2) corresponds to the P1 event (R = "+ / +", D = "- / -" or R = "- / -", D = "+ / +")), according to the probability density diagram, the InDel site is analyzed to obtain the initial test results of the donor content in the post-transplantation sample without a donor, and the deviation of the InDel site is analyzed at the same time, and the results are shown in Table 8 below;

[0145] Table 8 InDel site quantitative information and deviation

[0146]

[0147]

[0148] S2) selecting 1-2 InDel sites with small deviation in the above step S1), and continuing to detect using a dPCR quantitative kit to obtain a retest result of the donor content in the post-transplantation sample without a donor.

[0149] In this embodiment, the initial test was performed by NGS sequencing method, and then the dPCR quantitative kit was used for retesting, which significantly improved the detection efficiency and detection accuracy.

[0150] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0151] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A dPCR quantitative kit for detecting the donor ratio in a recipient, characterized in that: Includes primer sets and probe sets; Wherein, the primer set includes a first primer set having a nucleotide sequence as shown in SEQ ID NO.1-3 and amplifying a sequence of rs59841142 site, a second primer set having a nucleotide sequence as shown in SEQ ID NO.5-7 and amplifying a sequence of rs34076006 site, a third primer set having a nucleotide sequence as shown in SEQ ID NO.9-11 and amplifying a sequence of rs3049448 site, a fourth primer set having a nucleotide sequence as shown in SEQ ID NO.13-15 and amplifying a sequence of rs3029189 site, a fifth primer set having a nucleotide sequence as shown in SEQ ID NO.17-19 and amplifying a sequence of rs35309403 site, a sixth primer set having a nucleotide sequence as shown in SEQ ID NO.21-23 and amplifying a sequence of rs2307696 site, a seventh primer set having a nucleotide sequence as shown in SEQ ID NO.25-27 and amplifying a sequence of rs1610937 site, and a seventh primer set having a nucleotide sequence as shown in SEQ ID NO. The eighth primer set as shown in SEQ ID NOs.29-31 and amplifies the rs2308010 site sequence, the ninth primer set as shown in SEQ ID NOs.33-35 and amplifies the rs16663 site sequence, the tenth primer set as shown in SEQ ID NOs.37-39 and amplifies the rs34209360 site sequence, the eleventh primer set as shown in SEQ ID NOs.41-43 and amplifies the rs1610932 site sequence, the twelfth primer set as shown in SEQ ID NOs.45-47 and amplifies the rs35453727 site sequence, the thirteenth primer set as shown in SEQ ID NOs.49-51 and amplifies the rs5787309 site sequence, the fourteenth primer set as shown in SEQ ID NOs.53-55 and amplifies the rs60564093 site sequence, the nucleotide sequence as shown in SEQ ID NOs. No. 57-59 and amplifies the rs3038530 site sequence, the 15th primer set with nucleotide sequences as shown in SEQ ID NOs. 61-63 and amplifies the rs60867863 site sequence, the 17th primer set with nucleotide sequences as shown in SEQ ID NOs. 65-67 and amplifies the rs56024302 site sequence, the 18th primer set with nucleotide sequences as shown in SEQ ID NOs. 69-71 and amplifies the rs140790 site sequence, the 19th primer set with nucleotide sequences as shown in SEQ ID NOs. 73-75 and amplifies the rs769299 site sequence, the 20th primer set with nucleotide sequences as shown in SEQ ID NOs. 77-79 and amplifies the rs67405073 site sequence, the 21st primer set with nucleotide sequences as shown in SEQ ID NOs. 81-83 and amplifies the rs16458 site sequence, the 21st primer set with nucleotide sequences as shown in SEQ ID NOs.85-87 and amplify the rs4646006 site sequence, the 22nd primer set with nucleotide sequences as shown in SEQ ID NOs.89-91 and amplify the rs771783753 site sequence, the 24th primer set with nucleotide sequences as shown in SEQ ID NOs.93-95 and amplify the rs34419736 site sequence, the 25th primer set with nucleotide sequences as shown in SEQ ID NOs.97-99 and amplify the rs2308112 site sequence, the 26th primer set with nucleotide sequences as shown in SEQ ID NOs.101-103 and amplify the rs2067204 site sequence, the 27th primer set with nucleotide sequences as shown in SEQ ID NOs.105-107 and amplify the rs34855933 site sequence, the 28th primer set with nucleotide sequences as shown in SEQ ID NOs.109-111 and amplify the rs34076006 site sequence, the 29th primer set with nucleotide sequences as shown in SEQ ID NOs. The 29th primer set shown in IDNO.113-115 and amplifying the rs72031009 site sequence.

2. The dPCR quantitative kit according to claim 1, characterized in that The probe group includes a first probe whose nucleotide sequence is shown in SEQ ID NO.4 and detects the sequence of rs59841142 site, a second probe whose nucleotide sequence is shown in SEQ ID NO.8 and detects the sequence of rs34076006 site, a third probe whose nucleotide sequence is shown in SEQ ID NO.12 and detects the sequence of rs3049448 site, a fourth probe whose nucleotide sequence is shown in SEQ ID NO.16 and detects the sequence of rs3029189 site, a fifth probe whose nucleotide sequence is shown in SEQ ID NO.20 and detects the sequence of rs35309403 site, a sixth probe whose nucleotide sequence is shown in SEQ ID NO.24 and detects the sequence of rs2307696 site, a seventh probe whose nucleotide sequence is shown in SEQ ID NO.28 and detects the sequence of rs1610937 site, an eighth probe whose nucleotide sequence is shown in SEQ ID NO.32 and detects the sequence of rs2308010 site, a ninth probe whose nucleotide sequence is shown in SEQ ID NO.36 and detects the sequence of rs16663 site, and a NO.40 and detecting the rs34209360 site sequence, the eleventh probe whose nucleotide sequence is shown in SEQ ID NO.44 and detecting the rs1610932 site sequence, the twelfth probe whose nucleotide sequence is shown in SEQ ID NO.48 and detecting the rs35453727 site sequence, the thirteenth probe whose nucleotide sequence is shown in SEQ ID NO.52 and detecting the rs5787309 site sequence, the fourteenth probe whose nucleotide sequence is shown in SEQ ID NO.56 and detecting the rs60564093 site sequence, the fifteenth probe whose nucleotide sequence is shown in SEQ ID NO.60 and detecting the rs3038530 site sequence, the sixteenth probe whose nucleotide sequence is shown in SEQ ID NO.64 and detecting the rs60867863 site sequence, the seventeenth probe whose nucleotide sequence is shown in SEQ ID NO.68 and detecting the rs56024302 site sequence, the eighteenth probe whose nucleotide sequence is shown in SEQ ID NO.72 and detecting the rs140790 site sequence, the The nucleotide sequence of the nineteenth probe shown in SEQ ID NO.76 and detecting the sequence of the rs769299 site, the nucleotide sequence of the twentieth probe shown in SEQ ID NO.80 and detecting the sequence of the rs67405073 site, the nucleotide sequence of the twenty-first probe shown in SEQ ID NO.84 and detecting the sequence of the rs16458 site, the nucleotide sequence of the twenty-second probe shown in SEQ ID NO.88 and detecting the sequence of the rs4646006 site, the nucleotide sequence of the twenty-second probe shown in SEQ ID NO.92 and detecting the rs771783753 site sequence, the 23rd probe whose nucleotide sequence is shown in SEQ ID NO.96 and detecting the rs34419736 site sequence, the 25th probe whose nucleotide sequence is shown in SEQ ID NO.100 and detecting the rs2308112 site sequence, the 26th probe whose nucleotide sequence is shown in SEQ ID NO.104 and detecting the rs2067204 site sequence, the 27th probe whose nucleotide sequence is shown in SEQ ID NO.108 and detecting the rs34855933 site sequence, the 28th probe whose nucleotide sequence is shown in SEQ ID NO.112 and detecting the rs34076006 site sequence, and the 29th probe whose nucleotide sequence is shown in SEQ ID NO.116 and detecting the rs72031009 site sequence.

3. The dPCR quantitative kit according to claim 2, characterized in that The 5' end of each probe in the probe set contains a fluorescent group, and the 3' end contains a quenching group.

4. The dPCR quantitative kit according to claim 3, characterized in that The fluorescent group includes at least one of FAM, 6-FAM, HEX, VIC, Cy5, and ROX; the quenching group includes at least one of BHQ1, BHQ2, and MGB.

5. The dPCR quantitative kit according to claim 1, characterized in that It also includes at least one of a DNA extraction reagent and a PCR amplification reagent.

6. Use of the dPCR quantitative kit according to any one of claims 1 to 5 in detecting the donor ratio in a recipient.

7. A method for detecting the content of donor in a post-transplant sample with a donor, characterized in that: The steps include: S1. Using primer sequences such as nucleotide sequences shown in SEQ ID NO.117-176 to detect the InDel site combination of the transplant donor and the recipient, to obtain the differential sites of the donor and the recipient; S2. Use the dPCR quantitative kit described in any one of claims 1 to 8 to detect the differential sites to obtain the content of the donor in the post-transplantation sample with the donor.

8. The detection method according to claim 7, characterized in that: In step S2, the method for calculating the content of the donor in the transplanted sample with the donor is as follows: When the recipient's InDel site genotype is homozygous and the donor's differential InDel site genotype is homozygous, the donor's content = Q 供 / (Q 供 +Q 受 ); When the recipient's InDel site genotype is homozygous and the donor's differential InDel site genotype is heterozygous, the donor's content = 2×Q 供 / (Q 供 +Q 受 ); Among them, Q 供 is the gene content of the donor InDel site, Q 受 is the gene content of the recipient InDel site.

9. A method for detecting the content of donor in a donor-free post-transplantation sample, characterized in that: The steps include: 1) using primer sequences such as nucleotide sequences shown in SEQ ID NO.120-179 to detect the InDel site combination of the transplant recipient to obtain the InDel site with a homozygous genotype; 2) Using the dPCR quantitative kit according to any one of claims 1 to 8 to detect the homozygous InDel site, and obtaining the content of the donor in the donor-free post-transplantation sample.

10. The detection method according to claim 9, characterized in that: In step 2), the method for calculating the content of donor in the donor-free post-transplantation sample is as follows: When the number of homozygous InDel sites is N, and the genotype of the donor at the homozygous InDel site is heterozygous or homozygous, the donor InDel site ratio is set to X = Q 供 / (Q 供 +Q 受 ), if the genotype of the donor at the homozygous InDel site is all heterozygous, the proportion of the donor InDel site is 2X, if the genotype of the donor at the homozygous InDel site is all homozygous, the proportion of the donor InDel site is X, and the final content of the donor in the transplanted sample without a donor is 1.5X; When the number of homozygous InDel sites is N', and the genotype of the donor at the homozygous InDel site is heterozygous or homozygous, the donor InDel site ratio is set to X = Q 供 / (Q 供 +Q 受 ), if the donor's genotype at the homozygous InDel site is all heterozygous, the donor InDel site ratio is 2X, if the donor's genotype at the homozygous InDel site is all homozygous, the donor InDel site ratio is X, therefore, the donor InDel site ratio conforms to the probability distribution of X and 2X, the donor InDel site ratio values ​​are arranged from small to large, 1.6-2.4 times the minimum value is determined as homozygous, and the rest are heterozygous, and the average value of each donor InDel site ratio is obtained, which is the content of the donor in the transplanted sample without a donor; Where N and N' are positive integers greater than 3, Q 供 is the gene content of the donor InDel site, Q 受 is the gene content of the recipient InDel site.

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