A method and kit for detecting the presence or proportion of a donor in a receptor sample

Abstract

The present application relates to a method for detecting SNP sites in a donor-derived sample and a recipient-derived sample. Further, the present application also relates to a method for detecting the presence or proportion of a donor in a recipient sample, and a kit for implementing the method.

Description

Technical Field

[0001] This application relates to the field of molecular diagnostics. Specifically, this application relates to a method for detecting SNP sites in a donor-derived sample and a recipient-derived sample. Further, this application also relates to a method for detecting the presence or proportion of a donor in a recipient sample, and a kit for implementing the method. Background Art

[0002] Heterologous DNA refers to non-self DNA from one or more individuals present in an individual's own body. Relative to the individual's own DNA, non-self DNA from one or more individuals can be defined as heterologous DNA. The most common example is the presence of donor-derived DNA in the recipient's body during allogeneic transplantation. Currently, the detection methods for heterologous DNA can be applied to two major aspects: bone marrow transplantation and solid organ transplantation.

[0003] In bone marrow transplantation, allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is a major treatment for many malignant hematological diseases and some non-malignant diseases. The detection methods for the chimeric state of hematopoietic stem cells after transplantation mainly rely on polymorphic genetic markers in the population, such as red blood cell antigens, human leukocyte antigen typing, short tandem repeat analysis (STR-PCR), etc. Currently, the International Bone Marrow Transplant Registry has listed the STR-PCR analysis technology as the gold standard for quantitatively monitoring the chimeric state of donor cells after HSCT. However, its drawbacks include non-specific interference caused by competitive amplification and shadow (Stutter) bands caused by gene dropout. Some studies have found that when the ratio of donor and recipient cells is below 5%-10%, the sensitivity will be significantly reduced (Bone Marrow Transplant, 2001, 28(5): 511-8). Other types of specific marker-based chimerism detection methods have been reported (J Mol Diagn, 2009, 11(1): 66-74), but they still have disadvantages such as low sample throughput, high consumable costs, poor detection sensitivity, and complex experimental operations.

[0004] Currently, the monitoring of grafts after solid organ transplantation often involves blood sampling for kidney and liver function tests, or tissue sampling with a puncture needle for pathological examination. For routine blood function tests, the sensitivity and specificity of various indicators such as creatinine, ALT, AST, bilirubin, etc. are not high, and they cannot accurately reflect the condition of the graft. According to the current gold standard of tissue biopsy, although it can directly reflect the condition of the graft, there are problems such as infection or injury caused by invasive detection, the lag of injury compared to treatment when abnormalities are detected, and inaccurate sampling of the puncture lesion site. Stephen R. Quake et al. have shown (Proc Natl Acad Sci U S A. 2011;108(15):6229-6234.) that the proportion of donor-derived cell free DNA (dd-cfDNA) in the recipient's plasma can reflect the status of the graft to a certain extent.

[0005] Currently, the detection of dd-cfDNA content is mostly based on human genetic polymorphism information (Sci TranslMed. 2014; 6(241): 241ra77.), or based on changes in epigenetic modifications (Gut. 2018; 67(12): 2204-2212.). Beck J et al. reported in their research (Clin Chem, 2013, 59(12): 1732-41.) that in the early postoperative studies of liver, kidney, and heart transplant patients, the single nucleotide polymorphism information of the donor heterologous was analyzed by qPCR technology, and the proportion of dd-cfDNA in the recipient plasma after transplantation was measured by dPCR technology. Grskovic et al. further developed and improved on the Next-Generation Sequencing (NGS) platform for the synchronous detection of a large number of SNP loci, and obtained reliable verification in the real-time monitoring of the dd-cfDNA proportion in a large number of heart transplant patients after surgery (J Mol Diagn, 2016, 18(6): 890-902.). A method for determining the proportion of donor-derived cfDNA in a recipient cfDNA sample was disclosed in a Chinese invention patent (CN106544407A), which captured and sequenced the target region by NGS to obtain a large amount of SNP genotyping information of the recipient sample; at the same time, the plasma cfDNA sample of the recipient after transplantation was captured and sequenced in the target region to analyze the proportion of dd-cfDNA in the total cfDNA. However, the above methods still have the following problems when applied to the detection of heterologous genomic DNA or heterologous cell-free DNA: The NGS technical solution has cumbersome experimental operations, a long detection cycle (3-7 working days), and high detection costs, and is not suitable for regular monitoring after transplantation; while other conventional technologies have disadvantages such as low throughput, many operation steps, low detection sensitivity, and easy contamination when opening the lid during the detection of genetic polymorphism information. Summary of the Invention

[0006] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, to better understand the present invention, the following provides definitions and explanations of related terms.

[0007] As used herein, the term "donor" refers to an individual who has provided or intends to provide an organ, tissue, or cell for transplantation to another individual (recipient). In certain embodiments, the donor has provided or intends to provide an organ (e.g., kidney, heart, lung, liver, pancreas, or any combination thereof) for transplantation to another individual (recipient). In certain embodiments, the donor has provided or intends to provide hematopoietic stem cells (e.g., bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, cord blood hematopoietic stem cells) or tissues or organs containing hematopoietic stem cells (e.g., bone marrow) for transplantation to another individual (recipient).

[0008] As used herein, the term "recipient" refers to an individual who has received or is intended to receive or transplant an organ, tissue, or cell for transplantation provided by another individual (donor). In certain embodiments, the recipient has received or is intended to receive or transplant an organ (e.g., kidney, heart, lung, liver, pancreas, or any combination thereof) provided by another individual (donor). In certain embodiments, the recipient has received or is intended to receive or transplant hematopoietic stem cells (e.g., bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, umbilical cord blood hematopoietic stem cells) or a tissue or organ containing hematopoietic stem cells (e.g., bone marrow) provided by another individual (donor).

[0009] As used herein, the term "individual" refers to any biological individual. In certain embodiments, the individual is an animal individual, such as a mammalian (e.g., human, mouse, rabbit, horse, sheep, etc.) individual.

[0010] As used herein, the term "donor chimerism rate" or "donor cell chimerism rate" refers to the phenomenon in which donor and recipient cells migrate and coexist with each other after the recipient receives an allogeneic or xenogeneic graft, and it is a medical detection index that can be used to evaluate the efficacy of allogeneic hematopoietic stem cell transplantation. Its result has a warning effect on post-transplant recurrence and can prompt clinical intervention at an early stage.

[0011] As used herein, the term "donor-free DNA ratio" or "dd-cfDNA ratio" is a potential detection index that can be used to evaluate rejection after organ transplantation. It is derived from the free DNA released into the plasma during the apoptosis and necrosis of graft cells, and its result indicates the degree of graft damage and can guide clinical intervention at an early stage.

[0012] As used herein, the term "cluster analysis" refers to an analytical process of grouping a set of physical or abstract objects into multiple classes composed of similar objects. The goal of cluster analysis is to collect data for classification based on similarity. Cluster analysis has its origins in many fields, including mathematics, computer science, statistics, biology, and economics. In different application fields, these technical methods are used to describe data, measure the similarity between different data sources, and classify data sources into different clusters.

[0013] As used herein, the term "SNP (Single Nucleotide Polymorphism)" refers to nucleic acid sequence polymorphisms caused by variations of a single nucleotide at the genomic level. The term "SNP locus" refers to a locus in the genome that has a single nucleotide polymorphism. In this document, SNP loci include a single locus with a single nucleotide polymorphism and loci with insertions or deletions of 1 or more (e.g., 1, 2, 3, 4, 5, 6, or more) nucleotides. In this document, SNP loci are named by their reference numbers (e.g., rs ID). One can query SNP loci and their genotypes in public databases using the rs ID, e.g., through the NCBI's dbSNP database, ChinaMAP database, JSNP database, etc. In this application, the selected or used SNP loci are preferably biallelic SNP loci.

[0014] As used herein, when referring to the "genotype" of an SNP locus, it refers to the collective term for the gene combinations at that SNP locus in all homologous chromosomes (usually two homologous chromosomes) of a biological individual. In this document, the "genotype" of an SNP locus refers to the gene combination at that SNP locus in a pair of homologous chromosomes from a donor or recipient. For example, "the genotype of an individual at the rs5858210 locus is AG / -" means that the two homologous chromosomes of this individual have nucleotide sequences "AG" and "-" (where "-" represents a deletion) at the rs5858210 locus respectively. "The genotype of an individual at the rs5858210 locus is AG / AG" means that both homologous chromosomes of this individual have the nucleotide sequence "AG" at the rs5858210 locus. Correspondingly, a segment of a gene (i.e., a nucleotide segment) on a single chromosome that contains this SNP locus is called an "allele" containing this SNP locus. As used herein, for a certain SNP locus, except for the nucleotide differences at that SNP locus, different alleles usually have exactly the same nucleotide sequence. When a pair of homologous chromosomes of an individual have the same nucleotide sequence (i.e., have the same allele) at a certain SNP locus, the genotype of this individual at that SNP locus is homozygous. When a pair of homologous chromosomes of an individual have different nucleotide sequences (i.e., have different alleles) at a certain SNP locus, the genotype of this individual at that SNP locus is heterozygous.

[0015] As used herein, the term "Fst" refers to the fixation index of a population, which can reflect the level of allelic heterozygosity in a population and is used to measure the degree of population differentiation. The value of Fst ranges from 0 to 1. When Fst is 1, it indicates that the alleles are fixed in each local population and are completely differentiated; when Fst is 0, it indicates that the genetic structures of different local populations are completely identical and there is no differentiation between populations. In the present application, the selected SNP loci preferably have Fst < 0.01 among different human races. The differentiation degree of these loci among different human races is very small, and the level of gene heterozygosity is close.

[0016] As used herein, the term "complementary" means that two nucleic acid sequences can form hydrogen bonds with each other according to the base pairing principle (Waston-Crick principle) and thus form a duplex. In the present application, the term "complementary" includes "substantially complementary" and "fully complementary". As used herein, the term "fully complementary" means that each base in one nucleic acid sequence can pair with the base in another nucleic acid strand without mismatch or gap. As used herein, the term "substantially complementary" means that most of the bases in one nucleic acid sequence can pair with the bases in another nucleic acid strand, allowing for mismatch or gap (e.g., mismatch or gap of one or several nucleotides). Generally, under conditions allowing nucleic acid hybridization, annealing or amplification, two nucleic acid sequences that are "complementary" (e.g., substantially complementary or fully complementary) will selectively / specifically hybridize or anneal and form a duplex. Accordingly, the term "non-complementary" means that two nucleic acid sequences cannot hybridize or anneal and cannot form a duplex under conditions allowing nucleic acid hybridization, annealing or amplification. As used herein, the term "not fully complementary" means that the bases in one nucleic acid sequence cannot pair completely with the bases in another nucleic acid strand, with at least one mismatch or gap.

[0017] As used herein, the terms "hybridization" and "annealing" mean the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. In the present application, "hybridization" and "annealing" have the same meaning and can be used interchangeably. Generally, two nucleic acid sequences that are fully complementary or substantially complementary can undergo hybridization or annealing. The complementarity required for two nucleic acid sequences to hybridize or anneal depends on the hybridization conditions used, especially the temperature.

[0018] As used herein, the term "PCR reaction" has the meaning commonly understood by those skilled in the art, which refers to a reaction (polymerase chain reaction) using a nucleic acid polymerase and primers to amplify a target nucleic acid. As used herein, the term "multiplex amplification" refers to the amplification of multiple target nucleic acids in the same reaction system. As used herein, the term "asymmetric amplification" refers to an amplification product obtained by amplifying a target nucleic acid, in which the amounts of two complementary nucleic acid strands are not the same, and the amount of one nucleic acid strand is greater than that of the other nucleic acid strand.

[0019] As used herein, and as commonly understood by those skilled in the art, the terms "forward" and "reverse" are merely for convenience in describing and distinguishing the two primers in a primer pair; they are relative and do not have a special meaning.

[0020] As used herein, the term "melting curve analysis" has the meaning commonly understood by those skilled in the art, which refers to a method of analyzing the presence or identity of a double-stranded nucleic acid molecule by measuring the melting curve of the double-stranded nucleic acid molecule, which is generally used to evaluate the dissociation characteristics of the double-stranded nucleic acid molecule during heating. The methods for performing melting curve analysis are well known to those skilled in the art (see, for example, The Journal of Molecular Diagnostics 2009, 11(2): 93-101). In the present application, the terms "melting curve analysis" and "melting analysis" have the same meaning and can be used interchangeably.

[0021] In certain preferred embodiments of the present application, melting curve analysis can be performed by using a detection probe labeled with a reporter group and a quencher group. Briefly, at ambient temperature, the detection probe can form a duplex with its complementary sequence through base pairing. In this case, the reporter group (e.g., a fluorescent group) and the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (e.g., fluorescent signal) emitted by the reporter group. At this time, the strongest signal (e.g., fluorescent signal) can be detected. As the temperature increases, the two strands of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe assumes a single-stranded freely coiled state. In this case, the reporter group (e.g., a fluorescent group) and the quencher group on the dissociated detection probe approach each other, and thus the signal (e.g., fluorescent signal) emitted by the reporter group (e.g., a fluorescent group) is absorbed by the quencher group. Therefore, as the temperature increases, the detected signal (e.g., fluorescent signal) gradually weakens. When the two strands of the duplex are completely dissociated, all the detection probes assume a single-stranded freely coiled state. In this case, the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups) on all the detection probes are absorbed by the quencher groups. Therefore, the signal (e.g., fluorescent signal) emitted by the reporter group (e.g., a fluorescent group) is basically undetectable. Therefore, by detecting the signal (e.g., fluorescent signal) emitted by the duplex containing the detection probe during the heating or cooling process, the hybridization and dissociation process of the detection probe with its complementary sequence can be observed, forming a curve in which the signal intensity changes with temperature. Further, by performing a derivative analysis on the obtained curve, a curve with the rate of change of signal intensity as the ordinate and temperature as the abscissa can be obtained (i.e., the melting curve of the duplex). The peak in this melting curve is the melting peak, and the temperature corresponding thereto is the melting point (T m ). Generally speaking, the higher the degree of matching between the detection probe and the complementary sequence (e.g., the fewer the mismatched bases and the more the paired bases), the higher the T m of the duplex. Therefore, by detecting the T m of the duplex, the presence and identity of the sequence complementary to the detection probe in the duplex can be determined. In this article, the terms "melting peak", "melting point", and "T m " have the same meaning and can be used interchangeably.

[0022] Through in-depth research, the inventors of the present application established a method for detecting SNP sites in a donor-derived sample and a recipient-derived sample by using multiplex asymmetric PCR amplification and multi-color probe melting curve analysis. On this basis, in combination with a digital PCR system, the present application developed a method for detecting the presence and proportion of a donor in a recipient sample, as well as a kit for implementing the method.

[0023] Accordingly, in one aspect, the present application provides a method for detecting SNP sites with different genotypes between a donor and a recipient, which comprises the following steps:

[0024] (a) providing a first sample containing one or more target nucleic acids derived from the donor, and a second sample containing one or more target nucleic acids derived from the recipient, the target nucleic acids comprising one or more candidate SNP sites, and,

[0025] providing a first universal primer and a second universal primer, and, for each candidate SNP site, providing at least one pair of target-specific primers; wherein,

[0026] the first universal primer comprises a first universal sequence;

[0027] the second universal primer comprises a second universal sequence, the second universal sequence comprising the first universal sequence and additionally comprising at least one nucleotide at the 3'-end of the first universal sequence;

[0028] the pair of target-specific primers is capable of amplifying using the target nucleic acid as a template to generate a nucleic acid product containing the candidate SNP site, and the pair of target-specific primers comprises a forward primer and a reverse primer, wherein the forward primer comprises the first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3'-end of the first universal sequence; the reverse primer comprises the second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3'-end of the second universal sequence; and, the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer; and

[0029] (b) under conditions allowing nucleic acid amplification, using the first universal primer, the second universal primer and the pair of target-specific primers to amplify the target nucleic acids in the first sample and the second sample respectively, so as to obtain amplification products corresponding to the first sample and the second sample respectively;

[0030] (c) performing melting curve analysis on the amplification products corresponding to the first sample and the second sample obtained in step (b) respectively;

[0031] (d) according to the melting curve analysis results in step (c), determining such SNP sites: at which the first sample and the second sample have different genotypes.

[0032] In the method of the present application, the forward primer and the reverse primer respectively comprise a forward nucleotide sequence and a reverse nucleotide sequence specific to the target nucleic acid. Thus, during the PCR reaction, the target-specific primer pair (forward primer and reverse primer) will anneal to the target nucleic acid and initiate PCR amplification to produce an initial amplification product, which comprises two nucleic acid strands (nucleic acid strand A and nucleic acid strand B) complementary to the forward primer and the reverse primer respectively. Further, since both the forward primer and the first universal primer comprise a first universal sequence, the nucleic acid strand A complementary to the forward primer can also be complementary to the first universal primer. Similarly, the nucleic acid strand B complementary to the reverse primer can also be complementary to the second universal primer.

[0033] Therefore, as the PCR reaction proceeds, the first universal primer and the second universal primer will respectively anneal to nucleic acid strand A and nucleic acid strand B of the initial amplification product and further initiate PCR amplification. During this process, since the reverse primer / the second universal primer comprises the first universal sequence, the first universal primer can not only anneal to nucleic acid strand A (the nucleic acid strand complementary to the forward primer / the first universal primer) and synthesize its complementary strand, but also anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer / the second universal primer) and synthesize its complementary strand. That is to say, the first universal primer can amplify both nucleic acid strand A and nucleic acid strand B of the initial amplification product simultaneously. At the same time, the second universal primer comprises additional nucleotides at the 3'-end of the first universal sequence. Therefore, although the second universal primer may also anneal to nucleic acid strand A (the nucleic acid strand complementary to the forward primer / the first universal primer, which has a sequence complementary to the forward primer), it is mismatched with nucleic acid strand A at the 3'-end (i.e., it cannot be completely complementary at the 3'-end). Thus, during the amplification process, the second universal primer will preferentially anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer / the second universal primer) and synthesize its complementary strand, and basically cannot extend to synthesize the complementary strand of nucleic acid strand A (the nucleic acid strand complementary to the first forward primer / the first universal primer).

[0034] Therefore, as the PCR amplification proceeds, the synthesis efficiency of the complementary strand of nucleic acid strand A (nucleic acid strand B) will be significantly lower than that of the complementary strand of nucleic acid strand B (nucleic acid strand A), resulting in the complementary strand of nucleic acid strand B (nucleic acid strand A) being synthesized and amplified in large amounts, while the synthesis and amplification of the complementary strand of nucleic acid strand A (nucleic acid strand B) are inhibited, thereby producing a large amount of single-stranded products (nucleic acid strand A, which contains a sequence complementary to the forward primer / the first universal primer and the sequence of the reverse primer / the second universal primer), achieving asymmetric amplification of the target nucleic acid containing one or more SNP sites. Therefore, in steps (a) and (b) of the method of the present application, asymmetric amplification of one or more target nucleic acids in the sample is achieved.

[0035] In addition, since both the forward primer and the reverse primer contain the first universal sequence, during the PCR reaction, the primer dimer formed due to the non-specific amplification of the forward primer and the reverse primer will generate single-stranded nucleic acids with reverse sequences complementary to each other at their 5' and 3' ends after denaturation. This single-stranded nucleic acid is prone to self-annealing during the annealing stage, forming a stable panhandle structure that prevents the annealing and extension of the first universal primer and the second universal primer to this single-stranded nucleic acid, thereby inhibiting the further amplification of the primer dimer. Therefore, in the method of the present invention, the non-specific amplification of the primer dimer can be effectively inhibited.

[0036] In certain embodiments, in step (d) of the method, the genotypes of each candidate SNP locus of the first sample and the second sample are determined according to the melting curve analysis results, thereby detecting the SNP loci where the donor and the recipient have different genotypes.

[0037] In certain embodiments, the recipient has received or is intended to receive or transplant organs, tissues or cells from the donor.

[0038] In certain embodiments, the recipient has received or is intended to receive or transplant organs from the donor (e.g., kidneys, heart, lungs, liver, pancreas or any combination thereof).

[0039] In certain embodiments, the recipient has received or is intended to receive or transplant hematopoietic stem cells from the donor (e.g., bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, cord blood hematopoietic stem cells or any combination thereof) or tissues or organs containing hematopoietic stem cells (e.g., bone marrow).

[0040] In certain embodiments, the second sample substantially does not contain nucleic acids from the donor. In such embodiments, "substantially does not contain nucleic acids from the donor" means that it does not contain nucleic acids from the donor, or the total nucleic acids from the donor in the second sample do not exceed 10% (e.g., do not exceed 5%, do not exceed 3%, do not exceed 1%, or lower).

[0041] In certain embodiments, the first sample is from the donor; for example, the first sample contains cells or tissues from the donor; for example, the first sample is selected from the skin, saliva, urine, blood, hair, nails of the donor, or any combination thereof.

[0042] In certain embodiments, the second sample is from the recipient (e.g., the recipient who has or has not undergone a transplantation surgery); for example, the second sample contains cells or tissues from the recipient; for example, the second sample is selected from the skin, saliva, urine, blood, hair, nails of the recipient, or any combination thereof.

[0043] In certain embodiments, for a recipient who has not undergone a transplantation surgery, the second sample can be any cell or tissue (e.g., skin, saliva, urine, blood, etc.). For a recipient who has undergone a transplantation surgery, the second sample substantially does not contain nucleic acids from the donor.

[0044] In certain preferred embodiments, for a recipient who has undergone a hematopoietic stem cell transplantation, the second sample can be selected from skin, saliva, urine, hair, nails, or tissue, etc., but cannot be selected from blood, because the blood sample of a recipient who has undergone a hematopoietic stem cell transplantation may contain a large amount of donor nucleic acids. In certain preferred embodiments, for a recipient who has undergone a kidney transplantation, the second sample can be selected from skin, saliva, hair, nails, or tissue, etc., but cannot be selected from blood and urine, because the blood and urine samples of a recipient who has undergone a kidney transplantation may contain a large amount of donor nucleic acids. In certain preferred embodiments, for a recipient who has undergone a liver transplantation, the second sample can be selected from skin, saliva, hair, nails, urine, or tissue, etc., but cannot be selected from blood, because the blood sample of a recipient who has undergone a kidney transplantation may contain a large amount of donor nucleic acids.

[0045] In certain embodiments, in step (a), for each candidate SNP locus, a detection probe is further provided, the detection probe comprising a nucleotide sequence specific to the target nucleic acid and capable of annealing or hybridizing to the region of the target nucleic acid containing the candidate SNP locus, and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence;

[0046] And, in step (c), the melting curve analysis is respectively performed on the amplification products corresponding to the first sample and the second sample obtained in step (b) using the detection probe.

[0047] In certain embodiments, the first sample contains DNA (e.g., genomic DNA).

[0048] In certain embodiments, the second sample contains DNA (e.g., genomic DNA).

[0049] In a second aspect, the present application provides a method for detecting the presence or proportion of donor nucleic acids in a sample of a recipient who has undergone a transplantation surgery, wherein the method comprises the following steps:

[0050] (1) Providing a test sample containing nucleic acids from a recipient who has received a transplantation of cells, tissues, or organs from a donor;

[0051] (2) Identify one or more target SNP loci, wherein at the target SNP loci, the recipient has a first genotype containing a first allele, and the donor has a second genotype containing a second allele, wherein the first genotype is different from the second genotype, and the first allele is different from the second allele;

[0052] (3) Quantitatively detect the first allele and the second allele of each target SNP locus in the sample to be tested; then, determine the presence or proportion of the donor's nucleic acid in the sample to be tested according to the results of the quantitative detection of the first allele and the second allele.

[0053] In certain embodiments, in step (2), different alleles at a certain SNP locus can be distinguished by a mechanism selected from the following to identify the target SNP locus: probe hybridization, primer extension, hybridization ligation, and specific enzymatic digestion. In certain embodiments, in step (2), the target SNP locus can be identified by a method selected from the following: sequencing methods (e.g., first-generation sequencing method, pyrosequencing method, second-generation sequencing method), chip methods (e.g., using solid-phase chips or liquid-phase chips capable of detecting SNPs), qPCR-based detection methods (e.g., Taqman probe method), mass spectrometry (such as iPLEX TM Gold based on MassARRAY), chromatography (such as denaturing high-performance liquid chromatography dHPLC), electrophoresis (such as SNPshot method), and melting curve analysis-based detection methods. In certain embodiments, in step (2), the target SNP locus is identified by a detection method based on multiplex PCR combined with melting curve analysis.

[0054] In certain embodiments, the target SNP locus is identified by the method described above.

[0055] In certain embodiments, in step (3), digital PCR is used to quantitatively detect the first allele and the second allele of each target SNP locus in the sample.

[0056] In certain embodiments, step (3) is carried out by the following scheme:

[0057] (I) Select at least 1 (e.g., 1, 2, 3, or more) target SNP loci from step (2), and for each selected target SNP locus, provide an amplification primer set and a probe set, wherein,

[0058] (I-1) The amplification primer set contains at least one amplification primer (e.g., a pair of amplification primers or more amplification primers), which can specifically amplify a nucleic acid molecule containing the target SNP locus under conditions allowing nucleic acid hybridization or annealing;

[0059] (I-2) The probe set includes a first probe and a second probe; wherein,

[0060] (i) The first probe and the second probe are each independently labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and, the first probe and the second probe are labeled with different reporter groups (such as fluorescent groups); and

[0061] (ii) The first probe is capable of hybridizing or annealing (preferably completely complementary) with a nucleic acid molecule containing the first allele of the target SNP site, and the second probe is capable of hybridizing or annealing (preferably completely complementary) with a nucleic acid molecule containing the second allele of the target SNP site; and, the first probe and the second probe are specific for different alleles;

[0062] (II) Perform digital PCR on the recipient sample using the amplification primer set and the probe set, and quantitatively detect the nucleic acid molecules with the first allele and the nucleic acid molecules with the second allele;

[0063] (III) Determine the presence or proportion of the donor nucleic acid in the sample to be tested according to the quantitative detection result of step (II).

[0064] In some embodiments, the first probe specifically anneals or hybridizes with a nucleic acid molecule with the first allele during the digital PCR reaction; and, the second probe specifically anneals or hybridizes with a nucleic acid molecule with the second allele during the digital PCR reaction.

[0065] In some embodiments, the first probe does not anneal or hybridize with a nucleic acid molecule with the second allele during the digital PCR reaction; and / or, the second probe does not anneal or hybridize with a nucleic acid molecule with the first allele during the digital PCR reaction.

[0066] In some embodiments, before step (3), the sample to be tested from the recipient is pretreated.

[0067] In some embodiments, the pretreatment includes nucleic acid extraction of the sample and / or enrichment of the nucleic acid in the sample (for example, by concentration and / or amplification).

[0068] In some embodiments, the recipient has received or transplanted hematopoietic stem cells from a donor (such as bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, umbilical cord blood hematopoietic stem cells or any combination thereof) or a tissue or organ containing hematopoietic stem cells (such as bone marrow).

[0069] In certain embodiments, the sample to be tested comprises blood from a recipient after transplantation (e.g., peripheral blood) or a component thereof (e.g., blood cells, plasma, monocytes, granulocytes, T cells, or any combination thereof).

[0070] In certain embodiments, the target SNP locus is an SNP locus at which the recipient has a first genotype comprising a homozygous first allele and the donor has a second genotype comprising a homozygous second allele; or, the recipient has a first genotype comprising a heterozygous first allele and second allele and the donor has a second genotype comprising a homozygous second allele.

[0071] In certain embodiments, the target SNP locus is an SNP locus at which the recipient has a first genotype comprising a homozygous first allele and the donor has a second genotype comprising a homozygous second allele.

[0072] In certain embodiments, the proportion of the donor in the recipient sample is calculated by one or more of the following methods:

[0073] (1) When the target SNP locus is an SNP locus at which the recipient has a first genotype comprising a homozygous first allele (e.g., BB) and the donor has a second genotype comprising a homozygous second allele (e.g., AA), the proportion of the donor in the recipient sample is:

[0074] where N B is the copy number of allele B (which can be determined by digital PCR), and N A is the copy number of allele A (which can be determined by digital PCR);

[0075] (2) When the target SNP locus is an SNP locus at which the recipient has a first genotype comprising a heterozygous first allele and second allele (e.g., AB) and the donor has a second genotype comprising a homozygous second allele (e.g., AA), the proportion of the donor in the recipient sample is:

[0076] where N B is the copy number of allele B (which can be determined by digital PCR), and N A is the copy number of allele A (which can be determined by digital PCR).

[0077] In certain embodiments, the recipient has received or transplanted an organ from a donor (e.g., a kidney, heart, lung, liver, pancreas, or any combination thereof).

[0078] In certain embodiments, the recipient has received or transplanted a kidney from a donor.

[0079] In certain embodiments, the sample to be tested comprises blood (e.g., peripheral blood) or urine (especially in the case of kidney transplantation) from the recipient after transplantation.

[0080] In certain embodiments, the target SNP locus is an SNP locus at which the donor has a first genotype comprising a homozygous first allele and the recipient has a second genotype comprising a homozygous second allele; or, the donor has a first genotype comprising a heterozygous first allele and second allele and the recipient has a second genotype comprising a homozygous second allele.

[0081] In certain embodiments, the target SNP locus is an SNP locus at which the donor has a first genotype comprising a homozygous first allele and the recipient has a second genotype comprising a homozygous second allele.

[0082] In certain embodiments, the proportion of the recipient in the donor sample is calculated by one or more of the following methods:

[0083] (1) When the target SNP locus is an SNP locus at which the donor has a first genotype comprising a homozygous first allele (e.g., BB) and the recipient has a second genotype comprising a homozygous second allele (e.g., AA), the proportion of the donor in the recipient sample is:

[0084] where N B is the copy number of allele B (which can be determined by digital PCR), and N A is the copy number of allele A (which can be determined by digital PCR);

[0085] (2) When the target SNP locus is an SNP locus at which the donor has a first genotype comprising a heterozygous first allele and second allele (e.g., AB) and the recipient has a second genotype comprising a homozygous second allele (e.g., AA), the proportion of the recipient in the donor sample is:

[0086] where N B is the copy number of allele B (which can be determined by digital PCR), and N A is the copy number of allele A (which can be determined by digital PCR).

[0087] In certain embodiments, steps (a)-(b) of the method are carried out by a protocol comprising the following steps (I)-(VI):

[0088] (I) Provide the first sample, the second sample, the first universal primer and the second universal primer, and the target-specific primer pair; and optionally, the detection probe;

[0089] (II) Mix the samples with the first universal primer, the second universal primer, the target-specific primer pair, a nucleic acid polymerase, and optionally, the detection probe;

[0090] (III) Incubate the product of the previous step under conditions that permit nucleic acid denaturation;

[0091] (IV) Incubate the product of the previous step under conditions that permit nucleic acid annealing or hybridization;

[0092] (V) Incubate the product of the previous step under conditions that permit nucleic acid extension; and

[0093] (VI) Optionally, repeat steps (III)-(V) one or more times.

[0094] In certain embodiments, in step (III), the product of step (II) is incubated at a temperature of 80-105 °C to denature the nucleic acid.

[0095] In certain embodiments, in step (III), the product of step (II) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min.

[0096] In certain embodiments, in step (IV), the product of step (III) is incubated at a temperature of 35-40 °C, 40-45 °C, 45-50 °C, 50-55 °C, 55-60 °C, 60-65 °C, or 65-70 °C to permit nucleic acid annealing or hybridization.

[0097] In certain embodiments, in step (IV), the product of step (III) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min.

[0098] In certain embodiments, in step (V), the product of step (IV) is incubated at a temperature of 35-40 °C, 40-45 °C, 45-50 °C, 50-55 °C, 55-60 °C, 60-65 °C, 65-70 °C, 70-75 °C, 75-80 °C, 80-85 °C to permit nucleic acid extension.

[0099] In certain embodiments, in step (V), the product of step (IV) is incubated for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, 2 - 5 min, 5 - 10 min, 10 - 20 min, or 20 - 30 min.

[0100] In certain embodiments, steps (IV) and (V) are carried out at the same or different temperatures.

[0101] In certain embodiments, steps (III) - (V) are repeated at least once, for example at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times. In certain embodiments, when steps (III) - (V) are repeated one or more times, the conditions used for steps (III) - (V) in each cycle are independently the same or different.

[0102] In certain embodiments, the primers of the amplification primer set independently have lengths of 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt.

[0103] In certain embodiments, the primers of the amplification primer set or any of its components independently comprise or consist of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, non - natural nucleotides, or any combination thereof.

[0104] In certain embodiments, the amplification primer set independently includes primer pairs having nucleotide sequences selected from the following or any combination thereof (e.g., any combination of 5 pairs, 10 pairs, 15 pairs, 20 pairs, 23 pairs): SEQ ID NO:72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

[0105] In certain embodiments, each of the first probe and the second probe independently comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, unnatural nucleotides (e.g., peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof.

[0106] In certain embodiments, the lengths of the first probe and the second probe are independently 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 200 nt, 200 - 300 nt, 300 - 400 nt, 400 - 500 nt, 500 - 600 nt, 600 - 700 nt, 700 - 800 nt, 800 - 900 nt, 900 - 1000 nt.

[0107] In certain embodiments, each of the first probe and the second probe independently has a 3'-OH terminus; alternatively, the 3'-terminus of the probe is blocked; for example, by adding a chemical moiety (e.g., biotin or alkyl) to the 3'-OH of the last nucleotide of the probe, by removing the 3'-OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide, thereby blocking the 3'-terminus of the detection probe.

[0108] In certain embodiments, each of the first probe and the second probe is independently a self-quenching probe; for example, the probe is labeled with a reporter group at its 5'-end or upstream and with a quencher group at its 3'-end or downstream, or with a reporter group at its 3'-end or downstream and with a quencher group at its 5'-end or upstream. In certain embodiments, the reporter group and the quencher group are separated by a distance of 10 - 80 nt or more.

[0109] In certain embodiments, the reporter groups in the probes are independently fluorescent groups (e.g., ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and, the quencher groups are molecules or groups capable of absorbing / quenching the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA).

[0110] In certain embodiments, the first probe and the second probe are each independently linear or have a hairpin structure.

[0111] In certain embodiments, the first probe and the second probe have different reporter groups. In certain embodiments, the first probe and the second probe are degradable by a nucleic acid polymerase (e.g., DNA polymerase).

[0112] In certain embodiments, the probe set includes probes having nucleotide sequences selected from the following or any combination thereof (e.g., any combination of 5, 10, 20, 40, 60): SEQ ID NO:73, 74, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

[0113] In a third aspect, the present application provides a method for identifying an SNP locus in a receptor having a first genotype containing a homozygous first allele, comprising the following steps:

[0114] (a) Providing a fifth sample from the receptor, wherein the fifth sample contains one or more target nucleic acids derived from the receptor and substantially does not contain nucleic acids derived from a donor; the target nucleic acids contain one or more candidate SNP loci, and

[0115] providing a first universal primer and a second universal primer, and for each candidate SNP locus, providing at least one pair of target-specific primers; wherein

[0116] the first universal primer contains a first universal sequence;

[0117] the second universal primer contains a second universal sequence, the second universal sequence contains the first universal sequence and additionally contains at least one nucleotide at the 3' end of the first universal sequence;

[0118] The target-specific primer pair can amplify using the target nucleic acid as a template to generate a nucleic acid product containing the candidate SNP site, and the target-specific primer pair comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3'-end of the first universal sequence; the reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3'-end of the second universal sequence; and, the second universal sequence cannot be fully complementary to the complementary sequence of the forward primer; and

[0119] (b) Under conditions allowing nucleic acid amplification, use the first universal primer and the second universal primer and the target-specific primer pair to amplify the target nucleic acid in the fifth sample, thereby obtaining an amplification product corresponding to the fifth sample;

[0120] (c) Perform melting curve analysis on the amplification product corresponding to the fifth sample obtained in step (b);

[0121] (d) According to the melting curve analysis result of step (c), identify such SNP sites: at these sites, the receptor has a first genotype containing a homozygous first allele.

[0122] In the method of the present application, the forward primer and the reverse primer respectively comprise a forward nucleotide sequence and a reverse nucleotide sequence specific to the target nucleic acid. Thus, during the PCR reaction, the target-specific primer pair (forward primer and reverse primer) will anneal to the target nucleic acid and initiate PCR amplification to generate an initial amplification product, which contains two nucleic acid strands (nucleic acid strand A and nucleic acid strand B) complementary to the forward primer and the reverse primer respectively. Further, since both the forward primer and the first universal primer comprise the first universal sequence, the nucleic acid strand A complementary to the forward primer can also be complementary to the first universal primer. Similarly, the nucleic acid strand B complementary to the reverse primer can also be complementary to the second universal primer.

[0123] Therefore, as the PCR reaction proceeds, the first universal primer and the second universal primer will anneal to nucleic acid strand A and nucleic acid strand B of the initial amplification product respectively, and further initiate PCR amplification. During this process, since the reverse primer / second universal primer contains the first universal sequence, the first universal primer can not only anneal to nucleic acid strand A (the nucleic acid strand complementary to the forward primer / first universal primer) and synthesize its complementary strand, but also anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer / second universal primer) and synthesize its complementary strand. That is to say, the first universal primer can amplify both nucleic acid strand A and nucleic acid strand B of the initial amplification product simultaneously. At the same time, the second universal primer contains additional nucleotides at the 3' end of the first universal sequence. Therefore, although the second universal primer may also anneal to nucleic acid strand A (the nucleic acid strand complementary to the forward primer / first universal primer, which has a sequence complementary to the forward primer), it is mismatched with nucleic acid strand A at the 3' end (i.e., it cannot be completely complementary at the 3' end). Thus, during the amplification process, the second universal primer will preferentially anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer / second universal primer) and synthesize its complementary strand, and basically cannot extend to synthesize the complementary strand of nucleic acid strand A (the nucleic acid strand complementary to the first forward primer / first universal primer).

[0124] Therefore, as the PCR amplification proceeds, the synthesis efficiency of the complementary strand of nucleic acid strand A (nucleic acid strand B) will be significantly lower than that of the complementary strand of nucleic acid strand B (nucleic acid strand A), resulting in a large amount of synthesis and amplification of the complementary strand of nucleic acid strand B (nucleic acid strand A), while the synthesis and amplification of the complementary strand of nucleic acid strand A (nucleic acid strand B) are inhibited, thereby generating a large amount of single-stranded products (nucleic acid strand A, which contains a sequence complementary to the forward primer / first universal primer and the sequence of the reverse primer / second universal primer), achieving asymmetric amplification of the target nucleic acid containing one or more SNP sites. Therefore, in steps (a) and (b) of the method of the present application, asymmetric amplification of one or more target nucleic acids in the sample is achieved.

[0125] In addition, since both the forward primer and the reverse primer contain the first universal sequence, during the PCR reaction process, the primer dimer formed due to the non-specific amplification of the forward primer and the reverse primer will generate single-stranded nucleic acids with reverse sequences complementary to each other at their 5' end and 3' end after denaturation. This single-stranded nucleic acid is prone to self-annealing during the annealing stage, forming a stable panhandle structure, which prevents the first universal primer and the second universal primer from annealing to and extending this single-stranded nucleic acid, thereby inhibiting the further amplification of the primer dimer. Therefore, in the method of the present invention, the non-specific amplification of the primer dimer can be effectively inhibited.

[0126] In certain embodiments, "substantially free of nucleic acids from the donor" means free of nucleic acids from the donor, or the total nucleic acids from the donor in the fifth sample do not exceed 10% (e.g., do not exceed 5%, do not exceed 3%, do not exceed 1%, or lower).

[0127] In certain embodiments, the fifth sample is from the recipient (e.g., a recipient who has or has not undergone a transplantation surgery); for example, the fifth sample comprises cells or tissues from the recipient; for example, the fifth sample is selected from the skin, saliva, urine, blood, hair, nails, or any combination thereof from the recipient.

[0128] In certain embodiments, in step (a), for each candidate SNP locus, a detection probe is further provided, the detection probe comprising a nucleotide sequence specific to the target nucleic acid and capable of annealing or hybridizing to the region of the target nucleic acid containing the candidate SNP locus, and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence;

[0129] And, in step (c), melting curve analysis is performed on the amplification products corresponding to the fifth sample obtained in step (b) using the detection probe.

[0130] In certain embodiments, the fifth sample comprises DNA (e.g., genomic DNA).

[0131] In a fourth aspect, the present application provides a method for detecting the presence or proportion of nucleic acids from a donor in a recipient sample after transplantation surgery, wherein the method comprises the following steps:

[0132] (1) Providing a test sample containing nucleic acids from a recipient who has received transplantation of cells, tissues, or organs from a donor;

[0133] (2) Identifying a plurality (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more) such candidate SNP loci that exhibit at least a first allele and a second allele in the species to which the recipient belongs, and at the candidate SNP loci, the recipient has a first genotype comprising a homozygous first allele;

[0134] (3) Quantitatively detecting each allele of each candidate SNP locus of the test sample;

[0135] (4) Based on the quantitative detection results in step (3), select such target SNP sites from the candidate SNP sites: the test sample shows the signal of the first allele and the signal of the second allele at this site;

[0136] (5) Based on the results of quantitative detection of the first allele and the second allele of the target SNP site, determine the presence or proportion of the donor's nucleic acid in the test receptor sample.

[0137] In certain embodiments, in step (2), different alleles at a certain SNP site can be distinguished by mechanisms selected from the following to identify candidate SNP sites: probe hybridization, primer extension, ligation-mediated hybridization, and specific enzymatic cleavage. In certain embodiments, in step (2), candidate SNP sites can be identified by methods selected from the following: sequencing methods (e.g., first-generation sequencing, pyrosequencing, second-generation sequencing), chip methods (e.g., using solid-phase chips or liquid-phase chips capable of detecting SNPs), qPCR-based detection methods (e.g., Taqman probe method), mass spectrometry (such as iPLEX TM Gold based on MassARRAY), chromatography (such as denaturing high-performance liquid chromatography dHPLC), electrophoresis (such as SNPshot method), and melting curve analysis-based detection methods. In certain embodiments, in step (2), the candidate SNP sites are identified by a detection method based on multiplex PCR combined with melting curve analysis.

[0138] In certain embodiments, the candidate SNP sites are identified by the methods described above.

[0139] In certain embodiments, in step (3), digital PCR is used to quantitatively detect each allele of each candidate SNP site separately.

[0140] In certain embodiments, step (3) is carried out by the following scheme:

[0141] (I) Select a plurality (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more) of candidate SNP sites from step (2), and for each selected candidate SNP site, provide an amplification primer set and a probe set, wherein,

[0142] (I-1) The amplification primer set contains at least one amplification primer (e.g., a pair of amplification primers or more amplification primers), which can specifically amplify a nucleic acid molecule containing the candidate SNP site under conditions allowing nucleic acid hybridization or annealing;

[0143] (I-2) The probe set contains a first probe and a second probe; wherein,

[0144] (i) The first probe and the second probe are each independently labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the first probe and the second probe are labeled with different reporter groups (such as fluorescent groups); and

[0145] (ii) The first probe is capable of hybridizing or annealing (preferably completely complementary) with a nucleic acid molecule containing the first allele of the candidate SNP site, and the second probe is capable of hybridizing or annealing (preferably completely complementary) with a nucleic acid molecule containing the second allele of the candidate SNP site; and the first probe and the second probe are specific for different alleles;

[0146] (II) Using the amplification primer set and the probe set to perform digital PCR on the test sample from the recipient, and quantitatively detecting the nucleic acid molecules with the first allele and the nucleic acid molecules with the second allele;

[0147] In some embodiments, the first probe specifically anneals or hybridizes with a nucleic acid molecule having the first allele during the digital PCR reaction; and the second probe specifically anneals or hybridizes with a nucleic acid molecule having the second allele during the digital PCR reaction;

[0148] In some embodiments, the first probe does not anneal or hybridize with a nucleic acid molecule having the second allele during the digital PCR reaction; and / or the second probe does not anneal or hybridize with a nucleic acid molecule having the first allele during the digital PCR reaction;

[0149] In the method of the present application, taking the first probe in the probe set as an example, it can hybridize or anneal (preferably completely complementary) with a nucleic acid molecule having a first allele. Therefore, during the digital PCR reaction, during the annealing or extension process, the first probe will form a duplex with the nucleic acid molecule and be degraded by a nucleic acid polymerase (such as DNA polymerase) during amplification, releasing a reporter group (such as a fluorescent group). Thus, after the digital PCR amplification reaction is completed, the endpoint fluorescence of each droplet is detected by a droplet detector. According to the signal (such as the first fluorescence signal) intensity of the free first reporter group (such as the first fluorescent group), the number of positive droplets and negative droplets can be determined, and thus the amount of nucleic acid molecules having the first allele in the sample can be determined. Similarly, after the digital PCR amplification reaction is completed, the endpoint fluorescence of each droplet is detected by a droplet detector. According to the signal (such as the second fluorescence signal) intensity of the free second reporter group (such as the second fluorescent group), the number of positive droplets and negative droplets can be determined, and thus the amount of nucleic acid molecules having the second allele in the sample can be determined. Since the donor / recipient genotype is different, the corresponding first / second allele content is also different. Therefore, by comparing and analyzing the amounts of nucleic acid molecules containing the first / second allele, it can be determined whether a donor exists in the recipient sample, and optionally, the proportion of the donor can be determined.

[0150] In the method of the present application, in certain embodiments, the first probe does not anneal or hybridize with a nucleic acid molecule having a second allele during the digital PCR reaction; and / or, the second probe does not anneal or hybridize with a nucleic acid molecule having a first allele during the digital PCR reaction. It is easy to understand that the hybridization specificity of the first / second probe is particularly advantageous, which can help accurately determine the content of the first allele / second allele, and thus help calculate the respective proportions of the donor sample and the recipient sample. In certain embodiments, the hybridization specificity of the first / second probe can be obtained by controlling the annealing temperature and / or extension temperature of the digital PCR reaction. For example, the annealing temperature and / or extension temperature can be set lower than the melting point of the duplex formed by the first probe and a nucleic acid molecule having a first allele, but higher than the melting point of the duplex formed by the first probe and a nucleic acid molecule having a second allele, so that the first probe hybridizes with a nucleic acid molecule having a first allele during the digital PCR reaction, but does not hybridize with a nucleic acid molecule having a second allele. Similarly, the annealing temperature and / or extension temperature can be set lower than the melting point of the duplex formed by the second probe and a nucleic acid molecule having a second allele, but higher than the melting point of the duplex formed by the second probe and a nucleic acid molecule having a first allele, so that the second probe hybridizes with a nucleic acid molecule having a second allele during the digital PCR reaction, but does not hybridize with a nucleic acid molecule having a first allele.

[0151] In the method of the present application, the copy number of alleles can be detected by a digital PCR platform and directly output by software according to the principle of Poisson distribution. For the relevant principles and calculation methods, reference can be made to, for example, Milbury CA, Zhong Q, Lin J, et al. Determining lower limits of detection of digital PCR assays for cancer-related gene mutations. Biomol Detect Quantif. 2014;1(1):8 - 22. Published 2014 Aug 20. doi:10.1016 / j.bdq.2014.08.001.

[0152] In certain embodiments, in step (5), cluster analysis is performed on the quantitative detection results of the second alleles of multiple (such as at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more) target SNP loci; then, according to the results of the cluster analysis, the genotype of the donor at each target SNP locus is determined; then, according to the genotypes of the receptor and the donor at each target SNP locus, and the quantitative detection results of the first and second alleles in the test sample, the presence or proportion of the donor's nucleic acid in the test receptor sample is determined.

[0153] Since the receptor contains homozygous first alleles at the target SNP loci, the second allele signals detected in the test sample must come from the donor. In other words, the genotype of the donor at this target SNP locus may be homozygous second alleles or heterozygous first and second alleles. Theoretically, for the same sample, during the digital PCR quantitative detection process, the detection result (corresponding to the absolute copy number) of the second allele at a homozygous SNP locus will be twice that of the second allele at a heterozygous SNP locus. Therefore, by performing cluster analysis on the detection results of the second alleles of multiple target SNP loci, it can be determined which target SNP loci the donor has homozygous second alleles at, and which target SNP loci the donor has heterozygous first and second alleles at; among them, the detection result (corresponding to the absolute copy number) of the former will be twice that of the latter. In other words, by performing cluster analysis on the detection signals of the second alleles, the genotype of the donor at each target SNP locus can be determined. On this basis, according to the genotypes of the receptor and the donor at each target SNP locus, and the quantitative detection results of the first and second alleles in the test sample, the presence or proportion of the donor's nucleic acid in the test receptor sample can be easily determined.

[0154] In certain embodiments, before step (3), the test sample from the recipient is pre-treated.

[0155] In certain embodiments, the pre-treatment includes nucleic acid extraction of the sample and / or enrichment of nucleic acids in the sample (e.g., by concentration and / or amplification).

[0156] In certain embodiments, the recipient has received or transplanted hematopoietic stem cells from a donor (e.g., bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, cord blood hematopoietic stem cells, or any combination thereof) or a tissue or organ containing hematopoietic stem cells (e.g., spinal cord).

[0157] In certain embodiments, the test sample comprises blood (e.g., peripheral blood) or a component thereof (e.g., blood cells, plasma, monocytes, granulocytes, T cells, or any combination thereof) from the recipient after transplantation.

[0158] In certain embodiments, the recipient has received or transplanted an organ from a donor (e.g., kidney, heart, lung, liver, pancreas, or any combination thereof).

[0159] In certain embodiments, the recipient has received or transplanted a kidney from a donor.

[0160] In certain embodiments, the test sample comprises blood (e.g., peripheral blood) or urine (especially in the case of kidney transplantation) from the recipient after transplantation.

[0161] In certain embodiments, steps (a)-(b) of the method are carried out by a protocol comprising the following steps (I)-(VI):

[0162] (I) Provide the fifth sample, the first universal primer and the second universal primer, and the target-specific primer pair; and optionally, the detection probe;

[0163] (II) Mix the fifth sample with the first universal primer, the second universal primer, the target-specific primer pair, a nucleic acid polymerase, and optionally, the detection probe;

[0164] (III) Incubate the product of the previous step under conditions allowing nucleic acid denaturation;

[0165] (IV) Incubate the product of the previous step under conditions allowing nucleic acid annealing or hybridization;

[0166] (V) Incubate the product of the previous step under conditions allowing nucleic acid extension; and

[0167] (VI) Optionally, repeat steps (III)-(V) one or more times.

[0168] In certain embodiments, in step (III), the product of step (II) is incubated at a temperature of 80-105 °C to denature the nucleic acid.

[0169] In certain embodiments, in step (III), the product of step (II) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min.

[0170] In certain embodiments, in step (IV), the product of step (III) is incubated at a temperature of 35-40 °C, 40-45 °C, 45-50 °C, 50-55 °C, 55-60 °C, 60-65 °C, or 65-70 °C to allow annealing or hybridization of the nucleic acid.

[0171] In certain embodiments, in step (IV), the product of step (III) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min.

[0172] In certain embodiments, in step (V), the product of step (IV) is incubated at a temperature of 35-40 °C, 40-45 °C, 45-50 °C, 50-55 °C, 55-60 °C, 60-65 °C, 65-70 °C, 70-75 °C, 75-80 °C, 80-85 °C to allow extension of the nucleic acid.

[0173] In certain embodiments, in step (V), the product of step (IV) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, 2-5 min, 5-10 min, 10-20 min, or 20-30 min.

[0174] In certain embodiments, steps (IV) and (V) are carried out at the same or different temperatures.

[0175] In certain embodiments, steps (III)-(V) are repeated at least once, such as at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times. In certain embodiments, when steps (III)-(V) are repeated one or more times, the conditions used for each cycle of steps (III)-(V) are independently the same or different.

[0176] In certain embodiments, the primers of the amplification primer set each independently have a length of 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt.

[0177] In certain embodiments, the primers of the amplification primer set or any of its components each independently comprise or consist of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, non - natural nucleotides, or any combination thereof.

[0178] In certain embodiments, the amplification primer sets each independently include primer pairs having nucleotide sequences selected from the following or any combination thereof (e.g., any combination of 5 pairs, 10 pairs, 15 pairs, 20 pairs, 23 pairs): SEQ ID NO:72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

[0179] In certain embodiments, the first probe and the second probe each independently comprise or consist of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, non - natural nucleotides (such as peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof.

[0180] In certain embodiments, the first probe and the second probe each independently have a length of 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 200 nt, 200 - 300 nt, 300 - 400 nt, 400 - 500 nt, 500 - 600 nt, 600 - 700 nt, 700 - 800 nt, 800 - 900 nt, 900 - 1000 nt.

[0181] In certain embodiments, each of the first and second probes independently has a 3'-OH terminus; alternatively, the 3'-terminus of the probe is blocked; for example, by adding a chemical moiety (e.g., biotin or alkyl) to the 3'-OH of the last nucleotide of the probe, by removing the 3'-OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide, thereby blocking the 3'-terminus of the detection probe.

[0182] In certain embodiments, each of the first and second probes is independently a self-quenching probe; for example, the probe is labeled with a reporter group at its 5'-terminus or upstream and with a quenching group at its 3'-terminus or downstream, or is labeled with a reporter group at its 3'-terminus or downstream and with a quenching group at its 5'-terminus or upstream. In certain embodiments, the reporter group and the quenching group are separated by a distance of 10 - 80 nt or longer.

[0183] In certain embodiments, the reporter groups in the probes are independently fluorescent groups (e.g., ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CALFluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and the quenching group is a molecule or moiety capable of absorbing / quenching the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA).

[0184] In certain embodiments, each of the first and second probes is independently linear or has a hairpin structure.

[0185] In certain embodiments, the first and second probes have different reporter groups. In certain embodiments, the first and second probes are degradable by a nucleic acid polymerase (e.g., DNA polymerase).

[0186] In certain embodiments, the probe set includes probes having nucleotide sequences selected from the following or any combination thereof (e.g., any combination of 5, 10, 20, 40, 60): SEQ ID NO:73, 74, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

[0187] In a fifth aspect, the present application provides a method for detecting SNP sites where the donor and the recipient have different genotypes, which includes the following steps:

[0188] (a) Providing a third sample from the recipient and a fourth sample from the recipient after transplantation surgery, wherein the third sample contains one or more target nucleic acids derived from the recipient and substantially does not contain nucleic acids derived from the donor; the fourth sample contains one or more target nucleic acids derived from the donor, and the target nucleic acids contain one or more candidate SNP sites, and

[0189] Providing a first universal primer and a second universal primer, and for each candidate SNP site, providing at least one pair of target-specific primers; wherein,

[0190] The first universal primer contains a first universal sequence;

[0191] The second universal primer contains a second universal sequence, the second universal sequence contains the first universal sequence and additionally contains at least one nucleotide at the 3' end of the first universal sequence;

[0192] The pair of target-specific primers can amplify using the target nucleic acid as a template to produce a nucleic acid product containing the candidate SNP site, and the pair of target-specific primers contains a forward primer and a reverse primer, wherein the forward primer contains the first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3' end of the first universal sequence; the reverse primer contains the second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3' end of the second universal sequence; and the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer; and

[0193] (b) Under conditions allowing nucleic acid amplification, use the first universal primer, the second universal primer, and the target-specific primer pair to amplify the target nucleic acid in the third sample and the fourth sample, respectively, thereby obtaining amplification products corresponding to the third sample and the fourth sample, respectively;

[0194] (c) Perform melting curve analysis on the amplification products corresponding to the third sample and the fourth sample obtained in step (b), respectively;

[0195] (d) According to the melting curve analysis results of step (c), determine an SNP locus at which the third sample shows only the first allele and the fourth sample shows at least the second allele (e.g., shows the first and second alleles); the SNP locus is an SNP locus at which the donor and the recipient have different genotypes;

[0196] In the method of the present application, the forward primer and the reverse primer respectively contain a forward nucleotide sequence and a reverse nucleotide sequence specific to the target nucleic acid. Thus, during the PCR reaction, the target-specific primer pair (the forward primer and the reverse primer) will anneal to the target nucleic acid and initiate PCR amplification to produce an initial amplification product, which contains two nucleic acid strands (nucleic acid strand A and nucleic acid strand B) complementary to the forward primer and the reverse primer, respectively. Further, since both the forward primer and the first universal primer contain the first universal sequence, nucleic acid strand A complementary to the forward primer can also be complementary to the first universal primer. Similarly, nucleic acid strand B complementary to the reverse primer can also be complementary to the second universal primer.

[0197] Therefore, as the PCR reaction proceeds, the first universal primer and the second universal primer will anneal to nucleic acid strand A and nucleic acid strand B of the initial amplification product, respectively, and further initiate PCR amplification. During this process, since the reverse primer / second universal primer contains the first universal sequence, the first universal primer can not only anneal to nucleic acid strand A (the nucleic acid strand complementary to the forward primer / first universal primer) and synthesize its complementary strand, but also anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer / second universal primer) and synthesize its complementary strand. That is, the first universal primer can amplify nucleic acid strand A and nucleic acid strand B of the initial amplification product simultaneously. At the same time, the second universal primer contains additional nucleotides at the 3' end of the first universal sequence. Thus, although the second universal primer may also anneal to nucleic acid strand A (the nucleic acid strand complementary to the forward primer / first universal primer, which has a sequence complementary to the forward primer), it is mismatched with nucleic acid strand A at the 3' end (i.e., cannot be completely complementary at the 3' end). Therefore, during the amplification process, the second universal primer will preferentially anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer / second universal primer) and synthesize its complementary strand, and basically cannot extend to synthesize the complementary strand of nucleic acid strand A (the nucleic acid strand complementary to the first forward primer / first universal primer).

[0198] Therefore, as the PCR amplification progresses, the synthesis efficiency of the complementary strand of nucleic acid strand A (nucleic acid strand B) will be significantly lower than that of the complementary strand of nucleic acid strand B (nucleic acid strand A), resulting in the large-scale synthesis and amplification of the complementary strand of nucleic acid strand B (nucleic acid strand A), while the synthesis and amplification of the complementary strand of nucleic acid strand A (nucleic acid strand B) are inhibited, thereby generating a large number of single-stranded products (nucleic acid strand A, which contains sequences complementary to the forward primer / first universal primer and the reverse primer / second universal primer), achieving asymmetric amplification of the target nucleic acid containing one or more SNP sites. Therefore, in steps (a) and (b) of the method of the present application, asymmetric amplification of one or more target nucleic acids in the sample is achieved.

[0199] In addition, since both the forward primer and the reverse primer contain the first universal sequence, during the PCR reaction, the primer dimer formed due to non-specific amplification of the forward primer and the reverse primer will generate single-stranded nucleic acids with reverse sequences complementary to each other at their 5' and 3' ends after denaturation. This single-stranded nucleic acid is prone to self-annealing during the annealing stage, forming a stable panhandle structure, preventing the annealing and extension of the first universal primer and the second universal primer to this single-stranded nucleic acid, thereby inhibiting further amplification of the primer dimer. Therefore, in the method of the present invention, non-specific amplification of the primer dimer can be effectively inhibited.

[0200] In certain embodiments, in step (d) of the method, the genotypes of the respective candidate SNP sites of the third sample and the fourth sample are determined according to the melting curve analysis results, thereby determining such SNP sites: at this site, the third sample only shows the first allele, and the fourth sample shows the first and second alleles;

[0201] In certain embodiments, "substantially free of nucleic acids from the donor" means free of nucleic acids from the donor, or the total nucleic acids from the donor in the second sample do not exceed 10% (for example, do not exceed 5%, do not exceed 3%, do not exceed 1%, or lower).

[0202] In certain embodiments, the third sample is from the recipient (for example, a recipient who has or has not undergone a transplantation operation); for example, the third sample contains cells or tissues from the recipient; for example, the third sample is selected from the skin, saliva, urine, blood, hair, nails, or any combination thereof from the recipient;

[0203] In certain embodiments, for a recipient who has not undergone a transplantation operation, the third sample can be any cell or tissue (such as skin, saliva, urine, blood, etc.). For a recipient who has undergone a transplantation operation, the third sample is substantially free of nucleic acids from the donor.

[0204] In certain preferred embodiments, for a recipient who has undergone hematopoietic stem cell transplantation, the third sample can be selected from skin, saliva, urine, hair, nails, or tissue, etc., but cannot be selected from blood because the blood sample of a recipient who has undergone hematopoietic stem cell transplantation may contain a large amount of donor nucleic acids. In certain preferred embodiments, for a recipient who has undergone kidney transplantation, the third sample can be selected from skin, saliva, hair, nails, or tissue, etc., but cannot be selected from blood and urine because the blood and urine samples of a recipient who has undergone kidney transplantation may contain a large amount of donor nucleic acids. In certain preferred embodiments, for a recipient who has undergone liver transplantation, the third sample can be selected from skin, saliva, hair, nails, urine, or tissue, etc., but cannot be selected from blood because the blood sample of a recipient who has undergone kidney transplantation may contain a large amount of donor nucleic acids.

[0205] In certain embodiments, in the fourth sample, the amount of nucleic acid from the donor accounts for at least 20%, such as at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, or higher, of the total nucleic acid in the fourth sample;

[0206] In certain embodiments, the recipient has received or transplanted an organ, tissue, or cell from a donor;

[0207] For example, the recipient has received or transplanted an organ from a donor (e.g., kidney, heart, lung, liver, pancreas, or any combination thereof); in certain embodiments, the fourth sample comprises blood (e.g., peripheral blood) or urine (especially in the case of kidney transplantation) from the recipient after transplantation surgery; in certain embodiments, the fourth sample comprises blood (e.g., peripheral blood) or urine (especially in the case of kidney transplantation) from the recipient within no more than 5 days (e.g., no more than 3 days, 2 days, or 1 day) after transplantation surgery;

[0208] For example, the recipient has received or transplanted hematopoietic stem cells from a donor (e.g., bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, cord blood hematopoietic stem cells) or a tissue or organ containing hematopoietic stem cells (e.g., bone marrow); in certain embodiments, the fourth sample comprises blood (e.g., peripheral blood) or its components (e.g., blood cells) from the recipient after transplantation surgery; in certain embodiments, the fourth sample comprises blood (e.g., peripheral blood) or its components (e.g., blood cells) from the recipient at least 5 days (e.g., at least 10 days, at least 15 days, at least 20 days, at least 30 days) after transplantation surgery;

[0209] In certain embodiments, in step (a), for each candidate SNP locus, a detection probe is also provided, the detection probe comprising a nucleotide sequence specific to the target nucleic acid and capable of annealing or hybridizing to the region of the target nucleic acid containing the candidate SNP locus, and the detection probe being labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence;

[0210] And, in step (c), the amplification products corresponding to the third sample and the fourth sample obtained in step (b) are respectively subjected to melting curve analysis using the detection probe;

[0211] In certain embodiments, the third sample comprises DNA (such as genomic DNA).

[0212] In certain embodiments, the fourth sample comprises DNA (such as genomic DNA).

[0213] In a sixth aspect, the present application provides a method for detecting the presence or proportion of a donor's nucleic acid in a sample from a recipient who has undergone transplantation surgery, wherein the method comprises the following steps:

[0214] (1) Providing a test sample containing nucleic acid from a recipient who has received transplantation of donor cells, tissues or organs;

[0215] (2) Identifying a plurality (such as at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more) target SNP loci, wherein at the target SNP loci, the recipient has a first genotype containing a homozygous first allele and the donor has a second genotype containing a second allele, wherein the first genotype is different from the second genotype and the first allele is different from the second allele;

[0216] (3) Quantitatively detecting the first allele and the second allele of each target SNP locus in the test sample;

[0217] (4) Determining the presence or proportion of the donor's nucleic acid in the test recipient sample based on the results of the quantitative detection of the first allele and the second allele of the target SNP locus;

[0218] In certain embodiments, in step (2), different alleles at a certain SNP locus can be distinguished by mechanisms selected from the following to identify the target SNP locus: probe hybridization, primer extension, ligation hybridization, and specific enzymatic cleavage. In certain embodiments, in step (2), the target SNP locus can be identified by methods selected from the following: sequencing methods (e.g., first-generation sequencing, pyrosequencing, second-generation sequencing), chip methods (e.g., using solid-phase chips or liquid-phase chips capable of detecting SNPs), qPCR-based detection methods (e.g., Taqman probe method), mass spectrometry (such as iPLEX TM Gold based on MassARRAY), chromatography (such as denaturing high-performance liquid chromatography dHPLC), electrophoresis (such as SNPshot method), and melting curve analysis-based detection methods. In certain embodiments, in step (2), the target SNP locus is identified by a detection method based on multiplex PCR combined with melting curve analysis.

[0219] In certain embodiments, the target SNP locus is identified by the methods described above.

[0220] In certain embodiments, in step (3), digital PCR is used to quantitatively detect the first allele and the second allele of each target SNP locus in the sample.

[0221] In certain embodiments, step (3) is carried out by the following protocol:

[0222] (I) For each target SNP locus, provide an amplification primer set and a probe set, wherein,

[0223] (I-1) The amplification primer set includes at least one amplification primer (e.g., a pair of amplification primers or more amplification primers) that can specifically amplify a nucleic acid molecule containing the target SNP locus under conditions allowing nucleic acid hybridization or annealing;

[0224] (I-2) The probe set includes a first probe and a second probe; wherein,

[0225] (i) The first probe and the second probe are each independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and the first probe and the second probe are labeled with different reporter groups (e.g., fluorescent groups); and

[0226] (ii) The first probe is capable of hybridizing or annealing (preferably being completely complementary) to a nucleic acid molecule containing the first allele of the target SNP site, and the second probe is capable of hybridizing or annealing (preferably being completely complementary) to a nucleic acid molecule containing the second allele of the target SNP site; and, the first probe and the second probe are specific for different alleles;

[0227] (II) Using the amplification primer set and the probe set to perform digital PCR on the sample to be tested, and quantitatively detecting the nucleic acid molecules with the first allele and the nucleic acid molecules with the second allele.

[0228] In some embodiments, the first probe specifically anneals or hybridizes to a nucleic acid molecule with the first allele during the digital PCR reaction; and, the second probe specifically anneals or hybridizes to a nucleic acid molecule with the second allele during the digital PCR reaction.

[0229] In some embodiments, the first probe does not anneal or hybridize to a nucleic acid molecule with the second allele during the digital PCR reaction; and / or, the second probe does not anneal or hybridize to a nucleic acid molecule with the first allele during the digital PCR reaction.

[0230] In the method of the present application, taking the first probe in the probe set as an example, it is capable of hybridizing or annealing (preferably being completely complementary) to a nucleic acid molecule with the first allele. Therefore, during the digital PCR reaction, during the annealing or extension process, the first probe will form a duplex with the nucleic acid molecule and be degraded by a nucleic acid polymerase (such as DNA polymerase) during amplification, releasing a reporter group (such as a fluorescent group). Thus, after the digital PCR amplification reaction is completed, the end-point fluorescence of each droplet is detected by a droplet detector. According to the signal (such as the first fluorescence signal) intensity of the free first reporter group (such as the first fluorescent group), the number of positive droplets and negative droplets can be determined, thereby determining the amount of nucleic acid molecules with the first allele in the sample. Similarly, after the digital PCR amplification reaction is completed, the end-point fluorescence of each droplet is detected by a droplet detector. According to the signal (such as the second fluorescence signal) intensity of the free second reporter group (such as the second fluorescent group), the number of positive droplets and negative droplets can be determined, and the amount of nucleic acid molecules with the second allele in the sample can be determined. Since the donor / recipient genotype is different, the corresponding first / second allele content is also different. Therefore, by comparing and analyzing the amounts of nucleic acid molecules containing the first / second alleles, it is possible to determine whether a donor exists in the recipient sample, and optionally, determine the proportion of the donor.

[0231] In the method of the present application, in certain embodiments, the first probe does not anneal or hybridize with a nucleic acid molecule having a second allele during the digital PCR reaction; and / or, the second probe does not anneal or hybridize with a nucleic acid molecule having a first allele during the digital PCR reaction. It is readily understood that the hybridization specificity of the first / second probe is particularly advantageous, which can contribute to accurately determining the content of the first allele / second allele, thereby facilitating the calculation of the respective proportions of the donor sample and the recipient sample. In certain embodiments, the hybridization specificity of the first / second probe can be obtained by controlling the annealing temperature and / or the extension temperature of the digital PCR reaction. For example, the annealing temperature and / or the extension temperature can be set to be lower than the melting point of the double-stranded body formed by the first probe and the nucleic acid molecule having the first allele, but higher than the melting point of the double-stranded body formed by the first probe and the nucleic acid molecule having the second allele, so that the first probe hybridizes with the nucleic acid molecule having the first allele during the digital PCR reaction, but does not hybridize with the nucleic acid molecule having the second allele. Similarly, the annealing temperature and / or the extension temperature can be set to be lower than the melting point of the double-stranded body formed by the second probe and the nucleic acid molecule having the second allele, but higher than the melting point of the double-stranded body formed by the second probe and the nucleic acid molecule having the first allele, so that the second probe hybridizes with the nucleic acid molecule having the second allele during the digital PCR reaction, but does not hybridize with the nucleic acid molecule having the first allele.

[0232] In the method of the present application, the copy number of alleles can be detected by a digital PCR platform and directly output by software according to the Poisson distribution principle. For the relevant principle and calculation method, reference can be made to, for example, Milbury CA, Zhong Q, Lin J, et al. Determining lower limits of detection of digital PCR assays for cancer-related gene mutations. Biomol Detect Quantif. 2014;1(1):8 - 22. Published 2014 Aug 20. doi:10.1016 / j.bdq.2014.08.001.

[0233] In certain embodiments, in step (4), cluster analysis is performed on the quantitative detection results of the second alleles of multiple (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more) target SNP sites; then, based on the results of the cluster analysis, the genotype of the donor at each target SNP site is determined; then, based on the genotypes of the receptor and the donor at each target SNP site, and the quantitative detection results of the first allele and the second allele in the test sample, the presence or proportion of the donor's nucleic acid in the test receptor sample is determined.

[0234] Since the receptor contains a homozygous first allele at the target SNP site, the second allele signal detected in the test sample must originate from the donor. In other words, the genotype of the donor at this target SNP site may be homozygous for the second allele or heterozygous for the first and second alleles. Theoretically, for the same sample, during digital PCR quantitative detection, the detection result (corresponding to the absolute copy number) of the second allele at a homozygous SNP site will be twice that of the second allele at a heterozygous SNP site. Therefore, by performing cluster analysis on the detection results of the second alleles of multiple target SNP sites, it can be determined which target SNP sites the donor has a homozygous second allele at, and which target SNP sites the donor has a heterozygous first and second alleles at; among them, the detection result (corresponding to the absolute copy number) of the former will be twice that of the latter. In other words, by performing cluster analysis on the detection signals of the second alleles, the genotype of the donor at each target SNP site can be determined. On this basis, based on the genotypes of the receptor and the donor at each target SNP site, and the quantitative detection results of the first allele and the second allele in the test sample, the presence or proportion of the donor's nucleic acid in the test receptor sample can be easily determined.

[0235] In certain embodiments, before step (3), the test sample from the receptor is pre-treated.

[0236] In certain embodiments, the pre-treatment includes nucleic acid extraction from the sample and / or enrichment of the nucleic acid in the sample (e.g., by concentration and / or amplification).

[0237] In certain embodiments, wherein the receptor has received or transplanted hematopoietic stem cells from the donor (e.g., bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, cord blood hematopoietic stem cells or any combination thereof) or a tissue or organ containing hematopoietic stem cells (e.g., spinal cord);

[0238] In certain embodiments, the sample to be tested comprises blood (e.g., peripheral blood) or a component thereof (e.g., blood cells, plasma, monocytes, granulocytes, T cells, or any combination thereof) from a recipient after transplantation.

[0239] In certain embodiments, the recipient has received or transplanted an organ from a donor (e.g., a kidney, heart, lung, liver, pancreas, or any combination thereof).

[0240] In certain embodiments, the recipient has received or transplanted a kidney from a donor.

[0241] In certain embodiments, the sample to be tested comprises blood (e.g., peripheral blood) or urine (particularly in the case of kidney transplantation) from a recipient after transplantation.

[0242] In certain embodiments, steps (a)-(b) of the method are carried out by a protocol comprising the following steps (I)-(VI):

[0243] (I) Providing the third sample and the fourth sample, the first universal primer and the second universal primer, and the target-specific primer pair; and optionally, the detection probe;

[0244] (II) Mixing the samples with the first universal primer, the second universal primer, the target-specific primer pair, a nucleic acid polymerase, and optionally, the detection probe;

[0245] (III) Incubating the product of the previous step under conditions allowing nucleic acid denaturation;

[0246] (IV) Incubating the product of the previous step under conditions allowing nucleic acid annealing or hybridization;

[0247] (V) Incubating the product of the previous step under conditions allowing nucleic acid extension; and

[0248] (VI) Optionally, repeating steps (III)-(V) one or more times.

[0249] In certain embodiments, in step (III), the product of step (II) is incubated at a temperature of 80-105 °C to denature the nucleic acid.

[0250] In certain embodiments, in step (III), the product of step (II) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min.

[0251] In certain embodiments, in step (IV), the product of step (III) is incubated at a temperature of 35 - 40 °C, 40 - 45 °C, 45 - 50 °C, 50 - 55 °C, 55 - 60 °C, 60 - 65 °C, or 65 - 70 °C, thereby allowing nucleic acid annealing or hybridization.

[0252] In certain embodiments, in step (IV), the product of step (III) is incubated for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, or 2 - 5 min.

[0253] In certain embodiments, in step (V), the product of step (IV) is incubated at a temperature of 35 - 40 °C, 40 - 45 °C, 45 - 50 °C, 50 - 55 °C, 55 - 60 °C, 60 - 65 °C, 65 - 70 °C, 70 - 75 °C, 75 - 80 °C, 80 - 85 °C, thereby allowing nucleic acid extension.

[0254] In certain embodiments, in step (V), the product of step (IV) is incubated for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, 2 - 5 min, 5 - 10 min, 10 - 20 min or 20 - 30 min.

[0255] In certain embodiments, steps (IV) and (V) are carried out at the same or different temperatures.

[0256] In certain embodiments, steps (III)-(V) are repeated at least once, such as at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times. In certain embodiments, when steps (III)-(V) are repeated one or more times, the conditions used for each cycle of steps (III)-(V) are independently the same or different.

[0257] In certain embodiments, the lengths of the primers of the amplification primer set are each independently 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt.

[0258] In certain embodiments, the primers of the amplification primer set or any of its components each independently comprise or consist of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, non - natural nucleotides, or any combination thereof.

[0259] In certain embodiments, each of the amplification primer sets independently comprises a primer pair having a nucleotide sequence selected from the following or any combination thereof (e.g., any combination of 5 pairs, 10 pairs, 15 pairs, 20 pairs, 23 pairs): SEQ ID NO:72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

[0260] In certain embodiments, each of the first probe and the second probe independently comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., peptide nucleic acids (PNA) or locked nucleic acids), or any combination thereof.

[0261] In certain embodiments, the lengths of the first probe and the second probe are independently 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 200 nt, 200 - 300 nt, 300 - 400 nt, 400 - 500 nt, 500 - 600 nt, 600 - 700 nt, 700 - 800 nt, 800 - 900 nt, 900 - 1000 nt.

[0262] In certain embodiments, each of the first probe and the second probe independently has a 3'-OH terminus; alternatively, the 3'-terminus of the probe is blocked; for example, by adding a chemical moiety (e.g., biotin or an alkyl group) to the 3'-OH of the last nucleotide of the probe, by removing the 3'-OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide, thereby blocking the 3'-terminus of the detection probe.

[0263] In certain embodiments, each of the first probe and the second probe is independently a self-quenching probe; for example, the probe is labeled with a reporter group at its 5'-end or upstream and with a quencher group at its 3'-end or downstream, or with a reporter group at its 3'-end or downstream and with a quencher group at its 5'-end or upstream. In certain embodiments, the reporter group and the quencher group are separated by a distance of 10 - 80 nt or longer.

[0264] In certain embodiments, each of the reporter groups in the probes is independently a fluorophore (e.g., ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and the quenching group is a molecule or group capable of absorbing / quenching the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA).

[0265] In certain embodiments, each of the first probe and the second probe is independently linear or has a hairpin structure.

[0266] In certain embodiments, the first probe and the second probe have different reporter groups. In certain embodiments, the first probe and the second probe are degradable by a nucleic acid polymerase (e.g., DNA polymerase).

[0267] In certain embodiments, the probe set includes probes having nucleotide sequences selected from the following or any combination thereof (e.g., any combination of 5, 10, 20, 40, 60): SEQ ID NO:73, 74, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

[0268] In certain embodiments, the candidate SNP locus has one or more characteristics selected from the following:

[0269] (1) The Fst of the candidate SNP locus among different ethnic groups is less than 0.3 (e.g., less than 0.2, less than 0.1, less than 0.05, less than 0.01);

[0270] (2) The candidate SNP locus is located on different chromosomes;

[0271] (3) The allele frequency of the candidate SNP locus is between 0.2 and 0.8 (e.g., between 0.3 and 0.7, between 0.4 and 0.6).

[0272] In certain embodiments, the candidate SNP locus has one or more characteristics selected from the following:

[0273] (1) The Fst of the candidate SNP locus between different ethnic groups is less than 0.01;

[0274] (2) The candidate SNP locus is located on different chromosomes;

[0275] (3) The allele frequency of the candidate SNP locus is between 0.3 and 0.7.

[0276] In certain embodiments, the candidate SNP locus is an SNP locus with biallelic polymorphism.

[0277] In certain embodiments, the candidate SNP locus is an SNP locus in the human genome; for example, the target nucleic acid comprises a human genomic SNP locus selected from the following: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs5858210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, rs7160304, and any combination of the foregoing SNP loci (e.g., any combination of 5, 10, 15, 20, 23 of the foregoing SNP loci).

[0278] In certain embodiments, the target nucleic acid in the sample comprises the following human genomic SNP loci: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs5858210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, and rs7160304.

[0279] In certain embodiments, in step (b), the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, and a nucleic acid polymerase, and nucleic acid amplification (e.g., a PCR reaction) is carried out. Then, a detection probe is added to the product of step (b), and melting curve analysis is performed; alternatively, in step (b), the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, the detection probe, and a nucleic acid polymerase, and nucleic acid amplification (e.g., a PCR reaction) is carried out. Then, melting curve analysis is performed after the PCR reaction is completed.

[0280] In certain embodiments, the detection probe comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof. In certain preferred embodiments, the detection probe comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the detection probe comprises non-natural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acid (LNA).

[0281] In certain embodiments, the length of the detection probe is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 200 nt, 200 - 300 nt, 300 - 400 nt, 400 - 500 nt, 500 - 600 nt, 600 - 700 nt, 700 - 800 nt, 800 - 900 nt, 900 - 1000 nt.

[0282] In certain embodiments, the detection probe has a 3'-OH terminus; alternatively, the 3'-terminus of the detection probe is blocked; for example, by adding a chemical moiety (e.g., biotin or an alkyl group) to the 3'-OH of the last nucleotide of the detection probe, by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide, thereby blocking the 3'-terminus of the detection probe.

[0283] In some embodiments, the detection probe is a self-quenching probe; for example, the detection probe is labeled with a reporter group at its 5'-end or upstream and a quenching group at its 3'-end or downstream, or is labeled with a reporter group at its 3'-end or downstream and a quenching group at its 5'-end or upstream. In such embodiments, when the detection probe is not hybridized to other sequences, the quenching group is positioned such that it can absorb or quench the signal of the reporter group (e.g., the quenching group is in proximity to the reporter group), thereby absorbing or quenching the signal emitted by the reporter group. In this case, the detection probe does not emit a signal. Further, when the detection probe hybridizes to its complementary sequence, the quenching group is positioned such that it cannot absorb or quench the signal of the reporter group (e.g., the quenching group is at a distance from the reporter group), thereby not being able to absorb or quench the signal emitted by the reporter group. In this case, the detection probe emits a signal.

[0284] The design of such self-quenching detection probes is within the capabilities of those skilled in the art. For example, a reporter group can be labeled at the 5'-end of the detection probe and a quenching group at the 3'-end, or a reporter group can be labeled at the 3'-end of the detection probe and a quenching group at the 5'-end. Thus, when the detection probe exists alone, the reporter group and the quenching group are close to each other and interact such that the signal emitted by the reporter group is absorbed by the quenching group, thereby causing the detection probe not to emit a signal; while when the detection probe hybridizes to its complementary sequence, the reporter group and the quenching group are separated from each other such that the signal emitted by the reporter group cannot be absorbed by the quenching group, thereby causing the detection probe to emit a signal.

[0285] However, it should be understood that the reporter group and the quencher group are not necessarily labeled at the ends of the detection probe. The reporter group and / or the quencher group can also be labeled inside the detection probe, provided that the signal emitted by the detection probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence. For example, the reporter group can be labeled upstream (or downstream) of the detection probe, and the quencher group can be labeled downstream (or upstream) of the detection probe, and the two are separated by a sufficient distance (e.g., 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, or a longer distance). Thus, when the detection probe exists alone, due to the free curling of the probe molecule or the formation of the secondary structure of the probe (such as a hairpin structure), the reporter group and the quencher group are close to each other and interact, such that the signal emitted by the reporter group is absorbed by the quencher group, so that the detection probe does not emit a signal; and when the detection probe hybridizes to its complementary sequence, the reporter group and the quencher group are separated by a sufficient distance such that the signal emitted by the reporter group cannot be absorbed by the quencher group, so that the detection probe emits a signal. In some preferred embodiments, the reporter group and the quencher group are separated by 10-80 nt or a longer distance, such as 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt. In some preferred embodiments, the reporter group and the quencher group are separated by no more than 80 nt, no more than 70 nt, no more than 60 nt, no more than 50 nt, no more than 40 nt, no more than 30 nt, or no more than 20 nt. In some preferred embodiments, the reporter group and the quencher group are separated by at least 5 nt, at least 10 nt, at least 15 nt, or at least 20 nt.

[0286] Thus, the reporter group and the quencher group can be labeled at any suitable position of the detection probe, as long as the signal emitted by the detection probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence. However, in some preferred embodiments, at least one of the reporter group and the quencher group is located at the end of the detection probe (e.g., the 5' or 3' end). In some preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the detection probe or at a position 1-10 nt from the 5' end, and the reporter group and the quencher group are separated by a suitable distance such that the quencher group can absorb or quench the signal of the reporter group before the detection probe hybridizes to its complementary sequence. In some preferred embodiments, one of the reporter group and the quencher group is located at the 3' end of the detection probe or at a position 1-10 nt from the 3' end, and the reporter group and the quencher group are separated by a suitable distance such that the quencher group can absorb or quench the signal of the reporter group before the detection probe hybridizes to its complementary sequence. In some preferred embodiments, the reporter group and the quencher group can be separated by a distance as defined above (e.g., a distance of 10-80 nt or longer). In some preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the detection probe and the other is located at the 3' end.

[0287] In some embodiments, the reporter group in the detection probe is a fluorophore (e.g., ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL FluorRed 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and the quencher group is a molecule or group capable of absorbing / quenching the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA).

[0288] In some embodiments, the detection probe has resistance to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe contains modifications that resist nuclease activity, such as phosphorothioate bonds, alkyl phosphotriester bonds, aryl phosphotriester bonds, alkyl phosphonate bonds, aryl phosphonate bonds, hydrogen phosphonate bonds, alkyl aminophosphonate bonds, aryl aminophosphonate bonds, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, and 1-(4'-thio-PD-ribofuranosyl) modification.

[0289] In certain embodiments, the detection probe is linear or has a hairpin structure.

[0290] In certain embodiments, each of the detection probes independently has the same or different reporter groups. In certain embodiments, the detection probes have the same reporter group, and the product of step (b) is subjected to melting curve analysis, and then the presence of the target nucleic acid is determined according to the melting peak in the melting curve; or, the detection probes have different reporter groups, and the product of step (b) is subjected to melting curve analysis, and then the presence of the target nucleic acid is determined according to the signal type of the reporter group and the melting peak in the melting curve.

[0291] In certain embodiments, in step (c), the product of step (b) is gradually heated or cooled, and the signal emitted by the reporter group on each detection probe is monitored in real time, so as to obtain a curve showing the change of the signal intensity of each reporter group with temperature. For example, the product of step (2) can be gradually heated from a temperature of 45 °C or lower (e.g., not exceeding 45 °C, not exceeding 40 °C, not exceeding 35 °C, not exceeding 30 °C, not exceeding 25 °C) to a temperature of 75 °C or higher (e.g., at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C), and the signal emitted by the reporter group on the detection probe is monitored in real time, so as to obtain a curve showing the change of the signal intensity of the reporter group with temperature. The heating rate can be routinely determined by those skilled in the art. For example, the heating rate can be: heating 0.01 - 1 °C per step (e.g., 0.01 - 0.05 °C, 0.05 - 0.1 °C, 0.1 - 0.5 °C, 0.5 - 1 °C, 0.04 - 0.4 °C, e.g., 0.01 °C, 0.02 °C, 0.03 °C, 0.04 °C, 0.05 °C, 0.06 °C, 0.07 °C, 0.08 °C, 0.09 °C, 0.1 °C, 0.2 °C, 0.3 °C, 0.4 °C, 0.5 °C, 0.6 °C, 0.7 °C, 0.8 °C, 0.9 °C or 1.0 °C), and maintaining for 0.5 - 15 s per step (e.g., 0.5 - 1 s, 1 - 2 s, 2 - 3 s, 3 - 4 s, 4 - 5 s, 5 - 10 s, 10 - 15 s); or heating 0.01 - 1 °C per second (e.g., 0.01 - 0.05 °C, 0.05 - 0.1 °C, 0.1 - 0.5 °C, 0.5 - 1 °C, 0.04 - 0.4 °C, e.g., 0.01 °C, 0.02 °C, 0.03 °C, 0.04 °C, 0.05 °C, 0.06 °C, 0.07 °C, 0.08 °C, 0.09 °C, 0.1 °C, 0.2 °C, 0.3 °C, 0.4 °C, 0.5 °C, 0.6 °C, 0.7 °C, 0.8 °C, 0.9 °C or 1.0 °C).

[0292] Then, the derivative of the curve is taken to obtain the melting curve of the product of step (b).

[0293] In certain embodiments, the type of each SNP site is determined based on the melting peak (melting point) in the melting curve.

[0294] In certain embodiments, the detection probe includes a detection probe having a nucleotide sequence selected from the following or any combination thereof (e.g., any combination of 5, 10, 15, 20, 23): SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, and 69.

[0295] In certain embodiments, in step (a) of the method, 1 - 5, 5 - 10, 10 - 15, 15 - 20 or more target - specific primer pairs are provided.

[0296] In certain embodiments, in step (b) of the method, the working concentrations of the first universal primer and the second universal primer are higher than those of the forward primer and the reverse primer; for example, the working concentrations of the first universal primer and the second universal primer are 1 - 5 times, 5 - 10 times, 10 - 15 times, 15 - 20 times, 20 - 50 times or more times higher than those of the forward primer and the reverse primer.

[0297] In certain embodiments, in step (b) of the method, the working concentrations of the first universal primer and the second universal primer are the same; or, the working concentration of the first universal primer is lower than that of the second universal primer.

[0298] In certain embodiments, in step (b) of the method, the working concentrations of the forward primer and the reverse primer are the same or different.

[0299] In certain embodiments, the sample or target nucleic acid contains mRNA, and before performing step (b) of the method, a reverse transcription reaction is performed on the sample.

[0300] In certain embodiments, in step (b) of the method, a nucleic acid polymerase (particularly a template-dependent nucleic acid polymerase) is used to perform the PCR reaction. In certain embodiments, the nucleic acid polymerase is a DNA polymerase, such as a thermostable DNA polymerase. In certain embodiments, the thermostable DNA polymerase is obtained from Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermuschliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermusoshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus thermophllus, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Thermococcus litoralis, Thermococcus barossi, Thermococcusgorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictiumoccultum, Aquifexpyrophilus and Aquifex aeolieus. In certain embodiments, the DNA polymerase is Taq polymerase.

[0301] In certain embodiments, the first universal primer consists of a first universal sequence or comprises a first universal sequence and an additional sequence, the additional sequence being located at the 5' end of the first universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0302] In certain embodiments, the first universal sequence is located in or constitutes the 3' portion of the first universal primer.

[0303] In an embodiment of the present application, the first universal primer can be of any length as long as it can perform a PCR reaction. In certain embodiments, the length of the first universal primer is 5-15 nt, 15-20 nt, 20-30 nt, 30-40 nt, or 40-50 nt.

[0304] In certain embodiments, the first universal primer or any of its components comprises or consists of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, unnatural nucleotides, or any combination thereof. In certain preferred embodiments, the first universal primer (or any of its components) comprises or consists of natural nucleotides (such as deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the first universal primer (or any of its components) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the first universal primer (or any of its components) comprises unnatural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0305] In certain embodiments, the second universal primer consists of a second universal sequence or, comprises a second universal sequence and an additional sequence, the additional sequence being located at the 5' end of the second universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0306] In certain embodiments, the second universal sequence is located in or constitutes the 3' portion of the second universal primer.

[0307] In certain embodiments, the second universal sequence comprises the first universal sequence and additionally comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides at the 3' end of the first universal sequence.

[0308] In an embodiment of the present application, the second universal primer can be of any length as long as it can perform a PCR reaction. In certain embodiments, the length of the second universal primer is 8-15 nt, 15-20 nt, 20-30 nt, 30-40 nt, or 40-50 nt.

[0309] In certain embodiments, the second universal primer or any of its components comprises or consists of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, unnatural nucleotides, or any combination thereof. In certain preferred embodiments, the second universal primer (or any of its components) comprises or consists of natural nucleotides (such as deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the second universal primer (or any of its components) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the second universal primer (or any of its components) comprises unnatural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acid (LNA).

[0310] In certain embodiments, in the forward primer, the forward nucleotide sequence is directly linked to the 3'-end of the first universal sequence or is linked to the 3'-end of the first universal sequence via a nucleotide linker. In certain embodiments, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0311] In certain embodiments, the forward primer further comprises an additional sequence located at the 5'-end of the first universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0312] In certain embodiments, the forward primer comprises or consists of, from 5' to 3', the first universal sequence and the forward nucleotide sequence; or, from 5' to 3', the first universal sequence, the nucleotide linker, and the forward nucleotide sequence; or, from 5' to 3', the additional sequence, the first universal sequence, and the forward nucleotide sequence; or, from 5' to 3', the additional sequence, the first universal sequence, the nucleotide linker, and the forward nucleotide sequence.

[0313] In certain embodiments, the forward nucleotide sequence is located at or constitutes the 3'-portion of the forward primer.

[0314] In certain embodiments, the length of the forward nucleotide sequence is 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt.

[0315] In certain embodiments, the length of the forward primer is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt.

[0316] In certain embodiments, the forward primer or any of its components comprises or consists of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, unnatural nucleotides, or any combination thereof. In certain preferred embodiments, the forward primer (or any of its components) comprises or consists of natural nucleotides (such as deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the forward primer (or any of its components) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5 - methylcytosine or 5 - hydroxymethylcytosine. In certain preferred embodiments, the forward primer (or any of its components) comprises unnatural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β - D - ribofuranosyl)-3 - nitropyrrole, 5 - nitroindole, or locked nucleic acid (LNA).

[0317] In certain embodiments, in the reverse primer, the reverse nucleotide sequence is directly linked to the 3' end of the second universal sequence, or the reverse nucleotide sequence is linked to the 3' end of the second universal sequence through a nucleotide linker. In certain embodiments, the nucleotide linker comprises 1 - 5, 5 - 10, 10 - 15, 15 - 20, or more nucleotides.

[0318] In certain embodiments, the reverse primer further comprises an additional sequence located at the 5' end of the second universal sequence. In certain embodiments, the additional sequence comprises 1 - 5, 5 - 10, 10 - 15, 15 - 20, or more nucleotides.

[0319] In certain embodiments, the reverse primer comprises or consists of, from 5' to 3', the second universal sequence and the reverse nucleotide sequence; or, from 5' to 3', comprises or consists of the second universal sequence, the nucleotide linker, and the reverse nucleotide sequence; or, from 5' to 3', comprises or consists of the additional sequence, the second universal sequence, and the reverse nucleotide sequence; or, from 5' to 3', comprises or consists of the additional sequence, the second universal sequence, the nucleotide linker, and the reverse nucleotide sequence.

[0320] In certain embodiments, the reverse nucleotide sequence is located at or constitutes the 3' portion of the reverse primer.

[0321] In certain embodiments, the length of the reverse nucleotide sequence is 10 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt.

[0322] In certain embodiments, the length of the reverse primer is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt.

[0323] In certain embodiments, the reverse primer or any of its components comprises or consists of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, unnatural nucleotides, or any combination thereof. In certain preferred embodiments, the reverse primer (or any of its components) comprises or consists of natural nucleotides (such as deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the reverse primer (or any of its components) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5 - methylcytosine or 5 - hydroxymethylcytosine. In certain preferred embodiments, the reverse primer (or any of its components) comprises unnatural nucleotides, such as deoxyinosine, inosine, 1 - (2'-deoxy - β - D - ribofuranosyl)-3 - nitropyrrole, 5 - nitroindole or locked nucleic acid (LNA).

[0324] In certain embodiments, the second universal sequence cannot be fully complementary to the complementary sequence of the forward primer; for example, at least one nucleotide at the 3'-end of the second universal sequence, such as 1 - 5, 5 - 10, 10 - 15, 15 - 20 or more nucleotides, cannot be complementary to the complementary sequence of the forward primer.

[0325] In certain embodiments, the sequence of the first universal primer is as shown in SEQ ID NO:71.

[0326] In certain embodiments, the sequence of the second universal primer is as shown in SEQ ID NO:70.

[0327] In certain embodiments, the target-specific primer pairs include primer pairs having nucleotide sequences selected from the following or any combination thereof (e.g., any combination of 5 pairs, 10 pairs, 15 pairs, 20 pairs, 23 pairs): SEQ ID NO: 1 and 2; 4 and 5; 7 and 8; 10 and 11; 13 and 14; 16 and 17; 19 and 20; 22 and 23; 25 and 26; 28 and 29; 31 and 32; 34 and 35; 37 and 38; 40 and 41; 43 and 44; 46 and 47; 49 and 50; 52 and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68.

[0328] In certain embodiments, the target SNP sites are each independently selected from:

[0329] (1) SNP sites where the donor genotype is first homozygous and the recipient genotype is second homozygous;

[0330] (2) SNP sites where the donor genotype is homozygous and the recipient genotype is heterozygous.

[0331] In certain preferred embodiments, the proportion of the donor in the recipient sample is calculated by scheme (1).

[0332] In certain embodiments, the proportion of the recipient in the donor sample is calculated by one or more of the following methods:

[0333] (1) When the target SNP site is a SNP site where the donor genotype is first homozygous (e.g., AA) and the recipient genotype is second homozygous (e.g., BB), the proportion of the donor in the recipient sample is:

[0334] where N B is the copy number of allele B (which can be determined by digital PCR), and N A is the copy number of allele A (which can be determined by digital PCR);

[0335] (2) When the target SNP site is a SNP site where the donor genotype is homozygous (e.g., AA) and the recipient genotype is heterozygous (e.g., AB), the proportion of the donor in the recipient sample is:

[0336] where N B is the copy number of allele B (which can be determined by digital PCR), and N A is the copy number of allele A (which can be determined by digital PCR).

[0337] In certain embodiments, the transplantation is an organ transplantation.

[0338] In certain embodiments, the organ transplantation is selected from the group consisting of kidney, heart, lung, liver, pancreas, or any combination thereof.

[0339] In certain embodiments, the recipient sample is selected from the group consisting of blood (e.g., peripheral blood), urine, and any combination thereof from the recipient after transplantation.

[0340] In certain embodiments, the target SNP sites are each independently selected from:

[0341] (1) SNP sites where the donor genotype is homozygous for the first allele and the recipient genotype is homozygous for the second allele;

[0342] (2) SNP sites where the donor genotype is heterozygous and the recipient genotype is homozygous.

[0343] In certain preferred embodiments, the proportion of the donor in the recipient sample is calculated by scheme (1).

[0344] In certain embodiments, the proportion of the recipient in the donor sample is calculated by one or more of the following methods:

[0345] (1) When the target SNP site is a site where the donor genotype is homozygous for the first allele (e.g., BB) and the recipient genotype is homozygous for the second allele (e.g., AA), the proportion of the donor in the recipient sample is:

[0346] where N B is the copy number of allele B (which can be determined by digital PCR), and N A is the copy number of allele A (which can be determined by digital PCR);

[0347] (2) When the target SNP site is a site where the donor genotype is heterozygous (e.g., AB) and the recipient genotype is homozygous (e.g., AA), the proportion of the recipient in the donor sample is:

[0348] where N B is the copy number of allele B (which can be determined by digital PCR), and N A is the copy number of allele A (which can be determined by digital PCR).

[0349] In the method of the present application, taking the first probe in the probe set as an example, it can hybridize or anneal (preferably completely complementary) with a nucleic acid molecule having a first allele. Therefore, during the digital PCR reaction, during the annealing or extension process, the first probe will form a duplex with the nucleic acid molecule and be degraded by a nucleic acid polymerase (such as DNA polymerase) during amplification, releasing a reporter group (such as a fluorescent group). Thus, after the digital PCR amplification reaction ends, the endpoint fluorescence of each droplet is detected by a droplet detector. According to the signal intensity (such as the first fluorescence signal) of the free first reporter group (such as the first fluorescent group), the number of positive droplets and negative droplets can be determined, and thus the amount of nucleic acid molecules having the first allele in the sample can be determined. Similarly, after the digital PCR amplification reaction ends, the endpoint fluorescence of each droplet is detected by a droplet detector. According to the signal intensity (such as the second fluorescence signal) of the free second reporter group (such as the second fluorescent group), the number of positive droplets and negative droplets can be determined, and thus the amount of nucleic acid molecules having the second allele in the sample can be determined. Since the donor / recipient genotype is different, the corresponding first / second allele content is also different. Therefore, by comparing and analyzing the amounts of nucleic acid molecules containing the first / second allele, it can be determined whether a donor exists in the recipient sample, and optionally, the proportion of the donor can be determined.

[0350] In the method of the present application, in certain embodiments, the first probe does not anneal or hybridize with a nucleic acid molecule having a second allele during the digital PCR reaction; and / or, the second probe does not anneal or hybridize with a nucleic acid molecule having a first allele during the digital PCR reaction. It is easy to understand that the hybridization specificity of the first / second probe is particularly advantageous, which can contribute to accurately determining the content of the first allele / second allele, and thus contribute to calculating the respective proportions of the donor sample and the recipient sample. In certain embodiments, the hybridization specificity of the first / second probe can be obtained by controlling the annealing temperature and / or extension temperature of the digital PCR reaction. For example, the annealing temperature and / or extension temperature can be set lower than the melting point of the duplex formed by the first probe and the nucleic acid molecule having the first allele, but higher than the melting point of the duplex formed by the first probe and the nucleic acid molecule having the second allele, so that the first probe hybridizes with the nucleic acid molecule having the first allele during the digital PCR reaction, but does not hybridize with the nucleic acid molecule having the second allele. Similarly, the annealing temperature and / or extension temperature can be set lower than the melting point of the duplex formed by the second probe and the nucleic acid molecule having the second allele, but higher than the melting point of the duplex formed by the second probe and the nucleic acid molecule having the first allele, so that the second probe hybridizes with the nucleic acid molecule having the second allele during the digital PCR reaction, but does not hybridize with the nucleic acid molecule having the first allele.

[0351] In the method of the present application, the copy number of alleles can be detected by a digital PCR platform and directly output by software according to the principle of Poisson distribution. For the relevant principle and calculation method, reference can be made to, for example, Milbury CA, Zhong Q, Lin J, et al. Determining lower limits of detection of digital PCR assays for cancer-related gene mutations. Biomol Detect Quantif. 2014;1(1):8-22. Published 2014 Aug 20. doi:10.1016 / j.bdq.2014.08.001.

[0352] In a seventh aspect, the present application provides a kit, which includes an identification primer set capable of asymmetrically amplifying a target nucleic acid containing a candidate SNP site.

[0353] In certain embodiments, the identification primer set includes: a first universal primer and a second universal primer, and for each candidate SNP site, at least one pair of target-specific primers is provided, wherein,

[0354] the first universal primer contains a first universal sequence;

[0355] the second universal primer contains a second universal sequence, the second universal sequence contains the first universal sequence and additionally contains at least one nucleotide at the 3' end of the first universal sequence;

[0356] the pair of target-specific primers can amplify using the target nucleic acid as a template to generate a nucleic acid product containing the candidate SNP site, and the pair of target-specific primers includes a forward primer and a reverse primer, wherein the forward primer contains the first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3' end of the first universal sequence; the reverse primer contains the second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3' end of the second universal sequence; and the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer.

[0357] In certain embodiments, the kit further comprises one or more detection probes capable of detecting the candidate SNP locus, the detection probe comprising a nucleotide sequence specific to the target nucleic acid and capable of annealing or hybridizing to a region of the target nucleic acid containing the candidate SNP locus, and labeled with a reporter group and a quenching group, wherein the reporter group is capable of emitting a signal, and the quenching group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence.

[0358] In certain embodiments, the candidate SNP locus has one or more of the following characteristics:

[0359] (1) The Fst of the candidate SNP locus among different ethnic groups is less than 0.3 (e.g., less than 0.2, less than 0.1, less than 0.05, less than 0.01);

[0360] (2) The candidate SNP locus is located on different chromosomes;

[0361] (3) The allele frequency of the candidate SNP locus is between 0.2 and 0.8 (e.g., between 0.3 and 0.7, between 0.4 and 0.6).

[0362] In certain embodiments, the candidate SNP locus has one or more of the following characteristics:

[0363] (1) The Fst of the candidate SNP locus among different ethnic groups is less than 0.01;

[0364] (2) The candidate SNP locus is located on different chromosomes;

[0365] (3) The allele frequency of the candidate SNP locus is between 0.3 and 0.7.

[0366] In certain embodiments, the candidate SNP locus is a SNP locus with biallelic polymorphism.

[0367] In certain embodiments, the candidate SNP sites are SNP sites in the human genome; for example, the target nucleic acid comprises a human genomic SNP site selected from the following: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs5858210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, rs7160304, and any combination of the foregoing SNP sites (e.g., a combination of any 5, 10, 15, 20, 23 of the foregoing SNP sites).

[0368] In certain embodiments, the target nucleic acid comprises the following human genomic SNP sites: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs5858210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, and rs7160304.

[0369] In certain embodiments, the detection probe comprises a detection probe having a nucleotide sequence selected from the following or any combination thereof (e.g., a combination of any 5, 10, 15, 20, 23): SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, and 69.

[0370] In certain embodiments, the sequence of the first universal primer is as shown in SEQ ID NO:71.

[0371] In certain embodiments, the sequence of the second universal primer is as shown in SEQ ID NO:70.

[0372] In certain embodiments, the target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the following or any combination thereof (e.g., any combination of 5 pairs, 10 pairs, 15 pairs, 20 pairs, 23 pairs): SEQ ID NO: 1 and 2; 4 and 5; 7 and 8; 10 and 11; 13 and 14; 16 and 17; 19 and 20; 22 and 23; 25 and 26; 28 and 29; 31 and 32; 34 and 35; 37 and 38; 40 and 41; 43 and 44; 46 and 47; 49 and 50; 52 and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68.

[0373] It is understood that the first universal primer, the second universal primer, the target-specific primer pair, and the detection probe in the kit of the present application are used to implement the method described above. Therefore, the detailed descriptions of the first universal primer, the second universal primer, the target-specific primer pair, and the detection probe in the above text (including the descriptions of various preferred features and exemplary features) also apply here.

[0374] In certain embodiments, the kit further comprises one or more components selected from the following: an amplification primer set, a probe set, and reagents for digital PCR.

[0375] In certain embodiments, the amplification primer set comprises at least one amplification primer (e.g., a pair of amplification primers or more amplification primers) that can specifically amplify a nucleic acid molecule containing the SNP site under conditions allowing nucleic acid hybridization or annealing.

[0376] In certain embodiments, the probe set comprises a first probe and a second probe; wherein,

[0377] (i) The first probe and the second probe are each independently labeled with a reporter group and a quenching group, wherein the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the first probe and the second probe are labeled with different reporter groups (e.g., fluorescent groups); and

[0378] (ii) The first probe can hybridize or anneal (preferably completely complementary) to a nucleic acid molecule containing the first allele of the target SNP site, and the second probe can hybridize or anneal (preferably completely complementary) to a nucleic acid molecule containing the second allele of the target SNP site; and the first probe and the second probe are specific to different alleles.

[0379] In certain embodiments, the probe set comprises probes having nucleotide sequences selected from the following, or any combination thereof (e.g., any combination of 5, 10, 20, 40, 60): SEQ ID NO:73, 74, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

[0380] In certain embodiments, the amplification primer set comprises primer pairs having nucleotide sequences selected from the following, or any combination thereof (e.g., any combination of 5 pairs, 10 pairs, 15 pairs, 20 pairs, 23 pairs): SEQ ID NO:72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

[0381] In certain embodiments, the reagents for performing digital PCR are selected from one or more components including the following: reagents for preparing microdroplet samples, reagents for nucleic acid amplification, nucleic acid polymerases, reagents for detecting microdroplet samples, or any combination thereof.

[0382] In certain embodiments, the kit further comprises one or more components selected from the following: nucleic acid polymerases, reagents for nucleic acid amplification, reagents for performing melting curve analysis, or any combination thereof.

[0383] It is understood that the amplification primer set and probe set (first probe and second probe) in the kit of the present application are used to implement the methods described above. Therefore, the detailed descriptions of the amplification primer set and probe set (first probe and second probe) above (including the descriptions of various preferred and exemplary features) also apply here.

[0384] In certain embodiments, the nucleic acid polymerase is a template-dependent nucleic acid polymerase, such as a DNA polymerase, particularly a thermostable DNA polymerase; in certain embodiments, the nucleic acid polymerase is as defined above.

[0385] In certain embodiments, the reagents for nucleic acid amplification include a working buffer for an enzyme (such as a nucleic acid polymerase), dNTPs (labeled or unlabeled), water, a solution containing ions (such as Mg 2+ ), a single-stranded DNA binding protein, or any combination thereof.

[0386] In certain embodiments, the kit is used to determine whether a donor is present in a recipient sample, or to calculate the proportion of the donor in the recipient sample.

[0387] In certain embodiments, the digital PCR is selected from droplet digital PCR and chip digital PCR.

[0388] In certain embodiments, the present application provides the use of the identification primer set as described above for preparing a kit, the kit being used for asymmetric amplification of a target nucleic acid molecule, or for detecting the genotype of a candidate SNP site in a target nucleic acid molecule; or for identifying SNP sites at which the donor and the recipient have different genotypes; or for identifying SNP sites at which the recipient has homozygous alleles.

[0389] In certain embodiments, the kit further comprises a detection probe as defined above.

[0390] In certain embodiments, the kit is used to implement the method as described above.

[0391] In certain embodiments, the present application provides the use of the amplification primer set and the probe set as described above for preparing a kit, the kit being used for detecting the presence or proportion of the nucleic acid of the donor in a recipient sample after transplantation surgery.

[0392] In certain embodiments, the kit further comprises reagents for determining the genotype of one or more SNP sites in the genome of the recipient or the donor.

[0393] In certain embodiments, the kit further comprises the identification primer set and the detection probe as defined above.

[0394] In certain embodiments, the kit is used to implement the method as described above.

[0395] Advantages of the Invention

[0396] Compared with the prior art, the advantages of the present application are as follows: (1) Automated detection, with few manual operation steps and a short detection cycle. The unique SNP genotyping system of the present application can simultaneously perform genotyping of multiple SNPs, with a high degree of automation. The entire process from nucleic acid extraction to obtaining results can be completed within 1 day, enabling timely determination of the donor chimerism rate of patients who have completed bone marrow transplantation, evaluation of the hematopoietic stem cell chimeric state, and determination of the dd-cfDNA ratio of organ transplantation patients to reflect the health status of the graft; (2) Accurate and highly sensitive. It can absolutely quantify the copy number of heterologous DNA, accurately calculate the donor chimerism rate or dd-cfDNA ratio, and the detection sensitivity of heterologous DNA is as low as 0.1%. (3) Non-invasive and universal detection process: It does not rely on quantitative analysis of donor samples, has a low cost and intuitive digital quantitative results, making this method widely applicable.

[0397] The embodiments of the present invention will be described in detail below in conjunction with the drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and not to limit the scope of the present invention. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0398] Figure 1 An exemplary embodiment of the method of the present invention for detecting the presence or proportion of a donor in a recipient sample by SNP genotyping is schematically described to illustrate the basic principle of the method of the present invention.

[0399] Figure 1 A schematically describes the primer set and self-quenching fluorescence detection probe involved in this embodiment. Among them, the primer set includes: a first universal primer and a second universal primer, and a target-specific primer pair including a forward primer and a reverse primer; among them,

[0400] The first universal primer contains a first universal sequence (Tag1);

[0401] The second universal primer contains a second universal sequence (Tag2), and the second universal sequence contains the first universal sequence and additionally contains at least one nucleotide (such as 1-5, 5-10, 10-15, 15-20 or more nucleotides) at the 3' end of the first universal sequence;

[0402] The forward primer contains a first universal sequence and a forward nucleotide sequence specific to the target nucleic acid containing the SNP site, and the forward nucleotide sequence is located at the 3' end of the first universal sequence;

[0403] The reverse primer contains a second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid containing the SNP site, and the reverse nucleotide sequence is located at the 3'-end of the second universal sequence; and,

[0404] The forward primer and the reverse primer can specifically amplify the corresponding target nucleic acid containing the SNP site; and,

[0405] The second universal sequence cannot be completely complementary to the complementary sequence of the forward primer.

[0406] Figure 1 B schematically describes the principle of suppressing non-specific amplification of primer dimers when using the Figure 1 primer set of A for amplification. Among them, the primer dimers formed by non-specific amplification of the forward primer and the reverse primer will generate single-stranded nucleic acids with reverse sequences complementary to each other at their 5'-ends and 3'-ends after denaturation. This single-stranded nucleic acid will form a panhandle structure by itself during the annealing stage, preventing the annealing and extension of the first universal primer and the second universal primer to this single-stranded nucleic acid, thereby suppressing further amplification of the primer dimer.

[0407] Figure 1 C schematically describes the principle of simultaneously detecting multiple target nucleic acids containing SNP sites using the Figure 1 primer set of A and detection probes. In this embodiment, a pair of forward primer and reverse primer and a self-quenching fluorescence detection probe are designed for each target nucleic acid containing the SNP site respectively. The specific detection process is as follows:

[0408] First, PCR amplification is initiated by the target-specific primer pair with a low concentration to generate an initial amplification product, which contains two nucleic acid strands (nucleic acid strand A and nucleic acid strand B) complementary to the forward primer / first universal primer and the reverse primer / second universal primer respectively; subsequently, the subsequent PCR amplification of the initial amplification product is carried out by the first universal primer and the second universal primer with a high concentration.

[0409] Since the reverse primer / second universal primer contains the first universal sequence, the first universal primer can not only anneal to nucleic acid strand A (the nucleic acid strand complementary to the forward primer / first universal primer) and synthesize its complementary strand, but also anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer / second universal primer) and synthesize its complementary strand. That is to say, the first universal primer can amplify nucleic acid strand A and nucleic acid strand B simultaneously.

[0410] The second universal primer contains additional nucleotides at the 3'-end of the first universal sequence. Therefore, it is mismatched with nucleic acid strand A (the nucleic acid strand complementary to the forward primer / first universal primer) at the 3'-end (i.e., it cannot be fully complementary at the 3'-end). Thus, during the amplification process, the second universal primer will preferentially anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer / second universal primer) and synthesize its complementary strand, while it can basically not extend to synthesize the complementary strand of nucleic acid strand A (the nucleic acid strand complementary to the forward primer / first universal primer).

[0411] Therefore, as the PCR amplification proceeds, the synthesis efficiency of the complementary strand of nucleic acid strand A (nucleic acid strand B) will be significantly lower than that of the complementary strand of nucleic acid strand B (nucleic acid strand A), resulting in a large amount of synthesis and amplification of the complementary strand of nucleic acid strand B (nucleic acid strand A), while the synthesis and amplification of the complementary strand of nucleic acid strand A (nucleic acid strand B) are inhibited, thereby generating a large amount of target single-stranded products (nucleic acid strand A, which contains the sequence complementary to the forward primer / first universal primer and the sequence of the reverse primer / second universal primer), achieving asymmetric amplification. In addition, to further enhance the asymmetry of amplification, the ratio of the first universal primer to the second universal primer can also be adjusted so that the concentration of the first universal primer is lower than that of the second universal primer to better enrich the target single-stranded products. By simultaneously using multiple pairs of forward primers and reverse primers in the same reaction system, multiple target nucleic acids containing SNP sites can be simultaneously and asymmetrically amplified to generate a large amount of target nucleic acid single strands containing SNP sites.

[0412] After PCR amplification, multiple pre-added self-quenching fluorescence detection probes respectively bind to the corresponding target nucleic acid single strands containing SNP sites to form double-stranded hybrids of the detection probes and the target nucleic acid single strands. Due to the different stabilities of the formed double-stranded hybrids, after melting curve analysis, different melting peaks can be obtained, and then according to the melting point (T m ) and the type of fluorescent group labeled by the probe, the genotypes of SNPs in each target nucleic acid single strand can be determined.

[0413] Figure 2 Shows the flow chart of the method of the present invention for determining the donor chimerism rate in bone marrow transplantation.

[0414] Figure 3 Shows the flow chart of the method of the present invention for determining the dd-cfDNA ratio in organ transplantation.

[0415] Figure 4Shows the results of melting curve analysis after amplifying the genomic DNA (10 ng / μL) of the donors and recipients in the sample groups of bone marrow transplantation case 1 and case 2 using the system of the present invention in Example 2. Among them, the black solid line represents the detection result of the donor genomic DNA in the sample group of bone marrow transplantation case 1; the black dashed line represents the detection result of the recipient genomic DNA in the sample group of case 1; the gray solid line represents the detection result of the donor genomic DNA in the sample group of bone marrow transplantation case 2; the gray dashed line represents the detection result of the recipient genomic DNA in the sample group of case 2.

[0416] Figure 5 Shows the results of melting curve analysis after amplifying the genomic DNA (10 ng / μL) of the donors and recipients in the sample group of organ transplantation case 3 using the system of the present invention in Example 3. Among them, the black solid line represents the detection result of the donor genomic DNA in the sample group of organ transplantation case 3; the black dashed line represents the detection result of the recipient genomic DNA in the sample group of case 3; and, the gray solid line represents the results of melting curve analysis after amplifying the urine-free DNA (1 ng / μL) on the 3rd day after surgery in the sample group of organ transplantation case 3 using the system of the present invention in Example 4.

[0417] Figure 6 Shows the results of melting curve analysis after amplifying the urine-free DNA (1 ng / μL) after surgery and the genomic DNA (10 ng / μL) of the recipient in the sample group of organ transplantation case 4 using the system of the present invention in Example 5. Among them, the black solid line represents the detection result of the urine-free DNA after surgery in the sample group of organ transplantation case 4; the black dashed line represents the detection result of the recipient genomic DNA in the sample group of case 4; and, the results of melting curve analysis after amplifying the genomic DNA (10 ng / μL) of the recipient of organ transplantation case 5 (without donor sample) using the system of the present invention. Among them, the gray dashed line represents the detection result of the recipient genomic DNA of case 5. Detailed implementation mode

[0418] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention (but not to limit the present invention). It should be understood that these examples are only used to illustrate the principles and technical effects of the present invention, and do not represent all possibilities of the present invention. The present invention is not limited to the materials, reaction conditions or parameters mentioned in these examples. Those skilled in the art can use other similar materials or reaction conditions to implement other technical solutions according to the principles of the present invention. Such technical solutions do not depart from the basic principles and concepts described in the present invention and are covered by the scope of the present invention.

[0419] Example 1. Selection of Candidate SNP Loci

[0420] The SNP sites covered by the present invention are selected from the Single Nucleotide Polymorphism Database (dbSNP) of the National Center for Biotechnology Information (NCBI) in the United States. The SNP sites of the present invention preferably meet the following conditions: (1) Fst (population fixation coefficient) < 0.01 among different ethnic groups, that is, the differentiation degree of these sites in the populations of different ethnic groups is very small, and the gene heterozygosity levels are close; (2) the allele frequency is between 0.3 and 0.7; (3) the distribution in the Asian population follows the Hardy-Weinberg equilibrium; (4) the distance between every two SNPs is > 1 Mb; (5) in order to avoid linkage between different sites, sites located on different chromosomes are preferably selected. According to the above criteria, 23 preferred SNP sites are selected in this example, as shown in Table 1 specifically. The SNP site information and sequences are queried and downloaded from the dbSNP database of the National Center for Biotechnology Information (NCBI) in the United States. The allele frequencies refer to the Asian population frequencies sourced from the 1000 Genomes Project database. These sites are evenly distributed on each chromosome of the genome.

[0421] Table 1. SNP site information selected in Example 1

[0422]

[0423]

[0424] Example 2. Determination of Chimerism Rate of Bone Marrow Transplant Donors

[0425] The detection process of this example is as Figure 2 shown. Taking 2 groups of bone marrow transplantation samples as an example, the following two parts of samples are collected: 1. The donor samples and recipient samples before transplantation of the bone marrow transplantation patients are collected and extracted respectively for SNP genotyping. The principle of SNP genotyping is as Figure 1 shown. 2. The peripheral blood at each time point during the recipient monitoring period after transplantation is collected, and genomic DNA is extracted for the quantification of the target SNP sites, detecting the donor chimerism rate after bone marrow transplantation, and evaluating the chimeric status after allogeneic hematopoietic stem cell transplantation.

[0426] The specific operation steps of the above detection process are as follows:

[0427] 1. Collect 2 groups of bone marrow transplantation samples (each group includes the donor sample before transplantation, the recipient sample before transplantation, and the recipient samples at each time point after transplantation). Among them, the blood samples are collected using EDTA anticoagulant tubes (Zhejiang Gongdong Medical Instrument Co., Ltd., Taizhou) and stored at 4°C; the saliva samples are collected using a saliva collector (Xiamen Zhishan Biotechnology Co., Ltd., Xiamen) according to the requirements of its instruction manual and stored at room temperature.

[0428] 2. Use the Lab-Aid 824 nucleic acid extractor and the supporting genomic DNA extraction reagents for blood and saliva (Xiamen Zhishan Biotechnology Co., Ltd., Xiamen) to extract the genomic DNA of each of the above blood and saliva samples, and use a Nanodrop-2000 micro ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, USA) to measure the concentration and purity of the genomic DNA.

[0429] 3. SNP genotyping: According to the selected SNP loci, design the corresponding primers and probes, and use the multiplex asymmetric PCR system of the present invention (the principle is as Figure 1 shown) to simultaneously genotype 23 SNPs in 2 PCR reaction systems. The primer and probe sequences and the concentrations used are shown in Table 2.

[0430] Table 2. Primer, probe sequences and concentrations used in Example 2

[0431]

[0432]

[0433]

[0434] The specific configuration of the SNP genotyping system is as follows: A 25 μL PCR reaction system contains: 1×PCR buffer (TAKARA, Beijing), 5.0 mM MgCl 2 , 0.2 mM dNTPs, 1 U Taq DNA polymerase (TAKARA, Beijing), the primers and probes and their dosages are shown in Table 2, and 5 μL of human genomic DNA or negative control (water). The PCR amplification program is: pre-denaturation at 95°C for 5 min; 10 cycles of denaturation at 95°C for 15 s, annealing at 65°C - 56°C for 15 s (decreasing by 1°C for each cycle), and extension at 76°C for 20 s; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 76°C for 20 s, 50 cycles; then perform melting curve analysis, and the program is: denaturation at 95°C for 1 min, maintenance at 37°C for 3 min; then perform melting curve analysis by increasing the temperature from 40°C to 85°C at a rate of 0.04°C / s, and collect the fluorescence signals of the FAM, HEX, ROX, CY5, and Quasar705 channels. The instrument used in this experiment is a SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.). The typical results of SNP genotyping of the donor and recipient samples of the bone marrow transplantation cases in this example are as Figure 4 shown.

[0435] 4. Screening of target SNP loci: By comparing the genotypes of corresponding SNP loci in the donor DNA sample and the recipient DNA sample, the target SNP loci are obtained, that is, the same SNP locus in the donor DNA sample and the recipient DNA sample, where the SNP locus genotype of the donor sample is homozygous AA (or BB), and the SNP locus genotype of the recipient sample is the other homozygous BB (or AA); or the SNP locus genotype of the donor sample is homozygous AA (or BB), and the SNP locus genotype of the recipient sample is heterozygous AB. In this embodiment, taking the sample groups of bone marrow transplantation case 1 and case 2 as examples, the SNP typing results of the donor DNA sample and the recipient DNA sample are shown in Table 3 and Figure 4 . Among them, there are 6 target SNP loci in the sample group of case 1 (i.e., rs2307839, rs16363, rs12990278, rs4971514, rs9613376, rs7160304). Two preferred target SNP loci (i.e., rs12990278, rs4971514) are selected and the allele copy numbers are quantitatively analyzed using a digital PCR system to determine the donor chimerism rate; there are 10 target SNP loci in the sample group of case 2 (i.e., rs2307839, rs66960151, rs68076527, rs5789826, rs1611048, rs149809066, rs12990278, rs2122080, rs774763, rs9613776). Two preferred target SNP loci (i.e., rs5789826, rs2122080) are selected and the allele copy numbers are quantitatively analyzed using a digital PCR system to determine the donor chimerism rate.

[0436] Table 3: SNP typing results of the sample groups of bone marrow transplantation case 1 and case 2

[0437]

[0438]

[0439] 5. Quantitative detection of genomic DNA samples: Digital PCR quantitative analysis systems are established according to the selected target SNP loci. Each system contains a pair of primers and two fluorescent probes specific for different alleles of the SNP. The primers, probes and usage amounts used in the quantitative systems of each SNP locus are shown in Table 4. For the selected target SNP loci, the corresponding primer sets and probe sets in the digital PCR system are used to determine the proportions of each allele of the target SNP locus.

[0440] The digital PCR system configuration, PCR amplification program, operation process, and data analysis are as follows: The droplet digital PCR consists of a Drop Marker sample preparation instrument, a Chip Reader biochip reader (Novogene Bioinformatics Technology Co., Ltd., Beijing), and a LongGene A300 amplifier (LongGene Scientific Instruments Co., Ltd., Hangzhou) to form a complete digital PCR system. A microdroplet sample preparation general kit and a microdroplet sample detection general kit (Novogene Bioinformatics Technology Co., Ltd., Beijing) are used. The 30 μL PCR reaction solution contains a ddPCR general amplification reagent (Novogene Bioinformatics Technology Co., Ltd., Beijing) and a synthetic sequence (Shanghai Bioengineering Co., Ltd., Shanghai). Optionally, after pre-enriching the above free DNA samples, primer sets and probe sets corresponding to the target SNP sites in the digital PCR system are used. The upstream and downstream primer concentrations are 0.8 μmol / L, and the fluorescence probe concentration is 0.25 μmol / L to measure the SNP allele copy number. After adding the genomic DNA sample to the PCR premix, use the Drop Marker sample preparation instrument to prepare droplets with a volume in the nanoliter range. The PCR amplification program is pre-denaturation at 95°C for 10 min, 40 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 60 s, and incubation at 12°C after amplification. The overall temperature change rate is 1.5°C / s. After the PCR reaction is completed, use the Chip Reader biochip reader to quantitatively detect the microdroplets. The reading system exports the sample detection data in Excel format, including the number of negative and positive microdroplets and copy numbers in the FAM and HEX fluorescence channels.

[0441] 6. Calculation of donor chimerism rate: A quantitative analysis model for donor chimerism rate is derived based on the SNP molecular marker biallelic characteristics and the Hardy-Weinberg equilibrium law of genetic equilibrium.

[0442] 1) If the selected target SNP site has the donor SNP genotype of AA and the recipient SNP genotype of BB, and the number of recipient B alleles measured by digital PCR is N B , and the proportion of the number of donor A alleles measured is N A , then the percentage of donor genomic DNA in the total recipient genomic DNA is the donor chimerism rate:

[0443]

[0444] 2) If the selected target SNP site has the donor SNP genotype of BB and the recipient SNP genotype of AA, and the number of recipient A alleles measured by digital PCR is N A , and the proportion of the number of donor B alleles measured is N B , then the percentage of donor genomic DNA in the total recipient genomic DNA is the donor chimerism rate:

[0445]

[0446] 3) If the SNP genotype of the donor at the selected target SNP locus is AA, and the SNP genotype of the recipient is AB, and the number of B alleles of the recipient measured by digital PCR is N B , and the number of A alleles of the donor measured is in the proportion of N A , then the percentage of the donor genomic DNA in the total genomic DNA of the recipient is the donor chimerism rate:

[0447]

[0448] 4) If the SNP genotype of the donor at the selected target SNP locus is BB, and the SNP genotype of the recipient is AB, and the number of A alleles of the recipient measured by digital PCR is N A , and the number of B alleles of the donor measured is in the proportion of N B , then the percentage of the donor genomic DNA in the total genomic DNA of the recipient is the donor chimerism rate:

[0449]

[0450] For detecting multiple target SNP loci, first based on the donor chimerism rates detected at each target SNP locus, and then calculate their average value as the donor chimerism rate in the analysis report.

[0451] Table 4. Primers and probes used in the digital PCR quantitative analysis system

[0452]

[0453]

[0454]

[0455] 7. Detection results

[0456] The donor chimerism rates after transplantation of 2 cases of bone marrow transplantation were measured by the method of the present invention. Blood was collected at 4 time points after transplantation, and the detection results of the donor chimerism rates of the recipients at different time points are shown in Table 5. It can be seen from the results in the table that Case 1 and Case 2 were in a recovery state after transplantation, and the recipient chimerism rates at each time point were all greater than 95%.

[0457] Table 5: Detection results of donor chimerism rates after transplantation of 2 cases of bone marrow transplantation

[0458]

[0459] Example 3. Determination of the Proportion of Donor-Free DNA in Organ Transplantation (with Donor Information)

[0460] This example takes the determination of the proportion of donor cfDNA in plasma and urine samples after kidney transplantation as an example to monitor the organ damage of Case 3 of kidney transplantation, and to investigate the feasibility and detection performance of the method of the present invention for determining the proportion of dd-cfDNA in organ transplantation.

[0461] The detection process of this example case is as Figure 3 shown. Taking a group of kidney transplant samples as an example, the following two parts of samples need to be collected: (1) Collect and extract the donor samples and recipient samples before transplantation of kidney transplant patients for SNP genotyping. The principle of SNP genotyping is as Figure 1 shown; or collect the recipient blood cell precipitate, saliva, tissues outside the transplanted organ, skin, etc. after transplantation as the recipient sample before transplantation. (2) Collect the peripheral blood and urine at each time point during the recipient monitoring period after transplantation, separate the plasma and urine supernatant, and then extract cfDNA for target SNP quantification to detect the proportion of dd-cfDNA after organ transplantation and evaluate the degree of postoperative organ damage.

[0462] The specific operation steps of the above detection process are as follows:

[0463] 1. Collection of kidney transplant sample group

[0464] The sample group of Case 3 includes the donor peripheral blood sample, recipient peripheral blood sample before transplantation, and recipient samples (plasma and urine) at each time point after transplantation. Blood samples are collected using EDTA anticoagulant tubes (Zhejiang Gongdong Medical Instrument Co., Ltd., Taizhou), and plasma is separated within 2 hours after collection according to the standard separation process (1600g, centrifugation for 10 min, 16000g, centrifugation for 10 min). The plasma samples are stored frozen at -80°C; urine samples are collected using urine collection cups (Zhejiang Gongdong Medical Instrument Co., Ltd., Taizhou), and the supernatant is taken within 6 hours after collection according to the standard separation process (5000g, centrifugation for 20 min). The urine supernatant samples are stored frozen at -80°C.

[0465] 2. Extraction of genomic DNA and cell-free DNA

[0466] Use the Lab-Aid 824 nucleic acid extractor and the supporting blood genomic DNA extraction reagent (Xiamen Zesun Biotech Co., Ltd., Xiamen) to extract the genomic DNA of the above blood samples, and use the Nanodrop-2000 micro ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, USA) to measure the concentration and purity of the genomic DNA. Use Apostle MiniMax TMAn efficient cell-free DNA enrichment and isolation kit was used to extract cell-free DNA from plasma and urine samples (Apostle, USA), and the concentration of cell-free DNA was measured using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA).

[0467] 3. SNP genotyping

[0468] According to the selected SNP loci, corresponding primers and probes were designed. Using a multiplex asymmetric PCR genotyping system (the principle is as Figure 1 shown), 23 SNPs were genotyped simultaneously in 2 PCR reaction systems. The primer and probe sequences and their working concentrations are shown in Table 2. The specific configuration of the SNP genotyping system is the same as that in Example 2. The typical SNP genotyping results of the donor and recipient samples in the kidney transplantation cases in this example are as Figure 5 and Table 6 show.

[0469] Table 6. SNP genotyping results of the sample group of organ transplantation case 3

[0470]

[0471] 4. Screening of target SNP loci

[0472] By comparing the genotypes of the corresponding SNP loci of the donor genomic DNA and the recipient genomic DNA in the sample group of case 3, the target SNP loci were obtained, that is, the same SNP locus in the donor DNA sample and the recipient DNA sample. The SNP locus genotype of the donor sample is homozygous AA (or BB), and the SNP locus genotype of the recipient sample is the other homozygous BB (or AA), or the SNP locus genotype of the donor sample is heterozygous AB, and the SNP locus genotype of the recipient sample is homozygous AA (or BB). In this example, there are 3 target SNP loci in the sample group of case 3 (i.e., rs2122080, rs10779650, rs7160304). For these 3 target SNP loci, a digital PCR system was used to quantitatively analyze the allele copy number and determine the proportion of donor cell-free DNA.

[0473] 5. Pre-enrichment of cell-free DNA samples

[0474] According to the SNP loci selected in Example 1, pre-enrichment primers were designed. Each SNP enrichment primer pair is the same as the primer pair used in the SNP quantification system in digital PCR. See Table 4 in Example 1 for details. The pre-enrichment system is a 50 μL PCR reaction system, and the specific configuration is as follows: 1×PCR buffer (TAKARA, Beijing), 5.0 mM MgCl 2, 0.2 mM dNTPs, 2 U Taq DNA polymerase (TAKARA, Beijing), the dosage of each primer is shown in Table 4, add 1 - 10 ng of free DNA, and make up to 50 μL with ultrapure water. The PCR amplification program is as follows: pre-denaturation at 95°C for 5 min; 10 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 4 min, and extension at 72°C for 2 min. The instrument used in this experiment is an A300 type amplifier (Langji Scientific Instruments Co., Ltd., Hangzhou).

[0475] 6. Quantitative detection of free DNA samples

[0476] According to the SNP loci selected in Example 1, digital PCR quantitative analysis systems were established respectively. Each system contains a pair of primers and two probes specific for SNP alleles respectively. The primers, probes and their dosages used in the quantitative systems of each SNP locus are shown in Table 4. For the selected target SNP locus, the corresponding primer set and probe set in the digital PCR system were used to determine the proportion of each allele of the target SNP locus. The specific configuration of the quantitative detection system for free DNA samples is the same as that described for the quantitative detection of genomic DNA samples in Example 1.

[0477] 7. Calculation of the proportion of donor free DNA

[0478] The calculation method for Case 3 is as follows:

[0479] According to the SNP molecular marker biallelic characteristics and the Hardy-Weinberg equilibrium law of genetic equilibrium, a quantitative analysis model for dd-cfDNA can be deduced.

[0480] 1) If the selected target SNP locus has the donor's SNP genotype as AA and the recipient's SNP genotype as BB, and the number of donor A alleles determined by digital PCR is in the proportion of N A , and the number of recipient B alleles determined is N B , then the proportion of donor cfDNA in the total recipient cfDNA is:

[0481]

[0482] 2) If the selected target SNP locus has the donor's SNP genotype as BB and the recipient's SNP genotype as AA, and the number of donor B alleles determined by digital PCR is in the proportion of N B , and the number of recipient A alleles determined is N A , then the proportion of donor cfDNA in the total recipient cfDNA is:

[0483]

[0484] 3) If the SNP genotype of the donor at the selected target SNP locus is AB, and the SNP genotype of the recipient is AA, and the proportion of the number of donor B alleles measured by digital PCR is N B , and the number of recipient A alleles measured is N A , then the proportion of donor cfDNA in the total cfDNA of the recipient is:

[0485]

[0486] 4) If the SNP genotype of the donor at the selected target SNP locus is AB, and the SNP genotype of the recipient is BB, and the proportion of the number of donor A alleles measured by digital PCR is N A , and the number of recipient B alleles measured is N B , then the proportion of donor cfDNA in the total cfDNA of the recipient is:

[0487]

[0488] For the above cases where multiple target SNP loci are detected, first based on the dd-cfDNA ratios detected at each target SNP locus, and then calculate their average value as the dd-cfDNA ratio in the analysis report.

[0489] 8. Analysis of Detection Results

[0490] Using the method of the present invention to measure the postoperative dd-cfDNA ratio of kidney transplantation cases, blood and urine were collected at 7 time points after transplantation. After detection, the dd-cfDNA ratios of the samples collected from each recipient at different time points are shown in Table 7.

[0491] Table 7: Results of Measuring the dd-cfDNA Ratio after Kidney Transplantation in Case 3

[0492]

[0493]

[0494] Example 4. Urine-Free DNA after Kidney Transplantation for Screening Target SNP Loci

[0495] In organ transplantation monitoring, it may be impossible to collect donor samples. Taking the recipient samples of Example 3 as an example, this example simulates the situation where donor samples cannot be obtained, uses the free DNA in the urine of the recipient after kidney transplantation as the SNP genotyping template, and examines the feasibility of the method of this application for screening target SNP loci. Based on the urine dd-cfDNA ratio in Example 3 and the literature reports, the fluctuation range of the urine dd-cfDNA ratio is from 5% to 80%.

[0496] According to the SNP sites selected in Example 1, corresponding primers and probes were designed. Using a multiplex asymmetric PCR genotyping system (the principle is as shown in Figure 1 ), 23 SNPs were genotyped simultaneously in 2 PCR reaction systems. The primer and probe sequences and their usage concentrations are shown in Table 2. The specific configuration of the SNP genotyping system is the same as that in Example 2. The typical SNP genotyping results of the recipient urine-free DNA samples, donor and recipient genomic DNA samples after kidney transplantation in this case are as shown in Figure 5 and Table 7.

[0497] Table 7: SNP genotyping results of the sample group of organ transplantation case 3

[0498]

[0499]

[0500] By comparing the genotypes of the corresponding SNP sites of the recipient urine-free DNA on the 3rd day after surgery and the recipient genomic DNA in the sample group of case 3, target SNP sites were screened, that is, among the same SNP sites, the SNP sites where the recipient urine-free DNA sample on the 3rd day after surgery and the recipient genomic DNA sample have different alleles were screened. In this example, 3 target SNP sites (i.e., rs2122080, rs10779650, rs7160304) can be screened. This screening result is consistent with the result of the target SNP sites screened by using the donor genomic DNA sample and the recipient genomic DNA sample in Example 3, indicating that urine-free DNA after kidney transplantation can be used for the screening of target SNP sites when donor samples cannot be obtained.

[0501] The investigation results of Example 4 show that the free DNA extracted from the blood and urine samples collected after organ transplantation (such as cfDNA in peripheral blood on the 1st day after transplantation or urine cfDNA after kidney transplantation) contains some donor-derived free DNA. When the proportion of donor free DNA reaches a certain level (for example, reaches 20% and above), the cfDNA can be directly genotyped by using the SNP genotyping system, and by comparing the SNP genotyping results of the recipient's own genomic DNA, the target SNP sites can be screened.

[0502] Example 5. Determination of the Proportion of Donor-Free DNA in Organ Transplantation (without Donor Information)

[0503] In this example, taking the sample groups of kidney transplantation cases 4 and 5 as examples, the feasibility and detection performance of the method of the present invention for measuring the dd-cfDNA ratio after organ transplantation when donor samples cannot be obtained were investigated.

[0504] The specific operation steps are as follows:

[0505] 1. Collect 2 groups of kidney transplantation samples

[0506] Collect pre-transplant recipient samples (blood) and recipient samples (blood and urine) at each time point after transplantation of the sample group of Case 4, and pre-transplant recipient samples (blood) and recipient samples (blood) at each time point after transplantation of the sample group of Case 5. Among them, blood samples were collected using EDTA anticoagulant tubes (Zhejiang Gongdong Medical Instrument Co., Ltd., Taizhou), and plasma separation was performed within 2 hours after collection according to the standard separation process (1600g, centrifugation for 10 minutes, 16000g, centrifugation for 10 minutes), and the plasma samples were stored frozen at -80°C; urine samples were collected using urine collection cups (Zhejiang Gongdong Medical Instrument Co., Ltd., Taizhou), and the supernatant was taken within 6 hours after collection according to the standard separation process (5000g, centrifugation for 20 minutes), and the urine supernatant samples were stored frozen at -80°C.

[0507] 2. Extraction of genomic DNA and cell-free DNA

[0508] Use the Lab-Aid 824 nucleic acid extractor and the supporting blood extraction reagent (Xiamen Zesheng Biotechnology Co., Ltd., Xiamen) to extract the genomic DNA of each blood above, and use the Nanodrop-2000 micro ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, USA) to measure the concentration and purity of genomic DNA. Use the Apostle MiniMax TM High-efficiency cell-free DNA enrichment and separation kit to extract cell-free DNA from blood and urine (Apostle, USA), and use the Qubit3.0 fluorometer (Thermo Fisher Scientific, USA) to measure the concentration of cell-free DNA.

[0509] 3. SNP genotyping

[0510] According to the selected SNP sites, design the corresponding primers and probes, and use the multiplex asymmetric PCR genotyping system (the principle is as Figure 1 shown) to simultaneously genotype 23 SNPs in 2 PCR reaction systems. The primer and probe sequences and the use concentrations are shown in Table 2. The specific configuration of the SNP genotyping system is the same as that of Example 2. The typical SNP genotyping results of the urine samples and recipient samples after kidney transplantation in this case are as Figure 6 and Table 8 show.

[0511] Table 8: SNP genotyping results of the sample groups of Organ Transplantation Cases 4 and 5

[0512]

[0513] 4. Screening of target SNP sites

[0514] Compare the genotypes of the corresponding SNP sites of urine cfDNA and recipient genomic DNA in the sample group of Comparative Case 4 to obtain the target SNP sites, that is, the SNP genotype of the recipient is homozygous AA (or BB), and after transplantation, the SNP sites where the recipient urine cfDNA sample shows different alleles from the recipient genomic DNA sample. In this embodiment, 6 target SNP sites were screened in Case 4 (i.e., rs5858210, rs5789826, rs34765837, rs16363, rs1610937, rs149809066). Select 3 of the target SNP sites (i.e., rs5858210, rs149809066, rs1610937) and use a digital PCR system to quantitatively analyze the allele copy numbers thereof to determine the proportion of donor-free DNA.

[0515] For Case 5 without a donor sample, select SNP sites where the recipient sample genotype is homozygous (e.g., AA or BB). In Case 5, there are 11 SNP sites where the recipient sample is homozygous (i.e., rs2307839, rs112552066, rs5858210, rs66960151, rs68076527, rs34765837, rs1610937, rs2307533, rs98506667, rs10779650, rs9613776). Select 8 of the SNP sites and use a digital PCR system to quantitatively analyze the allele copy numbers of the 8 SNP sites in the postoperative blood cfDNA sample of Case 5 for the subsequent determination of the proportion of donor-free DNA.

[0516] 5. Pre-enrichment of free DNA samples

[0517] Design pre-enrichment primers according to the SNP sites selected in Example 1. Each SNP enrichment primer pair is the same as the primer pair used in the SNP quantitative system in digital PCR. See Table 4 in Example 1 for details. The pre-enrichment system is a 50 μL PCR reaction system, and the specific configuration is as follows: 1×PCR buffer (TAKARA, Beijing), 5.0 mM MgCl 2 , 0.2 mM dNTPs, 2 U Taq DNA polymerase (TAKARA, Beijing). The dosage of each primer is shown in Table 4. Add 1 - 10 ng of free DNA and make up to 50 μL with ultrapure water. The PCR amplification program is: pre-denaturation at 95°C for 5 min; 10 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 4 min, and extension at 72°C for 2 min. The instrument used in this experiment is an A300 type amplifier (Langji Scientific Instruments Co., Ltd., Hangzhou).

[0518] 6. Quantitative detection of free DNA samples

[0519] According to the SNP sites selected in Example 1, digital PCR quantitative analysis systems were established respectively. Each system included a pair of primers and two probes specific for SNP alleles respectively. The primers, probes and their usage amounts used in the quantitative systems of each SNP site are shown in Table 4. For the selected target SNP sites, the corresponding primer sets and probe sets in the digital PCR system were used to determine the proportions of each allele of the target SNP sites. The specific configuration of the quantitative detection system for the cell-free DNA sample was the same as that described for the quantitative detection of the genomic DNA sample in Example 1.

[0520] 7. Calculation of the proportion of donor cell-free DNA

[0521] 7.1 Calculation method for Case 4

[0522] After reading the digital PCR results of the target SNP sites, the absolute copy numbers of different alleles can be obtained. The copy number proportions of the donor-specific alleles can be divided into two categories by cluster analysis (K-means). There is a two-fold relationship between these two categories of values, that is, the two-fold copy number relationship between the heterozygous type and the homozygous type. Chi-square test was performed using the two categories of data after cluster analysis to determine whether there is a significant difference in the two-fold relationship. Taking the blood cfDNA sample on the first day after surgery of Case 4 as an example, the quantitative analysis of the specific allele ratios of the digital PCR systems for 3 target SNP sites is shown in Table 9. The corrected mean values of the 3 target SNP sites (rs5858210, rs149809066, rs1610937) were used as the dd-cfDNA ratio in the analysis report, that is, 36.41%.

[0523] Table 9: Analysis of postoperative blood cfDNA samples of organ transplantation Case 4

[0524]

[0525] 7.2 Calculation method for Case 5:

[0526] The SNP genotype of the recipient in Case 5 is homozygous AA or BB. Therefore, the alleles in the postoperative blood cfDNA sample of Case 5 that are different from those in the recipient's genomic DNA are considered to mostly come from the donor, and a very small part is due to signal interference below the blank detection limit of digital PCR. The SNP genotype of the donor may be heterozygous or homozygous, and the specific genotype is unknown. After reading the results of digital PCR, the absolute copy numbers of different alleles can be obtained. The copy number ratios of the donor-specific alleles can be classified into two categories by cluster analysis (K-means). There is a two-fold relationship between these two values, that is, the two-fold copy number relationship between heterozygous and homozygous types. The chi-square test is performed using the two categories of data after cluster analysis to determine whether there is a significant difference in the two-fold relationship. Taking the blood cfDNA sample of Case 5 on the second day after surgery as an example, quantitative analysis of the digital PCR system was performed on 8 SNP loci with homozygous genotypes in the recipient sample, and the specific allele ratios are shown in Table 10. The corrected mean of the 4 target SNP loci (rs2307839, rs66960151, rs10779650, rs9613776) was used as the dd-cfDNA ratio in the analysis report, that is, 3.76%.

[0527] Table 10: Analysis of postoperative blood cfDNA samples at homozygous loci of the recipient in Organ Transplantation Case 5

[0528]

[0529] 8. Analysis of Detection Results

[0530] The postoperative dd-cfDNA ratios of 2 cases of organ transplantation were measured using the method of the present invention. Blood was collected at 4 time points after transplantation. After testing, the dd-cfDNA ratios of the samples collected from each recipient at different time points are shown in Table 11.

[0531] Table 11: Measurement Results of Postoperative dd-cfDNA Ratios in 2 Cases of Organ Transplantation

[0532]

[0533] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings that have been disclosed, and these changes are within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for detecting SNP sites with different genotypes between a donor and a recipient for non-diagnostic and non-therapeutic purposes, comprising the following steps : (a) providing a first sample containing one or more target nucleic acids derived from the donor, and a second sample containing one or more target nucleic acids derived from the recipient, the target nucleic acids comprising one or more candidate SNP sites, and providing a first universal primer and a second universal primer, and for each candidate SNP site, providing at least one pair of target-specific primers; wherein, the first universal primer comprises a first universal sequence; the second universal primer comprises a second universal sequence, the second universal sequence comprising the first universal sequence and additionally comprising at least one nucleotide at the 3'-end of the first universal sequence; the pair of target-specific primers is capable of amplifying using the target nucleic acid as a template to produce a nucleic acid product containing the candidate SNP site, and the pair of target-specific primers comprises a forward primer and a reverse primer, wherein the forward primer comprises the first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3'-end of the first universal sequence; the reverse primer comprises the second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3'-end of the second universal sequence; and the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer; and (b) under conditions allowing nucleic acid amplification, using the first universal primer, the second universal primer and the pair of target-specific primers to amplify the target nucleic acids in the first sample and the second sample respectively, thereby obtaining amplification products corresponding to the first sample and the second sample respectively; (c) performing melting curve analysis on the amplification products corresponding to the first sample and the second sample obtained in step (b) respectively; (d) according to the results of the melting curve analysis in step (c), determining such SNP sites: at which the first sample and the second sample have different genotypes.

2. The method of claim 1, in step (d) of the method, determining the genotypes of each candidate SNP site of the first sample and the second sample according to the results of the melting curve analysis, thereby detecting SNP sites with different genotypes between the donor and the recipient.

3. The method of claim 1, wherein the recipient has received or is intended to receive or transplant organs, tissues or cells from the donor.

4. The method of claim 1, wherein the recipient has received or is intended to receive or transplant a kidney, heart, lung, liver, pancreas or any combination thereof from the donor.

5. The method of claim 1, wherein the recipient has received or is intended to receive or transplant hematopoietic stem cells from the donor or a tissue or organ containing hematopoietic stem cells.

6. The method of claim 1, wherein the recipient has received or is intended to receive or transplant bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, cord blood hematopoietic stem cells or any combination thereof from the donor; or bone marrow containing hematopoietic stem cells.

7. The method of claim 1, wherein the second sample does not contain nucleic acids from the donor.

8. The method of claim 1, wherein the first sample is from the donor.

9. The method of claim 1, wherein the first sample comprises cells or tissues from the donor.

10. The method of claim 1, wherein the first sample is selected from skin, saliva, urine, blood, hair, nails from the donor, or any combination thereof.

11. The method of claim 1, wherein the second sample is from a recipient who has undergone or not undergone transplantation surgery.

12. The method of claim 1, wherein the second sample comprises cells or tissues from the recipient.

13. The method of claim 1, wherein the second sample is selected from skin, saliva, urine, blood, hair, nails from the recipient, or any combination thereof.

14. The method of claim 1, wherein in step (a), for each candidate SNP locus, a detection probe is further provided, the detection probe comprising a nucleotide sequence specific to the target nucleic acid and capable of annealing or hybridizing to the region of the target nucleic acid containing the candidate SNP locus, and the detection probe is labeled with a reporter group and a quencher group, wherein, the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence; and in step (c), melting curve analysis is performed on the amplification products corresponding to the first sample and the second sample obtained in step (b) using the detection probe.

15. The method of claim 1, wherein the first sample comprises DNA.

16. The method of claim 1, wherein the second sample comprises DNA.

17. A method for detecting the presence or proportion of nucleic acids of a donor in a sample of a recipient who has undergone transplantation surgery for non-diagnostic and non-therapeutic purposes, wherein, the method comprises the following steps: (1) Providing a test sample containing nucleic acids from a recipient who has received transplantation of cells, tissues or organs from a donor; (2) Identifying one or more target SNP loci by the method of claim 1, wherein at the target SNP loci, the recipient has a first genotype containing a first allele, and the donor has a second genotype containing a second allele, wherein the first genotype is different from the second genotype, and the first allele is different from the second allele; (3) Quantitatively detecting the first allele and the second allele of each target SNP locus in the test sample; then, based on the results of the quantitative detection of the first allele and the second allele, determining the presence or proportion of the nucleic acids of the donor in the test sample.

18. The method of claim 17, wherein in step (2), different alleles at a certain SNP locus are distinguished by a mechanism selected from the following to identify the target SNP locus: probe hybridization, primer extension, hybridization ligation, and specific enzymatic cleavage.

19. The method of claim 17, wherein in step (2), the target SNP locus is identified by a method selected from the following: sequencing method, chip method, qPCR-based detection method, mass spectrometry method, chromatography method, electrophoresis method, and melting curve analysis-based detection method.

20. The method of claim 17, in step (2), the target SNP locus is identified by a method selected from the following: first-generation sequencing, pyrosequencing, second-generation sequencing, using a solid-phase chip capable of detecting SNPs, a liquid-phase chip, Taqman probe method, iPLEX based on MassARRAY TM Gold, denaturing high-performance liquid chromatography dHPLC, and SNPshot method.

21. The method of claim 17, in step (2), identifying the target SNP locus by a detection method based on multiplex PCR combined with melting curve analysis.

22. The method of claim 17, identifying the target SNP locus by the method described in claim 1.

23. The method of claim 17, in step (3), quantitatively detecting the first allele and the second allele of each target SNP locus in the sample by digital PCR.

24. The method of claim 17, performing step (3) by the following protocol: (I) Select at least 1 target SNP locus from step (2), and for each selected target SNP locus, provide an amplification primer set and a probe set, wherein, (I-1) The amplification primer set includes at least one amplification primer which, under conditions allowing nucleic acid hybridization or annealing, can specifically amplify a nucleic acid molecule containing the target SNP locus; (I-2) The probe set includes a first probe and a second probe; wherein, (i) The first probe and the second probe are each independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and the first probe and the second probe are labeled with different reporter groups; and (ii) The first probe can hybridize or anneal with a nucleic acid molecule containing the first allele of the target SNP locus, and the second probe can hybridize or anneal with a nucleic acid molecule containing the second allele of the target SNP locus; and the first probe and the second probe are specific to different alleles; (II) Performing digital PCR on the recipient sample using the amplification primer set and the probe set to quantitatively detect nucleic acid molecules with the first allele and nucleic acid molecules with the second allele; (III) Determining the presence or proportion of donor nucleic acid in the sample to be tested according to the quantitative detection result of step (II).

25. The method of claim 17, the first probe specifically anneals or hybridizes with a nucleic acid molecule with the first allele during the digital PCR reaction; and the second probe specifically anneals or hybridizes with a nucleic acid molecule with the second allele during the digital PCR reaction.

26. The method of claim 17, the first probe does not anneal or hybridize with a nucleic acid molecule with the second allele during the digital PCR reaction; and / or the second probe does not anneal or hybridize with a nucleic acid molecule with the first allele during the digital PCR reaction.

27. The method of claim 17, before step (3), preprocessing the sample to be tested from the recipient, the preprocessing including nucleic acid extraction of the sample and / or enrichment of the nucleic acid in the sample.

28. The method of claim 1, the recipient has received or transplanted hematopoietic stem cells or tissues or organs containing hematopoietic stem cells from a donor.

29. The method of claim 1, the sample to be tested contains blood or its components from the recipient after transplantation.

30. The method of claim 1, wherein the test sample comprises blood cells, plasma, monocytes, granulocytes, T cells, or any combination thereof, from a recipient after transplantation.

31. The method of claim 1, wherein the target SNP locus is an SNP locus at which the recipient has a first genotype comprising a homozygous first allele and the donor has a second genotype comprising a homozygous second allele.

32. The method of claim 1, wherein, the recipient has received or transplanted an organ from a donor.

33. The method of claim 1, wherein the test sample comprises blood or urine from a recipient after transplantation.

34. The method of claim 1, wherein when the transplantation is a kidney transplantation, the test sample comprises urine from a recipient after transplantation.

35. The method of claim 1, wherein the target SNP locus is an SNP locus at which the donor has a first genotype comprising a homozygous first allele and the recipient has a second genotype comprising a homozygous second allele.

36. The method of claim 1, wherein, steps (a)-(b) of the method are carried out by a protocol comprising the following steps (I)-(VI): (I) Providing the first sample, the second sample, the first universal primer and the second universal primer, and the target-specific primer pair; (II) Mixing the samples with the first universal primer, the second universal primer, the target-specific primer pair, and a nucleic acid polymerase; (III) Incubating the product of the previous step under conditions permitting nucleic acid denaturation; (IV) Incubating the product of the previous step under conditions permitting nucleic acid annealing or hybridization; and (V) Incubating the product of the previous step under conditions permitting nucleic acid extension; and (VI) Repeating steps (III)-(V) one or more times.

37. The method of claim 1, wherein, steps (a)-(b) of the method are carried out by a protocol comprising the following steps (I)-(VI): (I) Providing the first sample, the second sample, the first universal primer and the second universal primer, and the target-specific primer pair; and a detection probe; (II) Mixing the samples with the first universal primer, the second universal primer, the target-specific primer pair, a nucleic acid polymerase, and the detection probe; (III) Incubating the product of the previous step under conditions permitting nucleic acid denaturation; (IV) Incubating the product of the previous step under conditions permitting nucleic acid annealing or hybridization; (V) Incubating the product of the previous step under conditions permitting nucleic acid extension; and (VI) Repeating steps (III)-(V) one or more times.

38. The method of claim 36 or 37, wherein the method has one or more of the following technical features: (1) In step (III), incubating the product of step (II) at a temperature of 80-105 °C to denature the nucleic acid; (2) In step (III), incubating the product of step (II) for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min; (3) In step (IV), incubate the product of step (III) at a temperature of 35 - 40 °C, 40 - 45 °C, 45 - 50 °C, 50 - 55 °C, 55 - 60 °C, 60 - 65 °C, or 65 - 70 °C, thereby allowing nucleic acid annealing or hybridization; (4) In step (IV), incubate the product of step (III) for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, or 2 - 5 min; (5) In step (V), incubate the product of step (IV) at a temperature of 35 - 40 °C, 40 - 45 °C, 45 - 50 °C, 50 - 55 °C, 55 - 60 °C, 60 - 65 °C, 65 - 70 °C, 70 - 75 °C, 75 - 80 °C, 80 - 85 °C, thereby allowing nucleic acid extension; (6) In step (V), incubate the product of step (IV) for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, 2 - 5 min, 5 - 10 min, 10 - 20 min, or 20 - 30 min; (7) Steps (IV) and (V) are carried out at the same or different temperatures; and (8) Repeat steps (III) - (V) at least once; wherein, when repeating steps (III) - (V) one or more times, the conditions used for steps (III) - (V) in each cycle are independently the same or different.

39. The method according to claim 24, wherein, each primer of the amplification primer set independently has one or more technical features selected from the following: (1) The length of the primer is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt; (2) The primer or any of its components comprises or consists of naturally occurring nucleotides, modified nucleotides, non - natural nucleotides, or any combination thereof; (3) The amplification primer set includes primer pairs having nucleotide sequences selected from the following or any combination thereof: SEQ ID NO:72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

40. The method according to claim 24, wherein, each of the first probe and the second probe independently has one or more features selected from the following: (1) The first probe and the second probe each independently comprise or consist of naturally occurring nucleotides, modified nucleotides, unnatural nucleotides, or any combination thereof; (2) The lengths of the first probe and the second probe are each independently 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 200 nt, 200 - 300 nt, 300 - 400 nt, 400 - 500 nt, 500 - 600 nt, 600 - 700 nt, 700 - 800 nt, 800 - 900 nt, 900 - 1000 nt; (3) The first probe and the second probe each independently have a 3'-OH terminus; alternatively, the 3'-terminus of the probe is blocked; (4) The first probe and the second probe are each independently self-quenching probes; (5) The reporter group in the probe is a fluorescent group; and the quenching group is a molecule or group capable of absorbing / quenching the fluorescence; (6) The first probe and the second probe are each independently linear or have a hairpin structure; (7) The first probe and the second probe have different reporter groups; (8) The probe set includes probes having nucleotide sequences selected from the following or any combination thereof: SEQ ID NO:74, 75, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

41. The method of claim 40, the method having one or more technical features selected from the following: (1) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; (2) The unnatural nucleotide is a peptide nucleic acid or a locked nucleic acid; (3) Blocking the 3'-terminus of the probe by adding a chemical moiety to the 3'-OH of the last nucleotide of the probe, by removing the 3'-OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide; (4) The probe is labeled with a reporter group at its 5'-end or upstream and with a quenching group at its 3'-end or downstream, or labeled with a reporter group at its 3'-end or downstream and with a quenching group at its 5'-end or upstream; (5) The reporter group and the quenching group are separated by a distance of 10 - 80 nt or more; (6) The fluorescent group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705; (7) The quenching group is selected from DABCYL, BHQ, ECLIPSE and / or TAMRA; (8) The probe has 5'-nuclease activity; (9) The backbone of the probe contains modifications that resist nuclease activity.

42. A method for identifying a SNP locus in a receptor having a first genotype containing a homozygous first allele for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: (a) Providing a fifth sample from the receptor, wherein the fifth sample contains one or more target nucleic acids derived from the receptor and does not contain nucleic acids derived from a donor; the target nucleic acid contains one or more candidate SNP loci, and providing a first universal primer and a second universal primer, and for each candidate SNP locus, providing at least one pair of target-specific primers; wherein, the first universal primer contains a first universal sequence; the second universal primer contains a second universal sequence, the second universal sequence contains the first universal sequence and additionally contains at least one nucleotide at the 3'-end of the first universal sequence; the pair of target-specific primers can amplify using the target nucleic acid as a template to generate a nucleic acid product containing the candidate SNP locus, and the pair of target-specific primers contains a forward primer and a reverse primer, wherein the forward primer contains the first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3'-end of the first universal sequence; the reverse primer contains the second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3'-end of the second universal sequence; and the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer; and (b) Under conditions allowing nucleic acid amplification, using the first universal primer, the second universal primer and the pair of target-specific primers to amplify the target nucleic acid in the fifth sample respectively, thereby obtaining an amplification product corresponding to the fifth sample; (c) Performing melting curve analysis on the amplification product corresponding to the fifth sample obtained in step (b); (d) According to the melting curve analysis result of step (c), identifying such a SNP locus: at this locus, the receptor has a first genotype containing a homozygous first allele.

43. The method of claim 42, wherein the fifth sample is from a receptor who has undergone or not undergone a transplantation surgery.

44. The method of claim 42, wherein the fifth sample contains cells or tissues from the receptor.

45. The method of claim 42, wherein the fifth sample is selected from skin, saliva, urine, blood, hair, nails, or any combination thereof from the recipient.

46. The method of claim 42, wherein in step (a), for each candidate SNP locus, a detection probe is further provided, the detection probe comprising a nucleotide sequence specific to the target nucleic acid and capable of annealing or hybridizing to the region of the target nucleic acid containing the candidate SNP locus, and the detection probe is labeled with a reporter group and a quencher group, wherein, the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence; and in step (c), the melting curve analysis is respectively performed on the amplification products corresponding to the fifth sample obtained in step (b) using the detection probe.

47. The method of claim 42, wherein the fifth sample contains DNA.

48. A method for detecting the presence or proportion of nucleic acids of a donor in a recipient sample after transplantation surgery for non-diagnostic and non-therapeutic purposes, wherein, the method comprises the following steps: (1) providing a test sample containing nucleic acids from a recipient who has received transplantation of cells, tissues or organs from a donor; (2) identifying a plurality of such candidate SNP loci by the method of claim 1, the candidate SNP loci showing at least a first allele and a second allele in the species to which the recipient belongs, and at the candidate SNP loci, the recipient has a first genotype containing homozygous first alleles; (3) respectively performing quantitative detection on each allele of each candidate SNP locus of the test sample; (4) according to the quantitative detection results of step (3), selecting such target SNP loci from the candidate SNP loci: the test sample shows a signal of the first allele and a signal of the second allele at this locus; (5) determining the presence or proportion of nucleic acids of the donor in the test recipient sample according to the quantitative detection results of the first allele and the second allele of the target SNP locus.

49. The method of claim 48, wherein in step (2), different alleles at a certain SNP locus are distinguished by a mechanism selected from the following to identify candidate SNP loci: probe hybridization, primer extension, hybridization ligation, and specific enzymatic cleavage.

50. The method of claim 48, wherein in step (2), candidate SNP loci are identified by a method selected from the following: sequencing method, chip method, qPCR-based detection method, mass spectrometry method, chromatography method, electrophoresis method, melting curve analysis-based detection method.

51. The method of claim 48, in step (2), the target SNP locus is identified by a method selected from the following: first-generation sequencing, pyrosequencing, second-generation sequencing, using a solid-phase chip capable of detecting SNPs, a liquid-phase chip, Taqman probe method, iPLEX based on MassARRAY TM Gold, denaturing high-performance liquid chromatography dHPLC, and SNPshot method.

52. The method of claim 48, wherein in step (2), the candidate SNP loci are identified by a detection method based on multiplex PCR combined with melting curve analysis.

53. The method of claim 48, wherein the candidate SNP loci are identified by the method described in claim 8.

54. The method of claim 48, in step (3), each allele of each candidate SNP locus is quantitatively detected by digital PCR.

55. The method of claim 48, wherein step (3) is carried out by the following scheme: (I) Select a plurality of candidate SNP loci from step (2), and for each selected candidate SNP locus, provide an amplification primer set and a probe set, wherein, (I-1) The amplification primer set comprises at least one amplification primer which, under conditions allowing nucleic acid hybridization or annealing, can specifically amplify a nucleic acid molecule containing the candidate SNP locus; (I-2) The probe set comprises a first probe and a second probe; wherein, (i) The first probe and the second probe are each independently labeled with a reporter group and a quenching group, wherein the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the first probe and the second probe are labeled with different reporter groups; and (ii) The first probe can hybridize or anneal with a nucleic acid molecule containing the first allele of the candidate SNP locus, and the second probe can hybridize or anneal with a nucleic acid molecule containing the second allele of the candidate SNP locus; and the first probe and the second probe are specific for different alleles; (II) Use the amplification primer set and the probe set to perform digital PCR on the test sample from the recipient, and quantitatively detect the nucleic acid molecules with the first allele and the nucleic acid molecules with the second allele.

56. The method of claim 48, wherein the first probe specifically anneals or hybridizes with a nucleic acid molecule having the first allele during the digital PCR reaction; and the second probe specifically anneals or hybridizes with a nucleic acid molecule having the second allele during the digital PCR reaction.

57. The method of claim 48, wherein the first probe does not anneal or hybridize with a nucleic acid molecule having the second allele during the digital PCR reaction; and / or the second probe does not anneal or hybridize with a nucleic acid molecule having the first allele during the digital PCR reaction.

58. The method of claim 48, in step (5), perform cluster analysis on the quantitative detection results of the second alleles of a plurality of target SNP loci; then, according to the cluster analysis results, determine the genotype of the donor at each target SNP locus; then, according to the genotypes of the recipient and the donor at each target SNP locus, and the quantitative detection results of the first allele and the second allele in the test sample, determine the presence or proportion of the donor's nucleic acid in the test recipient sample.

59. The method of claim 48, before step (3), pre-treat the test sample from the recipient.

60. The method of claim 48, the pre-treatment includes nucleic acid extraction of the sample and / or enrichment of the nucleic acid in the sample.

61. The method according to any one of claims 42-60, wherein, the recipient has received or transplanted hematopoietic stem cells or tissues or organs containing hematopoietic stem cells from a donor. The method according to any one of claims 42-60, wherein, the recipient has received or transplanted bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, umbilical cord blood hematopoietic stem cells or any combination thereof from a donor; or spinal cord containing hematopoietic stem cells. The method according to any one of claims 42-60, wherein the test sample comprises blood or a component thereof from the recipient after transplantation, and the component is selected from blood cells, plasma, monocytes, granulocytes, T cells, or any combination thereof. The method according to any one of claims 42-60, wherein, the recipient has received or transplanted an organ from a donor. The method according to any one of claims 42-60, wherein the recipient has received or transplanted a kidney, heart, lung, liver, pancreas or any combination thereof from a donor. The method according to any one of claims 42-60, wherein the recipient has received or transplanted a kidney from a donor. The method according to any one of claims 42-60, wherein the test sample comprises blood or urine from the recipient after transplantation. The method according to any one of claims 42-60, when the transplantation is a kidney transplantation, the test sample comprises urine from the recipient after transplantation. The method according to claim 42, wherein, steps (a)-(b) of the method are carried out by a protocol comprising the following steps (I)-(VI): (I) providing the fifth sample, the first universal primer and the second universal primer, and the target-specific primer pair; (II) mixing the fifth sample with the first universal primer, the second universal primer, the target-specific primer pair, and a nucleic acid polymerase; (III) incubating the product of the previous step under conditions allowing nucleic acid denaturation; (IV) incubating the product of the previous step under conditions allowing nucleic acid annealing or hybridization; and (V) incubating the product of the previous step under conditions allowing nucleic acid extension; and (VI) repeating steps (III)-(V) one or more times. The method according to claim 42, wherein, steps (a)-(b) of the method are carried out by a protocol comprising the following steps (I)-(VI): (I) providing the fifth sample, the first universal primer and the second universal primer, and the target-specific primer pair; and a detection probe; (II) mixing the fifth sample with the first universal primer, the second universal primer, the target-specific primer pair, a nucleic acid polymerase, and the detection probe; (III) incubating the product of the previous step under conditions allowing nucleic acid denaturation; (IV) incubating the product of the previous step under conditions allowing nucleic acid annealing or hybridization; (V) incubating the product of the previous step under conditions allowing nucleic acid extension; and (VI) repeating steps (III)-(V) one or more times. The method according to claim 69 or 70, the method having one or more of the following technical features: (1) In step (III), incubating the product of step (II) at a temperature of 80-105 °C to denature the nucleic acid; (2) In step (III), incubate the product of step (II) for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, or 2 - 5 min; (3) In step (IV), incubate the product of step (III) at a temperature of 35 - 40 °C, 40 - 45 °C, 45 - 50 °C, 50 - 55 °C, 55 - 60 °C, 60 - 65 °C, or 65 - 70 °C to allow nucleic acid annealing or hybridization; (4) In step (IV), incubate the product of step (III) for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, or 2 - 5 min; (5) In step (V), incubate the product of step (IV) at a temperature of 35 - 40 °C, 40 - 45 °C, 45 - 50 °C, 50 - 55 °C, 55 - 60 °C, 60 - 65 °C, 65 - 70 °C, 70 - 75 °C, 75 - 80 °C, 80 - 85 °C to allow nucleic acid extension; (6) In step (V), incubate the product of step (IV) for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, 2 - 5 min, 5 - 10 min, 10 - 20 min or 20 - 30 min; (7) Steps (IV) and (V) are carried out at the same or different temperatures; and (8) Repeat steps (III) - (V) at least once; wherein, when steps (III) - (V) are repeated one or more times, the conditions used for each cycle of steps (III) - (V) are independently the same or different.

72. The method according to claim 55, wherein, each primer of the amplification primer set independently has one or more technical features selected from the following: (1) The length of the primer is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt; (2) The primer or any of its components comprises or consists of naturally occurring nucleotides, modified nucleotides, non - natural nucleotides, or any combination thereof; (3) The amplification primer set includes primer pairs having nucleotide sequences selected from the following or any combination thereof: SEQ ID NO:72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

73. The method according to claim 55, wherein, The first probe and the second probe each independently have one or more characteristics selected from the following: (1) The first probe and the second probe each independently comprise or consist of naturally occurring nucleotides, modified nucleotides, unnatural nucleotides, or any combination thereof; (2) The lengths of the first probe and the second probe are each independently 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 200 nt, 200 - 300 nt, 300 - 400 nt, 400 - 500 nt, 500 - 600 nt, 600 - 700 nt, 700 - 800 nt, 800 - 900 nt, 900 - 1000 nt; (3) The first probe and the second probe each independently have a 3'-OH terminus; alternatively, the 3'-terminus of the probe is blocked; (4) The first probe and the second probe are each independently self-quenching probes; (5) The reporter group in the probe is a fluorescent group; and the quenching group is a molecule or group capable of absorbing / quenching the fluorescence; (6) The first probe and the second probe are each independently linear or have a hairpin structure; (7) The first probe and the second probe have different reporter groups; (8) The probe set includes probes having nucleotide sequences selected from the following or any combination thereof: SEQ ID NO: 74, 75, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

74. The method of claim 73, the method having one or more technical characteristics selected from the following: (1) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; (2) The unnatural nucleotide is a peptide nucleic acid or a locked nucleic acid; (3) Blocking the 3'-terminus of the probe by adding a chemical moiety to the 3'-OH of the last nucleotide of the probe, by removing the 3'-OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide; (4) The detection probe is labeled with a reporter group at its 5'-end or upstream and with a quenching group at its 3'-end or downstream, or with a reporter group at its 3'-end or downstream and with a quenching group at its 5'-end or upstream; (5) The reporter group and the quenching group are separated by a distance of 10 - 80 nt or more; (6) The fluorescent group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705; (7) The quenching group is selected from DABCYL, BHQ, ECLIPSE, and / or TAMRA; (8) The probe has 5'-nuclease activity; (9) The backbone of the probe contains modifications that resist nuclease activity.

75. A method for detecting SNP sites with different genotypes of a donor and a recipient for non-diagnostic and non-therapeutic purposes, comprising the following steps : (a) providing a third sample from the recipient and a fourth sample from the recipient after transplantation surgery, wherein, the third sample contains one or more target nucleic acids derived from the recipient and does not contain nucleic acids derived from the donor; the fourth sample contains one or more target nucleic acids derived from the donor, and the target nucleic acids contain one or more candidate SNP sites, and, providing a first universal primer and a second universal primer, and for each candidate SNP site, providing at least one pair of target-specific primers; wherein, the first universal primer contains a first universal sequence; the second universal primer contains a second universal sequence, the second universal sequence contains the first universal sequence and additionally contains at least one nucleotide at the 3'-end of the first universal sequence; the pair of target-specific primers can amplify using the target nucleic acid as a template to generate a nucleic acid product containing the candidate SNP site, and the pair of target-specific primers contains a forward primer and a reverse primer, wherein the forward primer contains the first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3'-end of the first universal sequence; the reverse primer contains the second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3'-end of the second universal sequence; and the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer; and (b) under conditions allowing nucleic acid amplification, using the first universal primer, the second universal primer, and the pair of target-specific primers to amplify the target nucleic acids in the third sample and the fourth sample respectively, so as to obtain amplification products corresponding to the third sample and the fourth sample respectively; (c) performing melting curve analysis on the amplification products corresponding to the third sample and the fourth sample obtained in step (b) respectively; (d) according to the melting curve analysis results in step (c), determining such SNP sites: at these sites, the third sample only shows the first allele, and the fourth sample shows at least the second allele; the SNP sites are SNP sites with different genotypes of the donor and the recipient.

76. The method of claim 75, in step (d) of the method, determining the genotypes of the respective candidate SNP sites of the third sample and the fourth sample according to the melting curve analysis results, so as to determine such SNP sites: at this site, the third sample only shows the first allele, and the fourth sample shows the first and second alleles.

77. The method of claim 75, wherein the third sample is from a recipient who has or has not undergone a transplantation operation.

78. The method of claim 75, wherein the third sample contains cells or tissues from the recipient.

79. The method of claim 75, wherein the third sample is selected from the skin, saliva, urine, blood, hair, nails of the recipient, or any combination thereof.

80. The method of claim 75, in the fourth sample, the amount of nucleic acid from the donor accounts for at least 20% of the total nucleic acid in the fourth sample.

81. The method of claim 75, wherein the recipient has received or transplanted an organ, tissue or cell from a donor.

82. The method of claim 75, wherein the recipient has received or transplanted an organ from a donor.

83. The method of claim 75, wherein the recipient has received or transplanted a kidney, heart, lung, liver, pancreas from a donor, or any combination thereof.

84. The method of claim 75, wherein the fourth sample contains blood or urine from a recipient after transplantation surgery.

85. The method of claim 75, wherein the recipient has received or transplanted hematopoietic stem cells or a tissue or organ containing hematopoietic stem cells from a donor.

86. The method of claim 75, wherein the recipient has received or transplanted bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, umbilical cord blood hematopoietic stem cells or bone marrow from a donor.

87. The method of claim 75, wherein the fourth sample contains blood or a component thereof from a recipient after transplantation surgery.

88. The method of claim 75, wherein the fourth sample contains blood or a component thereof from a recipient at least 5 days, 10 days, 15 days, 20 days, or 30 days after transplantation surgery.

89. The method of claim 75, in step (a), for each candidate SNP site, a detection probe is further provided, the detection probe contains a nucleotide sequence specific to the target nucleic acid and can anneal or hybridize with the region containing the candidate SNP site in the target nucleic acid, and the detection probe is labeled with a reporter group and a quenching group, wherein, the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence; and, in step (c), using the detection probe to perform melting curve analysis on the amplification products corresponding to the third sample and the fourth sample obtained in step (b) respectively.

90. The method of claim 75, wherein the third sample contains DNA.

91. The method of claim 75, wherein the fourth sample contains DNA.

92. A method for detecting the presence or proportion of donor nucleic acid in a sample from a recipient who has undergone transplantation surgery for non-diagnostic and non-therapeutic purposes, wherein, the method comprises the following steps: (1) Providing a sample to be tested containing nucleic acid from a recipient who has received transplantation of donor cells, tissues or organs; (2) Identifying a plurality of target SNP sites by the method according to claim 1, wherein at the target SNP sites, the recipient has a first genotype containing a homozygous first allele, and the donor has a second genotype containing a second allele, wherein the first genotype is different from the second genotype, and the first allele is different from the second allele; (3) Quantitatively detecting the first allele and the second allele of each target SNP site in the sample to be tested; (4) Determining the presence or proportion of donor nucleic acid in the sample of the recipient to be tested according to the results of the quantitative detection of the first allele and the second allele of the target SNP site.

93. The method according to claim 92, in step (2), identifying 5, 6, 7, 8, 9, 10 or more target SNP sites.

94. The method according to claim 92, in step (2), differentiating different alleles at a certain SNP site by a mechanism selected from the following to identify the target SNP site: probe hybridization, primer extension, hybridization ligation and specific digestion.

95. The method according to claim 92, in step (2), identifying the target SNP site by a method selected from the following: sequencing method, chip method, qPCR-based detection method, mass spectrometry method, chromatography method, electrophoresis method, melting curve analysis-based detection method.

96. The method of claim 95, wherein in step (2), the target SNP locus is identified by a method selected from the following: Sanger sequencing, pyrosequencing, next-generation sequencing, using a solid-phase chip capable of detecting SNPs, a liquid-phase chip, TaqMan probe method, iPLEX based on MassARRAY TM Gold, denaturing high-performance liquid chromatography dHPLC, and SNPshot method.

97. The method according to claim 92, in step (2), identifying the target SNP site by a detection method based on multiplex PCR combined with melting curve analysis.

98. The method according to claim 92, identifying the target SNP site by the method described in any one of claims 75-91.

99. The method according to claim 92, in step (3), quantitatively detecting the first allele and the second allele of each target SNP site in the sample by digital PCR.

100. The method according to claim 92, performing step (3) by the following scheme: (I) For each target SNP site, providing an amplification primer set and a probe set, wherein, (I-1) The amplification primer set comprises at least one amplification primer which can specifically amplify a nucleic acid molecule containing the target SNP site under conditions allowing nucleic acid hybridization or annealing; (I-2) The probe set comprises a first probe and a second probe; wherein, (i) The first probe and the second probe are each independently labeled with a reporter group and a quenching group, wherein the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the first probe and the second probe are labeled with different reporter groups; and (ii) The first probe is capable of hybridizing or annealing to a nucleic acid molecule containing the first allele of the target SNP site, and the second probe is capable of hybridizing or annealing to a nucleic acid molecule containing the second allele of the target SNP site; and, the first probe and the second probe are specific for different alleles; (II) Using the amplification primer set and the probe set to perform digital PCR on the test sample to quantitatively detect nucleic acid molecules with the first allele and nucleic acid molecules with the second allele.

101. The method of claim 100, wherein the first probe specifically anneals or hybridizes to a nucleic acid molecule with the first allele during the digital PCR reaction; and, the second probe specifically anneals or hybridizes to a nucleic acid molecule with the second allele during the digital PCR reaction.

102. The method of claim 100, wherein the first probe does not anneal or hybridize to a nucleic acid molecule with the second allele during the digital PCR reaction; and / or, the second probe does not anneal or hybridize to a nucleic acid molecule with the first allele during the digital PCR reaction.

103. The method of claim 92, in step (4), performing cluster analysis on the quantitative detection results of the second alleles of multiple target SNP sites; then, according to the cluster analysis results, determining the genotype of the donor at each target SNP site; then, according to the genotypes of the recipient and the donor at each target SNP site, and the quantitative detection results of the first allele and the second allele in the test sample, determining the presence or proportion of the donor's nucleic acid in the test recipient sample.

104. The method of claim 92, pre-treating the test sample from the recipient before step (3).

105. The method of claim 104, wherein the pre-treatment includes nucleic acid extraction of the sample and / or enrichment of the nucleic acid in the sample.

106. The method of any one of claims 75 - 105, wherein, the recipient has received or transplanted hematopoietic stem cells from the donor or a tissue or organ containing hematopoietic stem cells.

107. The method of any one of claims 75 - 105, wherein the recipient has received or transplanted bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, cord blood hematopoietic stem cells, and / or spinal cord from the donor.

108. The method of any one of claims 75 - 105, wherein the test sample contains blood or its components from the recipient after transplantation.

109. The method of any one of claims 75 - 105, wherein the test sample contains blood cells, plasma, monocytes, granulocytes, T cells, or any combination thereof from the recipient after transplantation.

110. The method of any one of claims 75 - 105, wherein, the recipient has received or transplanted an organ from the donor.

111. The method of any one of claims 75 - 105, wherein, the recipient has received or transplanted a kidney, heart, lung, liver, pancreas, or any combination thereof from the donor.

112. The method of any one of claims 75 - 105, wherein the recipient has received or transplanted a kidney from the donor.

113. The method according to any one of claims 75 - 105, wherein the sample to be tested comprises blood or urine from a recipient after transplantation.

114. The method according to claim 75, wherein, steps (a) - (b) of the method are carried out by a protocol comprising the following steps (I) - (VI): (I) providing the third sample and the fourth sample, the first universal primer and the second universal primer, and the target - specific primer pair; (II) combining the samples with the first universal primer, the second universal primer, the target - specific primer pair, and a nucleic acid polymerase; (III) incubating the product of the previous step under conditions allowing nucleic acid denaturation; (IV) incubating the product of the previous step under conditions allowing nucleic acid annealing or hybridization; (V) incubating the product of the previous step under conditions allowing nucleic acid extension; and (VI) repeating steps (III) - (V) one or more times.

115. The method according to claim 75, wherein, steps (a) - (b) of the method are carried out by a protocol comprising the following steps (I) - (VI): (I) providing the third sample and the fourth sample, the first universal primer and the second universal primer, and the target - specific primer pair; and a detection probe; (II) combining the samples with the first universal primer, the second universal primer, the target - specific primer pair, a nucleic acid polymerase, and the detection probe; (III) incubating the product of the previous step under conditions allowing nucleic acid denaturation; (IV) incubating the product of the previous step under conditions allowing nucleic acid annealing or hybridization; (V) incubating the product of the previous step under conditions allowing nucleic acid extension; and (VI) repeating steps (III) - (V) one or more times.

116. The method according to claim 114 or 115, the method having one or more of the following technical features: (1) In step (III), incubating the product of step (II) at a temperature of 80 - 105 °C to denature the nucleic acid; (2) In step (III), incubating the product of step (II) for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, or 2 - 5 min; (3) In step (IV), incubating the product of step (III) at a temperature of 35 - 40 °C, 40 - 45 °C, 45 - 50 °C, 50 - 55 °C, 55 - 60 °C, 60 - 65 °C, or 65 - 70 °C to allow nucleic acid annealing or hybridization; (4) In step (IV), incubating the product of step (III) for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, or 2 - 5 min; (5) In step (V), incubating the product of step (IV) at a temperature of 35 - 40 °C, 40 - 45 °C, 45 - 50 °C, 50 - 55 °C, 55 - 60 °C, 60 - 65 °C, 65 - 70 °C, 70 - 75 °C, 75 - 80 °C, 80 - 85 °C to allow nucleic acid extension; (6) In step (V), incubate the product of step (IV) for 10 - 20 s, 20 - 40 s, 40 - 60 s, 1 - 2 min, 2 - 5 min, 5 - 10 min, 10 - 20 min, or 20 - 30 min; (7) Perform steps (IV) and (V) at the same or different temperatures; and (8) Repeat steps (III) - (V) at least once; wherein, when steps (III) - (V) are repeated one or more times, the conditions used for steps (III) - (V) in each cycle are independently the same or different.

117. The method according to any one of claims 75 - 105, wherein, each primer of the amplification primer set independently has one or more technical features selected from the following: (1) The length of the primer is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt; (2) The primer or any of its components comprises or consists of naturally occurring nucleotides, modified nucleotides, non - natural nucleotides, or any combination thereof; (3) The amplification primer set includes primer pairs having nucleotide sequences selected from the following or any combination thereof: SEQ ID NO: 72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

118. The method according to any one of claims 75 - 105, wherein, each of the first probe and the second probe independently has one or more features selected from the following: (1) Each of the first probe and the second probe independently comprises or consists of naturally occurring nucleotides, modified nucleotides, non - natural nucleotides, or any combination thereof; (2) The lengths of the first probe and the second probe are independently 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 200 nt, 200 - 300 nt, 300 - 400 nt, 400 - 500 nt, 500 - 600 nt, 600 - 700 nt, 700 - 800 nt, 800 - 900 nt, or 900 - 1000 nt; (3) Each of the first probe and the second probe independently has a 3'-OH terminus; or, the 3'-terminus of the probe is blocked; (4) The first probe and the second probe are each independently a self-quenching probe; (5) The reporter group in the probe is a fluorescent group; and the quenching group is a molecule or group capable of absorbing / quenching the fluorescence; (6) The first probe and the second probe are each independently linear or have a hairpin structure; (7) The first probe and the second probe have different reporter groups; (8) The first probe and the second probe are degradable by a nucleic acid polymerase; (9) The probe set includes a probe having a nucleotide sequence selected from the following or any combination thereof: SEQ ID NO:74, 75, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

119. The method of claim 118, the method having one or more technical features selected from the following: (1) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; (2) The unnatural nucleotide is a peptide nucleic acid or a locked nucleic acid; (3) By adding a chemical moiety to the 3'-OH of the last nucleotide of the probe, by removing the 3'-OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide, thereby blocking the 3'-end of the probe; (4) The probe is labeled with a reporter group at its 5'-end or upstream and with a quenching group at its 3'-end or downstream, or with a reporter group at its 3'-end or downstream and with a quenching group at its 5'-end or upstream; (5) The reporter group and the quenching group are separated by a distance of 10 - 80 nt or longer; (6) The fluorescent group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705; (7) The quenching group is selected from DABCYL, BHQ, ECLIPSE and / or TAMRA; (8) The probe has 5'-nuclease activity; (9) The backbone of the probe contains a modification that resists nuclease activity.

120. The method according to any one of claims 1 - 14, wherein, the candidate SNP locus has one or more characteristics selected from the following: (1) The Fst of the candidate SNP locus between different ethnic groups is less than 0.3; (2) The candidate SNP locus is located on different chromosomes; (3) The allele frequency of the candidate SNP locus is between 0.2 and 0.

8.

121. The method according to any one of claims 1-14, wherein the candidate SNP locus has one or more of the following characteristics: (1) The Fst of the candidate SNP locus between different human races is less than 0.01; (2) The candidate SNP locus is located on different chromosomes; (3) The allele frequency of the candidate SNP locus is between 0.3 and 0.

7.

122. The method according to any one of claims 1-14, wherein the candidate SNP locus is an SNP locus with bi-allelic polymorphism.

123. The method according to any one of claims 1-14, wherein the candidate SNP locus is an SNP locus in the human genome.

124. The method according to any one of claims 1-14, wherein the target nucleic acid comprises a human genome SNP locus selected from the following: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs5858210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, rs7160304, and any combination of the foregoing SNP loci.

125. The method according to any one of claims 1-14, wherein the target nucleic acid in the sample comprises the following human genome SNP loci: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs5858210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, and rs7160304.

126. The method according to claim 14, 37, 46 or 89, wherein, the method has one or more of the following technical characteristics: (1) In step (b), the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, and a nucleic acid polymerase, and nucleic acid amplification is performed. Then, a detection probe is added to the product of step (b), and melting curve analysis is performed; alternatively, in step (b), the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, the detection probe, and a nucleic acid polymerase, and nucleic acid amplification is performed, and then melting curve analysis is performed; (2) The detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, unnatural nucleotides, or any combination thereof; (3) The length of the detection probe is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 200 nt, 200 - 300 nt, 300 - 400 nt, 400 - 500 nt, 500 - 600 nt, 600 - 700 nt, 700 - 800 nt, 800 - 900 nt, or 900 - 1000 nt; (4) The detection probe has a 3'-OH terminus; or the 3'-terminus of the detection probe is blocked; (5) The detection probe is a self-quenching probe; (6) The reporter group in the detection probe is a fluorescent group; and the quenching group is a molecule or group capable of absorbing / quenching the fluorescence; (7) The detection probe has resistance to nuclease activity; (8) The detection probe is linear or has a hairpin structure; (9) The detection probes each independently have the same or different reporter groups; (10) In step (c), the product of step (b) is gradually heated or cooled, and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining a curve showing the change in the signal intensity of each reporter group with temperature; then, the derivative of the curve is taken to obtain the melting curve of the product of step (b); (11) According to the melting peaks in the melting curve, the types of each SNP site are determined; (12) The detection probes include detection probes having nucleotide sequences selected from the following or any combination thereof: SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, and 69.

127. The method of claim 126, the method having one or more technical features selected from the following: (1) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; (2) The unnatural nucleotide is a peptide nucleic acid or a locked nucleic acid; (3) Blocking the 3'-end of the detection probe by adding a chemical moiety to the 3'-OH of the last nucleotide of the detection probe, by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide; (4) The detection probe is labeled with a reporter group at its 5'-end or upstream and with a quencher group at its 3'-end or downstream, or is labeled with a reporter group at its 3'-end or downstream and with a quencher group at its 5'-end or upstream; (5) The reporter group and the quencher group are separated by a distance of 10 - 80 nt or longer; (6) The fluorophore is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705; (7) The quencher group is selected from DABCYL, BHQ, ECLIPSE, and / or TAMRA; (8) The detection probe has resistance to 5'-nuclease activity; (9) The backbone of the probe contains modifications that resist nuclease activity; (10) The detection probes have the same reporter group, and the melting curve analysis is performed on the product of step (b), and then the presence of the target nucleic acid is determined according to the melting peak in the melting curve; or, the detection probes have different reporter groups, and the melting curve analysis is performed on the product of step (b), and then the presence of the target nucleic acid is determined according to the signal type of the reporter group and the melting peak in the melting curve.

128. The method of claim 70, wherein, the method has one or more of the following technical features: (1) In step (a) of the method, 1 - 5, 5 - 10, 10 - 15, 15 - 20 or more target-specific primer pairs are provided; (2) In step (b) of the method, the working concentrations of the first universal primer and the second universal primer are higher than those of the forward primer and the reverse primer; (3) In step (b) of the method, the working concentrations of the first universal primer and the second universal primer are the same; or, the working concentration of the first universal primer is lower than that of the second universal primer; (4) In step (b) of the method, the working concentrations of the forward primer and the reverse primer are the same or different; (5) The sample or target nucleic acid contains mRNA, and before performing step (b) of the method, a reverse transcription reaction is performed on the sample; and (6) In step (b) of the method, a nucleic acid polymerase is used for nucleic acid amplification.

129. The method of claim 128, the method having one or more of the following features: (1) The nucleic acid polymerase is a DNA polymerase; (2) The nucleic acid polymerase is obtained from Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antraniildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus thermophllus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifex pyrophilus and Aquifex aeolicus; (3) The nucleic acid polymerase is Taq polymerase; (4) The working concentration of the first universal primer and the second universal primer is 1 - 5 times, 5 - 10 times, 10 - 15 times, 15 - 20 times, 20 - 50 times or more times higher than the working concentration of the forward primer and the reverse primer.

130. The method of claim 1, wherein, the method has one or more technical features selected from the following: (1) The first universal primer consists of a first universal sequence, or comprises a first universal sequence and an additional sequence, and the additional sequence is located at the 5' end of the first universal sequence; (2) The first universal sequence is located at or constitutes the 3' part of the first universal primer; (3) The length of the first universal primer is 5 - 15 nt, 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, or 40 - 50 nt; (4) The first universal primer or any of its components contains or consists of naturally occurring nucleotides, modified nucleotides, non - natural nucleotides, or any combination thereof; (5) The second universal primer consists of a second universal sequence, or comprises a second universal sequence and an additional sequence, where the additional sequence is located at the 5'-end of the second universal sequence; (6) The second universal sequence is located in or constitutes the 3'-portion of the second universal primer; (7) The second universal sequence contains the first universal sequence and additionally contains 1 - 5, 5 - 10, 10 - 15, 15 - 20 or more nucleotides at the 3'-end of the first universal sequence; (8) The length of the second universal primer is 8 - 15 nt, 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, or 40 - 50 nt; and (9) The second universal primer or any of its components contains or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

131. The method of claim 130, wherein the additional sequence contains 1 - 5, 5 - 10, 10 - 15, 15 - 20 or more nucleotides.

132. The method of claim 1, wherein, the method has one or more of the following technical features: (1) In the forward primer, the forward nucleotide sequence is directly linked to the 3'-end of the first universal sequence, or is linked to the 3'-end of the first universal sequence through a nucleotide linker; (2) The forward primer further contains an additional sequence located at the 5'-end of the first universal sequence; (3) The forward primer contains or consists of, from 5' to 3', the first universal sequence and the forward nucleotide sequence; or contains or consists of, from 5' to 3', the first universal sequence, a nucleotide linker, and the forward nucleotide sequence; or contains or consists of, from 5' to 3', an additional sequence, the first universal sequence, and the forward nucleotide sequence; or contains or consists of, from 5' to 3', an additional sequence, the first universal sequence, a nucleotide linker, and the forward nucleotide sequence; (4) The forward nucleotide sequence is located in or constitutes the 3'-portion of the forward primer; (5) The length of the forward nucleotide sequence is 10 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt; (6) The length of the forward primer is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt; (7) The forward primer or any of its components contains or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (8) In the reverse primer, the reverse nucleotide sequence is directly linked to the 3'-end of the second universal sequence, or the reverse nucleotide sequence is linked to the 3'-end of the second universal sequence through a nucleotide linker; (9) The reverse primer further comprises an additional sequence, which is located at the 5'-end of the second universal sequence; (10) The reverse primer comprises or consists of, from 5' to 3', the second universal sequence and a reverse nucleotide sequence; alternatively, from 5' to 3', it comprises or consists of the second universal sequence, a nucleotide linker, and a reverse nucleotide sequence; alternatively, from 5' to 3', it comprises or consists of the additional sequence, the second universal sequence, and a reverse nucleotide sequence; alternatively, from 5' to 3', it comprises or consists of the additional sequence, the second universal sequence, a nucleotide linker, and a reverse nucleotide sequence; (11) The reverse nucleotide sequence is located at or constitutes the 3'-portion of the reverse primer; (12) The length of the reverse nucleotide sequence is 10 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt; (13) The length of the reverse primer is 15 - 20 nt, 20 - 30 nt, 30 - 40 nt, 40 - 50 nt, 50 - 60 nt, 60 - 70 nt, 70 - 80 nt, 80 - 90 nt, 90 - 100 nt, 100 - 110 nt, 110 - 120 nt, 120 - 130 nt, 130 - 140 nt, 140 - 150 nt; (14) The reverse primer or any of its components comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and (15) The second universal sequence cannot be fully complementary to the complementary sequence of the forward primer.

133. The method of claim 132, the method having one or more features selected from the following: (1) The nucleotide linker comprises 1 - 5, 5 - 10, 10 - 15, 15 - 20 or more nucleotides; (2) The additional sequence comprises 1 - 5, 5 - 10, 10 - 15, 15 - 20 or more nucleotides; (3) At least one nucleotide at the 3'-end of the second universal sequence cannot be complementary to the complementary sequence of the forward primer; (4) 1 - 5, 5 - 10, 10 - 15, 15 - 20 or more nucleotides at the 3'-end of the second universal sequence cannot be complementary to the complementary sequence of the forward primer; (5) The sequence of the first universal primer is as shown in SEQ ID NO:71; (6) The sequence of the second universal primer is as shown in SEQ ID NO:70; (7) The target-specific primer pair includes a primer pair having a nucleotide sequence selected from the following or any combination thereof: SEQ ID NO: 1 and 2; 4 and 5; 7 and 8; 10 and 11; 13 and 14; 16 and 17; 19 and 20; 22 and 23; 25 and 26; 28 and 29; 31 and 32; 34 and 35; 37 and 38; 40 and 41; 43 and 44; 46 and 47; 49 and 50; 52 and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68.

134. A kit, the kit includes an identification primer set capable of asymmetrically amplifying a target nucleic acid containing a candidate SNP site; wherein, the identification primer set includes: a first universal primer and a second universal primer, and for each candidate SNP site, at least one target-specific primer pair is provided, wherein, the first universal primer includes a first universal sequence; the second universal primer includes a second universal sequence, the second universal sequence includes the first universal sequence and additionally includes at least one nucleotide at the 3'-end of the first universal sequence; the target-specific primer pair can amplify using the target nucleic acid as a template to generate a nucleic acid product containing the candidate SNP site, and the target-specific primer pair includes a forward primer and a reverse primer, wherein, the forward primer includes the first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3'-end of the first universal sequence; the reverse primer includes the second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3'-end of the second universal sequence; and, the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer.

135. The kit of claim 134, the kit further includes one or more detection probes capable of detecting the candidate SNP site, the detection probe includes a nucleotide sequence specific to the target nucleic acid and can anneal or hybridize with the region containing the candidate SNP site in the target nucleic acid, and is labeled with a reporter group and a quenching group, wherein, the reporter group can emit a signal, and, the quenching group can absorb or quench the signal emitted by the reporter group; and, the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence.

136. The kit of claim 134, the candidate SNP site has one or more of the following characteristics: (1) The Fst of the candidate SNP site between different ethnic groups is less than 0.3; (2) The candidate SNP site is located on different chromosomes; (3) The allele frequency of the candidate SNP site is between 0.2 and 0.

8.

137. The kit of claim 134, the candidate SNP site has one or more of the following characteristics: (1) The Fst of the candidate SNP site between different ethnic groups is less than 0.01; (2) The candidate SNP site is located on different chromosomes; (3) The allele frequency of the candidate SNP locus is between 0.3 and 0.

7.

138. The kit of claim 134, wherein the candidate SNP locus is a SNP locus with biallelic polymorphism.

139. The kit of claim 134, wherein the candidate SNP locus is a SNP locus in the human genome.

140. The target nucleic acid of the kit of claim 134 comprises a human genomic SNP locus selected from: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs5858210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, rs7160304, and any combination of the foregoing SNP loci.

141. The target nucleic acid of the kit of claim 134 comprises the following human genomic SNP loci: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs5858210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776 and rs7160304.

142. The kit of claim 134, wherein the detection probe comprises a detection probe having a nucleotide sequence selected from: SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 and 69, or any combination thereof.

143. The kit of claim 134, wherein the kit has one or more of the following characteristics: (1) The sequence of the first universal primer is as shown in SEQ ID NO:71; (2) The sequence of the second universal primer is as shown in SEQ ID NO:70; (3) The target-specific primer pair includes a primer pair having a nucleotide sequence selected from the following or any combination thereof: SEQ ID NO: 1 and 2; 4 and 5; 7 and 8; 10 and 11; 13 and 14; 16 and 17; 19 and 20; 22 and 23; 25 and 26; 28 and 29; 31 and 32; 34 and 35; 37 and 38; 40 and 41; 43 and 44; 46 and 47; 49 and 50; 52 and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68; (4) The kit further includes one or more components selected from the following: an amplification primer set, a probe set, and reagents for performing digital PCR.

144. The kit of claim 143, wherein the amplification primer set includes at least one amplification primer that can specifically amplify a nucleic acid molecule containing the SNP site under conditions allowing nucleic acid hybridization or annealing.

145. The kit of claim 143, wherein the probe set includes a first probe and a second probe; Wherein, (i) The first probe and the second probe are each independently labeled with a reporter group and a quenching group, wherein the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the first probe and the second probe are labeled with different reporter groups; and (ii) The first probe can hybridize or anneal with a nucleic acid molecule containing the first allele of the target SNP site, and the second probe can hybridize or anneal with a nucleic acid molecule containing the second allele of the target SNP site; and the first probe and the second probe are specific to different alleles.

146. The kit of claim 143, wherein the probe set includes a probe having a nucleotide sequence selected from the following or any combination thereof: SEQ ID NO: 74, 75, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

147. The kit of claim 143, wherein the amplification primer set includes a primer pair having a nucleotide sequence selected from the following or any combination thereof: SEQ ID NO: 72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

148. The kit of claim 144, wherein the reagents for digital PCR are selected from one or more components including: reagents for preparing microdroplet samples, reagents for nucleic acid amplification, nucleic acid polymerases, reagents for detecting microdroplet samples, or any combination thereof.

149. The kit of claim 143, wherein the kit further comprises one or more components selected from: nucleic acid polymerases, reagents for nucleic acid amplification, reagents for performing melting curve analysis, or any combination thereof.

150. The kit of claim 149, wherein the kit has one or more of the following characteristics: (1) The nucleic acid polymerase is a template-dependent nucleic acid polymerase; (2) The reagents for nucleic acid amplification include an enzyme working buffer, dNTPs, water, a solution containing ions, single-stranded DNA binding protein, or any combination thereof; (3) The kit is used to determine whether a recipient sample contains a donor, or to calculate the proportion of the donor in the recipient sample; (4) The digital PCR is selected from droplet digital PCR and chip digital PCR.

151. Use of the identification primer set defined in claim 134 for preparing a kit, wherein the kit is used for asymmetric amplification of a target nucleic acid molecule, or for detecting the genotype of a candidate SNP site in a target nucleic acid molecule; or for identifying SNP sites where the donor and the recipient have different genotypes; or for identifying SNP sites where the recipient has homozygous alleles.

152. The use of claim 151, wherein the kit further comprises the detection probe defined in claim 134.

153. The use of claim 151, wherein the kit is used to implement the method described in claim 1, 8 or 15.

154. Use of the amplification primer set and probe set defined in claim 134 for preparing a kit, wherein the kit is used to detect the presence or proportion of the donor's nucleic acid in a recipient sample after transplantation surgery.

155. The use of claim 154, wherein the kit further comprises reagents for determining the genotype of one or more SNP sites in the genome of the recipient or the donor.

156. The use of claim 154, wherein the kit further comprises the identification primer set and detection probe defined in claim 134.

157. The use of claim 154, wherein the kit is used to implement the method described in any one of claims 17-28, 48-60 or 92-105.

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