A method and kit for identifying biological individuals or performing kinship identification on biological individuals

Abstract

The present application relates to multiple, asymmetric amplification of nucleic acid molecules containing single nucleotide polymorphism (SNP) sites. In particular, by simultaneously and asymmetric amplification of nucleic acid molecules containing SNP sites derived from biological individuals, the present application provides a method and a kit for identifying biological individuals or performing kinship identification on biological individuals.

Description

Technical Field

[0001] The present application relates to multiple, asymmetric amplification of nucleic acid molecules containing single nucleotide polymorphism (SNP) sites. In particular, by simultaneously and asymmetric amplification of nucleic acid molecules containing SNP sites derived from biological individuals, the present application provides a method and a kit for identifying biological individuals or performing kinship identification on biological individuals. Background Art

[0002] With the increasing requirements for the scientificity, accuracy and objectivity of judicial proceedings, and the continuous development of the field of physical evidence identification, the requirements for the individual sources of the samples in the case have also increased. Usually, by testing the genetic material DNA, through testing the polymorphism of its sequence and length, it is possible to achieve individual identification and parentage identification. Due to the good accuracy and reliability of the test results, DNA analysis has become an important technical means in the field of physical evidence identification.

[0003] At present, forensic DNA identification mainly uses PCR-STR typing technology to perform typing based on STR (short tandem repeat) sites on samples of unknown individual origin. However, due to the different lengths of each STR site, it is often necessary to amplify a product of up to 400bp, and in actual cases, various factors often cause serious degradation of the sample. When PCR-STR typing technology is used to detect these samples, "dominant amplification" or "ineffective amplification" often occurs, resulting in the inability to effectively type the STR loci with larger fragments, thereby leading to erroneous typing results. In addition, STR-based typing technology has always relied on various PCR post-processing technologies, and currently it is mainly based on capillary electrophoresis of PCR products, but this uncapped analysis method has the risk of causing laboratory contamination, and the operation is complicated and time-consuming, and is also limited by special instruments. Therefore, PCR-STR typing technology cannot meet the current development needs of forensic DNA identification.

[0004] SNP (single nucleotide polymorphism) is the most widely distributed and most numerous DNA genetic polymorphism in the human genome, accounting for more than 90% of all known polymorphisms, with an average of 1 in 500 to 1000 base pairs. It is estimated that the total number can reach 3 million or even more, which is several orders of magnitude higher than STR. Since SNP is mainly a di-allelic polymorphism, its mutation rate is low, it is more stable and reliable than STR, and the PCR product required for SNP detection is shorter, which is more suitable for degraded samples in forensic DNA identification. However, SNP typing technology has not been widely used in forensic identification. The main reasons include: 1) The SNP recognition rate is lower than STR, and multiple highly polymorphic SNPs need to be detected to meet the needs of individual identification; 2) The incidence of SNP varies greatly in different countries and nationalities, and the same group of SNPs has different individual identification capabilities for different populations; 3) There is a lack of a simple, fast, and easy-to-automate SNP detection technology platform.

[0005] In summary, how to establish a simple, rapid, and easily automated SNP detection system that can simultaneously perform individual identification and paternity testing on trace and degraded DNA samples, and provide strong technical support for forensic DNA analysis, has become an urgent problem to be solved. Summary of the invention

[0006] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0007] As used herein, the term "SNP (single nucleotide polymorphism)" refers to a nucleic acid sequence polymorphism caused by the variation of a single nucleotide at the genome level. The site with a single nucleotide polymorphism in the genome is referred to as a "SNP site". In this article, the SNP site is named by its reference number (e.g., rs ID). The SNP site and its type can be inquired about in public databases using rs ID, for example, by the dbSNP database of NCBI, the ChinaMAP database, the JSNP database, etc.

[0008] As used herein, the terms "parentage testing" or "kinship testing" have the same meaning and can be used interchangeably. They refer to the use of monitoring and analysis of biological genetic markers to determine the relationship between the parent and offspring. In human paternity testing, it is divided into conventional paternity testing (e.g., parent-child testing), intergenerational paternity testing (e.g., grandparents and grandchildren testing) and difficult paternity testing (e.g., cousins, siblings whose parents are both suspected).

[0009] As used herein, the term “power of discrimination (DP)” refers to the probability that two individuals randomly selected from a population have different genetic markers.

[0010] As used herein, the term "triplet" refers to a father, a mother and a child, and the "triplet non-paternity exclusion rate" refers to the probability of excluding an individual who is not the biological father / mother of the child by detecting genetic markers.

[0011] As used herein, the term "complementary" means that two nucleic acid sequences can form hydrogen bonds between 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 "completely complementary". As used herein, the term "completely complementary" means that each base in a nucleic acid sequence can be paired with a base in another nucleic acid chain without mismatching or gaps. As used herein, the term "substantially complementary" means that most of the bases in a nucleic acid sequence can be paired with a base in another nucleic acid chain, and it allows the presence of mismatches or gaps (for example, mismatches or gaps of one or several nucleotides). Generally, under conditions that allow nucleic acid hybridization, annealing or amplification, two nucleic acid sequences of "complementary" (for example, substantially complementary or completely 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 under conditions that allow nucleic acid hybridization, annealing or amplification, and cannot form a duplex. As used herein, the term "not completely complementary" means that the bases in one nucleic acid sequence cannot be completely paired with the bases in another nucleic acid chain, and there is at least one mismatch or gap.

[0012] As used herein, the terms "hybridization" and "annealing" refer to the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Typically, two nucleic acid sequences that are fully complementary or substantially complementary can hybridize or anneal. The complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, particularly the temperature.

[0013] As used herein, the term "PCR reaction" has a meaning generally understood by those skilled in the art, and refers to a reaction (polymerase chain reaction) using a nucleic acid polymerase and a primer to amplify a target nucleic acid. As used herein, the term "multiplex amplification" refers to amplifying multiple target nucleic acids in the same reaction system. As used herein, the term "asymmetric amplification" refers to amplification products obtained by amplifying a target nucleic acid in which the amounts of two complementary nucleic acid chains are different, and the amount of one nucleic acid chain is greater than the amount of the other nucleic acid chain.

[0014] As used herein, and as generally understood by those skilled in the art, the terms "forward" and "reverse" are merely for the convenience of describing and distinguishing two primers in a primer pair; they are relative and have no special meaning.

[0015] As used herein, the term "melting curve analysis" has a meaning generally understood by those skilled in the art, which refers to a method for 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 assess the dissociation characteristics of the double-stranded nucleic acid molecule during heating. The method for performing melting curve analysis is well known to those skilled in the art (see, for example, The Journal of Molecular Diagnostics 2009, 11 (2): 93-101). In this application, the terms "melting curve analysis" and "melting analysis" have the same meaning and are used interchangeably.

[0016] 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. In short, 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., 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 rises, the two chains of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the detection probe under dissociation is in a single-stranded free curling state. In this case, the reporter group (e.g., fluorescent group) and the quencher group on the dissociated detection probe are close to each other, so that the signal (e.g., fluorescent signal) emitted by the reporter group (e.g., fluorescent group) is absorbed by the quencher group. Therefore, as the temperature rises, the detected signal (e.g., fluorescent signal) gradually weakens. When the two chains of the duplex are completely dissociated, all detection probes are in a single-stranded free curling state. In this case, the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups) on all detection probes are absorbed by the quenching group. Therefore, the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups) are basically undetectable. Therefore, by detecting the signals (e.g., fluorescent signals) emitted by the duplex containing the detection probe during the heating or cooling process, the hybridization and dissociation process of the detection probe and 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 signal intensity change rate as the ordinate and the temperature as the abscissa can be obtained (i.e., the melting curve of the duplex). The peak in the melting curve is the melting peak, and the temperature corresponding to it is the melting point (T m ). Generally speaking, the higher the matching degree between the detection probe and the complementary sequence (for example, the fewer mismatched bases and the more matched bases), the higher the T of the duplex. m Therefore, by detecting the T m , the presence and identity of the sequence in the duplex that is complementary to the detection probe can be determined. As used herein, the terms "melting peak", "melting point" and "T m ” have the same meaning and can be used interchangeably.

[0017] The inventors of the present application have developed a method for efficient asymmetric amplification of multiple target nucleic acids through in-depth research. Based on this, combined with melting curve analysis, the present application has developed a method for simply and quickly identifying biological individuals or performing kinship identification on biological individuals, as well as a kit for implementing the method.

[0018] Therefore, in one aspect, the present application provides a method for identifying an individual organism or performing kinship identification on an individual organism:

[0019] (a) for each individual to be analyzed, providing a sample derived from the individual and containing one or more target nucleic acids, wherein the target nucleic acids contain one or more SNP sites, and

[0020] A first universal primer and a second universal primer are provided, and at least one target-specific primer pair is provided for each SNP site; wherein,

[0021] The first universal primer comprises a first universal sequence;

[0022] The second universal primer comprises a second universal sequence, the second universal sequence comprises the first universal sequence and additionally comprises at least one nucleotide at the 3' end of the first universal sequence;

[0023] The target-specific primer pair can amplify the target nucleic acid as a template to generate a nucleic acid product containing the 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 completely complementary to the complementary sequence of the forward primer; and

[0024] (b) under conditions that allow nucleic acid amplification, using the first universal primer and the second universal primer and the target-specific primer pair, respectively amplifying the target nucleic acid in each sample by PCR reaction, thereby obtaining amplification products corresponding to each individual;

[0025] (c) performing melting curve analysis on the amplification products corresponding to each individual obtained in step (b);

[0026] (d) Identifying biological individuals or determining the relationship between individuals based on the melting curve analysis results of step (b).

[0027] 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, thereby, 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 contains two nucleic acid chains (nucleic acid chain A and nucleic acid chain B) complementary to the forward primer and the reverse primer, respectively. Further, since the forward primer and the first universal primer both contain the first universal sequence, the nucleic acid chain A complementary to the forward primer can also be complementary to the first universal primer. Similarly, the nucleic acid chain B complementary to the reverse primer can also be complementary to the second universal primer.

[0028] Therefore, as the PCR reaction proceeds, the first universal primer and the second universal primer will anneal to the nucleic acid chain A and nucleic acid chain B of the initial amplification product, respectively, and further initiate PCR amplification. In 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 chain A (nucleic acid chain complementary to the forward primer / first universal primer) and synthesize its complementary chain, but also anneal to nucleic acid chain B (nucleic acid chain complementary to the reverse primer / second universal primer) and synthesize its complementary chain. That is, the first universal primer can simultaneously amplify nucleic acid chain A and nucleic acid chain B of the initial amplification product. 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 chain A (nucleic acid chain complementary to the forward primer / first universal primer, which has a sequence complementary to the forward primer), it is not matched with nucleic acid chain A at the 3' end (that is, it cannot be completely complementary at the 3' end). Thus, during the amplification process, the second universal primer will preferentially anneal to nucleic acid chain B (the nucleic acid chain complementary to the reverse primer / second universal primer) and synthesize its complementary chain, while basically being unable to extend and synthesize the complementary chain of nucleic acid chain A (the nucleic acid chain complementary to the first forward primer / first universal primer).

[0029] Therefore, as the PCR amplification proceeds, the synthesis efficiency of the complementary chain of nucleic acid chain A (nucleic acid chain B) will be significantly lower than that of the complementary chain of nucleic acid chain B (nucleic acid chain A), resulting in the complementary chain of nucleic acid chain B (nucleic acid chain A) being synthesized and amplified in large quantities, while the synthesis and amplification of the complementary chain of nucleic acid chain A (nucleic acid chain B) are inhibited, thereby generating a large number of single-stranded products (nucleic acid chain A, which contains a sequence complementary to the forward primer / first universal primer and a 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.

[0030] In addition, since both the forward primer and the reverse primer contain the first universal sequence, during the PCR reaction, the primer dimer formed by the non-specific amplification of the forward primer and the reverse primer will generate a single-stranded nucleic acid whose 5' and 3' ends contain complementary reverse sequences after denaturation, and the single-stranded nucleic acid is easy to anneal itself in the annealing stage to form a stable panhandle structure, which prevents the first universal primer and the second universal primer from annealing and extending the single-stranded nucleic acid, thereby inhibiting 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.

[0031] In certain embodiments, the biological subject is selected from an animal subject, such as a mammalian subject, such as a human subject.

[0032] In certain embodiments, the SNP site is a SNP site in the human genome; for example, the target nucleic acid comprises a human genome SNP site selected from the following: rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs10779650, rs4971514, rs711725, rs2053911, rs9613776, rs7160304, rs1024676, rs1560193, rs10004744, rs6792367 , rs11856699, rs1561393, rs10820181, rs6504977, rs8027171, rs1110116, rs9621748, rs32853, rs4847034, rs2826949, r s8103778, rs1396009, rs1523537, rs1528460, rs7937238, rs2111980, rs7278737, rs591173, rs1358856, rs2730648, rs85 9400, rs876724, rs2270529, rs1463729, rs6857303, rs214955, rs7041158, rs6474513, rs964681, rs2237427, rs590162, r s560681, rs2342747, rs4796362, rs9307465, rs4288409, rs1027895, rs10098647, rs116187, rs7704770, rs2272998, rs90 1398, rs727811, rs3802268, rs1001389, rs4237677, rs1355366, rs3900, rs1019029, rs938283, rs464663, rs10776839, rs12997453, rs4606077, rs914165, rs722098, rs7104420, and any combination of the foregoing SNP sites (for example, a combination of any 20, 30, 40, 50, 60, or 70 of the foregoing SNP sites).

[0033] In certain embodiments, the target nucleic acid in the sample comprises the following human genomic SNP sites: rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs10779650, rs4971514, rs711725, rs2053911, rs9613776, rs7160304, rs1024676, rs1560193, rs10004744, rs6792367, s11856699, rs1561393, rs10820181, rs6504977, rs8027171, rs1110116, rs9621748, rs32853, rs4847034, rs2 826949, rs8103778, rs1396009, rs1523537, rs1528460, rs7937238, rs2111980, rs7278737, rs591173, rs13588 56, rs2730648, rs859400, rs876724, rs2270529, rs1463729, rs6857303, rs214955, rs7041158, rs6474513, rs 964681, rs2237427, rs590162, rs560681, rs2342747, rs4796362, rs9307465, rs4288409, rs1027895, rs100986 47, rs116187, rs7704770, rs2272998, rs901398, rs727811, rs3802268, rs1001389, rs4237677, rs1355366, rs3900, rs1019029, rs938283, rs464663, rs10776839, rs12997453, rs4606077, rs914165, rs722098, and rs7104420.

[0034] In certain embodiments, in step (a), for each SNP site, a detection probe is further provided, wherein the detection probe comprises a nucleotide sequence specific to the target nucleic acid and is capable of annealing or hybridizing with a region of the target nucleic acid containing the SNP site, 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 hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence;

[0035] Furthermore, in step (c), the detection probe is used to perform melting curve analysis on the amplification products corresponding to each individual obtained in step (b).

[0036] In certain embodiments, in step (b) of the method, the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, and a nucleic acid polymerase, and a PCR reaction is performed, and then, after the PCR reaction is completed, a detection probe is added to the product of step (b), and a melting curve analysis is performed; or, 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 a PCR reaction is performed, and then, after the PCR reaction is completed, a melting curve analysis is performed.

[0037] 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 acids (PNA) or locked nucleic acids), or any combination thereof. In certain preferred embodiments, the detection probe comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). 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 deoxyxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acids (LNA).

[0038] In the method of the present application, the detection probe is not limited by its length. In certain embodiments, the length of the detection probe is 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000nt.

[0039] 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.

[0040] In certain 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 quencher group at its 3' end or downstream, or a reporter group is labeled at its 3' end or downstream and a quencher group is labeled at the 5' end or upstream. In such embodiments, when the detection probe is not hybridized with other sequences, the quencher group is located at a position that can absorb or quench the signal of the reporter group (for example, the quencher group is located adjacent 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 is hybridized with its complementary sequence, the quencher group is located at a position that cannot absorb or quench the signal of the reporter group (for example, the quencher group is located away from the reporter group), thereby failing to absorb or quench the signal emitted by the reporter group. In this case, the detection probe emits a signal.

[0041] The design of such self-quenching detection probes is within the capabilities of those skilled in the art. For example, the 5' end of the detection probe may be labeled with a reporter group and the 3' end may be labeled with a quencher group, or the 3' end of the detection probe may be labeled with a reporter group and the 5' end may be labeled with a quencher group. Thus, when the detection probe exists alone, the reporter group and the quencher group are close to each other and interact with each other, so that the signal emitted by the reporter group is absorbed by the quencher group, thereby causing the detection probe to not emit a signal; and when the detection probe hybridizes with its complementary sequence, the reporter group and the quencher group are separated from each other, so that the signal emitted by the reporter group cannot be absorbed by the quencher group, thereby causing the detection probe to emit a signal.

[0042] However, it should be understood that the reporter group and the quencher group do not have to be marked at the end of the detection probe. The reporter group and / or the quencher group can also be marked inside the detection probe, as long as the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence. For example, the reporter group can be marked upstream (or downstream) of the detection probe, and the quencher group can be marked downstream (or upstream) of the detection probe, and the two are separated by a sufficient distance (e.g., 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 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 (e.g., hairpin structure) of the probe, the reporter group and the quencher group approach each other and interact with each other, so that the signal emitted by the reporter group is absorbed by the quencher group, thereby causing the detection probe to not emit a signal; and, when the detection probe hybridizes with its complementary sequence, the reporter group and the quencher group are separated by a sufficient distance from each other, so that the signal emitted by the reporter group cannot be absorbed by the quencher group, thereby causing the detection probe to emit a signal. In certain preferred embodiments, the reporter group and the quencher group are 10-80nt or longer apart, for example, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt. In certain preferred embodiments, the reporter group and the quencher group are no more than 80nt, no more than 70nt, no more than 60nt, no more than 50nt, no more than 40nt, no more than 30nt, or no more than 20nt apart. In certain preferred embodiments, the reporter group and the quencher group are at least 5 nt, at least 10 nt, at least 15 nt, or at least 20 nt apart.

[0043] Therefore, 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 with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence. However, in certain preferred embodiments, at least one of the reporter group and the quencher group is located at the end (e.g., 5' or 3' end) of the detection probe. In certain 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 1-10nt away from the 5' end, and the reporter group and the quencher group are at a suitable distance so that before the detection probe hybridizes with its complementary sequence, the quencher group can absorb or quench the signal of the reporter group. In certain 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 1-10nt away from the 3' end, and the reporter group and the quencher group are at a suitable distance so that before the detection probe hybridizes with its complementary sequence, the quencher group can absorb or quench the signal of the reporter group. In certain preferred embodiments, the reporter group and the quencher group may be at a distance as defined above (e.g., 10-80nt or longer). In certain 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.

[0044] In some embodiments, the reporter group in the detection probe is a fluorescent group (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 that can absorb / quench the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA).

[0045] In certain embodiments, the detection probe is resistant to nuclease activity (e.g., 5' nuclease activity, such as 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises a modification that resists nuclease activity, such as a thiophosphate bond, an alkyl phosphotriester bond, an aryl phosphotriester bond, an alkyl phosphoester bond, an aryl phosphoester bond, a hydrogenated phosphate bond, an alkyl phosphoramidate bond, an aryl phosphoramidate bond, a 2'-O-aminopropyl modification, a 2'-O-alkyl modification, a 2'-O-allyl modification, a 2'-O-butyl modification, and a 1-(4'-thio-PD-ribofuranosyl) modification.

[0046] In certain embodiments, the detection probe is linear, or has a hairpin structure.

[0047] In certain embodiments, the detection probes each independently have 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 a melting curve analysis, and then the presence of the target nucleic acid is determined based on the melting peak in the melting curve; or, the detection probes have different reporter groups, and the product of step (b) is subjected to a melting curve analysis, and then the presence of the target nucleic acid is determined based on the signal type of the reporter group and the melting peak in the melting curve.

[0048] 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, thereby obtaining a curve showing that the signal intensity of each reporter group changes with temperature. For example, the product of step (2) can be gradually heated from 45°C or lower (e.g., not more than 45°C, not more than 40°C, not more than 35°C, not more than 30°C, not more than 25°C) to 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, thereby obtaining a curve showing that the signal intensity of the reporter group changes with temperature. The rate of heating can be routinely determined by those skilled in the art. For example, the rate of temperature increase can be: 0.01-1°C (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, such as 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) per step, and each step is maintained for 0.5-15s (e.g., 0.5-1s , 1-2s, 2-3s, 3-4s, 4-5s, 5-10s, 10-15s); or the temperature rises by 0.01-1°C per second (for example, 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, for example, 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).

[0049] Then, the curve is derived to obtain the melting curve of the product of step (b).

[0050] In certain embodiments, the type of each SNP site is determined based on the melting peak (melting point) in the melting curve.

[0051] In certain embodiments, the detection probe comprises a detection probe having a nucleotide sequence selected from the following or any combination thereof (e.g., any combination of 20, 30, 40, 50, 60, 70): 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, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 12 0, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213 and 216.

[0052] In certain embodiments, in step (d) of the method, the SNP loci of each biological individual are determined based on the melting curve analysis results, thereby identifying the biological individuals or judging the relationship between individuals.

[0053] In certain embodiments, in step (d) of the method, the SNP loci of each biological individual are determined based on the melting curve analysis results, and compared with a reference database to identify the biological individual; or

[0054] In step (d) of the method, the SNP loci types of each biological individual are determined based on the melting curve analysis results, and the SNP loci types of two or more biological individuals are compared to determine the relationship between the two or more biological individuals.

[0055] In certain embodiments, the sample comprises 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids.

[0056] In certain embodiments, the target nucleic acid comprises 1-5, 5-10, 10-15, 15-20, 20-50 or more SNP sites.

[0057] In certain embodiments, the sample comprises genomic nucleic acid or fragments thereof, such as genomic DNA or fragments thereof, derived from the individual.

[0058] In certain embodiments, in step (a) of the method, 1-5, 5-10, 10-15, 15-20, 20-50 or more target-specific primer pairs are provided.

[0059] It is easy to understand that different forward primers and reverse primers can be used for different target nucleic acids. However, when there is sequence similarity between different target nucleic acids, different target-specific primer pairs may have the same forward primer or reverse primer.

[0060] In order to facilitate multiple asymmetric amplification and effectively inhibit non-specific amplification of primer dimers, in certain embodiments, in step (b) of the method, the working concentration of the first universal primer and the second universal primer is higher than the working concentration of the forward primer and the reverse primer; in certain preferred embodiments, the working concentration of the first universal primer and the second universal primer is at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 8 times, at least 10 times, at least 12 times, at least 15 times, at least 18 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times or more than the working concentration of the forward primer and the reverse primer. In certain preferred embodiments, 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 than the working concentration of the forward primer and the reverse primer.

[0061] In the method of the present application, the working concentrations of the first universal primer and the second universal primer may be the same or different. 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. As discussed in detail above, in the method of the present invention, asymmetric amplification is achieved by the difference in matching of nucleic acid chains A and B with the first and second universal primers. Therefore, the relative concentration of the first universal primer compared to the second universal primer can be varied. In certain preferred embodiments, the working concentrations of the first universal primer and the second universal primer are the same. In certain preferred embodiments, the working concentration of the first universal primer is higher than that of the second universal primer. In certain preferred embodiments, the working concentration of the first universal primer is lower than that of the second universal primer. As discussed in detail above, steps (a) and (b) of the method of the present invention can be used to achieve asymmetric amplification of a target nucleic acid containing one or more SNP sites. In some cases, higher asymmetry in amplification may be advantageous. Therefore, in certain preferred embodiments, the ratio of the first universal primer to the second universal primer can be adjusted so that the working concentration of the first universal primer is lower than that of the second universal primer, so as to further enhance the asymmetry of amplification and better enrich single-stranded products.

[0062] In certain embodiments, in the step (b) of the method, the working concentrations of the forward primer and the reverse primer are the same or different. In certain preferred embodiments, the working concentrations of the forward primer and the reverse primer are the same. In certain preferred embodiments, the working concentrations of the forward primer and the reverse primer are different. In certain preferred embodiments, the working concentration of the forward primer is lower than the working concentration of the reverse primer. In certain preferred embodiments, the working concentration of the forward primer is higher than the working concentration of the reverse primer.

[0063] In certain embodiments, the sample or target nucleic acid comprises mRNA, and prior to performing step (b) of the method, the sample is subjected to a reverse transcription reaction; and

[0064] In certain embodiments, in step (b) of the method, a nucleic acid polymerase (particularly a template-dependent nucleic acid polymerase) is used to carry out a 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, Thermus flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, 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, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga In some embodiments, the DNA polymerase is Taq polymerase.

[0065] In certain embodiments, the first universal primer consists of a first universal sequence, or comprises a first universal sequence and an additional sequence, wherein the additional sequence is located at the 5' end of the first universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0066] In certain embodiments, the first universal sequence is located at or constitutes the 3' portion of the first universal primer.

[0067] In the embodiments 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 5-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.

[0068] In certain embodiments, the first universal primer or any of its components comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the first universal primer (or any of its components) comprises or consists of natural nucleotides (e.g., 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 non-natural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0069] In certain embodiments, the second universal primer consists of the second universal sequence, or comprises the second universal sequence and an additional sequence, wherein the additional sequence is located at the 5' end of the second universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0070] In certain embodiments, the second universal sequence is located at or constitutes the 3' portion of the second universal primer.

[0071] 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 3' to the first universal sequence.

[0072] 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.

[0073] In certain embodiments, the second universal primer or any of its components comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the second universal primer (or any of its components) comprises or consists of natural nucleotides (e.g., 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 non-natural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0074] 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; preferably, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0075] 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.

[0076] In certain embodiments, the forward primer comprises or consists of a first universal sequence and a forward nucleotide sequence from 5' to 3'; or, comprises or consists of a first universal sequence, a nucleotide linker, and a forward nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, the first universal sequence, and a forward nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, the first universal sequence, a nucleotide linker, and a forward nucleotide sequence from 5' to 3'.

[0077] In certain embodiments, the forward nucleotide sequence is located at or constitutes the 3' portion of the forward primer.

[0078] In certain embodiments, the length of the forward nucleotide sequence is 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt.

[0079] In certain embodiments, the forward primer has a length of 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.

[0080] In certain embodiments, the forward primer or any of its components comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the forward primer (or any of its components) comprises or consists of natural nucleotides (e.g., 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 non-natural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0081] In certain embodiments, in the reverse primer, the reverse nucleotide sequence is directly connected to the 3' end of the second universal sequence, or the reverse nucleotide sequence is connected to the 3' end of the second universal sequence via a nucleotide linker. In certain embodiments, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0082] 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.

[0083] In certain embodiments, the reverse primer comprises or consists of a second universal sequence and a reverse nucleotide sequence from 5' to 3'; or, comprises or consists of a second universal sequence, a nucleotide linker, and a reverse nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, a second universal sequence, and a reverse nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, a second universal sequence, a nucleotide linker, and a reverse nucleotide sequence from 5' to 3'.

[0084] In certain embodiments, the reverse nucleotide sequence is located at or constitutes the 3' portion of the reverse primer.

[0085] In certain embodiments, the length of the reverse nucleotide sequence is 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt.

[0086] In certain embodiments, the length of the reverse primer is 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.

[0087] In certain embodiments, the reverse primer or any of its components comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the reverse primer (or any of its components) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the reverse primer (or any of its components) comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the reverse primer (or any of its components) comprises a non-natural nucleotide, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0088] In certain embodiments, the second universal sequence is not completely complementary to the complementary sequence of the forward primer; for example, at least one nucleotide located at the 3' end of the second universal sequence, such as 1-5, 5-10, 10-15, 15-20 or more nucleotides, is not complementary to the complementary sequence of the forward primer.

[0089] In certain embodiments, the sequence of the first universal primer is shown as SEQ ID NO:218.

[0090] In certain embodiments, the sequence of the second universal primer is shown as SEQ ID NO:217.

[0091] In certain embodiments, the target-specific primer pairs include primer pairs having nucleotide sequences selected from the group consisting of SEQ ID NOs: 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; 65; 67 and 68; 70 and 71; 73 and 74; 76 and 77; 79 and 80; 82 and 83; 85 and 86; 88 and 89; 91 and 92; 94 and 95; 97 and 98; 100 and 101; 103 and 104; 106 and 107; 109 and 110; 112 and 113; 115 and 116; 118 and 119; 12 1 and 122; 124 and 125; 127 and 128; 130 and 131; 133 and 134; 136 and 137; 139 and 140; 142 and 143; 1 45 and 146; 148 and 149; 151 and 152; 154 and 155; 157 and 158; 160 and 161; 163 and 164; 166 and 167; 1 69 and 170; 172 and 173; 175 and 176; 178 and 179; 181 and 182; 184 and 185; 187 and 188; 190 and 191; 193 and 194; 196 and 197; 199 and 200; 202 and 203; 205 and 206; 208 and 209; 211 and 212; 214 and 215.

[0092] In certain embodiments, the sample comprises DNA (e.g., genomic DNA or cDNA), RNA (e.g., mRNA), or any combination thereof. In certain preferred embodiments, the sample comprises or is DNA (e.g., genomic DNA or cDNA). In certain preferred embodiments, the sample comprises or is RNA (e.g., mRNA). In certain preferred embodiments, the sample comprises or is a mixture of nucleic acids (e.g., a mixture of DNA, a mixture of RNA, or a mixture of DNA and RNA).

[0093] In certain embodiments, the target nucleic acid to be amplified is DNA (eg, genomic DNA or cDNA), an RNA molecule (eg, mRNA), or any combination thereof.

[0094] In certain embodiments, the target nucleic acid to be amplified is single-stranded or double-stranded.

[0095] In certain embodiments, the target nucleic acid is obtained from a prokaryotic organism, a eukaryotic organism (e.g., a protozoan, a parasite, a fungus, a yeast, a plant, an animal including a mammal and a human) or a virus (e.g., Herpes virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, polio virus, etc.) or a viroid.

[0096] In certain embodiments, the target nucleic acid is obtained from a human.

[0097] In certain embodiments, the concentration of the target nucleic acid to be amplified (eg, human genomic DNA) is at least 0.05 ng / μL, such as at least 0.06 ng / μL, at least 0.07 ng / μL, or at least 0.08 ng / μL.

[0098] In certain embodiments, steps (a)-(b) of the method are performed by a scheme comprising the following steps (I)-(VI):

[0099] (I) providing a sample containing one or more target nucleic acids derived from the individual, wherein the target nucleic acids include one or more SNP sites; and providing a first universal primer and a second universal primer, and, for each SNP site, providing a target-specific primer pair; wherein the first universal primer, the second universal primer and the target-specific primer pair are as defined above;

[0100] (II) mixing the sample with the first universal primer and the second universal primer and a target-specific primer pair, and a nucleic acid polymerase;

[0101] (III) incubating the product of the previous step under conditions that allow nucleic acid denaturation;

[0102] (IV) incubating the product of the previous step under conditions that allow annealing or hybridization of the nucleic acids;

[0103] (V) incubating the product of the previous step under conditions permissive for nucleic acid extension; and (VI) optionally, repeating steps (III)-(V) one or more times.

[0104] In certain embodiments, in step (II) of the method, the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, and a nucleic acid polymerase, and a PCR reaction is performed, and then, after the PCR reaction is completed, a detection probe is added to the product of step (VI), and a melting curve analysis is performed; or, in step (II), 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 a PCR reaction is performed, and then, after the PCR reaction is completed, a melting curve analysis is performed.

[0105] In certain embodiments, steps (a)-(c) of the method are performed by a scheme comprising the following steps (I)-(VI):

[0106] (I) providing a sample containing one or more target nucleic acids derived from the individual, wherein the target nucleic acids include one or more SNP sites; and providing a first universal primer and a second universal primer, and, for each SNP site, providing a target-specific primer pair and a detection probe; wherein the first universal primer, the second universal primer, the target-specific primer pair and the detection probe are as defined above;

[0107] (II) mixing the sample with the first universal primer, the second universal primer, the target-specific primer pair and the detection probe, and a nucleic acid polymerase;

[0108] (III) incubating the product of the previous step under conditions that allow nucleic acid denaturation;

[0109] (IV) incubating the product of the previous step under conditions that allow annealing or hybridization of the nucleic acids;

[0110] (V) incubating the product of the previous step under conditions that allow nucleic acid extension;

[0111] (VI) optionally, repeating steps (III)-(V) one or more times; and

[0112] (VII) Perform melting curve analysis on the product of the previous step.

[0113] In certain embodiments, in step (III), the product of step (II) is incubated at a temperature of 80-105° C., thereby denaturing the nucleic acid.

[0114] 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.

[0115] 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 nucleic acid annealing or hybridization.

[0116] 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.

[0117] In certain embodiments, in step (V), the product of step (4) 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 nucleic acid extension.

[0118] 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.

[0119] In certain embodiments, steps (4) and (V) are performed at the same or different temperatures.

[0120] In certain embodiments, steps (III)-(V) are repeated at least once, e.g., 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; preferably, when steps (III)-(V) are repeated one or more times, the conditions used in steps (III)-(V) of each cycle are independently the same or different.

[0121] In certain embodiments, in step (VII), the product of step (VI) may be gradually heated and the signal emitted by the reporter group on the detection probe may be monitored in real time, thereby obtaining a curve showing that the signal intensity of the product of step (VI) changes with temperature. For example, the product of step (VI) may be gradually heated from a temperature of 45°C or lower (e.g., not more than 45°C, not more than 40°C, not more than 35°C, not more than 30°C, not more than 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 may be monitored in real time, thereby obtaining a curve showing that the signal intensity of the reporter group changes with temperature. The rate of heating may be routinely determined by a person skilled in the art. For example, the rate of temperature increase can be: 0.01-1°C (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, such as 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) per step, and each step is maintained for 0.5-15s (e.g., 0.5-1s , 1-2s, 2-3s, 3-4s, 4-5s, 5-10s, 10-15s); or the temperature rises by 0.01-1°C per second (for example, 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, for example, 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).

[0122] In certain embodiments, in step (VII), the product of step (VI) can be gradually cooled and the signal emitted by the reporter group on the detection probe can be monitored in real time, thereby obtaining a curve showing that the signal intensity of the product of step (VI) changes with temperature. For example, the product of step (VI) can be gradually cooled from 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) to a temperature of 45°C or lower (e.g., no more than 45°C, no more than 40°C, no more than 35°C, no more than 30°C, no more than 25°C), and the signal emitted by the reporter group on the detection probe can be monitored in real time, thereby obtaining a curve showing that the signal intensity of the reporter group changes with temperature. The rate of cooling can be routinely determined by those skilled in the art. For example, the cooling rate can be: cooling by 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, such as 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 each step is maintained for 0.5-15s (e.g., 0.5-1s , 1-2s, 2-3s, 3-4s, 4-5s, 5-10s, 10-15s); or cooling by 0.01-1°C per second (for example, 0.01-0.05°C, 0.05-0.1°C, 0.1-0.5°C, 0.5-1°C, 0.04-0.4°C, for example, 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).

[0123] Subsequently, the obtained curve can be differentiated to obtain the melting curve of the product of step (VI). According to the melting peak (melting point) in the melting curve, the presence of the mediator fragment corresponding to the melting peak (melting point) can be determined.

[0124] In another aspect, the present application provides a kit comprising a primer set capable of asymmetrically amplifying a target nucleic acid containing a SNP site.

[0125] In certain embodiments, the primer set comprises: a first universal primer and a second universal primer, and, for each SNP site, at least one target-specific primer pair is provided, wherein:

[0126] The first universal primer comprises a first universal sequence;

[0127] The second universal primer comprises a second universal sequence, the second universal sequence comprises the first universal sequence and additionally comprises at least one nucleotide at the 3' end of the first universal sequence;

[0128] The target-specific primer pair can amplify the target nucleic acid as a template to produce a nucleic acid product containing the 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 completely complementary to the complementary sequence of the forward primer.

[0129] In certain embodiments, the kit further comprises one or more detection probes capable of detecting the SNP site, wherein the detection probe comprises a nucleotide sequence specific to the target nucleic acid and is capable of annealing or hybridizing with a region of the target nucleic acid containing the SNP site, and 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 with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence.

[0130] In certain embodiments, the kit further comprises one or more components selected from the group consisting of: a nucleic acid polymerase, a reagent for performing nucleic acid amplification, a reagent for performing sequencing, a reagent for performing melting curve analysis, or any combination thereof.

[0131] In certain embodiments, the SNP site is selected from rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs10779650, rs4971514, rs711725, rs2053911, rs9613776, rs7160304, rs1024676, rs1560193, rs10004744, rs6792367, rs11856699, rs1561 393, rs10820181, rs6504977, rs8027171, rs1110116, rs9621748, rs32853, rs4847034, rs2826949, rs8103778, rs1 396009, rs1523537, rs1528460, rs7937238, rs2111980, rs7278737, rs591173, rs1358856, rs2730648, rs859400, r s876724, rs2270529, rs1463729, rs6857303, rs214955, rs7041158, rs6474513, rs964681, rs2237427, rs590162, r s560681, rs2342747, rs4796362, rs9307465, rs4288409, rs1027895, rs10098647, rs116187, rs7704770, rs227299 8, any combination (for example, any combination of 20, 30, 40, 50, 60, 70) of rs901398, rs727811, rs3802268, rs1001389, rs4237677, rs1355366, rs3900, rs1019029, rs938283, rs464663, rs10776839, rs12997453, rs4606077, rs914165, rs722098 and rs7104420.

[0132] In certain embodiments, the SNP sites include rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs10779650, rs4971514, rs711725, rs2053911, rs9613776, rs7160304, rs1024676, rs1560193, rs10004744, rs6792367, rs11856 699, rs1561393, rs10820181, rs6504977, rs8027171, rs1110116, rs9621748, rs32853, rs4847034, rs282694 9, rs8103778, rs1396009, rs1523537, rs1528460, rs7937238, rs2111980, rs7278737, rs591173, rs1358856, r s2730648, rs859400, rs876724, rs2270529, rs1463729, rs6857303, rs214955, rs7041158, rs6474513, rs964 681, rs2237427, rs590162, rs560681, rs2342747, rs4796362, rs9307465, rs4288409, rs1027895, rs10098647 , rs116187, rs7704770, rs2272998, rs901398, rs727811, rs3802268, rs1001389, rs4237677, rs1355366, rs3900, rs1019029, rs938283, rs464663, rs10776839, rs12997453, rs4606077, rs914165, rs722098, and rs7104420.

[0133] It is easy to understand 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 of identifying an individual organism or performing kinship identification on an individual organism as described above. Therefore, the detailed description of the first universal primer, the second universal primer, the target-specific primer pair and the detection probe above (including the description of various preferred features and exemplary features) is also applicable here.

[0134] In certain embodiments, the detection probe comprises a detection probe having a nucleotide sequence selected from the following or any combination thereof (e.g., any combination of 20, 30, 40, 50, 60, 70): 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, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120 , 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, and 216.

[0135] In certain embodiments, the sequence of the first universal primer is shown as SEQ ID NO:218.

[0136] In certain embodiments, the sequence of the second universal primer is as shown in SEQ ID NO:217.

[0137] In certain embodiments, the target-specific primer pairs include primer pairs having nucleotide sequences selected from the group consisting of SEQ ID NOs: 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; 65; 67 and 68; 70 and 71; 73 and 74; 76 and 77; 79 and 80; 82 and 83; 85 and 86; 88 and 89; 91 and 92; 94 and 95; 97 and 98; 100 and 101; 103 and 104; 106 and 107; 109 and 110; 112 and 113; 115 and 116; 118 and 119; 12 1 and 122; 124 and 125; 127 and 128; 130 and 131; 133 and 134; 136 and 137; 139 and 140; 142 and 143; 1 45 and 146; 148 and 149; 151 and 152; 154 and 155; 157 and 158; 160 and 161; 163 and 164; 166 and 167; 1 69 and 170; 172 and 173; 175 and 176; 178 and 179; 181 and 182; 184 and 185; 187 and 188; 190 and 191; 193 and 194; 196 and 197; 199 and 200; 202 and 203; 205 and 206; 208 and 209; 211 and 212; 214 and 215.

[0138] In certain embodiments, the kit has one or more technical features selected from the following:

[0139] (1) The nucleic acid polymerase is a template-dependent nucleic acid polymerase, such as a DNA polymerase, in particular a thermostable DNA polymerase; preferably, the nucleic acid polymerase is as defined above;

[0140] (2) The reagents for nucleic acid amplification include a working buffer for an enzyme (e.g., nucleic acid polymerase), dNTPs (labeled or unlabeled), water, and ions (e.g., Mg 2+ ), a single-stranded DNA binding protein, or any combination thereof;

[0141] In certain embodiments, the kit is used for individual identification or kinship testing of organisms.

[0142] It is easy to understand that the kit of the present application is used to implement the method of identifying an individual organism or performing kinship identification on an individual organism as described above. Therefore, the various preferred features and exemplary features described above for the method can also be applied to the kit of the present application and its components, and do not exceed the concept of the present invention and the scope of the present application.

[0143] In another aspect of the present application, there is provided use of the primer set as defined above for preparing a kit for asymmetric amplification of a target nucleic acid molecule, or for identifying an individual organism, or for performing kinship identification on an individual organism.

[0144] In certain embodiments, the kit further comprises a detection probe as defined above.

[0145] In certain embodiments, the kit is used to perform the method as described above.

[0146] Advantageous Effects of the Invention

[0147] Compared with similar technologies for individual identification or kinship testing in current forensic examinations, the present invention has the following advantages: (1) The method of the present invention can detect highly degraded samples; (2) The method of the present invention adopts multiple asymmetric amplification technology, which has strong specificity and can inhibit primer dimers, so that the detection system has high sensitivity and can detect trace amounts of DNA; (3) The present invention adopts a closed tube system throughout the process, without the risk of PCR product contamination, and the detection has a high degree of automation.

[0148] Taking the above advantages into consideration, the method of the present invention can be used to identify biological individuals and perform kinship identification on biological individuals with high accuracy and efficiency.

[0149] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 An exemplary embodiment of the method of the present invention for identifying an individual organism or performing kinship identification on an individual organism through SNP typing is schematically described to illustrate the basic principle of the method of the present invention.

[0151] Figure 1 A schematically describes the primer set and self-quenching fluorescence detection probe involved in this embodiment, wherein 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; wherein,

[0152] The first universal primer comprises a first universal sequence (Tag1);

[0153] The second universal primer comprises a second universal sequence (Tag2) comprising the first universal sequence and additionally comprising at least one nucleotide (e.g., 1-5, 5-10, 10-15, 15-20 or more nucleotides) at the 3' end of the first universal sequence;

[0154] The forward primer comprises 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;

[0155] The reverse primer comprises 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,

[0156] The forward primer and the reverse primer can specifically amplify the corresponding target nucleic acid containing the SNP site; and,

[0157] The second universal sequence may not be completely complementary to the complementary sequence of the forward primer.

[0158] Figure 1 B schematically depicts the use of Figure 1 The principle that when the primer set A is used for amplification, the nonspecific amplification of primer dimers is inhibited, wherein the primer dimers formed due to the nonspecific amplification of the forward primer and the reverse primer will produce a single-stranded nucleic acid whose 5' and 3' ends contain reverse sequences complementary to each other after denaturation, and the single-stranded nucleic acid itself will form a panhandle structure in the annealing stage, preventing the first universal primer and the second universal primer from annealing and extending the single-stranded nucleic acid, thereby inhibiting further amplification of the primer dimers.

[0159] Figure 1 C schematically depicts the use of Figure 1 The principle of the primer set and detection probe of A for simultaneous detection of multiple target nucleic acids containing SNP sites. In this embodiment, a pair of forward primers and reverse primers and a self-quenching fluorescent detection probe are designed for each target nucleic acid containing a SNP site. The specific detection process is as follows:

[0160] First, PCR amplification is initiated by a target-specific primer pair with a low concentration to produce an initial amplification product, which contains two nucleic acid chains (nucleic acid chain A and nucleic acid chain B) that are complementary to the forward primer / first universal primer and the reverse primer / second universal primer, respectively; then, the initial amplification product is subjected to subsequent PCR amplification by the first universal primer and the second universal primer with a high concentration.

[0161] Since the reverse primer / second universal primer contains the first universal sequence, the first universal primer can not only anneal to nucleic acid chain A (the nucleic acid chain complementary to the forward primer / first universal primer) and synthesize its complementary chain, but also anneal to nucleic acid chain B (the nucleic acid chain complementary to the reverse primer / second universal primer) and synthesize its complementary chain. That is, the first universal primer can amplify nucleic acid chain A and nucleic acid chain B at the same time.

[0162] The second universal primer contains an extra nucleotide at the 3' end of the first universal sequence, and therefore, it does not match the nucleic acid chain A (the nucleic acid chain complementary to the forward primer / first universal primer) 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 the nucleic acid chain B (the nucleic acid chain complementary to the reverse primer / second universal primer) and synthesize its complementary chain, and substantially cannot extend and synthesize the complementary chain of the nucleic acid chain A (the nucleic acid chain complementary to the forward primer / first universal primer).

[0163] Therefore, as the PCR amplification proceeds, the synthesis efficiency of the complementary chain of nucleic acid chain A (nucleic acid chain B) will be significantly lower than that of the complementary chain of nucleic acid chain B (nucleic acid chain A), resulting in a large amount of synthesis and amplification of the complementary chain of nucleic acid chain B (nucleic acid chain A), while the synthesis and amplification of the complementary chain of nucleic acid chain A (nucleic acid chain B) are inhibited, thereby generating a large amount of target single-stranded products (nucleic acid chain A, which contains a sequence complementary to the forward primer / first universal primer and a sequence of the reverse primer / second universal primer), achieving asymmetric amplification. In addition, in order 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 product. By using multiple pairs of forward primers and reverse primer pairs simultaneously in the same reaction system, multiple target nucleic acids containing SNP sites can be asymmetrically amplified at the same time, generating a large number of target nucleic acid single strands containing SNP sites.

[0164] After PCR amplification, the pre-added multiple self-quenching fluorescent detection probes are combined with the corresponding single-stranded target nucleic acid containing the SNP site to form a double-stranded hybrid of the detection probe and the target nucleic acid. Due to the different stabilities of the double-stranded hybrids formed, different melting peaks can be obtained after melting curve analysis. m ) and the type of fluorescent group labeled by the probe can determine the genotype of the SNP in each single strand of the target nucleic acid.

[0165] Figure 2The results of melting curve analysis after amplification of different genomic DNA samples using reaction systems 1-3 in the system of the present invention in Example 1 are shown. Among them, the black solid line (sample 1), gray solid line (sample 2), black dashed line (sample 3), gray dashed line (sample 4), and gray dotted line (no template control) respectively represent the results of melting curve analysis after amplification of samples 1-4 and no template control using the detection system of the present invention.

[0166] Figure 3 The results of melting curve analysis after amplification of different genomic DNA samples using reaction systems 4-6 in the system of the present invention in Example 1 are shown. Among them, the black solid line (sample 1), gray solid line (sample 2), black dashed line (sample 3), gray dashed line (sample 4), and gray dotted line (no template control) respectively represent the results of melting curve analysis after amplification of samples 1-4 and no template control using the detection system of the present invention.

[0167] Figure 4 The results of melting curve analysis after amplification of genomic DNA samples of different concentrations using reaction systems 1-3 in the system of the present invention in Example 2 are shown. Among them, the black solid line, gray solid line, black dashed line, gray dashed line, and gray dotted line represent the results of melting curve analysis after PCR amplification of samples with human genomic DNA concentrations of 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.05 ng / μL and no template control, respectively.

[0168] Figure 5 The results of melting curve analysis after amplification of genomic DNA samples of different concentrations using reaction systems 4-6 in the system of the present invention in Example 2 are shown. The black solid line, gray solid line, black dashed line, gray dashed line, and gray dotted line represent the results of melting curve analysis after PCR amplification of samples with human genomic DNA concentrations of 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.05 ng / μL and no template control, respectively. DETAILED DESCRIPTION

[0169] The present invention is now described with reference to the following embodiments which are intended to illustrate the present invention (but not to limit the present invention). It should be understood that these embodiments 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 embodiments. Those skilled in the art can implement other technical solutions based on the principles of the present invention using other similar materials or reaction conditions. Such technical solutions do not depart from the basic principles and concepts described in the present invention, and are encompassed within the scope of the present invention.

[0170] Example 1

[0171] In this example, the detection and differentiation capabilities of the system of the present invention for different SNP sites were investigated by taking the typing of 4 different human genomic DNA samples as an example. The instrument used in this example is a SLAN 96 real-time fluorescence PCR instrument (Xiamen Zhishan Biotechnology Co., Ltd., Xiamen).

[0172] In brief, in this example, 6 25 μL PCR reaction systems were used for PCR amplification and melting curve analysis, each of which included: 1×PCR buffer (TaKaRa, Beijing), 7.0 mM MgCl2, 0.3 mM dNTPs, 3.0 U Taq DNA polymerase (TaKaRa, Beijing), universal primers Tag1 and Tag2, and target-specific primer pairs and probes corresponding to the SNP to be detected (the sequences and amounts of primers and probes in each system are shown in Table 1), 5 μL human genomic DNA or negative control (water). The PCR amplification program was: 95°C pre-denaturation for 5 min; 10 cycles (95°C denaturation for 15 s, 65°C-56°C annealing for 15 s (1°C decrease per cycle), 76°C extension for 20 s); 50 cycles (95°C denaturation for 15 s, 55°C annealing for 15 s, 76°C extension for 20 s). After the PCR amplification, melting curve analysis was performed with the following procedures: denaturation at 95°C for 1 min; incubation at 37°C for 3 min; then, the temperature was increased from 40°C to 85°C at a rate of 0.04°C / s, and the fluorescence signals of FAM, HEX, ROX, CY5 and Quasar 705 channels were collected. Figure 2-3 shown.

[0173] Figure 2-3 The results of genotyping 72 SNP sites of 4 samples using the detection system of the present invention in Example 1 are shown. Among them, each sample is represented by a black solid line (sample 1), a gray solid line (sample 2), a black dotted line (sample 3), a gray dotted line (sample 4), and a gray dotted line (no template control). Figure 2-3 The melting curve analysis results are further summarized in Table 2.

[0174] The typing results of each SNP site of each sample in this example are completely consistent with the Sanger sequencing results. The above results show that the detection system of the present invention has good discrimination ability for each genotype of the 72 SNP sites.

[0175] Table 1. Sequences and amounts of primers and probes used in Example 1

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] Table 2. Typing results of 72 SNP loci

[0186]

[0187]

[0188]

[0189] Example 2

[0190] In this example, 1 known concentration and 72 SNP genotypes (rs6424243: A / G; rs12990278: T / T; rs2122080: G / G; rs98506667: G / G; rs774763: C / G; rs10779650: A / A; rs4971514: G / G; rs711725: T / T; rs2053911: A / G; rs9613776: G / A; rs7160304: T / T; rs1024676: T / C; rs1560193: C / C; rs10004744: G / G; rs6792367: T / A; rs11856699: A / A; rs1561393:G / C; rs10820181:T / C; rs6504977:A / G; rs8027171:G / G; rs1110116:A / G; rs9621748:C / C; rs32853:T / C; rs4847034:A / G; rs2826949 rs135885 6:A / A; rs2730648:G / A; rs859400:G / G; rs876724:T / C; rs2270529:T / T; rs1463729:A / A; rs6857303:G / G; rs214955:A / G; rs7041158:C / T; rs6474513 :A / A; rs964681:T / T; rs2237427:G / G; rs590162:G / G; rs560681:A / G; rs2342747:G / G; rs4796362:A / G; rs9307465:C / C; rs4288409:C / C; rs1027895: G / A; rs10098647:C / T; rs116187:G / G; rs7704770:A / G; rs2272998:G / C; rs901398:T / C; rs727811:A / A; rs3802268:A / G; rs1001389:C / C; rs4237677: A / A; rs1355366:T / T; rs3900:G; rs1019029:T / C; rs938283:T / T; rs464663:T / C; rs10776839:T / G; rs12997453:G / G; rs4606077:C / T; rs914165:A / G;rs722098:G / A; rs7104420:G / A) human genomic DNA (male) samples were gradiently diluted (the concentrations after dilution were 10ng / μL, 1ng / μL, 0.1ng / μL, 0.05ng / μL, respectively), and the analytical sensitivity of the system of the present invention was investigated. The instrument used in this embodiment is SLAN 96 real-time fluorescence PCR instrument (Xiamen Zhishan Biotechnology Co., Ltd., Xiamen). Six PCR reaction systems were used in this embodiment, and the PCR amplification program and melting curve analysis program were the same as in Example 1. ;

[0191] Figure 4-5 The results of melting curve analysis after amplification using the system of the present invention in Example 2 are shown. The black solid line, gray solid line, black dashed line, gray dashed line, and gray dotted line represent the results of melting curve analysis after amplification of genomic DNA concentrations of 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.05 ng / μL, and a no-template control, respectively. Figure 4-5 The results show that even if the concentration of human genomic DNA is as low as 0.05 ng / μL (gray dotted line), the system of the present invention can still stably and accurately detect the genotypes of all 72 SNP sites.

[0192] Example 3

[0193] In this example, 205 saliva samples were tested using the detection system of the present invention, DNA samples were extracted (using Lab-Aid820 nucleic acid extractor (Xiamen Zhishan Biotechnology Co., Ltd.) and supporting reagents for automatic extraction), and the genotype frequency, allele frequency, observed heterozygosity Ho, individual recognition rate DP, polymorphic information content PIC, and triplet non-paternal exclusion rate PE of each SNP locus in the collected area (Xiamen, Fujian) were calculated. trio , as well as the cumulative individual identification rate CDP and cumulative triplet non-paternal exclusion rate CPE of 72 SNPs loci trio In this example, six PCR reaction systems, PCR amplification procedures and melting curve analysis procedures were used, all the same as in Example 1.

[0194] The standard formula used to evaluate this system is as follows:

[0195] (1) Polymorphism Information Content (PIC)

[0196]

[0197] Where n is the number of alleles at a certain SNP site, Pi and P j are the frequencies of the i-th and j-th alleles, respectively.

[0198] (2) Observed Heterozygosity (Ho):

[0199]

[0200] Where n is the number of alleles at a certain SNP site, P i is the frequency of the ith allele.

[0201] (3) Individual recognition rate (Power of Discrimination, DP)

[0202]

[0203] Where n is the number of genotypes at a certain SNP site, P i is the frequency of the i-th genotype.

[0204] Cumulative Discrimination Power (CDP)

[0205]

[0206] Among them, m is the total number of SNP sites in the system, DP j is the individual recognition rate of the jth SNP site.

[0207] (4) Probability of Exclusion (PE)

[0208]

[0209] Where n is the number of alleles at a certain SNP site, Pi and P j are the frequencies of the i-th and j-th alleles, respectively.

[0210] Cumulative Excluding probability of Paternity (CPE)

[0211]

[0212] Among them, m is the total number of SNP sites in the system, PE j is the non-paternal exclusion rate of the j-th SNP site.

[0213] The specific statistical results are shown in Table 3. The heterozygosity of most SNP sites is between 0.3 and 0.7, which is relatively high. The actual cumulative individual recognition rate of the selected 72 SNP sites is 1-3.9×10-29 The actual cumulative non-paternity exclusion rate is 0.999999204, which meets the requirement that the cumulative non-paternity exclusion rate of the typing system used in forensic identification in the technical specifications for paternity identification issued by the Forensic Identification Administration of the Ministry of Justice of the People's Republic of China must be greater than 0.9999.

[0214]

[0215]

[0216]

[0217]

Claims

1. A method for identifying an individual organism or performing kinship testing on an individual organism: (a) for each individual to be analyzed, providing a sample derived from the individual and containing one or more target nucleic acids, wherein the target nucleic acids contain one or more SNP sites, and A first universal primer and a second universal primer are provided, and at least one target-specific primer pair is provided for each SNP site; wherein, The first universal primer comprises a first universal sequence; The second universal primer comprises a second universal sequence, the second universal sequence comprises the first universal sequence and additionally comprises at least one nucleotide at the 3' end of the first universal sequence; The target-specific primer pair can amplify the target nucleic acid as a template to generate a nucleic acid product containing the 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 completely complementary to the complementary sequence of the forward primer; and (b) under conditions that allow nucleic acid amplification, using the first universal primer and the second universal primer and the target-specific primer pair, respectively amplifying the target nucleic acid in each sample by PCR reaction, thereby obtaining amplification products corresponding to each individual; (c) performing melting curve analysis on the amplification products corresponding to each individual obtained in step (b); (d) Identifying biological individuals or determining the relationship between individuals based on the melting curve analysis results of step (b).

2. The method of claim 1, wherein the biological subject is selected from animal subjects.

3. The method of claim 2, wherein the animal subject is a mammalian subject.

4. The method of claim 3, wherein the mammalian subject is a human subject. The method of claim 1 , wherein the SNP site is a SNP site in the human genome.

6. The method of claim 1, wherein the target nucleic acid comprises a human genome SNP site selected from the group consisting of rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs10779650, rs4971514, rs711725, rs2053911, rs9613776, rs7160304, rs1024676, rs1560193, rs10004744, rs6792367, rs11856699, rs1561393, rs108201 81, rs6504977, rs8027171, rs1110116, rs9621748, rs32853, rs4847034, rs2826949, rs8103778, rs1396009, rs1523537, rs1528460, rs7937238, rs2111980, rs7278737, rs591173, rs1358856, rs2730648, rs859400, rs876724, rs2270529, rs1463729, rs6857303, rs214955, rs7041158, rs6474513, rs964681, rs2237427, rs590162, rs560681, rs2342747, rs4796362, rs9307465, rs4288409, rs1027895, rs10098647, rs116187, rs7704770, rs2272998, rs901398, rs727811, rs3802268, rs1001389, rs4237677, rs1355366, rs3900, rs1019029, rs938283, rs464663, rs10776839, rs12997453, rs4606077, rs914165, rs722098, rs7104420, and any combination of the foregoing SNP sites.

7. The method of claim 1, wherein the target nucleic acid in the sample comprises the following human genome SNP sites: rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs10779650, rs4971514, rs711725, rs2053911, rs9613776, rs7160304, rs1024676, rs1560193, rs10004744, rs6792367, rs11856699, rs1561393, rs10820181, rs6504977, rs8027171, rs1110116, rs9621748, rs32853, rs4847034, rs2826949, rs8103778, rs1396009, rs1523537, rs 1528460, rs7937238, rs2111980, rs7278737, rs591173, rs1358856, rs2730648, rs859400, rs876724, rs2270529, rs1463729, rs6857303, rs2149 55, rs7041158, rs6474513, rs964681, rs2237427, rs590162, rs560681, rs2342747, rs4796362, rs9307465, rs4288409, rs1027895, rs10098647, rs116187, rs7704770, rs2272998, rs901398, rs727811, rs3802268, rs1001389, rs4237677, rs1355366, rs3900, rs1019029, rs938283, rs464663, rs10776839, rs12997453, rs4606077, rs914165, rs722098, and rs7104420.

8. The method of claim 1, wherein in step (a), for each SNP site, a detection probe is further provided, wherein the detection probe comprises a nucleotide sequence specific to the target nucleic acid and is capable of annealing or hybridizing with a region of the target nucleic acid containing the SNP site, 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 with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence; Furthermore, in step (c), the detection probe is used to perform melting curve analysis on the amplification products corresponding to each individual obtained in step (b).

9. The method of claim 8, wherein The method has one or more technical features selected from the following: (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 a PCR reaction is carried out, and then, after the PCR reaction is completed, a detection probe is added to the product of step (b), and a melting curve analysis is performed; or, 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 a PCR reaction is carried out, and then, after the PCR reaction is completed, a melting curve analysis is performed; (2) The detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural 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, 60-70nt, 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; (4) The detection probe has a 3'-OH end; or, the 3'-end 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 quencher group is a molecule or group that can absorb / quench 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 of the signal intensity of each reporter group with the change of temperature; then, the curve is derived to obtain a melting curve of the product of step (b); (11) Determine the type of each SNP site based on the melting peak in the melting curve; (12) The detection probe comprises a detection probe having a nucleotide sequence selected from the following: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 96, SEQ ID NO: 99, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 138, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 147, SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 159, SEQ ID NO: 162, SEQ ID NO: 165, SEQ ID NO: 168, SEQ ID NO: 171, SEQ ID NO: 174, SEQ ID NO: 177, SEQ ID NO: 180, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 198, SEQ ID NO: 201, SEQ ID NO: 204,SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 213 and SEQ ID NO: 216., 10. The method of claim 9, wherein The method has one or more technical features selected from the following: (1) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; (2) The non-natural 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 a quencher group at its 3' end or downstream, or a reporter group at its 3' end or downstream and 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 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, TEXASRED, 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 nuclease activity is 5' nuclease activity; (9) The main chain of the detection probe contains a modification that resists nuclease activity; (10) The detection probes have the same reporter group, and a melting curve analysis is performed on the product of step (b), and then the presence of the target nucleic acid is determined based on the melting peak in the melting curve; or, the detection probes have different reporter groups, and a melting curve analysis is performed on the product of step (b), and then the presence of the target nucleic acid is determined based on the signal type of the reporter group and the melting peak in the melting curve.

11. The method of claim 10, wherein The modifications are phosphorothioate bonds, alkyl phosphotriester bonds, aryl phosphotriester bonds, alkyl phosphonate bonds, aryl phosphonate bonds, hydrogenated phosphoester bonds, alkyl phosphoramidate bonds, aryl phosphoramidate bonds, 2'-O-aminopropyl modifications, 2'-O-alkyl modifications, 2'-O-allyl modifications, 2'-O-butyl modifications, and 1-(4'-thio-PD-ribofuranosyl) modifications.

12. The method of claim 1, wherein In step (d) of the method, the SNP loci of each biological individual are determined based on the melting curve analysis results, thereby identifying the biological individuals or judging the relationship between individuals.

13. The method of claim 1, wherein In step (d) of the method, the SNP loci of each biological individual are determined according to the melting curve analysis results, and compared with the reference database to identify the biological individual; or, In step (d) of the method, the SNP loci types of each biological individual are determined based on the melting curve analysis results, and the SNP loci types of two or more biological individuals are compared to determine the relationship between the two or more biological individuals.

14. The method of claim 1, wherein The method has one or more technical features selected from the following: (1) The sample contains 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids; (2) The target nucleic acid contains 1-5, 5-10, 10-15, 15-20, 20-50 or more SNP sites; (3) The sample contains genomic nucleic acid or a fragment thereof derived from the individual; (4) in step (a) of the method, providing 1-5, 5-10, 10-15, 15-20, 20-50 or more target-specific primer pairs; (5) In step (b) of the method, the working concentrations of the first universal primer and the second universal primer are higher than the working concentrations of the forward primer and the reverse primer; (6) 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; (7) In step (b) of the method, the working concentrations of the forward primer and the reverse primer are the same or different; (8) the sample or target nucleic acid comprises mRNA, and the sample is subjected to a reverse transcription reaction before performing step (b) of the method; and (9) In step (b) of the method, a nucleic acid polymerase is used to perform a PCR reaction.

15. The method of claim 14, wherein The nucleic acid polymerase is a template-dependent nucleic acid polymerase.

16. The method of claim 14, wherein The nucleic acid polymerase is a DNA polymerase.

17. The method of claim 16, wherein The DNA polymerase is a thermostable DNA polymerase.

18. The method of claim 17, wherein 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, Thermussilvanus, Thermus thermophllus, Thermotoga maritima, Thermotoga neapolitana,Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi,Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana,Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcusabyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifex aeolieus.

19. The method of claim 16, wherein The DNA polymerase is Taq polymerase.

20. 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 the first universal sequence and an additional sequence, wherein the additional sequence is located at the 5' end of the first universal sequence; (2) the first universal sequence is located in or constitutes the 3' portion 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 component thereof comprises 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 the second universal sequence and an additional sequence, wherein 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 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; (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 component thereof comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

21. The method of claim 20, wherein The additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

22. The method of claim 20, wherein The naturally occurring nucleotides are deoxyribonucleotides or ribonucleotides.

23. The method of claim 1, wherein The method has one or more technical features selected from the following: (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 comprises an additional sequence located at the 5' end of the first universal sequence; (3) The forward primer comprises or consists of the first universal sequence and the forward nucleotide sequence from 5' to 3'; or, comprises or consists of the first universal sequence, a nucleotide linker and the forward nucleotide sequence from 5' to 3'; or, comprises or consists of the additional sequence, the first universal sequence and the forward nucleotide sequence from 5' to 3'; or, comprises or consists of the additional sequence, the first universal sequence, a nucleotide linker and the forward nucleotide sequence from 5' to 3'; (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, 60-70nt, 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 component thereof comprises 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 via a nucleotide linker; (9) the reverse primer further comprises an additional sequence located at the 5' end of the second universal sequence; (10) The reverse primer comprises or consists of the second universal sequence and the reverse nucleotide sequence from 5' to 3'; or, comprises or consists of the second universal sequence, a nucleotide linker and the reverse nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, the second universal sequence and the reverse nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, the second universal sequence, a nucleotide linker and the reverse nucleotide sequence from 5' to 3'; (11) the reverse nucleotide sequence is located in 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, 130-140nt, 140-150 nt; (14) the reverse primer or any component thereof comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and (15) The second universal sequence is not completely complementary to the complementary sequence of the forward primer.

24. The method of claim 23, wherein The method has one or more technical 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) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; (4) At least one nucleotide at the 3' end of the second universal sequence is not complementary to the complementary sequence of the forward primer.

25. The method of claim 1, wherein The sequence of the first universal primer is shown in SEQ ID NO:

218.

26. The method of claim 1, wherein The sequence of the second universal primer is shown in SEQ ID NO:

217.

27. The method of claim 1, wherein The target-specific primer pair includes a primer pair having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 11; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 23; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 28 and SEQ ID NO: 29; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 35; SEQ ID NO: 37 and SEQ ID NO: 38; SEQ ID NO: 40 and SEQ ID NO: 41; SEQ ID NO: SEQ ID NO: 82 and SEQ ID NO: 83; SEQ ID NO: 85 and SEQ ID NO: 86; SEQ ID NO: 88 and SEQ ID NO: 89; SEQ ID NO: 90 and SEQ ID NO: 91; SEQ ID NO: 92 and SEQ ID NO: 93; SEQ ID NO: 94 and SEQ ID NO: 95; SEQ ID NO: 96 and SEQ ID NO: 97; SEQ ID NO: 98 and SEQ ID NO: 99; SEQ ID NO: 100 and SEQ ID NO: 101; SEQ ID NO: 102 and SEQ ID NO: 103; SEQ ID NO: 105 and SEQ ID NO: 106; SEQ ID NO: 107 and SEQ ID NO: 108; SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 111 and SEQ ID NO: 112; SEQ ID NO: 113 and SEQ ID NO: 114; SEQ ID NO: 115 and SEQ ID NO: 116; 89; SEQ ID NO: 91 and SEQ ID NO: 92; SEQ ID NO: 94 and SEQ ID NO: 95; SEQ ID NO: 97 and SEQ ID NO: 98; SEQ ID NO: 100 and SEQ ID NO: 101;SEQ ID NO: 103 and SEQ ID NO: 104; SEQ ID NO: 106 and SEQ ID NO: 107; SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 112 and SEQ ID NO: 113; SEQ ID NO: 115 and SEQ ID NO: 116; SEQ ID NO: 118 and SEQ ID NO: 119; SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 124 and SEQ ID NO: 125; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 130 and SEQ ID NO: 131; SEQ ID NO: 133 and SEQ ID NO: 134; SEQ ID NO: 136 and SEQ ID NO: 137; SEQ ID NO: 139 and SEQ ID NO: 140; SEQ ID NO: 142 and SEQ ID NO: SEQ ID NO: 145 and SEQ ID NO: 146; SEQ ID NO: 148 and SEQ ID NO: 149; SEQ ID NO: 151 and SEQ ID NO: 152; SEQ ID NO: 154 and SEQ ID NO: 155; SEQ ID NO: 157 and SEQ ID NO: 158; SEQ ID NO: 160 and SEQ ID NO: 161; SEQ ID NO: 163 and SEQ ID NO: 164; SEQ ID NO: 166 and SEQ ID NO: 167; SEQ ID NO: 169 and SEQ ID NO: 170; SEQ ID NO: 172 and SEQ ID NO: 173; SEQ ID NO: 175 and SEQ ID NO: 176; SEQ ID NO: 178 and SEQ ID NO: 179; SEQ ID NO: 181 and SEQ ID NO: 182; SEQ ID NO: SEQ ID NO: 190 and SEQ ID NO: 191; SEQ ID NO: 193 and SEQ ID NO: 194; SEQ ID NO: 196 and SEQ ID NO: 197; SEQ ID NO: 199 and SEQ ID NO: 200;SEQ ID NO: 202 and SEQ ID NO: 203; SEQ ID NO: 205 and SEQ ID NO: 206; SEQ ID NO: 208 and SEQ ID NO: 209; SEQ ID NO: 211 and SEQ ID NO: 212; SEQ ID NO: 214 and SEQ ID NO:

215. ; 28. The method of claim 1, wherein The method has one or more technical features selected from the following: (1) The sample contains DNA, RNA, or any combination thereof; (2) The target nucleic acid to be amplified is a DNA, RNA molecule or any combination thereof; (3) The target nucleic acid to be amplified is single-stranded or double-stranded; (4) The target nucleic acid is obtained from a prokaryotic organism, a eukaryotic organism, a virus or a viroid; (5) The concentration of the target nucleic acid to be amplified is at least 0.05 ng / µL.

29. The method of claim 28, wherein The method has one or more technical features selected from the following: (1) The DNA is genomic DNA or cDNA; (2) The eukaryotic organism is selected from protozoa, parasites, fungi, yeast, plants, animals including mammals and humans, or any combination thereof; (3) The virus is selected from Herpes virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, polio virus, or any combination thereof; (4) The target nucleic acid is obtained from humans; (5) The target nucleic acid to be amplified is human genomic DNA.

30. The method of claim 1, wherein Steps (a)-(b) of the method are carried out by a scheme comprising the following steps (I)-(VI): (I) providing a sample containing one or more target nucleic acids derived from the individual, wherein the target nucleic acids include one or more SNP sites; and providing a first universal primer and a second universal primer, and providing a target-specific primer pair for each SNP site; wherein the first universal primer, the second universal primer and the target-specific primer pair are as defined in claim 1; (II) mixing the sample with the first universal primer and the second universal primer and a target-specific primer pair, and a nucleic acid polymerase; (III) incubating the product of the previous step under conditions that permit nucleic acid denaturation; (IV) incubating the product of the previous step under conditions that permit annealing or hybridization of the nucleic acids; (V) incubating the product of the previous step under conditions permissive for nucleic acid extension; and (VI) Repeat steps (III) to (V) one or more times.

31. The method of claim 30, wherein The method has one or more technical features selected from the following: (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 (4) 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), incubating 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) performing steps (4) and (V) at the same or different temperatures; and (8) Repeat steps (III) to (V) at least once.

32. The method of claim 30, wherein When steps (III)-(V) are repeated one or more times, the conditions used in steps (III)-(V) of each cycle are independently the same or different.

33. A kit comprising a primer set capable of asymmetrically amplifying a target nucleic acid containing a SNP site; in, The primer set comprises: a first universal primer and a second universal primer, and, for each SNP site, at least one target-specific primer pair is provided, wherein: The first universal primer comprises a first universal sequence; The second universal primer comprises a second universal sequence, the second universal sequence comprises the first universal sequence and additionally comprises at least one nucleotide at the 3' end of the first universal sequence; The target-specific primer pair can amplify the target nucleic acid as a template to produce a nucleic acid product containing the 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 completely complementary to the complementary sequence of the forward primer.

34. The kit of claim 33, further comprising one or more detection probes capable of detecting the SNP site, wherein the detection probes comprise a nucleotide sequence specific to the target nucleic acid and capable of annealing or hybridizing with a region of the target nucleic acid containing the SNP site, and are 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 hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence.

35. The kit of claim 33, further comprising one or more components selected from the group consisting of: a nucleic acid polymerase, a reagent for performing nucleic acid amplification, a reagent for performing sequencing, a reagent for performing melting curve analysis, or any combination thereof.

36. The kit of claim 33, wherein the SNP site is selected from rs6424243, rs12990278, rs2122080, rs98506667 of human chromosomes, rs774763, rs10779650, rs4971514, rs711725, rs2053911, rs9613776, rs7160304, rs1024676, rs1560193, rs10004744, rs6792367, rs118 56699, rs1561393, rs10820181, rs6504977, rs8027171, rs1110116, rs9621748, rs32853, rs4847034, rs2826949, rs8103778, rs1396009, rs1523537, rs1528460, rs7937238, rs2111980, rs7278737, rs591173, rs1358856, rs2730648, rs859400, rs876724, rs2270529, rs1463729, rs6857303, rs214955, rs7041158, rs6474513, rs964681, rs2237427, rs590162, rs560681, rs2342747, rs4796362, rs9307465, rs4288409, rs1027895, rs10098647, Any combination of rs116187, rs7704770, rs2272998, rs901398, rs727811, rs3802268, rs1001389, rs4237677, rs1355366, rs3900, rs1019029, rs938283, rs464663, rs10776839, rs12997453, rs4606077, rs914165, rs722098, and rs7104420.

37. The kit of claim 33, wherein the SNP sites include rs6424243, rs12990278, rs2122080, rs98506667 of human chromosomes, rs774763, rs10779650, rs4971514, rs711725, rs2053911, rs9613776, rs7160304, rs1024676, rs1560193, rs10004744, rs6792367, rs118 56699, rs1561393, rs10820181, rs6504977, rs8027171, rs1110116, rs9621748, rs32853, rs4847034, rs2826949, rs8103778, rs1396009, rs1523537, rs1528460, rs7937238, rs2111980, rs7278737, rs591173, rs1358856, rs2730648, rs859400, rs876724, rs2270529, rs1463729, rs6857303, rs214955, rs7041158, rs6474513, rs964681, rs2237427, rs590162, rs560681, rs2342747, rs4796362, rs9307465, rs4288409, rs1027895, rs10098647, rs116187, rs7704770, rs2272998, rs901398, rs727811, rs3802268, rs1001389, rs4237677, rs1355366, rs3900, rs1019029, rs938283, rs464663, rs10776839, rs12997453, rs4606077, rs914165, rs722098, and rs7104420.

38. The kit of claim 34, wherein the detection probe comprises a detection probe having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85 ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 96, SEQ ID NO: 99, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 138, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 147, SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 159, SEQ ID NO: 162, SEQ ID NO: 165, SEQ ID NO: 168, SEQ ID NO: 171, SEQ ID NO: 174, SEQ ID NO: 177, SEQ ID NO: 180, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 198, SEQ ID NO: 201,SEQ ID NO: 204, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 213, and SEQ ID NO: 216., 39. The kit of claim 33, wherein the sequence of the first universal primer is shown in SEQ ID NO:

218.

40. The kit of claim 33, wherein the sequence of the second universal primer is shown in SEQ ID NO:

217.

41. The kit of claim 33, wherein the target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 11; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 23; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 28 and SEQ ID NO: 29; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 35; SEQ ID NO: 37 and SEQ ID NO: 38; SEQ ID NO: 40 and SEQ ID NO: 41; SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 46 and SEQ ID NO: 47; SEQ ID NO: 49 and SEQ ID NO: 50; SEQ ID NO: 52 and SEQ ID NO: 53; SEQ ID NO: 55 and SEQ ID NO: 56; SEQ ID NO: 58 and SEQ ID NO: 59; SEQ ID NO: 61 and SEQ ID NO: 62; SEQ ID NO: 64 and SEQ ID NO: 65; SEQ ID NO: 67 and SEQ ID NO: 68; SEQ ID NO: 70 and SEQ ID NO: 71; SEQ ID NO: 73 and SEQ ID NO: 74; SEQ ID NO: 76 and SEQ ID NO: 77; SEQ ID NO: 79 and SEQ ID NO: 80; SEQ ID NO: 82 and SEQ ID NO: 83; SEQ ID NO: 85 and SEQ ID NO: 86; SEQ ID NO: SEQ ID NO: 88 and SEQ ID NO: 89; SEQ ID NO: 91 and SEQ ID NO: 92; SEQ ID NO: 94 and SEQ ID NO: 95; SEQ ID NO: 97 and SEQ ID NO: 98; SEQ ID NO: 100 and SEQ ID NO: 101;SEQ ID NO: 103 and SEQ ID NO: 104; SEQ ID NO: 106 and SEQ ID NO: 107; SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 112 and SEQ ID NO: 113; SEQ ID NO: 115 and SEQ ID NO: 116; SEQ ID NO: 118 and SEQ ID NO: 119; SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 124 and SEQ ID NO: 125; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 130 and SEQ ID NO: 131; SEQ ID NO: 133 and SEQ ID NO: 134; SEQ ID NO: 136 and SEQ ID NO: 137; SEQ ID NO: 139 and SEQ ID NO: 140; SEQ ID NO: 142 and SEQ ID NO: SEQ ID NO: 145 and SEQ ID NO: 146; SEQ ID NO: 148 and SEQ ID NO: 149; SEQ ID NO: 151 and SEQ ID NO: 152; SEQ ID NO: 154 and SEQ ID NO: 155; SEQ ID NO: 157 and SEQ ID NO: 158; SEQ ID NO: 160 and SEQ ID NO: 161; SEQ ID NO: 163 and SEQ ID NO: 164; SEQ ID NO: 166 and SEQ ID NO: 167; SEQ ID NO: 169 and SEQ ID NO: 170; SEQ ID NO: 172 and SEQ ID NO: 173; SEQ ID NO: 175 and SEQ ID NO: 176; SEQ ID NO: 178 and SEQ ID NO: 179; SEQ ID NO: 181 and SEQ ID NO: 182; SEQ ID NO: SEQ ID NO: 190 and SEQ ID NO: 191; SEQ ID NO: 193 and SEQ ID NO: 194; SEQ ID NO: 196 and SEQ ID NO: 197; SEQ ID NO: 199 and SEQ ID NO: 200;SEQ ID NO: 202 and SEQ ID NO: 203; SEQ ID NO: 205 and SEQ ID NO: 206; SEQ ID NO: 208 and SEQ ID NO: 209; SEQ ID NO: 211 and SEQ ID NO: 212; SEQ ID NO: 214 and SEQ ID NO:

215. ; 42. The kit of claim 35, wherein the kit has one or more technical features selected from the group consisting of: (1) The nucleic acid polymerase is as defined in any one of claims 15 to 19; (2) The reagents for nucleic acid amplification include enzyme working buffer, dNTPs, water, a solution containing ions, a single-stranded DNA binding protein, or any combination thereof.

43. The kit of claim 33, which is used for individual identification or kinship testing of organisms.

44. Use of the primer set defined in any one of claims 33 to 43 for preparing a kit for asymmetric amplification of a target nucleic acid molecule, or for identifying an individual organism, or for performing kinship identification on an individual organism.

45. The use according to claim 44, wherein the kit further comprises a detection probe as defined in any one of claims 8 to 10.

46. ​​The use according to claim 44, wherein the kit is used to implement the method described in any one of claims 1 to 32.

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