CN114645079A

CN114645079A - Method and kit for detecting existence or proportion of fetal free DNA in pregnant woman sample

Info

Publication number: CN114645079A
Application number: CN202011513441.3A
Authority: CN
Inventors: 黄秋英; 陈昕雯; 李庆阁
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2022-06-21

Abstract

The present application relates to the field of molecular diagnostics. Specifically, the method for detecting the SNP sites of the samples from the mother and the father utilizes multiple asymmetric PCR amplification and multicolor probe melting curve analysis. Further, the present application, in conjunction with a digital PCR system, develops a method and kit for detecting the presence or proportion of fetal free DNA in a maternal sample. Compared with the prior art, the method and the kit can realize automatic detection, and have the advantages of few manual operation steps, short detection period, accurate detection and high sensitivity.

Description

Method and kit for detecting existence or proportion of fetal free DNA in pregnant woman sample

Technical Field

The present application relates to the field of molecular diagnostics. In particular, the present application relates to a method for detecting SNP sites in samples of maternal origin and samples of paternal origin, and further relates to a method for detecting the presence or proportion of fetal free DNA in a sample of a pregnant woman, and a kit.

Background

As early as 1997, Lo YMD et al (Lancet. 1997; 350: 485-. The discovery of fetal free DNA enables noninvasive prenatal diagnosis of monogenic genetic diseases and noninvasive prenatal screening for chromosomal aneuploidies. However, the free DNA obtained in the plasma of pregnant women is mainly from the mother, and the Fetal free DNA Fraction (FF) is influenced by many factors including the gestational week, the Body Mass Index (BMI) of the pregnant woman, the age of the pregnant woman, etc., which may fluctuate from less than 3% to more than 30% (Prenat Diagn.2013; 33: 667-74). The proportion of free DNA in fetus has become an important factor influencing the accuracy of non-invasive prenatal diagnosis (NIPT) or prenatal screening (NIPS), and the detection accuracy of many molecular diagnosis methods is greatly reduced when the proportion of free DNA in fetus is lower than 4% (Ultrasound and oligonucleotide. 2013; 41: 26-32).

In view of the importance of the fetal free DNA ratio in noninvasive production screening, there are several methods available to determine the fetal free DNA ratio, which mainly include: (1) based on the Y chromosome estimation algorithm (BMJ.2011; 342: c 7401); (2) based on cff-DNA fragment number and length differences (PNAS.2018; 115(22): E5106-E5114); (3) deep-targeted or low-depth sequencing methods based on Single Nucleotide Polymorphisms (SNPs) (Bioinformatics 2012; 28,2883-2890; patent CN 103215350B); (4) SNP marker-based digital PCR methods (Clin chem.2018; 64(2): 336-; (5) a digital PCR method based on methylation labeling (patent CN 111154841A). The concentration and the proportion of cffDNA of the male fetus can be simply and accurately estimated based on the proportion of the Y chromosome fragment, and the method is not suitable for female fetus; most of the existing methods for estimating the concentration and proportion of cffDNA by an NIPS detection platform are based on the difference of the number and length of cffDNA fragments, and the fetal FF estimation is carried out by constructing an identification model, and the accuracy of the methods is yet to be verified by SNP and the like; although the second-generation sequencing method based on SNP and other methods can accurately estimate the concentration and proportion of cffDNA, the sequencing cost is high, and the method cannot be integrated into the existing NIPS detection process, so that the feasibility of large-scale clinical application is unavailable at present; the method based on digital PCR can accurately quantify the cffDNA proportion, but the cost for simultaneously carrying out digital PCR of a plurality of SNPs and methylation markers is too high, and the method is difficult to popularize in clinic. In summary, a simple, accurate, rapid and low-cost method for determining the concentration and proportion of fetal cfDNA is urgently needed clinically at present, so as to improve the accuracy of NIPS and reduce the false positive and false negative of NIPS.

Disclosure of Invention

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

As used herein, the term "SNP (single nucleotide polymorphism)" refers to a nucleic acid sequence polymorphism caused by a variation of a single nucleotide at the genomic level. The term "SNP site" is a site in the genome with a single nucleotide polymorphism. Herein, the SNP site includes a single site having a single nucleotide polymorphism and a site having an insertion or deletion of 1 or more (e.g., 1, 2, 3, 4, 5, 6, or more) nucleotides. Herein, a SNP site is named by its reference number (e.g., rs ID). The rs ID can be used to query SNP sites and their types in public databases, e.g., dbSNP database by NCBI, ChinaMAP database, JSNP database, etc.

As used herein, when referring to a "genotype" of a SNP site, it refers to a general term of a combination of genes at the SNP site in all homologous chromosomes (usually two homologous chromosomes) of a certain individual organism. As used herein, the "genotype" of an SNP site refers to the combination of genes at that SNP site in a pair of homologous chromosomes from a fetus or mother. For example, the genotype at rs5858210 site of an individual is "AG/-" indicating that a pair of homologous chromosomes of the individual have nucleotide sequences "AG" and "-" ("-" indicates a deletion) at rs5858210 site, respectively. "the genotype of the rs 58210 site of an individual is AG/AG" means that a pair of homologous chromosomes of the individual have a nucleotide sequence "AG" at the rs5858210 site. Accordingly, a segment of a gene (i.e., a nucleotide segment) on a single chromosome that contains the SNP site is referred to as an "allele" that contains the SNP site. As used herein, for a certain SNP site, the different alleles typically have identical nucleotide sequences except for the nucleotide differences at that SNP site. When a pair of homologous chromosomes of an individual has the same nucleotide sequence (i.e., has the same allele) at a SNP site, the individual is homozygous for the genotype at the SNP site. When a pair of homologous chromosomes of an individual has different nucleotide sequences (i.e., has different alleles) at a SNP site, the individual is heterozygous for the genotype at the SNP site. As used herein, the term "Fst" refers to a population fixation coefficient that reflects the level of heterozygosity for a population allele, as a measure of the degree of differentiation of the population. Fst is between 0 and 1, and when Fst is 1, the allele is fixed in each local population and is completely differentiated; when Fst is 0, the genetic structure of the populations in different places is completely consistent, and the populations are not differentiated. In the present application, the SNP sites selected are preferably among different human species with Fst < 0.01. These sites are poorly differentiated between different races of humans, with a near level of gene heterozygosity.

As used herein, the term "Fst" refers to a population fixation coefficient that reflects the level of heterozygosity for a population allele, as a measure of the degree of differentiation of the population. The value of Fst is between 0 and 1, and when the value of Fst is 1, the allele is fixed in each population and is completely differentiated; when Fst is 0, it indicates that the genetic structures of the populations are completely consistent from place to place and there is no differentiation between the populations. In the present application, the SNP sites selected are preferably among different human species with Fst < 0.01. These sites are differentiated to a small extent between different human races, with a close level of gene heterozygosity.

The term "complementary" as used herein means that two nucleic acid sequences are capable of forming hydrogen bonds between each other according to the base pairing principle (Watton-Crick principle) and thereby forming a duplex. In this application, the term "complementary" includes "substantially complementary" and "fully complementary". As used herein, the term "fully complementary" means that each base in one nucleic acid sequence is capable of pairing with a base in another nucleic acid strand without mismatches or gaps. As used herein, the term "substantially complementary" means that a majority of the bases in one nucleic acid sequence are capable of pairing with bases in another nucleic acid strand, which allows for the presence of mismatches or gaps (e.g., mismatches or gaps of one or several nucleotides). Typically, two nucleic acid sequences that are "complementary" (e.g., substantially complementary or fully complementary) will selectively/specifically hybridize or anneal and form a duplex under conditions that allow the nucleic acids to hybridize, anneal, or amplify. Accordingly, the term "non-complementary" means that two nucleic acid sequences do not hybridize or anneal under conditions that allow for hybridization, annealing, or amplification of the nucleic acids, and do not form a duplex. As used herein, the term "not being fully complementary" means that the bases in one nucleic acid sequence are not capable of fully pairing with the bases in another nucleic acid strand, at least one mismatch or gap being present.

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 the present application, "hybridization" and "annealing" have the same meaning and are used interchangeably. In general, two nucleic acid sequences that are completely or substantially complementary can hybridize or anneal. The complementarity required for two nucleic acid sequences to hybridize or anneal depends on the hybridization conditions used, particularly the temperature.

As used herein, the term "PCR reaction" has the meaning commonly understood by those skilled in the art, which refers to a reaction that uses a nucleic acid polymerase and primers to amplify a target nucleic acid (polymerase chain reaction). As used herein, the term "multiplex amplification" refers to the amplification of multiple target nucleic acids in the same reaction system. As used herein, the term "asymmetric amplification" refers to amplification of a target nucleic acid resulting in amplification products in which the two complementary nucleic acid strands are present in different amounts, one nucleic acid strand being present in a greater amount than the other nucleic acid strand.

As used herein, and as is generally understood by those of skill in the art, the terms "forward" and "reverse" are merely for convenience in describing and distinguishing the two primers of a primer pair; they are relative and do not have a particular meaning.

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

In certain preferred embodiments of the present application, the melting curve analysis may be performed by using a detection probe labeled with a reporter group and a quencher group. Briefly, at ambient temperature, the detection probe is capable of forming a duplex with its complementary sequence by base pairing. In this case, the reporter (e.g., fluorophore) and the quencher on the detection probe are separated from each other, and the quencher cannot absorb a signal (e.g., a fluorescent signal) emitted from the reporter, and at this time, the strongest signal (e.g., a fluorescent signal) can be detected. As the temperature is increased, both strands of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe is in a single-stranded free coiled-coil state. In this case, the reporter (e.g., fluorophore) and the quencher on the detection probe under dissociation are close to each other, and thus a signal (e.g., a fluorescent signal) emitted from the reporter (e.g., fluorophore) is absorbed by the quencher. Thus, as the temperature increases, the detected signal (e.g., the fluorescence signal) becomes progressively weaker. When both strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coiled-coil state. In this case, the signal (e.g., fluorescent signal) from the reporter (e.g., fluorophore) on all of the detection probes is absorbed by the quencher. Thus, a signal (e.g., a fluorescent signal) emitted by a reporter (e.g., a fluorophore) is substantially undetectable. Thus, by detecting the signal (e.g., fluorescent signal) emitted by the duplex containing the detection probe during the temperature increase or decrease, the hybridization and dissociation processes of the detection probe and its complementary sequence can be observed, forming a curve whose signal intensity varies with temperature. Further, to the obtainedThe resulting curve is subjected to derivative analysis to obtain a curve having the rate of change of signal intensity as ordinate and the temperature as abscissa (i.e., melting curve of the duplex). The peak in the melting curve is the melting peak and the corresponding temperature is the melting point (T) of the duplex_m). In general, the higher the degree of match of the detection probe to the complementary sequence (e.g., the fewer mismatched bases, the more bases paired), the T of the duplex_mThe higher. Thus, by detecting T of the duplex_mThe presence and identity of a 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"has the same meaning and is used interchangeably.

Through intensive research, the inventor establishes a method for detecting SNP sites of a mother sample and a father sample by utilizing multiple asymmetric PCR amplification and multicolor probe melting curve analysis. On this basis, in combination with a digital PCR system, the present application develops a method for detecting the presence and proportion of fetal free DNA in a maternal sample, and a kit for carrying out said method.

Thus, in one aspect, the present application provides a method of detecting SNP sites in a sample of maternal origin and a sample of paternal origin, comprising the steps of:

(a) providing a first sample comprising one or more target nucleic acids derived from the mother and a second sample comprising one or more target nucleic acids derived from the father, the target nucleic acids comprising one or more candidate SNP sites, and,

providing a first and a second universal primer and, for each candidate SNP site, providing at least one target-specific primer pair; wherein the content of the first and second substances,

the first universal primer comprises a first universal sequence;

the second universal primer comprises a second universal sequence comprising the first universal sequence and additionally comprising at least one nucleotide 3' to the first universal sequence;

the target-specific primer pair is capable of amplifying using the target nucleic acid as a template to produce a nucleic acid product containing the candidate SNP site, and comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific for the target nucleic acid, and the forward nucleotide sequence is 3' to 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 3' of the second universal sequence; and, the second universal sequence is not fully complementary to the complement of the forward primer; and

(b) amplifying the target nucleic acid in the first and second samples, respectively, by a PCR reaction using the first and second universal primers and the target-specific primer pair under conditions that allow nucleic acid amplification, thereby obtaining amplification products corresponding to the first and second samples, respectively;

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

(d) determining, from the melting curve analysis result of step (c), the SNP site: the first sample and the second sample have different genotype at the site.

In the method of the present application, the forward primer and the reverse primer comprise a forward nucleotide sequence and a reverse nucleotide sequence, respectively, specific for said target nucleic acid, whereby, during the PCR reaction, a target-specific primer pair (forward primer and reverse primer) will anneal to the target nucleic acid and initiate PCR amplification, resulting in an initial amplification product comprising two nucleic acid strands (nucleic acid strand a and nucleic acid strand B) complementary to the forward primer and reverse primer, respectively. Further, since the forward primer and the first universal primer both comprise the first universal sequence, the nucleic acid strand a that is complementary to the forward primer is also capable of being complementary to the first universal primer. Similarly, the nucleic acid strand B complementary to the reverse primer can also be complementary to the second universal primer.

Thus, as the PCR reaction proceeds, the first and second universal primers will anneal to nucleic acid strand A and nucleic acid strand B, respectively, of the initial amplification product and further initiate PCR amplification. In this process, since the reverse primer/second universal primer contains the first universal sequence, the first universal primer is capable of annealing not only to the nucleic acid strand a (the nucleic acid strand complementary to the forward primer/first universal primer) and synthesizing the complementary strand thereof, but also to the nucleic acid strand B (the nucleic acid strand complementary to the reverse primer/second universal primer) and synthesizing the complementary strand thereof. That is, the first universal primer can amplify both the nucleic acid strand A and the nucleic acid strand 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, and thus, although it is also possible for the second universal primer to anneal to nucleic acid strand a (the nucleic acid strand complementary to the forward primer/first universal primer, which has a sequence complementary to the forward primer), it is not matched at the 3' end (i.e., is not fully complementary at the 3' end) to nucleic acid strand a. Thus, during amplification, the second universal primer will preferentially anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer/second universal primer) and synthesize its complementary strand, while being substantially incapable of extending the complementary strand of synthetic nucleic acid strand A (the nucleic acid strand complementary to the first forward primer/first universal primer).

Therefore, as PCR amplification proceeds, the synthesis efficiency of the complementary strand of the nucleic acid strand a (nucleic acid strand B) will be significantly lower than that of the nucleic acid strand B (nucleic acid strand a), resulting in the synthesis and amplification of the complementary strand of the nucleic acid strand B (nucleic acid strand a) in large quantities, while the synthesis and amplification of the complementary strand of the nucleic acid strand a (nucleic acid strand B) is suppressed, thereby producing a large quantity of single-stranded products (nucleic acid strand a, which contains a sequence complementary to the forward primer/first universal primer and a sequence of the reverse primer/second universal primer), enabling asymmetric amplification of target nucleic acids containing one or more SNP sites. Thus, in steps (a) and (b) of the methods of the present application, asymmetric amplification of one or more target nucleic acids in a sample is achieved.

In addition, since both the forward primer and the reverse primer contain the first universal sequence, primer dimers formed by non-specific amplification of the forward primer and the reverse primer during the PCR reaction will, after denaturation, produce single-stranded nucleic acids whose 5 'and 3' ends contain reverse sequences complementary to each other, which are easily annealed to themselves at the annealing stage to form a stable panhandle structure, preventing annealing and extension of the single-stranded nucleic acids by the first universal primer and the second universal primer, thereby inhibiting further amplification of the primer dimers. Therefore, in the method of the present invention, nonspecific amplification of primer dimer can be effectively suppressed.

In certain embodiments, in step (d) of the method, the type of each candidate SNP site is determined from the melting curve analysis results and compared separately to identify such SNP sites: at this site the first sample has a first homozygous genotype and the second sample has a second homozygous genotype.

In certain embodiments, the first sample is selected from the group consisting of skin, saliva, hair, nails, and any combination thereof, from the mother.

In certain embodiments, the second sample is selected from the group consisting of skin, saliva, hair, nails, and any combination thereof, from the father.

In certain embodiments, the candidate SNP site has 1 or more features selected from:

(1) the candidate SNP site has an Fst of less than 0.3 (e.g., less than 0.2, less than 0.1, less than 0.05, less than 0.01) between different races;

(2) the candidate SNP loci are located on different chromosomes;

(3) the allele frequency of the candidate SNP site is between 0.2 and 0.8 (e.g., between 0.3 and 0.7, between 0.4 and 0.6).

(1) fst of the candidate SNP locus among different ethnic groups is less than 0.01;

(2) the candidate SNP loci are located on different chromosomes;

(3) the allele frequency of the candidate SNP site is between 0.3 and 0.7.

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

In certain embodiments, the target nucleic acid in the sample comprises the following human genomic SNP sites: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs 58210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776 and rs 7160304.

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

in step (c), the amplification products corresponding to the first sample and the second sample obtained in step (b) are subjected to melting curve analysis using the detection probe.

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

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 (PNAs) or locked nucleic acids), or any combination thereof. In certain preferred embodiments, the detection probe comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, for example 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the detection probe comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1- (2' -deoxy-. beta. -D-ribofuranosyl) -3-nitropyrrole, 5-nitroindole, or Locked Nucleic Acid (LNA).

In the methods of the present application, the detection probe is not limited by its length. In some 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, 700-800-900 nt, 900-1000 nt.

In certain embodiments, the detection probe has a 3' -OH terminus; alternatively, the 3' -end of the detection probe is blocked; for example, the 3' -end of the detection probe can be blocked 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.

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 is labeled with a reporter group at its 3 'end or downstream and a quencher group at its 5' end or upstream. In certain embodiments, the reporter and quencher are separated by a distance of 10-80nt or more.

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 is labeled with a reporter group at its 3 'end or downstream and a quencher group at its 5' end or upstream. In such embodiments, the quencher is positioned to absorb or quench the signal from the reporter (e.g., the quencher is positioned adjacent to the reporter) when the detection probe is not hybridized to the other sequence, thereby absorbing or quenching the signal from the reporter. In this case, the detection probe does not emit a signal. Further, when the detection probe hybridizes to its complement, the quencher is located at a position that is unable to absorb or quench the signal from the reporter (e.g., the quencher is located at a position remote from the reporter), and thus unable to absorb or quench the signal from the reporter. In this case, the detection probe emits a signal.

The design of such self-quenching detection probes is within the ability of those skilled in the art. For example, the detection probe may be labeled with a reporter group at the 5 'end and a quencher group at the 3' end, or the detection probe may be labeled with a reporter group at the 3 'end and a quencher group at the 5' end. Whereby, when the detection probe is present alone, the reporter and the quencher are in proximity to each other and interact such that a signal emitted by the reporter is absorbed by the quencher, thereby causing no signal to be emitted by the detection probe; and when the detection probe hybridizes to its complementary sequence, the reporter and the quencher are separated from each other such that a signal from the reporter is not absorbed by the quencher, thereby causing the detection probe to emit a signal.

However, it will be appreciated that the reporter and quencher need not be labeled at the terminus of the detection probe. The reporter and/or quencher may also be labeled within the detection probe, so long as the detection probe emits a signal upon hybridization to its complementary sequence that is different from the signal emitted without hybridization to its complementary sequence. For example, the reporter can be labeled upstream (or downstream) of the detection probe and the quencher can be labeled downstream (or upstream) of the detection probe at a sufficient distance (e.g., 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, or longer). Whereby, when the detection probe is present alone, the reporter and the quencher are in proximity to each other and interact due to free coil of the probe molecule or formation of a secondary structure (e.g., hairpin structure) of the probe such that the signal emitted by the reporter is absorbed by the quencher, thereby rendering the detection probe non-emitting a signal; and, when the detection probe hybridizes to its complement, the reporter and the quencher are separated from each other by a sufficient distance such that the signal from the reporter is not absorbed by the quencher, thereby causing the detection probe to emit a signal. In certain preferred embodiments, the reporter and quencher are separated by a distance of 10-80nt or more, e.g., 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80 nt. In certain preferred embodiments, the reporter and quencher are separated by 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 20 nt. In certain preferred embodiments, the reporter and quencher are separated by at least 5nt, at least 10nt, at least 15nt, or at least 20 nt.

Thus, the reporter and quencher can be labeled at any suitable location on the detection probe, so long as the detection probe emits a signal upon hybridization to its complementary sequence that is different from the signal emitted without hybridization to its complementary sequence. However, in certain preferred embodiments, at least one of the reporter and quencher is at a terminus (e.g., 5 'or 3' terminus) of the detection probe. In certain preferred embodiments, one of the reporter and the quencher is located at the 5 'end of the detection probe or 1-10nt from the 5' end, and the reporter and the quencher are suitably spaced apart such that the quencher is capable of absorbing or quenching the signal of the reporter prior to hybridization of the detection probe to its complementary sequence. In certain preferred embodiments, one of the reporter and the quencher is located at the 3 'end of the detection probe or 1-10nt from the 3' end, and the reporter and the quencher are suitably spaced apart such that the quencher is capable of absorbing or quenching the signal of the reporter prior to hybridization of the detection probe to its complementary sequence. In certain preferred embodiments, the reporter and quencher can be separated by a distance as defined above (e.g., a distance of 10-80nt or more). In certain preferred embodiments, one of the reporter and quencher is at the 5 'end of the detection probe and the other is at the 3' end.

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

In certain embodiments, the detection probe is resistant to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises modifications that are resistant to nuclease activity, such as phosphorothioate linkages, alkylphosphotriester linkages, arylphosphotriester linkages, alkylphosphonate linkages, arylphosphonate linkages, hydrogenphosphate linkages, alkylaminophosphate linkages, arylaminophosphate linkages, 2' -O-aminopropyl modifications, 2' -O-alkyl modifications, 2' -O-allyl modifications, 2' -O-butyl modifications, and 1- (4' -thio-PD-ribofuranosyl) modifications.

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

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 the presence of the target nucleic acid is determined from the melting peak in the melting curve; or, the detection probes have different reporter groups, and the product of step (b) is subjected to melting curve analysis, and then the presence of the target nucleic acid is determined based on the signal type of the reporter group and the melting peak in the melting curve.

In certain embodiments, in step (c), the product of step (b) is subjected to a gradual increase or decrease in temperature and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining a plot of the signal intensity of each reporter group as a function of temperature. For example, the signal intensity of the reporter group on the detection probe can be obtained by gradually increasing the temperature of the product of step (2) from a temperature of 45 ℃ or less (e.g., no more than 45 ℃, no more than 40 ℃, no more than 35 ℃, no more than 30 ℃, no more than 25 ℃) to a temperature of 75 ℃ or more (e.g., at least 75 ℃, at least 80 ℃, at least 85 ℃, at least 90 ℃, at least 95 ℃) and monitoring the signal emitted by the reporter group on the detection probe in real time. The rate of temperature rise may be routinely determined by one skilled in the art. For example, the rate of temperature increase may be: heating at 0.01-1 deg.C per step (such as 0.01-0.05 deg.C, 0.05-0.1 deg.C, 0.1-0.5 deg.C, 0.5-1 deg.C, 0.04-0.4 deg.C, such as 0.01 deg.C, 0.02 deg.C, 0.03 deg.C, 0.04 deg.C, 0.05 deg.C, 0.06 deg.C, 0.07 deg.C, 0.08 deg.C, 0.09 deg.C, 0.1 deg.C, 0.2 deg.C, 0.3 deg.C, 0.4 deg.C, 0.5 deg.C, 0.6 deg.C, 0.7 deg.C, 0.8 deg.C, 0.9 deg.C, or 1.0.0 deg.C), and maintaining at 0.5-15s per step (such as 0.5-1s, 1-2s, 2-3s, 3-4s, 4-5s, 5-10s, 10-15 s); or raising the temperature at 0.01-1 deg.C (e.g., 0.01-0.05 deg.C, 0.05-0.1 deg.C, 0.1-0.5 deg.C, 0.5-1 deg.C, 0.04-0.4 deg.C, e.g., 0.01 deg.C, 0.02 deg.C, 0.03 deg.C, 0.04 deg.C, 0.05 deg.C, 0.06 deg.C, 0.07 deg.C, 0.08 deg.C, 0.09 deg.C, 0.1 deg.C, 0.2 deg.C, 0.3 deg.C, 0.4 deg.C, 0.5 deg.C, 0.6 deg.C, 0.7 deg.8 deg.C, 0.9 deg.C, or 1.0 deg.C) per second.

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

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

In certain embodiments, the detection probes comprise detection probes having a nucleotide sequence selected from the group consisting of seq id no (e.g., any combination of 5, 10, 15, 20, 23): 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 and 69.

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

In certain embodiments, in step (b) of the method, the working concentration of the first and second universal primers is higher than the working concentration of the forward and reverse primers; for example, the working concentration of the first and second universal primers is 1-5 times, 5-10 times, 10-15 times, 15-20 times, 20-50 times or more higher than the working concentration of the forward and reverse primers.

In certain embodiments, in step (b) of the method, the working concentrations of the first and second universal primers are the same; alternatively, the first universal primer is at a lower working concentration than the second universal primer.

In certain embodiments, in step (b) of the method, the working concentration of the forward primer and the reverse primer is the same or different.

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

In certain embodiments, in step (b) of the method, a nucleic acid polymerase (particularly a template-dependent nucleic acid polymerase) is used to perform the PCR reaction. In certain embodiments, the nucleic acid polymerase is a DNA polymerase, e.g., a thermostable DNA polymerase. In certain embodiments, the thermostable DNA polymerase is obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filapormis, Thermus flavus, Thermococcus literalis, Thermus antandaani, Thermus caldophyllus, Thermus chloriphilus, Thermus flavus, Thermus igniterae, Thermus lacticus, Thermus osimai, Thermus ruber, Thermus rubens, Thermus scoodugutes, Thermus malcola, Thermus thermophilus, Thermotoga, Thermogoga neocalli, Thermomyces rhodobacter, Thermococcus aureus, Thermococcus nilotica, Thermomyces rhodobacter, Thermococcus paracola, Thermococcus flavocola, Thermomyces flavofaciens, Thermococcus afolicus, Thermococcus flavus, Thermococcus faecalis, Thermococcus faecalius, Thermococcus purpurea, Thermococcus purpura, Thermococcus purpurea, Thermococcus purpura, Thermococcus purpura, Thermococcus and Thermococcus. In certain embodiments, the DNA polymerase is Taq polymerase.

In certain embodiments, the first universal primer consists of the first universal sequence, or alternatively, comprises the first universal sequence and an additional sequence that is 5' to the first universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

In certain embodiments, the first universal sequence is located on or constitutes the 3' portion of the first universal primer.

In embodiments of the present application, the first universal primer may be any length as long as it is capable of performing a PCR reaction. In certain embodiments, the first universal primer is 5-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt in length.

In certain embodiments, the first universal primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof. In certain preferred embodiments, the first universal primer (or any component thereof) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the first universal primer (or any component thereof) comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, for example 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the first universal primer (or any component thereof) comprises a non-natural nucleotide, such as deoxyinosine, inosine, 1- (2' -deoxy-. beta. -D-ribofuranosyl) -3-nitropyrrole, 5-nitroindole, or Locked Nucleic Acid (LNA).

In certain embodiments, the second universal primer consists of the second universal sequence or, alternatively, comprises the second universal sequence and an additional sequence located 5' of the second universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

In certain embodiments, the second universal sequence is located on or forms the 3' portion of the second universal primer.

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

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

In certain embodiments, the second universal primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof. In certain preferred embodiments, the second universal primer (or any component thereof) comprises or consists of a natural nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide). In certain preferred embodiments, the second universal primer (or any component thereof) comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the second universal primer (or any component thereof) comprises a non-natural nucleotide, such as deoxyinosine, inosine, 1- (2' -deoxy-. beta. -D-ribofuranosyl) -3-nitropyrrole, 5-nitroindole, or Locked Nucleic Acid (LNA).

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

In certain embodiments, the forward primer further comprises an additional sequence located 5' to the first universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

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

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

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

In some embodiments, the length of the forward primer is 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-.

In certain embodiments, the forward primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof. In certain preferred embodiments, the forward primer (or any component thereof) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the forward primer (or any component thereof) comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the forward primer (or any component thereof) comprises a non-natural nucleotide, such as deoxyinosine, inosine, 1- (2' -deoxy-. beta. -D-ribofuranosyl) -3-nitropyrrole, 5-nitroindole, or Locked Nucleic Acid (LNA).

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

In certain embodiments, the reverse primer further comprises an additional sequence located 5' to the second universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

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

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

In certain embodiments, the inverted nucleotide sequence is 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt in length.

In some 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-.

In certain embodiments, the reverse primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof. In certain preferred embodiments, the reverse primer (or any component thereof) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the reverse primer (or any component thereof) comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, for example 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the reverse primer (or any component thereof) comprises a non-natural nucleotide, such as deoxyinosine, inosine, 1- (2' -deoxy-. beta. -D-ribofuranosyl) -3-nitropyrrole, 5-nitroindole, or Locked Nucleic Acid (LNA).

In certain embodiments, the second universal sequence is not fully complementary to the complement of the forward primer; for example, at least one nucleotide, e.g., 1-5, 5-10, 10-15, 15-20 or more nucleotides, at the 3' end of the second universal sequence is not complementary to the complement of the forward primer.

In certain embodiments, the sequence of the first universal primer is set forth in SEQ ID NO 71.

In certain embodiments, the sequence of the second universal primer is set forth in SEQ ID NO. 70.

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

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

(I) providing a first sample of one or more target nucleic acids derived from the mother and a second sample containing one or more target nucleic acids derived from the father, the target nucleic acids comprising one or more SNP sites; and, providing a first and a second universal primer, and, for each SNP site, providing a target-specific primer pair; and optionally, providing one detection probe for each SNP site; wherein the first and second universal primer and target-specific primer pairs are as defined above; the detection probe is as defined above;

(II) mixing the sample with the first and second universal primer and target-specific primer pairs, a nucleic acid polymerase, and optionally, a detection probe;

(III) incubating the product of the previous step under conditions that allow denaturation of the nucleic acids;

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

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

(VI) optionally, repeating steps (III) - (V) one or more times.

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

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

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

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

In certain embodiments, in step (V), the product of step (4) is incubated at a temperature of 35-40 ℃, 40-45 ℃, 45-50 ℃, 50-55 ℃, 55-60 ℃, 60-65 ℃, 65-70 ℃, 70-75 ℃, 75-80 ℃, 80-85 ℃ to allow nucleic acid extension.

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

In certain embodiments, steps (IV) and (V) are performed at the same or different temperatures.

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

In another aspect, the present application also provides a method of detecting the presence of fetal free DNA, or a proportion thereof, in a sample of a pregnant woman, wherein the method comprises the steps of:

(1) providing a pregnant woman sample to be detected;

(2) detecting the genotypes of one or more SNP sites of a maternal-derived sample and a paternal-derived sample, and identifying a target SNP site; wherein, at the target SNP site, the mother has a homozygous genotype comprising a first allele and the father has a homozygous genotype comprising a second allele, wherein the first allele is different from the second allele;

(3) respectively carrying out quantitative detection on the first allele and the second allele of each target SNP locus in the pregnant woman sample to be detected; then, the existence of the nucleic acid of the fetus in the pregnant woman sample to be detected or the proportion thereof is determined according to the quantitative detection result of the first allele and the second allele.

In certain embodiments, in step (2), the genotype of a certain SNP site may be detected by a mechanism selected from the group consisting of: probe hybridization, primer extension, hybridization connection and specific enzyme digestion.

In some casesIn embodiments, in step (2), the SNP site of interest may be identified by a method selected from the group consisting of: sequencing methods (e.g., first-generation sequencing, pyrosequencing, second-generation sequencing), chip methods (e.g., using solid-phase chips, liquid-phase chips capable of detecting SNPs), qPCR-based detection methods (e.g., Taqman probe method), mass spectrometry (e.g., MassARRAY-based iPLEX)^TMGold), chromatography (e.g., denaturing high performance liquid chromatography (hplc), electrophoresis (e.g., SNPshot), melting curve analysis-based assays.

In certain embodiments, in step (2), the SNP site of interest is identified by a multiplex PCR-based melting curve analysis assay.

In certain embodiments, the SNP site of interest is identified by the methods as described previously.

In certain embodiments, in step (3), the first allele and the second allele of each SNP site in the sample are each quantitatively detected by digital PCR.

In certain embodiments, step (3) is performed by the following scheme:

(I) selecting at least 1 (e.g., 1, 2, 3, or more) target SNP sites from step (2), and providing one amplification primer set and one probe set for each selected target SNP site, wherein,

(I-1) the amplification primer set comprises at least one amplification primer (e.g., a pair of amplification primers or more) capable of specifically amplifying a nucleic acid molecule containing the SNP site of interest under conditions that allow nucleic acid hybridization or annealing;

(I-2) the probe set comprises a first probe and a second probe; wherein the content of the first and second substances,

(i) the first probe and the second probe are respectively and independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; the first probe and the second probe are respectively marked with different reporter groups (such as fluorescent groups); and is

(ii) A first probe capable of hybridizing or annealing (preferably being fully complementary) to a nucleic acid molecule of a first allele containing said SNP site of interest, and a second probe capable of hybridizing or annealing (preferably being fully complementary) to a nucleic acid molecule of a second allele containing said SNP site of interest; and, the first and second probes are specific for different alleles;

(II) carrying out digital PCR on the pregnant woman sample to be detected by using the amplification primer group and the probe group, and quantitatively detecting nucleic acid molecules with a first allele and nucleic acid molecules with a second allele;

(III) determining the proportion of fetal free DNA in the pregnant woman sample to be detected according to the quantitative detection result in the step (II).

In certain embodiments, the maternal sample is blood (e.g., peripheral blood).

In certain embodiments, the first probe and the second probe are specific for different genotype of the same SNP site.

In certain embodiments, the SNP sites of interest are each independently selected from SNP sites with a mother genotype first homozygous and a father genotype second homozygous.

In certain embodiments, the maternal sample is pretreated prior to step (1).

In certain embodiments, the pretreatment comprises nucleic acid extraction of the sample and/or enrichment of nucleic acids in the sample.

In certain embodiments, the nucleic acid enrichment is by a method of nucleic acid concentration and/or nucleic acid amplification.

In certain embodiments, the nucleic acid amplification is performed by the amplification primer set.

In such embodiments, the fetal genotype is heterozygous at the SNP site of interest according to mendelian's law of inheritance, with the first allele being identical to the mother and the second allele being identical to the father.

In the method of the present application, the first probe in the probe set is exemplified as being capable of hybridizing or annealing (preferably being fully complementary) to a nucleic acid molecule having a first allele. Thus, in performing a digital PCR reaction, during annealing or extension, the first probe will form a duplex with the nucleic acid molecule and be degraded by a nucleic acid polymerase (e.g., DNA polymerase) during amplification, releasing a reporter group (e.g., a fluorophore). Thus, after the digital PCR amplification reaction is completed, the end point fluorescence of each droplet is detected by a droplet detector, and the number of positive droplets and the number of negative droplets can be determined based on the signal (e.g., first fluorescence signal) intensity of the free first reporter group (e.g., first fluorophore), thereby determining the amount of nucleic acid molecules having the first allele in the sample. Similarly, after the digital PCR amplification reaction is finished, the end point fluorescence of each droplet is detected by a droplet detector, and the number of positive droplets and negative droplets can be determined according to the signal (e.g., second fluorescence signal) intensity of the free second reporter group (e.g., second fluorophore), so that the amount of the nucleic acid molecule having the second allele in the sample can be determined. Since the maternal/fetal genotype differs, corresponding to the different first/second allele content, the proportion of fetal free DNA in the maternal sample can be calculated by comparing and analyzing the amount of nucleic acid molecules containing the first/second allele.

In the methods of the present application, in certain embodiments, the first probe does not anneal or hybridize to a nucleic acid molecule having a second allele during a digital PCR reaction; and/or the second probe does not anneal or hybridize to a nucleic acid molecule having the first allele during the digital PCR reaction. It will be readily appreciated that the hybridization specificity of the first/second probes is particularly advantageous, being able to facilitate the accurate determination of the first/second allele content and thus the calculation of the proportion of fetal free DNA in a maternal sample. In certain embodiments, the hybridization specificity of the first/second probe can be obtained by controlling the annealing temperature and/or the extension temperature of the digital PCR reaction. For example, the annealing temperature and/or the extension temperature may be set below the melting point of a duplex formed by the first probe and a nucleic acid molecule having a first allele but above the melting point of a duplex formed by the first probe and a nucleic acid molecule having a second allele, such that the first probe hybridizes to the nucleic acid molecule having the first allele but not to the nucleic acid molecule having the second allele during the digital PCR reaction. Similarly, the annealing temperature and/or the extension temperature may be set below the melting point of the duplex formed by the second probe and the nucleic acid molecule having the second allele but above the melting point of the duplex formed by the second probe and the nucleic acid molecule having the first allele, such that the second probe hybridizes to the nucleic acid molecule having the second allele but not to the nucleic acid molecule having the first allele during the digital PCR reaction.

In certain embodiments, the proportion of fetal free DNA in the maternal sample is calculated by: when the SNP site of interest is a SNP site with a maternal genotype of a first homozygous (e.g., AA) and a paternal genotype of a second homozygous (e.g., BB), the proportion of fetal free DNA in the maternal sample is: 2N_B/(N_A+N_B) Wherein N is_BIs the copy number of allele B (which can be determined by digital PCR), N_AIs the copy number of allele A (which can be determined by digital PCR).

In the method of the present application, the copy number of the allele can be detected by a digital PCR platform according to the poisson distribution principle and directly output by software, and the related principles and calculation methods thereof can be found in, for example, Milbury CA, Zhong Q, Lin J, et al. 1(1):8-22.

In certain embodiments, the first and second probes each independently comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., Peptide Nucleic Acids (PNAs) or locked nucleic acids), or any combination thereof.

In some embodiments, the length of the first probe and the length of the second probe are 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, respectively.

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

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

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

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

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

In certain embodiments, the set of probes comprises probes having a nucleotide sequence selected from the group consisting of seq id no:73, 74, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

In certain embodiments, the primers of the amplification primer set each independently contain a sequence specific for a target nucleic acid containing a SNP site.

In certain embodiments, the length of the primers of the amplification primer set is 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-minus 110nt, 110-minus 120nt, 120-minus 130nt, 130-minus 140nt, 140-minus 150 nt.

In certain embodiments, the primers of the amplification primer set, or any component thereof, each independently comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.

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

In another aspect, the present application also provides a kit comprising an identifying primer set capable of asymmetrically amplifying a target nucleic acid containing a candidate SNP site.

In certain embodiments, the identifying primer set comprises: a first and a second universal primer, and, for each candidate SNP site, providing at least one target-specific primer pair, wherein,

the first universal primer comprises a first universal sequence;

the second universal primer comprises a second universal sequence comprising the first universal sequence and additionally comprising at least one nucleotide 3' of the first universal sequence;

the target-specific primer pair is capable of amplifying using the target nucleic acid as a template to produce a nucleic acid product containing the candidate SNP site, and comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific for the target nucleic acid, and the forward nucleotide sequence is 3' to the first universal sequence; the reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific for the target nucleic acid, and the reverse nucleotide sequence is located 3' of the second universal sequence; and, the second universal sequence is not fully complementary to the complement of the forward primer.

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

(1) the candidate SNP site has an Fst of less than 0.3 (e.g., less than 0.2, less than 0.1, less than 0.05, less than 0.01) between different ethnic groups;

(2) the candidate SNP loci are located on different chromosomes;

(3) the allele frequency of the candidate SNP site is between 0.3 and 0.7.

In certain embodiments, the target nucleic acid comprises the following human genomic SNP sites: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs 58210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776 and rs 7160304.

It will be readily appreciated that the first universal primer, the second universal primer, the target-specific primer pair and the detection probe in the kits of the present application are used to perform the methods as described above. Thus, the detailed descriptions above for the first universal primer, the second universal primer, the target-specific primer pair, and the detection probe (including descriptions of various preferred and exemplary features) are equally applicable here.

In certain embodiments, the kit further comprises one or more components selected from the group consisting of: amplification primer set, probe set, reagents for performing digital PCR.

In certain embodiments, the amplification primer set comprises at least one amplification primer (e.g., a pair of amplification primers or more amplification primers) that is capable of specifically amplifying a nucleic acid molecule containing the SNP site under conditions that allow for nucleic acid hybridization or annealing.

In certain embodiments, the set of probes comprises a first probe and a second probe; wherein the content of the first and second substances,

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

(ii) A first probe capable of hybridizing or annealing (preferably being fully complementary) to a nucleic acid molecule of a first allele containing said SNP site of interest, and a second probe capable of hybridizing or annealing (preferably being fully complementary) to a nucleic acid molecule of a second allele containing said SNP site of interest; and, the first probe and the second probe are specific for different alleles.

In certain embodiments, the set of probes comprises probes having a nucleotide sequence selected from the group consisting of seq id no:73, 74, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163;

in certain embodiments, the amplification primer set comprises a primer pair having a nucleotide sequence selected from the group consisting of seq id no:72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

In certain embodiments, the reagents for performing digital PCR are selected from the group consisting of one or more components selected from the group consisting of: reagents for preparing the micro-droplet sample, reagents for performing nucleic acid amplification, nucleic acid polymerases, reagents for detecting the micro-droplet sample, or any combination thereof.

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 a melt curve analysis, or any combination thereof.

It will be readily appreciated that the amplification primer set and probe set (first probe and second probe) in the kits of the present application are used to perform the methods described above. Thus, the detailed descriptions above for the amplification primer set and probe set (first probe and second probe), including the description of various preferred and exemplary features, are equally applicable here.

In certain embodiments, the kit further comprises one or more components selected from the group consisting of: reagents for performing sequencing, reagents for performing melt curve analysis, or any combination thereof.

In certain embodiments, the nucleic acid polymerase is a template-dependent nucleic acid polymerase, e.g., a DNA polymerase, particularly a thermostable DNA polymerase. In certain embodiments, the nucleic acid polymerase is as defined above.

In certain embodiments, the reagents for performing nucleic acid amplification include, a working buffer for an enzyme (e.g., a nucleic acid polymerase), dNTPs (labeled or unlabeled), water, an ion-containing (e.g., Mg)²⁺) A single-stranded DNA binding protein, or any combination thereof.

In certain embodiments, the kit is used to determine whether a maternal sample contains fetal free DNA, or alternatively, calculate the proportion of fetal free DNA in the maternal sample.

In certain embodiments, the digital PCR is selected from the group consisting of a droplet-type digital PCR and a chip-type digital PCR.

In a further aspect, the present application provides the use of an identifying primer set as defined above for the preparation of a kit for asymmetric amplification of a target nucleic acid molecule, or for the detection of SNP sites in a maternal derived sample and a paternal derived sample.

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

In certain embodiments, the kit is for performing a method as described previously.

In a further aspect, the present application provides the use of an amplification primer set and a probe set as defined hereinbefore for the preparation of a kit for detecting the presence of fetal free DNA, or the proportion thereof, in a maternal sample.

In certain embodiments, the kit further comprises reagents for determining the genotype of one or more SNP sites in the genome of the pregnant woman or fetus.

In certain embodiments, the kit further comprises an identifying primer set and a detection probe as defined above.

Advantageous effects of the invention

Compared with the prior art, the application has the advantages that: (1) automatic detection, few manual operation steps and short detection period. The SNP typing system can realize the typing of a plurality of SNPs at the same time, and has high automation degree. The whole process from nucleic acid extraction to result acquisition can be completed within 1 day, the determination of the fetal free DNA proportion in the pregnant woman sample can be completed in time, and noninvasive prenatal screening and diagnosis (for example, screening chromosome aneuploid abnormality and monogenic genetic diseases) can be assisted; (2) high accuracy and high sensitivity. Can accurately quantify the proportion of the fetal free DNA in the pregnant woman sample, and even can detect the fetal free DNA as low as 0.1 percent.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 schematically depicts an exemplary embodiment of the method of the invention for detecting the presence or proportion of fetal free DNA in a maternal sample by SNP typing to illustrate the basic principles of the method of the invention.

FIG. 1A schematically depicts a primer set and a self-quenching fluorescent detection probe involved in this embodiment, wherein the primer set comprises: a first and a second universal primer, and a target-specific primer pair comprising a forward primer and a reverse primer; wherein the content of the first and second substances,

the first universal primer comprises a first universal sequence (Tag 1);

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;

the forward primer comprises a first universal sequence and a forward nucleotide sequence specific to a target nucleic acid containing the SNP site, 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 containing the SNP site, and the reverse nucleotide sequence is located at the 3' end of the second universal sequence; and the number of the first and second electrodes,

the forward primer and the reverse primer can specifically amplify corresponding target nucleic acid containing SNP sites; and the number of the first and second electrodes,

the second universal sequence is not fully complementary to the complementary sequence of the forward primer.

FIG. 1B schematically illustrates the principle that non-specific amplification of primer dimers is inhibited when amplification is performed using the primer set of FIG. 1A, wherein primer dimers formed by non-specific amplification of the forward primer and the reverse primer, after denaturation, will produce single-stranded nucleic acids whose 5 'and 3' ends contain reverse sequences complementary to each other, which will themselves form a panhandle structure during the annealing stage, preventing annealing and extension of the single-stranded nucleic acids by the first and second universal primers, thereby inhibiting further amplification of primer dimers.

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

first, PCR amplification is initiated by a low concentration of a target-specific primer pair to produce an initial amplification product comprising two nucleic acid strands (nucleic acid strand A and nucleic acid strand B) complementary to a forward primer/a first universal primer and a reverse primer/a second universal primer, respectively; subsequently, the initial amplification product is subjected to subsequent PCR amplification by the first and second universal primers at high concentrations.

Since the reverse primer/second universal primer contains the first universal sequence, the first universal primer is capable of annealing to not only the nucleic acid strand a (the nucleic acid strand complementary to the forward primer/first universal primer) and synthesizing the complementary strand thereof, but also the nucleic acid strand B (the nucleic acid strand complementary to the reverse primer/second universal primer) and synthesizing the complementary strand thereof. That is, the first universal primer can amplify both nucleic acid strand A and nucleic acid strand B.

The second universal primer contains additional nucleotides at the 3' end of the first universal sequence, and thus is mismatched (i.e., not fully complementary at the 3' end) to the nucleic acid strand a (the nucleic acid strand complementary to the forward primer/first universal primer) at the 3' end. Thus, during amplification, the second universal primer will preferentially anneal to nucleic acid strand B (the nucleic acid strand complementary to the reverse primer/second universal primer) and synthesize its complementary strand, while being substantially incapable of extending the complementary strand of the synthetic nucleic acid strand a (the nucleic acid strand complementary to the forward primer/first universal primer).

Therefore, as the PCR amplification proceeds, the synthesis efficiency of the complementary strand of the nucleic acid strand a (nucleic acid strand B) will be significantly lower than that of the nucleic acid strand B (nucleic acid strand a), resulting in the synthesis and amplification of the complementary strand of the nucleic acid strand B (nucleic acid strand a) in a large amount, while the synthesis and amplification of the complementary strand of the nucleic acid strand a (nucleic acid strand B) is suppressed, thereby producing a large amount of the target single-stranded product (nucleic acid strand a containing 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, to further enhance the asymmetry of amplification, the ratio of the first universal primer to the second universal primer can be adjusted such that the concentration of the first universal primer is lower than that of the second universal primer, thereby better enriching the target single-stranded product. By simultaneously using a plurality of pairs of forward primers and reverse primers in the same reaction system, a plurality of target nucleic acids containing SNP sites can be asymmetrically amplified simultaneously, and a large number of target nucleic acid single strands containing SNP sites can be generated.

After PCR amplification is finished, a plurality of self-quenching fluorescent detection probes which are added in advance are respectively combined with corresponding target nucleic acid single strands containing SNP sites to form double-strand hybrids of the detection probes and the target nucleic acid single strands, different melting peaks can be obtained after the formed double-strand hybrids are analyzed through a melting curve due to different stability, and then the melting points (T) are determined according to the melting points_m) The SNP genotype of each target nucleic acid single strand can be determined by the level of (2) and the type of the fluorophore labeled by the probe.

FIG. 2 shows a flow chart of the method of the present application for determining the proportion of fetal free DNA in a maternal sample.

FIG. 3 shows the SNP typing results of the NIPS-2001 sample group and the NIPS-2002 sample group using the method of the present invention in example 2. Wherein, the black solid line represents the detection result of the maternal genomic DNA in the NIPS-2001 sample group, and the black dotted line represents the detection result of the paternal genomic DNA in the NIPS-2001 sample group; the gray solid line represents the results of detection of maternal genomic DNA in the NIPS-2002 sample group; the gray dotted line represents the results of detection of the paternal genomic DNA in the NIPS-2002 sample group.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it. It is to be understood that these embodiments are merely illustrative of the principles and technical effects of the present invention, and do not represent all possibilities for the invention. The present invention is not limited to the materials, reaction conditions or parameters mentioned in these examples. Other embodiments may be practiced by those skilled in the art using other similar materials or reaction conditions in accordance with the principles of the invention. Such solutions do not depart from the basic principles and concepts described herein, and are intended to be within the scope of the invention.

Example 1 selection of candidate SNP sites

The SNP site of the application is selected from a single nucleotide polymorphism site library (dbSNP) of the National Center for Biotechnology Information (NCBI), and the preferred candidate SNP sites have the following conditions: (1) fst (population fixation coefficient) among different races is less than 0.01, namely the differentiation degree of the loci in the populations of different races is very small, and the level of gene heterozygosity is close; (2) allele frequency between 0.3 and 0.7; (3) distribution in asian populations follows the hardy-weinberg balance; (4) the distance between every two SNPs is >1 Mb; (5) to avoid linkage between different loci, it is endeavored to select loci located on different chromosomes. In this embodiment, 23 candidate SNP sites are selected according to the above preferred criteria, and specifically, as shown in table 1, SNP site information and sequences are queried and downloaded from dbSNP database of National Center for Biotechnology Information (NCBI), allele frequency refers to asian population frequency derived from thousand people genome database, and these sites are uniformly distributed on each chromosome of genome.

TABLE 1 information of candidate SNP sites selected in example 1

Name of SNP	Allelic type	Allele frequency	Chromosome of
				rs16363	TGTTT/-	0.4740/0.5260	22
rs1610937	AGGA/-	0.4300/0.5700	5
				rs5789826	GTAA/-	0.4440/0.5560	11
rs1611048	TAAG/-	0.4300/0.5700	7
				rs2307533	TGAC/-	0.5790/0.4210	14
rs112552066	AGAG/-	0.4940/0.5060	1
				rs5858210	AG/-	0.4380/0.5620	4
rs2307839	GA/-	0.4700/0.5300	6
				rs149809066	CT/-	0.4680/0.5320	2
rs66960151	TG/-	0.4900/0.5100	12
				rs34765837	ACAT/-	0.4554/0.5446	10
rs68076527	TT/-	0.5258/0.4742	7
				rs10779650	G/A	0.3353/0.6647	1
rs4971514	G/C	0.6399/0.3601	2
				rs6424243	G/A	0.5873/0.4127	1
rs12990278	T/C	0.7113/0.2887	2
				rs2122080	A/C	0.5009/0.4991	2
rs98506667	C/G	0.6348/0.3652	3
				rs774763	C/G	0.5654/0.4346	3
rs711725	A/T	0.3740/0.6260	3
				rs2053911	G/A	0.8392/0.1608	16
rs9613776	A/G	0.5645/0.4355	22
				rs7160304	G/T	0.5109/0.4891	14

Example 2 non-invasive prenatal screening of fetal proportion of free DNA

In the embodiment, 4 clinical non-invasive prenatal screening samples are collected and detected, the detection capability of the method is examined, and the overall experimental flow is shown in fig. 2.

3.1 Collection and extraction of samples

4 sets of non-invasive prenatal screening samples were collected, each set comprising a maternal (8-14 weeks gestation) peripheral blood sample and a saliva sample of the corresponding parent of the fetus (hereinafter referred to as "maternal sample" and "paternal sample", respectively). Saliva samples were collected according to the instructions of saliva collectors (Xiamen good Biotechnology Co., Ltd., Xiamen) and stored at room temperature. Collecting 5-10mL pregnant woman peripheral blood sample with EDTA anticoagulant tube (Taizhou, Ningdong medical instruments Co., Ltd.), separating plasma in 2 hr according to standard separation process (1600g, centrifugation for 10min, 16000g, centrifugation for 10min), and freezing at-80 deg.C for storage.

The genomic DNA of each sample was extracted using a Lab-Aid 824 nucleic acid extractor and a genomic DNA extraction reagent (Shiyagi, Xiamen) and the concentration and purity of the genomic DNA was determined using a Nanodrop-2000 micro-UV-visible spectrophotometer (Samerfei, USA). Using Apostle MiniMax^TMThe high efficiency free DNA enrichment isolation kit extracts free DNA (Apostle, USA) and the free DNA concentration was determined using a Qubit 3.0fluorometer (Thermo Fisher Scientific, USA).

3.2 detection of target SNP site

Corresponding primers and probes were designed according to the candidate SNP sites selected in example 1, and the specific sequences and the concentrations used are shown in Table 2. By using a multiplex asymmetric PCR system and a multicolor probe melting curve analysis technique (the technical principle is shown in FIG. 1), 23 SNPs were typed simultaneously in 2 PCR reaction systems.

The SNP typing system is specifically configured as follows: a25. mu.L PCR reaction contained: 1 XPCR buffer (TAKARA, Beijing), 5.0mM MgCl₂0.2mM dNTPs, 1U Taq DNA polymerase (TAKARA, Beijing), primers and probes and amounts shown in Table 2, 5. mu.L human genomic DNA or negative control (water). The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; 10 cycles of denaturation at 95 ℃ for 15s, annealing at 65-56 ℃ for 15s (1 ℃ per cycle), and extension at 76 ℃ for 20 s; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, and extension at 76 ℃ for 20s for 50 cycles; melting curve analysis was then performed, with the program: denaturation at 95 deg.C for 1min, and maintaining at 37 deg.C for 3 min; melting curve analysis was then performed with increasing ramp rates of 0.04 ℃/s from 40 ℃ to 85 ℃ and fluorescence signals were collected for FAM, HEX, ROX, CY5, Quasar705 channels. The instrument used in this experiment was a SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi medical science and technology Co., Ltd.).

TABLE 2 primers, Probe sequences used and concentrations used

3.3 selection of target SNP site

And comparing the genotypes of the corresponding SNP sites of the mother DNA sample and the father DNA sample to obtain the target SNP site. When the genotypes of the mother DNA sample and the father DNA sample are different homozygotes in the same SNP site (e.g., the genotype of the mother sample is homozygote AA, and the genotype of the father sample is homozygote BB), this site is the target SNP site. In this example, taking the NIPS-2001 sample set and the NIPS-2002 sample set as examples, the results of SNP typing of the maternal DNA sample and the paternal DNA sample are shown in Table 3 and FIG. 3. As shown in FIG. 3, the black solid line represents the results of detection of maternal genomic DNA in the NIPS-2001 sample group; the black dotted line represents the detection result of the father genomic DNA in the NIPS-2001 sample group; the gray solid line represents the results of detection of maternal genomic DNA in the NIPS-2002 sample group; the gray dotted line represents the results of detection of the paternal genomic DNA in the NIPS-2002 sample group. Wherein, the target SNP loci of the NIPS-2001 sample group are 5 (namely rs66960151, rs68076527, rs1610937, rs1611048 and rs9613776), 2 of the SNP loci are selected (namely rs66960151 and rs1610937), and each allele of the fetal cfDNA is quantitatively analyzed by adopting a digital PCR system; the target SNP loci of the NIPS-2002 sample group are 3 (namely rs 58210, rs149809066 and rs2307553), and 2 (namely rs5858210 and rs2307553) are selected to carry out quantitative analysis on each allele of fetal DNA by adopting a digital PCR system.

TABLE 3 SNP typing results

3.4 quantitative analysis of alleles of target SNP sites in free DNA samples

The digital PCR quantitative analysis systems were respectively established according to the SNP sites selected in example 1, each system comprising a pair of primers and two probes specific to SNP alleles, and the primers and probes used in each SNP site quantitative system and the amounts used are shown in Table 4. And for the selected target SNP locus, determining the ratio of each allele of the target SNP locus by adopting a corresponding primer group and a corresponding probe group in a digital PCR system.

Optionally, after the free DNA sample is pre-enriched, the allele proportion is determined by using digital PCR. Adding the free DNA sample or the pre-enriched free DNA sample into a PCR premix, preparing droplets with volume nanoliter level by using a Drop Marker sample preparation instrument (Xinyi organism, Beijing) (operating according to the instruction of a micro-droplet sample preparation universal kit (Xinyi organism, Beijing)), and carrying out a PCR amplification procedure: pre-denaturation at 95 ℃ for 10 min; 40 cycles of denaturation at 94 ℃ for 30s, annealing at 58 ℃ and extension for 60 s. After the PCR reaction is finished, according to the instruction of a universal kit for detecting the micro-droplet sample (Xinyi biology, Beijing), a Chip Reader biochip Reader is used for carrying out quantitative detection on the micro-droplet, and a reading system derives sample detection data in an Excel format, including the number of positive and negative micro-droplets, the copy number and the like of FAM and HEX fluorescent channels.

A fetal proportion quantitative analysis model can be deduced according to the Mendel rule. Assuming that the SNP genotype of the mother in the SNP site of interest is the first homozygous (e.g., AA) and the SNP genotype of the father is the second homozygous (e.g., BB), the number of the paternal B alleles determined by digital PCR is N_BDetermining the number of maternal A alleles as a ratio N_AThen the proportion of fetal free DNA to total free DNA of the mother (pregnant woman) is FF:

if a plurality of target SNP sites are detected, FF of each target SNP site is calculated by the method, and then the average value of the FF is calculated to be used as FF in an analysis report.

TABLE 4 primers and probes used in digital PCR quantitative analysis system

3.5 test results

The method of the invention was used to determine the proportion of fetuses in plasma samples from 4 pregnant women (between 8 and 14 weeks of pregnancy) and the results for each sample are shown in table 6. The results of the digital PCR quantitative analysis of the SNP sites of interest (rs66960151 and rs1610937) selected from the NIPS-2001 sample group and the SNP sites of interest (rs5858210 and rs2307553) selected from the NIPS-2002 sample group are shown in Table 5. Wherein N is_FatherRepresenting the copy number of the parent SNP allele, N_FemaleRepresents the copy number of the maternal SNP allele, and FF represents the proportion of fetal episomal DNA in total episomal DNA.

TABLE 5 detection results of digital PCR

TABLE 6.4 fetal ratio measurements of maternal plasma samples

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: many modifications and variations of the details are possible in light of the overall teachings of the disclosure, and such variations are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> university of mansion

<120> a method and kit for detecting the presence or proportion of fetal free DNA in a maternal sample

<130> IDC200388

<160> 163

<170> PatentIn version 3.5

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<223> primer

<400> 40

taggtgtgaa cgagcctg 18

<210> 41

<211> 17

<212> DNA

<213> artificial

<220>

<223> primer

<400> 41

cctgttagag ctcccac 17

<210> 42

<211> 27

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 42

cggtccccag ccctgtagcc acgaccg 27

<210> 43

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 43

tagtctcagt ggactttggt 20

<210> 44

<211> 21

<212> DNA

<213> artificial

<220>

<223> primer

<400> 44

caaacatcaa acaattcagc a 21

<210> 45

<211> 26

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 45

acctgagaat gtggttactt gcaggt 26

<210> 46

<211> 18

<212> DNA

<213> artificial

<220>

<223> primer

<400> 46

tccccaccca gaagaaac 18

<210> 47

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 47

gggaggagaa ggactgatg 19

<210> 48

<211> 33

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 48

cctcagctgt cctccccact tccgtcactg agg 33

<210> 49

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 49

ccccagtaat ggcagatca 19

<210> 50

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 50

tgccttccag atatgcattc 20

<210> 51

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 51

acagcaagtc aattcactgt 20

<210> 52

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 52

ctccagaatc aagctgtgt 19

<210> 53

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 53

tcatgtagga gtgcattgt 19

<210> 54

<211> 24

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 54

ccagtaagac agctgtacac tggt 24

<210> 55

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 55

cgtatcattc ggttatcaag 20

<210> 56

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 56

cccatctgag caaagaact 19

<210> 57

<211> 30

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 57

aatcggccgg atttccctcc aggtaccgat 30

<210> 58

<211> 25

<212> DNA

<213> artificial

<220>

<223> primer

<400> 58

taatttctct atgctcatag gttct 25

<210> 59

<211> 21

<212> DNA

<213> artificial

<220>

<223> primer

<400> 59

ttcaaacctc ctattccaca g 21

<210> 60

<211> 26

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 60

acagcacatg taacatatgg agtgct 26

<210> 61

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 61

gcacaggcaa ttgagaaga 19

<210> 62

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 62

ctcctttaaa agggtcggt 19

<210> 63

<211> 32

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 63

acagcccatt tgtttctcct gtcttgaggc tg 32

<210> 64

<211> 23

<212> DNA

<213> artificial

<220>

<223> primer

<400> 64

ccaaactcct ggatcataaa aca 23

<210> 65

<211> 23

<212> DNA

<213> artificial

<220>

<223> primer

<400> 65

ggaatcaggg ataatctcta tca 23

<210> 66

<211> 18

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 66

tccagggtgc ttacactg 18

<210> 67

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 67

tctaccgtct aacctgcaag 20

<210> 68

<211> 18

<212> DNA

<213> artificial

<220>

<223> primer

<400> 68

atctacgcct gagggaca 18

<210> 69

<211> 29

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 69

tgctgcctga gtgatgataa gtgtcagca 29

<210> 70

<211> 22

<212> DNA

<213> artificial

<220>

<223> Tag2

<400> 70

gtcgcaagca ctcacgtaga ga 22

<210> 71

<211> 20

<212> DNA

<213> artificial

<220>

<223> Tag1

<400> 71

tcgcaagcac tcacgtagag 20

<210> 72

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 72

aggtaatctg aggtggcatc 20

<210> 73

<211> 28

<212> DNA

<213> artificial

<220>

<223> primer

<400> 73

tgtaatttcc tacctaagta gttacagt 28

<210> 74

<211> 24

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 74

ggtgattatg agagaacaac cttc 24

<210> 75

<211> 22

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 75

ggtgattatg agaacaacct tc 22

<210> 76

<211> 30

<212> DNA

<213> artificial

<220>

<223> primer

<400> 76

atggaaaatg taatatttct gaatgaaaga 30

<210> 77

<211> 21

<212> DNA

<213> artificial

<220>

<223> primer

<400> 77

cctttcatct aaatgcgttg c 21

<210> 78

<211> 23

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 78

ttgaaactca gagagactac gag 23

<210> 79

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 79

tgaaactcag actacgagcc 20

<210> 80

<211> 21

<212> DNA

<213> artificial

<220>

<223> primer

<400> 80

cgctgggtca tctattaaca c 21

<210> 81

<211> 22

<212> DNA

<213> artificial

<220>

<223> primer

<400> 81

gaatgccagt attcacaaca gt 22

<210> 82

<211> 24

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 82

acaaaccaga gtcttcttat gaag 24

<210> 83

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 83

tccaacaaac cagtcttctt 20

<210> 84

<211> 22

<212> DNA

<213> artificial

<220>

<223> primer

<400> 84

tccaatccag tgtttcttct ga 22

<210> 85

<211> 17

<212> DNA

<213> artificial

<220>

<223> primer

<400> 85

cacccagaca agccacc 17

<210> 86

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 86

gacaacgctg tgaggctct 19

<210> 87

<211> 18

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 87

cgacaacgct gaggctct 18

<210> 88

<211> 23

<212> DNA

<213> artificial

<220>

<223> primer

<400> 88

agaaatgaga ttcatttgct gga 23

<210> 89

<211> 18

<212> DNA

<213> artificial

<220>

<223> primer

<400> 89

gtctaggcca cttccctc 18

<210> 90

<211> 23

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 90

cacctctttg agtgtcaatt tcc 23

<210> 91

<211> 22

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 91

cacctctgag tgtcaatttc cc 22

<210> 92

<211> 25

<212> DNA

<213> artificial

<220>

<223> primer

<400> 92

aattacacat ccctcattta tccag 25

<210> 93

<211> 26

<212> DNA

<213> artificial

<220>

<223> primer

<400> 93

tgcaattaaa atctattgag caatgg 26

<210> 94

<211> 23

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 94

ctgctattat ggtaagtgtc gga 23

<210> 95

<211> 21

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 95

gctattatgg tgtcggattc a 21

<210> 96

<211> 23

<212> DNA

<213> artificial

<220>

<223> primer

<400> 96

cgttagtcag tcttacccta aac 23

<210> 97

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 97

acacgagttt cgttctttgc 20

<210> 98

<211> 17

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 98

cggcacattc cgggagg 17

<210> 99

<211> 15

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 99

actgcccggc tccgg 15

<210> 100

<211> 18

<212> DNA

<213> artificial

<220>

<223> primer

<400> 100

agaagaacac agtggggc 18

<210> 101

<211> 21

<212> DNA

<213> artificial

<220>

<223> primer

<400> 101

gctggattga agtgcatttg a 21

<210> 102

<211> 24

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 102

ggacaacaaa acaaaacagg attc 24

<210> 103

<211> 21

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 103

gggacaacaa aacaggattc a 21

<210> 104

<211> 26

<212> DNA

<213> artificial

<220>

<223> primer

<400> 104

catttaggaa gccaaatagg atgtac 26

<210> 105

<211> 23

<212> DNA

<213> artificial

<220>

<223> primer

<400> 105

gtaaaagtct gcagaaaatg ggt 23

<210> 106

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 106

acgaggaagg aagggaaga 19

<210> 107

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 107

cacgaggaag ggaagacat 19

<210> 108

<211> 17

<212> DNA

<213> artificial

<220>

<223> primer

<400> 108

tgcatccttg ctgacga 17

<210> 109

<211> 25

<212> DNA

<213> artificial

<220>

<223> primer

<400> 109

agcttttatt tcagatacct gttga 25

<210> 110

<211> 26

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 110

tgcagtaact acaagtaagg aaagta 26

<210> 111

<211> 25

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 111

tgcagtaact acaaggaaag taatt 25

<210> 112

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 112

cccactgatc atctcccaaa 20

<210> 113

<211> 24

<212> DNA

<213> artificial

<220>

<223> primer

<400> 113

cactatggtg attcctagta cctt 24

<210> 114

<211> 21

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 114

gtgttgctct ctctcagttg g 21

<210> 115

<211> 18

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 115

gttgctctct cagttggg 18

<210> 116

<211> 25

<212> DNA

<213> artificial

<220>

<223> primer

<400> 116

gcatgcattt caaagtttat acctg 25

<210> 117

<211> 25

<212> DNA

<213> artificial

<220>

<223> primer

<400> 117

caaggagagc aataagtatg tatcg 25

<210> 118

<211> 22

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 118

catttgactg acagagtagg gg 22

<210> 119

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 119

agtgggcatt tgacagagta 20

<210> 120

<211> 18

<212> DNA

<213> artificial

<220>

<223> primer

<400> 120

agcagatcct tggtcagt 18

<210> 121

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 121

caaagggatg ggttcctct 19

<210> 122

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 122

gcagctacaa atgtacact 19

<210> 123

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 123

gcagctacaa atatacact 19

<210> 124

<211> 24

<212> DNA

<213> artificial

<220>

<223> primer

<400> 124

tccaccataa atctcaacta ttcg 24

<210> 125

<211> 17

<212> DNA

<213> artificial

<220>

<223> primer

<400> 125

gctcccacaa ccttcct 17

<210> 126

<211> 16

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 126

gttgccctgg tcatgg 16

<210> 127

<211> 16

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 127

gttgccctgg tcgtgg 16

<210> 128

<211> 24

<212> DNA

<213> artificial

<220>

<223> primer

<400> 128

gcattagctg aatcctttaa gaga 24

<210> 129

<211> 23

<212> DNA

<213> artificial

<220>

<223> primer

<400> 129

aatccttaaa aacaatgcag cag 23

<210> 130

<211> 22

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 130

ctgagaatgt tgttacttgc ag 22

<210> 131

<211> 22

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 131

ctgagaatgt ggttacttgc ag 22

<210> 132

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 132

gaaaccttgc catctccag 19

<210> 133

<211> 18

<212> DNA

<213> artificial

<220>

<223> primer

<400> 133

ggcatcagtg acggaagt 18

<210> 134

<211> 18

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 134

gttcccagct ctcctccc 18

<210> 135

<211> 17

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 135

gttcccagct gtcctcc 17

<210> 136

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 136

tctgctcagt gtgacaagt 19

<210> 137

<211> 27

<212> DNA

<213> artificial

<220>

<223> primer

<400> 137

gagtgtgatt tgatttttat gcttttg 27

<210> 138

<211> 22

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 138

tgtgaattga cttgctgagg aa 22

<210> 139

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 139

tgaattgact tggtgagga 19

<210> 140

<211> 21

<212> DNA

<213> artificial

<220>

<223> primer

<400> 140

gttacagata ttcccagagc a 21

<210> 141

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 141

ttctcccaat tctcaaagca 20

<210> 142

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 142

cagtaaggca gctgtacac 19

<210> 143

<211> 21

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 143

cagtaagaca gctgtacact g 21

<210> 144

<211> 24

<212> DNA

<213> artificial

<220>

<223> primer

<400> 144

cattcggtta tcaagtatta ccca 24

<210> 145

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 145

ctttctggct catgtctgac 20

<210> 146

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 146

gatttccctg caggtacct 19

<210> 147

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 147

cggatttccc tccaggtacc 20

<210> 148

<211> 29

<212> DNA

<213> artificial

<220>

<223> primer

<400> 148

gtctttaagg atgttctcta aatttttgt 29

<210> 149

<211> 24

<212> DNA

<213> artificial

<220>

<223> primer

<400> 149

acctcctatt ccacagaaga ttat 24

<210> 150

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 150

agcacatgta acatatggag 20

<210> 151

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 151

agcacatgta acataaggag 20

<210> 152

<211> 22

<212> DNA

<213> artificial

<220>

<223> primer

<400> 152

atgatctgaa cagagcttct ga 22

<210> 153

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 153

ggtctgagtt cacctcctc 19

<210> 154

<211> 17

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 154

ctcaagacag gagaaac 17

<210> 155

<211> 19

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 155

cctcaagaca agagaaaca 19

<210> 156

<211> 23

<212> DNA

<213> artificial

<220>

<223> primer

<400> 156

ccaaactcct ggatcataaa aca 23

<210> 157

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 157

caggaaaaga gctgggtca 19

<210> 158

<211> 17

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 158

tccagggtgc ttacact 17

<210> 159

<211> 18

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 159

atccagggtg ctcacact 18

<210> 160

<211> 22

<212> DNA

<213> artificial

<220>

<223> primer

<400> 160

acaagctctc tcatcctaca tc 22

<210> 161

<211> 20

<212> DNA

<213> artificial

<220>

<223> primer

<400> 161

ccctgagtct gtctgatctg 20

<210> 162

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 162

tgcctgagtg atgataagtg 20

<210> 163

<211> 20

<212> DNA

<213> artificial

<220>

<223> Probe

<400> 163

tgccttagtg atgataagtg 20

Claims

1. A method of detecting SNP sites in samples of maternal and paternal origin, comprising the steps of:

the first universal primer comprises a first universal sequence;

the target-specific primer pair is capable of amplifying using the target nucleic acid as a template to produce a nucleic acid product containing the candidate SNP site, and comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific for the target nucleic acid, and the forward nucleotide sequence is 3' to the first universal sequence; the reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific for the target nucleic acid, and the reverse nucleotide sequence is located 3' of the second universal sequence; and, the second universal sequence is not fully complementary to the complement of the forward primer; and

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

2. The method according to claim 1, wherein in step (d) of the method, the type of each candidate SNP site is determined from the melting curve analysis results and compared separately to identify such SNP sites: at the locus the first sample has a first homozygous genotype and the second sample has a second homozygous genotype;

preferably, the first sample is selected from the group consisting of skin, saliva, hair, nails from the mother, and any combination thereof;

preferably, the second sample is selected from the group consisting of skin, saliva, hair, nails, and any combination thereof, from the father;

preferably, the candidate SNP site has 1 or more features selected from:

(2) the candidate SNP loci are located on different chromosomes;

(3) the allele frequency of the candidate SNP site is between 0.2 and 0.8 (e.g., between 0.3 and 0.7, between 0.4 and 0.6);

preferably, the candidate SNP site has 1 or more features selected from:

(2) the candidate SNP loci are located on different chromosomes;

(3) (ii) the allele frequency of the candidate SNP site is between 0.3 and 0.7;

preferably, the candidate SNP sites are SNP sites in the human genome; for example, the target nucleic acid comprises a human genomic SNP site selected from the group consisting of: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs 58210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, rs7160304, and any combination of the foregoing SNP sites (e.g., any 5, 10, 15, 20, 23 combinations of the foregoing SNP sites);

preferably, the target nucleic acid in the sample comprises the following human genomic SNP sites: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs 58210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776 and rs 7160304.

3. The method of claim 1 or 2, wherein in step (a), for each SNP site, a detection probe is further provided, said detection probe comprising a nucleotide sequence specific to said target nucleic acid and being capable of annealing or hybridizing to a region of said target nucleic acid containing said SNP site, and said detection probe is labeled with a reporter group and a quencher group, wherein said reporter group is capable of emitting a signal, and said quencher group is capable of absorbing or quenching the signal emitted by said reporter group; and wherein the detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement;

and in step (c), performing a melting curve analysis on the amplification products corresponding to the first sample and the second sample obtained in step (b) using the detection probe;

preferably, the method has one or more technical features selected from the group consisting of:

(1) in the step (b), mixing the sample with the first universal primer, the second universal primer, the target-specific primer pair and a nucleic acid polymerase, carrying out a PCR reaction, then, after the PCR reaction is finished, adding a detection probe to the product of the step (b), and carrying out melting curve analysis; or, in the step (b), the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair and 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;

(2) the detection probes comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., Peptide Nucleic Acids (PNAs) or locked nucleic acids), or any combination thereof;

(3) 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, 900-1000 nt;

(4) the detection probe has a 3' -OH terminus; alternatively, the 3' -end of the detection probe is blocked; for example, the 3' -end of the detection probe can be blocked by adding a chemical moiety (e.g., biotin or alkyl) 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;

(5) 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 is labeled with a reporter group at its 3 'end or downstream and a quencher group at its 5' end or upstream; preferably, the reporter and quencher are separated by a distance of 10-80nt or more;

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

(7) the detection probe is resistant to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises modifications that are resistant to nuclease activity, such as phosphorothioate linkages, alkylphosphotriester linkages, arylphosphotriester linkages, alkylphosphonate linkages, arylphosphonate linkages, hydrogenphosphate linkages, alkylaminophosphate linkages, arylaminophosphate linkages, 2' -O-aminopropyl modifications, 2' -O-alkyl modifications, 2' -O-allyl modifications, 2' -O-butyl modifications, and 1- (4' -thio-PD-ribofuranosyl) modifications;

(8) the detection probe is linear or has a hairpin structure;

(9) the detection probes each independently have the same or different reporter groups; preferably, 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 from the melting peak in the melting curve; or, the detection probes have different reporter groups, and the product of step (b) is subjected to melting curve analysis, and then the presence of the target nucleic acid is determined according to the signal type of the reporter group and the melting peak in the melting curve;

(10) in the step (c), gradually heating or cooling the product of the step (b) and monitoring the signal emitted by the reporter group on each detection probe in real time, thereby obtaining a curve of the signal intensity of each reporter group changing along with the change of the temperature; then, deriving the curve to obtain a melting curve of the product of step (b);

(11) determining the type of each SNP site according to a melting peak (melting point) in a melting curve;

(12) the detection probes include detection probes having a nucleotide sequence selected from the group consisting of: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 and 69.

4. The method of any one of claims 1-3, wherein the method has one or more technical features selected from the group consisting of:

(1) in step (a) of the method, 1-5, 5-10, 10-15, 15-20 or more target-specific primer pairs are provided;

(2) in step (b) of the method, the working concentration of the first and second universal primers is higher than the working concentration of the forward and reverse primers; for example, the working concentration of the first and second universal primers is 1-5 times, 5-10 times, 10-15 times, 15-20 times, 20-50 times or more higher than the working concentration of the forward and reverse primers;

(3) in step (b) of the method, the working concentration of the first and second universal primers is the same; or, the working concentration of the first universal primer is lower than that of the second universal primer;

(4) in step (b) of the method, the working concentration of the forward and reverse primers is the same or different;

(5) the sample or target nucleic acid comprises mRNA and the sample is subjected to a reverse transcription reaction prior to performing step (b) of the method; and

(6) in step (b) of the method, a nucleic acid polymerase (particularly a template-dependent nucleic acid polymerase) is used to perform the PCR reaction; preferably, the nucleic acid polymerase is a DNA polymerase, such as a thermostable DNA polymerase; preferably, the thermostable DNA polymerase is obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, Thermus antalidanii, Thermus caldophlus, Thermus chloriphilus, Thermus flavus, Thermus agniterae, Thermus lacteus, Thermus osidamia, Thermus ruber, Thermus scodifuctus, Thermus silvanicus, Thermus thermophilus, Thermotogamarimaritima, Thermotoga neocolina, Thermosiperus africans, Thermococcus leucotrichuria, Thermococcus leucotrichum, Thermococcus thermophilus, Thermococcus maritima, Thermococcus purpurea, Thermococcus africans, Thermococcus flavus, Thermococcus purpurea, Thermococcus purpurea, Pyrococcus, Thermococcus purpurea, Pyrococcus, Thermococcus purpurea, Thermococcus pacifia, Thermocascus, Pyrococcus, Thermocascus purpurea, Thermocascus purpuria, Thermocascus, Thermoascus purpuria, Thermoascus, Pyrococcus, Thermoascus, Pyrococcus, Thermoascus, Pyrococcus, Thermoascus, Pyrococcus, Thermoascus, Pyrococcus; preferably, the DNA polymerase is Taq polymerase.

5. The method of any one of claims 1-4, wherein the method has one or more technical features selected from the group consisting of:

(1) the first universal primer consists of the first universal sequence or alternatively, comprises the first universal sequence and an additional sequence located 5' of the first universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(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 5-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50 nt;

(4) the first universal primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof;

(5) the second universal primer consists of or alternatively comprises a second universal sequence and an additional sequence, the additional sequence being located 5' of the second universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(6) the second universal sequence is located on 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 8-15nt, 15-20nt, 20-30nt, 30-40nt or 40-50 nt; and

(9) the second universal primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof.

6. The method of any one of claims 1-5, wherein the method has one or more technical features selected from the group consisting of:

(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; preferably, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(2) the forward primer further comprises an additional sequence located 5' to the first universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(3) the forward primer comprises or consists of, from 5 'to 3', a first universal sequence and a forward nucleotide sequence; alternatively, from 5 'to 3' comprises or consists of a first universal sequence, a nucleotide linker and a forward nucleotide sequence; alternatively, from 5 'to 3' comprises or consists of an additional sequence, a first universal sequence and a forward nucleotide sequence; alternatively, from 5 'to 3' comprises or consists of an additional sequence, a first universal sequence, a nucleotide linker and a forward nucleotide sequence;

(4) the forward nucleotide sequence is located in or constitutes the 3' portion of the forward primer;

(5) the length of the forward nucleotide sequence is 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt and 90-100 nt;

(6) the length of the forward 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 + 150 nt;

(7) the forward primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof;

(8) in the reverse primer, the reverse nucleotide sequence is directly linked to the 3 'end of the second universal sequence, or the reverse nucleotide sequence is linked to the 3' end of the second universal sequence through a nucleotide linker; preferably, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(9) the reverse primer further comprises an additional sequence located 5' to the second universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(10) the reverse primer comprises or consists of, from 5 'to 3', a second universal sequence and a reverse nucleotide sequence; alternatively, from 5 'to 3' comprises or consists of a second universal sequence, a nucleotide linker and an inverted nucleotide sequence; or, from 5 'to 3' comprises or consists of an additional sequence, a second universal sequence and an inverted nucleotide sequence; or, from 5 'to 3', comprises or consists of an additional sequence, a second universal sequence, a nucleotide linker, and an inverted nucleotide sequence;

(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 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt and 90-100 nt;

(13) 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 + 150 nt;

(14) the reverse primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof; and

(15) the second universal sequence is not fully complementary to the complement of the forward primer; for example, at least one nucleotide, e.g., 1-5, 5-10, 10-15, 15-20 or more nucleotides, at the 3' terminus of the second universal sequence is not complementary to the complement of the forward primer;

preferably, the sequence of the first universal primer is shown as SEQ ID NO. 71;

preferably, the sequence of the second universal primer is shown as SEQ ID NO. 70;

preferably, the target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of seq id no:1 and 2; 4 and 5; 7 and 8; 10 and 11; 13 and 14; 16 and 17; 19 and 20; 22 and 23; 25 and 26; 28 and 29; 31 and 32; 34 and 35; 37 and 38; 40 and 41; 43 and 44; 46 and 47; 49 and 50; 52 and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68.

7. The method of any one of claims 1-6, wherein steps (a) - (b) of the method are performed by a protocol comprising the following steps (I) - (VI):

(I) providing a first sample of one or more target nucleic acids derived from the mother, and a second sample containing one or more target nucleic acids derived from the father, the target nucleic acids comprising one or more SNP sites; and, providing a first and a second universal primer, and, for each SNP site, providing a target-specific primer pair; and optionally, providing one detection probe for each SNP site; wherein the first and second universal primer and target-specific primer pairs are as defined in claim 1; the detection probe is as defined in claim 3;

(IV) incubating the product of the previous step under conditions that allow annealing or hybridization of the nucleic acid;

(VI) optionally, repeating steps (III) - (V) one or more times;

(1) in step (III), incubating the product of step (II) at a temperature of 80-105 ℃ to denature the nucleic acid;

(2) incubating the product of step (II) in step (III) for 10-20s, 20-40s, 40-60s, 1-2min, or 2-5 min;

(3) in step (IV), incubating the product of step (III) at a temperature of 35-40 ℃, 40-45 ℃, 45-50 ℃, 50-55 ℃, 55-60 ℃, 60-65 ℃, or 65-70 ℃ to allow annealing or hybridization of the nucleic acid;

(4) incubating the product of step (III) in step (IV) for 10-20s, 20-40s, 40-60s, 1-2min, or 2-5 min;

(5) in step (V), incubating the product of step (IV) at a temperature of 35-40 ℃, 40-45 ℃, 45-50 ℃, 50-55 ℃, 55-60 ℃, 60-65 ℃, 65-70 ℃, 70-75 ℃, 75-80 ℃, 80-85 ℃ to allow nucleic acid extension;

(6) in step (V), incubating the product of step (IV) for 10-20s, 20-40s, 40-60s, 1-2min, 2-5min, 5-10min, 10-20min or 20-30 min;

(7) performing steps (IV) and (V) at the same or different temperatures; and

(8) repeating steps (III) - (V) at least once, such as at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times; preferably, when repeating steps (III) - (V) one or more times, the conditions used for steps (III) - (V) of each cycle are each independently the same or different.

8. A method of detecting the presence or proportion of fetal free DNA in a maternal sample, wherein the method comprises the steps of:

(1) providing a pregnant woman sample to be detected;

(2) detecting the genotypes of one or more SNP sites of a sample from a mother and a sample from a father, and identifying a target SNP site; wherein at the target SNP site, the mother has a homozygous genotype comprising a first allele and the father has a homozygous genotype comprising a second allele, wherein the first allele is different from the second allele;

(3) respectively carrying out quantitative detection on the first allele and the second allele of each target SNP locus in the pregnant woman sample to be detected; then, determining the existence of the nucleic acid of the fetus in the pregnant woman sample to be detected or the proportion of the nucleic acid according to the quantitative detection result of the first allele and the second allele;

preferably, in step (2), the genotype of a certain SNP site can be detected by a mechanism selected from the group consisting of: probe hybridization, primer extension, hybridization connection and specific enzyme digestion;

preferably, in step (2), the SNP site of interest may be identified by a method selected from the group consisting of: sequencing methods (e.g., first-generation sequencing, pyrosequencing, second-generation sequencing), chip methods (e.g., using solid-phase chips, liquid-phase chips capable of detecting SNPs), qPCR-based detection methods (e.g., Taqman probe method), mass spectrometry (e.g., MassARRAY-based iPLEX)^TMGold), chromatography (e.g., denaturing high performance liquid chromatography (hplc), electrophoresis (e.g., SNPshot), melting curve analysis-based assays;

preferably, in step (2), the target SNP site is identified by a multiplex PCR-based melting curve analysis-based assay;

preferably, the SNP site of interest is identified by the method described in any one of claims 1 to 7;

preferably, in the step (3), the first allele and the second allele of each target SNP site in the sample are respectively quantitatively detected by digital PCR;

preferably, step (3) is carried out by the following scheme:

(I-1) the amplification primer set comprises at least one amplification primer (e.g., a pair of amplification primers or more amplification primers) capable of specifically amplifying a nucleic acid molecule containing the SNP site of interest under conditions that allow nucleic acid hybridization or annealing;

(i) the first probe and the second probe are respectively and independently marked with a reporter group and a quencher group, wherein the reporter group can emit signals, and the quencher group can absorb or quench the signals emitted by the reporter group; and, the first probe and the second probe are labeled with different reporter groups (e.g., fluorescent groups), respectively; and is

(III) determining the proportion of fetal free DNA in the pregnant woman sample to be detected according to the quantitative detection result in the step (II);

preferably, the maternal sample is blood (e.g., peripheral blood);

preferably, the first probe specifically anneals or hybridizes to a nucleic acid molecule having a first allele during a digital PCR reaction; and, the second probe specifically anneals or hybridizes to a nucleic acid molecule having a second allele during a digital PCR reaction;

preferably, the first probe does not anneal or hybridize to a nucleic acid molecule having a second allele during a digital PCR reaction; and/or, the second probe does not anneal or hybridize to a nucleic acid molecule having a first allele during a digital PCR reaction;

preferably, the maternal sample is pre-treated prior to step (1);

preferably, the pre-treatment comprises nucleic acid extraction of the sample and/or enrichment of nucleic acids in the sample;

preferably, the nucleic acid enrichment is by a method of nucleic acid concentration and/or nucleic acid amplification;

preferably, the nucleic acid amplification is performed by the amplification primer set.

9. The method of claim 8, wherein the first probe and the second probe each independently have one or more characteristics selected from the group consisting of:

(1) the first and second probes each independently comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., Peptide Nucleic Acids (PNAs) or locked nucleic acids), or any combination thereof;

(2) the lengths of the first probe and the second probe are respectively 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-minus 200nt, 200-minus 300nt, 300-minus 400nt, 400-minus 500nt, 500-minus 600nt, 600-minus 700nt, 700-minus 800nt, 800-minus 900nt, 900-minus 1000 nt;

(3) the first probe and the second probe each independently have a 3' -OH terminus; alternatively, the 3' -end of the probe is blocked; for example, the 3' -end of the detection probe is blocked by adding a chemical moiety (e.g., biotin or alkyl) to the 3' -OH of the last nucleotide of the probe, by removing the 3' -OH of the last nucleotide of the probe, or by replacing the last nucleotide with a dideoxynucleotide;

(4) the first probe and the second probe are each independently a self-quenching probe; for example, the probe is labeled with a reporter group at its 5 'terminus or upstream and a quencher group at its 3' terminus or downstream, or is labeled with a reporter group at its 3 'terminus or downstream and a quencher group at its 5' terminus or upstream; preferably, the reporter and quencher are separated by a distance of 10-80nt or more;

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

(6) the first probe and the second probe are each independently linear or have a hairpin structure;

(7) the first probe and the second probe have different reporter groups; preferably, the first and second probes are degradable by a nucleic acid polymerase (e.g., a DNA polymerase);

(8) the set of probes includes probes having a nucleotide sequence selected from the group consisting of: 73, 74, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163.

10. The method of claim 8 or 9, wherein the primers of the amplification primer set each independently have one or more technical features selected from the group consisting of:

(1) a sequence specific for a target nucleic acid containing a SNP site;

(2) the length of the 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 + 150 nt;

(3) the primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof;

(4) the amplification primer set includes a primer pair having a nucleotide sequence selected from the group consisting of: 72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161.

11. A kit comprising, an identification primer set capable of asymmetrically amplifying a target nucleic acid containing a candidate SNP site;

preferably, the identifying primer set comprises: a first and a second universal primer, and, for each candidate SNP site, providing at least one target-specific primer pair, wherein,

the first universal primer comprises a first universal sequence;

the target-specific primer pair is capable of amplifying using the target nucleic acid as a template to produce a nucleic acid product containing the candidate SNP site, and comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific for the target nucleic acid, and the forward nucleotide sequence is 3' to the first universal sequence; the reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific for the target nucleic acid, and the reverse nucleotide sequence is located 3' of the second universal sequence; and, the second universal sequence is not fully complementary to the complement of the forward primer;

preferably, the kit further comprises one or more detection probes capable of detecting the candidate SNP site, the detection probes comprising a nucleotide sequence specific for the target nucleic acid and capable of annealing or hybridizing to a region of the target nucleic acid containing the candidate SNP site and labeled with a reporter and a quencher, wherein the reporter is capable of emitting a signal and the quencher is capable of absorbing or quenching the signal emitted by the reporter; and wherein the detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement;

preferably, the candidate SNP site has 1 or more features selected from:

(2) the candidate SNP loci are located on different chromosomes;

preferably, the candidate SNP site has 1 or more features selected from:

(1) fst of the candidate SNP locus among different races is less than 0.01;

(2) the candidate SNP loci are located on different chromosomes;

(3) (ii) the allele frequency of the candidate SNP site is between 0.3 and 0.7;

preferably, the candidate SNP sites are SNP sites in the human genome; for example, the target nucleic acid comprises a human genomic SNP site selected from: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs 58210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776, rs7160304, and any combination of the foregoing SNP sites (e.g., any 5, 10, 15, 20, 23 combinations of the foregoing SNP sites);

preferably, the target nucleic acid comprises the following human genomic SNP sites: rs16363, rs1610937, rs5789826, rs1611048, rs2307533, rs112552066, rs 58210, rs2307839, rs149809066, rs66960151, rs34765837, rs68076527, rs10779650, rs4971514, rs6424243, rs12990278, rs2122080, rs98506667, rs774763, rs711725, rs2053911, rs9613776 and rs 7160304;

preferably, the detection probes comprise detection probes having a nucleotide sequence selected from the group consisting of seq id no:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66 and 69;

preferably, the target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of seq id no:1 and 2; 4 and 5; 7 and 8; 10 and 11; 13 and 14; 16 and 17; 19 and 20; 22 and 23; 25 and 26; 28 and 29; 31 and 32; 34 and 35; 37 and 38; 40 and 41; 43 and 44; 46 and 47; 49 and 50; 52 and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68;

preferably, the kit further comprises one or more components selected from the group consisting of: an amplification primer set, a probe set, reagents for performing digital PCR;

preferably, the amplification primer set comprises at least one amplification primer (e.g., a pair of amplification primers or more amplification primers) that is capable of specifically amplifying a nucleic acid molecule containing the SNP site under conditions that allow nucleic acid hybridization or annealing;

preferably, the set of probes comprises a first probe and a second probe; wherein the content of the first and second substances,

(ii) A first probe capable of hybridizing to or annealing (preferably being fully complementary to) a nucleic acid molecule of a first allele containing said SNP site of interest, and a second probe capable of hybridizing to or annealing (preferably being fully complementary to) a nucleic acid molecule of a second allele containing said SNP site of interest; and, the first probe and the second probe are specific for different alleles;

preferably, the set of probes comprises probes having a nucleotide sequence selected from the group consisting of: 73, 74, 78, 79, 82, 83, 86, 87, 90, 91, 94, 95, 98, 99, 102, 103, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 127, 130, 131, 134, 135, 138, 139, 142, 143, 146, 147, 150, 151, 154, 155, 158, 159, 162, 163;

preferably, the amplification primer set comprises a primer pair having a nucleotide sequence selected from the group consisting of seq id no:72 and 73; 77 and 76; 80 and 81; 84 and 85; 88 and 89; 92 and 93; 96 and 97; 100 and 101; 104 and 105; 108 and 109; 112 and 113; 116 and 117; 120 and 121; 124 and 125; 128 and 129; 132 and 133; 136 and 137; 140 and 141; 144 and 145; 148 and 149; 152 and 153; 156 and 157; 160 and 161;

preferably, the reagents for performing digital PCR are selected from the group consisting of one or more components selected from the group consisting of: reagents for preparing a micro-droplet sample, reagents for performing nucleic acid amplification, nucleic acid polymerases, reagents for detecting a micro-droplet sample, or any combination thereof;

preferably, 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 melt curve analysis, or any combination thereof;

preferably, 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 in claims 4 and 5;

preferably, the reagents for performing nucleic acid amplification include, working buffers for enzymes (e.g., nucleic acid polymerases), dNTPs (labeled or unlabeled), water, ions (e.g., Mg) containing²⁺) A single-stranded DNA binding protein, or any combination thereof;

preferably, the kit is used for judging whether a pregnant woman sample contains fetal free DNA or not, or calculating the proportion of fetal free DNA in the pregnant woman sample;

preferably, the digital PCR is selected from the group consisting of a microdroplet digital PCR and a chip-based digital PCR.

12. Use of an identifying primer set as defined in claim 11 for the preparation of a kit for asymmetric amplification of a target nucleic acid molecule or for detection of SNP sites in a maternal-derived sample and a paternal-derived sample;

preferably, the kit further comprises a detection probe as defined in claim 3;

preferably, the kit is for carrying out the method described in any one of claims 1 to 10.

13. Use of an amplification primer set and a probe set as defined in claim 11 for the preparation of a kit for detecting the presence of fetal free DNA or a proportion thereof in a sample of a pregnant woman;

preferably, the kit further comprises reagents for determining the genotype of one or more SNP sites in the genome of the pregnant woman or fetus;

preferably, the kit further comprises an identifying primer set and a detection probe as defined in claim 11;