CN111154841A

CN111154841A - Method and kit for detecting absolute copy number of fetal free DNA in maternal plasma based on digital PCR

Info

Publication number: CN111154841A
Application number: CN202010082009.7A
Authority: CN
Inventors: 沈燕龙; 梁军; 景奉香; 吴东平
Original assignee: Jiangsu Shengji Gene Technology Co ltd
Current assignee: Jiangsu Shengji Gene Technology Co ltd
Priority date: 2020-02-06
Filing date: 2020-02-06
Publication date: 2020-05-15
Anticipated expiration: 2040-02-06
Also published as: CN111154841B

Abstract

The application provides a method for detecting the absolute copy number of free DNA of a fetus in maternal plasma based on digital PCR, which comprises the steps of obtaining total DNA from the peripheral plasma of a pregnant woman, and selecting a design primer and a probe containing a maternal-fetal methylation differential gene locus sequence and an internal reference gene sequence; carrying out methylation sensitive restriction endonuclease digestion treatment; carrying out PCR amplification on a gene locus sequence containing maternal-fetal methylation difference and an internal reference gene sequence in a digital PCR device, detecting a fluorescent signal to obtain the absolute copy number of fetal free DNA in maternal plasma, and eliminating false positive interference of maternal free DNA caused by incomplete enzyme cutting. The method combines a methylation sensitive restriction enzyme-PCR method and a digital PCR platform, detects the absolute copy number of the free DNA of the fetus in maternal plasma by using a maternal-fetal methylation differential gene as a marker, and is favorable for accurately and quickly carrying out early noninvasive prenatal screening or prenatal diagnosis on pregnant women.

Description

Method and kit for detecting absolute copy number of fetal free DNA in maternal plasma based on digital PCR

Technical Field

The application relates to the technical field of digital PCR, in particular to a method and a kit for detecting copy number of fetal free DNA in maternal plasma based on digital PCR.

Background

During non-invasive prenatal screening and prenatal diagnostic examinations of pregnant women, it is often necessary to know the copy number of fetal free DNA in maternal plasma.

Methods for measuring the copy number of fetal free DNA in maternal plasma exist at present, including Y chromosome estimation, SNP-based fetal specific SNP site method, SNP-based deep targeted sequencing, SNP-based low-depth sequencing, nucleosome blotting-based method, and the like. However, these methods in the prior art all have disadvantages, such as being unsuitable for female fetus, requiring very high sequencing depth, having poor accuracy, etc., and it is difficult to accurately obtain the absolute copy number of fetal free DNA in maternal plasma by the existing methods. There is also a large difference between the results of the various known methods, especially when the concentration of fetal free DNA in maternal plasma is low.

There is therefore an urgent need in the art to develop a method for detecting fetal free DNA copy number in maternal plasma for early, safe, noninvasive, accurate, rapid, appropriate noninvasive prenatal screening and prenatal diagnosis of pregnant women.

Disclosure of Invention

In view of the problems in the background art, the present application aims to provide a method and a kit for detecting fetal free DNA copy number in maternal plasma based on digital PCR, so as to accurately and rapidly perform early noninvasive prenatal screening or prenatal diagnosis on pregnant women.

In order to achieve the above object, the present application provides, in a first aspect, a method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR, comprising at least the following steps: (1) obtaining total DNA from the peripheral plasma of the pregnant woman as genome DNA to be detected; (2) selecting a gene locus sequence containing maternal-fetal methylation differences, and designing an amplification primer and a probe primer; (3) selecting an internal reference gene locus sequence containing non-maternal methylation difference, and designing an amplification primer and a probe primer, wherein the internal reference gene contains the same enzyme cutting locus of the maternal methylation difference gene in the step (2); (4) carrying out methylation sensitive restriction endonuclease digestion treatment on the genomic DNA to be detected in the step (1); (5) in a digital PCR device, taking the genome DNA after enzyme digestion as a template, adopting the amplification primer and the probe primer in the step (2) to perform PCR amplification on the gene locus sequence containing maternal-fetal methylation difference, detecting a fluorescent signal, and obtaining the copy number of the maternal-fetal methylation difference gene, namely the absolute copy number of fetal free DNA in maternal plasma; (6) and (3) in a digital PCR device, taking the genome DNA after enzyme digestion as a template, adopting the amplification primer and the probe primer in the step (3) to perform PCR amplification on the internal reference gene locus sequence containing the non-maternal fetal methylation difference, detecting a fluorescent signal, and eliminating false positive interference of maternal free DNA caused by incomplete enzyme digestion.

Furthermore, in the digital PCR device, the positive points obtained by the maternal-fetal methylation difference gene markers are the number of fetal free DNA in the peripheral plasma sample of the pregnant woman; when the methylation differential gene markers of the non-maternal fetus obtain positive points, the digestion treatment is not complete; when the methylation differential gene of the non-maternal fetus is marked, the number of non-positive points is obtained, which indicates that the digestion treatment is complete.

In the above method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR, the method for obtaining total DNA from the peripheral plasma of pregnant women in step (1) includes, but is not limited to, conventional methods such as ethanol precipitation, adsorption column method or magnetic bead method.

The amplification primers in the step (2) comprise a pair of nucleic acid amplification primers, the nucleic acid amplification primers can anneal and flank the amplification site, and the site comprises at least one enzyme cutting site of maternal-fetal methylation difference. The probe primer in step (2) comprises a primer or a probe to detect at least one sequence of maternal-fetal methylation differences.

In addition, the reference gene is a gene comprising non-maternal methylation differences of the same enzyme cutting site in step (2), and includes but is not limited to housekeeping genes, single copy genes and the like. The amplification primers in step (3) include, but are not limited to, a pair of nucleic acid amplification primers capable of annealing and flanking the amplification site comprising at least one enzyme cleavage site as in step (2). The probe primer in step (3) includes, but is not limited to, a primer or a probe to detect at least one sequence that is not a maternal methylation difference.

The division method of the PCR device into droplets used in the embodiments of the present application includes, but is not limited to, droplet generation methods such as "water-in-oil" droplet generation method, "physical division" droplet generation method, "capillary division" droplet generation method, "vibration injection" droplet generation method, and the like.

Preferably, the maternal-fetal methylation differential gene comprises at least one of NKX2-3, EGR, RASSF1A, APC, CASP8, RARB, SCGB3a1, DAB2IP, PTPN6, THY1, TMEEFF2, PYCARD. Wherein, NKX2-3 is a maternal-fetal methylation differential gene discovered for the first time in the application; EGR, RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEEFF2, PYCARD are known maternal-fetal methylation differential genes.

Preferably, the methylation sensitive restriction enzyme comprises at least one of AatII, AciI, AclI, AfeI, AgeI, AscI, AsiSI, AvaI, BceAI, BmgBI, BsaAI, BsiEI, BsiWI, BsmBI, BsrFI, BssHII, BstBI, BstUI, EagI, FauI, FspI, HaeII, HgaI, HhaI, HinP1I, HpaII, Hpy99I, HpyCH4IV, KasI, MluI, NaeI, NarI, NgoMIV, NotI, Nt.BsmAI, Nt.CviPII, PaeR7I, PmlI, PvuI, SasrII, SalI, SsaAI, SnaI, pMI, ZrI, TsgII.

Preferably, the maternal and fetal methylation difference gene is NKX2-3 gene, and primers and probes for amplifying and detecting a site sequence containing NKX2-3 gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 3, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

Preferably, the internal reference gene is an ACTB gene, and primers and probes for amplifying and detecting a locus sequence containing the internal reference ACTB gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 6, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

Preferably, the PCR amplification reaction system consists of: 10 x dPCR Buffer Mix 3.5 mul, upstream and downstream primers 1 mul, probe primer 1 mul, genome DNA template 1.5 mul, adding water to 35 mul; the PCR amplification reaction program comprises the following steps: performing pre-denaturation at 90 ℃ for 10min, performing PCR circulation, performing fluorescence detection on PCR amplification products at 95 ℃ for 20s, 60 ℃ for 40s, and 45 cycles.

The second aspect of the application provides a kit for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR, which at least comprises a primer and a probe for amplifying and detecting a locus sequence containing maternal-fetal methylation differences and a primer and a probe for amplifying and detecting a locus sequence containing non-maternal-fetal methylation differences reference genes.

Preferably, in the kit for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR provided by the present application, the maternal methylation difference gene is NKX2-3 gene, and primers and probes for amplifying and detecting the site sequence containing NKX2-3 gene are: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 3, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group; the internal reference gene is an ACTB gene, and primers and probes for amplifying and detecting a locus sequence containing the internal reference ACTB gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 6, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

A third aspect of the present application provides a kit for detecting RhD blood type genes of a fetus based on digital PCR, comprising at least: primers and probes for amplifying and detecting the NKX2-3 gene sequence; primers and probes for amplifying and detecting an internal reference ACTB gene sequence; primers and probes for amplifying and detecting the sequence of the RhD gene exon 7; primers and probes for amplifying and detecting the RhD gene exon 10 sequence; and (3) primers and probes for amplifying and detecting the intron 4 sequence of the RhD gene.

Preferably, in the kit for detecting RhD blood group genes of a fetus provided by the present application based on digital PCR:

the primers and the probes for amplifying and detecting the NKX2-3 gene locus sequence are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 3, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the site sequence of the internal reference ACTB gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 6, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the sequence of the RhD gene exon 7 are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 9, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the RhD gene exon 10 sequence are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 11, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 12, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the sequence of the intron 4 of the RhD gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 15, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quencher group;

the same reporter group is used for the probes for detecting the sequence of the RhD gene exon 7, the sequence of the RhD gene exon 10 and the sequence of the RhD gene intron 4.

A fourth aspect of the application provides a method for detecting fetal RhD blood type genes and distinguishing maternal RhD based on digital PCR^delA kit of genotypes comprising at least: primers and probes for amplifying and detecting the NKX2-3 gene sequence; primers and probes for amplifying and detecting an internal reference ACTB gene sequence; primers and probes for amplifying and detecting the sequence of the RhD gene exon 7; primers and probes for amplifying and detecting the RhD gene exon 10 sequence; primers and probes for amplifying and detecting the sequence of the intron 4 of the RhD gene; primers and probes for amplifying and detecting RHD1227A gene sequence.

Preferably, the digital PCR-based method for detecting fetal RhD blood type genes and distinguishing maternal RhD is provided in the embodiment of the application^delGenotype kits:

the primers and probes for amplifying and detecting the RHD1227A gene sequence are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 16, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 17, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 18, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quencher group;

The fifth aspect of the present application provides a kit for detecting fetal ABO blood group genes based on digital PCR, comprising at least: primers and probes for amplifying and detecting the NKX2-3 gene locus sequence; primers and probes for amplifying and detecting an internal reference ACTB gene sequence; primers and probes for amplifying and detecting the nt261 site sequence of the ABO gene; and primers and probes for amplifying and detecting the nt796 site sequence contained in the ABO gene.

Preferably, in the kit for detecting the fetal ABO blood group genes based on digital PCR provided in the embodiments of the present application:

the primers and probes for amplifying and detecting the sequence containing nt261 locus of ABO gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 21, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the sequence containing nt796 locus of ABO gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 22, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 23, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 24, wherein the 5 'end of the probe primer is labeled with a reporter group and the 3' end is labeled with a quencher group.

Compared with the prior art, the NKX2-3 gene methylation of the villus and maternal blood cells of early pregnant women is detected by combining the MSRE-PCR method and the dPCR platform, and the result shows that the methylation is high in the villus and low in the maternal blood cells, and the methylation detection rate is 100%. Furthermore, the applicant researches the plasma free DNA of the pregnant women, and the high-methylation NKX2-3 gene of the fetus is amplified from the maternal plasma for the first time by using the MSRE-PCR method, and the experimental system is stable and the experimental method is simple and reliable. In order to avoid the problem of false positive caused by incomplete enzyme digestion of plasma DNA, the applicant further optimizes the detection scheme and simultaneously amplifies the enzyme digestion internal reference ACTB gene to ensure the possibility of completely eliminating the false positive by enzyme digestion.

Compared with a fluorescence quantitative PCR method, the detection method provided by the application has extremely strong absolute quantitative capability, can directly carry out the quantification of the target molecules on the sample without depending on a standard curve and a standard substance, and has extremely high sensitivity, stability and repeatability. Compared with the NGS sequencing method, the detection method provided by the application has the advantages of low cost, short detection period, high sensitivity and convenience for clinical popularization.

In addition, the applicant does not have NKX2-3 target bands by researching blood cell and plasma DNA of a control group of healthy women who are not pregnant and women who are 3d postnatal, and the result indicates that hypermethylated fetal NKX2-3 genes exist in the plasma of pregnant women only, and fetal DNA is cleared quickly after delivery and is not influenced by previous pregnancy. The applicant further studied these samples, including the widely recognized comparison between RASSF1A gene located on chromosome 3 and DMR located in the ERG gene promoter region located on chromosome 21, with consistent effects, and thus further confirmed the important value of NKX2-3 gene in the judgment of this pregnancy.

Drawings

FIG. 1 is an experimental flow diagram according to an embodiment of the present application;

FIG. 2 is a schematic diagram of a Methylation Sensitive Restriction Enzyme (MSRE) digestion process according to an embodiment of the present application;

FIG. 3 is a schematic diagram of HpaII enzyme digestion detection according to an embodiment of the present application;

FIG. 4 is a schematic diagram of MspI enzymatic cleavage detection according to an embodiment of the present application;

FIG. 5 shows the detection of fetal RhD blood group genes and the discrimination of maternal RhD based on fetal free DNA concentration in maternal plasma according to embodiments of the present application^delDetection schematic of the method of genotype;

fig. 6 is a schematic detection diagram of a method for detecting a fetal ABO blood group gene based on a fetal free DNA concentration in maternal plasma according to an embodiment of the present application.

Detailed Description

The method for detecting fetal free DNA copy number in maternal plasma based on digital PCR according to the present application and the use thereof are described in detail below.

In some embodiments of the present application, there is provided a method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR, comprising at least the steps of: (1) obtaining total DNA from the peripheral plasma of the pregnant woman as genome DNA to be detected; (2) selecting a gene locus sequence containing maternal-fetal methylation differences, and designing an amplification primer and a probe primer; (3) selecting an internal reference gene locus sequence containing non-maternal methylation difference, and designing an amplification primer and a probe primer, wherein the internal reference gene contains the same enzyme cutting locus of the maternal methylation difference gene in the step (2); (3) carrying out methylation sensitive restriction endonuclease digestion treatment on the genomic DNA to be detected in the step (1); (4) in a digital PCR device, taking the genome DNA after enzyme digestion as a template, adopting the amplification primer and the probe primer in the step (2) to perform PCR amplification on the gene locus sequence containing maternal-fetal methylation difference, detecting a fluorescent signal, and obtaining the copy number of the maternal-fetal methylation difference gene, namely the absolute copy number of fetal free DNA in maternal plasma; (5) and (3) in a digital PCR device, taking the genome DNA after enzyme digestion as a template, adopting the amplification primer and the probe primer in the step (3) to perform PCR amplification on the internal reference gene locus sequence containing the non-maternal fetal methylation difference, detecting a fluorescent signal, and eliminating false positive interference of maternal free DNA caused by incomplete enzyme digestion. The experimental flow chart of the embodiment of the application is shown in the attached figure 1.

According to the method of the present invention, fetal DNA in a maternal plasma DNA sample is obtained by digesting plasma DNA with one or more methylation sensitive restriction enzymes based on maternal-fetal methylation difference genes. For example, plasma DNA is digested with an enzyme that cleaves only at methylated DNA recognition sites, or with an enzyme that cleaves only at unmethylated DNA recognition sites. Digestion with enzymes that cleave only unmethylated DNA recognition sites yields methylated DNA sequences in fetal DNA but not unmethylated DNA sequences in maternal DNA. Alternatively, digestion with an enzyme that cleaves only methylated DNA recognition sites can result in unmethylated DNA sequences in fetal DNA but not methylated DNA sequences in maternal DNA. Any enzyme that selectively cleaves maternal DNA but not the corresponding fetal DNA is suitable for use in the present invention.

In some embodiments of the present invention, in the method for detecting the absolute copy number of fetal free DNA in maternal plasma, the method for obtaining total DNA from the peripheral plasma of pregnant women in step (1) includes, but is not limited to, conventional methods such as ethanol precipitation, adsorption column method, or magnetic bead method.

In some embodiments of the invention, the maternal-fetal methylation differential gene comprises at least one of NKX2-3, EGR, RASSF1A, APC, CASP8, RARB, SCGB3a1, DAB2IP, PTPN6, THY1, TMEEFF2, PYCARD. Wherein, NKX2-3 is a maternal-fetal methylation differential gene disclosed for the first time in the application; EGR, RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEEFF2, PYCARD are known maternal-fetal methylation differential genes.

In some embodiments of the invention, the methylation sensitive restriction enzyme comprises at least one of AatII, AciI, AclI, AfeI, AgeI, AscI, AsiSI, AvaI, BceAI, BmgBI, BsaAI, BsiEI, BsiWI, BsmBI, BsrFI, BssHII, BstBI, BstUI, EagI, FauI, FspI, HaeII, HgaI, HhaI, HinP1I, HpaII, Hpy99I, HpyCH4IV, KasI, MluI, NaeI, NarI, NgoMIV, NotI, Nt.BsmAI, Nt.VicPII, PaeR7I, PmlI, PvuI, RsrII, SmaII, SalI, ScgAI, Sagri I, SnaI, SaaBI, TsI.

In some embodiments of the invention, the maternal-fetal methylation difference gene is NKX2-3 gene, and the primers and probes for amplifying and detecting the sequence comprising the NKX2-3 gene site are: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 3, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

In some embodiments of the invention, the reference gene is ACTB gene, and the primers and probes for amplifying and detecting the locus sequence comprising the reference ACTB gene are: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 6, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

In each probe primer provided in the present application, the reporter group used may be any one of FAM, HEX, TET, ROX, CY3, CY5, CY5.5, VIC, JOE, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor647, and Alexa Fluor 750; the quencher used may be any of TAMRA, Dabcyl, BHQ-1, BHQ-2, BHQ-3, MGB, Eclipse, etc.

In some embodiments of the present invention, the PCR amplification reaction system consists of: 10 XdPCRBuffer Mix 3.5. mu.l, upstream and downstream primers 1. mu.l each, probe primer 1. mu.l, genomic DNA template 1.5. mu.l, water to 35. mu.l; the PCR amplification reaction program comprises the following steps: performing pre-denaturation at 90 ℃ for 10min, performing PCR circulation, performing fluorescence detection on PCR amplification products at 95 ℃ for 20s, 60 ℃ for 40s, and 45 cycles.

Fig. 2 is a schematic diagram of a Methylation Sensitive Restriction Enzyme (MSRE) digestion process according to an embodiment of the present application. As shown in FIG. 2, HpaII is a methylation-sensitive restriction enzyme, and when the 2 nd C in the CCGG sequence is methylated, HpaII cannot cut the sequence, and the target band can be amplified by the designed primer. MspI is a methylation insensitive enzyme, and can cleave the CCGG sequence no matter whether the 2 nd C in the CCGG sequence is methylated or not, and can not amplify a target band, so that the MspI can be used as a control enzyme for HpaII enzyme digestion amplification.

FIG. 3 is a schematic diagram showing the HpaII cleavage assay of NKX2-3 gene and ACTB gene in fetus and mother. As shown in FIG. 3, the NKX2-3 gene of fetus contains methylation site, and can not be cut by HpaII enzyme, the target band can be amplified according to the designed primer, and the probe emits light; the parent NKX2-3 gene was unmethylated and cut by HpaII, the desired band could not be amplified, and the probe did not emit light. The ACTB genes of the fetus and the mother are not methylated and are cut by HpaII enzyme, the target band cannot be amplified, and the probe does not emit light.

FIG. 4 shows MspI enzymatic cleavage detection of NKX2-3 gene and ACTB gene in fetus and mother. As shown in FIG. 4, fetal NKX2-3 gene contains methylation sites and is cleaved by MspI; the parent NKX2-3 gene is not methylated and is cut by MspI, the target band cannot be amplified, and the probe does not emit light. The ACTB genes of the fetus and the mother are not methylated, and are cut by MspI enzyme, the target band cannot be amplified, and the probe does not emit light.

Based on the detection principle, the other embodiments of the application provide a kit for detecting the absolute copy number of the fetal free DNA in maternal plasma based on digital PCR, which at least comprises primers and probes for amplifying and detecting the locus sequence containing maternal-fetal methylation differences and primers and probes for amplifying and detecting the locus sequence containing non-maternal-fetal methylation differences reference genes.

In some embodiments of the present application, the kit for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR is provided, wherein the maternal methylation difference gene is NKX2-3 gene, and primers and probes for amplifying and detecting the sequence comprising the NKX2-3 gene site are: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 3, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group; the internal reference gene is an ACTB gene, and primers and probes for amplifying and detecting a locus sequence containing the internal reference ACTB gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 6, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

Further embodiments of the present application provide specific applications of the above-mentioned method for detecting copy number of fetal free DNA in maternal plasma based on digital PCR in non-invasive prenatal screening, for example, detection of fetal RhD blood group genes based on fetal free DNA concentration in maternal plasma and identification of maternal RhD^delGenotype, and detection of fetal ABO blood group genes based on fetal free DNA concentration in maternal plasma.

In some embodiments of the present application, there is provided a kit for detecting RhD blood group genes of a fetus based on digital PCR, comprising at least: primers and probes for amplifying and detecting the NKX2-3 gene sequence; primers and probes for amplifying and detecting an internal reference ACTB gene sequence; primers and probes for amplifying and detecting the sequence of the RhD gene exon 7; primers and probes for amplifying and detecting the RhD gene exon 10 sequence; and (3) primers and probes for amplifying and detecting the intron 4 sequence of the RhD gene.

In the kit for detecting RhD blood group genes of a fetus provided in some embodiments of the present application based on digital PCR:

In some embodiments of the present application, a method for detecting fetal RhD blood group genes and discriminating maternal RhD based on digital PCR is provided^delA kit of genotypes comprising at least: primers and probes for amplifying and detecting the NKX2-3 gene sequence; primers and probes for amplifying and detecting an internal reference ACTB gene sequence; primers and probes for amplifying and detecting the sequence of the RhD gene exon 7; primers and probes for amplifying and detecting the RhD gene exon 10 sequence; primers and probes for amplifying and detecting the sequence of the intron 4 of the RhD gene; primers and probes for amplifying and detecting RHD1227A gene sequence.

The digital PCR-based detection of fetal RhD blood group genes and the discrimination of maternal RhD provided in some embodiments of the present application^delGenotype kits:

In some embodiments of the present application, there is also provided a kit for detecting a fetal ABO blood group gene based on digital PCR, comprising at least: primers and probes for amplifying and detecting the NKX2-3 gene locus sequence; primers and probes for amplifying and detecting an internal reference ACTB gene sequence; primers and probes for amplifying and detecting the nt261 site sequence of the ABO gene; and primers and probes for amplifying and detecting the nt796 site sequence contained in the ABO gene.

In the kit for detecting the ABO blood group genes of a fetus based on digital PCR provided by some embodiments of the present application:

The method and kit for detecting the fetal free DNA concentration in maternal plasma by using the methylation difference of NKX2-3 gene between mothers and fetuses as a marker, the method and kit for detecting the fetal RhD blood type gene based on the fetal free DNA concentration in maternal plasma, and the method and kit for distinguishing the maternal RhD are further described in detail in the following with reference to specific test examples 1 to 4^delA method and a kit for genotype, and a method and a kit for detecting ABO blood group genes of a fetus based on the concentration of fetal free DNA in maternal plasma.

It is noted that, in each example, the 5 'end of the probe primer is labeled with a reporter group, and the 3' end is labeled with a quencher group, wherein the reporter group used may be any one of FAM, HEX, TET, ROX, CY3, CY5, CY5.5, VIC, JOE, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor647, Alexa Fluor 750, and the like; the quencher used may be any of TAMRA, Dabcyl, BHQ-1, BHQ-2, BHQ-3, MGB, Eclipse, etc. In order to avoid unnecessary repetition, the present application will not be described in detail in the following embodiments.

Test example 1

Materials (I) and (II)

Experimental samples: 10mL of peripheral blood (pregnant 7-39W) of healthy pregnant women are matched with fetal villi, amniotic fluid or placenta for 4 cases. All pregnant woman peripheral blood was collected before the induced abortion, amniocentesis and cesarean section.

Experimental apparatus and consumables: the invention uses a digital PCR system Biodigital HUA of Shanghai small chelonian science and technology Limited company, which comprises a microdroplet preparation instrument, a chip amplification instrument and a biochip reading instrument. The invention uses the digital PCR microdroplet preparation chip and the universal kit of Shanghai small chelonian science and technology Limited company.

Second, blood cell, plasma and villus tissue DNA extraction

(1) Extracting 3ml of fasting vein anticoagulation blood of experimental group women, centrifuging for 10min at 1600g within 3h, taking 16000g of supernatant, centrifuging for 10min, and extracting plasma DNA;

(2) extracting blood cell DNA by using the lower cell layer;

(3) washing the pregnant product obtained by negative pressure induced abortion with 4 deg.C physiological saline for 3 times, separating villus under microscope, removing decidua tissue and other connective tissue, and extracting villus tissue DNA;

(4) the extracted DNA is kept at-20 ℃ for later use.

DNA extraction was performed using the QIAamp DNA Mini and Blood Mini Kit (Qiagen, Germany), HpaII and MspI purchased from New England Biolabs (NEB, USA), and primers synthesized by Shanghai Biolabs.

Digestion with HpaII methylation sensitive restriction enzyme and MspI restriction enzyme

Mixing 1 μ g each of the above extracted leukocyte, villus and plasma DNA with HpaII 50U and MspI50U to form 50 μ l reaction system, and performing enzyme digestion at 37 deg.C for 1-2h to digest DNA. Heat-inactivating at 80 deg.C for 20 min.

PCR amplification of DNA from blood cells, villus genome and plasma

(1) And (3) respectively detecting the NG-016854.1 (4764) -4833) sequence of the NKX2-3 gene and the NG-007992.1 (5855) -5953) sequence of the ACTB gene by using genomic DNA before and after enzyme digestion as templates for amplification.

NKX2-3 amplification primer:

an upstream primer: 5'-CGCGGGAATCAATGGTTCAG-3' (SEQ ID NO: 1),

a downstream primer: 5'-AGAGTCCTGAAGTCCGAGACG-3' (SEQ ID NO: 2),

and (3) probe primer: 5'-ATCTGTGCAGCCTCGCC-3' (SEQ ID NO: 3).

ACTB amplification primers:

an upstream primer: 5'-CCTGGCGGCCTAAGGACT-3' (SEQ ID NO: 4),

a downstream primer: 5'-ATCATCCATGGTGAGCTGCG-3' (SEQ ID NO:);

and (3) probe primer: 5'-GGCGACCTCGGCTCACA-3' (SEQ ID NO: 6).

(2) And (3) PCR reaction system:

① blood cells and villi, 10 XdPCR Buffer Mix 3.5 mul, upstream and downstream primers 1 mul, probe primer 1 mul, enzyme digested plasma genome DNA 1.5 mul, adding water to 35 mul to form a reaction system;

② plasma, 10 XdPCR Buffer Mix 3.5. mu.l, upstream and downstream primers 1. mu.l each, probe primer 1. mu.l, enzyme digested plasma genome DNA 1.5. mu.l, adding water to 35. mu.l to form a reaction system;

(3) amplification conditions: pre-denaturation at 90 ℃ for 10min, and PCR circulation at 95 ℃ for 20s, 60 ℃ for 40s, and 45 cycles. And performing dPCR amplification and detection on the PCR amplification product.

The methylation identification results for each experimental group in this example are shown in table 1:

TABLE 1 identification of methylation in the experimental groups

In the embodiment, the methylation of NKX2-3 genes of villi and maternal blood cells of early pregnant women is detected by combining the MSRE-PCR method and the dPCR platform, and the result shows that the methylation is hypermethylated in the villi and hypomethylated in the maternal blood cells, and the methylation detection rate is 100%. Further research is carried out on the plasma free DNA of the pregnant women, the fetal hypermethylated NKX2-3 gene is amplified from maternal plasma for the first time by using the MSRE-PCR method, the experimental system is stable, and the experimental method is simple and reliable. In order to avoid the problem of false positive caused by incomplete enzyme digestion of plasma DNA, the ACTB gene in enzyme digestion is amplified simultaneously so as to ensure the possibility that false positive is completely eliminated by enzyme digestion.

Test example 2

Materials (I) and (II)

Experimental samples: 10mL of peripheral blood (pregnant 7-39W) of 4 healthy pregnant women.

Control samples: the peripheral blood of 4 women is 10mL determined by physical examination to be healthy women who are not pregnant, and the peripheral blood of 3 women who are 3d after birth is 10mL

Second, blood cell and plasma DNA extraction

(1) Extracting 3ml of fasting vein anticoagulation blood of experimental group and control group women, centrifuging for 10min at 1600g within 3h, taking 16000g of supernatant, centrifuging for 10min, and extracting plasma DNA;

(2) extracting blood cell DNA by using the lower cell layer;

(3) the extracted DNA is kept at-20 ℃ for later use.

Digestion with HpaII methylation sensitive restriction enzyme

Mixing 1 μ g of the above extracted leukocyte and plasma DNA with HpaII 50U to form 50 μ l reaction system, and performing enzyme digestion at 37 deg.C for 1-2h to digest DNA. Heat-inactivating at 80 deg.C for 20 min.

Fourth, PCR amplification of blood cell and plasma DNA

(1) The NKX2-3 gene NG-016854.1 (4764) -4833) sequence, the ERG gene NG-029732.1 (159731) -159808) sequence, the RASSF1A gene and the ACTB gene NG-007992.1 (5855) -5953) sequence are respectively detected by using genomic DNA before and after enzyme digestion as templates for amplification.

NKX2-3 amplification primer:

an upstream primer: 5'-CGCGGGAATCAATGGTTCAG-3' (SEQ ID NO: 1),

a downstream primer: 5'-AGAGTCCTGAAGTCCGAGACG-3' (SEQ ID NO: 2),

and (3) probe primer: 5'-ATCTGTGCAGCCTCGCC-3' (SEQ ID NO: 3).

ERG amplification primers:

upstream: 5'-CTGGGACCGGAATGTACGAC-3' (SEQ ID NO: 25),

downstream: 5'-ACCTTTGCATGTGAGAGGCAT-3' (SEQ ID NO: 26),

and (3) probe primer: 5'-GCCTCCTCCCATCTAGTTTGC-3' (SEQ ID NO: 27).

RASSF1A amplification primers:

upstream: 5'-CTGAGCTCATTGAGCTG-3' (SEQ ID NO: 28),

downstream: 5'-CCAGATGAAGTCGCCACAGA-3' (SEQ ID NO: 29),

and (3) probe primer: 5'-CACGCACACGTGGTGC-3' (SEQ ID NO: 30).

ACTB amplification primers:

an upstream primer: 5'-CCTGGCGGCCTAAGGACT-3' (SEQ ID NO: 4),

a downstream primer: 5'-ATCATCCATGGTGAGCTGCG-3' (SEQ ID NO: 5);

and (3) probe primer: 5'-GGCGACCTCGGCTCACA-3' (SEQ ID NO: 6).

Fifthly, PCR reaction system:

① blood cells, 10 XdPCR Buffer Mix 3.5. mu.l, upstream and downstream primers 1. mu.l each, probe primer 1. mu.l, enzyme digested plasma genome DNA 1.5. mu.l, adding water to 35. mu.l to form a reaction system;

sixthly, amplification conditions are as follows: pre-denaturation at 90 ℃ for 10min, and PCR circulation at 95 ℃ for 20s, 60 ℃ for 40s, and 45 cycles. And carrying out dPCR detection on the PCR amplification product.

The results of the methylation identification of blood cells and plasma in the experimental group and the control group in this example are shown in Table 2:

TABLE 2 identification of methylation of blood cells and plasma in the experimental and control groups

In this example, the study on blood cell and plasma DNA of the control group of healthy women who were not pregnant and women who were 3d postnatal was carried out, and the result showed that no NKX2-3 purpose band appeared, indicating that only the hypermethylated fetal NKX2-3 gene existed in the plasma of pregnant women, and that fetal DNA was cleared quickly postnatally without being affected by the previous pregnancy. Further research on the samples includes comparison of widely recognized methylation difference regions (DMR) located in the RASSF1A gene of chromosome 3 and the ERG gene promoter region of chromosome 21, the effects are consistent, and the important value of the NKX2-3 gene in judging the pregnancy is further confirmed.

Test example 3

Method for detecting RhD blood type gene of fetus and distinguishing RhD of mother based on concentration of free DNA of fetus in maternal plasma^delA method of genotyping comprising the steps of:

materials (I) and (II)

Experimental samples: and the peripheral blood (pregnancy 12-40W) of 4 healthy pregnant women which are diagnosed with RhD negative before delivery and identified as RhD positive after delivery is 10 mL. 2 cases of healthy pregnant women who are diagnosed with RhD negative before delivery and identified the fetus blood type as RhD negative after delivery as 10mL of peripheral blood (pregnancy 10-27W). 2 cases of prenatal diagnosis of RhD by absorption and diffusion technology^delAnd the blood type of the fetus is identified as 10mL of peripheral blood (pregnancy 12-30W) of a healthy pregnant woman which is RhD negative after delivery. 2 cases of prenatal diagnosis of RhD by absorption and diffusion technology^delAnd the blood type of the fetus is identified as 10mL of peripheral blood (pregnant 10-32W) of a healthy pregnant woman with positive RhD after delivery.

Control samples: the 4 women were examined by body and determined to be RhD negative non-pregnant healthy women with 10mL of peripheral blood.

Second, plasma DNA extraction

(2) the extracted DNA is kept at-20 ℃ for later use.

Digestion with HpaII methylation sensitive restriction enzyme

And (3) uniformly mixing 1 mu g of the extracted plasma DNA with HpaII 50U to form a 50 mu l reaction system, and carrying out enzyme digestion at 37 ℃ for 1-2h to digest the DNA so as to achieve complete digestion. Heat-inactivating at 80 deg.C for 20 min.

Fourth, PCR amplification of plasma DNA

The sequence of NKX2-3 gene NG _016854.1 (4764) -4833) and the sequence of ACTB gene NG _007992.1 (5855) -5953) are respectively detected by using genomic DNA before and after enzyme digestion as templates for amplification. The sequence of the RhD gene NC-000001.11: 25272393-25330445.

NKX2-3 amplification detection primer:

an upstream primer: 5'-CGCGGGAATCAATGGTTCAG-3' (SEQ ID NO: 1),

a downstream primer: 5'-AGAGTCCTGAAGTCCGAGACG-3' (SEQ ID NO: 2),

and (3) probe primer: 5'-ATCTGTGCAGCCTCGCC-3' (SEQ ID NO: 3).

ACTB amplification primers:

an upstream primer: 5'-CCTGGCGGCCTAAGGACT-3' (SEQ ID NO: 4),

a downstream primer: 5'-ATCATCCATGGTGAGCTGCG-3' (SEQ ID NO: 5);

and (3) probe primer: 5'-GGCGACCTCGGCTCACA-3' (SEQ ID NO: 6).

RhD exon 7 amplification primers:

an upstream primer: 5'-CTCCATCATGGGCTACAA-3' (SEQ ID NO: 7),

a downstream primer: 5'-CCGGCTCCGACGGTATC-3' (SEQ ID NO: 8),

and (3) probe primer: 5'-AGCAGCACAATGTAGATGATCTCTCCA-3' (SEQ ID NO: 9).

RhD exon 10 amplification primer:

an upstream primer: 5'-CCTCTCACTGTTGCCTGCATT-3' (SEQ ID NO: 10), downstream primer: 5'-AGTGCCTGCGCGAACATT-3' (SEQ ID NO: 11),

and (3) probe primer: 5'-TACGTGAGAAACGCTCATGACAGCAAAGTCT-3' (SEQ ID NO: 12).

RhD intron 4 amplification primers:

an upstream primer: 5'-GGTTGAAATCTGCATACCCCA-3' (SEQ ID NO: 13),

a downstream primer: 5'-ATGAATCATTTCTTTGAGTAGTGTTTGC-3' (SEQ ID NO: 14),

and (3) probe primer: 5'-CTCCTGAACCTGCTCTGTGAAGTGCTTAATTC-3' (SEQ ID NO: 15).

RHD1227A amplification primers:

an upstream primer: 5'-GATGACCAAGTTTTCTGGAAA-3' (SEQ ID NO: 16),

a downstream primer: 5'-CATAAACAGCAAGTCAACATATATACT-3' (SEQ ID NO: 17),

and (3) probe primer: TGATAGATTTTGAGTGCATGAAC-3' (SEQ ID NO: 18).

Fifthly, PCR reaction system: 10 x dPCR Buffer Mix 3.5 mul, upstream and downstream primers 1 mul, probe primer 1 mul, enzyme digested plasma genome DNA 1.5 mul, adding water to 35 mul to form a reaction system;

Seventhly, interpretation of results:

the principle of displaying fluorescence signals of the digital PCR instrument is shown in the attached figure 5, and the following results are interpreted according to the fluorescence signals:

(1) ACTB has no positive point, and the positive point shown by NKX2-3 is the absolute copy number of fetal free DNA in the sample.

(2) And if the ACTB has a positive point, the enzyme digestion is not complete in the experiment, false positive interference of the maternal free DNA exists in each amplification system, and the experiment needs to be carried out by enzyme digestion again.

(3) The positive points of the RhD genes E7, E10 and I4 are close to the ratio of the number of the positive points (the absolute copy number of free fetal DNA) of NKX2-3 to the number of the positive points (the absolute copy number of free fetal DNA) of the RhD genes, which indicates that the tested samples of the RhD genes E7, E10 and I4 are all from free fetal DNA, and the blood type of the fetus is RhD positive.

(4) The positive points of the RhD genes E7, E10 and I4 are between 0 and 1.5 in the ratio of the number of the positive points to the number of the positive points (the absolute copy number of the fetal free DNA) of NKX2-3, which indicates that the tested samples of the RhD genes E7, E10 and I4 are all derived from the fetal free DNA, but partial genes are deleted. Fetal blood type is RhD negative.

(5) The positive points of the RhD genes E7, E10 and I4 are far greater than 1.5 in the ratio of the number of the positive points (the absolute copy number of fetal free DNA) to the number of the positive points of NKX2-3, which indicates that the tested samples of the RhD genes E7, E10 and I4 are partially derived from maternal free DNA. But the pregnant woman is negative after RhD blood type detection. The fact shows that the parent RhD gene is partially deleted and needs to be further detected.

(6) When the above (5) occurs, there is a positive point in RHD1227A indicating that the blood type of mother is RHD^delTest example 4

A method for detecting ABO blood group genes of a fetus based on the concentration of free DNA of the fetus in maternal plasma comprises the following steps:

materials (I) and (II)

Experimental samples: the blood type of 3 cases of fetuses is identified as type A healthy O type blood pregnant woman peripheral blood (pregnancy 13-40W) 10mL after delivery. The blood type of 3 cases of fetuses is identified as 10mL of healthy O-type blood pregnant woman peripheral blood (pregnancy 11-27W) of type B after delivery. And 3, the blood type of the fetus is identified as 10mL of healthy O-type blood pregnant woman peripheral blood (pregnancy 12-30W) of the O-type after delivery.

Control samples: the 4 women were examined by physical examination to determine 10mL of peripheral blood from a group O infertile healthy woman.

Second, plasma DNA extraction

(2) the extracted DNA is kept at-20 ℃ for later use.

Digestion with HpaII methylation sensitive restriction enzyme

Fourth, PCR amplification of plasma DNA

Amplification detection of NKX2-3 gene NG _016854.1 by using genomic DNA before and after enzyme digestion as templates

(4764-4833) sequence, ACTB gene NG-007992.1 (5855-5953) sequence, ABO gene NC-000001.11: 25272393-25330445 sequence.

NKX2-3 amplification primer:

an upstream primer: 5'-CGCGGGAATCAATGGTTCAG-3' (SEQ ID NO: 1),

a downstream primer: 5'-AGAGTCCTGAAGTCCGAGACG-3' (SEQ ID NO: 2),

and (3) probe primer: 5'-ATCTGTGCAGCCTCGCC-3' (SEQ ID NO: 3).

ACTB amplification primers:

an upstream primer: 5'-CCTGGCGGCCTAAGGACT-3' (SEQ ID NO: 4),

a downstream primer: 5'-ATCATCCATGGTGAGCTGCG-3' (SEQ ID NO: 5);

and (3) probe primer: 5'-GGCGACCTCGGCTCACA-3' (SEQ ID NO: 6).

nt261 amplification primer:

an upstream primer: 5'-GCCTCTCTCCATGTGCAGTA-3' (SEQ ID NO: 19),

a downstream primer: ATGTGCCCTCCCAGACAATG-3' (SEQ ID NO: 20),

and (3) probe primer: 5'-GGAAGGATGTCCTCGTGGTgA-3' (SEQ ID NO: 21).

nt796 amplification primer:

an upstream primer: 5'-CAAGGACGAGGGCGATTTCT-3' (SEQ ID NO: 22),

a downstream primer: CATCATGGCCTGGTGGCA-3' (SEQ ID NO: 23),

and (3) probe primer: 5'-CGAAGAACCCCCCCATG-3' (SEQ ID NO: 24).

Fifthly, PCR reaction system:

10 x dPCR Buffer Mix 3.5 mul, upstream and downstream primers 1 mul, probe primer 1 mul, enzyme digested plasma genome DNA 1.5 mul, adding water to 35 mul to form a reaction system;

sixthly, amplification conditions are as follows:

pre-denaturation at 90 ℃ for 10min, and PCR circulation at 95 ℃ for 20s, 60 ℃ for 40s, and 45 cycles. And carrying out dPCR detection on the PCR amplification product.

Seventhly, interpretation of results:

the principle of displaying fluorescence signals of the digital PCR instrument is shown in the attached figure 6, and the following results are interpreted according to the fluorescence signals:

(3) nt261 has positive points, and the ratio of the number of the positive points to the number of positive points (absolute copy number of free DNA in fetus) of NKX2-3 is close to 1, which indicates that the blood type of the fetus is type A or type B.

(4) nt261 has no positive point, which indicates that the fetus blood type is O type

(5) nt796 has positive points, and the ratio of the number of the positive points to the number of positive points (the absolute copy number of free DNA of the fetus) of NKX2-3 is close to 1, which indicates that the blood type of the fetus is B type.

(6) nt796 has no positive point, which indicates that the blood type of the fetus is type A.

Variations and modifications to the above-described embodiments may occur to those skilled in the art based upon the disclosure and teachings of the above specification. Therefore, the present application is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present application should fall within the scope of the claims of the present application. In addition, although specific terms are used herein, they are used in a descriptive sense only and not for purposes of limitation.

SEQUENCE LISTING

<110> Jiangsu Shengji Gene science and technology Co., Ltd

<120> method and kit for detecting absolute copy number of fetal free DNA in maternal plasma based on digital PCR

<130>191937CN-CH-I

<160>30

<170>PatentIn version 3.3

<210>1

<211>20

<212>DNA

<213> Artificial sequence

<400>1

cgcgggaatc aatggttcag 20

<210>2

<211>21

<212>DNA

<213> Artificial sequence

<400>2

agagtcctga agtccgagac g 21

<210>3

<211>17

<212>DNA

<213> Artificial sequence

<400>3

atctgtgcag cctcgcc 17

<210>4

<211>18

<212>DNA

<213> Artificial sequence

<400>4

cctggcggcc taaggact 18

<210>5

<211>20

<212>DNA

<213> Artificial sequence

<400>5

atcatccatg gtgagctgcg 20

<210>6

<211>17

<212>DNA

<213> Artificial sequence

<400>6

ggcgacctcg gctcaca 17

<210>7

<211>18

<212>DNA

<213> Artificial sequence

<400>7

ctccatcatg ggctacaa 18

<210>8

<211>17

<212>DNA

<213> Artificial sequence

<400>8

ccggctccga cggtatc 17

<210>9

<211>27

<212>DNA

<213> Artificial sequence

<400>9

agcagcacaa tgtagatgat ctctcca 27

<210>10

<211>21

<212>DNA

<213> Artificial sequence

<400>10

cctctcactg ttgcctgcat t 21

<210>11

<211>18

<212>DNA

<213> Artificial sequence

<400>11

agtgcctgcg cgaacatt 18

<210>12

<211>31

<212>DNA

<213> Artificial sequence

<400>12

tacgtgagaa acgctcatga cagcaaagtc t 31

<210>13

<211>21

<212>DNA

<213> Artificial sequence

<400>13

ggttgaaatc tgcatacccc a 21

<210>14

<211>28

<212>DNA

<213> Artificial sequence

<400>14

atgaatcatt tctttgagta gtgtttgc 28

<210>15

<211>32

<212>DNA

<213> Artificial sequence

<400>15

ctcctgaacc tgctctgtga agtgcttaat tc 32

<210>16

<211>21

<212>DNA

<213> Artificial sequence

<400>16

gatgaccaag ttttctggaa a 21

<210>17

<211>27

<212>DNA

<213> Artificial sequence

<400>17

cataaacagc aagtcaacat atatact 27

<210>18

<211>23

<212>DNA

<213> Artificial sequence

<400>18

tgatagattt tgagtgcatg aac 23

<210>19

<211>20

<212>DNA

<213> Artificial sequence

<400>19

gcctctctcc atgtgcagta 20

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<400>20

atgtgccctc ccagacaatg 20

<210>21

<211>21

<212>DNA

<213> Artificial sequence

<400>21

ggaaggatgt cctcgtggtg a 21

<210>22

<211>20

<212>DNA

<213> Artificial sequence

<400>22

caaggacgag ggcgatttct 20

<210>23

<211>18

<212>DNA

<213> Artificial sequence

<400>23

catcatggcc tggtggca 18

<210>24

<211>17

<212>DNA

<213> Artificial sequence

<400>24

cgaagaaccc ccccatg 17

<210>25

<211>20

<212>DNA

<213> Artificial sequence

<400>25

ctgggaccgg aatgtacgac 20

<210>26

<211>21

<212>DNA

<213> Artificial sequence

<400>26

acctttgcat gtgagaggca t 21

<210>27

<211>21

<212>DNA

<213> Artificial sequence

<400>27

gcctcctccc atctagtttg c 21

<210>28

<211>17

<212>DNA

<213> Artificial sequence

<400>28

ctgagctcat tgagctg 17

<210>29

<211>20

<212>DNA

<213> Artificial sequence

<400>29

ccagatgaag tcgccacaga 20

<210>30

<211>16

<212>DNA

<213> Artificial sequence

<400>30

cacgcacacg tggtgc 16

Claims

1. A method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR, comprising at least the steps of:

(1) obtaining total DNA from the peripheral plasma of the pregnant woman as genome DNA to be detected;

(2) selecting a gene locus sequence containing maternal-fetal methylation differences, and designing an amplification primer and a probe primer;

(3) selecting an internal reference gene locus sequence containing non-maternal methylation difference, and designing an amplification primer and a probe primer, wherein the internal reference gene contains the same enzyme cutting locus of the maternal methylation difference gene in the step (2);

(4) carrying out methylation sensitive restriction endonuclease digestion treatment on the genomic DNA to be detected in the step (1);

(5) in a digital PCR device, taking the genome DNA after enzyme digestion as a template, adopting the amplification primer and the probe primer in the step (2) to perform PCR amplification on the gene locus sequence containing maternal-fetal methylation difference, detecting a fluorescent signal, and obtaining the copy number of the maternal-fetal methylation difference gene, namely the absolute copy number of fetal free DNA in maternal plasma;

(6) and (3) in a digital PCR device, taking the genome DNA after enzyme digestion as a template, adopting the amplification primer and the probe primer in the step (3) to perform PCR amplification on the internal reference gene locus sequence containing the non-maternal fetal methylation difference, detecting a fluorescent signal, and eliminating false positive interference of maternal free DNA caused by incomplete enzyme digestion.

2. The method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR according to claim 1,

positive points obtained by maternal-fetal methylation difference gene markers are the number of fetal free DNA in the peripheral plasma sample of the pregnant woman;

obtaining positive points by the methylation differential gene marker of the non-maternal fetus, and indicating that the digestion treatment is incomplete;

non-maternal methylation differential gene markers receive non-positive points, indicating complete digestion treatment.

3. The method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR according to claim 1, wherein: the maternal-fetal methylation differential gene comprises at least one of NKX2-3, EGR, RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEEFF2 and PYCARD.

4. The method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR as claimed in claim 1 wherein said methylation sensitive restriction enzyme comprises at least one of AatII, AciI, AfeI, AgeI, AscI, AsiSI, AvaI, bcei, bmgbbi, BsaAI, BsiEI, BsiWI, BsmBI, BsrFI, bshii, BstBI, EagI, FauI, FspI, HaeII, HgaI, HhaI, hindp 1I, HpaII, Hpy99I, HpyCH4IV, KasI, MluI, NaeI, narii, nmigov, NotI, nt.

5. The method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR according to claim 1, wherein the maternal-fetal methylation difference gene is NKX2-3 gene, and primers and probes for amplifying and detecting the site sequence comprising NKX2-3 gene are:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 3, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

6. The method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR of claim 1, wherein the internal reference gene is ACTB gene, and primers and probes for amplifying and detecting the site sequence containing the internal reference ACTB gene are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 6, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

7. The method for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR according to claim 1,

the PCR amplification reaction system comprises the following components: 10 x dPCR Buffer Mix 3.5 mul, upstream and downstream primers 1 mul, probe primer 1 mul, genome DNA template 1.5 mul, adding water to 35 mul;

the PCR amplification reaction program comprises the following steps: performing pre-denaturation at 90 ℃ for 10min, performing PCR circulation, performing fluorescence detection on PCR amplification products at 95 ℃ for 20s, 60 ℃ for 40s, and 45 cycles.

8. A kit for detecting the absolute copy number of free DNA of a fetus in maternal plasma based on digital PCR is characterized by at least comprising a primer and a probe for amplifying and detecting a locus sequence containing maternal-fetal methylation difference genes and a primer and a probe for amplifying and detecting a locus sequence containing non-maternal-fetal methylation difference reference genes.

9. The kit for detecting the absolute copy number of fetal free DNA in maternal plasma based on digital PCR according to claim 8,

the maternal-fetal methylation difference gene is NKX2-3 gene, and primers and probes for amplifying and detecting a locus sequence containing NKX2-3 gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 3, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the internal reference gene is an ACTB gene, and primers and probes for amplifying and detecting a locus sequence containing the internal reference ACTB gene are as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 6, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group.

10. A kit for detecting RhD blood type genes of a fetus based on digital PCR is characterized by at least comprising:

primers and probes for amplifying and detecting the NKX2-3 gene sequence;

primers and probes for amplifying and detecting an internal reference ACTB gene sequence;

primers and probes for amplifying and detecting the sequence of the RhD gene exon 7;

primers and probes for amplifying and detecting the RhD gene exon 10 sequence;

and (3) primers and probes for amplifying and detecting the intron 4 sequence of the RhD gene.

11. The kit for detecting RhD blood group genes of a fetus based on digital PCR as claimed in claim 10, wherein:

the primers and the probes for amplifying and detecting the NKX2-3 gene locus sequence are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 3, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the site sequence of the internal reference ACTB gene are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 6, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the sequence of the RhD gene exon 7 are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 9, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the RhD gene exon 10 sequence are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 11, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 12, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the sequence of the intron 4 of the RhD gene are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 15, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quencher group;

12. Digital PCR-based method for detecting fetus RhD blood type gene and distinguishing maternal RhD^delA kit of genotypes, characterized in that it comprises at least:

primers and probes for amplifying and detecting the NKX2-3 gene sequence;

primers and probes for amplifying and detecting the RhD gene exon 10 sequence;

primers and probes for amplifying and detecting the sequence of the intron 4 of the RhD gene;

primers and probes for amplifying and detecting RHD1227A gene sequence.

13. The digital PCR-based detection of fetal RhD blood group genes and resolution of maternal RhD according to claim 12^delA kit for genotyping, comprising:

the primers and probes for amplifying and detecting the RHD1227A gene sequence are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 16, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 17, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 18, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quencher group

14. A kit for detecting ABO blood group genes of a fetus based on digital PCR is characterized by at least comprising:

primers and probes for amplifying and detecting the NKX2-3 gene locus sequence;

primers and probes for amplifying and detecting the nt261 site sequence of the ABO gene;

and primers and probes for amplifying and detecting the nt796 site sequence contained in the ABO gene.

15. The kit for detecting the ABO blood group genes of a fetus based on digital PCR as claimed in claim 14, wherein:

the primers and probes for amplifying and detecting the sequence containing nt261 locus of ABO gene are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 21, wherein the 5 'end of the probe primer is marked with a reporter group, and the 3' end of the probe primer is marked with a quenching group;

the primers and probes for amplifying and detecting the sequence containing nt796 locus of ABO gene are as follows:

the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 22, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 23, the nucleotide sequence of the probe primer is shown as SEQ ID NO: 24, wherein the 5 'end of the probe primer is labeled with a reporter group and the 3' end is labeled with a quencher group.