CN105586392A - Method for evaluating maternal cell contamination degree in fetal sample - Google Patents
Method for evaluating maternal cell contamination degree in fetal sample Download PDFInfo
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- CN105586392A CN105586392A CN201410637811.2A CN201410637811A CN105586392A CN 105586392 A CN105586392 A CN 105586392A CN 201410637811 A CN201410637811 A CN 201410637811A CN 105586392 A CN105586392 A CN 105586392A
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Abstract
The invention discloses a method for evaluating whether a fetal nucleic acid sample contains maternal nucleic acid or not. The method comprises the following steps of obtaining the fetus nucleic acid sample and the maternal nucleic acid sample, wherein each of the fetus nucleic acid sample and the maternal nucleic acid sample contains DNA (Deoxyribonucleic Acid) fragments; respectively detecting the basic group types of sites, corresponding to at least five of the following 21 reference SNP (Single Nucleotide Polymorphism) sites, in the DNA fragments of the fetus nucleic acid sample and the maternal nucleic acid sample: rs1057079, rs3890011, rs13007735, rs10172036, rs2516835, rs1656922, rs2686817, rs11167136, rs70156, rs2275111, rs2293277, rs2292681, rs3742302, rs4981088, rs11737, rs7169981, rs3115438, rs2302836, rs1455556, rs2235862 and rs2296241; and judging whether the fetus nucleic acid sample contains the maternal nucleic acid or not on the basis of the differences of the basic group types of the sites, corresponding to the same reference SNP, in the DNA fragments in the fetus nucleic acid sample and the maternal nucleic acid sample.
Description
Technical field
The present invention relates to biology sample detection field, particularly, the present invention relates to assess mother cell pollution level in fetus sampleMethod, more specifically, the present invention relates to a kind of method and a kind of kit of whether containing parent nucleic acid in fetal nucleic acid sample of detecting.
Background technology
In pre-natal diagnosis at present, the obtain manner of fetus sample is mainly amniocentesis and fine hair puncture sampling art, and due to motherThe pollution of body cell or decidua, these are earned quadrat method and may cause existing mother cell to pollute (maternal in fetus sampleCellcontamination, MCC) possibility, be one of principal element affecting pre-natal diagnosis result accuracy. Nineteen eighty-three,BennandHsu investigates mother cell pollution condition in the amniotic fluid sample of U.S. nationwide, finds 0.3% amniotic fluid markSituation [BennPA, HsuLY.1983.Maternalcellcontaminationofamnioticfluid that this exists mother cell to polluteCellcultures:resultsofaU.S.nationwidesurvey.AmJMedGenet1 5 (2): 297 – 305.]. An Europe cooperationResearch center investigation finds, 0.34% through the amniotic fluid sample of cultivation exist the situation that mother cell pollutes [BuiTH, IseliusL,LindstenJ.1984.Europeancollaborativestudyonprenataldiagnosis:mosaicism,pseudomosaicismandsingleabnormalcellsinamnioticfluidcellcultures.PrenatDiagn4SpecNo:145–162.]。2005Year, Stojilkovic-Mikic etc. utilize quantitative fluorescence PCR (QF-PCR) technology to 307 routine pre-natal diagnosis sample (254 routine amniotic fluidSample) in mother cell pollution condition study, in uncultivated amniotic fluid sample, mother cell pollute incidence up to9.1%, in the amniotic fluid sample of cultivating, mother cell pollutes incidence up to 2.8%[Stojilkovic-MikicT, MannK,DochertyZ.2005.MaternalcellcontaminationofprenatalsamplesassessedbyQF-PCRgenotyping.PrenatDiagn2005；25:79–83.]。
In pre-natal diagnosis sample, diagnostic result and the pregnant woman gestation of the assessment of mother cell pollution level to antenatal sample is intervened to closingImportant. At present, in assessment pre-natal diagnosis sample, the method for mother cell pollution level mainly contains pcr amplification technology, quantitative fluorescent PCRTechnology (Quantitativefluorescence – polymerasechainreaction, QF-PCR), STR (STR)Typing method, efficient sex change liquid chromatography technology, gene microarray analysis technology etc. Pcr amplification technology be a kind of common, forDetect the method that in pre-natal diagnosis sample, mother cell pollutes, Antoniadi etc. use pcr amplification technology to fetus and parent sampleDNA carries out augmentation detection, in 135 routine fetus samples, find 4 routine samples exist mother cell pollute situation [AntoniadiT,YapijakisC,KaminopetrosP,MakatsorisC,VelissariouV,VassilopoulosD,PetersenMB.2002.PrenatDiagn2002；22:425–429.]。
Human genome DNA's SNP (SNP) typing method is to detect human genome DNA's mononucleotide polymorphicThe technology of property site base type, the free flight mass spectrum of main method (MALDI-TOFMS) technology, quantitative fluorescent PCRTechnology, biochip technology, distortion high performance liquid chromatography etc.
Summary of the invention
According to an aspect of of the present present invention, provide a kind of method that whether contains parent nucleic acid in fetal nucleic acid sample that detects, described parentNucleic acid, from the mother who nourishes described fetus, said method comprising the steps of: (a) obtain described fetal nucleic acid sample and described motherThe sample of nucleic acid of body, the each self-contained DNA fragmentation of sample of nucleic acid of described fetal nucleic acid sample and described parent, and optional obtainingObtain male parent sample of nucleic acid, described male parent sample of nucleic acid is from the father of described fetus, and described male parent sample of nucleic acid comprises DNA fragmentation;(b) detect respectively in the DNA fragmentation in the sample of nucleic acid of described fetal nucleic acid sample and described parent and following 21 referencesThe base type at least 5 corresponding sites in SNP: rs1057079, rs3890011, rs13007735, rs10172036,rs2516835、rs1656922、rs2686817、rs11167136、rs70156、rs2275111、rs2293277、rs2292681、Rs3742302, rs4981088, rs11737, rs7169981, rs3115438, rs2302836, rs1455556, rs2235862 andRs2296241, in the DNA fragmentation in the described male parent sample of nucleic acid of optional detection with described fetal nucleic acid sample and/or described inThe base type in site corresponding to described site in the sample of nucleic acid of parent; (c) described in detecting in (b) step based on the comparisonIn DNA fragmentation in the sample of nucleic acid of fetal nucleic acid sample and described parent and optional described male parent sample of nucleic acid corresponding toThe difference of the base type in the described site with reference to SNP, judges in described fetal nucleic acid sample, whether to contain described parent nucleic acid.Optionally, (b) step can be also, detects respectively the DNA sheet in the sample of nucleic acid of described fetal nucleic acid sample and described parentIn section and described 21 base types with reference at least 10 in SNP corresponding sites; Optionally, (b) step also canTo be, detect respectively in the DNA fragmentation in the sample of nucleic acid of described fetal nucleic acid sample and described parent and described 21 ginsengsExamine the base type at least 15 corresponding sites in SNP; Optionally, (b) step can also be, detects respectively described tireIn DNA fragmentation in the sample of nucleic acid of youngster's sample of nucleic acid and described parent with described 21 described with reference at least 20 in SNPThe base type in the site of individual correspondence; Optionally, (b) step can also be, detects respectively described fetal nucleic acid sample and described motherIn DNA fragmentation in the sample of nucleic acid of body and described 21 base types with reference to site corresponding to SNP. Aforesaid with " rs "To refer to SNP be according to ncbi database to numbering, and NCBI can provide after the snp of all submissions classify and investigatedOne No. rs, and provide the specifying information of this SNP, sequence, positional information, distribution frequency etc. before and after comprising. Profit in the present inventionWith NCBI, the numbering of SNP being indicated the SNP site of selected combination, is to utilize one name or the expression mode of SNP to refer toShowing certain (a bit) people's specific SNP, is to refer to for convenience of expressing, and can also there be other expression mode in a SNP site, thisField those of ordinary skill knows, adopt other naming method such as with this SNP the position on cDNA name and refer to and thisThe reference SNP that invention is the same or SNP combination also belong to the scope of the invention. A SNP, based on its No. rs, can determine this SNPFront and back sequence, according to primers before and after it, amplification sample to be tested, can be by comprising and that reference in sample to be testedThe nucleic acid fragment in the site that SNP is corresponding increases out, then, based on the amplification nucleic acid fragment that comprises above-mentioned site out, detects instituteThe base type of rheme point, the base type in acquisition site.
The method of this one side of the present invention, 21 SNP sites or its part of selected combination are based on HapMap dataStorehouse sample genotype, be optimized and choose meeting at utmost to distinguish on sample, few basis, combination site, have conservative degree low,The characteristic that variation frequency is high. By detecting these 21 SNP site of human genome or the base type of its part, logicalCross detect fetal nucleic acid sample, parent sample of nucleic acid and optional male parent's DNA sample with this 21 SNP sites or its portionThe base type in point corresponding site, comparative analysis parent sample of nucleic acid, optional male parent's sample of nucleic acid and fetus sample DNAThe base difference of above-mentioned site, can assess mother cell pollution level in approximately 95% pre-natal diagnosis sample, can be fastWhether speed assessment fetal nucleic acid sample is subject to mother cell pollution and is subject to the degree that mother cell pollutes.
In one embodiment of the invention, (b) detection of the base type in the site in step comprises: utilize mononucleotide primerExtension method obtains the extension products that comprises described site, and, detect described extension products, obtain the base type in described site.Mononucleotide primer extension method, also referred to as micrometering order, utilizes this method to carry out somatotype to SNP, the DNA that can first increase and contain SNPFragment is as template, then at upstream or the downstream design primer of described SNP, adds substrate ddNTP in reaction system, utilizesSNP and ddNTP complementary pairing, mix a base at 3 ' end of primer, produces allele-specific primers extension products,An end of this product is this SNP site, detects the difference of this site or the sequence based on before and after extending, and determines this positionThe base type of point, in one embodiment of the invention, utilizes Mass Spectrometer Method to extend the molecular weight difference of the sequence of front and back, determinesThe type of a base of mixing. More specifically, the base type of detection site is to utilize flight time mass spectrum (MALDI-TOFMS), utilizing MALDI-TOFMS method to detect SNP site base type is by detecting extension products and not extending primerBetween molecular weight difference, determine SNP site base type, there is automation, quick, sensitive, cost is low, spectrogram is simpleThe feature such as directly perceived is the Perfected process of a kind of SNP of detection.
In one embodiment of the invention, (b) detection of the base type in the site in step comprises, utilizes the first primer, pointDo not increase containing and institute in the sample of nucleic acid of described fetal nucleic acid sample and described parent and optional described male parent sample of nucleic acidState the DNA fragmentation in the site corresponding with reference to SNP, obtain fetus amplified production and parent amplified production and optional male parent amplification productThing; Utilize the second primer respectively in conjunction with described fetus amplified production and described parent amplified production and optional described male parent amplification productThing, extends a base separately, corresponding fetus extension products and parent extension products and the optional male parent extension products of obtaining; Based onDescribed fetus extension products and described parent extension products separately with the molecular weight difference of described the second primer, optional based on described fatherThe molecular weight difference of this extension products and described the second primer, determines the sample of nucleic acid of described fetal nucleic acid sample, described parent and appointsThe base type in the described site in the described male parent sample of nucleic acid of choosing. Described fetus extension products and described parent extension products and appoint3 ' end of the described male parent extension products of choosing is all described site, and this is by utilizing amplified production stencil design the second primer, makingWhen extension, the second primer can be attached to the position in the adjacent described site in template, and making an extended base is targetSite, and be ddNTP based on substrate, can not continue again to extend, obtain Single base extension product.
In one embodiment of the invention, in the time that described site is the corresponding described rs1057079 with reference in SNP, described firstPrimer is SEQIDNO:1 and SEQIDNO:2, and described the second primer is SEQIDNO:3; When described site is corresponding instituteWhile stating with reference to rs3890011 in SNP, described the first primer is SEQIDNO:4 and SEQIDNO:5, and described second drawsThing is SEQIDNO:6; In the time that described site is the corresponding described rs13007735 with reference in SNP, described the first primer is SEQIDNO:7 and SEQIDNO:8, described the second primer is SEQIDNO:9; When described site is corresponding described with reference to SNPIn rs10172036 time, described the first primer is SEQIDNO:10 and SEQIDNO:11, described the second primer is SEQIDNO:12; In the time that described site is the corresponding described rs2516835 with reference in SNP, described the first primer is SEQIDNO:13 and SEQIDNO:14, described the second primer is SEQIDNO:15; When described site is corresponding described with reference in SNPWhen rs1656922, described the first primer is SEQIDNO:16 and SEQIDNO:17, and described the second primer is SEQIDNO:18; In the time that described site is the corresponding described rs2686817 with reference in SNP, described the first primer be SEQIDNO:19 andSEQIDNO:20, described the second primer is SEQIDNO:21; When described site is corresponding described with reference in SNPWhen rs11167136, described the first primer is SEQIDNO:22 and SEQIDNO:23, and described the second primer is SEQIDNO:24; In the time that described site is the corresponding described rs70156 with reference in SNP, described the first primer is SEQIDNO:25 and SEQIDNO:26, described the second primer is SEQIDNO:27; When described site is corresponding described with reference to the rs2275111 in SNPTime, described the first primer is SEQIDNO:28 and SEQIDNO:29, described the second primer is SEQIDNO:30; WhenWhen described site is the corresponding described rs2293277 with reference in SNP, described the first primer is SEQIDNO:31 and SEQIDNO:32, described the second primer is SEQIDNO:33; In the time that described site is the corresponding described rs2292681 with reference in SNP,Described the first primer is SEQIDNO:34 and SEQIDNO:35, and described the second primer is SEQIDNO:36; When describedWhen site is the corresponding described rs3742302 with reference in SNP, described the first primer is SEQIDNO:37 and SEQIDNO:38, described the second primer is SEQIDNO:39; In the time that described site is the corresponding described rs4981088 with reference in SNP, instituteStating the first primer is SEQIDNO:40 and SEQIDNO:41, and described the second primer is SEQIDNO:42; When institute's rhemePoint is during for the corresponding described rs11737 with reference in SNP, and described the first primer is SEQIDNO:43 and SEQIDNO:44,Described the second primer is SEQIDNO:45; In the time that described site is the corresponding described rs7169981 with reference in SNP, described theOne primer is SEQIDNO:46 and SEQIDNO:47, and described the second primer is SEQIDNO:48; When described site isWhen the corresponding described rs3115438 with reference in SNP, described the first primer is SEQIDNO:49 and SEQIDNO:50, instituteStating the second primer is SEQIDNO:51; In the time that described site is the corresponding described rs2302836 with reference in SNP, described firstPrimer is SEQIDNO:52 and SEQIDNO:53, and described the second primer is SEQIDNO:54; When described site is rightShould be described during with reference to rs1455556 in SNP, described the first primer is SEQIDNO:55 and SEQIDNO:56, described inThe second primer is SEQIDNO:57; In the time that described site is the corresponding described rs2235862 with reference in SNP, described first drawsThing is SEQIDNO:58 and SEQIDNO:59, and described the second primer is SEQIDNO:60; When described site is correspondingWhen the described rs2296241 with reference in SNP, described the first primer is SEQIDNO:61 and SEQIDNO:62, describedTwo primers are SEQIDNO:63. To be inventor obtain through design experiment repeatedly above-mentioned concrete primer sequence, can be used in manyIn heavy PCR reaction system, in one embodiment of the invention, utilize many groups the first primer to carry out multiplex amplification, can be oneIn individual PCR reaction system, obtain multiple amplified productions that comprise different target site simultaneously, using described amplified production as template,In same extension system, add many groups the second primer, organize the second primer more and can non-interferingly be attached to separately target bitPut and carry out Single base extension, obtain extension products.
In one embodiment of the invention, (c) step comprises: the described fetal nucleic acid sample detecting in (b) step based on the comparisonThe base type corresponding to the identical described site with reference to SNP in DNA fragmentation in the sample of nucleic acid of this and described parent poorDifferent, the base type when arbitrary described site in described fetal nucleic acid sample and the sample of nucleic acid at described parent meets following (i)-(iii) one of at least, judge that described fetal nucleic acid sample contains described parent nucleic acid, (i) in the sample of nucleic acid of described parentBe two types of bases and described two types of base equivalent, be also described two types of bases and institute in described fetal nucleic acid sampleState two types of base inequalities, be (ii) one type of base, at described fetal nucleic acid sample in the sample of nucleic acid of described parentIn be the base of described one type of base and another kind of and its inequality, (iii) in the sample of nucleic acid of described parent, be two kindsType base and described two types of base equivalent are one of described two types of bases and another in described fetal nucleic acid samplePlant the base with its inequality. Here said " amount " can be the extension products that comprises described site base of reality quantity,Concentration or ratio can be also size or the mass spectra peak of the mass spectra peak of the base of reaction on site somatotype figure or site detection figureThe size of area.
In one embodiment of the invention, (c) step comprises: the described fetal nucleic acid sample based on detecting in (b) step andIn DNA fragmentation in the sample of nucleic acid of described parent and described male parent sample of nucleic acid corresponding to identical described with reference to SNP positionThe base type of point, first judges whether the base type in the described site of described fetal nucleic acid sample meets mendelian inheritance, (1)When meeting mendelian inheritance and this site, the base type in arbitrary described site meets (i), (ii) and (ii) one of at least,And/or (2) meet (iv) and (v) extremely when the base type mismatch in arbitrary described site closes mendelian inheritance and this siteOne of few, judge that described fetal nucleic acid sample contains described parent nucleic acid, in the sample of nucleic acid of described parent, be (i) two kindsType base and described two types of base equivalent are also described two types of bases and described two kinds in described fetal nucleic acid sampleType base inequality (ii) is one type of base in the sample of nucleic acid of described parent, in described fetal nucleic acid sample, be described inThe base of one type of base and another kind of and its inequality (iii) is two types of bases in the sample of nucleic acid of described parentAnd described two types of base equivalent, in described fetal nucleic acid sample, be one of described two types of bases and another kind of and its notThe base of equivalent is (iv) two types of bases and described two types of base equivalent, in institute in the sample of nucleic acid of described parentState and in fetal nucleic acid sample, also comprise described two types of bases and described two types of base inequalities, (v) at the core of described parentIn acid sample, be one type of base, in described fetal nucleic acid sample, comprise described one type of base and another kind of and its not etc.The base of amount.
According to another aspect of the present invention, the invention provides a kind of kit, it comprises primer pair, every pair of described primer pair comprisesOne primer and the second primer, described primer pair can in order to detect with following 21 with reference at least 5 in SNP corresponding sitesBase type: rs1057079, rs3890011, rs13007735, rs10172036, rs2516835, rs1656922, rs2686817,rs11167136、rs70156、rs2275111、rs2293277、rs2292681、rs3742302、rs4981088、rs11737、Rs7169981, rs3115438, rs2302836, rs1455556, rs2235862 and rs2296241. Optionally, described primer pairCan be in order to detect and described 21 base types with reference at least 10 in SNP corresponding sites. Optionally, described in, drawThing is to can be in order to detect and described 21 base types with reference at least 15 in SNP corresponding sites. Optionally, instituteStating primer pair can be in order to detect and described 21 base types with reference at least 20 in SNP corresponding sites. Optionally,Described primer pair can be in order to detect and described 21 base types with reference to site corresponding to SNP.
In one embodiment of the invention, the primer pair that described kit comprises is, when the site that will detect comprises described in correspondenceDuring with reference to the site of the rs1057079 in SNP, be SEQIDNO:1 and SEQID for detecting the first primer described in this siteNO:2, described the second primer is SEQIDNO:3; When for the corresponding described site with reference to the rs3890011 in SNP of detection,Described the first primer is SEQIDNO:4 and SEQIDNO:5, and described the second primer is SEQIDNO:6; When right for detectingWhile answering the described site with reference to the rs13007735 in SNP, described the first primer is SEQIDNO:7 and SEQIDNO:8,Described the second primer is SEQIDNO:9; In the time for the corresponding described site with reference to the rs10172036 in SNP of detection being, instituteStating the first primer is SEQIDNO:10 and SEQIDNO:11, and described the second primer is SEQIDNO:12; When being detection, described the first primer is SEQIDNO:13 and SEQIDNO when in corresponding the described site with reference to the rs2516835 in SNP:14, described the second primer is SEQIDNO:15; When for the corresponding described site with reference to the rs1656922 in SNP of detection,Described the first primer is SEQIDNO:16 and SEQIDNO:17, and described the second primer is SEQIDNO:18; When being inspectionWhile surveying the corresponding described site with reference to the rs2686817 in SNP, described the first primer is SEQIDNO:19 and SEQIDNO:20, described the second primer is SEQIDNO:21; When for the corresponding described site with reference to the rs11167136 in SNP of detection,Described the first primer is SEQIDNO:22 and SEQIDNO:23, and described the second primer is SEQIDNO:24; When being inspectionWhile surveying the corresponding described site with reference to the rs70156 in SNP, described the first primer is SEQIDNO:25 and SEQIDNO:26, described the second primer is SEQIDNO:27; When for detecting during corresponding to the described site with reference to the rs2275111 in SNP,Described the first primer is SEQIDNO:28 and SEQIDNO:29, and described the second primer is SEQIDNO:30; When being inspectionWhile surveying the corresponding described site with reference to the rs2293277 in SNP, described the first primer is SEQIDNO:31 and SEQIDNO:32, described the second primer is SEQIDNO:33; When for the corresponding described site with reference to the rs2292681 in SNP of detection,Described the first primer is SEQIDNO:34 and SEQIDNO:35, and described the second primer is SEQIDNO:36; When being inspectionWhile surveying the corresponding described site with reference to the rs3742302 in SNP, described the first primer is SEQIDNO:37 and SEQIDNO:38, described the second primer is SEQIDNO:39; When for the corresponding described site with reference to the rs4981088 in SNP of detection,Described the first primer is SEQIDNO:40 and SEQIDNO:41, and described the second primer is SEQIDNO:42; When being inspectionWhile surveying the corresponding described site with reference to the rs11737 in SNP, described the first primer is SEQIDNO:43 and SEQIDNO:44, described the second primer is SEQIDNO:45; When for the corresponding described site with reference to the rs7169981 in SNP of detection,Described the first primer is SEQIDNO:46 and SEQIDNO:47, and described the second primer is SEQIDNO:48; When being inspectionWhile surveying the corresponding described site with reference to the rs3115438 in SNP, described the first primer is SEQIDNO:49 and SEQIDNO:50, described the second primer is SEQIDNO:51; When for the corresponding described site with reference to the rs2302836 in SNP of detection,Described the first primer is SEQIDNO:52 and SEQIDNO:53, and described the second primer is SEQIDNO:54; When being inspectionWhile surveying the corresponding described site with reference to the rs1455556 in SNP, described the first primer is SEQIDNO:55 and SEQIDNO:56, described the second primer is SEQIDNO:57; When for the corresponding described site with reference to the rs2235862 in SNP of detection,Described the first primer is SEQIDNO:58 and SEQIDNO:59, and described the second primer is SEQIDNO:60; When being inspectionWhile surveying the corresponding described site with reference to the rs2296241 in SNP, described the first primer is SEQIDNO:61 and SEQIDNO:62, described the second primer is SEQIDNO:63. In method in aforementioned one aspect of the present invention or each detailed description of the inventionRelate to the technical characterictic of site Selection and Constitute and primer and the description of advantage, be applicable to too the reagent of this one side of the present inventionBox, does not repeat them here.
Utilize method of the present invention and/or kit, the method that in assessment fetal nucleic acid sample, mother cell pollutes, utilizesMALDI-TOFMS method, by SNP site base type in human genome is detected, assessment pre-natal diagnosis sampleMiddle mother cell pollution level, its advantage having mainly contains: 1) automation, sense cycle are short---can realize in the short timeMultiple samples are detected, in 2 days, can obtain testing result, can be thin to whether there is parent in pre-natal diagnosis sample in timeBorn of the same parents pollute and judge, auxiliary pre-natal diagnosis; 2) low, the spectrogram simple, intuitive of cost---utilize MALDI-TOFMS methodCan realize the detection to multiple samples, reduce testing cost, and detect mass spectra peak figure simple, intuitive, by comparing fetus sampleWhether peak figure and parent's sample mass spectra peak figure difference, can to existing mother cell to pollute in fetus sample judge rapidly.
Brief description of the drawings
Above-mentioned and/or additional aspect of the present invention and advantage, will become obviously and hold the description of embodiment in conjunction with accompanying drawing belowEasily understand, wherein:
Fig. 1 is the flow chart of the appraisal procedure of the mother cell pollution in the fetal nucleic acid sample in one embodiment of the present of invention;
Fig. 2 be in one embodiment of the present of invention exist mother cell pollute fetus and 21 SNP positions of parental nucleic acid samplePoint mass spectra peak figure.
Detailed description of the invention
Fig. 1 is the flow process of an embodiment that adopts the inventive method, and general flow comprises: the choosing and optimization, tire of SNP siteSNP site base type detection, the sample mass spectrogram analysis etc. of youngster and mother body D NA sample and optional male parent's DNA sample.
1,21 of human genome SNP sites
21 SNP sites are based on HapMap database sample genotype, in satisfied sample, the combination site at utmost distinguishedOn few basis, be optimized and choose, there is the characteristic that conservative degree is low, variation frequency is high. By detect pre-natal diagnosis DNA sample,These 21 SNP site base types of mother body D NA sample and male parent's DNA sample, relatively mass spectra peak figure difference, can be rightIn approximately 95% pre-natal diagnosis sample, mother cell pollution level is assessed. 21 SNP site informations refer to table 1.
Table 1
| No. | Chromosome | Position (Position) | Rs numbers (rs No) | Allele (Alleles) | Frequency (Frequencies) |
| 1 | chr1 | 11205058 | rs1057079 | A/G | 0.563/0.437 |
| 2 | chr1 | 47398743 | rs3890011 | C/G | 0.408/0.592 |
| 3 | chr2 | 141751592 | rs13007735 | A/G | 0.442/0.558 |
| 4 | chr2 | 204824283 | rs10172036 | G/T | 0.547/0.453 |
| 5 | chr2 | 99779131 | rs2516835 | C/T | 0.5/0.5 |
| 6 | chr3 | 186443018 | rs1656922 | C/T | 0.527/0.473 |
| 7 | chr7 | 47968927 | rs2686817 | A/C | 0.487/0.513 |
| 8 | chr8 | 143310815 | rs11167136 | A/G | 0.496/0.504 |
| 9 | chr9 | 125424507 | rs70156 | A/C | 0.606/0.394 |
| 10 | chr10 | 120917445 | rs2275111 | A/G | 0.516/0.483 |
| 11 | chr10 | 95279506 | rs2293277 | A/T | 0.421/0.579 |
| 12 | chr12 | 120995332 | rs2292681 | C/T | 0.433/0.557 |
| 13 | chr13 | 31233063 | rs3742302 | C/T | 0.506/0.494 |
| 14 | chr14 | 20665840 | rs4981088 | A/G | 0.472/0.528 |
| 15 | chr15 | 77344793 | rs11737 | A/T | 0.433/0.567 |
| 16 | chr15 | 90226947 | rs7169981 | A/C | 0.602/0.398 |
| 17 | chr16 | 20986506 | rs3115438 | C/T | 0.507/0.493 |
| 18 | chr17 | 5991344 | rs2302836 | A/G | 0.471/0.529 |
| 19 | chr18 | 61170721 | rs1455556 | A/G | 0.435/0.565 |
| 20 | chr20 | 50238545 | rs2235862 | C/T | 0.474/0.526 |
| 21 | chr20 | 52786219 | rs2296241 | A/G | 0.5/0.5 |
2, SNP site base type detection in pre-natal diagnosis sample
Pre-natal diagnosis sample SNP site base type detection is based on MALDI-TOFMS technology, utilizes mononucleotide primer to prolongThe method of stretching is carried out somatotype to SNP, and the DNA fragmentation that first amplification contains SNP, as template, utilizes SAP enzyme (shrimp alkaline phosphataseEnzyme) ddNTP is removed. At upstream or the downstream design primer of SNP, in reaction system, add corresponding ddNTP again, byIn SNP and ddNTP complementary pairing, primer 3 ' end mixes one or two base, produces allele-specific primers and extends productThing. After extension products is purified, detect for MALDI-TOFMS. According to the Different Alkali of primer institute's combination in extensionThe different quality of base shows different peaks and detects SNP type on mass spectrograph. Because extension is to comprise predetermined siteAmplified production is template, and carries out the described predetermined site of 3 ' end next-door neighbour of the extension primer of extension, and in the time of extensionUse ddNTP as raw material, thereby can ensure that extension only extends a base, i.e. corresponding predetermined site, based on respectivelyThere is comparatively marked difference in the molecular weight of individual different bases, thereby, detect Ke Yitong by the molecular weight to extension productsThe molecular weight of crossing obtained extension products is determined the base type of predetermined site.
3, DNASNP mass spectra peak map analysis
Utilize 21 the SNP positions of MALDI-TOFMS technology to pre-natal diagnosis sample, parent sample, male parent's sample DNAPoint base type detects, and testing result is divided into 4 kinds of situations:
(a) be best suited for mother cell and pollute the SNP point of judgement. Mother's sample DNA SNP site base is heterozygous, tireYoungster DNASNP site base is homozygous, and for example mother body D NASNP site base is AT, foetal DNA SNP site alkaliBase is AA, if exist mother cell to pollute, has the interference at " T " base peak in fetus sample DNA mass spectrogram.
(b) Asia is applicable to the SNP point of mother cell pollution judgement. Mother's sample DNA SNP site base is homozygous, tireYoungster DNASNP site base is heterozygous, and for example female DNASNP site base is AA, foetal DNA SNP site baseFor AT, judge by comparison mother and fetus sample DNA SNP site mass spectra peak area, if exist mother cell to pollute,In fetus mass spectrogram, the peak area of " A " will be higher than " T " peak area. Preferably, in actual applications such SNP site as auxiliaryJudgement, further searches (a) class site or searches multiple such sites further to confirm judged result.
(c) point that cannot judge. Fetus is identical with mother DNASNP site base type, for example: female DNASNPSite base is that AA, foetal DNA SNP site base are AA, or female DNASNP site base is AC, foetal DNASNP site base is AC, and such point can not be used for judging whether fetus sample exists mother cell to pollute.
(d) there is new sudden change in fetus sample DNA SNP site. According to father's basis, female parent and foetal DNA SNP base classType analysis, foetal DNA SNP base type mismatch closes mendelian inheritance, and there is new sudden change in fetus sample DNA. AsThere is allele heredity mother Yu of new sudden change in fruit foetal DNA, for example father DNASNP site base is AT, female DNASNP site base is that T, foetal DNA SNP site base are A, and it is new prominent that this situation shows that foetal DNA SNP occursBecome, according to the SNP site base type of the new sudden change of fetus, female DNASNP site base type and above-mentioned a and/or bDescribed in SNP site judge whether fetus sample exists mother cell pollution condition. If foetal DNA occur new sudden change etc.Position gene genetic father Yu, for example father DNASNP site base is that T, female DNASNP site base are AT, foetal DNASNP site base is A, and this situation shows that new sudden change occurs foetal DNA SNP, in this case according in a and/or bDescribed SNP site judges whether fetus sample exists mother cell pollution condition.
In the time of (d) situation, use father's base type, need the base type in the corresponding site of detecting male parent nucleic acid, the one, useWith auxiliary judgment fetus base type, verify the accuracy of mass spectrum result and get rid of the possibility of other sample contamination; The 2nd, rootJudge according to parent gene type whether the corresponding site of fetus meets mendelian inheritance whether new sudden change occurs.
By comparative analysis fetus sample, parent sample and male parent's sample DNA SNP mass spectra peak figure, according to fetus sample and motherIn the detected peaks figure diversity judgement fetus sample of sample body, whether exist mother cell to pollute.
The implementation and operation step that following describe, in general terms is concrete.
From parent, male parent's peripheral blood sample and fetus sample (amniotic fluid or fine hair etc.), extract the DNA of 1ug left and right. RightPcr amplification is carried out in the target SNPs site of sample DNA, and PCR is alkaline phosphatase (shrimpalkalinephosphatase for product(SAP)) process. SAP PCR product after treatment is carried out to extension, and extension product purifying resin, after purifyingProduct be transferred to SpectroCHIP chip, with MassARRAY analyzer carry out simpleness detect, result MassARRAYTYP-ER software is analyzed.
Male parent, parent and fetus sample DNA SNP somatotype and mass spectrogram are analyzed to fetus mass spectra peak figure result and SNPSomatotype result completes unanimously, shows that the possibility that exists mother cell to pollute in fetus sample is lower; If fetus mass spectra peak figure knotFruit is inconsistent with SNP somatotype result, if mother's sample DNA SNP site base is heterozygous, and foetal DNA SNP site alkaliBase is homozygous, and for example mother body D NASNP site base is that AT, foetal DNA SNP site base are AA, and fetus matterIn spectrum peak figure there is the interference at " T " base peak in corresponding SNP site, shows to exist mother cell to pollute in fetus samplePossibility.
Below in conjunction with concrete individual specimen, the operation result according to concrete detection method of the present invention is described in detail. Show belowExample, only for explaining the present invention, and can not be interpreted as limitation of the present invention. In description of the invention, except as otherwise noted,The implication of " multiple " is two or more.
Except as otherwise explaining, the reagent of not explaining especially, sequence (joint, label and primer), the software that in following examples, relate toAnd instrument, all conventional commercially available prod or increase income, such as the flight time mass spectrum kit purchased from Sequenom company entersRow Mass Spectrometer Method etc.
Embodiment
Utilize MALDI-TOFMS method to whether existing mother cell to pollute in 30 routine fetal nucleic acid samples (amniotic fluid sample)Detect, find to exist mother cell to pollute in 1 routine pre-natal diagnosis sample, sample is from Tianjin healthcare hospital for women & children.
1) design of primers
The PCR primer in 21 of human gene SNP sites and extension primer are designed, and primer sequence is as shown in table 2.
Table 2
2) sample DNA extracts 30 routine amniotic fluid samples, maternal peripheral blood and male parent's peripheral blood, utilize qiagen kit (Hilden,Germany) carry out sample DNA extraction, extracted amount is about 1ug left and right.
3) pcr amplification, by pcr amplification, obtains the amplified production of target SNP site sequence. PCR expands reaction system as table 3Shown in. All reagent is all bought the Sequenom company in the U.S., and PCR instrument is GeneAmpPCRSystem9700Dual384-WellSampleBlockModule. PCR reaction condition is as shown in table 4.
Table 3
| Reagent | Concentration | Reaction volume (ul) |
| Water | — | 1.8 |
| 10x PCR buffer solution | 0.2x/ul | 0.5 |
| MgCl2 | 0.4x/ul | 0.4 |
| DNTP mixture | 100uM/ul | 0.1 |
| Amplimer mixture | 20nM/ul | 1.0 |
| Tap enzyme | 0.1U/ul | 0.2 |
| Template DNA | — | 1.0 |
| Volume | — | 5.0 |
Table 4
Utilize SAP enzyme to process above-mentioned pcr amplification product, its reaction system is, 5.0ul amplified production 0.16ulSAPThe water (HPLE level) of enzyme buffer liquid and 0.3ulSAP enzyme and 1.54ul, total reaction volume is 7.0ul. SAP enzyme reaction condition is37 DEG C of incubation 40min, 85 DEG C of incubation 5min.
4) extension pcr amplification product, after SAP enzyme is processed, carries out the extension of single base, and reaction system is: 0.655ulWater, 0.2ul10xiPLEX buffer solution, 0.2ul termination mix, 0.041uliPLEX enzyme (Sequenom), 0.904ul10uMExtend primer. Extension condition is: 94 DEG C, 30s, 94 DEG C, 5s, 52 DEG C, 5s, 80 DEG C, a 5s5 circulation, totally 40Circulation; Last 72 DEG C, 3min. In cessation reaction, add 6mg cationic ion-exchange resin (Sequenom), 20ul water takes offSalt.
5) Mass Spectrometer Method is passed through point sample instrument (MassARRAYNanodispenserRS1000, Sequenom) after purifyingExtension products be transferred on 384 hole SpectroCHIP chips, utilize SequenomMALDI-TOF mass spectrograph to detect,Obtain the base mass spectra peak figure in parent, male parent and fetus sample DNA SNP site.
6) Analysis of test results, by 21 SNP mass spectrograms of comparison family sample male parent, parent and fetus sample DNA, is sent outIn existing 30 family samples (parent, male parent with fetus sample), detect in 1 routine fetus sample and exist mother cell to pollute.Fetus and 21 SNP site Mass Spectrometer Method results of parent's sample DNA that this example exists mother cell to pollute are as shown in table 5,Mass spectra peak figure as shown in Figure 2. Mass spectra peak figure result shows, in fetus sample mass spectrum result, and SNP site rs3890011, rs2275111There is the interference at the assorted peak of DNA base of source mother cell with rs7169981, show to have parent in the fetus sample of this familyCell contamination, the italic display line in corresponding table 5.
Table 5
| Position | rs No | Male parent | Parent | Fetus |
| chr1：11205058 | rs1057079 | T | T | T |
| chr1：47398743 | rs3890011 | GC | GC | CG |
| Chr2：141751592 | rs13007735 | AG | G | G |
| Chr2：204824283 | rs10172036 | G | GT | GT |
| Chr2：99779131 | rs2516835 | C | C | C |
| Chr3：186443018 | rs1656922 | CT | C | CT |
| Chr7：47968927 | rs2686817 | CA | CA | CA |
| Chr8：143310815 | rs11167136 | A | A | A |
| Chr9：125424507 | rs70156 | CA | C | CA |
| chr10：120917445 | rs2275111 | A | GA | AG |
| chr10：95279506 | rs2293277 | T | T | T |
| chr12：120995332 | rs2292681 | A | GA | GA |
| chr13：31233063 | rs3742302 | G | G | G |
| chr14：20665840 | rs4981088 | G | A | AG |
| chr15：77344793 | rs11737 | AT | T | AT |
| chr15：90226947 | rs7169981 | CA | CA | CA |
| chr16：20986506 | rs3115438 | CT | C | CT |
| chr17：5991344 | rs2302836 | T | TC | TC |
| chr18：61170721 | rs1455556 | C | TC | TC |
| Chr20：50238545 | rs2235862 | A | A | A |
| Chr20：52786219 | rs2296241 | G | G | G |
Claims (10)
1. detect a method that whether contains parent nucleic acid in fetal nucleic acid sample, described parent nucleic acid is from nourishing described fetusMother, said method comprising the steps of,
(a) obtain the sample of nucleic acid of described fetal nucleic acid sample and described parent, the core of described fetal nucleic acid sample and described parentThe each self-contained DNA fragmentation of acid sample, and optional acquisition male parent sample of nucleic acid, described male parent sample of nucleic acid is from described fetusFather, described male parent sample of nucleic acid comprises DNA fragmentation;
(b) detect respectively in the DNA fragmentation in the sample of nucleic acid of described fetal nucleic acid sample and described parent with following 21Base type with reference at least 5 in SNP corresponding sites: rs1057079, rs3890011, rs13007735, rs10172036,rs2516835、rs1656922、rs2686817、rs11167136、rs70156、rs2275111、rs2293277、rs2292681、Rs3742302, rs4981088, rs11737, rs7169981, rs3115438, rs2302836, rs1455556, rs2235862 andRs2296241, in the DNA fragmentation in the described male parent sample of nucleic acid of optional detection with described fetal nucleic acid sample and/or described inThe base type in site corresponding to described site in the sample of nucleic acid of parent;
(c) the described fetal nucleic acid sample detecting in (b) step based on the comparison and the sample of nucleic acid of described parent and optional instituteState the difference of the base type corresponding to the described site with reference to SNP in the DNA fragmentation in male parent sample of nucleic acid, described in judgementIn fetal nucleic acid sample, whether contain described parent nucleic acid;
Optionally, (b) in step, detect respectively the DNA fragmentation in the sample of nucleic acid of described fetal nucleic acid sample and described parentIn with described 21 base types with reference at least 10 in SNP corresponding sites;
Optionally, (b) in step, detect respectively the DNA fragmentation in the sample of nucleic acid of described fetal nucleic acid sample and described parentIn with described 21 base types with reference at least 15 in SNP corresponding sites;
Optionally, (b) in step, detect respectively the DNA fragmentation in the sample of nucleic acid of described fetal nucleic acid sample and described parentIn with described 21 described base types with reference at least 20 in SNP corresponding sites;
Optionally, (b) in step, detect respectively the DNA fragmentation in the sample of nucleic acid of described fetal nucleic acid sample and described parentIn with described 21 base types with reference to site corresponding to SNP.
2. the method for claim 1, is characterized in that, (b) detection of the base type in the site in step comprises,
Utilize mononucleotide primer extension method to obtain the extension products that comprises described site, and,
Detect described extension products, obtain the base type in described site.
3. the method for claim 1 or 2, is characterized in that, (b) detection of the base type in the site in step comprises,
Utilize the first primer, the sample of nucleic acid of increase respectively described fetal nucleic acid sample and described parent and optional described male parent coreContaining and the described DNA fragmentation with reference to site corresponding to SNP in acid sample, obtain fetus amplified production and parent amplified productionWith optional male parent amplified production;
Utilize the second primer respectively in conjunction with described fetus amplified production and described parent amplified production and optional described male parent amplification productThing, extends a base separately, corresponding fetus extension products and parent extension products and the optional male parent extension products of obtaining;
Based on described fetus extension products and described parent extension products separately with the molecular weight difference of described the second primer, optional baseIn the molecular weight difference of described male parent extension products and described the second primer, determine the nucleic acid of described fetal nucleic acid sample, described parentThe base type in the described site in sample and optional described male parent sample of nucleic acid.
4. the method for claim 3, is characterized in that, described fetus extension products and described parent extension products and optional institute3 ' the end of stating male parent extension products is all described site.
5. the method for claim 4, is characterized in that, in the time that described site is the corresponding described rs1057079 with reference in SNP,Described the first primer is SEQIDNO:1 and SEQIDNO:2, and described the second primer is SEQIDNO:3;
In the time that described site is the corresponding described rs3890011 with reference in SNP, described the first primer is SEQIDNO:4 and SEQIDNO:5, described the second primer is SEQIDNO:6;
In the time that described site is the corresponding described rs13007735 with reference in SNP, described the first primer be SEQIDNO:7 andSEQIDNO:8, described the second primer is SEQIDNO:9;
In the time that described site is the corresponding described rs10172036 with reference in SNP, described the first primer be SEQIDNO:10 andSEQIDNO:11, described the second primer is SEQIDNO:12;
In the time that described site is the corresponding described rs2516835 with reference in SNP, described the first primer be SEQIDNO:13 andSEQIDNO:14, described the second primer is SEQIDNO:15;
In the time that described site is the corresponding described rs1656922 with reference in SNP, described the first primer be SEQIDNO:16 andSEQIDNO:17, described the second primer is SEQIDNO:18;
In the time that described site is the corresponding described rs2686817 with reference in SNP, described the first primer be SEQIDNO:19 andSEQIDNO:20, described the second primer is SEQIDNO:21;
In the time that described site is the corresponding described rs11167136 with reference in SNP, described the first primer be SEQIDNO:22 andSEQIDNO:23, described the second primer is SEQIDNO:24;
In the time that described site is the corresponding described rs70156 with reference in SNP, described the first primer is SEQIDNO:25 and SEQIDNO:26, described the second primer is SEQIDNO:27;
In the time that described site is the corresponding described rs2275111 with reference in SNP, described the first primer be SEQIDNO:28 andSEQIDNO:29, described the second primer is SEQIDNO:30;
In the time that described site is the corresponding described rs2293277 with reference in SNP, described the first primer be SEQIDNO:31 andSEQIDNO:32, described the second primer is SEQIDNO:33;
In the time that described site is the corresponding described rs2292681 with reference in SNP, described the first primer be SEQIDNO:34 andSEQIDNO:35, described the second primer is SEQIDNO:36;
In the time that described site is the corresponding described rs3742302 with reference in SNP, described the first primer be SEQIDNO:37 andSEQIDNO:38, described the second primer is SEQIDNO:39;
In the time that described site is the corresponding described rs4981088 with reference in SNP, described the first primer be SEQIDNO:40 andSEQIDNO:41, described the second primer is SEQIDNO:42;
In the time that described site is the corresponding described rs11737 with reference in SNP, described the first primer is SEQIDNO:43 and SEQIDNO:44, described the second primer is SEQIDNO:45;
In the time that described site is the corresponding described rs7169981 with reference in SNP, described the first primer be SEQIDNO:46 andSEQIDNO:47, described the second primer is SEQIDNO:48;
In the time that described site is the corresponding described rs3115438 with reference in SNP, described the first primer be SEQIDNO:49 andSEQIDNO:50, described the second primer is SEQIDNO:51;
In the time that described site is the corresponding described rs2302836 with reference in SNP, described the first primer be SEQIDNO:52 andSEQIDNO:53, described the second primer is SEQIDNO:54;
In the time that described site is the corresponding described rs1455556 with reference in SNP, described the first primer be SEQIDNO:55 andSEQIDNO:56, described the second primer is SEQIDNO:57;
In the time that described site is the corresponding described rs2235862 with reference in SNP, described the first primer be SEQIDNO:58 andSEQIDNO:59, described the second primer is SEQIDNO:60;
In the time that described site is the corresponding described rs2296241 with reference in SNP, described the first primer be SEQIDNO:61 andSEQIDNO:62, described the second primer is SEQIDNO:63.
6. claim 1-5 either method, is characterized in that, (b) detection of the base type in the site in step utilizes mass spectrumCarry out.
7. claim 1-6 either method, is characterized in that, (c) step comprises,
In DNA fragmentation in the described fetal nucleic acid sample detecting in (b) step based on the comparison and the sample of nucleic acid of described parentThe difference of the base type corresponding to the identical described site with reference to SNP, when arbitrary described site is at described fetal nucleic acid sampleWith the base type in the sample of nucleic acid of described parent meet following one of at least, judge that described fetal nucleic acid sample contains described inParent nucleic acid,
(i) in the sample of nucleic acid of described parent, be two types of bases and described two types of base equivalent, at described fetal nucleusIn acid sample, be also described two types of bases and described two types of base inequalities,
(ii) in the sample of nucleic acid of described parent, be one type of base, be described one type in described fetal nucleic acid sampleThe base of base and another kind of and its inequality,
(iii) in the sample of nucleic acid of described parent, be two types of bases and described two types of base equivalent, at described fetal nucleusIt in acid sample, is the base of one of described two types of bases and another kind of and its inequality.
8. claim 1-6 either method, is characterized in that, (c) step comprises,
Described fetal nucleic acid sample based on detecting in (b) step and the sample of nucleic acid of described parent and described male parent sample of nucleic acidIn DNA fragmentation in the base type corresponding to the identical described site with reference to SNP, judge the institute of described fetal nucleic acid sampleWhether the base type of rheme point meets mendelian inheritance,
(1) when meeting mendelian inheritance and this site, the base type in arbitrary described site meets (i), (ii) and (ii) extremelyOne of few, and/or,
(2) when closing mendelian inheritance and this site, the base type mismatch in arbitrary described site meets (iv) and (v) at leastOne of,
Judge that described fetal nucleic acid sample contains described parent nucleic acid,
(i) in the sample of nucleic acid of described parent, be two types of bases and described two types of base equivalent, described fetusIn sample of nucleic acid, be also described two types of bases and described two types of base inequalities,
(ii) in the sample of nucleic acid of described parent, be one type of base, be described one in described fetal nucleic acid sampleThe base of type base and another kind of and its inequality,
(iii) in the sample of nucleic acid of described parent, be two types of bases and described two types of base equivalent, at described tireIn youngster's sample of nucleic acid, be the base of one of described two types of bases and another kind of and its inequality,
(iv) in the sample of nucleic acid of described parent, be two types of bases and described two types of base equivalent, at described tireIn youngster's sample of nucleic acid, also comprise described two types of bases and described two types of base inequalities,
(v) in the sample of nucleic acid of described parent, be one type of base, in described fetal nucleic acid sample, comprise described oneThe base of type base and another kind of and its inequality.
9. a kit, it comprises primer pair, every pair of described primer pair comprises the first primer and the second primer, described primer pairCan in order to detect with following 21 base types with reference at least 5 in SNP corresponding sites: rs1057079,rs3890011、rs13007735、rs10172036、rs2516835、rs1656922、rs2686817、rs11167136、rs70156、rs2275111、rs2293277、rs2292681、rs3742302、rs4981088、rs11737、rs7169981、rs3115438、Rs2302836, rs1455556, rs2235862 and rs2296241;
Optionally, described primer pair can be in order to detect and described 21 alkali with reference at least 10 in SNP corresponding sitesBase type;
Optionally, described primer pair can be in order to detect and described 21 alkali with reference at least 15 in SNP corresponding sitesBase type;
Optionally, described primer pair can be in order to detect and described 21 alkali with reference at least 20 in SNP corresponding sitesBase type;
Optionally, described primer can be in order to detect and described 21 base types with reference to site corresponding to SNP.
10. the kit of claim 9, is characterized in that, when described site is corresponding described with reference to the rs1057079 in SNPTime, described the first primer is SEQIDNO:1 and SEQIDNO:2, described the second primer is SEQIDNO:3;
In the time that described site is the corresponding described rs3890011 with reference in SNP, described the first primer is SEQIDNO:4 and SEQIDNO:5, described the second primer is SEQIDNO:6;
In the time that described site is the corresponding described rs13007735 with reference in SNP, described the first primer be SEQIDNO:7 andSEQIDNO:8, described the second primer is SEQIDNO:9;
In the time that described site is the corresponding described rs10172036 with reference in SNP, described the first primer be SEQIDNO:10 andSEQIDNO:11, described the second primer is SEQIDNO:12;
In the time that described site is the corresponding described rs2516835 with reference in SNP, described the first primer be SEQIDNO:13 andSEQIDNO:14, described the second primer is SEQIDNO:15;
In the time that described site is the corresponding described rs1656922 with reference in SNP, described the first primer be SEQIDNO:16 andSEQIDNO:17, described the second primer is SEQIDNO:18;
In the time that described site is the corresponding described rs2686817 with reference in SNP, described the first primer be SEQIDNO:19 andSEQIDNO:20, described the second primer is SEQIDNO:21;
In the time that described site is the corresponding described rs11167136 with reference in SNP, described the first primer be SEQIDNO:22 andSEQIDNO:23, described the second primer is SEQIDNO:24;
In the time that described site is the corresponding described rs70156 with reference in SNP, described the first primer is SEQIDNO:25 and SEQIDNO:26, described the second primer is SEQIDNO:27;
In the time that described site is the corresponding described rs2275111 with reference in SNP, described the first primer be SEQIDNO:28 andSEQIDNO:29, described the second primer is SEQIDNO:30;
In the time that described site is the corresponding described rs2293277 with reference in SNP, described the first primer be SEQIDNO:31 andSEQIDNO:32, described the second primer is SEQIDNO:33;
In the time that described site is the corresponding described rs2292681 with reference in SNP, described the first primer be SEQIDNO:34 andSEQIDNO:35, described the second primer is SEQIDNO:36;
In the time that described site is the corresponding described rs3742302 with reference in SNP, described the first primer be SEQIDNO:37 andSEQIDNO:38, described the second primer is SEQIDNO:39;
In the time that described site is the corresponding described rs4981088 with reference in SNP, described the first primer be SEQIDNO:40 andSEQIDNO:41, described the second primer is SEQIDNO:42;
In the time that described site is the corresponding described rs11737 with reference in SNP, described the first primer is SEQIDNO:43 and SEQIDNO:44, described the second primer is SEQIDNO:45;
In the time that described site is the corresponding described rs7169981 with reference in SNP, described the first primer be SEQIDNO:46 andSEQIDNO:47, described the second primer is SEQIDNO:48;
In the time that described site is the corresponding described rs3115438 with reference in SNP, described the first primer be SEQIDNO:49 andSEQIDNO:50, described the second primer is SEQIDNO:51;
In the time that described site is the corresponding described rs2302836 with reference in SNP, described the first primer be SEQIDNO:52 andSEQIDNO:53, described the second primer is SEQIDNO:54;
In the time that described site is the corresponding described rs1455556 with reference in SNP, described the first primer be SEQIDNO:55 andSEQIDNO:56, described the second primer is SEQIDNO:57;
In the time that described site is the corresponding described rs2235862 with reference in SNP, described the first primer be SEQIDNO:58 andSEQIDNO:59, described the second primer is SEQIDNO:60;
In the time that described site is the corresponding described rs2296241 with reference in SNP, described the first primer be SEQIDNO:61 andSEQIDNO:62, described the second primer is SEQIDNO:63.
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| CN116497106A (en) * | 2023-06-30 | 2023-07-28 | 北京大学第三医院（北京大学第三临床医学院） | Identification method for maternal pollution in prenatal diagnosis |
| CN117417994A (en) * | 2023-12-17 | 2024-01-19 | 广州嘉检医学检测有限公司 | Library pollution detection kit and detection method |
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| WO2020076474A1 (en) * | 2018-10-12 | 2020-04-16 | Nantomics, Llc | Prenatal purity assessments using bambam |
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| CN116497106B (en) * | 2023-06-30 | 2024-03-12 | 北京大学第三医院（北京大学第三临床医学院） | Identification method for maternal pollution in prenatal diagnosis |
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