CN101158632A - Down's syndrome diagnose kit - Google Patents

Abstract

A diagnostic reagent kit for the Downs syndrome is characterized in that the reagent kit comprises two independent sub reagent kits A and B, each sub kit comprises separately packed primer mixture, DNA polymerase, buffer solution, MgC1<SUB>2</SUB> and allele parting normal mixture. Wherein, the primer mixture of the sub reagent kit A comprises the multi-packed long sequence primers of six gene loci, namely LFG21, LFG24, LFG26, D21S1409, D21S2052 and PentaD; the primer mixture of the sub reagent kit B comprises the multi-packed long sequence primers of six gene loci, namely LFG20, LFG29, LFG33, LFG34, D21S1435 and D21S1436. The reagent kit of the invention saves detection time, specimen and consumable material as much as possible. Therefore, the invention is of great use value.

Description

Down's syndrome diagnose kit

Technical field

The present invention relates to a kind of kit that can make a definite diagnosis Down syndrome rapidly.

Background technology

Down syndrome is to gain public acceptance at first and one of most important serious chromosomal disorders.Britain doctor Langdon Down at first is described this disease, so be called Down syndrome.Nineteen fifty-nine, French cytogeneticist Lejeune confirms this sick cause of disease little G group chromosome that has been many, is defined as chromosome afterwards No. 21, so be also referred to as trisomy 21.The Clinical symptoms of Down syndrome is mainly growth retardation, feeblemindedness in various degree and comprises a series of unusual sign of women's head-ornaments portion feature, and this makes this disease unfortunately be called as mongolism in the past again.Among the neonate, the incidence of disease of trisomy 21 is about 1/600-900, is chromosome abnormality the most common among the neonate.95%Down syndrome patient's chromosome basis all is the trisomy 21 due to chromosome does not separate when being secondary to meiosis, promptly the whole body body cell is all many No. 21 chromosomes, and this class caryogram is called free type, usually clinical symptoms typical case and significantly.By the dna polymorphism analysis, can in most family, judge and not separate which side and maiotic which that occurs among the parents in step.The many initial meiosises of No. 21 extra chromosomes from mother.About 4% Down syndrome is the result of non-balanced translocation, this caryogram is easy bit-type, though the patient has only 46 chromosomes, but on a chromosome that translocates to another D group or G group for No. 21, add normal two No. 21 chromosomes, still many one extra No. 21 long-armed, and determine this sick key area band be No. 21 long-armed, so still show the symptom of trisomy 21 clinically.At last, the Down syndrome patient less than 3% is a chimera, has two kinds of normal and unusual clones simultaneously, and symptom depends on the ratio that abnormal cell is shared, but generally lighter.At present, except strengthening nursing and training, prolong patient's life-span, strengthen outside its self care ability, human do not have therapy measure for Down syndrome.The generation of this disease all brings great burden for patient family and society.Therefore, improve the pre-natal diagnosis level, reduce the birth of Down syndrome infant, extremely important.

It is existing biological aneuploid detection method that fetal cell is carried out karyotyping, this process comprise fetal cell in vitro culture and mid-term fetal cell karyotyping.Cells in vitro is cultivated needs the survivaling cell of some and suitable specialized technical knowledge, all possibly can't implement karyotyping under following several situations: 1, cellular incubation failure.According to statistics, the incidence of cellular incubation failure is about 1-2%.2, the cell number of Shou Jiing is limited, can not draw clear conclusions.For guaranteeing that enough cultures are used for karyotyping, must obtain amniotic fluid greater than 5m1.When the amniotic fluid amount of extracting out when amniocentesis is less than 5ml, just have to carry out amniocentesis repeatedly, infect and the risk of damage thereby increased.3, culture is contaminated.Bibliographical information is arranged, even in veteran testing laboratory, the pollution rate of mother cell also can reach 10-14%.4, because cellular incubation only limits to survivaling cell, so some special sample can't carry out karyotyping, as some product of conception and formalin fixed or paraffin embedding sample.In addition, the detection method of traditional biological aneuploid significant disadvantage the most is that cell cultivation process is consuming time oversize, roughly needs the time about two weeks, and this just is easy to cause the delay in the processing.Therefore, rapidly, detection technique is very valuable accurately.

In recent years, fluorescence in situ hybridization (fluorecent in situ hybridization, FISH) and quantitative fluorescence PCR (quantitative fluorecent polymerase chain reaction QF-PCR) has been applied to the detection of biological aneuploid.With regard to the method for the biological aneuploid of fluorescence in situ hybridization detection, interval the fluorescence in situ hybridization of cell can carry out cellular incubation, shortened the required time of diagnosing greatly; The application of commercialization probe has improved susceptibility and the specificity of FISH.But then, this diagnostic techniques still has certain limitation: 1, in the amniotic fluid of not cultivating, fluorescently-labeled probe can combine with cytoskeleton non-specificly, makes background unintelligible, and the validity of FISH reduces greatly.2, must there be complete cell to be used for analyzing.3, experimenter's professional skill is had relatively high expectations.4, the pollution of mother cell can cause the explanation of error to experimental result.5, compare QF-PCR, fish analysis length consuming time, cost height have limited its high throughput testing to sample.

The quantitative fluorescence PCR method has been utilized the STR (shorttandem repeats is hereinafter to be referred as STR) that extensively distributes in the genome.STR is repeated to form by the core sequence series connection of 2～6bp usually, has the polymorphism of height, is easy to adopt the composite amplification mode to increase.And fluorescent composite amplification is the most general composite amplification method of using in the world at present, normally at each target sequence fluorescent material on the end mark of a primer wherein, utilizes automatic laser fluorescence genetic analyzer that product is detected.The amplified production of STR separates by clip size when electrophoresis, determines each allelic copy number according to the number and the intensity of fluorescence peak, thereby reflects specific chromosomal copy number.A normal heterozygote cognition produces two highly close peaks, and homozygote then shows as an independent STR peak.Because the application of PCR (PCR) amplification method and fluoroscopic examination, this detection method are very sensitive, can be used for the detection of a small amount of sample, as fetal cell or the foetal DNA of collecting in the maternal blood, also can determine the pollution of mother cell.Particularly importantly, this method is very fast, and the extraction of DNA and amplification only need about 5 hours, and the analysis meeting of each fluorescence-causing substance was finished in half an hour.Easily be automated in addition, might detect simultaneously 36～96 samples, therefore tens, even the testing result of a hundreds of sample can obtain in one day.Identification for the testing result of quantitative fluorescence PCR test method is also very disputable at present, because this method itself remains in some problems.At first, composite amplification is carried out in 2-4 STR site that present quantitative fluorescence PCR test method has only been chosen on every chromosome.Concerning a specific chromosome, only the detection system that is made of 2-4 STR site can not guarantee to detect all aneuploids usually, especially when the heterozygosity in STR site is not high.In the detection of many utilization quantitative fluorescence PCR test methods, always there is the part sample that information can not be provided, promptly the several sites on chromosome all show as a STR peak, can't differentiate normally or the homozygote of aneuploid.Particularly importantly, quantitative fluorescence PCR test method is when detecting two peaks, with the ratio of peak height or peak area and the normal reference value contrast of setting up in advance, as judging whether one of euploid foundation of right and wrong, this reduces the validity of this method greatly.PCR is a complicated course of reaction, and amplified production amount and template amount are not simple linear relationship, when two allele peaks occurring, to judgement more complicated in practical operation of result.If the ratio of the height at two peaks or area will be equivocal to result's judgement near the dividing value of normal reference value; The composite amplification of a plurality of target sites, because fluorescence peak is overlapping, its position and intensity also are difficult to explain sometimes; There is the advantage pcr phenomenon in some site, is easy to cause mistaken diagnosis, especially in the template amount after a little while.When advantage pcr occurring, or because the little allele excessive amplification of fragment and the erroneous judgement of chromosomal aneuploid is normal dliploid, or owing to the low aneuploid that is mistaken for of an amplified allele efficient of normal diploid individuality.In practice, quantitative fluorescence PCR is normally united use as a kind of auxiliary detection method with traditional cytogenetic methods, and the last result that makes a definite diagnosis still need be by cytogenetic method acquisition.

The applicant once disclosed a kind of method for quick of No. 21 numerical abnormalities of chromosomess in No. 1 00218224, Chinese invention patent ZL2004.This method adopts the fluorescent composite amplification mode to be increased in a plurality of No. 21 chromosome specific STR sites in the sample DNA.The product of composite amplification carries out electrophoresis and analysis on the DNA genetic analyzer.At last, the number at allele peak is recognized, when observing plural No. 21 chromosome specific STR sites and have three allele peaks, assert this numerical abnormalities of chromosomes, be trisomy.Utilize the method for quick of this No. 21 numerical abnormalities of chromosomess, the commercialization diagnostic kit of exploitation Down syndrome, to promote this method applying in clinical practice, be very meaningful and practical value to realizing making a definite diagnosis fast of Down syndrome.

Summary of the invention

Purpose of the present invention promptly provides the kit that can make a definite diagnosis Down's syndrome fast.

Down's syndrome diagnose kit of the present invention comprises that two of A, B independently divide kit, and each minute, kit was by the primer mixture, archaeal dna polymerase, damping fluid, the MgCl that separate packing ₂Constitute with the allelic ladder potpourri; A divides the primer mixture of kit to be made of the long aligning primer that attaches together together LFG21, LFG24, LFG26, D21S1409, D21S2052 and six locus of Penta D, wherein:

The long aligning primer of LFG24 locus:

SA-P1：5’-TGTTCTT-AGGTTCTAGGAAACATGGCTG-3’

SB1-P2：5’-F1-TGTTCTT-TGCAAAACTGCTCTGGACT-3’，

The long aligning primer of LFG26 locus:

SA-P1：5’-TGTTCTT-TTAGAGTTGACAAACTCCATGTTG-3’

SB2-P2：5’-F2-TGTTCTT-TCTGAGCTAGTTGGGAGGATAG-3’，

The long aligning primer of D21S1409 locus:

SA-P1：5’-TGTTCTT-GGAGGGGAATACATTTGTG-3’

SB2-P2：5’-F2-TGTTCTT-TTGCCTCTGAATATCCCTAT-3’，

The long aligning primer of LFG21 locus:

SA-P1：5’-TGTTCTT-TTGGCGAATCATGACACTAA-3’

SB3-P2：5’-F3-TGTTCTT-GGGAAGCCTCAAGGAATAAC-3’，

The long aligning primer of D21S2052 locus:

SA-P1：5’-TGTTCTT-GCACCCTTTATACTTGGGTG-3’

SB3-P2：5’-F3-TGTTCTT-TAGTACTCTACCATCCATCTATCCC-3’；

The long aligning primer of Penta D locus:

SA-P1：5’-TGTTCTT-ATTAGAATTCTTTAATCTGGACACAAG-3’

SB3-P2：5’-F3-TGTTCTT-GAAGGTCGAAGCTGAAGTG-3’；

B divides the primer mixture of kit to be made of the long aligning primer that attaches together together LFG20, LFG29, LFG33, LFG34D21S1435 and six locus of D21S1436, wherein:

The long aligning primer of LFG20 locus:

SA-P1：5’-TGTTCTT-TGAGGTAGGTTCCTTAGCCC-3’

B1-P2：5’-F1-TGTTCTT-AAGGCCTCCCCTATGTTATG-3’，

The long aligning primer of D21S1436 locus:

SA-P1：5’-TGTTCTT-AGGAAAGAGAAAGAAAGGAAGG-3’

SB1-P2：5’-F1-TGTTCTT-TATATGATGAAAGTATATTGGGGG-3’，

The long aligning primer of LFG29 locus:

SA-P1：5’-TGTTCTT-TGGAAAATTTGCTTGAGAGG-3’

SB2-P2：5’-F2-TGTTCTT-TGAACCCAGGAGTCAGTTTG-3’，

The long aligning primer of LFG33 locus:

SA-P1：5’-TGTTCTT-CACCATACCCAGCCTTACTG-3’

SB2-P2：5’-F2-TGTTCTT-GCTCCCAAGCTACAGACCTA-3′，

The long aligning primer of LFG34 locus:

SA-P1：5’-TGTTCTT-CACAAGGCAGAATAAAGGGA-3’

SB3-P2：5’-F3-TGTTCTT-AGCATGTGTCCTGAATCTTTG-3’，

The long aligning primer of D21S1435 locus:

SA-P1：5’-TGTTCTT-CCCTCTCAATTGTTTGTCTACC-3’

SB3-P2：5’-F3-TGTTCTT-GCAAGAGATTTCAGTGCCAT-3’；

In the long aligning primer of described each locus, F1, F2 and F3 are respectively the fluorescent marker of different colours;

A divides the allelic ladder potpourri of kit to be made of the allelic ladder that attaches together together LFG21, LFG24, LFG26, D21S1409, D21S2052 and six locus of Penta D, and the allelic ladder of described each locus is the long aligning primer that utilizes each locus to this locus observed all allele in colony resulting product that increases;

B divides the allelic ladder potpourri of kit to be made of the allelic ladder that attaches together together LFG20, LFG29, LFG33, LFG34 D21S1435 and six locus of D21S1436, and the allelic ladder of described each locus is the long aligning primer that utilizes each locus to this locus observed all allele in colony resulting product that increases;

The amount ratio of each component is in each minute kit: each 0.25～0.35ml (40nM) of the long aligning primer of each locus in the primer mixture, archaeal dna polymerase 3000u (3u/ μ l); Damping fluid 3.25～4.25ml; MgCl ₂2.5～3.5ml (2.25mM); Allelic ladder amount of the mixture 6ml (40nM), the allelic ladder equivalent of contained each locus in the allelic ladder potpourri.

Below technical conceive of the present invention is further described.

Kit of the present invention comprises that two of A, B independently divide kit, and each divides kit by the primer mixture, archaeal dna polymerase, damping fluid, the MgCl that separate packing ₂Constitute with the allelic ladder potpourri, primer mixture is wherein mixed by 6 pairs of fluorescently-labeled long aligning primers respectively, and the long aligning primer of this 6 couple corresponds respectively to 6 specific STR sites on No. 21 chromosome.Because two branch kits of A, B detect 6 No. 21 chromosome specific STR sites respectively, therefore kit of the present invention detects 12 No. 21 chromosome specific STR sites altogether.When observing tested sample and in these 12 STR sites, have plural site to have three allele peaks, assert the individuality of this sample from Down syndrome.

In kit of the present invention, primer design has adopted the primer design method of the applicant's related multiple colour fluorescent composite amplification in Chinese invention patent ZL 200310104034.7 in the primer mixture.This method is with one section common sequence

SA(5’--TGTTCTT-3’)

With add fluorescent marker F at 5 of above-mentioned common sequence ' end after formed fluorescence labeling sequence

SB(5’--F-TGTTCTT-3’)

It is right to constitute short sequence; Above-mentioned short sequence to being added in 5 ' end of Oligonucleolide primers P1, the P2 that can combine with the human genomic sequence specificity, is constituted a pair of non-human genome sequence, promptly long aligning primer SA-P1, SB-P2.Wherein, Oligonucleolide primers P1, the P2 that can combine that is be the original primer of locus to be amplified with the human genomic sequence specificity.Because the composite amplification reaction is the pcr amplification that simultaneously a plurality of locus is carried out, with respect to the different genes seat, can be different with Oligonucleolide primers P1, the P2 of the human genomic sequence specificity combination of this locus, corresponding with it, after 5 of original primer P1, the P2 of different genes seat ' end added a pair of identical short sequence, the long aligning primer of formation was also inequality because of the difference of P1, P2.In the phase one of composite amplification, pcr amplification is incorporated into the genomic short sequence of non-human in the amplified production, makes it become the template of subordinate phase reaction.Therefore, the sequence of the amplified production of phase one with regard to having target gene fragment simultaneously and matching with short sequence.Like this, after the pcr amplification of phase one, long aligning primer promptly can be used as the reaction primer and plays a role.In the method, the interpolation of short sequence has played two aspects: at first, the short sequence and the specific original primer of artificial design have formed one section non-human genome sequence (promptly long aligning primer).As the reaction primer, the increase of primer length in addition significantly increases the PCR atopic with the non-human genome sequence.On the other hand, experiment showed, that this section sequence can promote 3 ' terminal the add adenylate A of Taq enzyme at amplified production, makes all amplified productions have same end.This is very crucial to fluorescent detection system.Because if it is incomplete that the PCR product adds adenylate A, have in the amplified production and add adenylate A and two kinds of molecules that do not add adenylate A, when fluoroscopic examination, can form double peak, make the Genotyping difficulty.In addition, 5 ' terminal short sequence of adding is identical, has avoided owing to add the interaction that different short sequences may occur.Because the 5 ' terminal base number that adds is few, in two stages of composite amplification, the Tm value of reaction primer is more or less the same.Therefore, optimizing reaction system is relative with amplification condition simpler.Since in a reaction system, have many to primer a plurality of target sequences that increase simultaneously, thereby can simplify procedures significantly, save the running time, reached the purpose that aneuploid is detected with a small amount of sample.

In the present invention, each to divide the composite amplification of 6 str locus seats in kit to relate to three pairs of different short sequences right.At common sequence SA

SA(5’--TGTTCTT?3’)

5 ' end add and just can obtain 3 fluorescence labeling sequences by fluorescent marker F1, F2, the F3 that 3 kinds of compositions and color have nothing in common with each other

SB1(5’--F1-TGTTCTT-3’)

SB2(5’--F2-TGTTCTT-3’)

SB3(5’--F3-TGTTCTT-3’)

These 3 fluorescence labeling sequences match with above-mentioned common sequence SA respectively, promptly constitute short sequence to " SA, SB1 ", short sequence to " SA, SB2 " and weak point sequence to " SA, SB3 ".Above-mentioned fluorescent marker F1, F2, F3 can adopt the fluorescent marker of existing different colours respectively, as existing commercially available indigo plant, green, red fluorescence label FAM, JOE and ROX and other similar fluorescent marker.

Long aligning primer described in this kit promptly be 5 ' end at above-mentioned original primer P1, the P2 that can combine with the human genomic sequence specificity add respectively the short sequence of this three couple to and constitute.Its concrete mode is:

5 ' end at Oligonucleolide primers P1, the P2 that can combine with the human genomic sequence specificity adds a pair of short sequence respectively

SA(5’--TGTTCTT-3’)

SB1(5’--F1-TGTTCTT-3’)，

Constitute a pair of non-human genome sequence, promptly long aligning primer SA-P1, SB1-P2;

5 ' end at Oligonucleolide primers P1, the P2 that can combine with the human genomic sequence specificity adds a pair of short sequence respectively

SA(5’--TGTTCTT-3’)

SB2(5’--F2-TGTTCTT-3’)

Constitute a pair of non-human genome sequence, promptly long aligning primer SA-P1, SB2-P2;

5 ' end at Oligonucleolide primers P1, the P2 that can combine with the human genomic sequence specificity adds a pair of short sequence respectively

SA(5’--TGTTCTT-3’)

SB3(5’--F3-TGTTCTT-3’)

Constitute a pair of non-human genome sequence, promptly long aligning primer SA-P1, SB3-P2;

In this kit, the selection in above-mentioned No. 21 chromosome specific STR sites is very crucial.Choosing of specific STR site is based on following consideration: 1, select the good STR site of polymorphism.So-called polymorphism is meant that there are two or more allelic phenomenons in a locus in the same colony.The polymorphism degree of a locus is high more, and using this locus, to carry out the usefulness of genetic analysis just high more.Most STR site all has the polymorphism of height, and its polymorphism comes from the individual difference of core sequence multiplicity.It has been generally acknowledged that duplicating slippage is the mechanism that this species diversity forms.The STR site that polymorphism is good can produce the different pcr amplification product of length in most of individuality of normal population, promptly concerning the good site of each polymorphism, most normal individual all can be a heterozygote, therefore the quantitative fluorescence analysis of PCR product can show as two fluorescence peaks, corresponding to two allele in this site.In chromosomal aneuploid, extra chromosome is many from parent.Loci polymorphism is good more, and it is just big more that parent has not homoallelic possibility.Simultaneously, the possibility different with male parent's allele is also big, and chromosomal aneuploid shows as three fluorescence peaks in this site possibility is also just big more.The polymorphism of heterozygosity loci commonly used is carried out qualitative assessment in Population Genetics.Therefore, when setting up detection system, should select for use heterozygosity greater than 0.6 STR site.2, increase selected STR site number.Concerning the detection system of specific chromosome trisome, its usefulness not only depends on the polymorphism in STR site, and is also relevant with the number in STR site.Undoubtedly, when the STR number of loci of analyzing increases, therefore the usefulness of system will increase, and observes chromosomal aneuploid in plural STR site and has three not homoallelic possibilities and will increase, and aneuploid determined also just more to have meaning on the statistics.According to our research, should select the STR site more than 6 at least for use.3, the STR site of selecting tetranucleotide or pentanucleotide to repeat, promptly the core sequence in site is made of 4bp or 5bp nucleotide.The little satellite of this two class not only has the polymorphism of height, and amplification is more loyal, and somatotype is also more accurate.

Based on mentioned above principle, we have selected the detection site of 12 No. 21 chromosome specific STR sites as kit of the present invention, the STR site that wherein has 3 pentanucleotides to repeat, the heterozygosity that the STR sites that 9 tetranucleotides repeat, mass survey confirm these sites in China Han colony all greater than 0.6.Because present public database lacks the microsatellite locus that the pentanucleotide of No. 21 chromosome specifics repeats, polymorphism No. 21 chromosome specific microsatellite locus good, that tetranucleotide repeats are also few, therefore we utilize the human genome sequencing result, 7 No. 21 new chromosome specific microsatellite locus have been developed, designed corresponding original primer, and be applied in this kit, comprising the microsatellite locus that 2 pentanucleotides repeat, 5 microsatellite locus that tetranucleotide repeats.Other 5 STR sites and original primer thereof can obtain by disclosed human genome database retrieval on the internet in the kit.

As previously mentioned, totally 7 in our independently developed No. 21 chromosome specific STR sites, called after LFG20, LFG21, LFG24, LFG26, LFG29, LFG33, LFG34 respectively.

Site LFG20 is little satellite that a pentanucleotide repeats, and core sequence is (CATAAxCATAG), and original primer is

P1：5’-TGAGGTAGGTTCCTTAGCCC-3’

P2：5’-AAGGCCTCCCCTATGTTATG-3’

Site LFG21 is little satellite that a pentanucleotide repeats, and core sequence is (GAATA), and original primer is

P1：5’-TTGGCGAATCATGACACTAA-3’

P2：5’-GGGAAGCCTCAAGGAATAAC-3’

Site LFG24 is little satellite that a tetranucleotide repeats, and core sequence is (TATGxCATGxTATC), and original primer is

P1：5’-AGGTTCTAGGAAACATGGCTG-3’

P2：5’-TGCAAAACTGCTCTGGACTT-3’

Site LFG26 is little satellite that a tetranucleotide repeats, and core sequence is (TATC), and original primer is

P1：5’-TTAGAGTTGACAAACTCCATGTTG-3’

P2：5’-TCTGAGCTAGTTGGGAGGATAG-3’

Site LFG29 is little satellite that a tetranucleotide repeats, and core sequence is (ACTTxATTT), and original primer is

P1：5’-TGGAAAATTTGCTTGAGAGG-3’

P2：5’-TGAACCCAGGAGTCAGTTTG-3’

Site LFG33 is little satellite that a tetranucleotide repeats, and core sequence is (ATAGxATAG), and original primer is

P1：5’-CACCATACCCAGCCTTACTG-3’

P2：5’-GCTCCCAAGCTACAGACCTA-3’

Site LFG34 is little satellite that a tetranucleotide repeats, and core sequence is (TAGGxTAGAxAGAT), and original primer is

P1：5’-CACAAGGCAGAATAAAGGGA-3’

P2：5’-AGCATGTGTCCTGAATCTTTG-3’

Other five STR sites are selected from public human genome database in this kit, are respectively D21S1435, D21S1436, D21S2052, Penta D.The original primer of the D21S1409 locus of announcing in the online GDB gene database is

P1：5’-GGAGGGGAATACATTTGTG-3’

P2：5’-TTGCCTCTGAATATCCCTAT-3’

The original primer of D21S1435 locus:

P1：5’-CCCTCTCAATTGTTTGTCTACC-3’

P2：5’-GCAAGAGATTTCAGTGCCAT-3’

The original primer of D21S1436 locus:

P1：5’-AGGAAAGAGAAAGAAAGGAAGG-3’

P2：5’-TATATGATGAAAGTATATTGGGGG-3’

The original primer of D21S2052 locus:

P1：5’-GCACCCTTTATACTTGGGTG-3’

P2：5’-TAGTACTCTACCATCCATCTATCCC-3’

The original primer of Penta D locus:

P1：5’ATTAGAATTCTTTAATCTGGACACAAG-3’

P2：5’GAAGGTCGAAGCTGAAGTG-3’；

Kit among the present invention is divided into two groups with above-mentioned 12 No. 21 chromosome specific STR sites, every group of 6 sites, and pack into two of A, B of the long aligning primer branch of each site correspondence independently divides kit.In testing process, if the testing result of a branch kit has reached the diagnostic criteria of Down's syndrome, be that sample shows as three allele peaks in plural No. 21 chromosome specific STR sites, just need not to detect with second branch kit further again, can save time as much as possible like this, sample and consumptive material.

In kit of the present invention, it is LFG21, LFG24, LFG26, D21S1435, D21S2052 and Penta D that A divides the detection site of kit, and these 6 sites are divided into I, II, III group, and the I group comprises site LFG24 and Penta D, and selected short sequence is:

SA(5/--TGTTCTT-3′)

SB1(5/--F1-TGTTCTT-3′)

Long aligning primer SA-P1, SB1-P2 are for adding the non-human genome sequence that SA, SB1 obtain at 5 of original primer P1, the P2 of locus LFG24 and D21S2054 ' end respectively.

The long aligning primer of LFG24 locus:

SA-P1：5/-TGTTCTT-AGGTTCTAGGAAACATGGCTG-3′

SB1-P2：5/-F1-TGTTCTT-TGCAAAACTGCTCTGGACT-3′

The long aligning primer of Penta D locus:

SA-P1：5’-TGTTCTT-ATTAGAATTCTTTAATCTGGACACAAG-3’

SB3-P2：5’-F3-TGTTCTT-GAAGGTCGAAGCTGAAGTG-3’；

The II group comprises site LFG26 and D21S1409, and selected short sequence is:

SA(5’--TGTTCTT-3’)

SB2(5’--F2-TGTTCTT-3’)

Long aligning primer SA-P1, SB2-P2 are for adding the non-human genome sequence that SA, SB2 obtain at 5 of original primer P1, the P2 of locus LFG26 and D21S1409 ' end respectively.

The long aligning primer of LFG26 locus:

SA-P1：5’-TGTTCTT-TTAGAGTTGACAAACTCCATGTTG-3’

SB2-P2：5’-F2-TGTTCTT-TCTGAGCTAGTTGGGAGGATAG-3’

The long aligning primer of D21S1409 locus:

SA-P1：5’-TGTTCTT-GGAGGGGAATACATTTGTG-3’

SB2-P2：5’-F2-TGTTCTT-TTGCCTCTGAATATCCCTAT-3’

The III group comprises site LFG21 and D21S2052, and selected short sequence is:

SA(5’--TGTTCTT-3’)

SB3(5’--F3-TGTTCTT-3’)

Long aligning primer SA-P1, SB3-P2 are for adding the non-human genome sequence that SA, SB3 obtain at 5 of original primer P1, the P2 of locus LFG21 and D21S2052 ' end respectively.

The long aligning primer of LFG21 locus:

SA-P1：5’-TGTTCTT-TTGGCGAATCATGACACTAA-3’

SB3-P2：5’-F3-TGTTCTT-GGGAAGCCTCAAGGAATAAC-3’

The long aligning primer of D21S2052 locus:

SA-P1：5’-TGTTCTT-GCACCCTTTATACTTGGGTG-3’

SB3-P2：5’-F3-TGTTCTT-TAGTACTCTACCATCCATCTATCCC-3’

It is LFG20, LFG29, LFG33, LFG34 D21S1435 and D21S1436 that B divides the detection site of kit, and these 6 sites are divided into I, II, III group, and the I group comprises site LFG20 and LFG29, and selected short sequence is:

SA(5’--TGTTCTT-3’)

SB1(5’--F1-TGTTCTT-3’)

Long aligning primer SA-P1, SB1-P2 are for adding the non-human genome sequence that SA, SB1 obtain at 5 of original primer P1, the P2 of locus LFG20 and D21S1436 ' end respectively.

The long aligning primer of LFG20 locus:

SA-P1：5’-TGTTCTT-TGAGGTAGGTTCCTTAGCCC-3’

B1-P2：5’-F1-TGTTCTT-AAGGCCTCCCCTATGTTATG-3’

The long aligning primer of D21S1436 locus:

SA-P1：5’-TGTTCTT-AGGAAAGAGAAAGAAAGGAAGG-3

SB1-P2：5’-F1-TGTTCTT-TATATGATGAAAGTATATTGGGGG-3’

The II group comprises site LFG29 and LFG33, and selected short sequence is:

SA(5’--TGTTCTT-3’)

SB2(5’--F2-TGTTCTT-3’)

Long aligning primer SA-P1, SB2-P2 are for adding the non-human genome sequence that SA, SB2 obtain at 5 of original primer P1, the P2 of locus LFG26 and D21S1409 ' end respectively.

The long aligning primer of LFG29 locus:

SA-P1：5’-TGTTCTT-TGGAAAATTTGCTTGAGAGG-3’

SB2-P2：5’-F2-TGTTCTT-TGAACCCAGGAGTCAGTTTG-3’

The long aligning primer of LFG33 locus:

SA-P1：5’-TGTTCTT-CACCATACCCAGCCTTACTG-3’

SB2-P2：5’-F2-TGTTCTT-GCTCCCAAGCTACAGACCTA-3’

The III group comprises site LFG34 and D21S1435, and selected short sequence is:

SA(5’--TGTTCTT-3’)

SB3(5’--F3-TGTTCTT-3’)

Long aligning primer SA-P1, SB3-P2 are for adding the non-human genome sequence that SA, SB3 obtain at 5 of original primer P1, the P2 of locus LFG34 and D21S1435 ' end respectively.

The long aligning primer of LFG34 locus:

SA-P1：5’-TGTTCTT-CACAAGGCAGAATAAAGGGA-3’

SB3-P2：5’-F3-TGTTCTT-AGCATGTGTCCTGAATCTTTG-3’

The long aligning primer of D21S1435 locus:

SA-P1：5’-TGTTCTT-CCCTCTCAATTGTTTGTCTACC-3’

SB3-P2：5’-F3-TGTTCTT-GCAAGAGATTTCAGTGCCAT-3’

In the present invention, described allelic ladder potpourri is mixed by the allelic ladder of six No. 21 chromosome specific str locus seats in each minute kit; Allelic ladder is meant the dna fragmentation group of the known array that is used for the STR somatotype, it is the tester that carries out result's somatotype when detecting, it allows uses the resulting composite amplification product of this kit can both carry out somatotype accurately, that is to say, the use of somatotype reference material helps to judge specific amplified production, get rid of the interference of non-specific amplification product, further guaranteed the reliability of testing result.In the present invention, divide the allelic ladder equivalent of each locus in the kit, be meant that promptly the gram molecular weight of the allelic ladder of each locus all equates.

In kit of the present invention, in the primer mixture each primer at the amplification site be the STR site that the good tetranucleotide of polymorphism or pentanucleotide repeat, promptly the detection site of kit has been passed through strict screening; Two independently A, B divide the primer setting in the kit to detect 6 STR sites respectively, thereby make the detection site sum of kit reach 12, guaranteed the usefulness of detection system, make detection that statistical meaning more be arranged.This kit has utilized the composite amplification technology, can carry out disposable amplification simultaneously, the purpose that real realization is quick, high flux ground is made a definite diagnosis Down syndrome to the site in minute kit.Simultaneously, this kit has adopted specific detection decision method, when promptly only plural specificity STR site has three allele peaks on observing specific chromosome, assert that just this chromosome is aneuploid, this just can make the judgement of testing result directly perceived more and clear and definite.In addition, in testing process, if a testing result of independently dividing kit has reached the diagnostic criteria of Down's syndrome, be that sample shows as three allele peaks in plural No. 21 chromosome specific STR sites, just need not independently to divide kit to detect with second further again, can save time as much as possible like this, sample and consumptive material.Therefore, the present invention has very high practical value.

Content of the present invention further illustrates with the following Examples, but content of the present invention is not limited only to content related among the embodiment.

Embodiment

Embodiment: the down's syndrome diagnose kit in the present embodiment comprises that two of A, B independently divide kit, and each minute, kit was by the primer mixture, archaeal dna polymerase, damping fluid, the MgCl that separate packing ₂Constitute with the allelic ladder potpourri; A divides the primer mixture of kit to be made of the long aligning primer that attaches together together LFG21, LFG24, LFG26, D21S1435, D21S2052 and six locus of Penta D, wherein:

The long aligning primer of LFG24 locus:

SA-P1：5/-TGTTCTT-AGGTTCTAGGAAACATGGCTG-3′

SB1-P2：5′-F1-TGTTCTT-TGCAAAACTGCTCTGGACT-3′，

The long aligning primer of Penta D locus:

SA-P1：5’-TGTTCTT-ATTAGAATTCTTTAATCTGGACACAAG-3’

SB3-P2：5-F3-TGTTCTT-GAAGGTCGAAGCTGAAGTG-3；

The long aligning primer of LFG26 locus:

SA-P1：5′-TGTTCTT-TTAGAGTTGACAAACTCCATGTTG-3′

SB2-P2：5/-F2-TGTTCTT-TCTGAGCTAGTTGGGAGGATAG-3′，

The long aligning primer of D21S1409 locus:

SA-P1：5′-TGTTCTT-GGAGGGGAATACATTTGTG-3′

SB2-P2：5/-F2-TGTTCTT-TTGCCTCTGAATATCCCTAT-3?′，

The long aligning primer of LFG21 locus:

SA-P1：5′-TGTTCTT-TTGGCGAATCATGACACTAA-3′

SB3-P2：5/-F3-TGTTCTT-GGGAAGCCTCAAGGAATAAC-3′，

The long aligning primer of D21S2052 locus:

SA-P1：5′-TGTTCTT-GCACCCTTTATACTTGGGTG-3′

SB3-P2：5/-F3-TGTTCTT-TAGTACTCTACCATCCATCTATCCC-3′；

The primer mixture that B divides kit constitutes by attaching together at-long the aligning primer of LFG20, the LFG29, LFG33, LFG34D21S1435 and six locus of D21S1436 that rise, wherein:

The long aligning primer of LFG20 locus:

SA-P1：5′-TGTTCTT-TGAGGTAGGTTCCTTAGCCC-3′

B1-P2：5/-F1-TGTTCTT-AAGGCCTCCCCTATGTTATG-3′，

The long aligning primer of D21S1436 locus:

SA-P1：5′-TGTTCTT-AGGAAAGAGAAAGAAAGGAAGG-3′

SB1-P2：5/-F1-TGTTCTT-TATATGATGAAAGTATATTGGGGG-3′，

The long aligning primer of LFG29 locus:

SA-P1：5′-TGTTCTT-TGGAAAATTTGCTTGAGAGG-3′

SB2-P2：5/-F2-TGTTCTT-TGAACCCAGGAGTCAGTTTG-3′，

The long aligning primer of LFG33 locus:

SA-P1：5′-TGTTCTT-CACCATACCCAGCCTTACTG-3′

SB2-P2：5/-F2-TGTTCTT-GCTCCCAAGCTACAGACCTA-3′，

The long aligning primer of LFG34 locus:

SA-P1：5′-TGTTCTT-CACAAGGCAGAATAAAGGGA-3′

SB3-P2：5/-F3-TGTTCTT-AGCATGTGTCCTGAATCTTTG-3′，

The long aligning primer of D21S1435 locus:

SA-P1：5/-TGTTCTT-CCCTCTCAATTGTTTGTCTACC-3′

SB3-P2：5′-F3-TGTTCTT-GCAAGAGATTTCAGTGCCAT-3′；

In the long aligning primer of described each locus, F1, F2 and F3 are respectively the fluorescent marker of different colours, promptly commercially available indigo plant, green, red fluorescence label FAM, JOE and ROX.

The structural formula of commercially available blue-fluorescence label FAM is

The structural formula of commercially available green fluorescence label JOE is

The structural formula of commercially available red fluorescence label ROX is

A divides the allelic ladder potpourri of kit to be made of the allelic ladder that attaches together together LFG21, LFG24, LFG26, D21S1435, D21S2052 and six locus of Penta D, and the allelic ladder of described each locus is that the long aligning primer of described each locus is to this locus observed all allele in colony resulting product that increases.When concrete the making, above-mentioned allelic ladder makes in the following manner: with increase the respectively individual specimen of 12 No. 21 chromosome specific str locus seat different genotype of the long aligning primer of unmarked fluorescence, electrophoretic separation, cma staining downcuts the whole allele purpose of each locus fragment gel; With increase the respectively allele gel soak solution of each locus of fluorescently-labeled long aligning primer, each allele PCR product of 6 locus that A, B two kits is related separately mixes respectively, promptly obtains the allelic ladder potpourri of two branch kits.

B divides the allelic ladder potpourri of kit to be made of the allelic ladder that attaches together together LFG20, LFG29, LFG33, LFG34 D21S1435 and six locus of D21S1436, and the allelic ladder of described each locus is that the long aligning primer of described each locus is to this locus observed all allele in colony resulting product that increases.B divides the concrete method for making of somatotype reference material potpourri in the kit to divide kit identical with A.

Divide in the kit at A, B, the consumption of each component is: each 0.25～0.35ml (40nM) of the long aligning primer of each locus in the primer mixture, archaeal dna polymerase 3000u (3u/ μ l); Damping fluid 3.25～4.25ml; MgCl ₂2.5～3.5ml (2.25mM); Allelic ladder amount of the mixture 6ml (40nM), the allelic ladder of contained each locus is 1ml (40nM) in the potpourri of allelic ladder.

Using method and result of use for further specifying kit in the present embodiment go on to say the detailed process that this kit is used for diagnosis of down syndrome below.

Testing process is carried out according to the following steps:

A, can utilize this kit to carry out antenatal exaination to the pregnant woman who meets following indication: 1, pregnant woman age surpasses 35 years old, belongs to the lying-in woman advanced in years.2, serology examination test is positive.3, fetal abnormality is found in ultrasonic examination.Family history or childbearing history that 4, chromosome abnormality is arranged.5, initiatively requirement of pregnant woman and family members thereof.In addition, diagnose doubting clinically to the also available kit of the infant of Down syndrome.

B, employing phenol-chloroform method or Chelex method are extracted DNA from sample.Be used for the amniotic fluid of the fetal cell sample of pre-natal diagnosis in the present embodiment, get its venous blood more doubting clinically to the infant of Down syndrome from parent.

C, employing divide kit A to be increased in 6 in the sample DNA No. 21 chromosome specific STR sites.

The consumption of each composition is as follows in the PCR reaction system:

DDH ₂O： 12.4μL

dNTP： 7.5μl

10Xbuffer 3.75μL、

6 couples of each 0.3 μ l of long aligning primer

Taq enzyme 1 μ l (3u)

BSA 3.75μL

MgCl2 3μl

Dna profiling: 2.5 μ L (about 3ng)

Cumulative volume: 37.5 μ L

Composite amplification is reflected in the PE-9600 amplification instrument and carries out, and adopts the warm start technology, circulates altogether 28 to take turns, and loop parameter is as follows:

Pre-sex change: 94 ℃ 3 minutes

Sex change: 94 ℃ 50 seconds

Renaturation: 54 ℃ 50 seconds

Extend: 72 ℃ of 4 circulations in 30 seconds

Sex change: 94 ℃ 50 seconds

Renaturation: 54 ℃ 30 seconds

Extend: 72 ℃ of 24 circulations in 30 seconds

Extend: 72 ℃ 10 minutes

D, the number of the corresponding fluorescence peak of allele is recognized, when observing plural No. 21 chromosome specific STR sites and have three allele peaks, assert that this sample is from the Down syndrome individuality.In the present embodiment, utilize ABI310 genetic analyzer (PE, the U.S.) that the resulting pcr amplification product of above-mentioned composite amplification process is carried out check and analysis.Number with PCR product 0.4 μ l and somatotype reference material GS500 ROX size standard 0.2 μ l, denaturant Hi-DiTMformamide3 μ l mixing, put into the auto injection dish.Electricity sample introduction 15000V, 5s. electrophoresis 15000V, 24 minutes.Collect data with DateCollection software, Genescan3.7 software analysis data use the AmpFISTR PLUS kit Kazam macro file of revising at Genetype3.7 software automatic parting direction.Allelic identification is by relatively confirming with allelic ladder, window ranges+/-0.5bp. is like this, whether have plural STR site to have three specific allele peaks in the parting line zone by Direct observation, whether we can detect examined samples from the Down syndrome individuality.

If divide kit A the testing result of sample to be reached the diagnostic criteria of Down's syndrome, be that sample shows as three allele peaks in plural No. 21 chromosome specific STR sites, just need not again further with dividing a kit B to detect, can save time as much as possible like this, sample and consumptive material.We detect 18 Down syndrome infants with the down's syndrome diagnose kit among the present invention, are goldstandard with traditional cytogenetic diagnosis method, and testing result has been carried out preliminary statistical analysis.Herein, said goldstandard is meant the current generally acknowledged the most reliable method that diagnoses the illness.The result shows that the susceptibility of this kit is 77.8%, and specificity is 100%, and accuracy is 94.9%.Divide kit A if use separately, susceptibility is 61.1%; Use separately and divide kit B, susceptibility is 50%.Therefore, there is the sample of half to need not to continue again to use branch kit B approximately.If divide the testing result of kit A negative, or the result is suspicious, when only having a str locus seat to show as three allele peaks, just must continues use branch kit B and do further detection.

Attached: genome sequence tabulation involved in the present invention:

<210>1

<211>339

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(122)...(265)

<400>1

TTAAAAGTTA?CAGGTTCTAG?GAAACATGGC?TGGTGAATAC?AATAATAATT?TATTAATTTT?60

AATGTCAGCT?TCAAATTTTT?GGTTCTTTGG?AAAATTGTTT?AGGCCCCCAA?ATATATAGGT?120

CTATGTATGT?ATGTATGTAT?GTATGTATGT?ATGTATGTAT?GTATGCATGC?ATGTATGCAT?180

GCATGCATGT?ATGTATGTAT?GTATGTATGT?ATGTATGTAT?CTATCTATCT?ATCTATCTAT?240

CTATCTATCT?ATCTATCTAT?CTATCTATAG?CTGCATTGAT?GCTGTAAGTC?CAGAGCAGTT?300

TTGCAAAATA?GGCTGTGTTG?CTAGAGAGAT?GCTATTTTC 339

<210>2

<211>249

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(87)...(134)

<400>2

ACCACTTTAG?AGTTGACAAA?CTCCATGTTG?GAACAGAGTA?TTCTTAAACA?GAACCCTTAA?60

AACCATATTT?TTCACCTCTC?TGTTTTTATC?TATCTATCTA?TCTATCTATC?TATCTATCTA?120

TCTATCTATC?TATCTATTGA?GACAGGGTCT?TGCTCTGTCA?CCCAGGCTGG?AGTGCAGTCG?180

TGTGATCACG?GTTCTCTGCA?GCCTCGATCT?CCTGGGCTCT?AGCTATCCTC?CCAACTAGCT?240

CAGATTACA 249

<210>3

<211>364

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(40)...(67)

<400>3

AGTTGATGTT?GGAGGGGAAT?ACATTTGTGT?AGGTAGGTAG?ATAGATAGAT?AGATAGATAG?60

ATAGATAGGT?CTGATTTATT?AAGAATCATT?CACCAGGTAT?TAGTGTTACA?TTTTAGAAAG?120

GGAGAAAGAG?AATAAGGGAG?AGAGCGTATG?TNCCAAGAGA?CAAAGAGACA?GAAAGATAGG?180

GATATTCAGA?GGCAAAAATA?CACGCATATG?AAGACTAAGA?GATAGANTCT?CAGAAACAAA?240

ACATAATTCA?TGAATCTCAA?CATTGGGAGA?TACTCATTGT?ACCTTATTTT?ACATGTCTAA?300

TCCTAACATT?ATAATACTAT?TAANCCTACA?ATGGTTAATT?GACTTTTAAG?CTTTCATTAA?360

GGCC 364

<210>4

<211>250

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(39)...(118)

<400>4

ATTGGCGAAT?CATGACACTA?ATTTTGGCCA?GCATTTACGA?ATAGAATAGA?ATAGAATAGA?60

ATAGAATAGA?ATAGAATAGA?ATAGAATAGA?ATAGAATAGA?ATAGAATAGA?ATAGAATAGA?120

ATGGATCAGA?GTAATATTAA?AGAGTATCTA?GATATTCATC?CACATTATAA?AGTTTTAGTT?180

CAGGCCTCTT?TTTCTTCTCC?ATCAAGTTAT?TCCTTGAGGC?TTCCCTGAAC?TTTTTATTTT?240

ATGCCTTTCT 250

<210>5

<211>297

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(26)...(92)

<400>5

GCACCCTTTA?TACTTGGGTG?TAGTNGATAG?ATAGATAGAT?AGATAGATAG?ATAGATAGAT?60

AGATGATAGA?TAGATAGATA?GACAGATAGA?TAGNTGGGAT?AGATGGATGG?TAGAGTACTA?120

TTATAAGGAA?TTGGCTCATG?TAATTCTGGA?GATCGGGCGT?GCTNAAAAAC?TGCAGTGAGN?180

AAGCTCGACA?TCCAGCAGAA?CTAACGGCCT?ACTTCCAGTC?TGNNNTCCAA?AGGCTTGAAA?240

NCCAGGAGNG?TTGATGNTGT?GNTTCTAGTC?TGATTCTAAN?GACCNGCNNG?NCCAGGG 297

<210>6

<211>430

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(95)...(159)

<400>6

GGAAGGTCGA?AGCTGAAGTG?AGCCATGATC?ACACCACTAC?ACTCCAGCCT?AGGTGACAGA?60

GCAAGACACC?ATCTCAAGAA?AGAAAAAAAA?GAAAGAAAAG?AAAAGAAAAG?AAAAGAAAAG?120

AAAAGAAAAG?AAAAGAAAAG?AAAAGAAAAG?AAAAGAAAAA?ACGAAGGGGA?AAAAAAGAGA?180

ATCATAAACA?TAAATGTAAA?ATTTCTCAAA?AAAATCGTTA?TGACCATAGG?TTAGGCAAAT?240

ATTTCTTAGA?TATCACAAAA?TCATGACCTA?TTAAAAAATA?ATAATAAAGT?AAGTTTCATC?300

AAAACTTAAA?AGTTCTACTC?TTCAAAAGAT?ACCTTATAAA?GAAAGTAAAA?AGACACGCCA?360

CAGGCTAAGA?GAAAGTACTT?CTAATCACAT?ATCTAAAAAA?GGACTTGTGT?CCAGATTAAA?420

GAATTCTTAC 430

<210>7

<211>300

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(149)...(228)

<400>7

ATTTATGAGG?TAGGTTCCTT?AGCCCCATTT?CACAGGCTGA?GAACCTGAGA?CTGTCAACAC?60

TAAGCTGATT?TTACTGTGAT?TACATCTTAG?TGAGTGACAG?ATCTAGCAAT?CATGGAATTG?120

AGAAGGCCCC?ACCTATGTTA?TGTTATAACA?TAACATAACA?TAACATAACA?TAACATAGCA?180

TAGCATAGCA?TAGCATAGCA?TAGCATAGCA?TAGCATAGCA?TAGCATAGCA?TGGCAACATA?240

ACATAACACA?GGGGGCCTTC?TCAATTATGT?TATAACATAA?CATAGGGGAG?GCCTTATAAT?300

<210>8

<211>386

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(104)...(155)

<400>8

AAAGCTTGCA?GTGAGCCTAG?ATTGGACCAC?CGTACTTCAG?CCTGGGCAAC?AGAGCAAGGC?60

TCTGTCTCAG?AAAAAAAAAA?AAAAAAAAGA?AAAGAAGGAA?AGAGAAAGAA?AGGAAGGAAG?120

GAAGGAAGGA?AGGAAGGAAG?GAAGGAAGGA?AGGAAGGGNG?AATGGAGTAG?GAAATAGGNC?180

ATAAATGGTA?AGCCTATATT?AATGTGTGTA?TATTATTCTG?CCTTTATACT?TTTCTGGNTA?240

ATTATCTTAC?CCCCAATATA?CTTTCATCAT?ATAATTTACC?TGAATGATAA?TAATAAAATC?300

CTTGCAATGA?NTTTANGCAG?CCACATATAT?TAGTGATACT?CTGAAAAAAA?TGTATCTGCA?360

AATNGGAAGN?TTAAATTGCT?TTNTTA 386

<210>9

<211>201

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(46)...(113)

<400>9

ACTTTCTGGT?TTGGAAAATT?TGCTTGAGAG?GTAAAAAGAA?AATTTACTTA?CTTACTTACT?60

TACTTACTTA?TTTATTTATT?TATTTATTTA?TTTATTTATT?TATTTATTTA?TTTAGTAGAG?120

ACAGAATCTC?GCTCTGTTGC?TCAGGCTGGA?GTGCAGTAGC?ACGATCCTGG?CTCACTGCAA?180

ACTGACTCCT?GGGTTCAGGT?G 201

<210>10

<211>167

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(36)...(98)

<400>10

CAGGTGTGAG?CCACCATACC?CAGCCTTACT?GTATCATAGA?TAGATAGATA?GATAGATAGA?60

TGATAGATAG?ATAGATAGAT?AGATAGATAG?ATAGATAGGA?ATATAGTTGC?CTACAATATT?120

CAGCACAGTA?ACATGCTATA?TAGGTCTGTA?GCTTGGGAGC?AATAGGC 167

<210>11

<211>250

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(42)...(132)

<400>11

CTATTTGGTA?CACAAGGCAG?AATAAAGGGA?TTATTGCTTG?ATAGGTAGGT?AGGTAGGTAG?60

GTAGGTAGGT?AGATAGATAG?ATAGATAGAA?GATAGATAGA?TAGATAGATA?GATAGATAGA?120

TAGATAGATA?GATGATAGAT?GAGACAAGAC?AAGACAGACT?ATGAATAAAT?TAATCATACT?180

GCTGTTTCTA?ACTCAAAGAT?TCAGGACACA?TGCTTTTGAC?TTAATTCTTC?CATGTAGCAT?140

CTATAATATT 250

<210>12

<211>367

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>repeat_region

<222>(56)...(106)

<400>12

TTGACATTCT?TCTGTAAGGA?AGAGNCCCAT?TCCCCTCTCA?ATTGTTTGTC?TACCCTATCT?60

ATCATCTATC?TATCTATCTA?TCTATCTATC?TATCTATCTA?TCTATCTAAT?CTTCTATTAT?120

CTATCTATCT?ATCTAAAAAT?CATTTCACAG?GACTCAATAA?TATACGTCAA?ATCTCATTTT?180

CCTTGATGGC?ACTGAAATCT?CTTGCTTTCC?TTTTGTTAGT?ATTTGACTTT?TAATATCTTT?240

GGCAAGCCTT?NTGTTACAGT?TTNACTATAT?GATNTCNTTT?CAGATGNGGT?ATATCNCAAA?300

CCTGGNTTTN?AAAAANANGG?GGTAATATGG?TNCACTTTNC?CCNTGGTAGG?NCTNGAGGTT?360

CTNGAAN 367

Claims (1)

1. a down's syndrome diagnose kit is characterized in that described kit comprises that two of A, B independently divide kit, and described each minute, kit was by the primer mixture, archaeal dna polymerase, damping fluid, the MgCl that separate packing ₂Constitute with the allelic ladder potpourri;

A divides the primer mixture of kit to be made of the long aligning primer that attaches together together LFG21, LFG24, LFG26, D21S1409, D21S2052 and six locus of Penta D, wherein,

The long aligning primer of LFG24 locus:

SA-P1：5’-TGTTCTT-AGGTTCTAGGAAACATGGCTG-3’

SB1-P2：5’-F1-TGTTCTT-TGCAAAACTGCTCTGGACT-3’，

The long aligning primer of LFG26 locus:

SA-P1：5’-TGTTCTT-TTAGAGTTGACAAACTCCATGTTG-3’

SB2-P2：5’-F2-TGTTCTT-TCTGAGCTAGTTGGGAGGATAG-3’，

The long aligning primer of D21S1409 locus:

SA-P1：5’-TGTTCTT-GGAGGGGAATACATTTGTG-3’

SB2-P2：5’-F2-TGTTCTT-TTGCCTCTGAATATCCCTAT-3’，

The long aligning primer of LFG21 locus:

SA-P1：5’-TGTTCTT-TTGGCGAATCATGACACTAA-3’

SB3-P2：5’-F3-TGTTCTT-GGGAAGCCTCAAGGAATAAC-3’，

The long aligning primer of D21S2052 locus:

SA-P1：5’-TGTTCTT-GCACCCTTTATACTTGGGTG-3’

SB3-P2：5’-F3-TGTTCTT-TAGTACTCTACCATCCATCTATCCC-3’；

The long aligning primer of Penta D locus:

SA-P1：5’-TGTTCTT-ATTAGAATTCTTTAATCTGGACACAAG-3’

SB3-P2：5’-F3-TGTTCTT-GAAGGTCGAAGCTGAAGTG-3’；

B divides the primer mixture of kit to be made of the long aligning primer that attaches together together LFG20, LFG29, LFG33, LFG34D21S1435 and six locus of D21S1436, wherein,

The long aligning primer of LFG20 locus:

SA-P1：5’-TGTTCTT-TGAGGTAGGTTCCTTAGCCC-3’

B1-P2：5’-F1-TGTTCTT-AAGGCCTCCCCTATGTTATG-3’，

The long aligning primer of D21S1436 locus:

SA-P1：5’-TGTTCTT-AGGAAAGAGAAAGAAAGGAAGG-3’

SB1-P2：5/-F1-TGTTCTT-TATATGATGAAAGTATATTGGGGG-3’，

The long aligning primer of LFG29 locus:

SA-P1：5’-TGTTCTT-TGGAAAATTTGCTTGAGAGG-3’

SB2-P2：5’-F2-TGTTCTT-TGAACCCAGGAGTCAGTTTG-3’，

The long aligning primer of LFG33 locus:

SA-P1：5’-TGTTCTT-CACCATACCCAGCCTTACTG-3’

SB2-P2：5’-F2-TGTTCTT-GCTCCCAAGCTACAGACCTA-3’，

The long aligning primer of LFG34 locus:

SA-P1：5’-TGTTCTT-CACAAGGCAGAATAAAGGGA-3’

SB3-P2：5’-F3-TGTTCTT-AGCATGTGTCCTGAATCTTTG-3’，

The long aligning primer of D21S1435 locus:

SA-P1：5’-TGTTCTT-CCCTCTCAATTGTTTGTCTACC-3’

SB3-P2：5’-F3-TGTTCTT-GCAAGAGATTTCAGTGCCAT-3’；

In the long aligning primer of described each locus, F1, F2 and F3 are respectively the fluorescent marker of different colours;

A divides the allelic ladder potpourri of kit to be made of the allelic ladder that attaches together together LFG21, LFG24, LFG26, D21S1409, D21S2052 and six locus of Penta D, and the allelic ladder of described each locus is the long aligning primer that utilizes each locus to this locus observed all allele in colony resulting product that increases;

B divides the allelic ladder potpourri of kit to be made of the allelic ladder that attaches together together LFG20, LFG29, LFG33, LFG34 D21S1435 and six locus of D21S1436, and the allelic ladder of described each locus is the long aligning primer that utilizes each locus to this locus observed all allele in colony resulting product that increases;

The amount ratio of each component is in each minute kit: each 0.25～0.35ml (40nM) of the long aligning primer of each locus in the primer mixture, archaeal dna polymerase 3000u (3u/ μ l); Damping fluid 3.25～4.25ml; MgCl ₂2.5～3.5ml (2.25mM); Allelic ladder amount of the mixture 6ml (40nM), the allelic ladder equivalent of contained each locus in the allelic ladder potpourri.