CN102337338B - Method for simultaneously and quickly detecting number of five types of chromosomes, and kit and application thereof - Google Patents
Method for simultaneously and quickly detecting number of five types of chromosomes, and kit and application thereof Download PDFInfo
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Abstract
The invention discloses a method for simultaneously and quickly detecting number of five types of chromosomes, and a kit using the method and application of the kit. The method disclosed by the invention comprises the following steps of: carrying out amplification on the following STR sites simultaneously: D21S1270, D21S1411, D21S1412, D21S1414, D21S1280, D18S535, D18S51, D18S1002, D18S391, D18S386, D13S136, D13S294, D13S305, D13S240, D13S256, DXS337, X22, DXS8377, DXS981, DXYS218, DYS448, SRY and AMEL; modifying the obtained fluorescently-labeled PCR (Polymerase Chain Reaction) products; through capillary electrophoresis detection, indirectly reflecting the amount of the PCR products through fluorescence peak value areas in the corresponding positions, thereby determining the number of a number 21 chromosome, a number 18 chromosome, a number 13 chromosome and sex chromosomes. The kit, which realizes the method disclosed by the invention, comprises the primers of the sites. The kit can be used for simultaneously detecting the number of five types of chromosomes, such as the number 13 chromosome, the number 18 chromosome, the number 21 chromosome, the sex chromosomes and the like; a little sample is needed; and villus, amniotic fluid and umbilical bleeding samples can be used for diagnosis. The kit has low detection cost is low, and is more applicable to large-scale detection.
Description
Technical field
The present invention relates to a kind of detection chromosome number purpose method, particularly method and the test kit of adopting said method and the application of this test kit of five kinds of chromosome numbers of a kind of while rapid detection (13,18,21 and sex chromosome x, y).
Background technology
Chromosomal disorder is congenital numerical abnormalities of chromosomes or structural aberration and the disease that causes, is the common disease therefore that causes inborn defect, account for 1/800 live-born infant, and the whole world is on the rise.Owing to existing thousands of even up to ten thousand genes on the karyomit(e), karyomit(e) generation numerical abnormality will cause being permitted polygenic increase or disappearance, therefore often show as serious, multiple birth defect and deformity, common clinical manifestation is mainly congenital non-carrying out property mental retardation, growth retardation, and with face, the deformity of the aspect such as four limbs and internal organ, it is the first heart, neural tube defects, one of Etiological of the main inborn defect such as harelip, bring spirit and the economical load of load-bearing for society and family, wherein, trisomy 21,18 trisomes, 13 trisomes and sex chromosome (x and y) numerical abnormality has accounted for more than 95% of chromosomal disorder.
Diagnostic method for chromosomal disorder mainly is divided into two classes at present: cytogenetic methods and molecular genetics method.Traditional cytogenetic methods, i.e. cultivation by cell, the complex steps such as aobvious band dyeing and microscopic examination are finished, the method is the gold standard of diagnosis chromosomal disorder, but there is certain limitation in it, and main manifestations is high to operator's technical requirements, makes a definite diagnosis the required cycle long, expense is high, and sending from the report of drawing materials needs the 2-3 time-of-week at least.The molecular genetics method mainly comprises fluorescence in situ hybridization (FISH, Fluorescence in situ hybridization) and quantitative fluorescence PCR (QF-PCR, Quantitative Fluorescence-PCR) analytical technology.FISH utilizes chromosome-specific probe to be combined with karyomit(e) to send fluorescence, under fluorescent microscope, show as the fluorescence spot, can judge chromosome number by the counting to the fluorescence spot, its operating process is unfavorable for that automatization, somewhat expensive (every routine testing cost is wanted thousands of units) all can not satisfy the needs of extensive detection.QF-PCR mainly utilizes fluorescent dye primer that STR on the karyomit(e) is increased in the site, obtain the karyomit(e) relevant information by the capillary electrophoresis fluoroscopic examination, thereby reach the purpose of diagnosis aneuploid disease, this method is highly sensitive, operation automation, consuming time short, is applicable to high throughput testing.This method is highly sensitive, operation automation, consuming time short, is applicable to high throughput testing, just the method for quick, simple, the accurate and economic antenatal diagnosis chromosomal disorder of clinical needs.Tandem repetitive sequence (short tandem repeat, STR) is that existence one class repeating unit length is the tumor-necrosis factor glycoproteins of 2bp～6bp in the genomic dna, is human dna fingerprint.In colony, present genetic polymorphism owing to the variation of core sequence repetition number, but hereditary in Mendelian's mode in same family.Be mainly used at present legal medical expert individual identification and paternity test.Use in the STR site has following characteristics and advantage: 1. the allelotrope fragment length is below 400bp, and the pcr amplification success ratio is high, and required sample is few, and nanogram level ultramicron DNA may increase successfully, is conducive to check; 2. a plurality of STR of composite amplification can save sample in the site, reduce cost, improve the quantity of information that single detects.
At present, composite fluorescence mark STR technology is used very ripe abroad, and the accuracy of common chromosomal disorder antenatal diagnosis can reach more than 98%.But because ethnic group is different, the selection in STR site has its skewed popularity.The research of domestic use composite fluorescence mark STR technology is less, and is crucial still in the selection in STR site.The patent No. is ZL200410021822.4, denomination of invention discloses a kind of No. 21 chromosome number purpose method for quick for the national inventing patent of " a kind of No. 21 chromosome number purpose method for quick ", its innovative point is to have 7 to be No. 21 new chromosome specific microsatellite locus in selected 12 STR sites, but the detection of also just carrying out for trisomy 21.The domestic method that detects simultaneously trisomy 21,18 trisomes, 13 trisomes and sex chromosome number that has no up to now.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides five kinds of chromosome number purposes of a kind of while rapid detection method with not enough.Described five kinds of karyomit(e)s refer to No. 21, No. 18, No. 13 and sex chromosome (x and y).
Another object of the present invention is to provide the test kit of using aforesaid method.
A further object of the present invention is to provide the application of described test kit.
Purpose of the present invention is achieved through the following technical solutions: five kinds of chromosome number purposes of a kind of while rapid detection method may further comprise the steps:
(1) use fluorescently-labeled primer mixture that pcr amplification is carried out in the STR site on No. 21 among the human gene group DNA, No. 18, No. 13 and the sex chromosome, wherein, No. 21 chromosomal STR sites are D21S1270, D21S1411, D21S1412, D21S1414 and D21S1280; No. 18 chromosomal STR site is D18S535, D18S51, D18S1002, D18S391 and D18S386; No. 13 chromosomal STR site is D13S136, D13S294, D13S305, D13S240 and D13S256; Heterosomal STR site DXS337, X22, DXS8377, DXS981, DXYS218, DYS448, SRY and AMEL.Above-mentioned STR site is known sequence, can find at the human genome database, the length of the fragment that the fluorescently-labeled selection of primer obtains based on amplification, the close primer of fragment length that amplification obtains need use different fluorescent marks, thus the fragment that amplification is obtained is distinguished;
Primer sequence concrete in the described primer mixture is as shown in table 1:
The primer in each STR site of table 1
(2) after the fluorescently-labeled PCR product sex change that step (1) is obtained, detect by capillary electrophoresis, the photoluminescence peak area height indirect reaction of the amount of PCR product by the corresponding position out, thereby determine No. 21, No. 18, No. 13 and heterosomal number, according to clinical biochemical association/clinical molecular genetic (ACC/CMGS best practice meeting was held on the 15th April of association, 2004) the regulation judging criterion is as follows: if be shown as three fluorescence peaks on the STR site behind the amplified allele, the peak area ratio was close to 1: 1: 1, can directly be judged as trisomy, if only be shown as two peaks, then calculate peak area ratio (the Area rate at two peaks, AR), all bimodal area ratio＞1.8 or＜0.65 can be judged to be trisome, normal bimodal area ratio interval is: 0.8～1.4, value between two intervals can't be judged, needs again to detect.
Fluorescently-labeled primer mixture described in the step (1) more preferably is made of fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II; The sequence of fluorescently-labeled primer mixture I is as shown in table 2, and the sequence of fluorescently-labeled primer mixture II is as shown in table 3; Fluorescently-labeled primer mixture is divided into fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II, carries out respectively PCR, capillary electrophoresis, the collection of illustrative plates that obtains is more clear;
The fluorescently-labeled primer mixture I of table 2
The fluorescently-labeled primer mixture II of table 3
The condition optimization of the pcr amplification described in the step (1) is: 94～95 ℃ of denaturations; (94～95 ℃ of sex change, 53～57 ℃ of annealing, 70～75 ℃ of extensions) * (20～40) individual circulation; 60～75 ℃ of last extensions;
The condition optimization of described denaturation is 94 ℃ of denaturation 5min;
The condition optimization of described sex change is 94 ℃ of sex change 30s;
Described circulation number is preferably 32;
The condition optimization of described last extension is 60 ℃ and extends 60min;
Sex change described in the step (2) is preferably carried out sex change by deionized formamide;
The operational condition of the capillary electrophoresis described in the step (2) is ABI 3100 genetic analysis user manual Standard operation procedure SOPs.
A kind of test kit of realizing aforesaid method, comprise fluorescently-labeled primer mixture, the length of the fragment that the fluorescently-labeled selection of primer obtains based on amplification, the close primer of fragment length that amplification obtains need use different fluorescent marks, thus the fragment that amplification is obtained is distinguished; The sequence of primer mixture is as shown in table 4:
Table 4
Described fluorescently-labeled primer mixture more preferably is made of fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II; The sequence of fluorescently-labeled primer mixture I is as shown in table 5, and the sequence of fluorescently-labeled primer mixture II is as shown in table 6; Fluorescently-labeled primer mixture is divided into fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II, carries out respectively PCR, capillary electrophoresis, the collection of illustrative plates that obtains is more clear;
The fluorescently-labeled primer mixture I of table 5
The fluorescently-labeled primer mixture II of table 6
Primer pair among the described fluorescently-labeled primer mixture I is 1: 5: 4.5 according to the molar ratio that following mole number carries out proportioning: AMEL, D13S136, DXS981, DYS448, D21S1412, D13S256, X22, D21S1414, D18S1002, SRY, D21S1280, DXY218 and D21S1270 preferably: 4.5: 1: 3: 4: 1.5: 3: 1: 2: 1: 3;
Described fluorescently-labeled primer mixture I more preferably contains AMEL primer pair 1pmol among per 7.5 μ l, D13S136 primer pair 5pmol, DXS981 primer pair 4.5pmol, DYS448 primer pair 4.5pmol, D21S1412 primer pair 1pmol, D13S256 primer pair 3pmol, X22 primer pair 4pmol, D21S1414 primer pair 1.5pmol, D18S1002 primer pair 3pmol, SRY primer pair 1pmol, D21S1280 primer pair 2pmol, DXY218 primer pair 1pmol and D21S1270 primer pair 3pmol;
Primer pair among the described fluorescently-labeled primer mixture II is 2.5: 5: 3 according to the molar ratio that following mole number carries out proportioning: D18S535, DXS337, D13S240, D13S305, D13S294, D18S391, DXS8377, D18S51, D18S386 and D21S1411 preferably: 1: 4: 1: 2: 1: 6: 4;
Described fluorescently-labeled primer mixture II more preferably contains D18S535 primer pair 2.5pmol, DXS337 primer pair 5pmol, D13S240 primer pair 3pmol, D13S305 primer pair 1pmol, D13S294 primer pair 4pmol, D18S391 primer pair 1pmol, DXS8377 primer pair 2pmol, D18S51 primer pair 1pmol, D18S386 primer pair 6pmol and D21S1411 primer pair 4pmol among per 7.5 μ l;
Described test kit also comprises rTaq enzyme, the PCR reaction buffer for the PCR reaction; Premix Taq (Premix Taq Version 2.0, precious biotechnology (Dalian) company limited) is used in suggestion;
The using method of described test kit may further comprise the steps:
(1) uses respectively fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II to carry out PCR, obtain respectively fluorescently-labeled PCR product;
(2) after the fluorescently-labeled PCR product difference sex change that step (1) is obtained, detect by capillary electrophoresis, the photoluminescence peak area height indirect reaction of the amount of PCR product by the corresponding position out, thereby determine No. 21, No. 18, No. 13 and heterosomal number, according to clinical biochemical association/clinical molecular genetic (ACC/CMGS best practice meeting was held on the 15th April of association, 2004) the regulation judging criterion is as follows: if be shown as three fluorescence peaks on the STR site behind the amplified allele, the peak area ratio was close to 1: 1: 1, can directly be judged as trisomy, if only be shown as two peaks, then calculate peak area ratio (the Area rate at two peaks, AR), all bimodal area ratio＞1.8 or＜0.65 can be judged to be trisome, normal bimodal area ratio interval is: 0.8～1.4, value between two intervals can't be judged, needs again to detect.
The condition optimization of the pcr amplification described in the step (1) is: 94～95 ℃ of denaturations; (94～95 ℃ of sex change, 53～57 ℃ of annealing, 70～75 ℃ of extensions) * (20～40) individual circulation; 60～75 ℃ of last extensions;
The condition optimization of described denaturation is 94 ℃ of denaturation 5min;
The condition optimization of described sex change is 94 ℃ of sex change 30s;
Described circulation number is preferably 32;
The condition optimization of described last extension is 60 ℃ and extends 60min;
Sex change described in the step (2) is preferably carried out sex change by deionized formamide;
The operational condition of the capillary electrophoresis described in the step (2) is ABI 3100 genetic analysis user manual Standard operation procedure SOPs.
Described test kit is applied in the antenatal diagnosis field.
The present invention has following advantage and effect with respect to prior art:
(1) the present invention can be simultaneously to 13,18,21 and five kinds of chromosomal numbers such as sex chromosome detect, required sample size is few, fine hair, amniotic fluid and bleeding of the umbilicus sample can be diagnosed.
(2) the present invention does not need cell cultures, and sense cycle is short, and whole testing process can detect in 24 hours and finish, and helped to alleviate pregnant woman's psychological pressure.
(3) the present invention carries out interpretation of result by means of capillary electrophoresis, and level of automation is high, good reproducibility.
(4) testing cost of the present invention is low, more is applicable to extensive detection.
(5) the STR site selected of the present invention is our right higher site in Chinese population, judge each STR site according to the fluorescence color of clip size and mark, according to the corresponding chromosomal number situation of the Analysis of test results in a plurality of STR site, the accuracy rate of diagnosis can reach 98.5%.
Description of drawings
Fig. 1 is the operating process schematic diagram of embodiment 1.
Fig. 2 is the capillary electrophoresis spectrogram in the normal people's that obtains of amplification AMEL, D13S136, DXS981, DYS448 and D21S1412 site.
Fig. 3 is the normal people's that obtains of amplification D18S1002 and the capillary electrophoresis spectrogram in D21S1414 site.
Fig. 4 is the capillary electrophoresis spectrogram in the normal people's that obtains of amplification SRY, DXYS218, D21S1270 and D21S1280 site.
Fig. 5 is the capillary electrophoresis spectrogram in the normal people's that obtains of amplification D13S294, D18S535, DXS337, D13S240 and D13S305 site.
Fig. 6 is the normal people's that obtains of amplification D18S391, DXS8377 and the capillary electrophoresis spectrogram in D18S51 site.
Fig. 7 is the normal people's that obtains of amplification D21S1411 and the capillary electrophoresis spectrogram in D18S386 site.
Fig. 8 is the capillary electrophoresis spectrogram in the D13S136 site of 13 trisomes that obtain of amplification.
Fig. 9 is the D13S294 of 13 trisomes that obtain of amplification and the capillary electrophoresis spectrogram in D13S305 site.
Figure 10 is the capillary electrophoresis spectrogram in the STR site of 18 trisomes that obtain of amplification, wherein:
A is D18S391 and D18S51 site; B is the D18S386 site.
Figure 11 is the capillary electrophoresis spectrogram in the D21S1414 site of the trisomy 21 that obtains of amplification.
Figure 12 is the capillary electrophoresis spectrogram in the D21S1412 site of the trisomy 21 that obtains of amplification.
Figure 13 is the capillary electrophoresis spectrogram in the D21S1411 site of the trisomy 21 that obtains of amplification.
Figure 14 is the capillary electrophoresis spectrogram in the site, AMEL site of the XYY that obtains of amplification.
Figure 15 is the capillary electrophoresis spectrogram in the site, X22 site of the XYY that obtains of amplification.
Figure 16 is the capillary electrophoresis spectrogram in the site, DXYS218 site of the XYY that obtains of amplification.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
Embodiment 1
Amniotic fluid samples to the pregnant woman detects, and concrete steps are as follows:
(1) extracts the human gene group DNA: the amniotic fluid sample is put into whizzer centrifugal, 2000rpm 10 minutes; The sample supernatant is poured into Glass tubing, and remaining general 200 μ l precipitation is pipetted into 1.5ml centrifuge tube prepare dna with the filter core suction nozzle and extracts.Use DNA extraction test kit (Quick Gene SP kit DNA whole blood, FUJIFILM) to carry out DNA extraction and obtain genomic dna.DNA concentration is in 10～30ng/ μ l, 280/260＞1.8,230/260＞2.5.
(2) use test kit of the present invention to carry out the PCR reaction, this test kit comprises fluorescently-labeled primer mixture I as shown in table 7 and fluorescently-labeled primer mixture II as shown in table 8:
The fluorescently-labeled primer mixture I of table 7
Annotate: upstream and downstream primer amount is the same, and take D13S136 as example, upstream primer and downstream primer are respectively 5pmol/7.5 μ l
The fluorescently-labeled primer mixture II of table 8
Use respectively fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II to carry out the PCR reaction, use the Premix Taq:Premix Taq Version 2.0 of precious biotechnology (Dalian) company limited, the PCR reaction system is that (total reaction volume 25 μ l) are as shown in table 9:
Table 9
| The composition of PCR | Dosage (μ l) |
| ?Premix?Taq | ?12.5 |
| Fluorescently-labeled primer mixture I or II | ?7.5 |
| Dna profiling | ?5 |
Reaction conditions is:
72 ℃ are extended 1min
60 ℃ are extended 60min.
(3) the fluorescently-labeled PCR product that step (2) is obtained (the PCR product that namely obtains by primer mixture I and the PCR product that obtains by primer mixture II) carries out respectively sex change and capillary electrophoresis:
1. with the sex change of fluorescently-labeled PCR product
PCR product sex change system:
Deionized formamide 9.0 μ l
Mark (Promega) 0.25 μ l in the Ils600
Fluorescently-labeled PCR product sex change 1.0 μ l
Mixing, instantaneous centrifugal, 95 ℃ of sex change 5 minutes are put in product rapidly 5 minutes on ice.
2. carry out capillary electrophoresis according to the fluorescently-labeled PCR product of ABI 3100 genetic analyzer user manual Standard operation procedure SOPs after with sex change.Genotype3.7 software analysis electrophoresis result.
(3) prenatal diagnosis judging criterion as a result
Specificity STR site is after the QF-PCR amplification on the karyomit(e), through the capillary electrophoresis analysis, the photoluminescence peak area height indirect reaction of the amount of PCR product by the corresponding position out, according to internationally recognized judging criterion (ACC/CMGS best practice meeting was held on the 15th April, 2004) analyze: if be shown as three fluorescence peaks on the STR site behind the amplified allele, the peak area ratio was close to 1: 1: 1, can directly be judged as trisomy, if only be shown as two peaks, then calculate peak area ratio (the Area rate at two peaks, AR), all bimodal area ratio＞1.8 or＜0.65 can be judged to be trisome, normal bimodal area ratio interval is: 0.8-1.4, the value between two intervals can't be judged, needs again to detect.
(4) detected result:
1. normal result is shown in Fig. 2～7: normal bimodal area ratio interval is: 0.8-1.4.
2. 13 trisome detected results are shown in Fig. 8 and 9:
D13S136 site peak area is approximately 1: 2 among Fig. 8.
Three peaks, D13S294 site among Fig. 9, the peak area ratio was close to 1: 1: 1; Site, D13S305 site peak area approximately 1: 2.
3. 18 trisome detected results are as shown in figure 10:
D18S391 site peak area approximately 2: 1; Three peaks, D18S51 site, the peak area ratio was close to 1: 1: 1.
Three peaks, D18S386 site, the peak area ratio was close to 1: 1: 1.
4. the trisomy 21 detected result is shown in Figure 11～13:
D21S1414 site peak area is approximately 2: 1 among Figure 11;
Three peaks, D21S1412 site among Figure 12, the peak area ratio was close to 1: 1: 1
Three peaks, D21S1411 site among Figure 13, the peak area ratio was close to 1: 1: 1.
5. sex chromosomal abnormality is described
Shown in XYY syndrome detected result Figure 14～16:
AMEL site peak area is approximately 1: 2 among Figure 14;
X22 site peak area is approximately 2: 1 among Figure 15;
Three peaks, DXYS218 site among Figure 16, the peak area ratio was close to 1: 1: 1
Empirical tests, the accuracy rate of method of the present invention and test kit diagnosis can reach 98.5%.As seen, method of the present invention and test kit can go out chromosomal disorder by rapid screening, and cost is low, and accuracy rate is high, can carry out on a large scale examination, significant to the prenatal and postnatal care of China.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (4)
1. realize the simultaneously test kit of five kinds of chromosome number purposes of rapid detection method for one kind, it is characterized in that: comprise following fluorescently-labeled primer mixture:
Primer sequence concrete in the primer mixture is as follows:
The upstream primer in D13S136 site is 5 '-GGT GCT TCA TGC ATT GCT AA-3 '; Downstream primer is 5 '-TTT AGTGCT TTA TGA TTA CAG TAG AGTGTG-3 ';
The upstream primer in D13S240 site is 5 '-CAA CGT TTA TTG ACT TAA AGG G-3 '; Downstream primer is 5 '-CGT GTA GTC CCA GCT ACT C-3 ';
The upstream primer in D13S256 site is 5 '-CCT GCG CAA CAA GAG CAA A-3 '; Downstream primer is 5 '-AGG AGA GAG ACA TAA TTG TG-3 ';
The upstream primer in D13S294 site is 5 '-CCG AGT GAG CAG CCT CTA AA-3 '; Downstream primer is 5 '-ATC AGA AAT CAG GCC AGT GTG-3 ';
The upstream primer in D13S305 site is 5 '-GCGTGTTTGAGGACCTGTCGTTA-3 '; Downstream primer is 5 '-TGCTAATAGAGCAGTTAAGGCAC-3 ';
The upstream primer in D18S51 site is 5 '-CGAACCCGACTACCAGCAAC-3 '; Downstream primer is 5 '-GACGCATGTTCATGCCACTG-3 ';
The upstream primer in D18S535 site is 5 '-TCTAGTGACAAAAGCCACAC-3 '; Downstream primer is 5 '-AGTCAGAAATATAGATGAGAATGCA-3 ';
The upstream primer in D18S386 site is 5 '-TCACCA GAA TCA CTT GGA AC-3 '; Downstream primer is 5 '-TCG ATG AAG TAG CTA AGC AG-3 ';
The upstream primer in D18S1002 site is 5 '-CAG AGAGTGAATGCTGTACAAACA GC-3 '; Downstream primer is 5 '-CATCATGTGAGTGTGCTTTTCAGGAG-3 ';
The upstream primer in D18S391 site is 5 '-GGAGTTACCACAGGCAATGTGACT-3 '; Downstream primer is 5 '-TAGTCTTCACTATTCCCATCTGAG-3 ';
The upstream primer in D21S1411 site is 5 '-ATCATGAATGCATAGATGGATG-3 '; Downstream primer is 5 '-ATT GTG TGT CCT TCC AGG C-3 ';
The upstream primer in D21S1412 site is 5 '-CGCTGGTTGCAGTGAGTTG-3 '; Downstream primer is 5 '-GGGAAGGCTATGGAGGAGA-3 ';
The upstream primer in D21S1414 site is 5 '-GCCACCCAGTAAAAAATTACT-3 '; Downstream primer is 5 '-CACTCTGTCTGTCTGTCTATC-3 ';
The upstream primer in D21S1280 site is 5 '-AGAGGGCATCAAAATTTGGA-3 '; Downstream primer is 5 '-GATGAAATAGGAGCCATAAAGC-3 ';
The upstream primer in D21S1270 site is 5 '-CTAAGCCACTGTATTATTCAGGGC-3 '; Downstream primer is 5 '-TGTGTCTCCAGGTTGCAGGTGACA-3 ';
The upstream primer in DXS337 site is 5 '-TGGATCATTCAGCTTTCAGG-3 '; Downstream primer is 5 '-GTGAGAGAGTGAGACCCTGTC-3 ';
The upstream primer in X22 site is 5 '-TATTGAGAGTTGGAAAGAAA-3 '; Downstream primer is 5 '-CGCATTGTTGCTACTTGAGA-3 ';
The upstream primer in DXS981 site is 5 '-CTCGATGTGGCCTTCCTTAAATG-3 '; Downstream primer is 5 '-TACTCTCCACTTTTCAGAGTCA-3 ';
The upstream primer in DYS448 site is 5 '-CAT GGA TCC AAA TAA AGA ACA GAG A-3 '; Downstream primer is 5 '-GCA TAT TTC TTG ATT CCC TGT G-3 ';
The upstream primer in AMEL site is 5 '-CCCTGGGCTCTGTAAAGAATAGT-3 '; Downstream primer is 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 ';
The upstream primer in SRY site is 5 '-AGTAAAGGCAACGTCCAGGAT-3 '; Downstream primer is 5 '-TTCCGACGAGGTCGATACTTA-3 ';
The upstream primer in DXS8377 site is 5 '-CAGATC ATG GCT TAC CAC AG-3 '; Downstream primer is 5 '-GAG CTT TGG AAA GCT AGT GT-3 ';
The upstream primer in DXY218 site is 5 '-TGTCTATGGGTTTCCTCTGT-3 '; Downstream primer is 5 '-CGTAACTCCGTCTCAAAATA-3 '.
One kind realize five kinds of chromosome number purposes of rapid detection method simultaneously test kit, it is characterized in that: consisted of by primer mixture I and primer mixture II;
The sequence of described primer mixture I is as follows:
The upstream primer in D13S136 site is 5 '-FAM-GGT GCT TCA TGC ATT GCT AA-3 '; Downstream primer is 5 '-TTT AGTGCT TTA TGA TTA CAG TAG AGTGTG-3 ';
The upstream primer in D13S256 site is 5 '-JOE-CCT GCG CAA CAA GAG CAA A-3 '; Downstream primer is 5 '-AGG AGA GAG ACA TAA TTG TG-3 ';
The upstream primer in D18S1002 site is 5 '-JOE-CAG AGAGTGAATGCTGTACAAACA GC-3 '; Downstream primer is 5 '-CATCATGTGAGTGTGCTTTTCAGGAG-3 ';
The upstream primer in D21S1412 site is 5 '-FAM-CGCTGGTTGCAGTGAGTTG-3 '; Downstream primer is 5 '-GGGAAGGCTATGGAGGAGA-3 ';
The upstream primer in D21S1414 site is 5 '-JOE-GCCACCCAGTAAAAAATTACT-3 '; Downstream primer is 5 '-CACTCTGTCTGTCTGTCTATC-3 ';
The upstream primer in D21S1280 site is 5 '-AGAGGGCATCAAAATTTGGA-3 '; Downstream primer is 5 '-GATGAAATAGGAGCCATAAAGC-3 ';
The upstream primer in D21S1270 site is 5 '-TAMRA-CTAAGCCACTGTATTATTCAGGGC-3 '; Downstream primer is 5 '-TGTGTCTCCAGGTTGCAGGTGACA-3 ';
The upstream primer in X22 site is 5 '-JOE-TATTGAGAGTTGGAAAGAAA-3 '; Downstream primer is 5 '-CGCATTGTTGCTACTTGAGA-3 ';
The upstream primer in DXS981 site is 5 '-FAM-CTCGATGTGGCCTTCCTTAAATG-3 '; Downstream primer is 5 '-TACTCTCCACTTTTCAGAGTCA-3 ';
The upstream primer in DYS448 site is 5 '-FAM-CAT GGA TCC AAA TAA AGA ACA GAG A-3 '; Downstream primer is 5 '-GCA TAT TTC TTG ATT CCC TGT G-3 ';
The upstream primer in AMEL site is 5 '-FAM-CCCTGGGCTCTGTAAAGAATAGT-3 '; Downstream primer is 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 ';
The upstream primer in SRY site is 5 '-TAMRA-AGTAAAGGCAACGTCCAGGAT-3 '; Downstream primer is 5 '-TTCCGACGAGGTCGATACTTA-3 ';
The upstream primer in DXY218 site is 5 '-TAMRA-TGTCTATGGGTTTCCTCTGT-3 '; Downstream primer is 5 '-CGTAACTCCGTCTCAAAATA-3 ';
The sequence of described primer mixture II is as follows:
The upstream primer in D13S240 site is 5 '-FAM-CAA CGT TTA TTG ACT TAA AGG G-3 '; Downstream primer is 5 '-CGT GTA GTC CCA GCT ACT C-3 ';
The upstream primer in D13S294 site is 5 '-FAM-CCG AGT GAG CAG CCT CTA AA-3 '; Downstream primer is 5 '-ATC AGAAAT CAG GCC AGT GTG-3 ';
The upstream primer in D13S305 site is 5 '-FAM-GCGTGTTTGAGGACCTGTCGTTA-3 '; Downstream primer is 5 '-TGCTAATAGAGCAGTTAAGGCAC-3 ';
The upstream primer in D18S51 site is 5 '-JOE-CGAACCCGACTACCAGCAAC-3 '; Downstream primer is 5 '-GACGCATGTTCATGCCACTG-3 ';
The upstream primer in D18S535 site is 5 '-FAM-TCTAGTGACAAAAGCCACAC-3 '; Downstream primer is 5 '-AGTCAGAAATATAGATGAGAATGCA-3 ';
The upstream primer in D18S386 site is 5 '-TAMRA-TCACCA GAA TCA CTT GGA AC-3 '; Downstream primer is 5 '-TCG ATG AAG TAG CTA AGC AG-3 ';
The upstream primer in D18S391 site is 5 '-JOE-GGAGTTACCACAGGCAATGTGACT-3 '; Downstream primer is 5 '-TAGTCTTCACTATTCCCATCTGAG-3 ';
The upstream primer in D21S1411 site is 5 '-TAMRA-ATCATGAATGCATAGATGGATG-3 '; Downstream primer is 5 '-ATT GTG TGT CCT TCC AGG C-3 ';
The upstream primer in DXS337 site is 5 '-FAM-TGGATCATTCAGCTTTCAGG-3 '; Downstream primer is 5 '-GTGAGAGAGTGAGACCCTGTC-3 ';
The upstream primer in DXS8377 site is 5 '-JOE-CAGATC ATG GCT TAC CAC AG-3 '; Downstream primer is 5 '-GAG CTT TGG AAA GCT AGT GT-3 '.
3. the simultaneously test kit of five kinds of chromosome number purposes of rapid detection method of realizing according to claim 2 is characterized in that:
Primer pair among the described primer mixture I is 1:5:4.5:4.5:1:3:4:1.5:3:1:2:1:3 according to the molar ratio that following mole number carries out proportioning: AMEL, D13S136, DXS981, DYS448, D21S1412, D13S256, X22, D21S1414, D18S1002, SRY, D21S1280, DXY218 and D21S1270;
Primer pair among the described primer mixture II is 2.5:5:3:1:4:1:2:1:6:4 according to the molar ratio that following mole number carries out proportioning: D18S535, DXS337, D13S240, D13S305, D13S294, D18S391, DXS8377, D18S51, D18S386 and D21S1411.
4. the simultaneously test kit of five kinds of chromosome number purposes of rapid detection method of realizing according to claim 3 is characterized in that:
Described primer mixture I contains AMEL primer pair 1pmol, D13S136 primer pair 5pmol, DXS981 primer pair 4.5pmol, DYS448 primer pair 4.5pmol, D21S1412 primer pair 1pmol, D13S256 primer pair 3pmol, X22 primer pair 4pmol, D21S1414 primer pair 1.5pmol, D18S1002 primer pair 3pmol, SRY primer pair 1pmol, D21S1280 primer pair 2pmol, DXY218 primer pair 1pmol and D21S1270 primer pair 3pmol among per 7.5 μ l;
Described primer mixture II contains D18S535 primer pair 2.5pmol, DXS337 primer pair 5pmol, D13S240 primer pair 3pmol, D13S305 primer pair 1pmol, D13S294 primer pair 4pmol, D18S391 primer pair 1pmol, DXS8377 primer pair 2pmol, D18S51 primer pair 1pmol, D18S386 primer pair 6pmol and D21S1411 primer pair 4pmol among per 7.5 μ l.
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| CN103074416B (en) * | 2012-06-20 | 2017-12-08 | 宁波海尔施基因科技有限公司 | A kind of method for detecting five numerical abnormalities of chromosomes |
| CN103103267B (en) * | 2013-01-19 | 2014-11-05 | 浙江大学医学院附属妇产科医院 | Kit for detecting genotype of human chromosome 21 STR (short tandem repeat) |
| CN103966207B (en) * | 2013-02-01 | 2017-10-13 | 天昊生物医药科技（苏州）有限公司 | New short nucleotides tandem repetitive sequence site and its application |
| CN104099326B (en) * | 2013-04-02 | 2018-02-02 | 天昊生物医药科技（苏州）有限公司 | New short nucleotides tandem repetitive sequence site and its application |
| CN104099327B (en) * | 2013-04-02 | 2018-06-05 | 天昊生物医药科技（苏州）有限公司 | New short nucleotide tandem repetitive sequence site and its application |
| CN104099328B (en) * | 2013-04-02 | 2018-07-06 | 天昊生物医药科技（苏州）有限公司 | New short nucleotide tandem repetitive sequence site and its application |
| CN104099325B (en) * | 2013-04-02 | 2018-06-26 | 天昊生物医药科技（苏州）有限公司 | New short nucleotide tandem repetitive sequence site and its application |
| CN104099324B (en) * | 2013-04-02 | 2018-01-19 | 天昊生物医药科技（苏州）有限公司 | New short nucleotides tandem repetitive sequence site and its application |
| CN104651488B (en) * | 2014-11-25 | 2017-08-08 | 北京阅微基因技术有限公司 | Detect the Amplification thing and quick detection kit of chromosome aneuploid numerical abnormality |
| CN104818323B (en) * | 2015-04-10 | 2017-08-29 | 上海五色石医学研究有限公司 | The mankind 13, the gene parting detecting reagent of 18 and No. 21 chromosome, 20 str locus seats |
| CN108866175A (en) * | 2017-05-10 | 2018-11-23 | 杭州中翰金诺医学检验所有限公司 | A kind of Amplification object and quick detection kit detecting numerical abnormalities of chromosomes |
| CN113046430B (en) * | 2021-03-15 | 2022-02-01 | 北京阅微基因技术股份有限公司 | Amplification composition for chromosome aneuploid number abnormality and application thereof |
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