CN102337338A - 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, method and the test kit of adopting said method and the application of this test kit of five kinds of chromosome numbers of particularly 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 exist thousands of even up to ten thousand genes on the karyomit(e); Karyomit(e) generation numerical abnormality will cause the increase or the disappearance of many genes, so often show as serious, multiple birth defect and deformity, the common clinical manifestation is mainly congenital non-carrying out property mental retardation; Growth retardation; And with the deformity of aspects such as face, four limbs and internal organ, be the main diseases therefore of main inborn defect such as the first heart, NTD, harelip, bring the 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 types at present: cytogenetic methods and molecular genetics method.Traditional cytogenetic methods promptly through the cultivation of cell, shows complex steps such as band dyeing and microscopic examination and accomplishes; This method is the gold standard of diagnosis chromosomal disorder; But there is certain limitation in it, mainly shows as operator's technical requirements highly, and it is long to make a definite diagnosis the required cycle; 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 combine to send fluorescence with karyomit(e); Under fluorescent microscope, show as the fluorescence spot; Counting through to the fluorescence spot can judge that its operating process is unfavorable for that robotization, expensive (every example detects cost and wants thousands of units) all can not satisfy the needs of extensive detection to chromosome number.QF-PCR mainly utilizes fluorescent dye primer that STR on the karyomit(e) is increased in the site; Obtain the karyomit(e) relevant information through the capillary electrophoresis fluoroscopic examination; Thereby reach the purpose of diagnosis aneuploid disease, this method is highly sensitive, operation automation, weak point consuming time, is applicable to high throughput testing.This method is highly sensitive, operation automation, weak point consuming time, is applicable to high throughput testing, just the method for quick, simple, the accurate and economic antenatal diagnosis chromosomal disorder of clinical needs.(short tandem repeat is to have one type of Tumor-necrosis factor glycoproteins that repeating unit's length is 2bp～6bp in the genomic dna STR) to tandem repetitive sequence, is human dna fingerprint.In colony, demonstrate genetic polymorphism owing to the variation of core sequence repetition number, but hereditary with Mendelian's mode in same family.Be mainly used in legal medical expert's individual identification and paternity test at present.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 the nanogram level ultramicron DNA success of possibly increasing helps check; 2. a plurality of STR of composite amplification can practice thrift sample in the site, reduce cost, and 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.Domestic use composite fluorescence mark STR Study on 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 has 7 for No. 21 new chromosome specific microsatellite locus for institute selects for use in 12 STR sites, but also just is directed against the detection that trisomy 21 carries out.Domesticly do not see the method that detects trisomy 21,18 trisomes, 13 trisomes and sex chromosome number simultaneously 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 purpose more of the present invention is to provide the application of said test kit.
The object of the invention is realized through following technical proposals: 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 sequences; Can find at the human genome DB; The segmental length 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 able to difference;
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 through capillary electrophoresis, the amount of PCR product is come out through the photoluminescence peak area height indirect reaction of corresponding position; Thereby confirm No. 21, No. 18, No. 13 and heterosomal number; Following according to clinical biochemical association/clinical molecular genetic association (ACC/CMGS best practice meeting was held on the 15th April, 2004) regulation judgement criteria: if be shown as three fluorescence peaks on the STR site behind the amplified allele, the peak area ratio approached 1: 1: 1; Can directly be judged as trisomy; If only be shown as two peaks, then calculate two peaks peak area ratio (Area rate, AR); All bimodal area ratio＞1.8 perhaps＜0.65 decidable are trisome; Normal bimodal area ratio interval is: 0.8～1.4, and the value between two intervals can't be judged, needs to detect again.
Fluorescently-labeled primer mixture described in the step (1) more preferably is made up 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 PCR respectively, 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 preparatory sex change; (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 preparatory sex change is 94 ℃ of preparatory sex change 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 through 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 segmental length 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 able to difference; The sequence of primer mixture is as shown in table 4:
Table 4
Described fluorescently-labeled primer mixture more preferably is made up 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 PCR respectively, 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 among the described fluorescently-labeled primer mixture I is 1: 5: 4.5 to the molar ratio that preferably carries out proportioning: AMEL, D13S136, DXS981, DYS448, D21S1412, D13S256, X22, D21S1414, D18S1002, SRY, D21S1280, DXY218 and D21S1270 according to following mole number: 4.5: 1: 3: 4: 1.5: 3: 1: 2: 1: 3;
Contain the AMEL primer to 1pmol among the more preferably per 7.5 μ l of described fluorescently-labeled primer mixture I; The D13S136 primer is to 5pmol; The DXS981 primer is to 4.5pmol; The DYS448 primer is to 4.5pmol; The D21S1412 primer is to 1pmol; The D13S256 primer is to 3pmol; The X22 primer is to 4pmol; The D21S1414 primer is to 1.5pmol; The D18S1002 primer is to 3pmol; The SRY primer is to 1pmol; The D21S1280 primer is to 2pmol; The DXY218 primer to 1pmol and D21S1270 primer to 3pmol;
Primer among the described fluorescently-labeled primer mixture II is 2.5: 5: 3 to the molar ratio that preferably carries out proportioning: D18S535, DXS337, D13S240, D13S305, D13S294, D18S391, DXS8377, D18S51, D18S386 and D21S1411 according to following mole number: 1: 4: 1: 2: 1: 6: 4;
Contain among the more preferably per 7.5 μ l of described fluorescently-labeled primer mixture II the D18S535 primer to 2.5pmol, DXS337 primer to 5pmol, D13S240 primer to 3pmol, D13S305 primer to 1pmol, D13S294 primer to 4pmol, D18S391 primer to 1pmol, DXS8377 primer to 2pmol, D18S51 primer to 1pmol, D18S386 primer to 6pmol and D21S1411 primer to 4pmol;
Described test kit also comprises rTaq enzyme, the PCR reaction buffer that is used for the PCR reaction; Premix Taq (Premix Taq Version 2.0, precious biotechnology (Dalian) ltd) is used in suggestion;
The method of use of described test kit may further comprise the steps:
(1) uses fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II to carry out PCR respectively, obtain fluorescently-labeled PCR product respectively;
(2) after the fluorescently-labeled PCR product that step (1) is obtained is distinguished sex change, detect through capillary electrophoresis, the amount of PCR product is come out through the photoluminescence peak area height indirect reaction of corresponding position; Thereby confirm No. 21, No. 18, No. 13 and heterosomal number; Following according to clinical biochemical association/clinical molecular genetic association (ACC/CMGS best practice meeting was held on the 15th April, 2004) regulation judgement criteria: if be shown as three fluorescence peaks on the STR site behind the amplified allele, the peak area ratio approached 1: 1: 1; Can directly be judged as trisomy; If only be shown as two peaks, then calculate two peaks peak area ratio (Area rate, AR); All bimodal area ratio＞1.8 perhaps＜0.65 decidable are trisome; Normal bimodal area ratio interval is: 0.8～1.4, and the value between two intervals can't be judged, needs to detect again.
The condition optimization of the pcr amplification described in the step (1) is: 94～95 ℃ of preparatory sex change; (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 preparatory sex change is 94 ℃ of preparatory sex change 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 through 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.
Said 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 completion in 24 hours, help 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) to detect cost low in the present invention, more is applicable to extensive detection.
(5) the STR site selected for use 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; Analyze corresponding chromosomal number situation according to the detected result in a plurality of STR site, accurate rate of diagnosis can reach 98.5%.
Description of drawings
Fig. 1 is the operating process synoptic 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 the 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 capillary electrophoresis spectrogram in the normal people's that obtains of amplification D18S391, DXS8377 and D18S51 site.
Fig. 7 is the normal people's that obtains of amplification the 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
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail, but embodiment of the present invention is not limited thereto.
Embodiment 1
Amniotic fluid sample to the pregnant woman detects, and concrete steps are following:
(1) extract the human gene group DNA: it is centrifugal that the amniotic fluid sample is put into whizzer, 2000rpm 10 minutes; Pour the sample supernatant into Glass tubing, remaining general 200 μ l deposition is pipetted into 1.5ml centrifuge tube prepare dna with the filter core suction nozzle and extracts.(Quick Gene SP kit DNA whole blood FUJIFILM) carries out DNA extraction and obtains genomic dna to use the DNA extraction test kit.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, is example with D13S136, and upstream primer and downstream primer are respectively 5pmol/7.5 μ l
The fluorescently-labeled primer mixture II of table 8
Use fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II to carry out the PCR reaction respectively; Use the Premix Taq:Premix Taq Version 2.0 of precious biotechnology (Dalian) ltd, 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 promptly obtains through primer mixture I and the PCR product that obtains through primer mixture II) carries out sex change and capillary electrophoresis respectively:
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 5 minutes on ice rapidly.
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) chromosome aneuploid disease judgement criteria as a result
Specificity STR site is after the QF-PCR amplification on the karyomit(e), and through the capillary electrophoresis analysis, the amount of PCR product is come out through the photoluminescence peak area height indirect reaction of corresponding position; Analyze according to internationally recognized judgement criteria (ACC/CMGS best practice meeting was held on the 15th April, 2004): if be shown as three fluorescence peaks on the STR site behind the amplified allele, the peak area ratio approached 1: 1: 1; Can directly be judged as trisomy; If only be shown as two peaks, then calculate two peaks peak area ratio (Area rate, AR); All bimodal area ratio＞1.8 perhaps＜0.65 decidable are trisome; Normal bimodal area ratio interval is: 0.8-1.4, the value between two intervals can't be judged, needs to detect again.
(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 about 1: 2 among Fig. 8.
Three peaks, D13S294 site among Fig. 9, the peak area ratio approached 1: 1: 1; About 1: 2 of site, D13S305 site peak area.
3. 18 trisome detected results are shown in figure 10:
About 2: 1 of D18S391 site peak area; Three peaks, D18S51 site, the peak area ratio approached 1: 1: 1.
Three peaks, D18S386 site, the peak area ratio approached 1: 1: 1.
4. the trisomy 21 detected result is shown in Figure 11～13:
D21S1414 site peak area is about 2: 1 among Figure 11;
Three peaks, D21S1412 site among Figure 12, the peak area ratio approached 1: 1: 1
Three peaks, D21S1411 site among Figure 13, the peak area ratio approached 1: 1: 1.
5. sex chromosomal abnormality is described
Shown in XYY syndrome detected result Figure 14～16:
AMEL site peak area is about 1: 2 among Figure 14;
X22 site peak area is about 2: 1 among Figure 15;
Three peaks, DXYS218 site among Figure 16, the peak area ratio approached 1: 1: 1
Empirical tests, method of the present invention and test kit accurate rate of diagnosis can reach 98.5%.It is thus clear that method of the present invention and test kit can go out chromosomal disorder by rapid screening, cost is low, and accuracy rate is high, can carry out examination on a large scale, significant to the prenatal and postnatal care of China.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is 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 (10)
1. five kinds of chromosome number purposes of rapid detection method simultaneously is characterized in that 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;
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 '
(2) after the fluorescently-labeled PCR product sex change that step (1) is obtained, detect through capillary electrophoresis, the amount of PCR product is come out through the photoluminescence peak area height indirect reaction of corresponding position; Thereby confirm No. 21, No. 18, No. 13 and heterosomal number; Following according to molecular genetic association of clinical biochemical association/clinical regulation judgement criteria: if be shown as three fluorescence peaks on the STR site behind the amplified allele, the peak area ratio approached 1: 1: 1, can directly be judged as trisomy; If only be shown as two peaks; Then calculate the peak area ratio at two peaks, all bimodal area ratio＞1.8 perhaps＜0.65 decidable are trisome, and normal bimodal area ratio interval is: 0.8～1.4; Value between two intervals can't be judged, needs to detect again.
2. five kinds of chromosome number purposes of while rapid detection according to claim 1 method, it is characterized in that: the fluorescently-labeled primer mixture described in the step (1) is for to be made up of fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II;
The sequence of fluorescently-labeled 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 fluorescently-labeled 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 site is 5 '--3 '; Downstream primer is 5 '--3 ';
The upstream primer in D13S294 site is 5 '-FAM-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 '-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. five kinds of chromosome number purposes of while rapid detection according to claim 1 method is characterized in that:
Sex change described in the step (2) is for to carry out sex change through 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.
4. a test kit of realizing five kinds of chromosome number purposes of the described while rapid detection of claim 1 method is characterized in that: comprise the fluorescently-labeled primer mixture described in the claim 1.
5. the test kit of five kinds of chromosome number purposes of while rapid detection according to claim 4 method, it is characterized in that: described fluorescently-labeled primer mixture is for to be made up of fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II;
The sequence of fluorescently-labeled 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 fluorescently-labeled 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 site is 5 '--3 '; Downstream primer is 5 '--3 ';
The upstream primer in D13S294 site is 5 '-FAM-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 '-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 '.
6. the test kit of five kinds of chromosome number purposes of while rapid detection according to claim 5 method is characterized in that:
Primer among the described fluorescently-labeled primer mixture I is 1: 5: 4.5 to the molar ratio that carries out proportioning: AMEL, D13S136, DXS981, DYS448, D21S1412, D13S256, X22, D21S1414, D18S1002, SRY, D21S1280, DXY218 and D21S1270 according to following mole number: 4.5: 1: 3: 4: 1.5: 3: 1: 2: 1: 3;
Primer among the described fluorescently-labeled primer mixture II is 2.5: 5: 3 to the molar ratio that carries out proportioning: D18S535, DXS337, D13S240, D13S305, D13S294, D18S391, DXS8377, D18S51, D18S386 and D21S1411 according to following mole number: 1: 4: 1: 2: 1: 6: 4.
7. the test kit of five kinds of chromosome number purposes of while rapid detection according to claim 6 method is characterized in that:
Described fluorescently-labeled primer mixture I contains the AMEL primer to 1pmol among per 7.5 μ l; The D13S136 primer is to 5pmol; The DXS981 primer is to 4.5pmol; The DYS448 primer is to 4.5pmol; The D21S1412 primer is to 1pmol; The D13S256 primer is to 3pmol; The X22 primer is to 4pmol; The D21S1414 primer is to 1.5pmol; The D18S1002 primer is to 3pmol; The SRY primer is to 1pmol; The D21S1280 primer is to 2pmol; The DXY218 primer to 1pmol and D21S1270 primer to 3pmol;
Described fluorescently-labeled primer mixture II be contain among per 7.5 μ l the D18S535 primer to 2.5pmol, DXS337 primer to 5pmol, D13S240 primer to 3pmol, D13S305 primer to 1pmol, D13S294 primer to 4pmol, D18S391 primer to 1pmol, DXS8377 primer to 2pmol, D18S51 primer to 1pmol, D18S386 primer to 6pmol and D21S1411 primer to 4pmol.
8. the method for use of the test kit of five kinds of chromosome number purposes of the described while rapid detection of claim 7 method is characterized in that may further comprise the steps:
(1) uses fluorescently-labeled primer mixture I and fluorescently-labeled primer mixture II that the human gene group DNA is carried out PCR respectively, obtain fluorescently-labeled PCR product respectively;
(2) after the fluorescently-labeled PCR product that step (1) is obtained is distinguished sex change, detect through capillary electrophoresis, the amount of PCR product is come out through the photoluminescence peak area height indirect reaction of corresponding position; Thereby confirm No. 21, No. 18, No. 13 and heterosomal number; Following according to molecular genetic association of clinical biochemical association/clinical regulation judgement criteria: if be shown as three fluorescence peaks on the STR site behind the amplified allele, the peak area ratio approached 1: 1: 1, can directly be judged as trisomy; If only be shown as two peaks; Then calculate the peak area ratio at two peaks, all bimodal area ratio＞1.8 perhaps＜0.65 decidable are trisome, and normal bimodal area ratio interval is: 0.8～1.4; Value between two intervals can't be judged, needs to detect again.
9. the method for use of the test kit of five kinds of chromosome number purposes of while rapid detection according to claim 8 method, it is characterized in that: the condition of the pcr amplification described in the step (1) is: 94～95 ℃ of preparatory sex change; (94～95 ℃ of sex change, 53～57 ℃ of annealing, 70～75 ℃ of extensions) * (20～40) individual circulation; 60～75 ℃ of last extensions;
Sex change described in the step (2) is for to carry out sex change through 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.
10. the method for use of the test kit of five kinds of chromosome number purposes of while rapid detection according to claim 9 method is characterized in that: the condition of described preparatory sex change is 94 ℃ of preparatory sex change 5min;
The condition of described sex change is 94 ℃ of sex change 30s;
Described circulation number is 32;
The condition of described last extension is 60 ℃ and extends 60min.
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| CN103074416A (en) * | 2012-06-20 | 2013-05-01 | 海尔施生物医药股份有限公司 | Method for detecting abnormal numbers of five chromosomes |
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| CN104099328A (en) * | 2013-04-02 | 2014-10-15 | 天昊生物医药科技（苏州）有限公司 | New short nucleotide tandem repeat site and application thereof |
| CN104651488A (en) * | 2014-11-25 | 2015-05-27 | 北京阅微基因技术有限公司 | Amplification composition for detecting abnormal number of chromosomal aneuploid and rapid detection kit |
| CN104818323A (en) * | 2015-04-10 | 2015-08-05 | 上海五色石医学研究有限公司 | Gene typing detection kit for human 13,18 and 21 chromosome 20 STR locus |
| 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 |
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