CN103074416A - Method for detecting abnormal numbers of five chromosomes - Google Patents
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Abstract
The present invention relates to a method for detecting abnormal numbers of five chromosomes, wherein the method is provided for concurrently detecting aneuploidy of numbers of five chromosomes such as chromosome 21, chromosome 13, chromosome 18, chromosome X and chromosome Y, and comprises: (1) respectively selecting 7 genes from the five chromosomes, and designing 38 pairs of primers based on the selected genes; (2) adopting combinations of the primers to carry out multiplex PCR and capillary electrophoresis; and (3) adopting a software to analyze electrophoresis results.
Description
Technical field
The present invention relates to a kind of detection method, specifically, the present invention relates to the method for five numerical abnormalities of chromosomes of a kind of detection.
Background technology
At present, means for detection of mongolism mainly are to extract pregnancy serum clinically, detect PAPP-A (PAPP-A) in the maternal serum, the index of free hCGB subunit (early stage two) or first type fetus albumen (AFP) and chorionic gonadotropin (HCG) and FE3 (uE3) (three of mid-terms), in conjunction with pregnant woman expected date of childbirth, body weight, pregnant week when age and blood sampling, calculate the danger coefficient of " Tang Shi ", can find like this 80% Tang Shi and part 18 trisomes, but whether can not make a definite diagnosis fetus ill.Tang Shi examination index exceeds normal pregnant woman should carry out amniocentesis inspection or fine hair inspection, then carries out the fetal cell chromosome karyotype analysis.And the shortcoming of chromosome karyotype analysis is length consuming time (more than the week), and technical requirements is relatively high, charges also higher.
Molecular diagnosis claims again gene diagnosis, is that a kind of application high-tech means detects DNA and RNA, and then discloses the structure of gene and the technology of expression.This technology at present development is rapid, and Application Areas is very extensive.Our method is used the method for multiplex PCR and electrocapillary phoresis, have sensitivity, quantitatively, fast, the technical superiority such as high-throughput.Our invention can provide a kind of superior molecular diagnosis method for clinical obstetrics.
Summary of the invention
The invention provides (the male sex 38 sites, 38 Human genome sites of a kind of synchronous amplification, women 31 sites) multiplex PCR scheme, and energy synchronous detection 13,18,21, X and the method for five numerical abnormalities of chromosomes of Y and the purposes of the method.
According to a first aspect of the invention, the method of five numerical abnormalities of chromosomes of a kind of detection, be used for detecting simultaneously comprise 21,13,18, five chromosome number purpose dysploidy of X and Y chromosome, comprise: (1), on described five karyomit(e)s, choose respectively seven genes, the gene design for selected 38 38 pairs of primers (table 1); (2), utilize above combination of primers to carry out multiplex PCR and electrocapillary phoresis; (3), utilize software that electrophoresis result is analyzed.
Preferably, described chromosome number purpose dysploidy refers to that the chromosome number purpose that is different from the normal human increases or disappearance.
Preferably, step (1) also is included on other karyomit(e)s outside these five karyomit(e)s and chooses three genes in contrast (CK).
Preferably, the pcr template in the described step (2) is the human genome DNA.
Preferably, same chromosomal several genes are positioned at this chromosomal different zones as far as possible; The design clip size is between 100bp and 400bp, and the difference of adjacent two clip size is generally 3～10bp.
According to a further aspect in the invention, a kind of purposes that adopts preceding method, described method are applied to adopt wound and need the antenatal diagnosis of the clinical sample of detection without creating two kinds of means acquisitions.
Preferably, described have the wound means to obtain amniotic fluid or embryo villi theca cell by amniocentesis.
Preferably, describedly obtain fetal cell without the wound means by flow cytometer screening maternal blood.
Owing to having adopted technique scheme, the present invention to have following beneficial effect:
The inventor is through deep research, and " human chromosomal dysploidy multiple molecular detection kit " (hereinafter to be referred as " test kit ") that combines PCR and electrocapillary phoresis advantage produced in first research and development.This test kit adopts the method for molecular diagnosis, not only sensitivity and accuracy rate high, consuming time short (8 hours), and also it is sick to detect simultaneously 5 kinds of different congenital heredities, and expense is also relatively cheap.This is the diagnostic means of at present urgent clinical needs just, has good market application foreground.
Table 1, human chromosomal dysploidy are analyzed each site and primer sequence table
Description of drawings
The analysis of cases of Fig. 1 chromosomal aneuploidy.
Chromosome number purpose quantitative analysis in Fig. 2 normal specimens.
Chromosome number purpose quantitative analysis in Fig. 3 normal specimens and the case sample.
Embodiment
Embodiments of the invention are as follows:
One, DNA extraction
Sample DNA extracts Chelex-100 (BioRad) method that adopts.
1.1 amniotic fluid and cell culture thereof
Get 1ml amniotic fluid or 200 μ l cell cultures, to the 1.5ml centrifuge tube, add people's 500 μ l pure water, thermal agitation was placed 15 minutes under the room temperature.Centrifugal 3 minutes of 13,000rpm removes supernatant, collecting precipitation.Add 200 μ l, 5% Chelex-100 solution (5%Chelex-100 is suspension liquid, wants shake well before the use, makes the Chelex-100 particle suspension) in the precipitation, repeatedly vibration on vibrator was put into 56 ℃ of insulations more than 30 minutes.Vibration after taking out, 100 ℃ of insulations 8 minutes, after the vibration, centrifugal 3 minutes of 13,000rpm, supernatant is used for pcr amplification, or puts 4 ℃ and save backup.
1.2 fetus chorion tissue
Get the tissue block of soya bean size, put into the 1.5ml centrifuge tube, add pure water and wash once, then add the Chelex-100 of 200 μ l 5% and the Proteinase K of 5 μ l 5mg/ml, put into 56 ℃ of insulations more than 3 hours.Take out vibration, 100 ℃ are incubated 8 minutes, vibration, and centrifugal 3 minutes of 13000rpm, supernatant are used for pcr amplification or put 4 ℃ saving backup.
Two, test kit forms (PCR reagent, 100 secondary responses)
The composition of this test kit comprises:
The H that does not contain DNA enzyme/RNA enzyme
2O, 1000 μ l
10 * PCR damping fluid, 220 μ l
5 * PCR primer mixture, 440 μ l
MgCl
2，440μl，25mM
The Taq archaeal dna polymerase, 55 μ l
Solution X，220μl
The DNA contrast, 100 μ l, 20ng/ μ l
Three, experimentation
3.1PCR reaction:
3.1.1 add reagent and sample (table 2) in 96 hole sample panel (PCR plate) in proportion
Table 2, PCR reaction system
| The PCR reaction reagent | Amount/ |
| 10 * PCR damping fluid | 2μl |
| 25mM MgCl 2 | 4μl |
| 5 * PCR primer mixture | 4μl |
| HotStart Taq archaeal dna polymerase | 0.7μl |
| Solution X | 2μl |
| Dna profiling | 50～100ng |
| The H that does not contain DNA enzyme/RNA enzyme 2O | Add to 20 μ l |
| Cumulative volume | 20μl |
3.1.2 carry out thermal cycle reaction (table 3) by following temperature
Table 3, PCR program
| | Temperature | Time | |
| 1 | 94℃ | 3min | |
| 2 | 94℃ | 30s | |
| 3 | 60℃ | 30s | |
| 4 | 70℃ | 1min | |
| 5 | N/A | Repeat 2-4 step 34 time (totally 35 times) | |
| 6 | 70℃ | 1min | |
| 7 | 4℃ | Continue: until collect the PCR product |
3.2 electrocapillary phoresis (take the GeXP genetic analyzer as example)
3.2.1 preparation GeXP sample (table 4)
Table 4, GeXP loading system
| The GeXP sample | Amount/hole |
| The upper foreign damping fluid of GeXP | 38.75μl |
| Dna fragmentation size criteria-400 | 0.25μl |
| The PCR product | 1μl |
| Cumulative volume | |
| Mineral oil | |
| 1 |
3.2.2 preparation parting liquid: about 250 μ l parting liquids are added in the hole of proper number on the 96 hole parting liquid plates.
3.2.3GenomeLab GeXP genetic analyzer electrocapillary phoresis sample separation
(seeing GenomeLab GeXP genetic analyzer specification sheets)
Four, data analysis
4.1 data derive
The file of " File " → " Export Fragments/Genotypes As " → preservation CSV form.
4.2 data analysis
This test kit comprises 38 pairs of primers, its amplified production size between 151 and 372bp between.Wherein 35 pairs of primers are evenly distributed in 13,18,21, X and Y chromosome, seven pairs of primers of every karyomit(e).Other 3 pairs of primer amplification fragments be respectively 150bp (Chr.11, RRM1), 239bp (Chr.11/16, β-Globin) and 282bp (Chr.10, RNaseP), in contrast.
4.2.1 the comparison in the sample
Take the XY sample as example, we are divided into seven groups with 35 genes of 35 pairs of primer amplifications, and each group comprises respectively a gene on 13,18,21, X and the Y chromosome.When analyzing the relative signal of certain gene, the peak area (Peak Area) that we calculate the GeXP peak that this gene pairs answers accounts for the summation of five gene peak areas of this group.Seven gene peak area accounting values on each karyomit(e) can be averaged, and calculate variation value (Stdev).The advantage of this method of calculation is farthest to reduce the impact that fragment analysis baseline (Baseline) brings; Its shortcoming is when running into the trisome sample, such as trisomy 21, gene peak area accounting on No. 21 karyomit(e) increases, and gene peak area accounting will have to some extent and reduce on other karyomit(e) simultaneously, and this situation about reducing may be with corresponding karyomit(e) monomer the time is obscured.Otherwise, when running into the monomer sample.Such as the X monomer, the gene peak area accounting on the X chromosome reduces, and simultaneously gene peak area accounting will have to some extent and increase on other karyomit(e), and the situation when this increase may be with corresponding karyomit(e) trisome is obscured.This just needs us further to analyze.
4.2.2 the comparison of sample room
Seven gene peak area accounting values on each karyomit(e) that relatively draws in the above sample can the accounting value corresponding with normal control compare.In addition, when the relative signal of analyzing gene, except peak area value that each gene pairs is answered divided by crt gene (RRM1, β-Globin and the RNaseP) peak area value, also need each gene peak of each gene peak and a normal control sample is compared.We have introduced such method of calculation:
X represents unknown DNA sample to be analyzed, X
G1-2, X
G3-4And X
G5-7Represent respectively the peak area of 1-2 group, 3-4 group and 5-7 group gene, X
RRM1, X
β-GlobinAnd X
RNasePBe the peak area of each crt gene; The normal contrast of Ctrl representative DNA sample (sex that X and Ctrl are corresponding is consistent).
Seven gene pairss are answered on each karyomit(e) seven log values; Log value between the coloured differently body can be carried out significance of difference analysis and be analyzed chromosome number purpose dysploidy.
Here, normal control seems particularly important.This normal control sample need to meet certain requirement, namely with analytic sample from similar tissue samples, use the same method and extract DNA, carry out simultaneously PCR and GeXP and analyze.When comparing with the normal control sample, must follow with sex principle relatively.
Five, sample analysis
The inventor uses test kit and a collection of fetus chorionic cells sample has been carried out analyzing (table 5) analytical results and MLPA result coincide.This test kit is working properly, shows as: peak number meets design; Peak type normal (Fig. 1); The primer of upper each gene of karyomit(e) (13,18 and No. 21 karyomit(e)) is working properly, can reach quantitative purpose (Fig. 2); The sex recall rate reaches 100%; Each chromosomal aneuploidy case all can clear detecting (Fig. 3).
The analysis of cases of Fig. 1 chromosomal aneuploidy.Diagram DNA sample (50-100ng) is behind multiplex PCR, and the result of GeXP electrocapillary phoresis analysis is from top to bottom successively: normal female DNA sample; The X chromosome monomer; No. 13 karyomit(e) trisomes; No. 18 karyomit(e) trisomes and No. 21 karyomit(e) trisomes.Chromosomal aneuploidy can detect by quantitative analysis.For example can clearly illustrate that by the relative height of observing each karyomit(e) specific peak in the black surround difference of peak height is corresponding with chromosomal aneuploidy or sex; In addition by relatively and the relative peak area of calculating each karyomit(e) specific peak can show more accurately chromosomal aneuploidy (seeing " embodiment 4, data analysis ")
Chromosome number purpose quantitative analysis in Fig. 2 normal specimens.The relative copy number of upper each gene (primer pair) of each karyomit(e) (13,18 with No. 21 karyomit(e)s) has represented quantitative result, and wherein 50% sample is positioned at the cylindricality box; 95% sample is positioned at error line.(n＝205，SD＝0.07～0.24)
Chromosome number purpose quantitative analysis in Fig. 3 normal specimens and the case sample.The cylindricality box has represented the relative copy number of upper each gene (primer pair) of each karyomit(e) in the normal specimens (13,18,21, X and Y chromosome).50% sample is positioned at the cylindricality box; 95% sample is positioned at error line.Heterosomal statistics is from two gender groups (n=102 and 77, SD=0.05～0.09).Horizontal line has represented the mean value of the relative copy number of upper each gene (primer pair) of each karyomit(e) in the case sample (13,18,21, X and Y chromosome).
Table 5, chromosomal aneuploidy sample analysis result.
| Chromosomal aneuploidy | Statistics numbers |
| Normally | 179 |
| The X monomer | 22 |
| 13 trisomes | 8 |
| 18 trisomes | 4 |
| |
12 |
| |
1 |
| |
1 |
| 21 monomers | 4 |
| Add up to | 231 |
Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make on the basis of the above description other multi-form variation and changes.Here can't give all embodiments exhaustive.Everyly belong to the row that apparent variation that technical scheme of the present invention amplifies out or change still are in protection scope of the present invention.
Claims (8)
1. method that detects five numerical abnormalities of chromosomes, be used for detecting simultaneously comprise 21,13,18, five chromosome number purpose dysploidy of X and Y chromosome, comprise: (1), on described five karyomit(e)s, choose respectively seven genes, the gene design for selected 38 38 pairs of primers; (2), utilize above combination of primers to carry out multiplex PCR and electrocapillary phoresis; (3), utilize software that electrophoresis result is analyzed.
2. the method for claim 1, described chromosome number purpose dysploidy refer to that the chromosome number purpose that is different from the normal human increases or disappearance.
3. the method for claim 1, step (1) also are included on other karyomit(e)s outside these five karyomit(e)s and choose three genes in contrast (CK).
4. the method for claim 1, the pcr template in the described step (2) is the human genome DNA.
5. the method for claim 1, same chromosomal several genes are positioned at this chromosomal different zones as far as possible; The design clip size is between 100bp and 400bp, and the difference of adjacent two clip size is generally 3～10bp.
6. the purposes of each the described method in a kind as the aforementioned claim, described method are applied to adopt wound and the antenatal diagnosis of the clinical sample that need to obtain without two kinds of means of wound to detect.
7. purposes as claimed in claim 6, described have the wound means to obtain amniotic fluid or embryo villi theca cell by amniocentesis.
8. purposes as claimed in claim 6 describedly obtains fetal cell without the wound means by flow cytometer screening maternal blood.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3008215A4 (en) * | 2013-06-13 | 2017-01-25 | Ariosa Diagnostics, Inc. | Statistical analysis for non-invasive sex chromosome aneuploidy determination |
| US11270781B2 (en) | 2011-01-25 | 2022-03-08 | Ariosa Diagnostics, Inc. | Statistical analysis for non-invasive sex chromosome aneuploidy determination |
| RU2777072C1 (en) * | 2021-06-15 | 2022-08-01 | Общество с ограниченной ответственностью «Хроматест» | Method for identifying fetal aneuploidy in a blood sample of the pregnant woman |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11270781B2 (en) | 2011-01-25 | 2022-03-08 | Ariosa Diagnostics, Inc. | Statistical analysis for non-invasive sex chromosome aneuploidy determination |
| EP3008215A4 (en) * | 2013-06-13 | 2017-01-25 | Ariosa Diagnostics, Inc. | Statistical analysis for non-invasive sex chromosome aneuploidy determination |
| EP3663414A1 (en) * | 2013-06-13 | 2020-06-10 | Ariosa Diagnostics, Inc. | Statistical analysis for non-invasive sex chromosome aneuploidy determination |
| RU2777072C1 (en) * | 2021-06-15 | 2022-08-01 | Общество с ограниченной ответственностью «Хроматест» | Method for identifying fetal aneuploidy in a blood sample of the pregnant woman |
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