CN109371121A - A kind of kit for pre-natal diagnosis chromosome abnormality - Google Patents
A kind of kit for pre-natal diagnosis chromosome abnormality Download PDFInfo
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- CN109371121A CN109371121A CN201811459551.9A CN201811459551A CN109371121A CN 109371121 A CN109371121 A CN 109371121A CN 201811459551 A CN201811459551 A CN 201811459551A CN 109371121 A CN109371121 A CN 109371121A
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Abstract
The invention discloses a kind of kits for pre-natal diagnosis chromosome abnormality, it include: human peripheral extracting genome DNA reagent, DNA deposition and purification reagent, the end DNA is repaired and linker reagents, fetus dissociative DNA in maternal plasma is detected by high-flux sequence method, analyze the difference of No. 21, No. 18 and No. 13 chromosome quantitatives of the fetus dissociative DNA in sample, it is contemplated that for carrying out Prenatal Screening to foetal chromosome aneuploidy disease 21- patau syndrome, E trisomy and 13- patau syndrome.Detection accuracy of the present invention is up to 99.9% or more；2 steps sample-adding, the purifying of 1 step, single tube reaction reduce the potential pollution of manual operation and fault；It can be achieved to be sequenced while for up to 96 samples；With splendid repeatability.
Description
Technical field
This application involves medical instruments fields, in particular to a kind of reagent for pre-natal diagnosis chromosome abnormality
Box.
Background technique
Birth defect refers to the exception of embryo or fetus in growth course in developmental anatomy or function.Birth defect disease
The complexity of cause is the difficult point studied at present.Chromosome abnormality is the most common inherent cause for leading to birth defect.
Trisomy 21,18 three-bodies, 13 in foetal chromosome aneuploidy (Fetal Chromosome Aneuploidies)
Three-body (Trisomies 21, Trisomies 18, Trisomies13, i.e. T21, T18, T13) is the clinically most common dyeing
Body aneuploid disease.It is respectively that (also known as Down syndrome, mongolism or Down are comprehensive for 21- patau syndrome that its is corresponding
Sign), E trisomy (also known as Edwards syndrome) and 13- patau syndrome (also known as Patau syndrome), disease incidence divides
Not Yue Wei 1/700,1/6000,1/10000, there are serious dysnoesia and malformations by the infant overwhelming majority.
Prenatal Screening and pre-natal diagnosis are the means to avoid generation genetic defect infant from providing.Conventional Prenatal Screening side
Method includes: the ultrasound and serology United screening and middle pregnancy period maternal serum screening of trimester.Screening results are high risk
Pregnant woman is suggested finally being confirmed through pre-natal diagnosis, by pregnant woman's informed choice.The pre-natal diagnosis gold mark of fetal chromosomal abnormalities
Standard refers to through Chorionic villus sampling art/amniocentesis/percutaneous Umbilical blood art to intervene preceding pre-natal diagnosis operation, takes phase
Cell is answered to carry out karyotyping to fetal chromosomal using cell biology method.
Above-mentioned prenatal gene diagnosis be built upon it is invasive on the basis of cytogenetic diagnosis, due to operation have wound
Property, easily cause intrauterine infection, miscarriage, even have certain influence to fetus, it is corresponding therefore, it is necessary to be carried out to noninvasive inspection
Research.
Summary of the invention
The main purpose of the application is to provide a kind of atraumatic, the reagent for pre-natal diagnosis chromosome abnormality
Box.
The principle of the present invention is: there are fetus dissociative DNA (cell-free DNA, cfDNA) in maternal blood plasma, long
Degree is about 75bp -250bp, and almost all derives from the trophocyte of placenta, and concentration and pregnant week are closely related and with certain
Ratio (5% -30%) is stable in the presence of in maternal peripheral blood plasma.High-flux sequence method by take Maternal plasma extraction include
The plasma DNA of normal parent and fetus, through library construction, fragment amplification etc. finally carries out machine sequencing, passes through software point
It analyses data and obtains chromosome number evaluation result.For detecting trisomy 21 syndrome, when to the Maternal plasma for nourishing T21 fetus
When dissociative DNA data are analyzed, No. 21 chromosome dissociative DNA sums have the raising of small scale, pass through statistical algorithms
This fine difference is distinguished to realize the Prenatal Screening for carrying out foetal chromosome aneuploidy using cfDNA.
Kit provided by the present invention for pre-natal diagnosis chromosome abnormality, comprising: human peripheral genomic DNA mentions
Reagent, DNA deposition and purification reagent, the reparation of the end DNA and linker reagents are taken, passes through high-flux sequence method and detects maternal blood blood
Fetus dissociative DNA in slurry analyzes No. 21, No. 18 and No. 13 chromosome quantitatives of the fetus dissociative DNA in sample
Difference, it is contemplated that for comprehensive to foetal chromosome aneuploidy disease 21- patau syndrome, E trisomy and 13- three-body
Sign carries out Prenatal Screening.
Specifically, the high-flux sequence method, refers to and carries out upper machine sequencing by library construction, fragment amplification
Sequencing approach.
Specifically, the DNA extracts the mixing that reagent is SDS, NP40 surfactant, Proteinase K, Tris saturated phenol
Liquid.
Specifically, the DNA deposition and purification reagent is spermine.Reagent of the present invention is more special, conventional skill
DAN precipitation reagent and DNA purified reagent can be respectively adopted in art.And present invention employs spermine.Spermine is not organic solvent, but
DNA can quickly and effectively be precipitated.After spermine is in conjunction with DNA, making DNA, structure condenses and precipitates in the solution, and can make monokaryon
Thuja acid and protein impurities are opened with DNA points, achieve the purpose that purify DNA.
Specifically, the end DNA repair and linker reagents include: T4DNA polymerase, T4 polynueleotide kinase and
KlenowDNA polymerase.It further include buffer.
Specifically, the kit further include: PCR reaction solution.The PCR reaction solution includes that PCR increases primer, drop
Stabilizer, ultrapure water.The PCR increasing primer length is 18~22 bases, G/C content is 50~60%, TM value is 55~65
Degree.The PCR increases the dosage of primer and is greater than 3.2pmol/ μ l for 10~20 μ l, concentration.
Specifically, the kit further include: bead suspension.
The utility model has the advantages that the present invention has the following advantages that 1) Detection accuracy is up to 99.9% or more；2) 2 steps sample-adding, 1 step are pure
Change, single tube reaction, reduces the potential pollution of manual operation and fault；3) it can be achieved to be sequenced while for up to 96 samples；4)
Splendid repeatability；5) minimum only to need 1ng DNA that build library.
The present invention is a kind of antenatal detection technique of novel foetal chromosome aneuploidy disease, is surveyed using high-throughput gene
Sequence technology and analysis of biological information method need to only extract pregnant woman's 5-10ml vein peripheral blood (without on an empty stomach), can accurately sentence
Whether disconnected fetus suffers from Down syndrome (21- patau syndrome), Edward's syndrome (E trisomy), pa Tao Shi
Three kinds of common prenatal diagnosis of syndrome (13- patau syndrome).This product application high throughput sequencing technologies, each
Sample contains unique identification sequence and anti-error mechanism, and single detection can carry out quick sequencing analysis to 96 samples.Detection takes
Sample process is noninvasive, on pregnant woman and fetus without influence.
Specific embodiment
It is right below in conjunction with the embodiment of the present application in order to make those skilled in the art more fully understand application scheme
Technical solution in the embodiment of the present application is clearly and completely described, it is clear that described embodiment is only the application one
Partial embodiment, instead of all the embodiments.
Kit:
DNA extract reagent: SDS, NP40 surfactant, Proteinase K, Tris saturated phenol mixed liquor.
DNA deposition and purification reagent: spermine.
The end DNA is repaired and linker reagents include: T4DNA polymerase, T4 polynueleotide kinase and KlenowDNA polymerization
Enzyme, buffer.
PCR reaction solution: PCR increases primer, droplets stable agent, ultrapure water.
PCR increases primer: length is 18~22 bases, G/C content is 50~60%, TM value is 55~65 degree.
PCR increases the dosage of primer and is greater than 3.2pmol/ μ l for 10~20 μ l, concentration.
Bead suspension.
Embodiment
Plasma dna extracts, and needs that negative setting is arranged in extraction process and repeats control group, by the plasma dna after extraction
It is placed in be checked in -80 DEG C of incubators.
Plasma dna after extraction carries out T4 archaeal dna polymerase, T4 polynueleotide kinase and KlenowDNA polymerase and repairs
It is multiple, to guarantee that DNA fragmentation is connect with 5 ends with the special joint of " T " base；Row PCR amplification operates again, selects after amplification
Agilent2100Bioanalyzer detects library yield and size；After the completion of paced work, select
The sequencing of IlluminaHiSeq2000 high-flux sequence instrument.
The chromosomal nucleic acid number of fragments of acquisition is subjected to bioinformatic analysis, and calculates that every chromosome is corresponding to be covered
Lid depth value；Depth value is converted into risk index, to determine fetus E trisomy, 21- patau syndrome, 13- tri-
The relative risk of the diseases such as body syndrome.
All collection data inputting tables, it is for statistical analysis in SPSS 10.0.Measurement data indicates with (x ± s),
Using two independent samples t tests, enumeration data uses Chi-square Test, and inspection level takes a=0.05.
Choose separate unit gravid woman 1000, the age 25~40 years old of early, the middle pregnancy period of antenatal exaination；Pregnant week 13~26
Week.
Aneuploid fetus 8 are found altogether to 1500 pregnant woman blood plasma cffDNA high throughput testing results, specific as follows:
CffDNA concentration estimated by NIFTY Y chromosome is 0.01；CffDNA concentration estimated by NIFTY Y chromosome
0.03；T value is 4.51.Caryogram: 21- three-body.
CffDNA concentration estimated by NIFTY Y chromosome is 0.03；CffDNA concentration estimated by NIFTY Y chromosome
0.03；T value is 4.60.Caryogram: 21- three-body.
CffDNA concentration estimated by NIFTY Y chromosome is 0.39；CffDNA concentration estimated by NIFTY Y chromosome
0.05；T value is 3.58.Caryogram: 18- three-body.
CffDNA concentration estimated by NIFTY Y chromosome is 0.05；CffDNA concentration estimated by NIFTY Y chromosome
0.08；T value is 3.52.Caryogram: 13- three-body.
CffDNA concentration estimated by NIFTY Y chromosome is 0.01；CffDNA concentration estimated by NIFTY Y chromosome
0.06；T value is -0.87.Caryogram: 45, XO.
CffDNA concentration estimated by NIFTY Y chromosome is 0.25；CffDNA concentration estimated by NIFTY Y chromosome
0.02；T value is 2.68.Caryogram: 47, XXX.
CffDNA concentration estimated by NIFTY Y chromosome is 0.15；CffDNA concentration estimated by NIFTY Y chromosome
0.01；T value is 2.54.Caryogram: 47, XXX.
CffDNA concentration estimated by NIFTY Y chromosome is 0.38；CffDNA concentration estimated by NIFTY Y chromosome
0.07；T value is 8.64.Caryogram: 47, XYY.
Including 2 21- three-bodies；1 18- three-body；1 13- three-body；1 45, XO；1 47, XYY；2 47,
XXX。
Comparative test
There are 1 47, XYY and FISH testing result and chromosome karyotype analysis result to exist in high-throughput testing result poor
Different, high-throughput antenatal exaination is 99.9% or more with the coincidence rate of FISH testing result and chromosome karyotype analysis result.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application.
Claims (10)
1. a kind of kit for pre-natal diagnosis chromosome abnormality, which is characterized in that the kit reagent box includes: people
Peripheral blood extracting genome DNA reagent, DNA deposition and purification reagent, the end DNA is repaired and linker reagents, passes through high-flux sequence
Method detects fetus dissociative DNA in maternal plasma, No. 21, No. 18 for analyzing the fetus dissociative DNA in sample
And the difference of No. 13 chromosome quantitatives, it is contemplated that for foetal chromosome aneuploidy disease 21- patau syndrome, 18- three-body
Syndrome and 13- patau syndrome carry out Prenatal Screening.
2. the kit according to claim 1 for pre-natal diagnosis chromosome abnormality, which is characterized in that the high pass
PCR sequencing PCR is measured, refers to the sequencing approach for carrying out upper machine sequencing by library construction, fragment amplification.
3. the kit according to claim 1 for pre-natal diagnosis chromosome abnormality, which is characterized in that the DNA
Extraction reagent is the mixed liquor of SDS, Proteinase K, Tris saturated phenol, NP40 surfactant.
4. the kit according to claim 1 for pre-natal diagnosis chromosome abnormality, which is characterized in that the DNA
Deposition and purification reagent is spermine, and spermine is not organic solvent, but can quickly and effectively precipitate DNA；After spermine is in conjunction with DNA, make DNA
Structure condenses and precipitates in the solution, and mononucleotide and protein impurities can be made to open with DNA points, reaches purifying DNA's
Purpose.
5. the kit according to claim 1 for pre-natal diagnosis chromosome abnormality, which is characterized in that the DNA
End is repaired and linker reagents include: T4DNA polymerase, T4 polynueleotide kinase and KlenowDNA polymerase.
6. the kit according to claim 1 for pre-natal diagnosis chromosome abnormality, which is characterized in that the reagent
Box further include: PCR reaction solution.
7. the kit according to claim 6 for pre-natal diagnosis chromosome abnormality, which is characterized in that the PCR
Reaction solution includes that PCR increases primer, droplets stable agent, ultrapure water.
8. the kit according to claim 7 for pre-natal diagnosis chromosome abnormality, which is characterized in that the PCR
Increasing primer length is 18~22 bases, G/C content is 50~60%, TM value is 55~65 degree.
9. the kit according to claim 7 for pre-natal diagnosis chromosome abnormality, which is characterized in that the PCR
The dosage for increasing primer is 10~20 μ l, concentration is greater than 3.2pmol/ μ l.
10. the kit according to claim 1 for pre-natal diagnosis chromosome abnormality, which is characterized in that the examination
Agent box further include: bead suspension.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111286529A (en) * | 2019-07-22 | 2020-06-16 | 常州市妇幼保健院 | Kit for prenatal screening of false positive by using free DNA of peripheral blood fetus |
| CN111500704A (en) * | 2020-04-28 | 2020-08-07 | 广州市金圻睿生物科技有限责任公司 | Human fetus chromosome aneuploidy detection kit and method |
| CN112662756A (en) * | 2021-01-26 | 2021-04-16 | 上海博奥颐和医学检验所有限公司 | Fetal chromosome aneuploid genome sequencing detection method |
| WO2022167017A1 (en) * | 2021-02-08 | 2022-08-11 | Bioinova, A.S. | Liquid composition for molecular diagnostics by pcr |
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| CN111500704A (en) * | 2020-04-28 | 2020-08-07 | 广州市金圻睿生物科技有限责任公司 | Human fetus chromosome aneuploidy detection kit and method |
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