CN105087809A - Method and kit for rapidly detecting fetal cell VHL gene mutation in amniotic fluid - Google Patents
Method and kit for rapidly detecting fetal cell VHL gene mutation in amniotic fluid Download PDFInfo
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Abstract
The invention provides a method and kit for rapidly detecting fetal cell VHL gene mutation in amniotic fluid and relates to the field of gene detection. The method comprises the following steps: detecting whether fetal cell DNA extracted from amniotic fluid is polluted by maternal genetic materials or not; if maternal genetic material pollution does not exist, carrying out VHL gene mutation detection on fetal cells directly; if maternal genetic material pollution exists, subculturing the fetal cells and then carrying out VHL gene mutation detection on fetal cells, wherein VHL gene mutation detection comprises the followings: carrying out VHL gene large fragment deletion detection on fetal cells through a fluorescence in-situ hybridization probe and carrying out VHL gene point mutation, small fragment deletion and splice site mutation detection on fetal DNA by adopting amplification primers of three exons of a VHL gene. By adopting the method and kit provided by the invention, whether fetal cell VHL gene mutates or not can be detected accurately and efficiently, the operation is simple and the cost is low.
Description
Technical field
The present invention relates to field of gene detection, particularly relate to method and the test kit of amniotic fluid fetal cell vhl gene abrupt climatic change.
Background technology
Vhl gene (MIM numbering 608537) is positioned 3p25-26, total length 10KD, comprises three exons and two introns, transcribed formation two kinds of mRNA.The mRNA comprising three exon transcription products translates p30 (213 amino acid) and p19 (159 amino acid) albumen.P19 is the isomer that vhl gene second transcription initiation site (No. 54 codons) transcribes formation, with p30 functional similarity.
Vhl gene mutational formats is various, comprises point mutation, large fragment deletion, small segment disappearance or insertion, shearing site sudden change etc.Current detection vhl gene the most frequently used method of suddenling change is PCR direct Sequencing, accuracy rate is 38% ~ 80%, this is that detection technique can only check point suddenlys change, small segment lacks or inserts, shearing site sudden change because PCR checks order, can not detect vhl gene large fragment deletion, but the incidence of VHL large fragment deletion is 11% ~ 40%.The domestic and international method for the detection of vhl gene large fragment deletion comprises marking hybrid method SouthernBlot, multiplex ligation-dependent probe amplification MLPA, reverse transcription method RT-PCR and universal primer fluorescence quantitative PCR method UPQFM-PCR etc. at present.SouthernBlot and MLPA has detection method complex operation, expensive, be unsuitable for defects such as applying, and the detection sample of above-mentioned vhl gene sudden change mostly is peripheral blood, at present also not to the method that the vhl gene of fetal cell in amniotic fluid detects, time on the one hand owing to extracting amniotic fluid, the pollution of maternal inheritance thing may be subject in amniotic fluid, affect detected result; On the other hand, current is applied to the vhl gene detection method that peripheral blood etc. detects sample, and sense cycle is long, and testing cost is high, is unfavorable for the detection fast and accurately of vhl gene.
Summary of the invention
Object of the present invention is exactly to overcome above-mentioned prior art Problems existing, provide a kind of simple to operate, detect fast, fetal cell vhl gene mutation detection methods that result is accurate and with low cost and test kit.
For realizing object of the present invention, one aspect of the present invention provides the method for fetal cell vhl gene sudden change in a kind of rapid detection amniotic fluid, comprising:
By extracting the DNA of fetal cell in amniotic fluid, obtain DNA solution;
Described DNA solution is carried out to the detection of maternal inheritance Substances Pollution, judge whether there is maternal inheritance Substances Pollution in DNA solution;
If there is not maternal inheritance Substances Pollution in judgement DNA solution, then direct vhl gene abrupt climatic change is carried out to fetal cell;
If there is maternal inheritance Substances Pollution in judgement DNA solution, then carry out vhl gene abrupt climatic change again after subculture being carried out to fetal cell in amniotic fluid.
Wherein, described maternal inheritance Substances Pollution detects and comprises:
3 STR DXS6797 on synthetic mankind sex determining gene SRY and X chromosome, the amplimer of DXS6807, AR are to 1-4;
Utilize described amplimer to set up pcr amplification reaction system to 1-4 and described DNA solution, carry out PCR reaction, obtain pcr amplification product;
1.5% agarose gel electrophoresis is carried out to the sry gene amplified production in described pcr amplification product, STR analysis is carried out to the amplified production of DXS6797, DXS6807 and AR gene in described pcr amplification product, judges whether there is maternal inheritance Substances Pollution in described DNA solution according to the electrophoresis result of described sry gene amplified production and STR analytical results.
Wherein, the described pcr amplification reaction system of described amplimer to 1-4 and the foundation of described DNA solution that utilize is: cumulative volume is 25 μ l, comprises the DNA profiling of PCRmix, 20-80ng of 12.5 μ l, the amplimer of 0.5 μ l and the ddH of surplus
2o; Reaction conditions is: denaturation 5min under 95 DEG C of temperature condition; 95 DEG C of sex change 20s, sry gene, DXS6797, DXS680755 DEG C annealing 20s, AR58 DEG C annealing 20s, 72 DEG C extend 20s, 30 rear 72 DEG C of renaturation 5min of circulation, 4 DEG C of preservations.
Particularly, described amplimer, is respectively 1-4 as shown in SEQIDNO.1-8:
Amplimer is to 1:SRY-F:5 '-gagtgaagcgacccatgaac-3 '
SRY-R：5’-tcttgagtgtgtggctttcg-3’
Amplimer is to 2:DXS6797-F:5 '-ttccctctctccctctgtct-3 '
DXS6797-R：5’-acacacacccaaaaccagat-3’
Amplimer is to 3:DXS6807-F:5 '-gagcaatgatctcatttgca-3 '
DXS6807-R：5’-aagtaaacatgtataggaaaaagct-3’
Amplimer is to 4:AR-F:5 '-tccagaatctgttccagagcgtgc-3 '
AR-R：5’-gctgtgaaggttgctgttctccat-3’
Wherein, described amplimer is to 5 ' end flag F AM fluorescence of DXS6797-F primer in 2.
Wherein, described amplimer is to 5 ' end flag F AM fluorescence of DXS6807-F primer in 3.
Wherein, described amplimer is to 5 ' end flag F AM fluorescence of AR-F primer in 4.
Wherein, described vhl gene abrupt climatic change comprises:
Adopt fluorescence in situ hybridization probe to carry out the detection of vhl gene large fragment deletion to fetal cell in described amniotic fluid, obtain the detected result whether described fetal cell exists large fragment deletion.
Particularly, described fluorescence in situ hybridization probe is marked with fluorescent signal, carries out PCR prepare by the probe primer with the base sequence shown in SEQIDNO.15-20 to 8-10.
Wherein, described probe primer, to the base sequence of 8-10 as shown in SEQIDNO.15-20, comprising:
Probe primer 85 '-agtaacgagttggcctagcctcg
5’-gttcctccgggccggac
Probe primer 95 '-gtactgacgttttactttttaaaaagataagg
5’-catgctctacacattgttctcctgg
Probe primer 105 '-gcattgcacatcaacggat
5’-cagtcttcccaaagcaggag
Wherein, the size that the described probe primer by having the base sequence shown in SEQIDNO.15 ~ 20 carries out the hybridization probe that PCR prepares is 300-1000bp.
Further preferably, the size that the described probe primer by having the base sequence shown in SEQIDNO.15 ~ 20 carries out the hybridization probe that PCR prepares is 300-800bp.
Further preferably, the size that the described probe primer by having the base sequence shown in SEQIDNO.15 ~ 20 carries out the hybridization probe that PCR prepares is 300-500bp.
Wherein, described employing fluorescence in situ hybridization probe comprises the detection that described fetal cell carries out vhl gene large fragment deletion:
Fetal cell in the amniotic fluid gathered is utilized to prepare cell smear;
Described fluorescence in situ hybridization probe is placed in hybridization solution and described cell generation hybridization, obtains hybrid product;
Wash described hybrid product, and carry out nuclear targeting, obtain the cell smear after nuclear staining;
By the cell smear after nuclear staining described in fluorescence microscope, judge whether the DNA of sample to be tested there occurs vhl gene large fragment deletion.
Wherein, in the amniotic fluid that gathered of described utilization, fetal cell is prepared cell smear and is also comprised: carry out subculture to fetal cell in the amniotic fluid collected.
Particularly, describedly carry out subculture to the cell in amniotic fluid and refer to carry out subculture in the fetal cell nutrient solution prepared in advance, wherein culture temperature is 37 DEG C, CO in air
2volume percent be 5%.
Particularly, described fetal cell nutrient solution is formulated by the hyclonedmem/f12 substratum of 70-79%, the penicillin of 20-28% foetal calf serum FBS and 1-2% and the dual anti-PS of Streptomycin sulphate.
Preferably, described fetal cell nutrient solution by 74% hyclonedmem/f12 substratum, 25% foetal calf serum FBS, 1% penicillin and the dual anti-PS of Streptomycin sulphate formulated.
Wherein, the described step preparing cell smear comprises: by the cell in centrifugal acquisition sample to be tested, after the centrifugal and KCl Hypotonic treatment of phosphate buffered saline buffer, fixes the form of described cell, make cell smear with stationary liquid; Described cell smear is carried out burin-in process, after gastric pepsin digestion process, carries out alcohol serial dehydration, obtain cell smear.
Wherein, described stationary liquid is that the methyl alcohol of 3:1 and Glacial acetic acid are formulated by volume ratio.
Wherein, described hybridization solution is that the methane amide of 5:1:1:3,20xSSC, T 500 and water are formulated by volume ratio.
Wherein, described burin-in process is that described cell smear is processed 20min under 56 DEG C of conditions on roasting sheet machine.
Wherein, described burin-in process can also be spend the night to place 12-16h under the room temperature condition of 15-30 DEG C.
Particularly, described employing fluorescence in situ hybridization probe also comprises the detection that described fetal cell carries out vhl gene large fragment deletion: the preparation of fluorescence in situ hybridization probe.
Wherein, the preparation of described fluorescence in situ hybridization probe comprises: in human genome, screening is used as the vhl gene sequence of FISH probe, obtains goal gene fragment by cloning primer to 11-13; The gene fragment obtained is connected in plasmid, obtains the plasmid containing goal gene fragment; Described plasmid is increased in intestinal bacteria, after extracting plasmid, obtains plasmid solution; Probe primer is utilized to have the fluorescence in situ hybridization probe of fluorochrome label to the preparation of 8-10, fluorescence raw material and described plasmid solution.
Wherein, described cloning primer, to the base sequence of 11-13 as shown in SEQIDNO.21-26, comprising:
Cloning primer is to 11F:5 '-aaccttagaggggcgaaaaa
R:5’-gcttcagaccgtgctatcgt
Cloning primer is to 12F:5 '-aacctttgcttgtcccgata
R:5’-ttatcagagtgggtggcaca
Cloning primer is to 13F:5 '-gcaaagcctcttgttcgttc
R:5’-cagtcttcccaaagcaggag。
Wherein, describedly by cloning primer to the reaction system that 11-13 obtains goal gene fragment be
Reactant volume
PCRmix25μl
DNA profiling 100ng
Primer 2 μ l
DdH
2o surplus
Cumulative volume 50 μ l.
Wherein, describedly by cloning primer, goal gene fragment reaction conditions is obtained to 11-13 and be: 94 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 60s, 30 circulations, 72 DEG C of renaturation 7min, 4 DEG C of preservations.
Wherein, describedly by the reaction system that the gene fragment obtained is connected in plasmid be: T-vector0.7 μ l, PCR primer 5 μ l, T4 ligase enzyme 1 μ l, T4B μ ffer1 μ l, ddH
2o surplus, cumulative volume 10 μ l.
Wherein, describedly by the reaction conditions that the gene fragment obtained is connected in plasmid be: react 2h at ambient temperature.
Wherein, the described reaction system utilizing the preparation of probe primer, fluorescence raw material and described plasmid solution to have the fluorescence in situ hybridization probe of fluorochrome label is:
Wherein, the described reaction conditions utilizing the preparation of probe primer, fluorescence raw material and described plasmid solution to have the fluorescence in situ hybridization probe of fluorochrome label is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 45s, 30 circulations, 72 DEG C of renaturation 10min, 4 DEG C of preservations.Obtain fluorescence in situ hybridization probe.
Wherein, the concentration of described fluorescence in situ hybridization probe is about 8-20ng/ μ l.
Preferably, the concentration of described fluorescence in situ hybridization probe is about 10-15ng/ μ l.
Wherein, the common denaturation temperature of described hybridization is 70-80 DEG C, and the co-variation reaction times is 5-10min.
Preferably, the common denaturation temperature of described hybridization is 73-76 DEG C, and the co-variation reaction times is 7-8min.
Wherein, the hybridization temperature of described hybridization is 40-46 DEG C, and hybridization time is 14-18h.
Preferably, the hybridization temperature of described hybridization is 42-44 DEG C, and hybridization time is 16h.
Wherein, described vhl gene abrupt climatic change also comprises:
Adopt vhl gene three exons VHL-1, VHL-2 and VHL-3 amplimer to 5-7 to described DNA solution carry out vhl gene point mutation, small segment disappearance or insert, shearing site sudden change detection, obtain in described DNA solution whether exist point mutation, small segment disappearance or insert, shearing site sudden change detected result.
Wherein, the amplimer of three exons VHL-1, VHL-2 and VHL-3 of described vhl gene, is respectively 5-7 as shown in SEQIDNO.9-14:
Amplimer is to 5:VHL-1F5 '-ggtggtctggatcgcgga-3 '
VHL-1R5’-ggcttcagaccgtgctatcg-3’
Amplimer is to 6:VHL-2F5 '-gtggctctttaacaacctttgc-3 '
VHL-2R5’-cctgtacttaccacaacaaccttatc-3’
Amplimer is to 7:VHL-3F5 '-gcaaagcctcttgttcgttc-3 '
VHL-3R5’-caaaaatgccaccaccttct-3’。
Especially, the amplimer of three exons VHL-1, VHL-2 and VHL-3 of described employing vhl gene carries out vhl gene point mutation to described DNA solution, small segment lacks or inserts, the detection of shearing site sudden change comprises the following steps:
The amplimer of three exons VHL-1, VHL-2 and VHL-3 of synthetic vhl gene is to 5-7;
Utilize described amplimer to set up pcr amplification reaction system to 5-7 and described DNA solution, carry out PCR reaction and obtain PCR primer;
The PCR primer obtained is carried out purification process and sequencing analysis, judges whether described DNA there occurs vhl gene point mutation, small segment disappearance or insert and shearing site sudden change.
Wherein, the described pcr amplification reaction system utilizing described primer pair 5-7 and described DNA solution to set up is: cumulative volume is 25 μ l, comprises the DNA profiling of PCRmix, 100ng of 12.5 μ l, the primer of 1 μ l and the ddH of surplus
2o; Reaction conditions is: 94 DEG C of 10min denaturations; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations; 72 DEG C of renaturation 5min; 4 DEG C of preservations, obtain PCR primer.
For realizing object of the present invention, the present invention provides a kind of test kit for detecting vhl gene sudden change on the other hand, comprising:
For extracting the reagent of cell DNA in amniotic fluid;
Comprising the amplimer of sex determining gene as shown in SEQIDNO.1-8 and X chromosome STR, whether there is the reagent of maternal inheritance Substances Pollution for detecting described amniocyte DNA;
Comprise the amplimer as shown in SEQIDNO.9-14, for carry out vhl gene point mutation, small segment disappearance or insert, the detection of shearing site sudden change is with the reagent of detected result obtaining fetal cell and whether there is vhl gene point mutation, small segment disappearance or insert, shearing site suddenlys change;
The amplimer comprising the hybridization probe as shown in SEQIDNO.15-20 prepares by PCR reaction the hybridization probe indicating fluorescent signal, also comprises other for carrying out the detection of vhl gene large fragment deletion to obtain the reagent whether fetal cell exists the detected result of vhl gene large fragment deletion; And,
For carrying out the reagent of subculture to the fetal cell in described amniotic fluid.
Wherein, in described extraction amniotic fluid, the reagent of cell DNA can obtain from commercially available, other that can also be obtained by preparation can extract arbitrarily the reagent of DNA from amniocyte, according to one embodiment of present invention, reagent used in the present invention is purchased from the A1120 genome DNA extracting reagent kit of Promega company.
Wherein, the described reagent that whether there is maternal inheritance material for detecting described amniocyte comprises: for carrying out PCRmix and the amplimer of pcr amplification reaction.
Wherein, described primer be as shown in SEQNO.1-8 for amplifying human sex determining gene SRY amplimer to 1 and 3 STR DXS6797 on the X chromosome that increases, DXS6807, AR amplimer to 2-4:
Amplimer is to 1:SRY-F:5 '-gagtgaagcgacccatgaac-3 '
SRY-R：5’-tcttgagtgtgtggctttcg-3’
Amplimer is to 2:DXS6797-F:5 '-ttccctctctccctctgtct-3 '
DXS6797-R：5’-acacacacccaaaaccagat-3’
Amplimer is to 3:DXS6807-F:5 '-gagcaatgatctcatttgca-3 '
DXS6807-R：5’-aagtaaacatgtataggaaaaagct-3’
Amplimer is to 4:AR-F:5 '-tccagaatctgttccagagcgtgc-3 '
AR-R：5’-gctgtgaaggttgctgttctccat-3’
Wherein, described amplimer is to 5 ' end flag F AM fluorescence of DXS6797-F primer in 2.
Wherein, described amplimer is to 5 ' end flag F AM fluorescence of DXS6807-F primer in 3.
Wherein, described amplimer is to 5 ' end flag F AM fluorescence of AR-F primer in 4.
Wherein, described for carry out vhl gene point mutation, small segment disappearance or insert, the reagent of shearing site abrupt climatic change comprises: for carrying out PCRmix and the amplimer of pcr amplification reaction.
Wherein, described primer is the amplimer 5-7 of three exons for the vhl gene that increases comprised as shown in SEQNO.9-14:
Amplimer is to 5:VHL-1F5 '-ggtggtctggatcgcgga-3 '
VHL-1R5’-ggcttcagaccgtgctatcg-3’
Amplimer is to 6:VHL-2F5 '-gtggctctttaacaacctttgc-3 '
VHL-2R5’-cctgtacttaccacaacaaccttatc-3’
Amplimer is to 7:VHL-3F5 '-gcaaagcctcttgttcgttc-3 '
VHL-3R5’-caaaaatgccaccaccttct-3’。
Wherein, the reagent of the described detection for carrying out vhl gene large fragment deletion comprises: comprising the described probe primer by the base sequence shown in SEQIDNO.15-20 and carry out to 8-10 the hybridization probe indicating fluorescent signal that PCR prepares, also comprising: for sample to be tested being prepared into the reagent I of cell smear; For making described fluorescence in situ hybridization probe and described cell generation hybridization, obtain the reagent II of hybrid product; For washing described hybrid product, above-mentioned hybrid product being carried out nuclear targeting, obtaining the reagent III of the cell smear of nuclear staining.
Wherein, described probe primer to 8-10 is:
Probe primer 85 '-agtaacgagttggcctagcctcg
5’-cgtcttcttcagggccgtactc
Probe primer 95 '-gtactgacgttttactttttaaaaagataagg
5’-catgctctacacattgttctcctgg
Probe primer 105 '-gcattgcacatcaacggat
5’-cagtcttcccaaagcaggag
Wherein, described reagent I cell smear being prepared by sample to be tested comprises: be the formulated stationary liquid of the methyl alcohol of 3:1 and Glacial acetic acid by volume ratio;
Wherein, described for making described fluorescence in situ hybridization probe and described cell generation hybridization, obtain the reagent II of hybrid product and comprise: be the formulated hybridization solution of the methane amide of 5:1:1:3,20 × SSC, T 500 and water by volume ratio.
Wherein, described for washing described hybrid product, hybrid product is carried out nuclear targeting, and the reagent III of cell smear obtaining nuclear staining comprises: 0.4 × SSC solution of washing hybrid product prepare 0.3% NP40,2 × SSC solution NP40 of 0.1%, the spirituous solution of 70% and the DAPI to nuclear targeting that prepare.
Wherein, the described reagent for carrying out subculture to the fetal cell in described amniotic fluid comprises: the hyclonedmem/f12 substratum of 70-79%, the penicillin of 20-28% foetal calf serum FBS and 1-2% and the dual anti-PS of Streptomycin sulphate.
Preferably, the described reagent for carrying out subculture to the fetal cell in described amniotic fluid comprises: the hyclonedmem/f12 substratum of 74%, the penicillin of 25% foetal calf serum FBS and 1% and the dual anti-PS of Streptomycin sulphate
Beneficial effect of the present invention embodies in the following areas:
1, detection method provided by the invention can judge whether there is maternal inheritance Substances Pollution in the fetal cell DNA extracted from amniotic fluid fast and accurately, thus ensures the accuracy of fetus vhl gene abrupt climatic change result.
2, detection method provided by the invention can detect whether fetus vhl gene exists the sudden changes such as point mutation, small segment disappearance or insertion, large fragment deletion and shearing site sudden change at short notice, sense cycle is short, can time cost be reduced, the fastest once week in can go out result.
3, detection method simple operation provided by the invention, accurately and reliably, detection efficiency is high for detected result, and testing cost is low, practical.
4, test kit provided by the invention can reach above-mentioned testing goal, test kit is with low cost, detected result accurately and reliably.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of the pcr amplification product of sex determining gene SRY of the present invention, and wherein, the first swimming lane is DNAMarker, and size is 100-700, and swimming lane 2,3,4 is respectively the PCR primer electrophorogram of fetus, father and mother SRY;
Fig. 2 is STR analytical results figure of the present invention, and wherein, figure A is the peak figure of DXS6797, the peak figure of figure B to be the peak figure of DXS6807, figure C be AR, is respectively the peak figure of mother, father and fetus in figure from top to bottom;
Fig. 3 is the vhl gene sequencing result figure of fetal cell in amniotic fluid of the present invention;
Fig. 4 is the fluorescent hybridization signal graph of fetal cell in amniotic fluid of the present invention, and the white point in figure is fluorescence bright spot;
Fig. 5 is the fluorescent hybridization signal graph of positive control of the present invention, and the white point in figure is fluorescence bright spot;
Fig. 6 is the fluorescent hybridization signal graph of negative control sample of the present invention, and the white point in figure is fluorescence bright spot.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
It should be noted that, before carrying out the present embodiment, need the detection first peripheral blood of fetus M & F being carried out to vhl gene sudden change, its detection method can by PCR check order detection technique check point sudden change, small segment disappearance or insertion, shearing site suddenlys change, also SouthernBlot can be passed through, MLPA, RT-PCR and UPQFM-PCR carries out the detection of vhl gene large fragment deletion, its detection method is unrestricted, according to embodiments of the invention, the detection that the peripheral blood to fetus M & F that the inventive method provides carries out vhl gene sudden change is identical with the detection method of fetus.
It should be noted that, the extraction of the DNA mentioned in the application, comprise the extraction of foetal DNA, fetus mother peripheral blood DNA and fetus father peripheral blood DNA and normal human peripheral blood DNA, can be realized by any one approach, according to one embodiment of present invention, the extraction of the DNA that the present invention carries out is all adopt the A1120 genome DNA extracting reagent kit of Promega company to extract, and extracting method carries out according to the step of specification sheets.
The detection of maternal inheritance Substances Pollution whether is there is in embodiment 1 fetal cell DNA
1, the collection of sample
Extract the amniotic fluid of the gestation pregnant woman of 14 ~ 22 weeks with pricking device, obtain amniotic fluid fetal cell.
It should be noted that, detection method provided by the invention is equally applicable to use pricking device and extracts the fine hair sample that gestation is the pregnant woman of 11 ~ 14 weeks.
It should be noted that, pricking device used in this application is obtained by commercially available, and there is no particular limitation, can be that any one can realize the pricking device of amniocentesis.
2, the detection of maternal inheritance Substances Pollution
2.1, the design of primer and synthesis
According to the base sequence of 3 STR DXS6797 on mankind's sex determining gene SRY, X chromosome, DXS6807, AR, design and synthesize the pcr amplification primer pair of sry gene, DXS6797, DXS6807, AR sequence:
Amplimer is to 1:SRY-F:5 '-gagtgaagcgacccatgaac-3 '
SRY-R：5’-tcttgagtgtgtggctttcg-3’
Amplimer is to 2:DXS6797-F:5 '-ttccctctctccctctgtct-3 '
DXS6797-R：5’-acacacacccaaaaccagat-3’
Amplimer is to 3:DXS6807-F:5 '-gagcaatgatctcatttgca-3 '
DXS6807-R：5’-aagtaaacatgtataggaaaaagct-3’
Amplimer is to 4:AR-F:5 '-tccagaatctgttccagagcgtgc-3 '
AR-R：5’-gctgtgaaggttgctgttctccat-3’
Wherein, 5 ' the end flag F AM fluorescence of amplimer DXS6797-F, DXS6807-F, AR-F.
2.2, increase sry gene and DXS6797, DXS6807, AR gene
Extract the DNA of amniotic fluid fetal cell, fetus mother peripheral blood DNA and fetus father peripheral blood DNA, and set up the pcr amplification reaction system that cumulative volume is 25 μ l with the DNA of the amniotic fluid foetal DNA extracted and father and mother respectively for template: the PCRmix solution (2xTaqPCRMasterMixKT201-02 that described PCRmix solution selects Tian Gen biochemical technology company limited to produce) of 12.5 μ l, 70ngDNA (wherein, the consumption of described DNA can also be any consumption within the scope of 40-70ng, it can also be any consumption within the scope of 20-80ng, object of the present invention can be realized), the each 0.5 μ l of above-mentioned primer pair, primer concentration is 10pmol/ μ l, surplus ddH
2o.The reaction conditions of above-mentioned reaction is: denaturation 5min under 95 DEG C of temperature condition; 95 DEG C of sex change 20s, sry gene, DXS6797, DXS680755 DEG C annealing 20s, AR58 DEG C annealing 20s, 72 DEG C extend 20s, 30 rear 72 DEG C of renaturation 5min of circulation; 4 DEG C of preservations, obtain the amplified production of SRY, DXS6797, DXS6807, AR gene.
2.3, analysis judges gene amplification product
Sry gene amplified production step 2.2 obtained carries out 1.5% agarose gel electrophoresis (as shown in Figure 1), judge sex of foetus, again STR analysis (as shown in Figure 2) is carried out to DXS6797, DXS6807 and AR gene fragment in described pcr amplification product, judge whether there is maternal inheritance Substances Pollution in described DNA solution according to STR analytical results.
Agarose gel electrophoresis figure as is shown in fig. 1, first swimming lane is DNAMarker, size is 100-700bp, swimming lane 2,3,4 is respectively the PCR primer electrophorogram of fetus, father and mother SRY, only be present on Y chromosome according to the known sex determining gene SRY of the common practise of this area, the amplified production band of sry gene as shown in Figure 1 appears on the swimming lane of expression father exactly, and band is clear, without assorted band, the primer pair SRY sex determining gene high specificity that the present invention designs is described, can accurately increase sry gene; And represent in figure that the swimming lane of fetus does not have band to produce, illustrate that fetus does not exist sry gene, therefore judge that fetus is as girl.
STR analytical results figure as shown in Figure 2, known, the analytical results figure that the primer of the present invention's design obtains is accurately clear, and the sequencing result obtained is single-minded, illustrates that the specificity of primer is good.Wherein, figure A is the peak figure of DXS6797, the peak figure of figure B to be the peak figure of DXS6807, figure C be AR, in figure, is respectively the peak figure of mother, father and fetus from top to bottom.In figure, A can find out, the appearance of fetus only two peaks, appear at 294.5 and 300 places respectively, identical with the peak at 294.6 places of mother, identical with the peak at father 299.9 place, and there is not another peak of mother, illustrate that fetus has two X chromosomes respectively from father and mother, for girl, and there is not the pollution of mother's genetic material; In figure, B can find out, a peak only appears in mother, and is same position with the peak of father, and same peak has also appearred in fetus, illustrates that two peaks of mother there occurs overlap, and is positioned at same position with father, and this is gene-determined due to father and mother; In figure, C can find out, fetus only there are two peaks, appear at 294.5 places respectively, identical with the peak position at 294.7 places of mother, the peak of 300 positions is identical with the peak at father 300.2 place, there is not another peak of mother, illustrate that fetus has two X chromosomes respectively from father and mother, be girl, and there is not the pollution of mother's genetic material, show there is not maternal inheritance Substances Pollution in fetal cell according to this test-results; If foetal DNA is contaminated, then can there is two peaks of mother and a peak of father simultaneously.
The detection that the point mutation of embodiment 2 amniotic fluid fetal cell vhl gene, small segment disappearance or insertion and shearing site suddenly change
The present embodiment is three exons by vhl gene in the DNA to be measured that increases, the exon genes sequence of sample to be tested is obtained through sequenator, compare with the vhl gene in gene pool, whether analytical sequence there occurs point mutation, small segment disappearance or the sudden change such as insertion and shearing site sudden change.Concrete grammar is as follows:
1, design of amplification primers
According to three exon VHL-1, VHL-2, VHL-3 design of amplification primers of vhl gene to 5-6:
Amplimer is to 5:VHL-1F5 '-ggtggtctggatcgcgga-3 '
VHL-1R5’-ggcttcagaccgtgctatcg-3’
Amplimer is to 6:VHL-2F5 '-gtggctctttaacaacctttgc-3 '
VHL-2R5’-cctgtacttaccacaacaaccttatc-3’
Amplimer is to 7:VHL-3F5 '-gcaaagcctcttgttcgttc-3 '
VHL-3R5’-caaaaatgccaccaccttct-3’。
2, detect vhl gene point mutation, small segment disappearance or insert and shearing site sudden change
With the DNA of the amniocyte obtained in embodiment 1 for template, with described amplimer to 5-7 for primer sets up amplification reaction system, wherein, the cumulative volume of reaction system is 25 μ l, and the PCRmix comprising 12.5 μ l (selects Beijing Quanshijin Biotechnology Co., Ltd to produce
pCRSuperMixAS111), the DNA profiling of 100ng, concentration are primer each 1 μ l, the ddH of 5pmol/ μ l
2o surplus.Reaction conditions is: 94 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, after 30 circulations, 72 DEG C of renaturation 5min, 4 DEG C of preservations, obtain the amplified production of three exons of the vhl gene of foetal DNA, the exon product of the vhl gene after amplification is delivered to Sinogenomax Co., Ltd. and carries out purification process, and use ABI3730 sequencer, obtain sequencing result, as shown in Figure 3, judge whether vhl gene there occurs point mutation according to sequencing result analysis, shearing site suddenlys change, small segment disappearance or insertion etc.
3, detected result
Sequencing result figure as shown in Figure 3, the peak figure of often pair of base is high-visible, and occur without assorted peak, show that the primer specificity that the present invention designs is good, as can be seen from Figure 3, in amniotic fluid, c.499C>Tp.Arg167Trp suddenling change has appearred in the vhl gene of fetal cell, and the 499bp place namely in the 3rd exon of vhl gene exists the sudden change by C to T, causes VHL albumen the 167th amino acids to become tryptophane Trp from arginine Arg.
According to the detected result that embodiment 1 obtains, find the pollution that there is not maternal inheritance material in amniotic fluid in fetal cell, therefore the detected result of the present embodiment is accurately.
The preparation of embodiment 3 fluorescence in situ hybridization probe
1, the screening of fluorescence in situ hybridization probe gene order
In human genome, screening is used as the vhl gene sequence of fluorescence in situ hybridization probe, selects the probe sequence taking into account specificity and feasibility as fluorescence in situ hybridization probe gene order.
Vhl gene is positioned euchromosome 3p25-26 district, comprise three exons, the all sequences containing vhl gene of retrieval Μ CSCgenomebrowser, NCBICloneRegistry, EnsemblGenomeBrowser database, the optimum gene order of screening containing above-mentioned exon, and be numbered 1 exon VHL-Exon1,2 exon VHL-Exon2,3 exon VHL-Exon3.
2, the preparation of hybridization probe
The clone of 2.1 goal gene sequences
According to the gene order of numbering VHL-Exon1, VHL-Exon2, VHL-Exon3, design cloning primer respectively to 11-13:
Cloning primer is to 11F:5 '-aaccttagaggggcgaaaaa
R:5’-gcttcagaccgtgctatcgt
Cloning primer is to 12F:5 '-aacctttgcttgtcccgata
R:5’-ttatcagagtgggtggcaca
Cloning primer is to 13F:5 '-gcaaagcctcttgttcgttc
R:5’-cagtcttcccaaagcaggag。
To extract DNA solution in normal human blood for template, with cloning primer to 11-13 for primer, utilize PCR to react amplifying target genes respectively, wherein, reaction system is: cumulative volume 50 μ l, PCRmix25 μ l, DNA profiling 100ng, each 2 μ l of cloning primer, ddH2O surplus, reaction conditions is: 94 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 60s, 30 circulations, 72 DEG C of renaturation 7min, 4 DEG C of preservations, obtain gene fragment.
2.2, the plasmid containing goal gene is built
Gene fragment step 2.1 obtained is connected in pBl μ escript plasmid, and wherein the reaction system of ligation is as shown in table 1.
Table 1 builds the reaction system of plasmid
Above-mentioned reaction system is reacted 2h at ambient temperature, obtains the plasmid containing goal gene fragment.
2.3, plasmid conversion, cultivate with extraction
Changed by the intestinal bacteria cultivated in advance on LB substratum and cultivate to new LB substratum, culture condition is 28 DEG C, and described LB substratum is by Tryptones 10g/L, and yeast extract 5g/L, sodium-chlor 10g/L, agar powder 15g/L form, for subsequent use, the mode (these intestinal bacteria are prepared competent cell by calcium chloride process adopts thermal excitation to carry out conversion and be equally applicable to the present invention) adopting electricity to turn the plasmid containing goal gene is again transformed in intestinal bacteria, intestinal bacteria coating is inoculated on LB solid medium and cultivates, choose single bacterium colony and be inoculated in 10mlLB liquid nutrient medium and carry out shaking bacterium cultivation, described LB liquid culture based component is by Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L forms, wherein, culture temperature is 37 DEG C, rotating speed is 220r/min, 8 ~ 16h cultivated by shaking table, plasmid purification kit is adopted to extract this plasmid subsequently, operating process is undertaken by the specification sheets of test kit.
It should be noted that, described plasmid purification kit can be that any one can carry out the test kit of Isolation and purification to plasmid, according to embodiments of the invention, the plasmid purification kit that plasmid purification kit used in the present invention uses GeneMark company to produce.
The qualification of goal gene in 2.4 plasmids
By the method amplification plasmid of PCR, then electrophoresis checking is carried out to amplified production.Wherein reaction system is:
Reaction conditions is:
The PCR primer of acquisition is carried out the agarose gel electrophoresis of 1%, under ultraviolet lamp, observe electrophoresis result, judge that amplified production size is whether correct, thus checking goal gene whether correction in intestinal bacteria.
Empirical tests, finds that amplified production is between 600-700bp, in the same size with expection amplified production, therefore this goal gene known correction in intestinal bacteria.Single bacterium colony intestinal bacteria of correction goal gene in picking step 2.4, and be inoculated in 2mlLB nutrient solution, under 37 DEG C of temperature condition, carry out shaking table with the rotating speed of 220r/min and cultivate 8 ~ 16h, get 100 μ l Escherichia coli bacteria liquids and deliver to Sinogenomax Co., Ltd.'s order-checking, thus whether verify further in plasmid containing right-on goal gene sequence fragment.
The preparation of 2.5 fluorescence in situ hybridization probe solution
Get the plasmid containing right-on goal gene that step 2.4 obtains, as the template preparing fluorescent probe after diluting 1000 times with ultrapure water, use Fl μ orescein-dUTP reagent, by PCR legal system for fluorescent probe.
Wherein, the probe primer of design to 8-10 is:
Probe primer 85 '-agtaacgagttggcctagcctcg
5’-cgtcttcttcagggccgtactc
Probe primer 95 '-gtactgacgttttactttttaaaaagataagg
5’-catgctctacacattgttctcctgg
Probe primer 105 '-gcattgcacatcaacggat
5’-cagtcttcccaaagcaggag。
Reaction system is:
Reaction conditions is:
The content of 2.6 mensuration fluorescence in situ hybridization probe
The in situ hybridization probe of preparation not with fluorescence, as standard substance, adopts the concentration of polyacrylamide gel vertical electrophoresis technology quantitative fluorescent probe.
The preparation of standard substance adopts the reaction system of step 2.5 to carry out PCR, obtains PCR primer, wherein, not containing fluorescent dUTP reagent in reaction system.After the PCR primer of acquisition is carried out purification process, prepare the probe standard substance that DNA content is respectively 100ng, 50ng, 25ng, 12.5ng, get the PCR primer 5 μ l that step 2.5 obtains, acrylamide gel vertical electrophoresis is carried out with probe standard substance, the amount of the fluorescent probe contained in the 5 μ lPCR products obtained by the optical density value calculation procedure 2.5 measuring electrophoresis product, knows the content of fluorescent probe in the PCR primer solution of remaining 45 μ by inference.
The purifying of 2.7 fluorogenic hybridization probe and the preparation of hybridization probe solution
100 μ lTE are added in the PCR primer obtained through step 2.6 to remaining 45 μ l, 15 μ l sodium acetates are added after mixing, 410 μ l dehydrated alcohols, after mixing, under the temperature condition of-20 DEG C, lucifuge places 30min, 4 DEG C of centrifugal 15min of 13500r/min, abandon supernatant, add 500 μ l again, 75% ethanol, put upside down centrifugal 2min for several times, abandon supernatant, lucifuge of uncapping is placed and is dried, adding with volume ratio is the methane amide of 5:1:1:3, 20 × SSC, T 500 and the formulated hybridization solution of water make concentration and probe concentration reach 8-20ng/ μ l, and preserve under the temperature condition of-20 DEG C, obtain fluorogenic hybridization probe.
It should be noted that, this step also can make concentration and probe concentration reach concentration range at 10 ~ 15ng/ μ l.
The detection of embodiment 4 fetal cell vhl gene large fragment deletion
The making of 1 cell smear
Amniocyte embodiment 1 obtained carries out Secondary Culture, obtains enough fetal cells, as sample to be tested.Wherein, amniotic fluid cell culture is in the fetal cell nutrient solution comprising the hyclonedmem/f12 substratum of 74%, the penicillin of 25% foetal calf serum FBS and 1% and the dual anti-PS of Streptomycin sulphate, and culture temperature is 37 DEG C, and air conditions is the CO of 5%
2.Fetal cell is carried out centrifugal after, through rinsing, hypotonic, fixing after make cell smear.For verifying the accuracy of this experiment, extraction has been diagnosed as the peripheral blood 3ml of vhl gene large fragment deletion patient as positive control, extract the peripheral blood 3ml of normal people as negative control, with the peripheral blood of lymphocyte separation medium process positive control and negative control, obtain cellular layer, after cellular layer is carried out rinsing process and Hypotonic treatment, fix with stationary liquid, drip sheet and make cell smear.Concrete operation step is as follows:
The preparation of 1.1 check sample cell suspending liquids
Get 3ml lymphocyte separation medium in 15ml plastic pipe, slowly be added on lymphocyte separation medium along described plastic pipe tube wall after the peripheral blood of 3ml and 1.5mlPBS damping fluid are mixed, after the speed centrifugal treating 30min of 2500r/min, sucking-off is positioned at the cotton-shaped suspension lymphocyte of middle portion, obtains cell suspending liquid.
Process and the sheet of 1.2 amniocytes and compared with control cells suspension
The amniocyte of acquisition and compared with control cells suspension are placed in new 15ml plastic pipe, and add the PBS of 3 times of volumes wherein, after mixing, with the speed centrifugal treating 10min of 1800r/min, abandon supernatant, then add 5mlPBS, mixing, after the speed centrifugal treating 10min of 1500r/min, abandon most supernatant.Add pre-temperature again and carry out Hypotonic treatment to the KCl6-8ml of the 0.075M of 37 DEG C, under 37 DEG C of temperature condition after piping and druming mixing 20min, add 2ml again by methyl alcohol and Glacial acetic acid by the formulated stationary liquid of 3:1 volume, mix again, with the centrifugal 10min of the rotating speed of 1000r/min, abandon supernatant, add the stationary liquid that 5-8ml is same again, described stationary liquid first adds on a small quantity, blow even after again full dose add, again with the centrifugal 10min of the speed of 1000r/min after mixing, abandon supernatant, lay equal stress on and be added with stationary liquid and centrifugation step 2-3 time, to cell in white, finally add a small amount of stationary liquid again, mixing, when in managing, material is rare thin rice gruel sample, the cleaning of it being crossed in alcohol immersion is without on fat slide glass.
Aging and saturatingization of 1.3 amniocytes and compared with control cells processes
By above-mentioned slide glass on roasting sheet machine after 56 DEG C of roasting sheet 20min (this slide is spent the night at ambient temperature aging be equally applicable to the present invention), being placed in pre-temperature is more successively the 2 × SSC10min of 37 DEG C, the gastric enzyme 2min of pre-temperature to 37 DEG C, finally rinse in 2 × SSC, stand upside down and blot on toilet paper, put into 70%, 85%, 100% alcohol more respectively respectively to dewater 3min, stand upside down and dry on toilet paper, obtain cell smear that is aging and saturatingization process.
2, the hybridization of fluorescence in situ hybridization probe and cell
Get the fluorescence in situ hybridization probe solution of 10ul, drip on cell smear, and covered immediately, use mounting adhesive edge, be positioned in hybridization instrument and hybridize, the common denaturation temperature of hybridization is 70-80 DEG C, and the co-variation reaction times is 5-10min, hybridization temperature is 40-46 DEG C, and hybridization time is 14-18h.
It should be noted that, the common denaturation temperature of hybridization condition can also be 73-76 DEG C, and denaturation time can also be 7-8min altogether; Hybridization temperature can also be 42-44 DEG C, and hybridization time can also be 16h.
3, the process of developing a film after hybridization
After carefully throwing off mounting glue and cover glass, slide glass is placed in successively preheating be the use 0.4 × SSC of 68 DEG C prepare 0.3% NP40 solution rinsing 1.5 ~ 2min minute (same, being placed on preheating temperature is that in the NP40 solution of 0.3% of 0.4 × SSC preparation of 46 DEG C, rinsing 3min is still applicable to the present invention), under room temperature condition 2 × SSC preparation 0.1% NP40 solution in rinsing 30s, finally rinsing 3min in the alcohol of 70%, after washing away unconjugated fluorescent probe, slide glass is dried, obtain the pure Cell sheet glass be combined with fluorescent probe.
4, observation of cell and fluorescent probe are in conjunction with situation
In above-mentioned Cell sheet glass, add 3 μ lDAPI, immediately covered, by the endonuclear fluorescent hybridization RST of fluorescence microscope and the per-cent counted in 200 cells shared by abnormal cells, obtain paracytic per-cent.
Endonuclear fluorescent hybridization signal as shown in the figure, Fig. 4 is the fluorescent hybridization signal graph of sample to be tested, there are two fluorescence bright spots, Fig. 5 is the fluorescent hybridization signal graph of positive control, only has a fluorescence bright spot, Fig. 6 is the fluorescent hybridization signal graph of negative control sample, has two fluorescence bright spots.
5, level threshold value is set up and result interpretation
5.1 level threshold values are set up
Level threshold value according to routine detects establishment method, and gather blood sample 20 example of healthy person, every part of sample view 200 cells, add up paracytic number and percentage; What show two fluorescent signals is normal cell, and what display was less than two greens is abnormal cells; Calculate mean value and the standard deviation of abnormal cells per-cent in 20 increments bases, and then calculated threshold (threshold value=mean value+3 × standard deviation).
5.2 result interpretations
Count 200 clear decipherable cells of hybridization signal, by abnormal cells ratio and threshold value multilevel iudge vhl gene abnormal conditions, if equal threshold value, need mast cell counts be added.
If the per-cent of the shared total cell of the observable abnormal cells of sample to be tested is less than threshold value, then show that sample to be tested cell does not have producer large fragment deletion; Otherwise, if the per-cent of the shared total cell of the observable abnormal cells of sample to be tested is greater than threshold value, then show sample to be tested cell producer large fragment deletion.
Shared by the sample to be tested abnormal cells that the present invention calculates, the per-cent of total cell is 4.8%, level threshold value is 5.6%, therefore the abnormal cells percentage of sample to be tested is less than level threshold value, judges that vhl gene generation large fragment deletion does not occur sample to be tested.
According to the detected result that embodiment 1 obtains, find the pollution that there is not maternal inheritance material in amniocyte, the result of therefore carrying out vhl gene abrupt climatic change to amniocyte is accurately.
Although above-mentioned to invention has been detailed description; but be not limited thereto; those skilled in the art can principle according to the present invention modify, and therefore, all various amendments carried out according to principle of the present invention all should be understood to fall into protection scope of the present invention.
Claims (9)
1. a method for fetal cell vhl gene sudden change in rapid detection amniotic fluid, is characterized in that, comprising:
By extracting the DNA of fetal cell in amniotic fluid, obtain DNA solution;
The whether detection of maternal inheritance Substances Pollution is carried out to described DNA solution, judges whether there is maternal inheritance Substances Pollution in DNA solution;
If there is not maternal inheritance Substances Pollution in judgement DNA solution, then direct vhl gene abrupt climatic change is carried out to fetal cell;
If there is maternal inheritance Substances Pollution in judgement DNA solution, then carry out vhl gene abrupt climatic change again after subculture being carried out to fetal cell in amniotic fluid;
Described vhl gene abrupt climatic change comprises:
Adopt fluorescence in situ hybridization probe to carry out the detection of vhl gene large fragment deletion to fetal cell in described amniotic fluid, obtain the detected result whether described fetal cell exists large fragment deletion.
2. method according to claim 1, is characterized in that, described vhl gene abrupt climatic change also comprises:
Adopt vhl gene three exons VHL-1, VHL-2 and VHL-3 amplimer to 5-7 to described DNA solution carry out vhl gene point mutation, small segment disappearance or insert, shearing site sudden change detection, obtain in described DNA solution whether exist point mutation, small segment disappearance or insert, shearing site sudden change detected result.
3. the method for claim 1, is characterized in that, described maternal inheritance Substances Pollution detects and comprises:
3 STR DXS6797 on synthetic mankind sex determining gene SRY and X chromosome, the amplimer of DXS6807, AR are to 1-4;
Utilize described amplimer to set up pcr amplification reaction system to 1-4 and described DNA solution, carry out PCR reaction, obtain pcr amplification product;
1.5% agarose gel electrophoresis is carried out to the sry gene amplified production in described pcr amplification product, STR analysis is carried out to the amplified production of DXS6797, DXS6807 and AR gene in described pcr amplification product, judges whether there is maternal inheritance Substances Pollution in described DNA solution according to the electrophoresis result of described sry gene amplified production and STR analytical results.
4. method as claimed in claim 3, is characterized in that, 3 STR DXS6797 on described mankind's sex determining gene SRY and X chromosome, the amplimer of DXS6807, AR, are respectively 1-4 as shown in SEQIDNO.1-8:
Amplimer is to 1:SRY-F:5 '-gagtgaagcgacccatgaac-3 '
SRY-R：5’-tcttgagtgtgtggctttcg-3’
Amplimer is to 2:DXS6797-F:5 '-ttccctctctccctctgtct-3 '
DXS6797-R：5’-acacacacccaaaaccagat-3’
Amplimer is to 3:DXS6807-F:5 '-gagcaatgatctcatttgca-3 '
DXS6807-R：5’-aagtaaacatgtataggaaaaagct-3’
Amplimer is to 4:AR-F:5 '-tccagaatctgttccagagcgtgc-3 '
AR-R：5’-gctgtgaaggttgctgttctccat-3’
Wherein, 5 ' end flag F AM fluorescence of DXS6797-F primer in described primer pair 2;
Wherein, 5 ' end flag F AM fluorescence of DXS6807-F primer in described primer pair 3;
Wherein, 5 ' end flag F AM fluorescence of AR-F primer in described primer pair 4.
5. detection method of gene mutation as claimed in claim 1, it is characterized in that, described employing fluorescence in situ hybridization probe comprises the detection that described fetal cell carries out vhl gene large fragment deletion:
Fetal cell in the amniotic fluid gathered is utilized to prepare cell smear;
Described fluorescence in situ hybridization probe is placed in hybridization solution and described cell generation hybridization, obtains hybrid product;
Wash described hybrid product, and carry out nuclear targeting process, obtain the cell smear after nuclear staining;
By the cell smear after nuclear staining described in fluorescence microscope, judge whether the DNA of sample to be tested there occurs gene large deletion.
6. method as claimed in claim 5, it is characterized in that, described fluorescence in situ hybridization probe is marked with fluorescent signal, carries out PCR prepare by the probe primer with the base sequence shown in SEQIDNO.15-20.
7. method as claimed in claim 2, it is characterized in that, the amplimer of three exons VHL-1, VHL-2 and VHL-3 of described vhl gene, is respectively 5-7 as shown in SEQIDNO.9-14:
Amplimer is to 5:VHL-1F5 '-ggtggtctggatcgcgga-3 '
VHL-1R5’-ggcttcagaccgtgctatcg-3’
Amplimer is to 6:VHL-2F5 '-gtggctctttaacaacctttgc-3 '
VHL-2R5’-cctgtacttaccacaacaaccttatc-3’
Amplimer is to 7:VHL-3F5 '-gcaaagcctcttgttcgttc-3 '
VHL-3R5’-caaaaatgccaccaccttct-3’。
8. method as claimed in claim 2, it is characterized in that, the detection that amplimer carries out vhl gene point mutation to described DNA solution, small segment lacks or inserts, shearing site suddenlys change of three exons VHL-1, VHL-2 and VHL-3 of described employing vhl gene comprises the following steps:
The amplimer of three exons VHL-1, VHL-2 and VHL-3 of synthetic vhl gene is to 5-7;
Utilize described primer pair 5-7 and described DNA solution to set up pcr amplification reaction system, carry out PCR reaction and obtain PCR primer;
The PCR primer obtained is carried out purification process and sequencing analysis, judges whether described DNA there occurs vhl gene point mutation, small segment disappearance or insert and shearing site sudden change.
9., for detecting a test kit for vhl gene sudden change, it is characterized in that, comprise:
For extracting the reagent of cell DNA in amniotic fluid;
Comprising the amplimer of sex determining gene as shown in SEQIDNO.1-8 and X chromosome STR, whether there is the reagent of maternal inheritance material for detecting described amniocyte DNA;
Comprise the amplimer as shown in SEQIDNO.9-14, for carry out vhl gene point mutation, small segment disappearance or insert, the detection of shearing site sudden change is with the reagent of detected result obtaining foetal DNA and whether there is vhl gene point mutation, small segment disappearance or insert, shearing site suddenlys change;
Comprise by the amplimer of the hybridization probe shown in SEQIDNO.15-20, prepare by PCR reaction the hybridization probe indicating fluorescent signal.
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