CN105087809A - Method and kit for rapidly detecting fetal cell VHL gene mutation in amniotic fluid - Google Patents

Abstract

The invention provides a method and kit for rapidly detecting VHL gene mutation of fetal cells in amniotic fluid, and relates to the field of gene detection. Including: First, detect whether the fetal cell DNA extracted from the amniotic fluid is contaminated by maternal genetic material; if there is no maternal genetic material contamination, then directly conduct VHL gene mutation detection on fetal cells; if there is maternal genetic material contamination, then test the fetus The VHL gene mutation detection is carried out after the cells are cultured and passaged; the VHL gene mutation detection includes: using fluorescence in situ hybridization probes to detect the large fragment deletion of the VHL gene on fetal cells and using the amplification of three exons of the VHL gene. Amplifiers are used to detect VHL gene point mutations, small fragment deletions or insertions, and splicing site mutations on fetal DNA. The invention can accurately and efficiently detect whether the VHL gene of fetal cells in the amniotic fluid is mutated, and the operation is simple and the cost is low.

Description

A method and kit for rapidly detecting VHL gene mutation of fetal cells in amniotic fluid

technical field

The invention relates to the field of gene detection, in particular to a method and a kit for detecting VHL gene mutation of amniotic fluid fetal cells.

Background technique

The VHL gene (MIM No. 608537) is located at 3p25-26, with a full length of 10KD, including three exons and two introns, and can be transcribed to form two mRNAs. The mRNA containing three exon transcripts translates the p30 (213 amino acids) and p19 (159 amino acids) proteins. p19 is an isoform formed by the transcription of the second transcription initiation site (codon 54) of VHL gene, and has similar functions to p30.

There are various ways of VHL gene mutation, including point mutation, large fragment deletion, small fragment deletion or insertion, splicing site mutation and so on. At present, the most commonly used method for detecting VHL gene mutations is direct PCR sequencing, with an accuracy rate of 38% to 80%. VHL gene large fragment deletion, however, the incidence of VHL large fragment deletion is 11% to 40%. At present, the methods used to detect large fragment deletions of VHL gene at home and abroad include imprint hybridization method SouthernBlot, multiple ligation probe amplification technology MLPA, reverse transcription method RT-PCR and universal primer fluorescence quantitative PCR method UPQFM-PCR, etc. SouthernBlot and MLPA have the disadvantages of cumbersome detection methods, high price, and not suitable for popularization and application, and the above-mentioned VHL gene mutation detection samples are mostly peripheral blood. At present, there is no method for detecting the VHL gene of fetal cells in amniotic fluid. When the amniotic fluid is extracted, the amniotic fluid may be contaminated by maternal genetics, which will affect the test results; on the other hand, the current VHL gene detection method applied to peripheral blood and other samples has a long detection cycle and high detection cost, which is not conducive to VHL. Rapid and accurate detection of genes.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned problems in the prior art, and provide a fetal cell VHL gene mutation detection method and kit with simple operation, rapid detection, accurate results and low cost.

To achieve the purpose of the present invention, the present invention provides a method for rapidly detecting VHL gene mutation of fetal cells in amniotic fluid on the one hand, comprising:

DNA solution is obtained by extracting the DNA of fetal cells in amniotic fluid;

Carrying out the detection of maternal genetic material contamination to the DNA solution, and judging whether there is maternal genetic material contamination in the DNA solution;

If it is judged that there is no maternal genetic material contamination in the DNA solution, the fetal cells are directly tested for VHL gene mutations;

If it is judged that there is maternal genetic material contamination in the DNA solution, the fetal cells in the amniotic fluid are cultured and passaged before the VHL gene mutation detection is performed.

Wherein, said maternal genetic material contamination detection includes:

Artificially synthesized human sex-determining gene SRY and three short tandem repeat sequences DXS6797, DXS6807, and AR amplification primer pairs 1-4 on the X chromosome;

Using the amplification primer pairs 1-4 and the DNA solution to establish a PCR amplification reaction system, perform a PCR reaction, and obtain a PCR amplification product;

Carry out 1.5% agarose gel electrophoresis to the SRY gene amplification product in the PCR amplification product, carry out STR analysis to the amplification product of DXS6797, DXS6807 and AR gene in the described PCR amplification product, according to the SRY The electrophoresis results and STR analysis results of the gene amplification products determine whether there is maternal genetic material contamination in the DNA solution.

Wherein, the PCR amplification reaction system established by using the amplification primer pairs 1-4 and the DNA solution is: a total volume of 25 μl, including 12.5 μl of PCRmix, 20-80ng of DNA template, 0.5 μl of amplification Add primers and the rest of ddH ₂ O; the reaction conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 20s, SRY gene, DXS6797, DXS680755°C annealing for 20s, AR58°C for 20s, extension at 72°C for 20s, 30°C After each cycle, renature at 72°C for 5 minutes and store at 4°C.

In particular, the amplification primer pair 1-4 is shown in SEQ ID NO.1-8, respectively:

Amplification primer pair 1: SRY-F: 5'-gagtgaagcgacccatgaac-3'

SRY-R: 5'-tcttgagtgtgtggctttcg-3'

Amplification primer pair 2: DXS6797-F: 5'-ttccctctctccctctgtct-3'

DXS6797-R: 5'-acacacacccaaaaccagat-3'

Amplification primer pair 3: DXS6807-F: 5'-gagcaatgatctcatttgca-3'

DXS6807-R: 5'-aagtaaacatgtatagggaaaaagct-3'

Amplification primer pair 4: AR-F: 5'-tccagaatctgttccagagcgtgc-3'

AR-R: 5'-gctgtgaaggttgctgttctccat-3'

Wherein, the 5' end of the DXS6797-F primer in the amplification primer pair 2 is labeled with FAM fluorescence.

Wherein, the 5' end of the DXS6807-F primer in the amplification primer pair 3 is labeled with FAM fluorescence.

Wherein, the 5' end of the AR-F primer in the amplification primer pair 4 is labeled with FAM fluorescence.

Wherein, the VHL gene mutation detection includes:

Using fluorescence in situ hybridization probes to detect the large fragment deletion of the VHL gene in the fetal cells in the amniotic fluid, and obtain a detection result of whether the fetal cells have a large fragment deletion.

In particular, the fluorescent in situ hybridization probe is marked with a fluorescent signal, and is prepared by performing PCR on the probe primer pair 8-10 having the base sequence shown in SEQ ID NO.15-20.

Wherein, the probe primer pair 8-10 has the base sequence shown in SEQ ID NO.15-20, including:

Probe primer 85'-agtaacgagttggcctagcctcg

5'-gttcctccgggccggac

Probe primer 95'-gtactgacgttttactttttaaaaagataagg

5'-catgctctacacattgttctcctgg

Probe primer 105'-gcattgcacatcaacggat

5'-cagtcttcccaaagcaggag

Wherein, the size of the hybridization probe prepared by PCR with the probe primers having the base sequences shown in SEQ ID NO.15-20 is 300-1000bp.

Further preferably, the size of the hybridization probe prepared by PCR with the probe primers having the base sequences shown in SEQ ID NO.15-20 is 300-800bp.

Further preferably, the size of the hybridization probe prepared by PCR with the probe primers having the base sequences shown in SEQ ID NO. 15-20 is 300-500 bp.

Wherein, the detection of the large fragment deletion of VHL gene on the fetal cells by means of fluorescent in situ hybridization probes comprises:

Cell smears were prepared using the collected fetal cells in amniotic fluid;

placing the fluorescent in situ hybridization probe in a hybridization solution to undergo a hybridization reaction with the cells to obtain a hybridization product;

washing the hybridization product, and performing nuclear staining to obtain a cell smear after nuclear staining;

The cell smear after nuclear staining is observed by a fluorescence microscope to determine whether a large fragment of the VHL gene has been deleted in the DNA of the sample to be tested.

Wherein, the preparation of the cell smear by using the collected fetal cells in amniotic fluid further includes: culturing the collected fetal cells in amniotic fluid.

In particular, the culture and passage of the cells in the amniotic fluid refers to the culture and passage of the cells in the pre-prepared fetal cell culture medium, wherein the culture temperature is 37° C., and the volume percentage of CO ₂ in the air is 5%.

In particular, the fetal cell culture medium is prepared from 70-79% hyclonedmem/f12 medium, 20-28% fetal bovine serum FBS and 1-2% penicillin and streptomycin double-antibody PS.

Preferably, the fetal cell culture medium is prepared by 74% hyclonedmem/f12 medium, 25% fetal bovine serum FBS, 1% penicillin and streptomycin double antibody PS.

Wherein, the step of preparing the cell smear includes: obtaining the cells in the sample to be tested by centrifugation, after centrifugation with phosphate buffer solution and KCl hypotonic treatment, fixing the morphology of the cells with a fixative to make a cell smear ; aging the cell smear, digesting it with pepsin, and dehydrating it with alcohol gradient to obtain the cell smear.

Wherein, the fixative is prepared from methanol and glacial acetic acid with a volume ratio of 3:1.

Wherein, the hybridization solution is prepared from formamide, 20xSSC, dextran sulfate and water in a volume ratio of 5:1:1:3.

Wherein, the aging treatment is to process the cell smear on a toaster at 56° C. for 20 minutes.

Wherein, the aging treatment may also be placed overnight at a room temperature of 15-30° C. for 12-16 hours.

In particular, the detection of the deletion of large fragments of the VHL gene on the fetal cells by using the fluorescent in situ hybridization probe also includes: the preparation of the fluorescent in situ hybridization probe.

Wherein, the preparation of the fluorescent in situ hybridization probe includes: screening the VHL gene sequence used as a FISH probe in the human genome, obtaining the target gene fragment by cloning primer pairs 11-13; connecting the obtained gene fragment into a plasmid , obtain a plasmid containing the target gene fragment; amplify the plasmid in Escherichia coli, extract the plasmid, and obtain a plasmid solution; use probe primer pairs 8-10, fluorescent raw materials and the plasmid solution to prepare fluorescent dye-labeled fluorescent in situ hybridization probes.

Wherein, the cloning primer pair 11-13 has the base sequence shown in SEQ ID NO.21-26, including:

Cloning primer pair 11F: 5'-aaccttagaggggcgaaaaa

R: 5'-gcttcagaccgtgctatcgt

Cloning primer pair 12F: 5'-aacctttgcttgtcccgata

R: 5'-ttatcagagtgggtggcaca

Cloning primer pair 13F: 5'-gcaaagcctcttgttcgttc

R: 5'-cagtcttcccaaagcaggag.

Wherein, the reaction system for obtaining the target gene fragment by cloning primer pairs 11-13 is

reactant volume

PCR mix25μl

DNA template 100ng

Primer 2μl

ddH ₂ O balance

Total volume 50 μl.

Wherein, the reaction conditions for obtaining the target gene fragment by cloning primer pairs 11-13 are: pre-denaturation at 94°C for 10 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 60 seconds, 30 cycles, renaturation at 72°C for 7 minutes, Store at 4°C.

Wherein, the reaction system for ligating the obtained gene fragment into the plasmid is: T-vector 0.7 μl, PCR product 5 μl, T4 ligase 1 μl, T4B μffer 1 μl, ddH ₂ O balance, the total volume is 10 μl.

Wherein, the reaction condition for linking the obtained gene fragment into the plasmid is: react at room temperature for 2 hours.

Wherein, the reaction system for preparing fluorescent in situ hybridization probes labeled with fluorescent dyes using probe primers, fluorescent raw materials and the plasmid solution is:

Wherein, the reaction conditions for preparing fluorescent in situ hybridization probes labeled with fluorescent dyes using probe primers, fluorescent raw materials and the plasmid solution are: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30s, annealing at 58°C for 30s, and 72°C. Extend at ℃ for 45s, 30 cycles, anneal at 72℃ for 10min, and store at 4℃. Fluorescence in situ hybridization probes were obtained.

Wherein, the concentration of the fluorescent in situ hybridization probe is about 8-20 ng/μl.

Preferably, the concentration of the fluorescent in situ hybridization probe is about 10-15 ng/μl.

Wherein, the co-denaturation temperature of the hybridization reaction is 70-80° C., and the co-denaturation reaction time is 5-10 min.

Preferably, the co-denaturation temperature of the hybridization reaction is 73-76° C., and the co-denaturation reaction time is 7-8 minutes.

Wherein, the hybridization temperature of the hybridization reaction is 40-46°C, and the hybridization time is 14-18h.

Preferably, the hybridization temperature of the hybridization reaction is 42-44° C., and the hybridization time is 16 hours.

Wherein, the VHL gene mutation detection also includes:

Use amplification primer pairs 5-7 of the three exons VHL-1, VHL-2 and VHL-3 of the VHL gene to perform VHL gene point mutations, small fragment deletions or insertions, and cut site mutations on the DNA solution detection, to obtain the detection results of point mutations, small fragment deletions or insertions, and cut site mutations in the DNA solution.

Wherein, the amplification primer pairs 5-7 of the three exons VHL-1, VHL-2 and VHL-3 of the VHL gene are shown in SEQ ID NO.9-14, respectively:

Amplification primer pair 5: VHL-1F5'-ggtggtctggatcgcgga-3'

VHL-1R5'-ggcttcagaccgtgctatcg-3'

Amplification primer pair 6: VHL-2F5'-gtggctctttaacaacctttgc-3'

VHL-2R5'-cctgtacttaccacaacaaccttatc-3'

Amplification primer pair 7: VHL-3F5'-gcaaagcctcttgttcgttc-3'

VHL-3R5'-caaaaatgccaccaccttct-3'.

In particular, the use of amplification primers of the three exons VHL-1, VHL-2 and VHL-3 of the VHL gene is used to carry out VHL gene point mutation, small fragment deletion or insertion, and cut site Detection of mutations involves the following steps:

Amplification primer pairs 5-7 of the three exons VHL-1, VHL-2 and VHL-3 of the artificially synthesized VHL gene;

Using the amplification primer pairs 5-7 and the DNA solution to establish a PCR amplification reaction system, perform a PCR reaction to obtain a PCR product;

The obtained PCR product is subjected to purification treatment and sequencing analysis to determine whether the DNA has a VHL gene point mutation, a small fragment deletion or an insertion and splicing site mutation.

Wherein, the PCR amplification reaction system established by using the primer pair 5-7 and the DNA solution is: a total volume of 25 μl, including 12.5 μl of PCRmix, 100 ng of DNA template, 1 μl of primers and the remainder of ddH ₂ O; the reaction conditions are: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 30 cycles; renaturation at 72°C for 5 min; storage at 4°C to obtain PCR products.

For realizing the purpose of the present invention, the present invention provides a kind of test kit for detecting VHL gene mutation on the other hand, comprising:

Reagents for extracting cell DNA from amniotic fluid;

An amplification primer comprising a sex-determining gene and an X chromosome short tandem repeat sequence as shown in SEQ ID NO.1-8, a reagent for detecting whether there is maternal genetic material contamination in the amniocyte DNA;

Contains amplification primers as shown in SEQ ID NO.9-14, for the detection of VHL gene point mutations, small fragment deletions or insertions, and splicing site mutations to obtain whether fetal cells have VHL gene point mutations, small fragment deletions or Reagents for the detection of insertion and cleavage site mutations;

The amplification primer comprising the hybridization probe shown in SEQ ID NO.15-20 is prepared by PCR reaction to obtain the hybridization probe marked with fluorescent signal, and also includes other detections for large fragment deletion of VHL gene to obtain whether fetal cells exist Reagents for the detection of large deletions in the VHL gene; and,

Reagents for culturing the fetal cells in the amniotic fluid.

Wherein, the reagents for extracting cell DNA in amniotic fluid can be obtained commercially, and any other reagents that can extract DNA from amniotic fluid cells can also be prepared. According to an embodiment of the present invention, the reagents used in the present invention can be purchased from A1120 Genomic DNA Extraction Kit from Promega Company.

Wherein, the reagents for detecting whether there is maternal genetic material in the amniocytes include: PCRmix and amplification primers for PCR amplification reaction.

Wherein, the primers are amplification primer pair 1 for amplifying the human sex-determining gene SRY as shown in SEQNO.1-8 and for amplifying three short tandem repeat sequences DXS6797, DXS6807, AR on the X chromosome Amplification primer pairs 2-4:

Amplification primer pair 1: SRY-F: 5'-gagtgaagcgacccatgaac-3'

SRY-R: 5'-tcttgagtgtgtggctttcg-3'

Amplification primer pair 2: DXS6797-F: 5'-ttccctctctccctctgtct-3'

DXS6797-R: 5'-acacacacccaaaaccagat-3'

Amplification primer pair 3: DXS6807-F: 5'-gagcaatgatctcatttgca-3'

DXS6807-R: 5'-aagtaaacatgtataggaaaaagct-3'

Amplification primer pair 4: AR-F: 5'-tccagaatctgttccagagcgtgc-3'

AR-R: 5'-gctgtgaaggttgctgttctccat-3'

Wherein, the 5' end of the DXS6797-F primer in the amplification primer pair 2 is labeled with FAM fluorescence.

Wherein, the 5' end of the DXS6807-F primer in the amplification primer pair 3 is labeled with FAM fluorescence.

Wherein, the 5' end of the AR-F primer in the amplification primer pair 4 is labeled with FAM fluorescence.

Wherein, the reagents for detecting VHL gene point mutations, small fragment deletions or insertions, and splicing site mutations include: PCRmix and amplification primers for performing PCR amplification reactions.

Wherein, the primers include amplification primers 5-7 for amplifying three exons of the VHL gene as shown in SEQNO.9-14:

Amplification primer pair 5: VHL-1F5'-ggtggtctggatcgcgga-3'

VHL-1R5'-ggcttcagaccgtgctatcg-3'

Amplification primer pair 6: VHL-2F5'-gtggctctttaacaacctttgc-3'

VHL-2R5'-cctgtacttaccacaacaaccttatc-3'

Amplification primer pair 7: VHL-3F5'-gcaaagcctcttgttcgttc-3'

VHL-3R5'-caaaaatgccaccaccttct-3'.

Wherein, the reagents for detecting the deletion of large fragments of the VHL gene include: the probe primer pairs 8-10 comprising the base sequence shown in SEQ ID NO.15-20 are prepared by PCR and labeled with fluorescent signals The hybridization probe also includes: reagent I for preparing the sample to be tested into a cell smear; reagent II for hybridizing the fluorescent in situ hybridization probe with the cell to obtain a hybridization product; After the hybridization product is washed, the above hybridization product is subjected to nuclear staining to obtain reagent III of a nuclear stained cell smear.

Wherein, the probe primer pair 8-10 is:

Probe primer 85'-agtaacgagttggcctagcctcg

5'-cgtcttcttcagggccgtactc

Probe primer 95'-gtactgacgttttactttttaaaaagataagg

5'-catgctctacacattgttctcctgg

Probe primer 105'-gcattgcacatcaacggat

5'-cagtcttcccaaagcaggag

Wherein, the reagent I for preparing a cell smear from the sample to be tested includes: a fixative prepared from methanol and glacial acetic acid at a volume ratio of 3:1;

Wherein, the reagent II for hybridizing the fluorescent in situ hybridization probe with the cells to obtain the hybridization product comprises: formamide, 20×SSC, Hybridization solution prepared from dextran sulfate and water.

Wherein, the reagent III for washing the hybridization product, performing nuclear staining on the hybridization product, and obtaining a nuclear-stained cell smear includes: 0.3% NP40 prepared by washing the hybridization product in 0.4×SSC solution, 2×SSC solution Prepared 0.1% NP40, 70% ethanol solution and DAPI for nuclei staining.

Wherein, the reagents for culturing the fetal cells in the amniotic fluid include: 70-79% hyclonedmem/f12 medium, 20-28% fetal bovine serum FBS and 1-2% penicillin and streptomycin Antibodies against PS.

Preferably, the reagents used to culture and passage the fetal cells in the amniotic fluid include: 74% hyclonedmem/f12 medium, 25% fetal bovine serum FBS and 1% penicillin and streptomycin double antibody PS

The beneficial effects of the present invention are reflected in the following aspects:

1. The detection method provided by the present invention can quickly and accurately judge whether there is maternal genetic material contamination in fetal cell DNA extracted from amniotic fluid, thereby ensuring the accuracy of fetal VHL gene mutation detection results.

2. The detection method provided by the present invention can detect whether there are mutations such as point mutations, small fragment deletions or insertions, large fragment deletions, and splicing site mutations in the fetal VHL gene in a short period of time. The detection cycle is short, and the time cost can be reduced. Results can be available within a week at the earliest.

3. The detection method provided by the present invention is easy to operate, accurate and reliable in detection results, high in detection efficiency, low in detection cost and strong in practicability.

4. The kit provided by the present invention can achieve the above-mentioned detection purpose, the kit is low in cost, and the detection result is accurate and reliable.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of the PCR amplification product of sex-determining gene SRY of the present invention, wherein, the first swimming lane is DNAMarker, and size is 100-700, and swimming lanes 2, 3, 4 are fetus, father and mother respectively SRY PCR product electrophoresis;

Figure 2 is a diagram of the STR analysis results of the present invention, wherein Figure A is the peak diagram of DXS6797, Figure B is the peak diagram of DXS6807, and Figure C is the peak diagram of AR, and the diagrams are respectively mother, father and fetus from top to bottom Peak diagram;

Fig. 3 is the result figure of the VHL gene sequencing of fetal cells in amniotic fluid of the present invention;

Fig. 4 is a fluorescent hybridization signal diagram of fetal cells in amniotic fluid of the present invention, and the white dots in the figure are fluorescent bright spots;

Fig. 5 is the fluorescence hybridization signal diagram of the positive control sample of the present invention, and the white dot in the figure is the fluorescent bright spot;

Fig. 6 is a fluorescent hybridization signal graph of the negative control sample of the present invention, and the white dots in the graph are fluorescent bright spots.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

It should be noted that, before carrying out this embodiment, the peripheral blood of the fetal mother and father must be detected for VHL gene mutations. The detection method can detect point mutations, small fragment deletions or insertions, and splicing by PCR sequencing detection technology. Site mutations can also be detected by SouthernBlot, MLPA, RT-PCR and UPQFM-PCR for the large fragment deletion of VHL gene, and its detection method is not limited. The father's peripheral blood was tested for VHL gene mutation in the same way as the fetus.

It should be noted that the extraction of DNA mentioned in this application, including the extraction of fetal DNA, fetal mother's peripheral blood DNA, fetal father's peripheral blood DNA and normal human peripheral blood DNA, can be achieved by any method. In one embodiment, the extraction of DNA in the present invention is carried out by using the A1120 Genomic DNA Extraction Kit of Promega Company, and the extraction method is carried out according to the steps in the instructions.

Example 1 Detection of maternal genetic material contamination in fetal cell DNA

1. Collection of samples

The amniotic fluid of pregnant women at 14 to 22 weeks of pregnancy is extracted with a puncture device to obtain amniotic fluid fetal cells.

It should be noted that the detection method provided by the present invention is also applicable to using a puncture device to extract villi samples of pregnant women whose pregnancy is 11-14 weeks.

It should be noted that the puncture device used in this application is commercially available, and is not particularly limited, and may be any puncture device that can realize amniocentesis.

2. Detection of maternal genetic material contamination

2.1. Design and synthesis of primers

According to the base sequences of the human sex-determining gene SRY and the three short tandem repeat sequences DXS6797, DXS6807, and AR on the X chromosome, a primer pair for PCR amplification of the SRY gene, DXS6797, DXS6807, and AR sequences was designed and synthesized:

Amplification primer pair 1: SRY-F: 5'-gagtgaagcgacccatgaac-3'

SRY-R: 5'-tcttgagtgtgtggctttcg-3'

Amplification primer pair 2: DXS6797-F: 5'-ttccctctctccctctgtct-3'

DXS6797-R: 5'-acacacacccaaaaccagat-3'

Amplification primer pair 3: DXS6807-F: 5'-gagcaatgatctcatttgca-3'

DXS6807-R: 5'-aagtaaacatgtatagggaaaaagct-3'

Amplification primer pair 4: AR-F: 5'-tccagaatctgttccagagcgtgc-3'

AR-R: 5'-gctgtgaaggttgctgttctccat-3'

Among them, the 5' ends of the amplification primers DXS6797-F, DXS6807-F, and AR-F were labeled with FAM fluorescence.

2.2. Amplify SRY gene and DXS6797, DXS6807, AR gene

Extract the DNA of the amniotic fluid fetal cells, the peripheral blood DNA of the fetal mother and the peripheral blood DNA of the fetal father, and use the extracted amniotic fluid fetal DNA and parental DNA as templates to establish a PCR amplification reaction system with a total volume of 25 μl: 12.5 μl of PCRmix solution (the PCRmix solution selects 2xTaqPCCRMasterMixKT201-02 produced by Tiangen Biochemical Technology Co., Ltd.), 70ngDNA (wherein, the amount of the DNA can also be any amount within the 40-70ng range, or any amount within the 20-80ng range. The amount used can achieve the purpose of the present invention), each of the above primer pairs is 0.5 μl, the primer concentration is 10 pmol/μl, and the balance is ddH ₂ O. The reaction conditions of the above reaction are: pre-denaturation at 95°C for 5min; denaturation at 95°C for 20s, SRY gene, DXS6797, DXS680755°C annealing for 20s, AR58°C for 20s, extension at 72°C for 20s, and renaturation at 72°C after 30 cycles. 5min; store at 4°C to obtain amplification products of SRY, DXS6797, DXS6807, and AR genes.

2.3. Analysis and judgment of gene amplification products

Perform 1.5% agarose gel electrophoresis on the SRY gene amplification product obtained in step 2.2 (as shown in Figure 1), determine the sex of the fetus, and then perform STR analysis on the DXS6797, DXS6807 and AR gene fragments in the PCR amplification product (As shown in FIG. 2 ), judge whether there is maternal genetic material contamination in the DNA solution according to the STR analysis result.

As shown in Figure 1, the agarose gel electrophoresis figure, the first swimming lane is DNAMarker, the size is 100-700bp, and swimming lanes 2, 3, and 4 are respectively the PCR product electrophoresis figures of fetus, father and mother SRY, according to the standards in the art It is known from common knowledge that the sex-determining gene SRY only exists on the Y chromosome, and the amplified product band of the SRY gene as shown in Figure 1 accurately appears on the swimming lane representing the father, and the band is clear and free of miscellaneous bands, which illustrates the present invention The designed primers have strong specificity to the SRY sex-determining gene, and can accurately amplify the SRY gene; and there is no band in the lane showing the fetus in the figure, indicating that the fetus does not have the SRY gene, so it is judged that the fetus is a girl.

As shown in the graph of STR analysis results in FIG. 2 , it can be seen that the graph of the analysis results obtained by the primers designed in the present invention is clear and accurate, and the obtained sequencing results are specific, indicating that the specificity of the primers is good. Among them, Fig. A is the electropherogram of DXS6797, Fig. B is the electropherogram of DXS6807, and Fig. C is the electropherogram of AR. In the figure, from top to bottom are the electropherograms of mother, father and fetus respectively. It can be seen in Figure A that there are only two peaks in the fetus, which appear at 294.5 and 300 respectively, which are the same as the mother's peak at 294.6 and the father's peak at 299.9, and there is no other peak for the mother. It shows that the fetus has two X chromosomes from the father and mother respectively, and is a girl, and there is no pollution of the mother's genetic material; it can be seen in Figure B that the mother only has one peak, and it is at the same position as the father's peak, and the fetus is also The same peak appeared, indicating that the two peaks of the mother overlapped and were located at the same position as the father, which was determined by the genes of the father and mother; it can be seen from C in the figure that only two peaks appeared in the fetus, They appear at 294.5, which is the same as the mother's peak at 294.7, and the peak at 300 is the same as the father's peak at 300.2. There is no other peak for the mother, indicating that the fetus has two X chromosomes from the father and mother , is a girl, and there is no pollution of the mother's genetic material. According to the results of this test, there is no pollution of the mother's genetic material in the fetal cells; if the fetal DNA is contaminated, two peaks of the mother and one peak of the father will appear at the same time.

Example 2 Detection of VHL gene point mutations, small fragment deletions or insertions, and splicing site mutations in amniotic fluid fetal cells

In this embodiment, by amplifying the three exons of the VHL gene in the DNA to be tested, the exon gene sequence of the sample to be tested is obtained by a sequencer, compared with the VHL gene in the gene bank, and analyzed whether the sequence has occurred Point mutations, small fragment deletions or insertions, and splice site mutations. The specific method is as follows:

1. Design amplification primers

According to the three exons VHL-1, VHL-2, VHL-3 of the VHL gene, the amplification primer pair 5-6 is designed:

Amplification primer pair 5: VHL-1F5'-ggtggtctggatcgcgga-3'

VHL-1R5'-ggcttcagaccgtgctatcg-3'

Amplification primer pair 6: VHL-2F5'-gtggctctttaacaacctttgc-3'

VHL-2R5'-cctgtacttaccacaacaaccttatc-3'

Amplification primer pair 7: VHL-3F5'-gcaaagcctcttgttcgttc-3'

VHL-3R5'-caaaaatgccaccaccttct-3'.

2. Detection of VHL gene point mutations, small fragment deletions or insertions, and splicing site mutations

Using the amniotic fluid cell DNA obtained in Example 1 as a template, and using the amplification primer pair 5-7 as primers, an amplification reaction system was established, wherein the total volume of the reaction system was 25 μl, including 12.5 μl of PCRmix (selected from Beijing Produced by Quanshijin Biotechnology Co., Ltd. PCRSuperMixAS111), 100 ng of DNA template, 1 μl each of primers at a concentration of 5 pmol/μl, and the remainder of ddH ₂ O. The reaction conditions are: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, after 30 cycles, renaturation at 72°C for 5 min, and storage at 4°C to obtain three exosomes of the VHL gene of fetal DNA. For the amplified product of the exon, the exon product of the amplified VHL gene was sent to Beijing Nuosai Genome Research Center Co., Ltd. for purification, and sequenced using the ABI3730 sequencer to obtain the sequencing result, as shown in Figure 3. According to the analysis of the sequencing results, it is judged whether there are point mutations, splicing site mutations, small fragment deletions or insertions, etc. in the VHL gene.

3. Test results

As shown in Fig. 3, the peak diagram of each pair of bases is clearly visible, and no miscellaneous peaks appear, indicating that the primers designed by the present invention have good specificity. As can be seen from Fig. 3, the VHL gene of fetal cells in amniotic fluid The c.499C>Tp.Arg167Trp mutation appeared, that is, there was a mutation from C to T at the 499bp in the 3rd exon of the VHL gene, resulting in the 167th amino acid of the VHL protein changing from arginine Arg to tryptophan Sour Trp.

According to the test results obtained in Example 1, it was found that there is no contamination of maternal genetic material in the fetal cells in the amniotic fluid, so the test results in this example are accurate.

The preparation of embodiment 3 fluorescent in situ hybridization probes

1. Screening of fluorescent in situ hybridization probe gene sequences

The VHL gene sequence used as a fluorescent in situ hybridization probe is screened in the human genome, and the probe sequence with both specificity and feasibility is selected as the fluorescent in situ hybridization probe gene sequence.

The VHL gene is located in the autosome 3p25-26 region and contains three exons. Search all the sequences containing the VHL gene in the MCSCgenomebrowser, NCBICloneRegistry, and EnsemblGenomeBrowser databases, screen the optimal gene sequence containing the above exons, and number it as exon 1. Sub-VHL-Exon1, No. 2 exon VHL-Exon2, No. 3 exon VHL-Exon3.

2. Preparation of hybridization probes

2.1 Cloning of target gene sequence

According to the gene sequences numbered VHL-Exon1, VHL-Exon2, and VHL-Exon3, respectively design cloning primer pairs 11-13:

Cloning primer pair 11F: 5'-aaccttagaggggcgaaaaa

R: 5'-gcttcagaccgtgctatcgt

Cloning primer pair 12F: 5'-aacctttgcttgtcccgata

R: 5'-ttatcagagtgggtggcaca

Cloning primer pair 13F: 5'-gcaaagcctcttgttcgttc

R: 5'-cagtcttcccaaagcaggag.

Using DNA solution extracted from normal human blood as a template and cloning primer pairs 11-13 as primers, PCR reactions were used to amplify target genes respectively. The reaction system was: total volume 50 μl, PCR mix 25 μl, DNA template 100ng, cloning primers each 2 μl, ddH2O balance, the reaction conditions are: 94°C pre-denaturation for 10 minutes, 94°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 60 seconds, 30 cycles, 72°C renaturation for 7 minutes, and 4°C storage to obtain gene fragments.

2.2. Construction of a plasmid containing the target gene

The gene fragment obtained in step 2.1 was ligated into the pBlμescript plasmid, wherein the reaction system of the ligation reaction is shown in Table 1.

Table 1 Reaction system for constructing plasmids

The above reaction system was reacted at room temperature for 2 hours to obtain a plasmid containing the target gene fragment.

2.3 Transformation, cultivation and extraction of plasmids

The Escherichia coli pre-cultured on the LB medium was replaced with a new LB medium for cultivation, and the culture condition was 28°C. The LB medium was composed of tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, composed of agar powder 15g/L, for subsequent use; then the plasmid containing the target gene is transformed by electroporation (the Escherichia coli is treated with calcium chloride to prepare competent cells and transformed by heat excitation, which is also applicable to the present invention) transformation In Escherichia coli, spread and inoculate Escherichia coli on LB solid medium for culture, pick a single colony and inoculate it in 10ml LB liquid medium for shaking culture, the LB liquid medium composition consists of tryptone 10g/L, yeast The extract is composed of 5g/L and sodium chloride 10g/L. The culture temperature is 37°C, the rotation speed is 220r/min, and the shaking table is cultivated for 8-16h, and then the plasmid is extracted with a plasmid purification kit. The operation process is as follows: kit instructions.

It should be noted that the plasmid purification kit can be any kit that can extract and purify the plasmid. According to an embodiment of the present invention, the plasmid purification kit used in the present invention uses the plasmid purification kit produced by GeneMark. Reagent test kit.

2.4 Identification of the target gene in the plasmid

The plasmid was amplified by PCR, and the amplified product was verified by electrophoresis. Wherein the reaction system is:

The reaction conditions are:

Perform 1% agarose gel electrophoresis on the obtained PCR products, and observe the electrophoresis results under ultraviolet light to judge whether the size of the amplified product is correct, thereby verifying whether the target gene is correctly expressed in E. coli.

After verification, it was found that the amplified product was between 600-700bp, which was consistent with the size of the expected amplified product, so it was known that the target gene had been correctly expressed in E. coli. Pick a single colony of Escherichia coli that correctly expresses the target gene in step 2.4, inoculate it in 2ml of LB culture medium, and culture it on a shaking table at a speed of 220r/min at a temperature of 37°C for 8-16 hours, take 100μl of the Escherichia coli liquid and send it to Sequenced by Beijing Nuosai Genome Research Center Co., Ltd., so as to further verify whether the plasmid contains completely correct sequence fragments of the target gene.

2.5 Preparation of fluorescence in situ hybridization probe solution

Take the plasmid containing the completely correct target gene obtained in step 2.4, dilute it 1000 times with ultrapure water, and use it as a template for preparing fluorescent probes, and use Flμorescein-dUTP reagent to prepare fluorescent probes by PCR.

Wherein, the designed probe primer pair 8-10 is:

Probe primer 85'-agtaacgagttggcctagcctcg

5'-cgtcttcttcagggccgtactc

Probe primer 95'-gtactgacgttttactttttaaaaagataagg

5'-catgctctacacattgttctcctgg

Probe primer 105'-gcattgcacatcaacggat

5'-cagtcttcccaaagcaggag.

The reaction system is:

The reaction conditions are:

2.6 Determination of fluorescence in situ hybridization probe content

In situ hybridization probes without fluorescence were prepared as standards, and the concentration of fluorescent probes was quantified by polyacrylamide gel vertical electrophoresis.

The preparation of the standard product is carried out by PCR using the reaction system in step 2.5 to obtain a PCR product, wherein the reaction system does not contain fluorescent dUTP reagent. After purifying the obtained PCR products, prepare probe standards with DNA contents of 100ng, 50ng, 25ng, and 12.5ng respectively, take 5 μl of the PCR products obtained in step 2.5, and perform vertical electrophoresis on acrylamide gel with the probe standards. Calculate the amount of fluorescent probe contained in 5 μl of PCR product obtained in step 2.5 by measuring the optical density value of the electrophoresis product, and infer the content of fluorescent probe in the remaining 45 μl of PCR product solution.

2.7 Purification of fluorescent hybridization probes and preparation of hybridization probe solutions

Add 100 μl TE to the remaining 45 μl of the PCR product obtained in step 2.6, mix well, add 15 μl sodium acetate and 410 μl absolute ethanol, mix well, place in the dark at -20°C for 30 minutes, and centrifuge at 13500 r/min at 4°C 15min, discard the supernatant, then add 500μl, 75% ethanol, invert several times and centrifuge for 2min, discard the supernatant, open the cover and place it in the dark to dry, add formamide with a volume ratio of 5:1:1:3, 20 The hybridization solution prepared by ×SSC, dextran sulfate and water makes the concentration of the probe reach 8-20ng/μl, and is stored at -20°C to obtain the fluorescent hybridization probe.

It should be noted that this step can also make the probe concentration reach a concentration range of 10-15 ng/μl.

Example 4 Detection of Large Fragment Deletion of Fetal Cell VHL Gene

1 Preparation of cell smears

Subculture the amniotic fluid cells obtained in Example 1 to obtain a sufficient amount of fetal cells as samples to be tested. Among them, the amniotic fluid cells were cultured in fetal cell culture medium containing 74% hyclonedmem/f12 medium, 25% fetal bovine serum FBS and 1% penicillin and streptomycin double antibody PS, the culture temperature was 37°C, and the air condition was 5% CO ₂ . After the fetal cells are centrifuged, they are rinsed, hypotonic, and fixed to make a cell smear. In order to verify the accuracy of this experiment, 3ml of peripheral blood from patients diagnosed with large fragment deletion of VHL gene was taken as positive control, 3ml of peripheral blood from normal people was taken as negative control, and the peripheral blood of positive control and negative control were treated with lymphocyte separation medium. For the blood, the cell layer is obtained, and after the cell layer is rinsed and treated with hypotonicity, it is fixed with a fixative solution, and the cell smear is made into a drop sheet. The specific operation steps are as follows:

1.1 Preparation of control sample cell suspension

Take 3ml of lymphocyte separation solution into a 15ml plastic tube, mix 3ml of peripheral blood with 1.5ml of PBS buffer, then slowly add to the lymphocyte separation solution along the wall of the plastic tube, and centrifuge at a speed of 2500r/min After 30 minutes, the flocculent suspended lymphocytes located in the middle part were sucked out to obtain a cell suspension.

1.2 Treatment and dripping of amniocytes and control cell suspensions

Put the obtained amniotic fluid cell and control cell suspension into a new 15ml plastic tube, and add 3 times the volume of PBS to it, mix well, centrifuge at 1800r/min for 10min, discard the supernatant, and then add 5ml of PBS , mix well, centrifuge at 1500r/min for 10min, discard the supernatant. Then add 0.075M KCl 6-8ml pre-warmed to 37°C for hypotonic treatment, blow and mix at 37°C for 20 minutes, then add 2ml of fixative solution prepared by methanol and glacial acetic acid in a volume ratio of 3:1 , then mix well, centrifuge at a speed of 1000r/min for 10min, discard the supernatant, and then add 5-8ml of the same fixative, the fixative is first added in a small amount, blown and then added in full, and then mixed with 1000r/min Centrifuge at a speed of 10 min, discard the supernatant, and repeat the steps of adding fixative and centrifugation 2-3 times until the cells turn white, and finally add a small amount of fixative, mix well, and when the substance in the tube is like dilute rice soup, remove it Drop onto clean, fat-free glass slides soaked in ethanol.

1.3 Aging and permeabilization of amniocytes and control cells

After the above-mentioned slides were baked at 56°C for 20 minutes on a roaster (the overnight aging of the slides at room temperature is also applicable to the present invention), they were then placed in 2×SSC with a preheating temperature of 37°C for 10 minutes, and then preheated to 37°C. 2 minutes in gastric enzymes at 37°C, rinsed in 2×SSC at last, blotted upside down on toilet paper, then dehydrated in 70%, 85% and 100% alcohol for 3 minutes each, stood upside down on toilet paper to dry, and obtained Aged and permeabilized cell smear.

2. Hybridization of fluorescence in situ hybridization probes with cells

Take 10ul of fluorescent in situ hybridization probe solution, drop it on the cell smear, cover it with a cover glass immediately, seal the edge with mounting glue, and place it in a hybridization instrument for hybridization. The co-denaturation temperature of the hybridization reaction is 70- 80°C, the co-denaturation reaction time is 5-10min, the hybridization temperature is 40-46°C, and the hybridization time is 14-18h.

It should be noted that the co-denaturation temperature of the hybridization reaction conditions can also be 73-76°C, and the co-denaturation time can also be 7-8min; the hybridization temperature can also be 42-44°C, and the hybridization time can also be 16h.

3. Washing after hybridization

After carefully peeling off the mounting glue and cover glass, rinse the slides in the 0.3% NP40 solution prepared with 0.4×SSC that has been preheated to 68°C for 1.5-2 minutes (similarly, put them in the In the 0.3% NP40 solution prepared by 0.4×SSC at a preheating temperature of 46° C., rinsing for 3 minutes is still applicable to the present invention), rinsing for 30 seconds in the 0.1% NP40 solution prepared by 2×SSC at room temperature, and finally at 70% Rinse in ethanol for 3 minutes to wash off unbound fluorescent probes, and then dry the slides to obtain pure cell slides bound to fluorescent probes.

4. Observe the combination of cells and fluorescent probes

Add 3 μl DAPI to the above cell slide, immediately cover with a cover glass, observe the fluorescent hybridization signal in the nucleus with a fluorescence microscope and calculate the percentage of abnormal cells among the 200 cells to obtain the percentage of abnormal cells.

The fluorescent hybridization signal in the nucleus is shown in the figure. Figure 4 is the fluorescent hybridization signal diagram of the sample to be tested, with two fluorescent bright spots. Figure 5 is the fluorescent hybridization signal diagram of the positive control sample, with only one fluorescent bright spot, and Figure 6 is negative. Fluorescent hybridization signal plot of a control sample, with two fluorescent bright spots.

5. Standard threshold establishment and result interpretation

5.1 Standard Threshold Establishment

According to the conventional standard threshold detection method, collect 20 blood samples from healthy people, observe 200 cells in each sample, and count the number and percentage of abnormal cells; normal cells that show two fluorescent signals are normal cells, and those that show less than two green signals abnormal cells; calculate the mean and standard deviation of the percentage of abnormal cells in 20 samples, and then calculate the threshold (threshold = mean + 3 × standard deviation).

5.2 Interpretation of results

Count 200 cells with clear and readable hybridization signals, and compare the proportion of abnormal cells with the threshold value to determine the abnormality of the VHL gene. If it is equal to the threshold value, increase the cell count.

If the percentage of abnormal cells in the total cells observed in the sample to be tested is less than the threshold value, it indicates that the cells in the sample to be tested have no gene fragment deletion; on the contrary, if the percentage of abnormal cells in the total cells observed in the sample to be tested is greater than the threshold value , it indicates that a large gene fragment deletion occurs in the sample cells to be tested.

The percentage of abnormal cells in the sample to be tested calculated according to the present invention is 4.8% of the total cells, and the standard threshold is 5.6%. Therefore, the percentage of abnormal cells in the sample to be tested is less than the standard threshold, and it is determined that the sample to be tested has no VHL gene occurrence. Large fragments are missing.

According to the test results obtained in Example 1, it is found that there is no contamination of maternal genetic material in the amniotic fluid cells, so the result of the VHL gene mutation detection of the amniotic fluid cells is accurate.

Although the present invention has been described in detail above, it is not limited thereto. Those skilled in the art can make modifications according to the principle of the present invention. scope of protection.

Claims (9)

1. A method for rapid detection of fetal cell VHL gene mutation in amniotic fluid, characterized in that, comprising: DNA solution is obtained by extracting the DNA of fetal cells in amniotic fluid; Carrying out the detection of maternal genetic material contamination to the DNA solution, and judging whether there is maternal genetic material contamination in the DNA solution; If it is judged that there is no maternal genetic material contamination in the DNA solution, the fetal cells are directly tested for VHL gene mutations; If it is judged that there is maternal genetic material contamination in the DNA solution, the fetal cells in the amniotic fluid are cultured and passaged before the VHL gene mutation detection is performed; The VHL gene mutation detection includes: Using fluorescence in situ hybridization probes to detect the large fragment deletion of the VHL gene in the fetal cells in the amniotic fluid, and obtain a detection result of whether the fetal cells have a large fragment deletion. 2. method according to claim 1, is characterized in that, described VHL gene mutation detection also comprises: Use amplification primer pairs 5-7 of the three exons VHL-1, VHL-2 and VHL-3 of the VHL gene to perform VHL gene point mutations, small fragment deletions or insertions, and cut site mutations on the DNA solution detection, to obtain the detection results of point mutations, small fragment deletions or insertions, and cut site mutations in the DNA solution. 3. the method for claim 1, is characterized in that, described maternal genetic material pollution detection comprises: Artificially synthesized human sex-determining gene SRY and three short tandem repeat sequences DXS6797, DXS6807, and AR amplification primer pairs 1-4 on the X chromosome; Using the amplification primer pairs 1-4 and the DNA solution to establish a PCR amplification reaction system, perform a PCR reaction, and obtain a PCR amplification product; Carry out 1.5% agarose gel electrophoresis to the SRY gene amplification product in the PCR amplification product, carry out STR analysis to the amplification product of DXS6797, DXS6807 and AR gene in the described PCR amplification product, according to the SRY The electrophoresis results and STR analysis results of the gene amplification products determine whether there is maternal genetic material contamination in the DNA solution. 4. the method as claimed in claim 3 is characterized in that, the amplification primer pair 1-4 of 3 short tandem repeat sequences DXS6797, DXS6807, AR on the human sex-determining gene SRY and X chromosome is as SEQIDNO.1 -8, respectively: Amplification primer pair 1: SRY-F: 5'-gagtgaagcgacccatgaac-3' SRY-R: 5'-tcttgagtgtgtggctttcg-3' Amplification primer pair 2: DXS6797-F: 5'-ttccctctctccctctgtct-3' DXS6797-R: 5'-acacacacccaaaaccagat-3' Amplification primer pair 3: DXS6807-F: 5'-gagcaatgatctcatttgca-3' DXS6807-R: 5'-aagtaaacatgtatagggaaaaagct-3' Amplification primer pair 4: AR-F: 5'-tccagaatctgttccagagcgtgc-3' AR-R: 5'-gctgtgaaggttgctgttctccat-3' Wherein, the 5' end of the DXS6797-F primer in the primer pair 2 is marked with FAM fluorescence; Wherein, the 5' end of the DXS6807-F primer in the primer pair 3 is marked with FAM fluorescence; Wherein, the 5' end of the AR-F primer in the primer pair 4 is labeled with FAM fluorescence. 5. gene mutation detection method as claimed in claim 1, is characterized in that, described employing fluorescence in situ hybridization probe to carry out the detection of VHL gene large fragment deletion to described fetal cell comprises: Cell smears were prepared using the collected fetal cells in amniotic fluid; placing the fluorescent in situ hybridization probe in a hybridization solution to undergo a hybridization reaction with the cells to obtain a hybridization product; washing the hybridization product, and performing nuclear staining to obtain a nuclear stained cell smear; The cell smear after nuclear staining is observed through a fluorescence microscope to determine whether the DNA of the sample to be tested has a large gene fragment deletion. 6. The method according to claim 5, wherein the fluorescent in situ hybridization probe is marked with a fluorescent signal, and is prepared by PCR with a probe primer having a base sequence shown in SEQ ID NO.15-20 . 7. the method for claim 2 is characterized in that, the amplification primer pair 5-7 of three exons VHL-1, VHL-2 and VHL-3 of described VHL gene is as SEQIDNO.9-14 As shown, they are: Amplification primer pair 5: VHL-1F5'-ggtggtctggatcgcgga-3' VHL-1R5'-ggcttcagaccgtgctatcg-3' Amplification primer pair 6: VHL-2F5'-gtggctctttaacaacctttgc-3' VHL-2R5'-cctgtacttaccacaacaaccttatc-3' Amplification primer pair 7: VHL-3F5'-gcaaagcctcttgttcgttc-3' VHL-3R5'-caaaaatgccaccaccttct-3'. 8. The method according to claim 2, characterized in that, the amplification primers of three exons VHL-1, VHL-2 and VHL-3 of the VHL gene are used to carry out VHL gene pointing to the DNA solution. The detection of mutations, small fragment deletions or insertions, and splicing site mutations includes the following steps: Amplification primer pairs 5-7 of the three exons VHL-1, VHL-2 and VHL-3 of the artificially synthesized VHL gene; Using the primer pairs 5-7 and the DNA solution to establish a PCR amplification reaction system, perform a PCR reaction to obtain a PCR product; The obtained PCR product is subjected to purification treatment and sequencing analysis to determine whether the DNA has a VHL gene point mutation, a small fragment deletion or an insertion and splicing site mutation. 9. A test kit for detecting VHL gene mutation, characterized in that, comprising: Reagents for extracting cell DNA from amniotic fluid; An amplification primer comprising a sex-determining gene and an X chromosome short tandem repeat sequence as shown in SEQ ID NO.1-8, a reagent for detecting whether there is maternal genetic material in the amniocyte DNA; Including amplification primers as shown in SEQ ID NO.9-14, used for the detection of VHL gene point mutations, small fragment deletions or insertions, and splicing site mutations to obtain fetal DNA whether there are VHL gene point mutations, small fragment deletions or Reagents for the detection of insertion and cleavage site mutations; Amplification primers comprising hybridization probes shown in SEQ ID NO.15-20 are prepared by PCR reaction to obtain hybridization probes marked with fluorescent signals.