CN113046435B - Specific primer for preparing PCR reaction system for detecting prenatal fetal 21-trisomy syndrome - Google Patents
Specific primer for preparing PCR reaction system for detecting prenatal fetal 21-trisomy syndrome Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6851—Quantitative amplification
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
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Abstract
The invention relates to a detection method for prenatal fetal 21-trisomy syndrome, which comprises the steps of sample acquisition, methylation DNA immunoprecipitation, enzyme digestion, preparation of a PCR reaction system, PCR reaction, analysis and the like. The invention relates to detection of 8 sites, and the results can be mutually verified, so that the diagnosis accuracy is improved. The invention can rapidly detect the prenatal fetus trisomy, has short detection time and low cost, is hopeful to replace a second generation sequencing link in noninvasive DNA detection, reduces the detection expense expenditure of puerperal families, reduces the economic pressure of young families, and brings good economic and social benefits.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a specific primer for preparing a PCR reaction system for detecting prenatal fetal 21-trisomy syndrome.
Background
21-trisomy syndrome, down's syndrome, is currently the most common genetic disorder. In newborns, 21-trisomy syndrome has a high incidence of 1/700, and the incidence increases with the age of the parturient. Because the disease can not be treated, the prevention means is mainly carried out by prenatal screening, but traditional invasive techniques, including early chorionic biopsy, middle-stage amniocentesis, middle-stage cord blood puncture and the like, have higher risk of intrauterine infection, premature birth and abortion of pregnant women. In addition, cytogenetic diagnosis takes about 4 weeks, and has disadvantages of time consuming, easy culture failure, inability to detect minor structural aberrations of chromosomes, and the like. Down screening techniques are based on serological tests, but have a relatively low accuracy and therefore are only useful as a pre-screening means.
With the discovery of fetal free DNA in the peripheral blood of pregnant women, researchers have found that it can be used to indicate the risk of fetal genetic disease, i.e., fetal free DNA contains all the genetic information of the fetus. The university of hong Kong Lu, the Ming's education and the university of Hopkins cooperate to develop a prenatal detection method based on free fetal DNA (cfDNA) in the peripheral blood of pregnant women based on the second generation sequencing technology, and the method is mainly based on the second generation sequencing technology, compares the differences of gene abundance or mutation conditions on different chromosomes, and determines the risk of trisomy and genetic diseases. The technology has high detection sensitivity and accurate data analysis result, so the technology is widely popularized and applied clinically at home and abroad at present. However, the second generation sequencing operation procedure is complicated, the single operation cost is high, in addition, cfDNA fragmentation is serious, the extraction yield is low, and the like, so that the preparation difficulty of the DNA library is increased.
Disclosure of Invention
Further research results show that the methylation level of partial sites of pregnant women and fetuses are different, so that the method of utilizing specific endonuclease or methylation immunoprecipitation can be used for enriching fetal free DNA in the peripheral blood of the pregnant women, and the risk of detecting fetal trisomy is detected under the condition of removing maternal DNA. However, considering the errors of the molecular detection techniques such as fluorescent quantitative PCR and the like, individual differences of the fetal/maternal DNA content and the like, it is necessary to improve the diagnosis accuracy through the combined detection of a plurality of groups of sites.
The invention provides a detection method for prenatal fetal 21-trisomy syndrome, which comprises the following steps:
(1) Collecting a sample to be tested:
collecting 500 mu L-1mL of peripheral blood of a mother body to be detected, extracting free DNA of the blood by adopting a general nucleic acid column type extraction kit, detecting the obtained DNA sample by a micro ultraviolet spectrophotometer to obtain the concentration and purity, diluting the DNA sample to obtain the DNA sample to be detected, wherein the purity of the DNA sample is 1.8-2.0;
(2) Methylated DNA co-immunoprecipitation:
according to the requirements of the instruction of the methylation DNA co-precipitation kit, the methylation DNA is separated from a DNA Sample to be detected (Sample), a Positive control (Positive control) and a Negative control (Negative control), and ddH is added in the final step of eluting the methylation DNA 2 O-dissolution eluting methylated DNA;
(3) And (3) enzyme cutting:
preparing an enzyme digestion reaction system from the methylated DNA of the DNA sample to be detected, the positive control and the negative control according to the HpaII enzyme digestion reaction system, carrying out enzyme digestion reaction on the prepared enzyme digestion reaction system in a water bath at 37 ℃ for 2 hours, and inactivating HpaII enzyme in the water bath at 80-90 ℃ for 20-30 minutes after the reaction to obtain a cut DNA sample to be detected, a cut positive control and a cut negative control;
(4) Preparing a PCR reaction system:
preparing a PCR reaction system by using 9 groups of specific primers of the obtained cut DNA sample to be detected, the cut positive control and the cut negative control, wherein each group of primers of each sample is configured with 3-6 individuals repeatedly, and the 9 groups of specific primers comprise 1 group of conventional chromosome locus detection primers and 8 groups of chromosome locus detection primers No. 21;
(5) And (3) PCR reaction:
the PCR reaction and the fluorescence measurement are carried out by using a real-time fluorescence quantitative PCR instrument, and the PCR process is as follows:
first stage, pre-denaturation: the PCR reaction system reacts for 5min at 95 ℃;
and (3) a second stage, a cyclic reaction: the PCR reaction system was reacted at 95℃for 10sec and then at 60℃for 30sec;
third stage, obtaining dissolution curve: the PCR reaction system was reacted at 95℃for 15sec and then at 60℃for 30sec; then heating to 95 ℃ to react for 15sec to obtain a dissolution curve;
(6) Analysis:
analyzing the experimental result by using analysis software of a real-time fluorescence quantitative PCR instrument to obtain a Cq value, and analyzing the obtained Cq value, wherein the Cq value comprises the Cq value of a DNA sample to be detected: cq Sample of Positive control Cq value: cq Positive control Negative control Cq value: cq Negative control The method comprises the steps of carrying out a first treatment on the surface of the Each of the Cq values of 8 sites on chromosome 21 is compared with the Cq value of a conventional chromosome to obtain a ratio R; if more than 4 sites R Positive control ≈R Sample of ＞R Negative control The probability of the trisomy 21 of the fetus is extremely high, and the amniotic fluid puncture is recommended to be performed for the analysis and confirmation of the karyotype; if 1-4 sites R Positive control ≈R Sample of ＞R Negative control Then suggest review; if all sites R Positive control ＞R Sample of ≈R Negative control The fetal 21 trisomy probability is extremely low, and the birth can be guaranteed.
Preferably, in the step of collecting the sample to be measured (1), the DNA sample concentration of the sample to be measured is diluted to 50 ng/. Mu.l with deionized water.
Preferably, in the step of (2) methylation DNA co-precipitation, the positive control is a standard substance obtained by mixing DNA extracted from fetal amniotic fluid puncture cells of trisomy 21 with blood cell DNA obtained from peripheral blood of a healthy adult human body according to a ratio of 1:20, and the concentration is 50 ng/. Mu.l; the negative control is blood cell DNA obtained from peripheral blood of adult human body of healthy female, and the concentration is 50 ng/. Mu.l; the DNA content of each group in the methylation DNA separation process is not less than 0.8 mug.
Preferably, in the cleavage reaction system in the step (3), the DNA sample, hpaII enzyme, 10 Xbuffer, ddH 2 O volume ratio of 40:1:5:4, per sampleThe volume of the prepared enzyme digestion reaction system is 50 μl respectively; the concentration of the DNA sample in the enzyme digestion reaction system is 50 ng/. Mu.l; the HpaII enzyme concentration was 5U/. Mu.l.
Preferably, in the step of (4) preparing a PCR reaction system, in the PCR reaction system, a fluorescent quantitative PCR 2X premix, an upstream primer, a downstream primer, a sample/control, ddH 2 The volume ratio of O is 25:1:1:1:22; the volume of the PCR reaction system prepared for each sample was 20. Mu.l.
Preferably, in the step of (4) preparing a PCR reaction system, the 9 sets of specific primers are as follows:
group 1 was used for detection of conventional chromosomal locus No. 3:
the upstream primer Ch3-F: AGCTGGCACCCGCTGG
The downstream primer Ch3-R: GTGTGGGGTTGCACGCG
Group 2 was used for detection of chromosome 21 site 1:
the upstream primer ERG-F: CAGGAGGCTGAGGCAGGG
The downstream primer ERG-R: GGTACAGGTTGGGAGTTTGGG
Group 3 was used for detection of chromosome 21 site 2:
the upstream primer AIRE-F: TAGTAAGGGAGGGCCGAGAA
The downstream primer AIRE-R: CAGAGCCAGAACGCACAGAG
Group 4 was used for detection of chromosome 21 site 3:
the upstream primer SIM-F: ACCGGGCCTTCTGTCTGTC
Downstream primer SIM-R: TCTGCACGCTTCAATCCTTC
Group 5 was used for detection of chromosome 21 site 4:
the upstream primer NIPK4-F: GAGCCAGGTCTGTTCTCCACG
Downstream primer NIPK4-R: AGCCGATGCCCTTGTTGC
Group 6 was used for detection of chromosome 21 site 5:
the upstream primer YBEY-1F: GGTTCGTGCACTCGCTGAG
Downstream primer YBEY-1R: GCCTTCTCCTTCTGGAACATCT
Group 7 was used for detection of chromosome 21 site 6:
upstream primer JAM2-F: TTTTGGGCCACATCATTCT
Downstream primer JAM2-R: GCTGGGATTACAGGCGTGAG
Group 8 was used for detection of chromosome 21 site 7:
the upstream primer DSCAM-F: CCTAAGACCTTTGCCTAGCTTCT
Downstream primer DSCAM-R: TCCTTTATGGGATTTCTGTTGTG
Group 9 was used for detection of chromosome 21 site 8:
the upstream primer PCNT-F: AGCAGACACGGGCTCGGT
Downstream primer PCNT-R: CGGTGCTCAGCAACCACC
The concentration of each set of upstream and downstream primers was 10. Mu.M.
The invention provides a group of fetal specific methylation sites and corresponding primer sequences thereof, wherein the sites are respectively positioned on chromosome 21 and chromosome 3, and the precursors on the sites are correspondingly unmethylated, so that the risk of fetal 21-trisomy can be determined by comparing the relative abundance of genes on chromosome 21 and chromosome 3 after specific enrichment. The invention relates to detection of 8 sites, and the results can be mutually verified, so that the diagnosis accuracy is improved.
The site on the chromosome 3 related to the specific primer provided by the invention has basically stable content and methylation level in the peripheral blood of different pregnant women, and the 8 sites on the chromosome 21 related to the specific primer can ensure that more than 4 of the sites in the peripheral blood of different pregnant women have obvious fetal-pregnant woman differences; the conventional chromosome 3 locus has extremely strong abundance consistency with 8 loci of the chromosome 21, and ensures the accuracy of relative abundance detection.
The invention has the beneficial effects that:
the invention can rapidly detect the prenatal fetus trisomy, has short detection time and low cost, is hopeful to replace a second generation sequencing link in noninvasive DNA detection, reduces the detection expense expenditure of puerperal families, reduces the economic pressure of young families, and brings good economic and social benefits.
Drawings
FIG. 1 is a graph showing the comparison of R values of a detection result in the case of the present invention;
FIG. 2 is a graph showing the comparison of R values of the second detection result in the case of the present invention;
FIG. 3 is a graph showing the comparison of R values of the detection results of the third case of the present invention;
FIG. 4 is a graph showing the comparison of R values of the fourth detection result in the case of the present invention;
FIG. 5 is a graph showing the comparison of R values of the fifth detection result in the case of the present invention;
fig. 6 is a graph showing comparison of R values of the detection results of the sixth embodiment of the present invention.
Detailed Description
The invention provides a detection method for prenatal fetal 21-trisomy syndrome, which comprises the following steps:
(1) Collecting a sample to be tested:
collecting 500 mu L-1mL of peripheral Blood of a mother body to be detected, extracting free DNA of the Blood by using a general nucleic acid column extraction kit (FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit) and detecting the obtained DNA sample by using a micro ultraviolet spectrophotometer (Nanodrop One) to obtain the concentration and purity, wherein the purity of the DNA sample is 1.8-2.0, and diluting the concentration of the DNA sample of the sample to be detected to 50 ng/. Mu.L by using deionized water to obtain the DNA sample to be detected;
(2) Methylated DNA co-immunoprecipitation:
according to Eimer methylation DNA co-precipitation kit (methyl lamp) TM Methylated DNA Capture Kit) carrying out methylation DNA separation on a DNA Sample (Sample) to be tested, a Positive control (Positive control) and a Negative control (Negative control), wherein the Positive control is a standard product obtained by mixing DNA extracted from fetal amniotic fluid puncture cells of trisomy 21 with blood cell DNA obtained from peripheral blood of a healthy adult human body according to a ratio of 1:20, and the concentration is 50 ng/. Mu.l; the negative control is blood cell DNA obtained from peripheral blood of adult human body of healthy female, and the concentration is 50 ng/. Mu.l; the DNA content of each group during the methylation DNA separation process was 0.8. Mu.g; at the final elution of methylated DNA, 10. Mu. LddH was added 2 O-dissolution elutes methylated DNA.
(3) And (3) enzyme cutting:
preparing a digestion reaction system by using methylated DNA of a DNA sample to be detected, a positive control and a negative control according to HpaII digestion reaction system treatment, wherein the digestion reaction system is provided with the following components in table 1:
table 1:
performing enzyme digestion reaction on the prepared enzyme digestion reaction system in water bath at 37 ℃ for 2 hours, and inactivating HpaII enzyme in water bath at 80-90 ℃ for 20-30 minutes after the reaction to obtain a cut DNA sample to be detected, a cut positive control and a cut negative control; the volume of the enzyme digestion reaction system prepared by each sample is 50 μl respectively; wherein 10 Xbuffer is supplied by enzyme company and is an existing commercial reagent for the purpose of maximizing enzyme activity.
(4) Preparing a PCR reaction system:
preparing a PCR reaction system by using 9 groups of specific primers respectively for the obtained cut DNA sample to be detected, the cut positive control and the cut negative control, wherein each group of primers of each sample is configured with 3-6 individual system repeats, and the PCR reaction system is configured with the following components in table 2:
table 2:
wherein 2xUniversal SYBR qPCR Master Mix is fluorescent quantitative PCR 2X premix available from Nanjinouzan Biotechnology Co., ltd, and is a commercial reagent. Primer F is the upstream Primer and Primer R is the downstream Primer.
The 9 groups of specific primers comprise 1 group of conventional chromosome locus detection primers and 8 groups of chromosome locus detection primers No. 21, and the specific primers are as follows:
group 1 was used for detection of conventional chromosomal locus No. 3:
the upstream primer Ch3-F: AGCTGGCACCCGCTGG
The downstream primer Ch3-R: GTGTGGGGTTGCACGCG
Group 2 was used for detection of chromosome 21 site 1:
the upstream primer ERG-F: CAGGAGGCTGAGGCAGGG
The downstream primer ERG-R: GGTACAGGTTGGGAGTTTGGG
Group 3 was used for detection of chromosome 21 site 2:
the upstream primer AIRE-F: TAGTAAGGGAGGGCCGAGAA
The downstream primer AIRE-R: CAGAGCCAGAACGCACAGAG
Group 4 was used for detection of chromosome 21 site 3:
the upstream primer SIM-F: ACCGGGCCTTCTGTCTGTC
Downstream primer SIM-R: TCTGCACGCTTCAATCCTTC
Group 5 was used for detection of chromosome 21 site 4:
the upstream primer NIPK4-F: GAGCCAGGTCTGTTCTCCACG
Downstream primer NIPK4-R: AGCCGATGCCCTTGTTGC
Group 6 was used for detection of chromosome 21 site 5:
the upstream primer YBEY-1F: GGTTCGTGCACTCGCTGAG
Downstream primer YBEY-1R: GCCTTCTCCTTCTGGAACATCT
Group 7 was used for detection of chromosome 21 site 6:
upstream primer JAM2-F: TTTTGGGCCACATCATTCT
Downstream primer JAM2-R: GCTGGGATTACAGGCGTGAG
Group 8 was used for detection of chromosome 21 site 7:
the upstream primer DSCAM-F: CCTAAGACCTTTGCCTAGCTTCT
Downstream primer DSCAM-R: TCCTTTATGGGATTTCTGTTGTG
Group 9 was used for detection of chromosome 21 site 8:
the upstream primer PCNT-F: AGCAGACACGGGCTCGGT
Downstream primer PCNT-R: CGGTGCTCAGCAACCACC
The concentration of each set of upstream and downstream primers was 10. Mu.M.
(5) And (3) PCR reaction:
the PCR reaction and fluorescence assay were performed using a LightCycler96 real-time fluorescent quantitative PCR instrument, the PCR procedure was as follows:
first stage, pre-denaturation: the PCR reaction system reacts for 5min at 95 ℃;
and (3) a second stage, a cyclic reaction: the PCR reaction system was reacted at 95℃for 10sec and then at 60℃for 30sec;
third stage, obtaining dissolution curve: the PCR reaction system was reacted at 95℃for 15sec and then at 60℃for 30sec; then heating to 95 ℃ to react for 15sec to obtain a dissolution curve;
the real-time fluorescence quantitative PCR instrument of the LightCycler96 is the existing equipment.
(6) Analysis:
analyzing the experimental result by using analysis software of a LightCycler96 real-time fluorescence quantitative PCR instrument to obtain a Cq value, and analyzing the obtained Cq value, wherein the Cq value comprises the Cq value of a DNA sample to be detected: cq Sample of Positive control Cq value: cq Positive control Negative control Cq value: cq Negative control The method comprises the steps of carrying out a first treatment on the surface of the Each of the Cq values of 8 sites on chromosome 21 is compared with the Cq value of a conventional chromosome to obtain a ratio R; if more than 4 sites R Positive control ≈R Sample of ＞R Negative control The probability of the trisomy 21 of the fetus is extremely high, and the amniotic fluid puncture is recommended to be performed for the analysis and confirmation of the karyotype; if 1-4 sites R Positive control ≈R Sample of ＞R Negative control Then suggest review; if all sites R Positive control ＞R Sample of ≈R Negative control The fetal 21 trisomy probability is extremely low, and the birth can be guaranteed.
Six fetal cases were actually detected using the method of the above example, with the following results:
as shown in fig. 1-2, the detection results of cases one and two show that: more than 4 loci R Positive control ≈R Sample of ＞R Negative control The fetal probability is shown to be a three-body fetus, and the follow-up amniotic fluid puncture test result shows that the two cases are three-body fetuses;
as shown in figures 3-4 of the drawings,the detection results of the third and fourth cases show that: 1-4 sites R Positive control ≈R Sample of ＞R Negative control The proposal is reexamined, and the subsequent amniotic fluid puncture test result shows that one example is a three-body fetus and one example is a normal fetus;
as shown in fig. 5-6, the detection results of cases five and six show that: all sites R Positive control ＞R Sample of ≈R Negative control The three-body probability of the fetus is extremely low, and the subsequent amniotic fluid puncture test results show that both cases are normal fetuses.
Sequence listing
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<120> a method for detecting prenatal fetal 21-trisomy syndrome
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Claims (2)
1. A specific primer for preparing a PCR reaction system for the detection of prenatal fetal 21-trisomy syndrome, characterized in that:
preparing a PCR reaction system by using 9 groups of specific primers, wherein the 9 groups of specific primers comprise 1 group of conventional chromosome locus detection primers and 8 groups of chromosome locus detection primers No. 21;
the 9 groups of specific primers are as follows:
group 1 was used for detection of conventional chromosomal locus No. 3:
the upstream primer Ch3-F: AGCTGGCACCCGCTGG
The downstream primer Ch3-R: GTGTGGGGTTGCACGCG
Group 2 was used for detection of chromosome 21 site 1:
the upstream primer ERG-F: CAGGAGGCTGAGGCAGGG
The downstream primer ERG-R: GGTACAGGTTGGGAGTTTGGG
Group 3 was used for detection of chromosome 21 site 2:
the upstream primer AIRE-F: TAGTAAGGGAGGGCCGAGAA
The downstream primer AIRE-R: CAGAGCCAGAACGCACAGAG
Group 4 was used for detection of chromosome 21 site 3:
the upstream primer SIM-F: ACCGGGCCTTCTGTCTGTC
Downstream primer SIM-R: TCTGCACGCTTCAATCCTTC
Group 5 was used for detection of chromosome 21 site 4:
the upstream primer NIPK4-F: GAGCCAGGTCTGTTCTCCACG
Downstream primer NIPK4-R: AGCCGATGCCCTTGTTGC
Group 6 was used for detection of chromosome 21 site 5:
the upstream primer YBEY-1F: GGTTCGTGCACTCGCTGAG
Downstream primer YBEY-1R: GCCTTCTCCTTCTGGAACATCT
Group 7 was used for detection of chromosome 21 site 6:
upstream primer JAM2-F: TTTTGGGCCACATCATTCT
Downstream primer JAM2-R: GCTGGGATTACAGGCGTGAG
Group 8 was used for detection of chromosome 21 site 7:
the upstream primer DSCAM-F: CCTAAGACCTTTGCCTAGCTTCT
Downstream primer DSCAM-R: TCCTTTATGGGATTTCTGTTGTG
Group 9 was used for detection of chromosome 21 site 8:
the upstream primer PCNT-F: AGCAGACACGGGCTCGGT
Downstream primer PCNT-R: CGGTGCTCAGCAACCACC.
2.9 sets of specific primers are applied to the preparation of a PCR reaction system for detecting the 21-trisomy syndrome of a fetus before production, wherein the 9 sets of specific primers are as follows:
group 1 was used for detection of conventional chromosomal locus No. 3:
the upstream primer Ch3-F: AGCTGGCACCCGCTGG
The downstream primer Ch3-R: GTGTGGGGTTGCACGCG
Group 2 was used for detection of chromosome 21 site 1:
the upstream primer ERG-F: CAGGAGGCTGAGGCAGGG
The downstream primer ERG-R: GGTACAGGTTGGGAGTTTGGG
Group 3 was used for detection of chromosome 21 site 2:
the upstream primer AIRE-F: TAGTAAGGGAGGGCCGAGAA
The downstream primer AIRE-R: CAGAGCCAGAACGCACAGAG
Group 4 was used for detection of chromosome 21 site 3:
the upstream primer SIM-F: ACCGGGCCTTCTGTCTGTC
Downstream primer SIM-R: TCTGCACGCTTCAATCCTTC
Group 5 was used for detection of chromosome 21 site 4:
the upstream primer NIPK4-F: GAGCCAGGTCTGTTCTCCACG
Downstream primer NIPK4-R: AGCCGATGCCCTTGTTGC
Group 6 was used for detection of chromosome 21 site 5:
the upstream primer YBEY-1F: GGTTCGTGCACTCGCTGAG
Downstream primer YBEY-1R: GCCTTCTCCTTCTGGAACATCT
Group 7 was used for detection of chromosome 21 site 6:
upstream primer JAM2-F: TTTTGGGCCACATCATTCT
Downstream primer JAM2-R: GCTGGGATTACAGGCGTGAG
Group 8 was used for detection of chromosome 21 site 7:
the upstream primer DSCAM-F: CCTAAGACCTTTGCCTAGCTTCT
Downstream primer DSCAM-R: TCCTTTATGGGATTTCTGTTGTG
Group 9 was used for detection of chromosome 21 site 8:
the upstream primer PCNT-F: AGCAGACACGGGCTCGGT
Downstream primer PCNT-R: CGGTGCTCAGCAACCACC.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202110527026.1A CN113046435B (en) | 2021-05-14 | 2021-05-14 | Specific primer for preparing PCR reaction system for detecting prenatal fetal 21-trisomy syndrome |
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| CN109022541A (en) * | 2018-07-16 | 2018-12-18 | 苏州大学附属第二医院 | A kind of kit of concentration fetus dissociative DNA, method and its application |
| CN109689896A (en) * | 2015-11-10 | 2019-04-26 | 科戴克斯生命股份公司 | Fetal chromosomal aneuploidy is detected using the region of DNA domain of the differential methylation between fetus and pregnant female animal |
| CN113061653A (en) * | 2021-05-07 | 2021-07-02 | 济南国科医工科技发展有限公司 | PCR amplification composition for prenatal noninvasive detection of trisomy 21 syndrome and detection kit |
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| CN109689896A (en) * | 2015-11-10 | 2019-04-26 | 科戴克斯生命股份公司 | Fetal chromosomal aneuploidy is detected using the region of DNA domain of the differential methylation between fetus and pregnant female animal |
| CN109022541A (en) * | 2018-07-16 | 2018-12-18 | 苏州大学附属第二医院 | A kind of kit of concentration fetus dissociative DNA, method and its application |
| CN113061653A (en) * | 2021-05-07 | 2021-07-02 | 济南国科医工科技发展有限公司 | PCR amplification composition for prenatal noninvasive detection of trisomy 21 syndrome and detection kit |
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| Noninvasive Prenatal Detection of Trisomy 21 by an Epigenetic–Genetic Chromosome-Dosage Approach;Yu K.Tong等;Clinical Chemistry;第56卷(第1期);90-98 * |
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