CN113897427A - Reagent and kit for detecting fetal FGA gene mutation - Google Patents
Reagent and kit for detecting fetal FGA gene mutation Download PDFInfo
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- 101150083830 FGA gene Proteins 0.000 title claims abstract description 38
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 29
- 230000001605 fetal effect Effects 0.000 title claims abstract description 23
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 20
- 238000012408 PCR amplification Methods 0.000 claims abstract description 14
- 210000003754 fetus Anatomy 0.000 claims abstract description 14
- 230000035772 mutation Effects 0.000 claims abstract description 13
- 108700039887 Essential Genes Proteins 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 6
- 238000012986 modification Methods 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 230000026731 phosphorylation Effects 0.000 claims description 3
- 238000006366 phosphorylation reaction Methods 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 238000002123 RNA extraction Methods 0.000 claims description 2
- 238000001821 nucleic acid purification Methods 0.000 claims description 2
- 238000010839 reverse transcription Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 14
- 108020004707 nucleic acids Proteins 0.000 abstract description 10
- 150000007523 nucleic acids Chemical class 0.000 abstract description 10
- 102000039446 nucleic acids Human genes 0.000 abstract description 10
- 238000013459 approach Methods 0.000 abstract description 3
- 230000008774 maternal effect Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 238000012163 sequencing technique Methods 0.000 description 20
- 238000000034 method Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 5
- 208000036830 Normal foetus Diseases 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 206010010356 Congenital anomaly Diseases 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 102200042452 rs121909605 Human genes 0.000 description 2
- 102200042453 rs121909606 Human genes 0.000 description 2
- 102200042537 rs121909608 Human genes 0.000 description 2
- 102200042527 rs121909610 Human genes 0.000 description 2
- 102220005692 rs121909611 Human genes 0.000 description 2
- 102200042533 rs121909612 Human genes 0.000 description 2
- 102200042530 rs121909613 Human genes 0.000 description 2
- 102200042539 rs121909614 Human genes 0.000 description 2
- 102220005697 rs121909615 Human genes 0.000 description 2
- 102220005695 rs146387238 Human genes 0.000 description 2
- 102200042529 rs6050 Human genes 0.000 description 2
- 102220005696 rs606231225 Human genes 0.000 description 2
- 102200042531 rs78506343 Human genes 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012070 whole genome sequencing analysis Methods 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 206010008805 Chromosomal abnormalities Diseases 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
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- 102100031752 Fibrinogen alpha chain Human genes 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 102200042538 rs121909607 Human genes 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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Abstract
The invention discloses a reagent and a kit for detecting fetal FGA gene mutation. The reagent for detecting the fetal FGA gene mutation comprises six groups of FGA gene specific primers, the sequences of the primers are the sequences shown by SEQ ID NO.1 to 12 in sequence, wherein two groups of primers are combined into one group of PCR amplification primers in sequence and used for detecting the fetal FGA gene mutation. The reagent of the invention can directly detect the free nucleic acid of the fetus in the plasma of the pregnant woman and obtain the mutation information of the fetus FGA gene, solves the problem that the fetus gene can not be subjected to mutation detection due to a large number of maternal genome backgrounds in the plasma of the pregnant woman, and provides a new scheme and approach for the fetus FGA gene mutation detection.
Description
Technical Field
The invention relates to the field of fetal FGA gene mutation detection, in particular to a reagent and a kit for detecting fetal FGA gene mutation.
Background
Studies have shown that congenital fibrinogenemia is associated with mutations in the FGA gene, which is rarely expressed in pregnant women but only in fetuses. Therefore, detection of fetal FGA genes is an important method and approach for early diagnosis and prediction of congenital fibrinogen-free disease.
Current genetic testing techniques for fetuses are mainly down's screening, e.g., based on whole genome sequencing. However, this technique is mainly intended for the detection of chromosomal abnormalities, and cannot detect single gene mutations, such as FGA gene mutations. Moreover, there is a problem that the detection cost is high when only a single gene mutation is detected by whole genome sequencing.
Disclosure of Invention
The invention aims to provide a novel reagent and a kit for detecting fetal FGA gene mutation.
The invention specifically adopts the following technical scheme:
the invention discloses a reagent for detecting fetal FGA gene mutation, which comprises six groups of FGA gene specific primers, wherein the primer sequences of the six groups of primers are the sequences shown by SEQ ID NO.1 and SEQ ID NO.12 in sequence, wherein SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 and the like are combined in pairs to form a group of PCR amplification primers for amplifying fetal FGA genes.
Studies have shown that pregnant women contain free nucleic acids of the fetus in their blood; however, because of the low content of fetal free nucleic acids in the blood of pregnant women, no technical means for accurately and effectively detecting these nucleic acids is available. Even if research reports claim that the fetal free nucleic acid can be detected from the blood of pregnant women, the detection sensitivity and specificity are poor, and the clinical use requirement is difficult to meet. Therefore, the invention creatively develops a reagent which has high sensitivity and good specificity and is specially used for detecting the fetal FGA genes, namely six groups of FGA gene specific primers. The six groups of specific FGA gene primers can detect the common 17 SNP loci on the fetal FGA gene, and well meet the clinical use requirement.
Preferably, the reagent of the present invention further comprises a housekeeping gene specific primer.
Preferably, the upstream primer of the housekeeping gene specific primer is a sequence shown by SEQ ID NO.13, and the downstream primer is a sequence shown by SEQ ID NO. 14.
Preferably, the reagent also comprises a universal primer, wherein the universal primer consists of a first primer and a second primer, the first primer is a sequence shown by SEQ ID NO.15, and the second primer is a sequence shown by SEQ ID NO. 16.
Preferably, the 5' end of the first primer has a phosphorylation modification.
The invention also discloses a kit for detecting the fetal FGA gene mutation, which comprises the reagent.
Preferably, the kit also comprises a free RNA extraction reagent in the blood of the pregnant woman.
Preferably, the kit further comprises a reverse transcription reagent.
Preferably, PCR amplification reagents are also included in the kit.
Preferably, nucleic acid purification reagents are also included in the kit.
The above reagents are added mainly for convenience of use, and may be separately purchased if not added to the kit.
The invention has the beneficial effects that:
the reagent of the invention can directly detect the free nucleic acid of the fetus in the plasma of the pregnant woman and obtain the mutation information of the fetus FGA gene, solves the problem that the fetus gene can not be subjected to mutation detection due to a large number of maternal genome backgrounds in the plasma of the pregnant woman, and provides a new scheme and approach for the fetus FGA gene mutation detection.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The following examples are merely illustrative of the present invention and should not be construed as limiting thereof.
Examples
The experiment aims at FGA genes, covers common FGA mutation sites, and designs six groups of FGA gene specific primers. Meanwhile, specific primers are designed aiming at housekeeping gene beta-actin. All primer sequences are shown in Table 1, and all primers were synthesized in Shanghai.
TABLE 1 primer sequences
In Table 1, F represents the forward primer, and R represents the reverse primer. The sequences in table 1 are primers obtained by final screening that can meet the use requirements of multiplex PCR amplification; in particular, the selection and primers for housekeeping genes were also specifically designed in view of performing multi-tier PCR amplification using six sets of FGA gene-specific primers.
The six groups of specific FGA gene primers cover 17 kinds of SNP mutation of FGA genes, and specifically comprise: rs78506343, rs121909613, rs121909612, rs 5877761, rs121909611, rs121909610, rs121909615, rs6050, rs606231225, rs146387238, rs121909614, rs121909608, rsl21909609, rs121909607, rs121909606, rs121909605 and rs 121909604.
Experimental samples: the test adopts two pregnant woman plasma samples, one is a pregnant woman sample of a normal fetus, the other is a pregnant woman sample of which the fetus is congenital fibrinogen-free, and the blood of each pregnant woman sample is 5 mL.
1. Pregnant woman plasma free nucleic acid extraction
The test adopts a kit for extracting the plasma free nucleic acid of the pregnant women from Qigen company, and the extraction is carried out according to the standard operation flow, and the finally obtained free DNA is dissolved in 20 mu L of water.
2. Specific PCR amplification
And (3) PCR reaction system: 25. mu.L of 2 Xkappa mixture, 5. mu.L of 10. mu.M mixed primer, 5. mu.L of free DNA, and sterilized ddH2O to 50. mu.L. Each sample was repeated 3 times.
The mixed primers comprise six groups of FGA gene specific primers and housekeeping gene specific primers, and the concentration of all the upstream primers and the downstream primers is 10 mu M.
And (3) PCR reaction conditions: at 95 deg.C for 2min, at 95 deg.C for 5s, at 58 deg.C for 1min, and at 72 deg.C for 20s for 15 cycles, and then at 72 deg.C for 10min, and at 4 deg.C for storage.
The PCR amplification product was purified using equivalent volume of Agencourt AMPure XP magnetic beads and sterilized ddH at 25. mu.L2O redissolving the purified DNA.
3. Sequencing library construction
And (3) amplifying the amplification product obtained in the step (2) of specific PCR amplification by adopting a universal primer.
The universal primer consists of a first primer and a second primer, wherein the first primer is a sequence shown by SEQ ID NO.15, the second primer is a sequence shown by SEQ ID NO.16, and the 5' end of the first primer has phosphorylation modification.
SEQ ID NO.15：5’-ACACACGACGCTCTTCCGAT-3’
SEQ ID NO.16：
5’-AGCCAAGGAGTTGCTGCGTACATTTGTCTTCCTAGACGTGTGCTCTTCCGATC-3’。
Reaction system: 2 Xkappa mixture 25. mu.L, 10. mu.M first primer 2. mu.L, 10. mu.M second primer 2. mu.L, purified product of PCR amplification of "2. specific PCR amplification" 10. mu.L, supplemented with sterilized ddH2O to 50. mu.L.
Reaction conditions are as follows: at 95 deg.C for 2min, at 95 deg.C for 5s, at 58 deg.C for 1min, and at 72 deg.C for 20s for 15 cycles, and then at 72 deg.C for 10min, and at 4 deg.C for storage.
The reaction product was purified using equivalent volume of Agencour AMPure XP magnetic beads and sterilized ddH at 25. mu.L2O redissolving the purified DNA.
4. Sequencing
Sequencing the product of the 3. sequencing library construction by adopting BGISEQ-500, wherein the sequencing type is double-ended by 50 bp.
5. Data analysis
Filtering sequencing off-line data by adopting a conventional filtering scheme, and comparing the data to a genome; and (4) counting the comparison rate, the proportion of the target region, the depth of the target region and the base distribution of the target site. The statistical results are shown in Table 2.
TABLE 2 statistical results of sequencing data
In table 2, the original reads number refers to the number of reads obtained after conventional filtering of the offline data, the remaining reads number after removing the linker refers to the number of reads obtained after removing the linker, the reads number in the comparison refers to the number of reads compared to the genome, and the reads number of the FGA gene refers to the number of reads of the FGA gene in the target region. The results in table 2 show that the primers used in the experiment for amplifying and sequencing the fetal FGA gene can achieve a data utilization rate of more than 97%, and a comparison rate and a target region ratio of more than 98%.
Statistics is carried out on the coverage rate of each specific primer pair and housekeeping gene amplification primers in six sequencing samples, and the results show that the depth distribution obtained after sequencing of all the primer pairs is uniform, and the depth difference of different primer pairs is small and is within 5 times.
The repeatability of 3 repeated sequencing samples of the plasma sample of the pregnant woman with the normal fetus and 3 repeated sequencing samples of the plasma sample of the pregnant woman with the infant is counted, and the result shows that the depth stability of each primer obtained by different experiments of the same sample is good, and the R of 3 repeated samples of the plasma sample of the pregnant woman with the normal fetus is good20.98, R of 3 replicates of a pregnant woman plasma sample of an infant2The reproducibility of both samples is good, say 0.99.
Mutation analysis is carried out on the six sequencing samples, and the result shows that no mutation is detected in 3 repeated sequencing samples of the pregnant woman plasma sample of a normal fetus; the C.1039C > T mutation is detected in 3 replicates of a pregnant woman sample of the infant; the above results are in agreement with expectations. Indicating that the assay is capable of detecting fetal FGA gene mutations.
6. Sensitivity detection
Free DNA obtained from a pregnant woman plasma sample of an infant patient is adopted for sensitivity detection, and concentration sequencing and dilution are carried out on the DNA to obtain free DNA samples with the concentrations of 10 ng/mu L, 5 ng/mu L, 2.5 ng/mu L and 1 ng/mu L respectively. According to the method, 2, specific PCR amplification, 3, sequencing library construction, 4, sequencing and 5, data analysis are carried out, and fetal FGA gene mutation detection conditions under different concentrations are analyzed.
The results show that the mutation information of FGA gene can be accurately detected by samples with five concentrations, namely, the C.1039C > T mutation can be detected. The primers of this experiment were shown to be capable of performing fetal FGA gene mutation detection on free DNA at lower concentrations, i.e., 1 ng/. mu.L.
7. Actual sample detection
In the test, plasma samples of 30 pregnant women with children patients are respectively obtained, and nucleic acid extraction, specific PCR amplification, sequencing library construction and data analysis are carried out according to the method, wherein the nucleic acid extraction is carried out according to the method, and the method comprises the steps of 2, specific PCR amplification, 3, sequencing library construction, 4, sequencing and 5; the 17 SNP sites covered by the test were detected, and the results are shown in Table 3.
TABLE 3 actual sample test results
The results in table 3 show that the primers in this test can indeed detect 17 SNPs of fetal FGA gene, such as rs78506343, rs121909613, rs121909612, rs 5877761, rs121909611, rs121909610, rs121909615, rs6050, rs606231225, rs146387238, rs121909614, rs121909608, rs121909609, rsl21909607 29607, rs121909606, rs121909605 and rs 121909604.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Sequence listing
<110> Shenzhen Zhiyin cause cell Biotech, Ltd
<120> reagent and kit for detecting fetal FGA gene mutation
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Claims (10)
1. A reagent for detecting fetus FGA gene mutation is characterized by comprising six groups of FGA gene specific primers, wherein the primer sequences of the six groups of primers are sequences shown as SEQ ID NO.1 and SEQ ID NO.12 in sequence, wherein SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 are analogized, and the six groups of primers are combined into a group of PCR amplification primers in pairs for amplifying the fetus FGA gene;
SEQ ID NO.1：
5’-GACGTGTGCTCTTCCGATCTGCTGTAACTTGAAGATTTAC-3’
SEQ ID NO.2：
5’-ACACGACGCTCTTCCGATCTCACCTATGTTAGGAGAG-3’
SEQ ID NO.3：
5’-GACGTGTGCTCTTCCGATCTGCCTCGGGACAGTCAGAACCA-3’
SEQ ID NO.4：
5’-ACACGACGCTCTTCCGATGACTGGTAAAGAGAAGGTCACC-3’
SEQ ID NO.5：
5’-GACGTGTGCTCTTCCGATCCAGGATTCCAGGTTCCGGTAC-3’
SEQ ID NO.6：
5’-ACACGACGCTCTTCCGATGACTGGAGGGACTGCAACCTG-3’
SEQ ID NO.7：
5’-GACGTGTGCTCTTCCGATCAGTAATCTCATTTCCACCAGGTCT-3’
SEQ ID NO.8：
5’-ACACGACGCTCTTCCGATGAACAGGTCATTGCCAAAGACT-3’
SEQ ID NO.9：
5’-GACGTGTGCTCTTCCGATCACTTTTCTCTGCATATAGTAGAC-3’
SEQ ID NO.10：
5’-ACACGACGCTCTTCCGATTGAAGTCCTGAAGCGCAAAG-3’
SEQ ID NO.11：
5’-GACGTGTGCTCTTCCGATCACTGCTTACCCAGTCTTCAT-3’
SEQ ID NO.12：
5’-ACACGACGCTCTTCCGATGTGAATGAATCTTTAAAGACTG-3’。
2. the reagent of claim 1, further comprising a housekeeping gene specific primer.
3. The reagent of claim 2, wherein the upstream primer of the housekeeping gene specific primer is a sequence shown by SEQ ID No.13, and the downstream primer is a sequence shown by SEQ ID No. 14;
SEQ ID NO.13：
5’-GACGTGTGCTCTTCCGATCACCCCTCGTAGATGGGCA-3’
SEQ ID NO.14：
5’-ACACGACGCTCTTCCGATTCCCCAGTGTGACATGGT-3’。
4. the reagent of claim 1, further comprising a universal primer, wherein the universal primer comprises a first primer and a second primer, the first primer is a sequence shown in SEQ ID No.15, and the second primer is a sequence shown in SEQ ID No. 16;
SEQ ID NO.15：5’-ACACACGACGCTCTTCCGAT-3’
SEQ ID NO.16：
5’-AGCCAAGGAGTTGCTGCGTACATTTGTCTTCCTAGACGTGTGCTCTTCCGATC-3’。
5. the reagent according to claim 4, wherein the 5' end of the first primer has a phosphorylation modification.
6. A kit for detecting mutations in fetal FGA gene, comprising the reagent of any one of claims 1-5.
7. The kit of claim 6, wherein: the kit also comprises a pregnant woman blood free RNA extraction reagent.
8. The kit of claim 6, wherein: the kit also comprises a reverse transcription reagent.
9. The kit of claim 6, wherein: the kit also comprises a PCR amplification reagent.
10. The kit of claim 6, wherein: the kit also comprises a nucleic acid purification reagent.
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| KR101533792B1 (en) * | 2015-02-24 | 2015-07-06 | 대한민국 | Method for Autosomal Analysing Human Subject of Analytes based on a Next Generation Sequencing Technology |
| US20200332287A1 (en) * | 2017-11-28 | 2020-10-22 | Emendobio Inc. | Differential knockout of an allele of a heterozygous fibrinogen alpha chain (fga) gene |
| CN112996926A (en) * | 2018-12-07 | 2021-06-18 | 深圳华大生命科学研究院 | Construction method, detection device and application of target gene library |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101533792B1 (en) * | 2015-02-24 | 2015-07-06 | 대한민국 | Method for Autosomal Analysing Human Subject of Analytes based on a Next Generation Sequencing Technology |
| US20200332287A1 (en) * | 2017-11-28 | 2020-10-22 | Emendobio Inc. | Differential knockout of an allele of a heterozygous fibrinogen alpha chain (fga) gene |
| CN112996926A (en) * | 2018-12-07 | 2021-06-18 | 深圳华大生命科学研究院 | Construction method, detection device and application of target gene library |
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