CN116814766A - Deafness noninvasive prenatal diagnostic kit based on single-cell whole genome amplification - Google Patents
Deafness noninvasive prenatal diagnostic kit based on single-cell whole genome amplification Download PDFInfo
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- 206010011878 Deafness Diseases 0.000 title claims abstract description 34
- 208000016354 hearing loss disease Diseases 0.000 title claims abstract description 34
- 231100000895 deafness Toxicity 0.000 title claims abstract description 33
- 238000013412 genome amplification Methods 0.000 title claims abstract description 26
- 238000009007 Diagnostic Kit Methods 0.000 title claims description 11
- 230000003321 amplification Effects 0.000 claims abstract description 74
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 74
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 238000012163 sequencing technique Methods 0.000 claims abstract description 14
- 238000003793 prenatal diagnosis Methods 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 239000012634 fragment Substances 0.000 claims abstract description 8
- 210000000349 chromosome Anatomy 0.000 claims abstract description 7
- 206010064571 Gene mutation Diseases 0.000 claims abstract description 4
- 102100035278 Pendrin Human genes 0.000 claims description 20
- 108091006507 SLC26A4 Proteins 0.000 claims description 20
- 210000004027 cell Anatomy 0.000 claims description 20
- 239000011259 mixed solution Substances 0.000 claims description 15
- 102100037156 Gap junction beta-2 protein Human genes 0.000 claims description 9
- 101000954092 Homo sapiens Gap junction beta-2 protein Proteins 0.000 claims description 9
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 102000004317 Lyases Human genes 0.000 claims description 6
- 108090000856 Lyases Proteins 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 6
- 108700028369 Alleles Proteins 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000001605 fetal effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000004075 alteration Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 231100000888 hearing loss Toxicity 0.000 description 1
- 230000010370 hearing loss Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
The application discloses a common deafness noninvasive prenatal diagnosis kit based on single-cell whole genome amplification, which relates to the technical field of bioengineering and has the technical key points that: the kit comprises about 8000 SNP loci, wherein the SNPMAF is more than 0.35 (the frequency of east Asia population of a thousand human genome), and the loci are evenly distributed on 1-23 chromosomes; the kit also comprises the total fragment length of the common deafness gene. The detection kit comprises about 8000 SNP loci, wherein the SNP loci are evenly distributed on 1-23 chromosomes, the amplification effect can be determined through comparison of the results after second-generation sequencing, and the allele release rate (ADO), the False Positive Rate (FPR) and the copy number variation Coefficient (CV) of the SNP loci are evaluated. Meanwhile, the kit also comprises all fragment lengths of common deafness genes, can realize simultaneous detection of the deafness gene mutations, and has high detection sensitivity and low cost.
Description
Technical Field
The application relates to the technical field of bioengineering, in particular to a deafness noninvasive prenatal diagnosis kit based on single-cell whole genome amplification.
Background
Noninvasive prenatal diagnosis of congenital genetic diseases is currently the subject of hot spot research, and particularly mutation detection for monogenic diseases is still a technical bottleneck. At present, noninvasive prenatal detection based on fetal free nucleated red blood cells is theoretically feasible, and the specific implementation process comprises fetal nucleated red blood cell capture, single cell whole genome amplification, second generation sequencing and Sanger sequencing for detecting pathogenic mutation, and verification with the invasive prenatal diagnosis. Among them, the current research of DNA products amplified by whole genome of single cell in the subsequent gene sequencing and belief analysis stage still has many difficulties to break through, and a simple and effective kit is needed to complete the above process. Based on the above, the inventor provides a novel single-cell whole genome amplification-based noninvasive prenatal diagnosis kit for deafness.
In addition, single-cell whole genome amplification products have unstable amplification of the product and deletion of the effective fragment DNA compared with DNA derived from peripheral blood, and sometimes the target of diagnosis cannot be accomplished by sequencing based on the product. Therefore, the detection and evaluation of the DNA product is particularly important, and the sample side meeting the detection standard can be used for later application. Therefore, the detection kit designed by the inventor comprises about 8000 SNP loci which are evenly distributed on 1-23 chromosomes, the amplification effect can be determined through comparison of the results after second generation sequencing, and the allele release rate and the copy number variation coefficient are evaluated. Meanwhile, the kit also comprises all fragment lengths of common deafness genes, and the mutation of the deafness genes can be detected simultaneously.
Disclosure of Invention
The application aims to solve the problems and provide a non-invasive prenatal diagnosis kit for deafness based on single-cell whole genome amplification.
In order to achieve the above purpose, the technical scheme of the application is as follows: the kit comprises about 8000 SNP loci, wherein the SNP loci are evenly distributed on 1-23 chromosomes; the kit also comprises the total fragment length of the common deafness gene; the kit also comprises PicoPlex single-cell whole genome amplification reagents, wherein the reagents comprise cell lyase, a lyase slow-release solution, an amplification buffer solution, an amplification enzyme and nuclease-free water.
Further, the specific amplification procedure in the kit is as follows:
(1) Taking 5ul of freshly prepared extraction mixed solution for every 5ul of cell samples;
(2) Samples were incubated in a thermocycler as follows:
| 1cycle | 75℃ | 10min |
| 1cycle | 95℃ | 4min |
| 1cycle | room temperature | Hold |
；
(3) The tube was slightly centrifuged;
(4) Adding the components of the pre-amplification mixed solution and fully and uniformly mixing; adding 5ul of pre-amplification mixed solution to each cell sample;
(5) Samples were incubated in a thermocycler as follows:
(6) Slightly centrifuging, and placing the pre-amplified incubation product on ice;
(7) Adding the components of the amplification mixed solution and fully and uniformly mixing; amplification mixture composition: amplification buffer, amplification enzyme, nuclease-free water;
(8) Mixing 60ul of amplification mixed solution and 15ul of pre-amplification product, and uniformly mixing by a blowing pipe;
(9) Samples were amplified as follows:
the PicoPlex amplification product was stored at-20deg.C;
the kit is used for establishing a library of single peripheral blood lymphocyte amplification products, and the MGISEQ-2000 (PE 100) sequencing mode is used for comparing the single peripheral blood lymphocyte amplification products with a reference genome GRCh37/hg19 by BWA, and the single peripheral blood lymphocyte amplification products are calculated according to the number of reads; the comparison rate of the captured data is more than 85%, and the capturing efficiency of the target area is more than 90%.
Further, the site information includes: GJB2:235delC &299_300delA, SLC26A4:919-2A > G &917insG, SLC26A4:2168A > G.
Further, the GJB2: the primer and sequence for one amplification of 235delC &299_300delA are as follows:
GJB2-2F-36：AGCAAACCGCCCAGAGTA；
GJB2-2R-496：CGAAGATGACCCGGAAGA。
further, the GJB2: the second amplification primer and sequence of 235delC &299_300delA are as follows:
GJB2-2F-207：CTTTGTCTGCAACACCCTG；
GJB2-2R-364：CCCTTGATGAACTTCCTCT。
further, the primary amplification primer and the sequence of SLC26A4:919-2A > G &917insG are as follows:
SLC26A4-7-8F-17：TTTTATAGACGCTGGTTGAG；
SLC26A4-7-8R-545：AAATGGCTTGACGTTTATCT。
further, the secondary amplification primer and the sequence of the SLC26A4:919-2A > G & 91kinsG are as follows:
SLC26A4-7-8F-144'：AATCCCAGTCCCTATTCCTA；
SLC26A4-7-8R-335：GGGATGGATTTAACAATGCC。
further, the primary amplification primer and the sequence of the SLC26A4:2168A > G are as follows:
SLC26A4-19F-33：CACTTGAACTTGGGACGC；
SLC26A4-19R-464：ACTGATGAAAAAACTGAGGCT。
further, the secondary amplification primer and the sequence of the SLC26A4:2168A > G are as follows:
SLC26A4-19F-89：CTGGGCAATAGAATGAGACT；
SLC26A4-19R-464：ACTGATGAAAAAACTGAGGCT。
the application also provides application of the single-cell whole genome amplification-based noninvasive prenatal diagnosis kit for deafness, and the kit is applied to detection of common deafness gene mutation.
Compared with the prior art, the beneficial effect of this scheme:
1. the detection kit can realize one-time detection to realize amplification result evaluation of amplification products and mutation site detection.
2. The kit has the characteristics of high SNP locus amplification efficiency and high sensitivity.
3. The kit can greatly reduce the detection cost of the prior art. For example, the prior art needs to perform second generation targeted sequencing (2000 yuan) +whole genome amplification effect detection (1000 yuan) or whole exon sequencing (3000 yuan). The detection kit of the present application predicts about 1000 yuan per detection sample.
Drawings
FIG. 1 is a schematic illustration of a technical route of a cartridge according to an embodiment of the present application;
FIG. 2 is a specific amplification scheme in a reagent cartridge according to an embodiment of the present application.
Detailed Description
The advantages and various effects of the present application will be more clearly presented by the following description of the present application in conjunction with the examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the application, not to limit the application.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification will control.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present application are commercially available or may be obtained by existing methods. In the present application, "about" means within 10%, preferably within 5% of a given value or range.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the general idea is as follows.
Examples:
the scheme provided by the embodiment of the application is as follows: the kit comprises about 8000 SNP loci, wherein the SNP loci are evenly distributed on 1-23 chromosomes; the kit also comprises the total fragment length of the common deafness gene; the kit also comprises PicoPlex single-cell whole genome amplification reagents, wherein the reagents comprise cell lyase, a lyase slow-release solution, an amplification buffer solution, an amplification enzyme and nuclease-free water.
In this example, the specific amplification procedure in the kit is (see fig. 2):
(1) Taking 5ul of freshly prepared extraction mixed solution for every 5ul of cell samples;
(2) Samples were incubated in a thermocycler as follows:
| 1cycle | 75℃ | 10min |
| 1cycle | 95℃ | 4min |
| 1cycle | room temperature | Hold |
；
(3) The tube was slightly centrifuged;
(4) Adding the components of the pre-amplification mixed solution and fully and uniformly mixing; adding 5ul of pre-amplification mixed solution to each cell sample;
(5) Samples were incubated in a thermocycler as follows:
(6) Slightly centrifuging, and placing the pre-amplified incubation product on ice;
(7) Adding the components of the amplification mixed solution and fully and uniformly mixing; amplification mixture composition: amplification buffer, amplification enzyme, nuclease-free water;
(8) Mixing 60ul of amplification mixed solution and 15ul of pre-amplification product, and uniformly mixing by a blowing pipe;
(9) Samples were amplified as follows:
the PicoPlex amplification product was stored at-20deg.C;
pooling single peripheral blood lymphocyte amplification products by using a kit, and performing MGISEQ-2000 (PE 100) sequencing mode, and comparing the single peripheral blood lymphocyte amplification products to a reference genome GRCh37/hg19 by using BWA, wherein the single peripheral blood lymphocyte amplification products are calculated according to reads; the comparison rate of the captured data is more than 85%, and the capturing efficiency of the target area is more than 90%.
In the present embodiment, the site information includes: GJB2:235delC &299_300delA, SLC26A4:919-2A > G &917insG, SLC26A4:2168A > G.
In this embodiment, GJB2: the primer and sequence for one amplification of 235delC &299_300delA are as follows:
GJB2-2F-36：AGCAAACCGCCCAGAGTA；
GJB2-2R-496：CGAAGATGACCCGGAAGA。
in this embodiment, GJB2: the second amplification primer and sequence of 235delC &299_300delA are as follows:
GJB2-2F-207：CTTTGTCTGCAACACCCTG；
GJB2-2R-364：CCCTTGATGAACTTCCTCT。
in this example, the primary amplification primers and sequences for SLC26A4:919-2A > G &917insG are:
SLC26A4-7-8F-17：TTTTATAGACGCTGGTTGAG；
SLC26A4-7-8R-545：AAATGGCTTGACGTTTATCT。
in this example, the secondary amplification primers for SLC26A4:919-2A > G & 91kinsG and the sequences are:
SLC26A4-7-8F-144'：AATCCCAGTCCCTATTCCTA；
SLC26A4-7-8R-335：GGGATGGATTTAACAATGCC。
in this example, the primary amplification primers and sequences of SLC26A4:2168A > G are:
SLC26A4-19F-33：CACTTGAACTTGGGACGC；
SLC26A4-19R-464：ACTGATGAAAAAACTGAGGCT。
in this example, the secondary amplification primer and sequence of SLC26A4:2168A > G are:
SLC26A4-19F-89：CTGGGCAATAGAATGAGACT；
SLC26A4-19R-464：ACTGATGAAAAAACTGAGGCT。
the non-invasive prenatal diagnosis kit for deafness based on single-cell whole genome amplification in the embodiment is applied to detection of common deafness gene mutation.
The following are specific test examples of the embodiment of the present application:
the 082-1 hereditary hearing loss family is selected, the mother is SLC26A4 c.919-2A > G heterozygous mutation, and the father is SLC26A4 c.919-2A > G heterozygous mutation. Taking peripheral blood of a mother during 19 weeks of pregnancy, capturing fetal nucleated red blood cells, carrying out whole genome amplification according to product specification by using a Pi COPLEX single-cell whole genome kit, carrying out targeted capturing by using the single-cell whole genome deafness prenatal diagnosis kit, and comparing the results by second generation sequencing, wherein the result proves that the sample does not carry SLC26SLC26A4 c.919-2A > G deafness mutation sites, and the result is consistent with Sanger sequencing and amniotic fluid puncture DNA results. Physical control data are shown in tables 1 and 2 below:
TABLE 1
TABLE 2
Through the embodiment of the application, the detection kit comprises about 8000 SNP loci which are evenly distributed on 1-23 chromosomes, the amplification effect is defined through comparison of the results after second-generation sequencing, and the allele release rate and the copy number variation coefficient are evaluated. Meanwhile, the kit also comprises all fragment lengths of common deafness genes, can realize simultaneous detection of the mutation of the deafness genes, and can realize amplification result evaluation of amplification products and detection of mutation sites by one-time detection. In addition, the kit has the characteristics of high SNP locus amplification efficiency and high sensitivity. In addition, the kit greatly reduces the detection cost of the prior art. For example, the prior art needs to perform second generation targeted sequencing (2000 yuan) +whole genome amplification effect detection (1000 yuan) or whole exon sequencing (3000 yuan). Our test kit predicts around 1000 yuan per test sample.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present application have been described above, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application. It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. A non-invasive prenatal diagnosis kit for deafness based on single-cell whole genome amplification is characterized in that: the kit comprises about 8000 SNP loci, wherein the SNP loci are evenly distributed on 1-23 chromosomes; the kit also comprises the total fragment length of the common deafness gene; the kit also comprises PicoPlex single-cell whole genome amplification reagents, wherein the reagents comprise cell lyase, a lyase slow-release solution, an amplification buffer solution, an amplification enzyme and nuclease-free water.
2. The single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to claim 1, wherein: the specific amplification flow in the kit is as follows:
(1) Taking 5ul of freshly prepared extraction mixed solution for every 5ul of cell samples;
(2) Samples were incubated in a thermocycler as follows:
；
(3) The tube was slightly centrifuged;
(4) Adding the components of the pre-amplification mixed solution and fully and uniformly mixing; adding 5ul of pre-amplification mixed solution to each cell sample;
(5) Samples were incubated in a thermocycler as follows:
(6) Slightly centrifuging, and placing the pre-amplified incubation product on ice;
(7) Adding the components of the amplification mixed solution and fully and uniformly mixing; amplification mixture composition: amplification buffer, amplification enzyme, nuclease-free water;
(8) Mixing 60ul of amplification mixed solution and 15ul of pre-amplification product, and uniformly mixing by a blowing pipe;
(9) Samples were amplified as follows:
，
preserving PicoPlex amplification products at-20deg.C;
the kit is used for establishing a library of single peripheral blood lymphocyte amplification products, and the MGISEQ-2000 (PE 100) sequencing mode is used for comparing the single peripheral blood lymphocyte amplification products with a reference genome GRCh37/hg19 by BWA, and the single peripheral blood lymphocyte amplification products are calculated according to the number of reads; the comparison rate of the captured data is more than 85%, and the capturing efficiency of the target area is more than 90%.
3. The single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to claim 1, wherein: the site information includes: GJB2:235delC &299_300delA, SLC26A4:919-2A > G &917insG, SLC26A4:2168A > G.
4. A single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to claim 3, characterized in that: the GJB2: the primer and sequence for one amplification of 235delC &299_300delA are as follows:
GJB2-2F-36：AGCAAACCGCCCAGAGTA；
GJB2-2R-496：CGAAGATGACCCGGAAGA。
5. a single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to claim 3, characterized in that: the GJB2: the second amplification primer and sequence of 235delC &299_300delA are as follows:
GJB2-2F-207：CTTTGTCTGCAACACCCTG；
GJB2-2R-364：CCCTTGATGAACTTCCTCT。
6. a single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to claim 3, characterized in that: the primary amplification primer and the sequence of SLC26A4:919-2A > G &917insG are as follows:
SLC26A4-7-8F-17：TTTTATAGACGCTGGTTGAG；
SLC26A4-7-8R-545：AAATGGCTTGACGTTTATCT。
7. a single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to claim 3, characterized in that: the secondary amplification primer and the sequence of SLC26A4:919-2A > G &917insG are as follows:
SLC26A4-7-8F-144'：AATCCCAGTCCCTATTCCTA；
SLC26A4-7-8R-335：GGGATGGATTTAACAATGCC。
8. the single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to claim 2, wherein: the primary amplification primer and the sequence of the SLC26A4:2168A > G are as follows:
SLC26A4-19F-33：CACTTGAACTTGGGACGC；
SLC26A4-19R-464：ACTGATGAAAAAACTGAGGCT。
9. a single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to claim 3, characterized in that: the secondary amplification primer and the sequence of the SLC26A4:2168A > G are as follows:
SLC26A4-19F-89：CTGGGCAATAGAATGAGACT；
SLC26A4-19R-464：ACTGATGAAAAAACTGAGGCT。
10. the single cell whole genome amplification-based noninvasive prenatal diagnostic kit for deafness according to any one of claims 1 to 9, wherein: the kit is applied to detection of common deafness gene mutation.
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