CN116555417A - Site for detecting congenital deafness before embryo implantation, primer combination and application thereof - Google Patents
Site for detecting congenital deafness before embryo implantation, primer combination and application thereof Download PDFInfo
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- 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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Abstract
The invention relates to the field of biotechnology, in particular to a group of loci for detecting congenital deafness before embryo implantation, a primer combination and application thereof. In addition, the SNP locus related to congenital deafness can be used for preparing a diagnostic kit, a diagnostic reagent or a diagnostic product for testing single genetic disease before congenital deafness embryo implantation, and the diagnostic kit, the diagnostic reagent or the diagnostic product can improve the accuracy of diagnosis of congenital deafness before clinical embryo implantation, thereby avoiding embryo implantation carrying pathogenic genotype and blocking the occurrence of congenital deafness family inheritance.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a group of sites and primer combinations for detecting congenital deafness before embryo implantation and application thereof.
Background
Congenital deafness refers to hearing loss at birth, is a common hearing disorder, and has a morbidity of 1-3 per mill. Congenital deafness may be caused by genetic or non-genetic factors. Hereditary hearing loss is the most common one of congenital hearing loss, accounting for more than 60%. At present, various gene mutations are known to be associated with congenital deafness, such as GJB2, SLC26A4, MYO7A, CDH23 and other genes, and the mutation of the genes can cause abnormal ear structure and auditory function, thereby causing congenital deafness.
Single gene genetic disease detection (PGT-M) prior to embryo implantation is a technique that targets the detection of whether an embryo carries a pathogenic gene mutation prior to embryo implantation. This technique aims at examining the genetic information of embryos to find abnormal gene mutations that may lead to infants suffering from a certain genetic disease at birth. The technology starts to be researched and explored at the end of the 80 s of the last century, and the early pre-pregnancy diagnosis mode is widely accepted at present. Traditional PGT-M assays are typically performed using capillary electrophoresis-based detection of nucleic acid short tandem repeats (short tandom repeats, STR) or PCR-based first generation sequencing techniques, which have the disadvantages of long detection time, low throughput, complex operation, general inability to achieve simultaneous detection and analysis of pathogenic variations and polymorphic sites, and relatively high risk of misdiagnosis due to allele-tripping (ADO). Detection techniques for nucleotide polymorphism (SNP) sites can solve some of the above problems. In recent years, SNP chip technology is applied to the field of gene detection more, but in the auxiliary reproduction field, the method has high cost and long detection time, and has poor direct detection effect on pathogenic variation, so that the method is limited to be widely applied to single-gene genetic disease detection before embryo implantation.
The second generation sequencing technology (NGS) has the advantages of rapidness, accuracy and low cost, and can detect the chromosome number, structure, single-gene diseases and other information of the embryo by carrying out second generation sequencing on the embryo blastula stage cells, thereby improving the success rate of embryo implantation and healthy birth rate and reducing the risks of abortion and genetic diseases.
Allele tripping (ADO) refers to the dominant amplification of one of the two alleles of a heterozygote with about 5% -20% occurrence of complete amplification failure when single cell PCR amplification is performed. This is also one of the main reasons for misdiagnosis. For autosomal recessive genetic disease, the occurrence of ADO may affect pregnancy rate by misidentifying heterozygous embryos as diseased embryos; however, in the case of autosomal dominant genetic disease, the occurrence of ADO may lead to misidentification of the diseased embryo as a normal embryo, ultimately leading to misdiagnosis.
Disclosure of Invention
To overcome the shortcomings of the prior art, a first object of the present invention is to provide a set of sites for detecting congenital deafness before embryo implantation, which can be applied to the preparation of reagents for detecting and diagnosing congenital deafness before embryo implantation.
A second object of the present invention is to provide the use of the above-mentioned sites for preparing a diagnostic reagent or kit for detecting congenital deafness before embryo implantation.
It is a third object of the present invention to provide a primer composition for detecting a site for detecting congenital deafness before embryo implantation.
A fourth object of the present invention is to provide a kit comprising the above primer composition.
It is a fifth object of the present invention to provide a specific primer combination for the site detection for detecting congenital deafness before embryo implantation.
A sixth object of the present invention is to provide a kit comprising the above specific primer combination.
A seventh object of the present invention is to provide an application of the above kit in preparing a diagnostic product for detecting congenital deafness before embryo implantation.
In order to achieve the first object of the invention, the present invention adopts the following technical scheme:
the invention provides a group of sites for detecting congenital deafness before embryo implantation, which comprises 74 sites carried by chromosome 13, wherein the sites comprise a GJB2 mutation site, 35 SNP sites linked with the GJB2 mutation site are arranged at the upstream of the GJB2 mutation site, and 38 SNP sites linked with the GJB2 mutation site are arranged at the downstream of the GJB2 mutation site;
the 74 sites are as follows:
the analysis of the plurality of SNP effective loci can be used for detecting GJB2 mutation (NM_ 004004.6 (GJB2): c.109G > A) before embryo implantation, and can further construct haplotypes better.
In order to achieve the second object of the invention, the present invention adopts the following technical scheme:
the invention provides application of the site in preparing a diagnostic reagent or a kit for detecting congenital deafness before embryo implantation.
In order to achieve the third object of the present invention, the present invention adopts the following technical scheme:
the invention provides a primer composition for detecting a site for detecting congenital deafness before embryo implantation, which is characterized in that the information of each primer in the primer composition is as follows:
in order to achieve the fourth object of the present invention, the present invention adopts the following technical scheme:
the invention provides a kit for detecting congenital deafness before embryo implantation, which contains the primer composition.
In order to achieve the fifth object of the present invention, the present invention adopts the following technical scheme:
the invention provides a specific primer combination for detecting congenital deafness before embryo implantation, which comprises 35 pairs of upstream primers, 1 pair of specific primers containing pathogenic mutation sites and 38 pairs of downstream primers;
the sequence information of the specific primer combination is as follows:
among these specific primers are the following: (1) sequence unique on the target chromosome; (2) The Tm values of the primers are similar, and the primers have no complementary or partially complementary sequences with each other, so that a dimer or hairpin structure can be avoided; (3) The upstream and downstream targeted amplification interval contains at least one SNP site selected.
In order to achieve the sixth object of the present invention, the present invention adopts the following technical scheme:
the invention provides a kit for detecting congenital deafness before embryo implantation, which contains the specific primer combination.
In order to achieve the seventh object of the present invention, the present invention adopts the following technical scheme:
the invention provides application of the kit in preparing a diagnostic product for detecting congenital deafness before embryo implantation.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention detects a group of SNP loci genetically linked with congenital deafness pathogenic loci (NM_ 004004.6 (GJB 2): c.109G > A) and proves that genotype information of the congenital deafness related pathogenic loci of the embryo can be detected by detecting genotype information of the group of SNP loci, so that the group of SNP loci can be used as biomarkers for detecting congenital deafness before clinical embryo implantation. In addition, the SNP locus related to congenital deafness and the related primer combination can be used for preparing a diagnostic kit, a diagnostic reagent or a diagnostic product for testing single genetic disease before congenital deafness embryo implantation, and the diagnostic kit, the diagnostic reagent or the diagnostic product can improve the accuracy of detecting congenital deafness diagnosis before clinical embryo implantation, thereby avoiding embryo implantation carrying pathogenic genotype and blocking the occurrence of congenital deafness family inheritance.
(2) The SNP locus for detecting congenital deafness linkage before embryo implantation is based on a second-generation high-throughput sequencing technology, can detect the locus of congenital deafness before embryo implantation, has the advantages of universality, high throughput, low cost, high sensitivity and strong specificity, and can rapidly and accurately detect the congenital deafness before embryo implantation by sequencing multi-locus SNP and preventing detection misdiagnosis caused by amplified allele tripping in the practical application process.
(3) The SNP locus for detecting congenital deafness linkage before embryo implantation is a linkage region from a GJB2 gene mutation locus and 1Mb upstream and downstream of the GJB2 gene mutation locus, so that a haplotype for detecting congenital deafness mutation is constructed according to genotypes of the detected SNP loci, and the SNP locus has the advantages of strong specificity and high linkage.
(4) The primer composition for detecting the locus for detecting congenital deafness before embryo implantation has relatively good universality because 74 SNP are adopted for analysis, and the primer composition can be used for diagnosis before embryo transfer of different families. In addition, because a plurality of SNP loci are detected simultaneously, the method can be used for detecting the congenital deafness of a plurality of families combined embryo transfer, and can also be used for fetal detection and abortive tissue detection.
(5) The specific primer combination for detecting the locus for detecting congenital deafness before embryo implantation comprises 35 pairs of upstream primers, 1 pair of specific primers containing pathogenic mutation loci and 38 pairs of downstream primers, wherein the specific primer combination can amplify a targeted deletion region and a linkage locus simultaneously, a targeted locus result and a linkage locus haplotype result are mutually verified, the accuracy is higher, and the cost is saved. That is, because the specific primer combination is used for detecting instead of a single site, misdiagnosis caused by allele tripping (ADO) is reduced, the accuracy and the reliability of detection can be effectively improved, and the cost and the time for detecting congenital deafness can be greatly reduced.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this application, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
To aid in a better understanding of the invention, the following concepts are explained:
SNP chip (SNP chip) technology refers to technology in which high-density DNA fragments are attached to a solid phase surface such as a film, a glass plate or the like in a certain sequence or arrangement manner by a high-speed robot or an in-situ synthesis manner through a Microarray technology, and a large number of SNP locus genotypes are detected by using DNA probes labeled with isotopes or fluorescence and by means of a base complementary hybridization principle.
Haplotypes (Haplotype) are genotypes of a set of closely related alleles on a chromosome, usually inherited as a unit. Haplotypes may reflect the combination and distribution of Single Nucleotide Polymorphisms (SNPs) on a chromosome.
The sequencing depth refers to the ratio of the total number of bases sequenced to the size of the genome to be tested, and can be understood as the average number of times each base in the genome is sequenced. The higher the sequencing depth, the higher the quality and reliability of the sequencing data, but also increases the cost and data volume of sequencing. For example, in one embodiment, a sequencing depth of 1000x represents that the strip of specific PCR amplification product is sequenced 1000 times.
The effective site refers to a SNP site or an STR site of both man and woman, wherein one of the SNP site and the STR site is homozygous and the other is heterozygous, and the site is called as the effective site.
The following description is made with reference to specific embodiments.
Example 1
A group of sites for detecting congenital deafness before embryo implantation, comprising 74 sites carried by chromosome 13, wherein the sites comprise a GJB2 mutation site, 35 SNP sites linked with the GJB2 mutation site are arranged at the upstream of the GJB2 mutation site, and 38 SNP sites linked with the GJB2 mutation site are arranged at the downstream of the GJB2 mutation site;
the 74 sites are as follows:
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the analysis of the plurality of SNP effective loci can be used for detecting GJB2 mutation (NM_ 004004.6 (GJB2): c.109G > A) before embryo implantation, and can further construct haplotypes better.
Wherein, a group of the invention is used for detecting the locus of congenital deafness before embryo implantation, and the corresponding coordinates are as follows:
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example 2
The use of the set of sites for detecting congenital deafness before embryo implantation of example 1 for the preparation of a diagnostic reagent or kit for congenital deafness before embryo implantation.
Designing a primer composition for detecting the locus for detecting congenital deafness before embryo implantation, wherein the information of each primer in the designed primer composition is as follows:
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wherein, a kit for detecting the locus of congenital deafness before embryo implantation is designed, and the kit contains the primer composition.
In addition, a specific primer combination for detection is designed for the group of sites for detecting congenital deafness before embryo implantation, wherein the specific primer combination comprises 35 pairs of upstream primers, 1 pair of specific primers containing pathogenic mutation sites and 38 pairs of downstream primers; the sequence information of this specific primer combination is as follows:
/>
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wherein, a kit for detecting the locus of congenital deafness before embryo implantation is designed, and the kit contains the specific primer combination.
In addition, the kit is applied to preparing a diagnostic product for detecting congenital deafness before embryo implantation.
Experiment:
1. single cell whole genome amplification
Based on a group of loci for detecting congenital deafness before embryo implantation, the single-cell whole genome amplification technology is adopted to increase the detection DNA initial quantity, the pathogenic locus genotype and the pathogenic locus linked SNP locus genotype are detected by the targeted multiplex PCR capture sequencing technology, and a haplotype method related to the pathogenic locus is constructed by adopting the haplotype linkage analysis to detect whether the embryo carries pathogenic mutation.
Among them, single-cell whole genome amplification (SCWGA) technology used for pretreatment of a sample is a genome amplification technology capable of amplifying enough genomic DNA from a Single cell to allow comprehensive analysis of the genome of the Single cell. The specific operation of the single-cell whole genome amplification is that random primers with reverse sequence tails are adopted to amplify single-cell DNA for 6 rounds to generate a semi-amplified product. The two ends of the semi-amplified products are provided with reverse complementary sequences, the reverse complementary sequences are mutually combined to form a circular ring, and the circular ring cannot be used as a template for the next round of PCR amplification, so that excessive copying of DNA is avoided, exponential amplification after DNA replication is prevented to a great extent, and the uniformity of whole genome amplification is improved. In addition, the pre-amplified product is exponentially amplified by a pair of primers, so that the amplified product yield is increased. Wherein, the pair of primers used for carrying out exponential amplification on the pre-amplified product is matched with a commercial SCWGA kit.
The specific method comprises the following steps:
the targeted capturing method comprises the following steps: multiplex PCR capture sequencing is a target region capture sequencing method based on multiplex PCR technology. The multiplex PCR technology is a technology for simultaneously amplifying a plurality of targets by one PCR reaction, and detecting an amplification product by combining a certain detection means to thereby realize diagnosis of the plurality of targets. Multiplex PCR capture sequencing is an integrated solution for rapid targeted linear amplification of a target region of interest using pre-designed primers, i.e., the specific primer combination for site design detection for pre-embryo implantation detection of congenital deafness, followed by mass sample parallel detection and depth analysis using a high throughput sequencing platform. Has the advantages of high efficiency, high flux and low cost, and is suitable for determining the overspeed detection mode of the gene hotspot at clinical level. The invention designs the specific primer combination for detecting the locus of congenital deafness before embryo implantation, and 74 pairs of primer combination are SNP loci aiming at the congenital deafness of Chinese, and can avoid the occurrence of allele tripping.
High throughput sequencing method: the designed multiplex primer Panel is used to match with a specific bar code and a DNA library, so as to amplify the target sequence rapidly and efficiently; quality inspection before loading is carried out on the successfully constructed library, so that the quality and reliability of the library are ensured; homogenizing the library passing the quality inspection, and then preparing and enriching templates to obtain a sufficient template quantity; and amplifying the template, and performing a sequencing experiment on a high-throughput sequencing platform such as Ion Torrent by using the positive template.
The biological information analysis method comprises the following steps: filtering the second generation sequencing result to obtain a filtered sequencing result, wherein the filtering conditions comprise the proportion of N contained in the sequence and the proportion of low-quality base numbers, wherein N is the base which is obtained by sequencing and cannot be judged, and filtering a sequence with the proportion of N being more than 5% in the sequence and a sequence with the proportion of mass value being less than 20 being more than 40% in the sequence. Comparing the filtered sequencing result with a reference human genome sequence to obtain a sequencing data set subjected to comparison; the software for comparison adopts BWA-MEM; the reference human genome is GRCh38, the genotype of the polymorphic site of the target region is determined, the effective site is screened, and finally the haplotype is analyzed through the family relation among samples.
2. The criteria for screening SNP effective loci are:
(1) The loci are located in the 1Mb region on the upstream and downstream of the GJB2 mutation region and in the specific region in the three mutation regions, and the SNP loci are uniformly distributed on the gene on the upstream and downstream, and one SNP locus is evenly distributed every 4-5 Kb.
(2) SNP loci are recorded in a thousand genome or dbSNP database, loci with higher allele frequency are selected, and SNP with MAF larger than 0.2.
(3) The sequences near the SNP site have no homology in the human genome.
(4) Because the MAF value difference of the same SNP locus among different people is larger, a thousand-person genome MAF database and a self-built 15 ten thousand Chinese crowd-based SNP database are combined when the SNP locus selection is carried out, so that the method is more suitable for Chinese crowd.
Experimental cases:
experimental example 1: determination of haplotype by upstream and downstream SNP loci
1. Method of operation
(1) Sample acquisition: selecting parent parties and a diseased child generation (a forensic person) and adopting a whole blood sample; extracting blood genome DNA, measuring DNA concentration by using Qubit 4.0, and taking a certain amount as a DNA sample to be measured. Then 6 cases of embryo to be detected are selected, the embryo develops to the blastula stage, 3-5 external trophoblast cells are taken out by adopting a conventional embryo biopsy technology, and the trophoblast cells of the blastula cultured in vitro enrich genomic DNA in the cells through whole genome amplification, and the whole genome amplification adopts Fapon Single Cell genome-Ampli Kit (NK 023).
(2) The obtained sample DNA was subjected to a Qubit 4.0 concentration measurement. In this experimental example, the concentration of the DNA sample was measured using a dsDNA HS Assay Kit kit, and the DNA sample was detected according to the procedure described in the kit.
(3) Amplification of the target fragment: mixing the DNA with a multiplex PCR primer and a PCR amplification reagent, and obtaining a target gene DNA fragment through multiplex PCR reaction;
wherein, the reaction system is as follows:
wherein, the reaction conditions are as follows: pre-denaturation at 99 ℃ for 2min,1 cycle; denaturation at 99℃for 15s, annealing and extension at 60℃for 4min,22 cycles; terminating at 10 ℃.
(4) Primer elimination: adding the DNA fragment obtained in the step (3) into FuPa Reagent primer elimination Reagent of 2 mu L Life Technologines company, mixing and reacting to degrade and remove primer sequences in the PCR amplified fragment, so as to obtain the primer-removed DNA fragment.
(5) Library construction: mixing the DNA fragment obtained in the step (4) after the primer removal with a P1 joint, a specific joint and a connecting reaction reagent for connection reaction to obtain a DNA fragment added with the joint;
wherein, the reaction system is as follows:
| reagent(s) | Volume (mu L) |
| The product of step (4) | 22 |
| Connection buffer solution | 4 |
| P1 joint | 4 |
| Specific linkers | 4 |
| DNA ligase | 2 |
| Total volume of | 30 |
When the reaction is carried out, the reaction system is vibrated and mixed uniformly, and then the mixture is centrifuged and then put into a PCR instrument, and the reaction conditions are as follows: heating to 68deg.C for 5min at 22deg.C for 30min, heating to 72deg.C for 5min, and cooling to 10deg.C.
(6) Purifying magnetic beads: and (3) performing magnetic bead purification on the spliced DNA fragment, and removing redundant small fragment splices to obtain a purified DNA fragment.
(7) Library detection: detecting the library concentration of the purified DNA fragments obtained in the step (6) by adopting a fluorescence quantitative PCR method.
(8) High throughput sequencing: and (3) carrying out high-throughput sequencing on the library obtained in the step (7) by adopting an Ion Torrent PGM sequencing platform.
(9) Bioinformatics analysis: capturing and sequencing a WGA product (DNA) obtained by amplifying the whole genome of an embryo biopsy cell and a family sample, performing bioinformatics SNP analysis to obtain linkage haplotype information of couples and pathogenic genes, judging the genotype of a pathogenic site related to congenital deafness according to the sequencing information of the embryo sample, and classifying and diagnosing. The original data is filtered by using fastp-0.21.0 software, and compared to a human GRCh38 reference genome by using BWA-MEM software, low-quality sequences are removed, the sequencing depth of target sites is counted, sites with the depth less than 100 are removed, and then the genotype of SNP sites in the target region is determined. And screening effective loci to construct haplotypes by utilizing genotypes of SNP loci of parent and embryo target regions. And then judging whether the embryo carries mutation or not by using the established haplotype.
2. Judgment result
The 18 effective sites of the target region were selected to construct haplotypes, and the results are shown in Table 1.
TABLE 1 upstream and downstream SNP locus determination monomer type result data sheet
Upstream and downstream SNP locus judgment monomer type result data sheet (upper sheet)
Wherein, the column of chrB of female genotype, the column of chrD of male genotype, the column of chrB and the column of chrD of forensic genotype, the column of chrD of embryo 1, the column of chrB and the column of chrD of embryo 2, the column of chrA of embryo 3, the column of chrB and the column of chrD of embryo 4, the column of chrB and the column of chrD of embryo 5 are all staining monomers carrying pathogenic deletion genes. Wherein chrA is an normal chromosome monomer, chrB is a risk dyeing monomer carrying a parent source, chrC is an normal chromosome monomer, and chrD is a risk dyeing monomer carrying a parent source.
The judgment result shows that: the male dyeing monomers are divided into chrC and chrD, wherein chrD is a dyeing monomer carrying pathogenic mutation, and chrC is an normal chromosome monomer; female dyeing monomers are divided into chrA and chrB, wherein chrA is an normal chromosome monomer, and chrB carries pathogenic mutation dyeing monomers; the chromosome monomers of the first-mentioned individuals are chrysB and chrysD, respectively, and are homozygous mutation. Embryo 1 is heterozygous, embryo 2 is homozygous, embryo 3 is heterozygous, embryo 4 is homozygous, embryo 5 is homozygous, and embryo 6 is null.
Under the condition that the embryo pathogenic site is subjected to allele tripping (ADO) due to SCWGA, the pathogenic site can be typed only through a SNP site genotype detection structure screened at the upstream and downstream of the pathogenic site, and the congenital deafness GJB2 pathogenic site is not needed. And combining the screened SNP loci with other SNP loci related to the targeted amplification interval to perform haplotype analysis, increasing the number of effective loci, helping haplotype typing and helping congenital deafness detection to obtain better judgment.
In addition, the invention carries out linkage analysis according to the upstream and downstream polymorphic Sites (SNP) of the pathogenic gene, and when carrying out SNP linkage analysis, in order to avoid the tripping problem affecting the diagnosis result, more polymorphic sites are selected, even if few sites are tripped, the last diagnosis result is not affected, thereby improving the accuracy of genetic diagnosis and reducing the misdiagnosis rate.
Experimental example 2: panel capture performance index detection
The experimental example uses the same 6 cases of genome-wide amplification products of trophoblast cells at embryo blastocyst stage, and DNA samples of peripheral blood cells of parents and precursors as in experimental example 1, uses the primer composition (74 pairs of SNP primers) designed by the invention, performs library construction according to the Fapon Single Cell genome-Ampli Kit (NK 023) standard library construction flow, and performs sequencing on Ion Torrent PGM to obtain the DNA fragment sequence of the target region with the fragment length distributed in 100-150 bp. And performing quality control and analysis of conventional parameters on the obtained sequencing off-machine data.
Detection result:
the samples in the experimental example 1 are tested, so that the full coverage of the target interval can be obtained, the target full coverage is realized, the lowest sequencing depth is 100x,mapping rate-99%, the ontarget rate is 95% or more, and the uniformity of 100x is 95% or more. Specific information is shown in table 2 below.
Table 2 sample test information table
In table 2, "Mapping rate" represents the comparison rate at the time of resequencing; "Ontarget rate" represents the mid-target rate, representing the ratio of sequencing data aligned to the targeted amplicon; ">0.2x Average Depth Rate" represents a proportion of sites greater than 0.2x average sequencing depth, the higher the value, the better the amplification uniformity; ">30 xLepth Rate" represents coverage of target sites, the higher this value, the higher the target site coverage.
The foregoing description of the preferred embodiments of the present application is not intended to be limiting, but is intended to cover any and all modifications, equivalents, and alternatives falling within the spirit and principles of the present application.
Claims (7)
1. A set of sites for detecting congenital deafness before embryo implantation, comprising 74 sites carried by chromosome 13, wherein the sites comprise a GJB2 mutation site, 35 SNP sites linked with the GJB2 mutation site are arranged at the upstream of the GJB2 mutation site, and 38 SNP sites linked with the GJB2 mutation site are arranged at the downstream of the GJB2 mutation site;
the 74 sites are as follows:
2. use of the locus according to claim 1 for the preparation of a diagnostic reagent or kit for the detection of congenital deafness prior to embryo implantation.
3. The set of primer compositions for detecting a site for congenital deafness before embryo implantation according to claim 1, wherein the information of each primer in said primer composition is as follows:
4. a kit for detecting a site of congenital deafness before embryo implantation, comprising the primer composition according to claim 3.
5. The set of specific primer combinations for site detection for pre-embryo implantation detection of congenital deafness according to claim 1, wherein said specific primer combinations comprise 35 pairs of upstream primers, 1 pair of specific primers containing pathogenic mutation sites, 38 pairs of downstream primers;
the sequence information of the specific primer combination is as follows:
6. a kit for detecting a site of congenital deafness prior to embryo implantation, comprising a specific primer combination according to claim 5.
7. Use of the kit according to any one of claims 4 or 6 for the preparation of a diagnostic product for detecting congenital deafness prior to embryo implantation.
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| KR20140000439A (en) * | 2012-06-22 | 2014-01-03 | 주식회사 진인 | Oligonucleotide for detection of mutations in genes associated with korean nonsyndromic hearing loss, dna chip, diagnositic kit and method for detection of the mutations using the oligonucleotides |
| CN108220403A (en) * | 2017-12-26 | 2018-06-29 | 北京科迅生物技术有限公司 | Detection method, detection device, storage medium and the processor in specific mutation site |
| CN109055532A (en) * | 2018-09-12 | 2018-12-21 | 苏州贝康医疗器械有限公司 | Hereditary hearing impairment genetic test Primer composition, kit and application before embryo implantation |
| CN111705122A (en) * | 2020-05-19 | 2020-09-25 | 江苏先声医学诊断有限公司 | Genetic deafness screening method based on MassArray nucleic acid mass spectrometry platform and application thereof |
| CN115678976A (en) * | 2022-10-31 | 2023-02-03 | 广州凯普医药科技有限公司 | Kit for simultaneously detecting 26 deafness susceptibility gene mutation sites based on time-of-flight mass spectrometry and application thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140000439A (en) * | 2012-06-22 | 2014-01-03 | 주식회사 진인 | Oligonucleotide for detection of mutations in genes associated with korean nonsyndromic hearing loss, dna chip, diagnositic kit and method for detection of the mutations using the oligonucleotides |
| CN108220403A (en) * | 2017-12-26 | 2018-06-29 | 北京科迅生物技术有限公司 | Detection method, detection device, storage medium and the processor in specific mutation site |
| CN109055532A (en) * | 2018-09-12 | 2018-12-21 | 苏州贝康医疗器械有限公司 | Hereditary hearing impairment genetic test Primer composition, kit and application before embryo implantation |
| CN111705122A (en) * | 2020-05-19 | 2020-09-25 | 江苏先声医学诊断有限公司 | Genetic deafness screening method based on MassArray nucleic acid mass spectrometry platform and application thereof |
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