Primer group for high myopia gene detection, application of primer group, kit and high myopia gene detection method

Abstract

The invention provides a primer group for high myopia gene detection, application of the primer group, a kit and a high myopia gene detection method, and relates to the technical field of gene detection. Nucleic acid sequences of the primer group for high myopia gene detection are shown as SEQ ID NO.1-296, and every two primers sequentially form a forward primer and a reverse primer according to the sequence numbers of the nucleotide sequences from small to large. The primer group is high in specificity and sensitivity, 89 high-myopia genes and 166 loci can be detected through one-time PCR amplification in the same tube, the detection efficiency of the high-myopia genes is effectively improved, meanwhile, the detection cost is reduced, and convenience is provided for prediction of high-myopia susceptible people.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a primer set for high myopia gene detection, an application thereof, a kit, and a high myopia gene detection method. Background Art

[0002] High myopia (HM) refers to a symptom of refractive error with a refractive power exceeding -6.00D or an axial length ≥26mm, which is often accompanied by a series of fundus changes, including posterior scleral staphyloma, myopic maculopathy (such as fundus atrophy, choroidal neovascularization, myopic maculoschisis, etc.), peripheral retinal degeneration, etc. It may also be accompanied by other eye complications such as cataracts, glaucoma, retinal detachment, etc., and is one of the important causes of blindness.

[0003] For high myopia, especially early-onset high myopia and pathological myopia, the role of genetic factors is more obvious.

[0004] The nine non-syndromic high myopia pathogenic genes (ZNF644, LOXL3, LRPAP1, CCDC111, P4HA2, CPSF1, SLC39A5, SCO2, and ARR3) included in the OMIM database can directly cause high myopia.

[0005] Myopia, especially high myopia, has a clear familial clustering pattern, and multiple lines of evidence suggest that genetic factors play an important role in its pathogenesis. Genes associated with high myopia prevalence in the Chinese population include: ACAN, BLID / LOC399959 intergenic region, BMP2K, C1QTNF9B, CRYBA4, CTNND2, HGF, IGF1, KCNQ5, LAMA1, MET, MIPEP, MYOC, MYP10, MYP11, MYP15, SNTB1, TGFB1, UMODL1, VIPR2, and ZFHX1B.

[0006] Myopic maculopathy is one of the most common complications of high myopia. Excessive axial elongation of the eye leads to structural damage to the posterior retina, choroid, and sclera, ultimately causing irreversible damage to the macular region and is the leading cause of blindness in high myopia. Numerous studies have shown that myopic maculopathy is highly heritable, making it crucial to assess genetic susceptibility in individuals highly susceptible to high myopia. This can inform prognostic strategies for patients with high myopia or those susceptible to it, reducing the risk of blindness through early screening, control of axial length growth, and targeted treatment. Genes highly associated with myopic maculopathy include CCDC102B, LINC00461, RDH5, SLC6A20, and TSPAN10.

[0007] Axial length (AL) is the straight-line distance from the front surface (cornea) to the back surface (retina) of the eyeball. It is a core indicator for assessing eye development and the pathological mechanisms of myopia, and is particularly directly related to axial myopia (myopia caused by excessive axial length). Axial length growth leads to increased refractive power. Axial length is a trait regulated by multiple genes, and genes associated with AL regulation include APLP2, C3orf26, CD55, GJD2, LAMA2, RSPO1, TGFB2, WNT7B, ZC3H, ZC3H11B, and ZNRF3.

[0008] Refractive error is a general term for visual impairment that prevents the eye from correctly focusing light onto the retina. Myopia is one of the most common types of refractive error. Genome-wide association analysis (GWAS) found that the following 43 genes are highly correlated with refractive error: ARID2-SNAT1 fusion gene, BICC1, BMP3, BMP4, CACNA1D, CHD7, CHRNG, CNDP2, CYP26A1, DCN, DIS3L-MAP2K1 fusion gene, DLG2, DLX1, FAM150B-ACP1 fusion gene, FBN1, GRI A4, HERC2 / OCA2 intergenic region, KCNJ2, KCNMA1, LINC00340, LRRC4C, LTBP1, MYO1D, NPLOC4 / TSPAN10 intergenic region, PABPCP2, PCCA, PDE11A, PRSS56, RASGRF1, RBFOX1, RGR, RORB, SFRP1, SHISA6, SLC14A2, STAG1, THBS2, TJP2, TOX, TOX / CA8 intergenic region, ZBTB38, ZIC2, ZMAT4.

[0009] High myopia is a complex multi-gene genetic disease driven by the cumulative effects of multiple low-effect gene loci. A more comprehensive analysis incorporating common risk loci can significantly improve prediction accuracy. However, existing high myopia screening kits only detect one or a few variant sites in a small number of genes. Detecting all of the high myopia-related genes mentioned above requires multiple PCR tests, which is cumbersome.

[0010] In view of this, the present invention is proposed. Summary of the Invention

[0011] The first purpose of the present invention is to provide a primer set for high myopia gene detection, which can detect the above-mentioned 89 genes and 166 sites through a single PCR amplification to solve the above-mentioned technical problems.

[0012] The second object of the present invention is to provide the use of the above primer set in the preparation of products for high myopia gene detection.

[0013] The third object of the present invention is to provide a kit for genetic detection of high myopia.

[0014] The fourth object of the present invention is to provide a method for detecting high myopia genes for non-disease diagnosis and treatment purposes.

[0015] In order to achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides a primer set for genetic detection of high myopia, wherein the nucleic acid sequence of the primer set is shown in SEQ ID NO. 1 to 296, and each two primers constitute a forward primer and a reverse primer in sequence according to the nucleotide sequence numbering from small to large.

[0016] As a further technical solution, the high myopia genes include ZNF644, LOXL3, LRPAP1, CCDC111, P4HA2, CPSF1, SLC39A5, SCO2, ARR3, CD55, PRSS56, CHRNG, CACNA1D, BMP3, LAMA2, CHD7, TOX, ZMAT4, RORB, CYP26A1, BICC1, GRIA4, RDH5, PCCA, ZIC2, GJD2, RASGRF1, MYO1D, KCNJ2, CNDP2, LRRC4C, RBFOX1, KCNQ5, TOX / CA8 intergenic region, SFRP1, SHISA6, PABPCP2, TJP2, RGR, DLG2, ZBTB38, PDE11A, DLX1, KCNMA1, BMP4, APLP2, FAM150B-ACP1 fusion gene, L INC00340, FBN1, DIS3L-MAP2K1 fusion gene, ARID2-SNAT1 fusion gene, SLC14A2, ZC3H11B, HERC2 / OCA2 intergenic region, NPLOC4 / TSPAN10 intergenic region, DCN, LTBP1, STAG1, THBS2, BLID / LOC399959 intergenic region, RSPO1, ZC3H, C3orf26, ZNRF3, TGFB2, WNT7B, LINC00461, TSPAN10, SLC6A20, CCDC102B, HGF, TGFB1, UMODL1, LAMA1, MYP11, MYP10, MYP15, CRYBA4, ZFHX1B, SNTB1, VIPR2, ACAN, MET, MYOC, BMP2K, CTNND2, MIPEP, C1QTNF9B, and IGF1.

[0017] In a second aspect, the present invention provides the use of the above primer set in preparing a product for genetic detection of high myopia.

[0018] In a third aspect, the present invention provides a kit for genetic detection of high myopia, comprising the primer set.

[0019] As a further technical solution, the kit further includes a multiplex PCR amplification reaction premix; The primer set is contained in the multiplex PCR amplification reaction premix, and the multiplex PCR amplification reaction premix further includes a multiplex amplification enzyme and a multiplex amplification buffer.

[0020] As a further technical solution, the sample detected by the kit includes blood or saliva.

[0021] As a further technical solution, it also includes reagents for constructing a sequencing library of the amplified products, including E1 enzyme: an enzyme used for end repair and adding A tails; adapter: used for complementary pairing and ligation with the "A" tails at the ends of DNA fragments, and subsequent hybridization with oligonucleotides (P5 / P7) on the sequencer flow cell); E2 ligase: a ligase used to connect the adapter adapter to the two ends of the DNA fragment; B1 ligase buffer; PB sorting magnetic beads: sorting magnetic beads used to purify PCR products and remove small fragment impurities such as primer dimers; E3 amplification enzyme and amplification buffer premix; P5 and P7 primer premix.

[0022] In a fourth aspect, the present invention provides a method for detecting a high myopia gene for non-disease diagnosis and treatment purposes, comprising the following steps: The primer set is used to perform multiple PCR amplification on the DNA sample to be tested, and then the amplified products are sequenced to obtain the sequence of the high myopia gene.

[0023] As a further technical solution, the program of the multiplex PCR amplification is: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 30 s, annealing at 60°C for 4 min, extension at 72°C for 30 s, cycle number 35-45; extension at 72°C for 10 min.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The primer set provided by the present invention has strong specificity and high sensitivity. It can detect 89 high myopia genes and 166 loci through a single PCR amplification in the same tube, effectively improving the detection efficiency of high myopia genes while reducing detection costs, providing convenience for the prediction of people susceptible to high myopia. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The results of gel electrophoresis. DETAILED DESCRIPTION

[0027] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0028] In a first aspect, the present invention provides a primer set for genetic detection of high myopia, wherein the nucleic acid sequence of the primer set is shown in SEQ ID NO. 1 to 296, and each two primers constitute a forward primer and a reverse primer in sequence according to the nucleotide sequence numbering from small to large.

[0029] The primer set provided by the present invention can detect 89 high myopia genes and 166 loci through a single PCR amplification in the same tube. Among them, 89 high myopia genes include ZNF644, LOXL3, LRPAP1, CCDC111, P4HA2, CPSF1, SLC39A5, SCO2, ARR3, CD55, PRSS56, CHRNG, CACNA1D, BMP3, LAMA2, CHD7, TOX, ZMAT4, RORB, CYP26A1, BICC1, GRIA4, RDH5, PCCA, ZIC2, GJD2, RASGRF1, MYO1D, KCNJ2, CNDP2, LRRC4C, RBFOX1, KCNQ5, TOX / CA8 intergenic region, SFRP1, SHISA6, PABPCP2, TJP2, RGR, DLG2, ZBTB38, PDE11A, DLX1, KCNMA1, BMP4, APLP2, FAM150B-ACP1 fusion gene, LINC 00340, FBN1, DIS3L-MAP2K1 fusion gene, ARID2-SNAT1 fusion gene, SLC14A2, ZC3H11B, HERC2 / OCA2 intergenic region, NPLOC4 / TSPAN10 intergenic region, DCN, LTBP1, STAG1, THBS2, BLID / LOC399959 intergenic region, RSPO1, ZC3H, C3orf26, ZNRF3, TGFB2, WNT7B, LINC00461, TSPAN10, SLC6A20, CCDC102B, HGF, TGFB1, UMODL1, LAMA1, MYP11, MYP10, MYP15, CRYBA4, ZFHX1B, SNTB1, VIPR2, ACAN, MET, MYOC, BMP2K, CTNND2, MIPEP, C1QTNF9B, and IGF1.

[0030] The primers of the present invention and the genes or sites they detect are shown in Table 1.

[0031] Table 1

[0032] In a second aspect, the present invention provides the use of the above primer set in preparing a product for genetic detection of high myopia.

[0033] The primer set provided by the present invention can be used for amplifying high myopia genes, and thus can be used to prepare products for high myopia gene detection.

[0034] In a third aspect, the present invention provides a kit for genetic detection of high myopia, comprising the primer set.

[0035] This test kit can be used to detect genes for high myopia, predict the susceptibility of the sample to be tested to high myopia, and be used for early monitoring and regular follow-up of high-risk susceptible populations for high myopia, to carry out key prevention and control and reduce their vision damage.

[0036] In some optional embodiments, the kit further comprises a multiplex PCR amplification reaction premix; The primer set is contained in the multiplex PCR amplification reaction premix, and the multiplex PCR amplification reaction premix further includes a multiplex amplification enzyme and a multiplex amplification buffer.

[0037] In some optional embodiments, the sample detected by the kit includes blood or saliva.

[0038] In some optional embodiments, reagents for constructing a sequencing library of the amplified product are also included, including E1 enzyme: an enzyme for end repair and adding A tails; adapters: used for complementary pairing with and ligating the "A" tails at the ends of DNA fragments, and subsequent hybridization with oligonucleotides (P5 / P7) on the sequencer flow cell); E2 ligase: a ligase used to connect the adapters to the two ends of the DNA fragments; B1 ligase buffer; PB sorting magnetic beads: sorting magnetic beads used to purify PCR products and remove small fragment impurities such as primer dimers; E3 amplification enzyme and amplification buffer premix; P5 and P7 primer premix.

[0039] In a fourth aspect, the present invention provides a method for detecting a high myopia gene for non-disease diagnosis and treatment purposes, comprising the following steps: The primer set is used to perform multiple PCR amplification on the DNA sample to be tested, and then the amplified products are sequenced to obtain the sequence of the high myopia gene.

[0040] This method is simple to operate and highly efficient, and can be used for theoretical research on the relationship between these genes and high myopia.

[0041] In some optional embodiments, the program of the multiplex PCR amplification is: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 30 s, annealing at 60°C for 4 min, extension at 72°C for 30 s, cycle number 35-45; extension at 72°C for 10 min.

[0042] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0043] Example 1: Genotyping and sequencing of 89 genes and 166 loci 1. Detection method (I) Extraction of genomic DNA from 18 blood samples A total of 18 venous whole blood samples (denoted as LaneHM-1~18) were included in the test. They were collected in EDTA anticoagulant tubes with a sample volume of 3mL / tube. All samples were centrifuged at 3000 rpm for 10 minutes within 1 hour after collection to separate plasma and blood cells, and DNA was extracted from the blood cells using the phenol-chloroform method.

[0044] (II) Genotyping of 89 genes and 166 loci 1. Multiplex PCR Amplification 1.1 Preparation of amplification mixture a. Prepare 0.2ml PCR tubes in advance and label them accordingly.

[0045] b. Thaw the extracted DNA at room temperature. Vortex and mix the DNA and primers (e.g., SEQ ID NOs. 1-296) until fully mixed.

[0046] c. Melt the reagents listed in the table below on ice, and prepare the primer amplification mix on ice.

[0047]

[0048] d. Configure the PCR system according to the above configuration system.

[0049] e. After vortex mixing, centrifuge the PCR tube in a tabletop centrifuge to remove any liquid from the tube cap and sidewalls.

[0050] f. Place the amplification mixture on ice and prepare it for loading onto the instrument.

[0051] g. Computer Procedure:

[0052] 1.2 Agarose gel electrophoresis a. Prepare 1% agarose gel according to the sample volume.

[0053] b. Once the agarose gel is ready for use, perform electrophoresis on 5 μl of the first-round PCR product at 200 V for 10 minutes.

[0054] c. After the electrophoresis process is completed, observe the maker product band between 200-300bp on the gel imager ( Figure 1 ).

[0055] 1.3 PCR product purification a. After removing the PB (magnetic beads) from the refrigerator, equilibrate them at room temperature for at least 30 minutes and vortex thoroughly to mix before use.

[0056] b. Add 40.5 μL (0.9X) of PB (magnetic beads) to the first-round PCR amplification for purification. Pipette up and down 30 times to mix, and centrifuge briefly.

[0057] c. Place on a PCR plate and let stand at room temperature for 5 minutes. Place on a magnetic separation stand for 5 minutes until the supernatant is clear. Carefully discard the supernatant.

[0058] d. Keep the tube on the magnetic separation rack and add 200 μL of freshly prepared 80% ethanol. Let it stand at room temperature for 30 seconds and discard the supernatant.

[0059] e. Repeat step d and use a 10 μL pipette to remove as much residual ethanol as possible.

[0060] f. Add 35 μL of ddH2O, vortex to mix, and centrifuge briefly. Place on a PCR plate, incubate at room temperature for 5 minutes, and then place on a magnetic stand for 5 minutes.

[0061] g. Transfer 32 μL of the supernatant to a 1.5 ml centrifuge tube for later use. Then, take 2 μL of the supernatant and use the dsDNA HS Assay Kit for Qubit to determine the DNA concentration.

[0062] 2. Library Construction 2.1 DNA fragmentation / end repair / dA tail addition a. Prepare 0.2 mL PCR tubes in advance and label them accordingly.

[0063] b. Thaw the extracted DNA at room temperature and vortex to mix thoroughly.

[0064] c. Melt the reagents listed in the table below on ice and prepare Library Mix 1 on ice.

[0065]

[0066] d. Add 10 μL of E1 enzyme to a labeled 0.2 mL PCR tube.

[0067] e. Add the purified PCR product (250 ng DNA input) to a 0.2 mL PCR tube.

[0068] f. Use ddH2O to make up the 0.2 mL PCR aliquot to 60 μL.

[0069] g. Use a pipette to gently pipette up and down the contents of the 0.2 mL PCR tube 30 times, then centrifuge immediately to remove any liquid from the tube cap and sidewalls.

[0070] h. Place the library mixture 1 in a PCR thermal cycler and incubate under the following conditions:

[0071] Note: After loading library mixture 1 onto the machine, take out PB (sorting magnetic beads) from the refrigerator in advance, vortex thoroughly to mix, and equilibrate at room temperature for 30 minutes before use.

[0072] 2.2 Adding connectors a. When the module temperature drops to 4°C, remove Library Mix 1 and place it on ice. Place the reagents listed in the table below and the required adapters on ice. (Vortex the adapters and thaw B1 (ligase buffer) by inverting and mixing. Do not vortex E2 (ligase).)

[0073] b. Prepare Library Mix 2 by adding the corresponding reagents from the kit to the reaction tube containing Library Mix 1 according to the table below (perform this preparation on ice).

[0074]

[0075] Note: Because this kit uses Illumina long adapters, different libraries in the same batch need to be added with adapters with different numbers. When adding adapters to each library, add them below the liquid level to ensure that the reagents are completely added to the system.

[0076] c. Use a pipette to gently pipette Library Mix 2 30 times. Centrifuge quickly to remove the reaction mixture from the tube. Place the reaction tube in a PCR thermal cycler and incubate according to the following conditions (wait until the block cools to 4°C before removing the sample).

[0077]

[0078] Note: Prepare 80% ethanol in advance according to the sample volume (preparation volume = sample volume × 1 mL).

[0079] 2.3 Magnetic Bead Purification (Nucleic Acid Purification Reagent) a. After removing the PB (magnetic beads) from the refrigerator, equilibrate them at room temperature for at least 30 minutes and vortex thoroughly to mix before use.

[0080] b. After the ligation reaction is complete, add 60 μL of PB (sorting magnetic beads) to library mixture 2. Use a pipette to mix 30 times and centrifuge briefly.

[0081] c. Place on a PCR plate and let stand at room temperature for 5 minutes. Place on a magnetic separation stand for 5 minutes until the supernatant is clear. Carefully discard the supernatant.

[0082] d. Keep the tube on the magnetic separation rack and add 200 μL of freshly prepared 80% ethanol. Let it stand at room temperature for 30 seconds and discard the supernatant.

[0083] e. Repeat step d and use a 10 μL pipette to aspirate again to remove as much residual ethanol as possible.

[0084] f. Add 22 μL of ddH2O, vortex to mix, and centrifuge briefly. Place on a PCR plate, incubate at room temperature for 5 minutes, and then place on a magnetic stand for 5 minutes. (You can remove the PCR amplification reagents for the next step in advance and thaw them on ice. Prepare newly labeled 0.2 mL PCR tubes for later use.)

[0085] g. Take 20 μL of supernatant and perform PCR amplification.

[0086] 2.4 PCR amplification a. Prepare Library Mix 3 by adding the reagents as shown in the table below to a new 0.2 mL PCR tube (perform this preparation on ice).

[0087]

[0088] b. Mix by pipetting up and down or vortexing. Centrifuge briefly to collect the liquid.

[0089] c. Place the prepared library mix 3 in a PCR thermal cycler and amplify according to the following reaction schedule:

[0090] 2.5 Magnetic Bead Purification (Nucleic Acid Purification Reagent) a. After removing the PB (magnetic beads) from the refrigerator, equilibrate them at room temperature for at least 30 minutes and vortex thoroughly to mix before use.

[0091] b. After the amplification reaction is complete, add 45 μL of PB (magnetic beads) to the library mixture 3. Pipette up and down 30 times to mix thoroughly, and centrifuge briefly.

[0092] c. Place on a PCR plate and let stand at room temperature for 5 minutes. Place on a magnetic separation stand for 5 minutes until the supernatant is clear. Carefully discard the supernatant.

[0093] d. Keeping the tube on the magnetic separation stand, add 200 μL of freshly prepared 80% ethanol to the 0.2 mL PCR tube. Let it stand at room temperature for 30 seconds, then discard the supernatant.

[0094] e. Repeat step d and use a 10 μL pipette to remove as much residual ethanol as possible.

[0095] f. Add 22 μL of ddH2O, vortex to mix, and centrifuge briefly. Place on a PCR plate, incubate at room temperature for 5 minutes, and then place on a magnetic stand for 5 minutes.

[0096] g. Transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube for library capture. (If the next step is not yet completed, the library can be frozen at -20°C.)

[0097] 2.6 Preliminary library quality control Take 2 μL of the sample and perform DNA quantification quality control using the dsDNA HS Assay Kit for Qubit®. If the pre-library DNA concentration is >15 ng / μL, the quality control passes.

[0098] 3. Sequencing Analysis Sequencing analysis was performed using an Illumina sequencer.

[0099] 2. Test Results To verify the reliability of the test results from the primer set for high myopia gene detection provided by this invention, we used whole-exome sequencing (WES) results as a comparison to validate the genotyping results of 18 collected samples at 166 gene loci. The results showed that the genotyping results of all 18 samples at 166 comparable loci were completely consistent with the whole-exome sequencing (WES) results. The specific test results for the 18 samples are shown in Table 2.

[0100] Table 2 Example of 165 gene sequencing results of clinical samples

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for high myopia gene detection, characterized in that: The nucleic acid sequences of the primer set are shown in SEQ ID NO. 1 to 296. According to the nucleotide sequence numbering from small to large, every two primers sequentially constitute a forward primer and a reverse primer.

2. The primer set according to claim 1, characterized in that The high myopia genes include ZNF644, LOXL3, LRPAP1, CCDC111, P4HA2, CPSF1, SLC39A5, SCO2, ARR3, CD55, PRSS56, CHRNG, CACNA1D, BMP3, LAMA2, CHD7, TOX, ZMAT4, RORB, CYP26A1, BICC1, GRIA4, RDH5, PCCA, ZIC2, GJD2, RASGRF1, MYO1D, KCNJ2, CNDP2, LRRC4C, RBFOX1, KCNQ5, TOX / CA8 intergenic region, SFRP1, SHISA6, PABPCP2, TJP2, RGR, DLG2, ZBTB38, PDE11A, DLX1, KCNMA1, BMP4, APLP2, FAM150B-ACP1 fusion gene, and LINC00. 340, FBN1, DIS3L-MAP2K1 fusion gene, ARID2-SNAT1 fusion gene, SLC14A2, ZC3H11B, HERC2 / OCA2 intergenic region, NPLOC4 / TSPAN10 intergenic region, DCN, LTBP1, STAG1, THBS2, BLID / LOC399959 intergenic region, RSPO1, ZC3H, C3orf26, ZNRF3, TGFB2, WNT7B, LINC00461, TSPAN10, SLC6A20, CCDC102B, HGF, TGFB1, UMODL1, LAMA1, MYP11, MYP10, MYP15, CRYBA4, ZFHX1B, SNTB1, VIPR2, ACAN, MET, MYOC, BMP2K, CTNND2, MIPEP, C1QTNF9B, and IGF1.

3. Use of the primer set according to claim 1 in preparing a product for genetic detection of high myopia.

4. A kit for genetic detection of high myopia, characterized in that: The kit comprises the primer set according to claim 1.

5. The kit according to claim 4, characterized in that The kit also includes a multiplex PCR amplification reaction premix; The primer set is contained in the multiplex PCR amplification reaction premix, and the multiplex PCR amplification reaction premix further includes a multiplex amplification enzyme and a multiplex amplification buffer.

6. The kit according to claim 4, wherein The samples detected by the kit include blood or saliva.

7. The kit according to claim 4, characterized in that It also includes reagents for constructing a sequencing library of the amplified products, including E1 enzyme, adapter, E2 ligase, B1 ligase buffer, PB sorting magnetic beads, E3 amplification enzyme and amplification buffer premix, and P5 and P7 primer premix.

8. A method for detecting high myopia genes for non-disease diagnosis and treatment purposes, characterized in that: The steps include: The primer set according to claim 1 is used to perform multiplex PCR amplification on the DNA sample to be tested, and then the amplified products are sequenced to obtain the sequence of the high myopia gene.

9. The detection method according to claim 8, wherein The program of the multiplex PCR amplification was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 30 s, annealing at 60°C for 4 min, extension at 72°C for 30 s, cycle number 35-45; and extension at 72°C for 10 min.

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