High-risk myopia risk prediction model, method for constructing the same and application thereof

Abstract

The application provides a high-risk myopia risk prediction model and a construction method and application thereof, and relates to the technical field of molecular diagnosis.The construction method of the high-risk myopia risk prediction model is simple and convenient, the sensitivity and specificity of the model constructed are high, and the model can accurately realize prediction of high-risk myopia risk of a to-be-tested sample.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostic technology, and in particular to a high-risk myopia risk prediction model, its construction method, and its application. Background Technology

[0002] Myopia is the most common refractive error. Depending on the refractive power, it is usually divided into low to moderate myopia (spherical refractive power less than -6.00 D) and high myopia (spherical refractive power equal to or greater than -6.00 D).

[0003] High-risk myopia is currently considered a multifactorial or single-gene inherited disease, with inheritance patterns including autosomal dominant, autosomal recessive, and X-linked recessive inheritance. Currently, 26 myopia genetic loci have been approved by the International Committee on Human Genome Nomenclature and mapped in the journal *Human Mendelian Inheritance*. Most of these loci were located in high myopia families or twin cohorts through linkage analysis and sequencing studies using microsatellite markers. Using exome sequencing and automated analysis, 15 SNP loci from 15 genes have been found to be highly associated with high myopia. These are: rs11200647 of the HTRA1 gene, rs9318086 of the MIPEP gene, rs1003483 of the IGF2 gene, rs4459940 of the SOX2-OT gene, rs4697489 of the CCDC149 gene, rs56995061 of the CHRM1 gene, rs11210537 of the HIVEP3 gene, rs4778879 of the RASGRF1 gene, rs7744813 of the KCNQ5 gene, rs782555528 of the CPSF1 gene, rs6214 of the IGF1 gene, rs3743123 of the GJD2 gene, rs6469937 of the SNTB1 gene, rs587777625 of the SLC39A5 gene, and rs1550094 of the PRSS56 gene.

[0004] Gene chips are a type of biochip. By immobilizing designed probes on a special carrier, they enable the detection of gene mutation sites through various methods such as hybridization, primer extension, and ligation. Compared to qPCR, this method offers advantages in speed and efficiency, and allows for parallel analysis of polymorphic information. However, conventional gene chip technology involves PCR products hybridizing in an open environment, which can easily cause aerosol contamination, affecting the accuracy of detection and limiting its application in clinical diagnostics.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for constructing a high-risk myopia prediction model to solve the aforementioned technical problems.

[0007] The second objective of this invention is to provide a high-risk myopia prediction model.

[0008] A third objective of this invention is to provide an application of the above-mentioned high-risk myopia risk prediction model in the preparation of a high-risk myopia risk prediction device.

[0009] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a method for constructing a high-risk myopia prediction model, comprising the following steps: a. Obtain nucleotide information and average daily outdoor time for high-risk myopia susceptibility gene SNP sites in myopic and emmetropic populations; the high-risk myopia susceptibility gene SNP sites include: rs11200647 of the HTRA1 gene, rs9318086 of the MIPEP gene, rs1003483 of the IGF2 gene, rs4459940 of the SOX2-OT gene, rs4697489 of the CCDC149 gene, and rs56995061 of the CHRM1 gene. The rs11210537 site of the HIVEP3 gene, the rs4778879 site of the RASGRF1 gene, the rs7744813 site of the KCNQ5 gene, the rs782555528 site of the CPSF1 gene, the rs6214 site of the IGF1 gene, the rs3743123 site of the GJD2 gene, the rs6469937 site of the SNTB1 gene, the rs587777625 site of the SLC39A5 gene, and the rs1550094 site of the PRSS56 gene; b. Calculate the weight W of the risk mutation at each high-risk myopia susceptibility gene SNP site on the impact of myopia using the following formula: W = ln((Number of samples with risky alleles of target SNP sites in the myopia group / Number of samples without risky alleles of target SNP sites in the myopia group) / (Number of samples with risky alleles of target SNP sites in the emmetropic group / Number of samples without risky alleles of target SNP sites in the emmetropic group)); c. Calculate the weight W of the average daily outdoor time on the impact of myopia using the following formula: W = ln((Number of samples in the myopia group with an average daily outdoor time of less than 2 hours / Number of samples in the myopia group with an average daily outdoor time of more than 2 hours) / (Number of samples in the emmetropic group with an average daily outdoor time of less than 2 hours / Number of samples in the emmetropic group with an average daily outdoor time of more than 2 hours)); d. The high-risk myopia prediction model is constructed as follows: P=W t t+W HTRA1 HTRA1+W MIPEP MIPEP+W IGF2 IGF2+W SOX2-OT SOX2-OT+W CCDC149 CCDC149+W CHRM1 CHRM1+W HIVEP3 HIVEP3+W RASGRF1 RASGRF1+W KCNQ5 KCNQ5+W CPSF1 CPSF1+W IGF1 IGF1+W GJD2 GJD2+W SNTB1 SNTB1+W SLC39A5 SLC39A5+W PRSS56 PRSS56; Among them, W t The weight W for the impact of average daily outdoor time on myopia is given; when outdoor activity time is less than 2 hours, t is assigned a value of 1, and when outdoor activity time is greater than 2 hours, t is assigned a value of 0; W HTRA1 W MIPEP W IGF2 W SOX2OT W CCDC149 W CHRM1 W HIVEP3 W RASGRF1 W KCNQ5 W CPSF1 W IGF1 W GJD2 W SNTB1 W SLC39A5 and W PRSS56 The values ​​W represent the weights of risk mutations at each high-risk myopia susceptibility gene SNP site on the impact of myopia. If a risk allele exists at a high-risk myopia susceptibility gene SNP site, the gene is assigned a value of 1; if no risk allele exists at a high-risk myopia susceptibility gene SNP site, the gene is assigned a value of 0. e. Set a threshold for P, and predict whether the sample to be tested has a high risk of myopia based on the P value.

[0010] As a further technical solution, the high-risk myopia risk is defined as myopia progression ≥1.00D / year.

[0011] As a further technical solution, gene chips are used to obtain nucleotide information of high-risk myopia susceptibility gene SNP sites in myopic and emmetropic groups; The gene chip is closed, comprising an inner ring region and an outer ring region with concentric circle structures; The inner ring area includes an inlet and an amplification chamber; The amplification chamber is pre-filled with amplification lyophilization reagents; The amplification lyophilized reagent contains primers for detecting high-risk myopia susceptibility gene SNP sites and optional internal reference primers; The outer ring area includes a color development incubation chamber and a waste liquid recovery chamber; The colorimetric incubation chamber is pre-filled with a nucleic acid probe array membrane and a lyophilized reagent for ligation reaction; The nucleic acid probe array membrane and the lyophilized ligation reaction reagent each contain an upstream or downstream probe for detecting high-risk myopia susceptibility gene SNP sites. Along the direction of liquid flow, the sample inlet, amplification chamber, color development incubation chamber and waste liquid recovery chamber are connected in sequence via microfluidic channels; the connected amplification chamber, color development incubation chamber and waste liquid recovery chamber correspond to a high-risk myopia susceptibility gene SNP locus; When the gene chip is facing upwards, in the vertical position, the sample inlet is not lower than the sample inlet of the amplification chamber, the sample inlet of the amplification chamber is lower than the sample outlet of the amplification chamber, the sample outlet of the amplification chamber is not lower than the sample inlet of the color development incubation chamber, the sample inlet of the color development incubation chamber is higher than the sample outlet of the color development incubation chamber, and the sample outlet of the color development incubation chamber is not lower than the sample inlet of the waste liquid recovery chamber.

[0012] As a further technical solution, the inner ring area also includes a reaction solution transfer chamber, and the amplification chamber and the reaction solution transfer chamber are equipped with one-way valves; The sample inlet, amplification chamber, reaction solution transfer chamber, color development incubation chamber, and waste liquid recovery chamber are sequentially connected via a microfluidic channel.

[0013] As a further technical solution, the amplification primer sequences for the high-risk myopia susceptibility genes are shown in SEQ ID NO. 1~60, respectively; The upstream and downstream probe sequences of the high-risk myopia susceptibility gene are shown in SEQ ID NO. 65~94, respectively; The internal reference gene is the β-actin gene; The amplification primer sequences for the internal reference gene are shown in SEQ ID NO. 61~64; The upstream and downstream probe sequences of the internal reference gene are shown in SEQ ID NO.95~96, respectively.

[0014] The upstream probe is labeled with an amino group at its 5' end, and the downstream probe is labeled with biotin at its 3' end.

[0015] As a further technical solution, the amplification lyophilization reagent also includes a lyophilization protectant, an isothermal amplification enzyme, dNTPs, betaine, buffer ion pairs, magnesium ions, and potassium ions; The probe immobilization carrier in the nucleic acid probe array membrane includes aldehyde-modified nylon membrane, glass slide, silicon wafer, cellulose acetate, polypropylene membrane or nitrocellulose membrane; The lyophilization reagent for the ligation reaction also includes a lyophilization protectant, streptavidin-modified spherical gold nanoparticles, Taq DNA Ligase, and NAD. + dNTPs, buffer ion pairs, magnesium ions, and potassium ions.

[0016] Secondly, the present invention provides a high-risk myopia prediction model, which is constructed using the above-mentioned construction method.

[0017] As a further technical solution, the high-risk myopia prediction model is: P=1.415 t+1.37 HTRA1+1.74 MIPEP+1.22 IGF2+ 1.579 SOX2-OT+1.82 CCDC149+1.34 CHRM1+1.64 HIVEP3+1.87 RASGRF1+1.68 KCNQ5+1.92 CPSF1+1.41 IGF1+1.32 GJD2+1.55 SNTB1+1.36 SLC39A5+1.28 PRSS56.

[0018] As a further technical solution, the threshold of P is set to 12.249. If the sample to be tested has P>12.249, then the sample has a high risk of myopia.

[0019] Thirdly, the present invention provides the application of the above-mentioned high-risk myopia risk prediction model in the preparation of a high-risk myopia risk prediction device.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The method for constructing a high-risk myopia prediction model provided by this invention is simple and convenient. The constructed model has high sensitivity and specificity, and can accurately predict the high-risk myopia risk of the test sample. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the mutation detection principle of the present invention; Figure 2 This is a schematic diagram of the structure of a gene chip for high-risk myopia screening provided in an embodiment of the present invention; Figure 3 A schematic diagram of the internal pathway of a gene chip for high-risk myopia screening provided in an embodiment of the present invention; Figure 4 This is an ROC curve of the P-value of high-risk myopia in the training set samples of this invention.

[0023] Icons: 1-Inlet; 2-Amplification chamber; 2a-Isothermal amplification reaction chamber; 2b-Reaction solution transfer chamber; 2c-One-way valve; 3-Colorimetric incubation chamber; 3a-Built-in nucleic acid probe array membrane structure; 3b-Incubation and colorimetric reaction chamber; 4-Waste liquid recovery chamber. Detailed Implementation

[0024] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0025] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In a first aspect, the present invention provides a method for constructing a high-risk myopia prediction model, comprising the following steps: a. Obtain nucleotide information and average daily outdoor time for high-risk myopia susceptibility gene SNP sites in myopic and emmetropic populations; the high-risk myopia susceptibility gene SNP sites include: rs11200647 of the HTRA1 gene, rs9318086 of the MIPEP gene, rs1003483 of the IGF2 gene, rs4459940 of the SOX2-OT gene, rs4697489 of the CCDC149 gene, rs56995061 of the CHRM1 gene, and rs1 of the HIVEP3 gene. The following are the SNP loci for high-risk myopia susceptibility genes: rs4778879 (1210537), rs4778879 (RASGRF1), rs7744813 (KCNQ5), rs782555528 (CPSF1), rs6214 (IGF1), rs3743123 (GJD2), rs6469937 (SNTB1), rs587777625 (SLC39A5), and rs1550094 (PRSS56). Specific information on these SNP loci is shown in Table 1. Table 1. Detection loci of high-risk myopia susceptibility genes

[0028] b. Calculate the weight W of the risk mutation at each high-risk myopia susceptibility gene SNP site on the impact of myopia using the following formula: W = ln((Number of samples with risky alleles of target SNP sites in the myopia group / Number of samples without risky alleles of target SNP sites in the myopia group) / (Number of samples with risky alleles of target SNP sites in the emmetropic group / Number of samples without risky alleles of target SNP sites in the emmetropic group)); c. Calculate the weight W of the average daily outdoor time on the impact of myopia using the following formula: W = ln((Number of samples in the myopia group with an average daily outdoor time of less than 2 hours / Number of samples in the myopia group with an average daily outdoor time of more than 2 hours) / (Number of samples in the emmetropic group with an average daily outdoor time of less than 2 hours / Number of samples in the emmetropic group with an average daily outdoor time of more than 2 hours)); d. The high-risk myopia prediction model is constructed as follows: P=W t t+W HTRA1 HTRA1+W MIPEP MIPEP+W IGF2 IGF2+W SOX2-OT SOX2-OT+W CCDC149 CCDC149+W CHRM1 CHRM1+W HIVEP3 HIVEP3+W RASGRF1 RASGRF1+W KCNQ5 KCNQ5+W CPSF1 CPSF1+W IGF1 IGF1+W GJD2 GJD2+W SNTB1 SNTB1+W SLC39A5 SLC39A5+W PRSS56 PRSS56; Among them, W t The weight W for the impact of average daily outdoor time on myopia is given; when outdoor activity time is less than 2 hours, t is assigned a value of 1, and when outdoor activity time is greater than 2 hours, t is assigned a value of 0; W HTRA1 W MIPEP W IGF2 W SOX2OT W CCDC149 W CHRM1W HIVEP3 W RASGRF1 W KCNQ5 W CPSF1 W IGF1 W GJD2 W SNTB1 W SLC39A5 and W PRSS56 The values ​​W represent the weights of risk mutations at each high-risk myopia susceptibility gene SNP site on the impact of myopia. If a risk allele exists at a high-risk myopia susceptibility gene SNP site, the gene is assigned a value of 1; if no risk allele exists at a high-risk myopia susceptibility gene SNP site, the gene is assigned a value of 0. e. Set a threshold for P, and predict whether the sample to be tested has a high risk of myopia based on the P value.

[0029] The method for constructing a high-risk myopia prediction model provided by this invention is simple and convenient. The constructed model has high sensitivity and specificity, and can accurately predict the high-risk myopia risk of the test sample.

[0030] It should be noted that this invention does not impose specific limitations on the sample size for myopic and emmetropic groups. Those skilled in the art will understand that a larger sample size generally leads to higher accuracy in model prediction. The threshold value of P in this invention can be selected based on the prediction accuracy, preferably the threshold value with the highest accuracy.

[0031] In some alternative implementations, the myopia group includes a low-myopia group and a high-myopia group.

[0032] In some alternative implementations, the high risk of myopia is defined as myopia progression ≥1.00D / year.

[0033] In some alternative implementations, the average daily outdoor time can be, for example, the average daily outdoor time of the sample over the past few years.

[0034] For example, within three years (such as the three years of junior high school), based on the actual situation, the average daily cumulative outdoor activity time of the target group or personnel is statistically analyzed. Specifically, from Monday to Friday, the average daily outdoor time is about 3 hours, and on Saturday and Sunday, the average daily outdoor time is about 4 hours, with a comprehensive average of 3.3 hours of outdoor time per day.

[0035] In some alternative implementations, gene chips are used to obtain nucleotide information of high-risk myopia susceptibility gene SNP sites in myopic and emmetropic populations; The gene chip is closed, comprising an inner ring region and an outer ring region with concentric circle structures; The inner ring area includes an inlet and an amplification chamber; The outer ring area includes a color development incubation chamber and a waste liquid recovery chamber; Along the direction of liquid flow, the sample inlet, amplification chamber, color development incubation chamber, and waste liquid recovery chamber are connected sequentially via microfluidic channels; the connected amplification chamber, color development incubation chamber, and waste liquid recovery chamber correspond to a high-risk myopia susceptibility gene. When the gene chip is facing upwards, in the vertical position, the sample inlet is not lower than the sample inlet of the amplification chamber, the sample inlet of the amplification chamber is lower than the sample outlet of the amplification chamber, the sample outlet of the amplification chamber is not lower than the sample inlet of the color development incubation chamber, the sample inlet of the color development incubation chamber is higher than the sample outlet of the color development incubation chamber, and the sample outlet of the color development incubation chamber is not lower than the sample inlet of the waste liquid recovery chamber.

[0036] The sample inlet can be single, connecting to multiple amplification chambers simultaneously for sample injection; alternatively, there can be multiple inlet ports, such as one inlet port per amplification chamber, or one inlet port for two or more amplification chambers. Preferably, one inlet port corresponds to multiple amplification chambers, reducing sample error and simplifying operation. When selecting one inlet port for multiple amplification chambers, it is preferable to install an injection equalization valve between the inlet port and the functional chambers to ensure consistency in the addition of the sample to be tested.

[0037] By defining the vertical position of the sample inlet and sample outlet of each functional chamber, the reaction solution in the gene chip can enter the sample inlet through each sample outlet under the action of gravity. At the same time, the reaction solution flows into only one functional chamber during a single centrifugation, ensuring uniform reaction under the same reaction conditions. This also ensures that the gene chip provided by this invention can be used by multiple inversion centrifugations.

[0038] The gene chip for high-risk myopia screening provided by this invention has concentric inner and outer ring regions. This structure facilitates centrifugation, and by positioning each functional compartment in a specific vertical position, centrifugation allows the sample and reaction reagents to be transferred to each compartment. Isothermal amplification and colorimetric reactions are then completed within the gene chip, eliminating the need for additional detection equipment. The colorimetric results displayed in the colorimetric incubation chamber enable rapid, simple, and accurate screening for high-risk myopia-related genes. Furthermore, the inner and outer ring regions can be configured with multiple interconnected functional compartments in equal numbers according to detection needs, offering high throughput. Moreover, the gene chip provided by this invention is pre-loaded with amplification lyophilized reagents, nucleic acid probe array membranes, and ligation reaction lyophilized reagents, allowing for room temperature storage and closed-loop operation, thus avoiding sample cross-contamination and aerosol contamination of amplification products. Additionally, based on the one-to-one correspondence between each functional compartment and high-risk myopia susceptibility genes, the inner and outer ring regions can be configured with multiple interconnected functional compartments in equal numbers according to detection needs, further enhancing throughput.

[0039] It should be noted that the color development incubation chamber is transparent to facilitate observation of the color development results. For color development determination, if color develops in the incubation chamber, the corresponding gene to be tested is considered positive; otherwise, it is considered negative.

[0040] To further reduce reaction solution backflow, a reaction solution transfer chamber is preferably included in the inner ring region. This transfer chamber features a redundant design to prevent backflow, effectively preventing reaction solution backflow caused by incorrect chip flipping. One-way valves are installed inside the amplification chamber and the reaction solution transfer chamber. The sample inlet, amplification chamber, reaction solution transfer chamber, color development incubation chamber, and waste liquid recovery chamber are sequentially connected via microfluidic channels.

[0041] Optionally, to guide waste liquid recovery, the waste liquid recovery chamber preferably has a water absorption function, such as a highly absorbent resin layer. Through capillary siphon and centrifugal force, it can adsorb waste liquid (aqueous solution, and nucleic acid and protein substances containing amino groups) from the color development incubation chamber. This absorbent layer has a large water absorption capacity and good water retention, which can effectively prevent the backflow of waste liquid (aqueous solution, and nucleic acid and protein substances containing amino groups) and thus affect the subsequent elution and color development reaction.

[0042] The gene chip provided by this invention can perform amplification reactions including isothermal amplification reactions, such as, but not limited to, any one of loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), rolling circle amplification (RCA), and helicase-dependent amplification (HAD).

[0043] In the gene chip provided by the present invention, the amplification chamber is pre-filled with amplification lyophilization reagent; The colorimetric incubation chamber is pre-filled with a nucleic acid probe array membrane and a lyophilized reagent for ligation reaction; The gene chip contains pre-installed amplification lyophilization reagents, nucleic acid probe array membranes, and ligation reaction lyophilization reagents. It is stored at room temperature and operated in a closed system to avoid cross-contamination of samples and aerosol contamination of amplification products. For ease of preparation, the amplification lyophilization reagents and ligation reaction lyophilization reagents are preferably in the form of lyophilized beads.

[0044] The amplification lyophilized reagent contains primers for detecting high-risk myopia susceptibility genes and optional internal reference primers. It should be noted that the amplification lyophilized reagent may or may not contain internal reference primers. To further ensure the accuracy of the test results, it is preferred to include internal reference primers.

[0045] The nucleic acid probe array membrane and the lyophilized ligation reaction reagent each contain upstream or downstream probes for detecting high-risk myopia susceptibility genes; for example, the nucleic acid probe array membrane may contain the upstream probe and the lyophilized ligation reaction reagent may contain the downstream probe, or vice versa. A schematic diagram of the detection principle is shown below. Figure 1 As shown, the upstream ligation probe consists of a 5' amino-terminal modification group, a poly-T sequence, and a complementary region to the amplification product. The first base at the 3' end of the upstream ligation probe is designed at the target SNP site. The upstream ligation probe is fixed by reacting with the aldehyde group on the substrate through the 5' amino-terminal modification group under certain conditions. When the target SNP site is present in the amplification product, the upstream and downstream ligation probes hybridize with the amplification product and form a stable structure under the action of DNA ligase, resulting in a clear color signal during the washing and color development stage. When the target SNP site is absent in the amplification product, the first base at the 3' end of the upstream ligation probe mismatches with the amplification product, and the DNA ligase cannot connect the upstream and downstream probes. Consequently, the biotin-modified downstream probe cannot form a stable structure with the amplification product and is removed during the washing stage, resulting in no clear color signal during the color development stage.

[0046] In this invention, the internal reference genes include: glyceraldehyde-3-phosphate dehydrogenase gene, β-actin gene, microtubule protein gene or 18S rRNA gene, preferably β-actin gene; The upstream probe is labeled with an amino group at its 5' end, and the downstream probe is labeled with biotin at its 3' end.

[0047] In some preferred embodiments, the amplification primer sequences for high-risk myopia susceptibility genes are shown in SEQ ID NO. 1~60, and the upstream and downstream probe sequences are shown in SEQ ID NO. 65~94, respectively.

[0048] The internal reference gene is the β-actin gene; its amplification primer sequences are shown in SEQ ID NO. 61~64, and the upstream and downstream probe sequences are shown in SEQ ID NO. 95~96, respectively.

[0049] In some preferred embodiments, the amplification lyophilization reagent further includes a lyophilization protectant, an isothermal amplification enzyme, dNTPs, betaine, buffer ion pairs, magnesium ions, and potassium ions.

[0050] In some preferred embodiments, the probe immobilization carrier in the nucleic acid probe array membrane includes an aldehyde-modified nylon membrane, a glass slide, a silicon wafer, cellulose acetate, a polypropylene membrane, or a cellulose nitrate membrane.

[0051] In some preferred embodiments, the lyophilization reagent for the ligation reaction further includes a lyophilization protectant, streptavidin-modified spherical gold nanoparticles, Taq DNA Ligase, and NAD. + dNTPs, buffer ion pairs, magnesium ions, and potassium ions.

[0052] By combining the gene chip structure and specific pre-set reagents of this invention, the application of the gene chip provided by this invention to screen for high-risk myopia genes has the characteristics of high accuracy, high throughput, pollution prevention, simple operation and result visualization.

[0053] In addition, the present invention provides a method for using the above-mentioned gene chip, the method comprising: adding the nucleic acid sample to be tested into the gene chip through the injection port, placing the gene chip in a centrifugation system, and transferring the sample to be tested and the reaction reagent through a microfluidic channel by at least two inverted centrifugations, and developing color in a colorimetric incubation chamber to achieve high-risk myopia gene screening.

[0054] The method of using the gene chip provided by the present invention can be completed by simple centrifugation and flipping after sample addition. It is simple and convenient to operate and does not require specific technical personnel or expensive equipment, which can effectively save costs.

[0055] To ensure good centrifugation results, the gene chip is embedded in a suitable centrifugation system for centrifugation, enabling unidirectional closed transfer of the reaction solution, incubation solution, and elution solution. The suitable centrifugation system, such as the Super MiniDancer (Sangon Biotech), requires removing the centrifuge rotor, aligning the chip's pre-drilled center hole with the centrifuge shaft, and fixing the chip onto the centrifuge shaft. Centrifugation at 1000 rpm for 5-10 seconds is sufficient.

[0056] The phrase "at least two inverted centrifugations" refers to the following: First, the sample is transferred from the inlet to the amplification chamber via centrifugation. Then, after inversion, a second centrifugation transfers the reaction solution from the amplification chamber to the colorimetric incubation chamber. Thus, the target gene in the sample can be detected through these two inverted centrifugations. A third inverted centrifugation can be performed after color development in the colorimetric incubation chamber, allowing for waste liquid recovery. It is important to note that during the first centrifugation, the gene chip is face up; during the second centrifugation, the reverse side is face up; and during the third centrifugation, the gene chip is face up. The "face up" refers to the side visible in the colorimetric incubation chamber where color development occurs.

[0057] The nucleic acid samples to be tested are purified nucleic acid samples, and the extraction methods include magnetic bead extraction, column extraction, and solution extraction. After extraction, the samples can be transferred to the respective functional compartments through the injection port and microfluidic channel using a pipette.

[0058] Secondly, the present invention provides a high-risk myopia prediction model, which is constructed using the above-mentioned construction method.

[0059] The model has high sensitivity and specificity, and can accurately predict the high risk of myopia in the test samples.

[0060] In some optional implementations, the high-risk myopia prediction model is: P=1.415 t+1.37 HTRA1+1.74 MIPEP+1.22 IGF2+ 1.579 SOX2-OT+1.82 CCDC149+1.34 CHRM1+1.64 HIVEP3+1.87 RASGRF1+1.68 KCNQ5+1.92 CPSF1+1.41 IGF1+1.32 GJD2+1.55 SNTB1+1.36 SLC39A5+1.28 PRSS56.

[0061] In some alternative implementations, the threshold for P is set to 12.249. If the sample to be tested has P > 12.249, then the sample has a high risk of myopia.

[0062] Thirdly, the present invention provides the application of the above-mentioned high-risk myopia risk prediction model in the preparation of a high-risk myopia risk prediction device.

[0063] The model provided by this invention can be used to prepare a high-risk myopia risk prediction device, and then used to predict the risk of high-risk myopia.

[0064] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0065] Example 1 This embodiment provides a gene chip for high-risk myopia screening, such as... Figure 2 As shown, the gene chip includes an upper cover and a lower cover that can be interlocked. When the upper and lower covers are interlocked, they form a closed structure. The upper and lower covers form an inner ring area and an outer ring area with concentric circles. The inner ring area includes an inlet 1 and an amplification chamber 2; The amplification chamber 2 is pre-loaded with amplification lyophilized bulbs; The amplified lyophilized pellets contain primers for detecting high-risk myopia susceptibility genes and optional internal reference primers; The outer ring area includes a color development incubation chamber 3 and a waste liquid recovery chamber 4; The colorimetric incubation chamber 3 is pre-loaded with a nucleic acid probe array membrane and a lyophilized reaction ball; The nucleic acid probe array membrane and the lyophilized reagent for the ligation reaction contain upstream or downstream probes for detecting high-risk myopia susceptibility genes, respectively. The sample inlet 1, amplification chamber 2, color development incubation chamber 3, and waste liquid recovery chamber 4 are connected in sequence via a microfluidic channel; the connected amplification chamber 2, color development incubation chamber 3, and waste liquid recovery chamber 4 correspond to a high-risk myopia susceptibility gene.

[0066] The internal pathways of the gene chip used for high-risk myopia screening are as follows: Figure 3 As shown, 1 is the sample inlet; 2a is the isothermal amplification reaction chamber, containing a built-in isothermal amplification reaction lyophilized bulb; 2b is the reaction solution transfer chamber; 2c is a one-way valve; 3a is a transparent membrane structure containing a built-in nucleic acid probe array; 3b is the incubation and colorimetric reaction chamber, containing a connected lyophilized reagent; and 4 is a waste liquid recovery chamber with water absorption function. The sample inlet 1 is connected to the isothermal amplification reaction chamber 2a, the reaction solution transfer chamber 2b is connected to the nucleic acid probe array membrane 3a, and the incubation and colorimetric reaction chamber 3b is connected to the waste liquid recovery chamber 4 via microfluidic channels.

[0067] When using the gene chip for high-risk myopia screening provided by this invention for high-risk myopia gene screening, firstly, the extracted nucleic acid sample to be tested is added to the gene chip through the injection port 1 using a pipette. Then, the gene chip is placed face up in a centrifuge system for the first centrifugation. After centrifugation, the sample to be tested is transferred from the injection port 1 to the isothermal amplification reaction chamber 2a through a microfluidic channel and mixed and reacted with the pre-placed amplification lyophilized bulbs in the isothermal amplification reaction chamber 2a. After the reaction, the gene chip is turned face down, and the reaction solution enters the reaction solution transfer chamber 2b under the action of gravity. Then, a second centrifugation is performed, and the reaction solution in the reaction solution transfer chamber 2b is transferred to the array membrane structure 3a through the microfluidic channel. After reacting with the nucleic acid probes built into the array membrane structure, it reacts with the connected reaction lyophilized bulbs in the incubation colorimetric reaction chamber 3b and develops color. At this time, the colorimetric reaction can be observed through the transparent 3a structure to determine whether there are high-risk myopia genes in the sample to be tested. Then, the gene chip is centrifuged for the third time with the front side facing up, so that the reaction liquid enters the waste liquid recovery chamber 4 through the microfluidic channel to realize waste liquid recovery.

[0068] Example 2 This embodiment provides the amplification system, ligation system, and color development system of the gene chip used for high-risk myopia screening in Example 1, specifically involving amplification primers, ligation probes, isothermal amplification buffer, and ligation system buffer.

[0069] 1. Primer and probe design Using an online primer and probe design website, amplification primers for 15 high-risk myopia detection targets (Table 1) and human internal reference genes and upstream and downstream ligation probes for the target genes were designed. The specific amplification primers are shown in Table 2, and the upstream and downstream ligation probes are shown in Table 3.

[0070] Table 2 Primers for detecting high-risk myopia susceptibility genes and internal reference genes

[0071] Table 3. Upstream and downstream linkage probes for high-risk myopia susceptibility genes and internal reference genes.

[0072] 2. Preparation of isothermal amplification lyophilized beads and ligation system lyophilized beads The main components of the amplification lyophilized pellets were trehalose, strand displacement DNA polymerase, dNTPs, betaine, buffer ion pairs, magnesium ions, potassium ions, primers for detecting high-risk myopia susceptibility genes, and human internal control primers (Table 5). The main components of the ligation reaction lyophilized pellets were sucrose, trehalose, Taq DNA Ligase, and NAD+. +The optimized system was prepared using lyophilized microspheres (dNTPs, buffer ion pairs, magnesium ions, potassium ions, and a 3' biotin-labeled downstream linker probe) (Table 4). Lyophilized microspheres were fabricated from the optimized system for subsequent pre-placement in gene chips.

[0073] Table 4. Main components and final concentrations of each component in the lyophilized pellets for the bonding reaction.

[0074] Table 5. Main components and final concentrations of each component in the amplified lyophilized bulbs.

[0075] 3. Preparation of nucleic acid probe array membranes The amino-modified upstream probe was diluted to 25 μM using 150 mM phosphate-buffered saline (PBS; pH: 7.8). Using a micropipette, the probe solution was spotted onto the predetermined sites on a commercially available aldehyde-modified nylon membrane (Suzhou Betty Biotechnology Co., Ltd.), and incubated at room temperature for 5 min. The membrane was then incubated at 37°C for 10 h for fixation. The fixed membrane was then transferred to 0.5% sodium borohydride solution and incubated at 25°C for 1 h for blocking. The membrane was then washed three times alternately with 0.1% SDS solution and ultrapure water. Next, it was incubated in 30 mM freshly prepared sodium borohydride solution at 25°C for 30 min. Finally, the membrane was washed 3–5 times with ultrapure water to remove all chemical reagents. The washed membrane was then air-dried at room temperature for later use.

[0076] Example 3 This embodiment provides a gene chip for rapid screening of high-risk myopia, which includes a specific method for using an implanted amplification reaction microsphere, a connecting reaction microsphere and a gene chip membrane.

[0077] 1. Isothermal amplification Select a magnetic bead extraction kit, such as the Magbead Blood DNA Kit (Jiangsu Kangwei Century), to extract and purify the DNA from the test sample (sample type: blood sample). Dilute the test sample DNA and positive sample DNA separately with DNA diluent (10 mM Tris-HCl, pH 8.0) to the range of 0.05 ng / μL~2 ng / μL, constructing 100 μL of test sample DNA and positive sample DNA. Using a pipette, separately aspirate the test sample DNA and positive sample DNA and inject them into the sample distribution valve through the sample inlet 1 of the two gene chips. Then, centrifuge at 1000 r / min for 5 s using an adapter centrifuge to complete the transfer of template solution to amplification chamber 2. Mix the pre-prepared lyophilized microspheres containing Bst DNA polymerase, amplification primers, and magnesium ions with the template solution and react at 62℃ for 35 min.

[0078] Select a magnetic bead extraction kit, such as the Magbead Blood DNA Kit (Jiangsu Kangwei Century), to extract and purify human blood samples. Then, dilute the extracted and purified DNA to a concentration of 0.05 ng / μL~2 ng / μL using DNA dilution buffer (10 mM Tris-HCl, pH 8.0) to construct a 100 μL DNA sample. Using a pipette, aspirate the DNA sample and inject it into the sample aliquot valve through the injection port. Then, centrifuge the sample at 1000 r / min for 5 s using an appropriate centrifuge to transfer the DNA sample to the isothermal amplification reaction chamber. Mix the pre-prepared lyophilized microspheres containing Bst DNA polymerase, amplification primers, and magnesium ions with the template solution and incubate at 62 degrees Celsius for 35 min.

[0079] 2. Ligation and Hybridization The chip was flipped and centrifuged at 1000 r / min for 5 s using an adapter centrifuge to transfer the isothermal amplification reaction product to the colorimetric incubation chamber 3. The product was then incubated at 50℃ for 30 min to complete the hybridization and ligation reaction. The chip was then flipped again and centrifuged at 1000 r / min for 30 s using the adapter centrifuge to remove the waste liquid from the colorimetric incubation chamber, transferring it to the waste liquid recovery chamber 4.

[0080] 3. Washing and color development Using elution buffer (1×TBS, 0.1% SDS, 0.05% Tween 20), the excess residual free fragments were washed off using the above-described gene chip multiple-turn centrifugation method, with three washes. Then, streptavidin-modified spherical gold nanoparticles were added, and the mixture was subjected to multiple-turn centrifugation for colorimetric qualitative analysis. The results showed that for positive samples, clear and distinct red spots were observed in the chromogenic area.

[0081] Example 4 This embodiment provides a high-risk myopia risk early warning model based on a gene chip for rapid screening of high-risk myopia.

[0082] Clinical sample collection: This embodiment strictly adheres to the Declaration of Helsinki of the World Medical Association. The blood clinical samples required for this clinical trial were obtained from Beijing Zhide Medical Laboratory, covering hospitals in Beijing, Wuxi, and other regions, and were approved by the ethics committees of the hospitals in the aforementioned regions.

[0083] This clinical trial collected a training set of 352 cases, covering emmetropia, low myopia, and high myopia, aged 6-18 years. Specific sample details are shown in Table 6. This clinical trial conducted dynamic monitoring for 3 years on high-risk myopia individuals identified in the low myopia and emmetropia samples of the training set, validating the high-risk myopia risk model through dynamic risk warning. In addition, 105 new low myopia and emmetropia samples were included in the validation set.

[0084] Table 6. Myopia status and age group of effective samples in the training set

[0085] The patients who provided the above samples agreed to participate in the questionnaires related to this study and to donate blood, and agreed to complete the "Informed Consent Form". The subjects were not related to each other by blood.

[0086] Referring to Example 3, sample testing was conducted. The number of samples with and without the target risk mutation allele in the myopia and emmetropia groups were counted respectively. The weight of the risk mutation at each test gene locus on the impact of myopia was determined using the formula W=ln((number of samples with the target risk mutation in the myopia group / number of samples without the target risk mutation in the myopia group) / (number of samples with the target risk mutation in the emmetropia group / number of samples without the target risk mutation in the emmetropia group)), as shown in Table 7. Furthermore, the average daily outdoor time of myopia patients and controls in the training set was statistically analyzed. Using 2 hours of daily outdoor activity time as a dividing point, the number of myopia and emmetropia samples with daily outdoor activity time greater than 2 hours and less than 2 hours were counted respectively. The weight of the outdoor activity time factor is determined to be 1.415 according to the formula W=ln((number of samples with an average daily outdoor time of less than 2 hours in the myopia group / number of samples with an average daily outdoor time of more than 2 hours in the myopia group) / (number of samples with an average daily outdoor time of less than 2 hours in the emmetropic group / number of samples with an average daily outdoor time of more than 2 hours in the emmetropic group)). The number of samples with myopia and emmetropic vision corresponding to daily outdoor activity time in the training set is shown in Table 8.

[0087] Table 7. Risk allele weights of target gene loci in the training set samples.

[0088] Table 8. Daily outdoor activity time in the training set corresponding to myopia and emmetropia samples.

[0089] Note: W=log e ((Number of samples with target risk mutation alleles in the myopia group / Number of samples without target risk mutation alleles in the myopia group) / (Number of samples with target risk mutation alleles in the emmetropic group / Number of samples without target risk mutation alleles in the emmetropic group)).

[0090] Based on the risk alleles of 15 SNP loci identified in the training set and the weights of daily outdoor activity time on the impact of myopia, a predictive model for high-risk myopia was constructed, with P=1.415. t+1.37 HTRA1+1.74 MIPEP+1.22 IGF2+ 1.579 SOX2-OT+1.82 CCDC149+1.34 CHRM1+1.64 HIVEP3+1.87 RASGRF1+1.68 KCNQ5+1.92 CPSF1+1.41 IGF1+1.32 GJD2+1.55 SNTB1+1.36 SLC39A5+1.28 PRSS56. Specifically, when the outdoor activity time is less than 2 hours, t is assigned a value of 1; when the outdoor activity time is greater than 2 hours, t is assigned a value of 0. If a risk allele exists at the target gene locus, the corresponding gene is assigned a value of 1; if no risk allele exists at the target gene locus, the corresponding gene is assigned a value of 0.

[0091] Based on the aforementioned risk prediction model, the detection results of the training set samples were statistically analyzed to determine the P-value for each sample. Then, using the clinical diagnosis of myopia as the standard, an ROC curve for the risk P-value was constructed. The sample included 96 cases of high myopia and 256 cases of emmetropia and low myopia. The results showed an AUC value of 0.9921, a detection sensitivity of 94.95%, and a specificity of 98.05%. The myopia risk P-value was 5.06, meaning that when P > 5.06, the sample was considered to have a myopia risk.

[0092] A ROC curve for the P-value of high-risk myopia was constructed, with a value of 1 assigned to those whose myopia progressed ≥1.00D / year within the past year, and a value of 0 assigned to those with emmetropia or slow myopia progression. Figure 4 Of these, 47 cases showed rapid progression, and 305 cases showed emmetropia or slow progression. The results showed an AUC value of 0.9815, a detection sensitivity of 95.74%, and a specificity of 93.77%. The P-value for high-risk myopia was 12.249, meaning that when P > 12.249, the sample was considered to have a high risk of myopia.

[0093] This clinical trial included 47 individuals with low myopia and emmetropia in the training set who were identified as high-risk for myopia. An additional 105 individuals with low myopia and emmetropia served as a control group, with p-values ​​less than 12.249 for all cases. Dynamic monitoring was conducted on these samples for three years to validate the high-risk myopia risk model. Statistically, within three years, among the 47 samples, 40 showed a progression greater than 1.00D / year in any given year, and 7 showed a progression less than 1.00D / year in any given year. In the control group, 11 samples showed a progression greater than 1.00D / year in any given year, and 94 showed a progression less than 1.00D / year in any given year. The accuracy of the early warning was 85.11%, and the specificity was 89.52%.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 method for constructing a high-risk myopia prediction model, characterized in that, Includes the following steps: a. Obtain nucleotide information of high-risk myopia susceptibility gene SNP sites and average daily outdoor time in myopic and emmetropic populations; The high-risk myopia susceptibility gene SNP loci include: rs11200647 of the HTRA1 gene, rs9318086 of the MIPEP gene, rs1003483 of the IGF2 gene, rs4459940 of the SOX2-OT gene, rs4697489 of the CCDC149 gene, rs56995061 of the CHRM1 gene, and rs11210537 of the HIVEP3 gene. The rs4778879 site of the RASGRF1 gene, the rs7744813 site of the KCNQ5 gene, the rs782555528 site of the CPSF1 gene, the rs6214 site of the IGF1 gene, the rs3743123 site of the GJD2 gene, the rs6469937 site of the SNTB1 gene, the rs587777625 site of the SLC39A5 gene, and the rs1550094 site of the PRSS56 gene; b. Calculate the weight W of the risk mutation at each high-risk myopia susceptibility gene SNP site on the impact of myopia using the following formula: W = ln((Number of samples with risky alleles of target SNP sites in the myopia group / Number of samples without risky alleles of target SNP sites in the myopia group) / (Number of samples with risky alleles of target SNP sites in the emmetropic group / Number of samples without risky alleles of target SNP sites in the emmetropic group)); c. Calculate the weight W of the average daily outdoor time on the impact of myopia using the following formula: W = ln((Number of samples in the myopia group with an average daily outdoor time of less than 2 hours / Number of samples in the myopia group with an average daily outdoor time of more than 2 hours) / (Number of samples in the emmetropic group with an average daily outdoor time of less than 2 hours / Number of samples in the emmetropic group with an average daily outdoor time of more than 2 hours)); d. The high-risk myopia prediction model is constructed as follows: P=W t t+W HTRA1 HTRA1+W MIPEP MIPEP+W IGF2 IGF2+W SOX2-OT SOX2-OT+W CCDC149 CCDC149+W CHRM1 CHRM1+W HIVEP3 HIVEP3+W RASGRF1 RASGRF1+W KCNQ5 KCNQ5+W CPSF1 CPSF1+W IGF1 IGF1+W GJD2 GJD2+W SNTB1 SNTB1+W SLC39A5 SLC39A5+W PRSS56 PRSS56; Among them, W t The weight W for the impact of average daily outdoor time on myopia is given; when outdoor activity time is less than 2 hours, t is assigned a value of 1, and when outdoor activity time is greater than 2 hours, t is assigned a value of 0; W HTRA1 W MIPEP W IGF2 W SOX2OT W CCDC149 W CHRM1 W HIVEP3 W RASGRF1 W KCNQ5 W CPSF1 W IGF1 W GJD2 W SNTB1 W SLC39A5 and W PRSS56 The values ​​W represent the weights of risk mutations at each high-risk myopia susceptibility gene SNP site on the impact of myopia. If a risk allele exists at a high-risk myopia susceptibility gene SNP site, the gene is assigned a value of 1; if no risk allele exists at a high-risk myopia susceptibility gene SNP site, the gene is assigned a value of 0. e. Set a threshold for P, and predict whether the sample to be tested has a high risk of myopia based on the P value.

2. The construction method according to claim 1, characterized in that, The high-risk myopia is defined as myopia progression ≥1.00D / year.

3. The construction method according to claim 1, characterized in that, Nucleotide information of high-risk myopia susceptibility gene SNP sites in myopic and emmetropic populations was obtained using gene chips. The gene chip is closed, comprising an inner ring region and an outer ring region with concentric circle structures; The inner ring area includes an inlet and an amplification chamber; The amplification chamber is pre-filled with amplification lyophilization reagents; The amplification lyophilized reagent contains primers for detecting high-risk myopia susceptibility gene SNP sites and optional internal reference primers; The outer ring area includes a color development incubation chamber and a waste liquid recovery chamber; The colorimetric incubation chamber is pre-filled with a nucleic acid probe array membrane and a lyophilized reagent for ligation reaction; The nucleic acid probe array membrane and the lyophilized ligation reaction reagent each contain an upstream or downstream probe for detecting high-risk myopia susceptibility gene SNP sites. Along the direction of liquid flow, the sample inlet, amplification chamber, color development incubation chamber and waste liquid recovery chamber are connected in sequence via microfluidic channels; the connected amplification chamber, color development incubation chamber and waste liquid recovery chamber correspond to a high-risk myopia susceptibility gene SNP locus; When the gene chip is facing upwards, in the vertical position, the sample inlet is not lower than the sample inlet of the amplification chamber, the sample inlet of the amplification chamber is lower than the sample outlet of the amplification chamber, the sample outlet of the amplification chamber is not lower than the sample inlet of the color development incubation chamber, the sample inlet of the color development incubation chamber is higher than the sample outlet of the color development incubation chamber, and the sample outlet of the color development incubation chamber is not lower than the sample inlet of the waste liquid recovery chamber.

4. The construction method according to claim 3, characterized in that, The inner ring area also includes a reaction solution transfer chamber, and the amplification chamber and the reaction solution transfer chamber are equipped with one-way valves; The sample inlet, amplification chamber, reaction solution transfer chamber, color development incubation chamber, and waste liquid recovery chamber are sequentially connected via a microfluidic channel.

5. The construction method according to claim 3, characterized in that, The amplification primer sequences for the high-risk myopia susceptibility genes are shown in SEQ ID NO.1~60, respectively; The upstream and downstream probe sequences of the high-risk myopia susceptibility gene are shown in SEQ ID NO. 65~94, respectively; The internal reference gene is the β-actin gene; The amplification primer sequences for the internal reference gene are shown in SEQ ID NO. 61~64; The upstream and downstream probe sequences of the internal reference gene are shown in SEQ ID NO. 95~96, respectively; The upstream probe is labeled with an amino group at its 5' end, and the downstream probe is labeled with biotin at its 3' end.

6. The construction method according to claim 3, characterized in that, The amplification lyophilization reagent also includes a lyophilization protectant, an isothermal amplification enzyme, dNTPs, betaine, buffer ion pairs, magnesium ions, and potassium ions. The probe immobilization carrier in the nucleic acid probe array membrane includes aldehyde-modified nylon membrane, glass slide, silicon wafer, cellulose acetate, polypropylene membrane or nitrocellulose membrane; The lyophilization reagent for the ligation reaction also includes a lyophilization protectant, streptavidin-modified spherical gold nanoparticles, Taq DNA Ligase, and NAD. + dNTPs, buffer ion pairs, magnesium ions, and potassium ions.

7. A high-risk myopia risk prediction model, characterized in that, It is constructed using the construction method described in any one of claims 1 to 6.

8. The high-risk myopia prediction model according to claim 7, characterized in that, The high-risk myopia prediction model is: P=1.415 t+1.37 HTRA1+1.74 MIPEP+1.22 IGF2+ 1.579 SOX2-OT+1.82 CCDC149+1.34 CHRM1+1.64 HIVEP3+1.87 RASGRF1+1.68 KCNQ5+1.92 CPSF1+1.41 IGF1+1.32 GJD2+1.55 SNTB1+1.36 SLC39A5+1.28 PRSS56.

9. The high-risk myopia prediction model according to claim 7, characterized in that, The threshold for P is set to 12.

249. If the P value of the sample to be tested is greater than 12.249, then the sample has a high risk of myopia.

10. The application of the high-risk myopia risk prediction model according to any one of claims 7 to 9 in the preparation of a high-risk myopia risk prediction device.

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