CN116287199A - Primer combination and kit for detecting high myopia risk and application of primer combination and kit - Google Patents
Primer combination and kit for detecting high myopia risk and application of primer combination and kit Download PDFInfo
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Abstract
The invention provides a primer combination for detecting high myopia risk, a kit and application thereof, and relates to the technical field of biological detection. The primer combination comprises a primer for amplifying SNP loci related to high myopia risk, wherein the SNP loci related to high myopia risk comprise 17 SNP loci in total of SNP1-SNP 17. The primer combination has the advantages of high accuracy, simplicity, convenience and cost saving when being applied to the high myopia risk prediction process; according to the invention, the fluorescent PCR technology is utilized to combine SNP loci and specific primers thereof, so that simultaneous detection of a plurality of target SNP loci can be realized within two to three hours, and then the genotype of each locus of a sample is accurately and rapidly judged through the difference of Tm values of detection channels, so that genotyping of highly myopic susceptibility genes is realized, the detection flux is remarkably improved, the detection cost is reduced, and the detection accuracy is improved.
Description
Technical Field
The application relates to the technical field of biological detection, in particular to a primer combination, a kit and application for detecting high myopia risk.
Background
High Myopia (HM) is a common complex characteristic ocular disease. There is evidence that genetic factors play an important role in the development of high myopia, i.e. the identification of genes susceptible to high myopia will reveal the risk of developing it.
Single Nucleotide Polymorphisms (SNPs) refer to single base-altered deoxyribonucleic acid (DNA) sequence polymorphisms that occur at the genomic level. In recent years, it has been reported in literature that SNPs at related gene loci are changed in close relation to the occurrence and development of myopia. Along with the rapid development of modern detection technology, technical methods such as whole genome SNP chip, whole genome sequencing, exome sequencing, target region re-sequencing and the like are widely applied to high myopia gene research, but the detection methods have the defects of long detection period, multiple sites, complex analysis method, high cost, insufficient prediction accuracy and the like, and influence the popularization and the use of the detection methods.
The high resolution melting analysis technique (High Resolution Melting analysis), abbreviated as HRMA, is based on the physical properties of nucleic acid molecules, and the length, GC content, GC distribution, etc. of different nucleic acid molecules are different, so that any double-stranded DNA molecule will have its own shape and position of melting curve when denatured by heating. This item is a technique for analysis by monitoring DNA melting curve change by means of a saturated intercalating dye based on the physical properties of nucleic acids. The key points of the technology are as follows: the designed melting curve Tm values of the DNA amplification products are different and can be detected, and double-stranded DNA intercalating saturated fluorescent dyes such as Eva Green are needed, so that the saturated dyes cannot inhibit PCR at saturated concentration, meanwhile, high-concentration dyes are saturated and inserted into minor grooves in a DNA double-helix structure, rearrangement cannot occur in the DNA melting process, and the melting curve has higher resolution; finally, an instrument with an accurate temperature control device and high-density data acquisition is required to perform experiments.
In summary, there is a need for a method and a product for detecting a gene for high myopia that has high detection throughput, high accuracy, a small number of required sites, and is convenient and fast.
Disclosure of Invention
The invention aims to provide an SNP locus combination for detecting high myopia risk and a primer combination and application thereof, and the SNP locus combination and the primer combination have the effects of high accuracy, less required locus number, convenience and rapidness.
The high-myopia high-frequency variation locus (SNP locus) obtained based on the pre-exon sequencing method is optimized to obtain the SNP locus combination based on the high-resolution fusion analysis technology, and the primer combination, the reaction system and the reaction conditions thereof, can be used for rapidly screening high-myopia risk groups, can be used for preventing myopia prevention and control work of children and teenagers high myopia, and has important significance in reducing the prevalence rate of high myopia in China.
In one aspect, the present application provides a primer combination for detecting a high myopia risk, the primer combination comprising a primer pair for specifically amplifying a high myopia risk related SNP site comprising at least one SNP site from SNP1 site to SNP17 site,
wherein, the rs number of SNP1 site is rs12767252, the rs number of SNP2 site is rs942871, the rs number of SNP3 site is rs2728433, the rs number of SNP4 site is rs4927578, the rs number of SNP5 site is rs72843921, the rs number of SNP6 site is rs2278884, the rs number of SNP7 site is rs2271496, the rs number of SNP8 site is rs9854418, the rs number of SNP9 site is rs4927663, the rs number of SNP10 site is rs76859481, the rs number of SNP11 site is rs17009618, the rs number of SNP12 site is rs11833187, the rs number of SNP13 site is rs1800159, the rs number of SNP14 site is rs17828632, the rs number of SNP15 site is rs11553598, the rs number of SNP16 site is rs72790445, and the rs number of SNP17 site is rs145819902.
Wherein, the rs number of SNP1 locus is rs12767252, the SNP is C/T polymorphism, the reference allele is C, the mutant allele is T;
rs number of SNP2 locus is rs942871, the SNP is G/A polymorphism, the reference allele is G, the mutant allele is A;
rs number of SNP3 locus is rs2728433, the SNP is T/G polymorphism, the reference allele is T, the mutant allele is G;
rs number of SNP4 locus is rs4927578, the SNP is C/T polymorphism, reference allele is C, mutant allele is T;
rs number of SNP5 locus is rs72843921, the SNP is T/C polymorphism, the reference allele is T, the mutant allele is G;
rs number of SNP6 locus is rs2278884, the SNP is T/C polymorphism, the reference allele is T, and the mutant allele is C;
rs number of SNP7 locus is rs2271496, the SNP is A/G polymorphism, reference allele is A, mutant allele is G;
rs number of SNP8 locus is rs9854418, the SNP is C/T polymorphism, reference allele is C, mutant allele is T;
rs number of SNP9 locus is rs4927663, the SNP is G/C polymorphism, the reference allele is G, and the mutant allele is C;
rs number of SNP10 locus is rs76859481, the SNP is T/C polymorphism, the reference allele is T, and the mutant allele is C;
rs number of SNP11 locus is rs17009618, the SNP is C/T polymorphism, reference allele is C, mutant allele is T;
rs number of SNP12 locus is rs11833187, the SNP is T/C polymorphism, the reference allele is T, the mutant allele is C;
rs number of SNP13 locus is rs1800159, the SNP is G/A polymorphism, the reference allele is G, the mutant allele is A;
rs number of SNP14 locus is rs17828632, the SNP is A/G polymorphism, reference allele is A, mutant allele is G;
rs number of SNP15 locus is rs11553598, the SNP is A/G polymorphism, reference allele is A, mutant allele is G;
rs number of SNP16 locus is rs72790445, the SNP is G/C polymorphism, the reference allele is G, and the mutant allele is C;
rs number of SNP17 locus is rs145819902, the SNP is A/C polymorphism, reference allele is A, mutant allele is C;
each SNP site mutant allele is a high myopia risk related gene, and the wild type allele (i.e., can be considered as a reference allele) is a non-high myopia risk related gene.
Preferably, the primer combination comprises a primer pair for specifically amplifying the SNP1-SNP17 site associated with high myopia risk.
Further, the primer combination includes at least one of the following SNP1 site primer pair-SNP 17 site primer pair,
the SNP1 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.1 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 2;
the SNP2 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4;
the SNP3 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.5 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 6;
the SNP4 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.7 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 8;
the SNP5 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.9 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 10;
the SNP6 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.11 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 12;
the SNP7 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.13 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 14;
the SNP8 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.15 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 16;
the SNP9 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.17 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 18;
the SNP10 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.19 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 20;
the SNP11 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.21 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 22;
the SNP12 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.23 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 24;
the SNP13 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.25 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 26;
the SNP14 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.27 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 28;
the SNP15 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.29 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 30;
the SNP16 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.31 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 32;
the SNP17 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.33 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 34.
Preferably, the primer combination includes the following SNP1 locus primer pair-SNP 17 locus primer pair total 17 pair primers.
Preferably, the primer combination comprises the nucleotide sequence primers shown as SEQ ID NO.1 to SEQ ID NO.34 or variants having at least more than 90% sequence identity to the shown sequence.
The melting curve Tm value of the designed DNA amplification product is different and can be detected through the double-strand DNA embedded saturated fluorescent dye, rearrangement can not occur in the DNA melting process, and higher resolution can be realized through detecting the melting curve, so that higher sensitivity and specificity are achieved.
On the other hand, the application also provides application of the primer combination in preparing a product for detecting high myopia risk.
In another aspect, the present application also provides a kit for detecting high myopia risk, the kit comprising the primer combination described above.
Further, the kit further comprises reagents used in any one of a fluorescent PCR method, a PCR amplification method, a digital PCR method, a liquid chip method, a second generation sequencing method, a third generation sequencing method or a combination thereof.
Preferably, the kit further comprises reagents used in a fluorescent PCR method; more preferably, real-time fluorescent PCR.
For example, the detection reagent may be selected from the following: bisulfite and derivatives thereof, PCR buffer, polymerase, dNTP, primer, probe, enzyme digestion buffer, fluorescent dye, fluorescence quenching agent, fluorescent reporter, exonuclease, alkaline phosphatase, internal standard, control and the like.
More preferably, the kit further comprises a saturated fluorescent dye; more preferably, the double-stranded DNA intercalating saturated fluorescent dye.
More preferably, the kit comprises: the SNP1-17 locus primer pair, HRM qPCR Master Mix and a saturated fluorescent dye.
The kit is added with template genome DNA and then reacts.
The real-time fluorescence PCR reaction conditions of the reaction system are as follows: pre-denaturation at 95℃for 1min; denaturation at 95℃for 15s, annealing at 60℃for 15s, extension at 72℃for 45s and a total of 40 cycles;
the conditions for preparing the melting curve are as follows: denaturation at 95℃for 1min; annealing and hybridizing for 1min at 40 ℃; the melting curve was recorded 5 times at each temperature rise of 1℃from 65℃to 97 ℃.
On the other hand, the application also provides a construction method of the high myopia risk assessment model, which comprises the following steps:
(a) Collecting samples of high myopia and low myopia; (b) Extracting DNA of a sample, and sequencing the sample to obtain sample data; (c) Correcting the sample data, constructing an algorithm model, and screening out SNP loci related to the high myopia risk according to the sample data.
Preferably, the algorithm model comprises a machine learning model; more preferably, the machine learning model includes any one of a principal component analysis model, a logistic regression analysis model, a nearest neighbor analysis model, a support vector machine, and a neural network model.
In a preferred embodiment, the sample is a mammalian blood and other biologically derived liquid sample, such as peripheral blood, serum, plasma, ascites, urine, cerebral spinal fluid, sputum, saliva, and the like. The mammal includes rats, mice and humans; preferably a human.
More preferably, the sample is peripheral blood, and genomic DNA of the sample is extracted.
In another aspect, the present application also provides a method for assessing risk of high myopia, the method comprising:
(a) Obtaining genotypes of at least one of the SNP1 locus to the SNP17 locus in a sample to be detected; (b) typing the SNP site; (c) A high myopia risk of the individual is determined based on the typing result.
Wherein the typing result comprises wild type, mutation heterozygous type and mutation homozygous type, and the risk is judged to be sequentially from low to high.
In a preferred embodiment, the present application uses qPCR methods to obtain a melting curve of 17 SNP sites of a sample, to obtain 17 SNP site genotypes thereof, and to predict the risk of developing high myopia, as compared to known genotypes.
On the other hand, the application also provides a high myopia risk assessment model, which is constructed by adopting the method, and comprises the following steps: the data acquisition module is at least used for acquiring a sample data set; a sequencing module at least for obtaining the genotype of at least one of the SNP1 locus to the SNP17 locus; the data comparison module is at least used for typing the genotype; a result determination module determines a high myopia risk of the individual based on the typing result.
Further, the typing results comprise wild type, mutation heterozygous type and mutation homozygous type, and risks are judged to be sequentially low to high.
Preferably, the high risk group is determined by the SNP site being homozygous for the mutation, and the low risk group is determined by the SNP site being wild type.
Wherein, the specific determination method is to sort the SNP locus by comparing whether the melting curve peak of the amplified product exists corresponding to the positive peak type, and the specific typing of SNP1-17 is shown in figures 2-18.
Preferably, the predictive high myopia risk model employs a multigenic risk scoring (Polygenic Risk Score, PRS) method to construct a model for predicting high myopia risk, specifically calculated using PRSice software, wherein PRS values are calculated as follows:
wherein i refers to the number of SNP sites, n refers to the number of SNP sites, gj refers to genotype, the value is 0, 1 OR 2, and beta represents the OR value of the SNP sites;
PT represents a threshold value (1 e-6) of the P value in the analysis result of the association study, and SNP sites with the P value smaller than the threshold value are selected for calculation. The model aims at the defects of the prior detection technology and the prior high myopia susceptibility prediction model, and can rapidly and accurately detect the high myopia susceptibility gene prediction model.
And taking out one part of data of the patients with the high myopia phenotype as a verification data set, taking the other part of data as a training data set, carrying out statistical analysis after data quality control to screen SNP sites with significant differences in frequency distribution in the patients and normal controls, obtaining p values and OR values of the sites, carrying out weighted sum calculation on risk scores by taking the OR values as weights on the sites, and checking the specificity and the sensitivity of a risk scoring method in the verification data set.
In a preferred embodiment, the present application provides a model sensitivity of 72.6%, a specificity of 66.4%, and an AUC of 0.734 (95% CI: 0.679-0.788).
And constructing a risk scoring model by screening SNP loci highly associated with the phenotype in the training data set, and checking the accuracy and sensitivity of the model in the verification data set. The model provided by the application is not highly dependent on the whole genome association analysis data like a polygenic risk scoring method, the number of sites required by the model is small, the model has the effects of simplicity, convenience and cost saving, and on the other hand, the model has good performance in verification data sets, and the risk scoring efficacy is not lost on the premise of saving the cost.
In another aspect, the present application further provides an information data processing terminal, the information data processing terminal including a memory, a processor, and a computer program stored on the memory and executable on the processor, the processor implementing the following steps when executing the computer program:
(a) Obtaining genotypes of at least one of the SNP1 locus to the SNP17 locus in a sample to be detected; (b) typing the SNP site; (c) A high myopia risk of the individual is determined based on the typing result.
A computer readable storage medium having stored thereon a computer program which, when executed by a processor, performs the steps of:
(a) Obtaining genotypes of at least one of the SNP1 locus to the SNP17 locus in a sample to be detected; (b) typing the SNP site; (c) A high myopia risk of the individual is determined based on the typing result.
The invention has the following beneficial effects:
1. the application provides 17 SNP loci which can be used for detecting the high myopia risk, namely SNP1 locus to SNP17 locus and free combination thereof, has the advantages of high accuracy, convenience and rapidness and cost saving when being applied to the high myopia risk prediction process, can realize higher detection sensitivity, well balances model specificity, and can reduce misdiagnosis rate;
2. the application also provides a specific primer for detecting the SNP locus and a combination thereof;
3. according to the high-resolution melting curve analysis technology combining the specific primer combination of the application by utilizing the fluorescence PCR technology, the simultaneous detection of a plurality of target SNP loci can be realized within two to three hours, the detection flux is obviously improved, the detection cost is reduced, the detection accuracy is improved, the genotype of each locus of a sample can be accurately and rapidly judged according to the difference of Tm values of each detection channel, compared with other technologies, the reaction cost is lower, the instrument requirement is relatively low, and the method is more suitable for popularization and application.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
FIG. 1 is a ROC graph;
FIG. 2 is a peak diagram of SNP1 loci of different genotypes;
FIG. 3 is a peak diagram of SNP2 loci of different genotypes;
FIG. 4 is a peak diagram of SNP3 loci of different genotypes;
FIG. 5 is a peak diagram of SNP4 loci of different genotypes;
FIG. 6 is a peak diagram of SNP5 loci of different genotypes;
FIG. 7 is a peak diagram of SNP6 loci of different genotypes;
FIG. 8 is a peak diagram of SNP7 loci of different genotypes;
FIG. 9 is a peak diagram of SNP8 loci of different genotypes;
FIG. 10 is a peak diagram of SNP9 loci of different genotypes;
FIG. 11 is a peak diagram of SNP10 loci of different genotypes;
FIG. 12 is a peak diagram of SNP11 loci of different genotypes;
FIG. 13 is a peak diagram of SNP12 loci of different genotypes;
FIG. 14 is a peak diagram of SNP13 loci of different genotypes;
FIG. 15 is a peak diagram of SNP14 loci of different genotypes;
FIG. 16 is a peak diagram of SNP15 loci of different genotypes;
FIG. 17 is a peak diagram of SNP16 loci of different genotypes;
FIG. 18 is a peak diagram of SNP17 loci of different genotypes.
Detailed Description
In order to more clearly illustrate the general concepts of the present application, the following detailed description is made by way of example with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the invention may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the invention.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
In the following embodiments, unless specified otherwise, the reagents or apparatus used are conventional products available commercially without reference to the manufacturer.
Example 1
The SNP used in the present embodiment is preferably a SNP registered in a public database, and can be determined from its reference number (reference number). For example, a SNP determined by the reference number rs of the SNP database of NCBI (National center for Biotechnology Information) (db SNP BUILD 135).
And, through analyzing the exome sequencing data of 572 patients with high myopia and 272 low myopia controls, removing the rare variation of low quality sites, tri-state sites and crowd frequency lower than 0.05 through data filtering, and carrying out case-control association research on the obtained common variation data.
The specific data analysis method is that SNP locus combinations are screened by adopting a polygenic risk scoring (Polygenic Risk Score, PRS) method, a predicted high myopia risk model is constructed, and the PRSice software is specifically used for calculation, wherein the PRS value is calculated as follows:
wherein i refers to the number of SNP sites, n refers to the number of SNP sites, gj refers to genotype, the value is 0, 1 OR 2, and beta represents the OR value of the SNP sites;
PT represents a threshold value (0.01) of the P value in the analysis result of the association study, and SNP sites with the P value smaller than the threshold value are selected for calculation.
After quality control of sequencing data, logistic regression analysis is carried out to screen SNP loci with obvious difference in frequency distribution between patients and normal controls, and p values and OR values of the loci are obtained. Polymorphic sites which still have significant differences in frequency distribution (p value less than 0.01) between the case group and the control group after correction by the Benjamini-Hochberg method are considered to have statistically significant correlation with the risk of high myopia. Sorting according to the p value and the OR value, and removing linkage sites with r2 more than 0.8 according to linkage relations of the polymorphic sites in Chinese people in thousands of people genome.
And respectively taking one part of data from the sample of the case group phenotype and the sample of the control group phenotype as a training data set, taking the other part of data as a verification data set, carrying out weighted summation on the sites by taking an OR value as a weight to calculate a risk score, and checking the specificity and the sensitivity of the risk scoring method in the verification data set. And (3) carrying out qPCR experimental optimization on the screened loci, and finally selecting 17 polymorphic loci as the most obvious loci with the highest relative risk of the disease, wherein the specific loci are shown in table 1.
The calculation formula of the sensitivity is as follows: sensitivity = true positive number/(true positive number + false negative number) ×100%. The specific calculation formula is as follows: specificity = true negative population/(true negative population + false positive population) ×100%.
TABLE 1
The sensitivity of the model in this example was 72.6%, the specificity was 66.4% and the AUC was 0.734 (95% CI: 0.679-0.788), as shown in FIG. 1. The model aims at the defects of the prior detection technology and the prior high myopia susceptibility prediction model, and can rapidly and accurately detect the high myopia susceptibility gene prediction model.
Example 2
1. Extracting genome DNA of a sample to be detected:
peripheral blood of 100 volunteers was collected, extracted using a root blood extraction kit whole genome gDNA, and DNA concentrations were measured with onedrop and Qubit3.0 instruments, and the corresponding concentrations were recorded and absorbance was measured at 260nm, 280nm, 230nm, respectively, to calculate the ratio of A260/A280 and A260/A230.
2. The specific design method is to design a primer by taking the sites in the table 1 in the example 1 as target sequences, wherein the primer set has the following characteristics:
(1) The amplicon has the size of 100-500bp, the optimal size of 120bp plus or minus 80bp, and the range has the optimal amplification efficiency;
(2) The GC content of the primer is controlled to be 40% -60%, and too high or too low is unfavorable for initiating the reaction. The Tm value is 58-62 ℃, and the activity of the 5' -end exonuclease in the temperature range is the highest;
(3) The length is 19-25bp, and the quenching effect is not good when the length is too long.
(4) SNP loci are in the middle of the sequences, so that the complementary pairing of the sequences on two sides and the template is ensured.
The primers with high specificity obtained by preference are shown in Table 2.
TABLE 2
3. The above-mentioned extracted genomic DNA was used as a template, and a fluorescent quantitative PCR system was prepared as described in Table 3, and the PCR reaction was performed using a LightCycler480 PCR apparatus.
TABLE 3 composition of the reaction system
4. Each tube of the reaction system was amplified using the same amplification conditions, and the specific amplification conditions are shown in Table 4 below.
TABLE 4 fluorescent quantitative PCR reaction conditions
5. Analysis of PCR products by fluorescence dissolution curve
Determining peak types of a melting curve of a sample according to collected fluorescent signals, comparing peak shapes of the sample with internal reference peak types, and typing SNP loci according to the peak types of the melting curve to judge the high myopia risk of the sample to be detected, wherein the typing result of each locus is shown in figures 2-18, the peak types of different genotypes, mutation heterozygotes (namely genotype 0/1 and REF/ALT), wild types (namely genotype 0/0 and REF/REF) and mutation homozygotes (namely genotype 1/1 and ALT/ALT) are marked in the figures. One skilled in the art can determine the genotype based on the genotyping result map comparison and determine whether the sample-derived individual is at high risk of myopia.
After detection, the results were compared with pathological results, and 100 volunteers included 36 control groups and 64 patient groups, 23 of the 36 control groups were predicted to be low risk, 52 of the 64 patient groups were predicted to be high risk, and the model sensitivity was about 81.25% and the specificity was about 63.89% in this example. The model provided by the application can accurately predict the high myopia risk, and has higher sensitivity and better specificity.
The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.
Claims (10)
1. A primer combination for detecting high myopia risk, characterized in that the primer combination comprises a primer pair for specifically amplifying high myopia risk related SNP sites comprising at least one SNP site from SNP1 site to SNP17 site,
wherein, the rs number of SNP1 site is rs12767252, the rs number of SNP2 site is rs942871, the rs number of SNP3 site is rs2728433, the rs number of SNP4 site is rs4927578, the rs number of SNP5 site is rs72843921, the rs number of SNP6 site is rs2278884, the rs number of SNP7 site is rs2271496, the rs number of SNP8 site is rs9854418, the rs number of SNP9 site is rs4927663, the rs number of SNP10 site is rs76859481, the rs number of SNP11 site is rs17009618, the rs number of SNP12 site is rs11833187, the rs number of SNP13 site is rs1800159, the rs number of SNP14 site is rs17828632, the rs number of SNP15 site is rs11553598, the rs number of SNP16 site is rs72790445, and the rs number of SNP17 site is rs145819902.
2. The primer combination according to claim 1, wherein the primer combination comprises at least one of the following SNP1 site primer set-SNP 17 site primer set,
the SNP1 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.1 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 2;
the SNP2 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4;
the SNP3 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.5 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 6;
the SNP4 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.7 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 8;
the SNP5 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.9 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 10;
the SNP6 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.11 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 12;
the SNP7 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.13 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 14;
the SNP8 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.15 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 16;
the SNP9 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.17 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 18;
the SNP10 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.19 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 20;
the SNP11 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.21 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 22;
the SNP12 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.23 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 24;
the SNP13 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.25 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 26;
the SNP14 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.27 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 28;
the SNP15 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.29 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 30;
the SNP16 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.31 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 32;
the SNP17 locus primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.33 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 34.
3. Use of a primer combination according to claim 1 or 2 for the preparation of a product for detecting a high risk of myopia.
4. A kit for detecting a high risk of myopia, comprising a primer combination according to claim 1 or 2.
5. The kit according to claim 4, further comprising a reagent used in any one of a fluorescent PCR method, a PCR amplification method, a digital PCR method, a liquid chip method, a second generation sequencing method, a third generation sequencing method, or a combination thereof.
6. The method for constructing the high myopia risk assessment model is characterized by comprising the following steps of:
(a) Collecting samples of high myopia and low myopia; (b) Extracting DNA of a sample, and sequencing the sample to obtain sample data; (c) Correcting the sample data, constructing an algorithm model, and screening out SNP loci related to the high myopia risk according to the sample data.
7. A high myopia risk assessment model constructed using the method of claim 6, comprising: the data acquisition module is at least used for acquiring a sample data set; a sequencing module for obtaining at least the genotype of at least one of the SNP1 locus to the SNP17 locus as defined in claim 1; the data comparison module is at least used for typing the genotype; the result judging module judges the high myopia risk of the individual based on the parting result.
8. The high myopia risk assessment model according to claim 7, wherein the typing results comprise wild type, mutant heterozygous, mutant homozygous, and the risk is determined to be sequentially low to high.
9. An information data processing terminal comprising a memory, a processor and a computer program stored on the memory and executable on the processor, characterized in that the processor when executing the computer program performs the steps of:
(a) Obtaining the genotype of at least one of the SNP1 locus to the SNP17 locus according to claim 1 in a sample to be tested; (b) typing the SNP site; (c) A high myopia risk of the individual is determined based on the typing result.
10. A computer readable storage medium, wherein the storage medium has stored thereon a computer program which, when executed by a processor, performs the steps of:
(a) Obtaining the genotype of at least one of the SNP1 locus to the SNP17 locus according to claim 1 in a sample to be tested; (b) typing the SNP site; (c) A high myopia risk of the individual is determined based on the typing result.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101892306A (en) * | 2009-04-03 | 2010-11-24 | 四川省医学科学院（四川省人民医院） | Screening test kit for detecting pathologic moyopia |
| CN113462771A (en) * | 2021-09-03 | 2021-10-01 | 山东中医药大学附属眼科医院 | SNP marker combination, primer probe combination, kit and application in preparation thereof |
| CN113637742A (en) * | 2021-09-29 | 2021-11-12 | 成都二十三魔方生物科技有限公司 | High myopia gene detection kit, and high myopia genetic risk assessment system and method |
| CN114395620A (en) * | 2021-12-20 | 2022-04-26 | 温州谱希医学检验实验室有限公司 | Biomarker combination for detecting high-myopia susceptible population |
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| CN101892306A (en) * | 2009-04-03 | 2010-11-24 | 四川省医学科学院（四川省人民医院） | Screening test kit for detecting pathologic moyopia |
| CN113462771A (en) * | 2021-09-03 | 2021-10-01 | 山东中医药大学附属眼科医院 | SNP marker combination, primer probe combination, kit and application in preparation thereof |
| CN113637742A (en) * | 2021-09-29 | 2021-11-12 | 成都二十三魔方生物科技有限公司 | High myopia gene detection kit, and high myopia genetic risk assessment system and method |
| CN114395620A (en) * | 2021-12-20 | 2022-04-26 | 温州谱希医学检验实验室有限公司 | Biomarker combination for detecting high-myopia susceptible population |
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