CN113462771A

CN113462771A - SNP marker combination, primer probe combination, kit and application in preparation thereof

Info

Publication number: CN113462771A
Application number: CN202111029132.3A
Authority: CN
Inventors: 毕宏生; 蒋文君; 赵海强; 王兴荣; 季鹏; 郭滨; 吴建峰
Original assignee: Affiliated Ophthalmology Hospital Of Shandong University Of Traditional Chinese Medicine
Current assignee: Affiliated Ophthalmology Hospital Of Shandong University Of Traditional Chinese Medicine
Priority date: 2021-09-03
Filing date: 2021-09-03
Publication date: 2021-10-01

Abstract

The application discloses an SNP marker combination, a primer probe combination, a kit and application thereof in preparation. The SNP marker combination for judging the high myopia risk comprises 8 SNP loci including SNP1-SNP8, wherein the SNP loci are 1-SNP8 as follows: the rs numbers of the sites of SNP1-SNP8 are rs35771565, rs2076486, rs2301753, rs3130573, rs1065356, rs3134605, rs3131290 and rs2076523 respectively, and the sites of the SNP1-SNP8 of the rs numbers are C/T polymorphism, A/G polymorphism, G/A polymorphism, T/C polymorphism, G/A polymorphism and T/C polymorphism respectively.

Description

SNP marker combination, primer probe combination, kit and application in preparation thereof

Technical Field

The invention relates to the field of genetic disease gene detection, in particular to an SNP marker combination, a primer probe combination, a kit and application thereof in preparation.

Background

Myopia with diopters > -6.00D or axial length of the eye exceeding 26mm is called high myopia. The prevalence of high myopia is clearly population specific. The main factors causing the myopia are generally considered to be environmental and genetic factors, and family researches show that the prevalence rate of the high myopia of children with the myopia of parents is higher than that of children without the myopia of parents, and the genetic factors are prompted to play an important role in the onset of the high myopia.

Traditional methods for detecting SNPs associated with high myopia include first-generation sequencing, allele-specific PCR, genotyping based on fluorescent-labeled single-base extension, conventional fluorescent quantitative PCR, and the like. With the rapid development of modern detection technologies such as gene chip technology and high-throughput sequencing method, a plurality of new technical methods such as whole genome SNP chip, whole genome sequencing, exome sequencing and target region resequencing are widely applied to the research of high myopia genes. However, these detection methods have disadvantages.

For example, the period of detecting SNP by the first-generation sequencing technology is long, and if a plurality of SNP sites are detected, the cost of artificial reagents is relatively high and the time is relatively consumed; genotyping based on fluorescence labeling single base extension is suitable for detecting SNPs of a large number of samples, and if the number of detected samples is small, the cost is obviously increased; according to the conventional fluorescent quantitative PCR method, a single system detects a single site, the flux is low, the method is only suitable for the research of a small amount of sites, and the cost for detecting the single site of a single sample is high; the whole genome SNP chip and the second-generation sequencing method have the advantages of longer detection period, high detection cost, relatively complex operation in the whole experimental process and higher requirement on the capability of data analysts. The conventional multigene risk scoring method is generally based on whole genome association analysis data, needs a large number of sites to establish a scoring model, is limited to the whole genome association data, and is not suitable for whole exome sequencing data.

Disclosure of Invention

In order to solve the problems, an SNP marker combination, a primer probe combination, a kit and application thereof in preparation are provided. The high myopia risk is predicted based on the SNP marker combination, the accuracy is high, the number of required sites is small, and the method has the effects of simplicity, convenience and cost saving; by utilizing a multicolor probe melting curve analysis technology combining multiple fluorescent PCR with the specific primer probe combination, the simultaneous detection of a plurality of target SNP sites can be realized within two to three hours, the detection flux is obviously improved, the detection cost is reduced, and the detection accuracy is improved; and accurately and quickly judging the genotype of each site of the sample according to the difference of the Tm values of all the detection channels.

According to one aspect of the application, a SNP marker combination for determining the high myopia risk is provided, and comprises 8 SNP sites of SNP1-SNP8, wherein the SNP1-SNP8 sites are as follows:

the rs number of the SNP1 site is rs35771565, and the SNP is C/T polymorphism;

the rs number of the SNP2 locus is rs2076486, and the SNP is A/G polymorphism;

the rs number of the SNP3 site is rs2301753, and the SNP is G/T polymorphism;

the rs number of the SNP4 site is rs3130573, and the SNP is A/G polymorphism;

the rs number of the SNP5 site is rs1065356, and the SNP is G/A polymorphism;

the rs number of the SNP6 site is rs3134605, and the SNP is T/C polymorphism;

the rs number of the SNP7 site is rs3131290, and the SNP is G/A polymorphism;

the rs number of the SNP8 site is rs2076523, and the SNP is T/C polymorphism.

Alternatively, the SNP1 polymorphism is C/T, T is the risk of high myopia, and C is the risk of non-high myopia; the SNP2 polymorphism is A/G, G is the high myopia risk, and A is the non-high myopia risk; the SNP3 polymorphism is G/T, T is the risk of high myopia, and G is the risk of non-high myopia; the SNP4 polymorphism is A/G, G is the high myopia risk, and A is the non-high myopia risk; the SNP5 polymorphism is G/A, A is the high myopia risk, and G is the non-high myopia risk; the SNP6 polymorphism is T/C, C is the risk of high myopia, and T is the risk of non-high myopia; the SNP7 polymorphism is G/A, A is the high myopia risk, and G is the non-high myopia risk; the SNP8 polymorphism is T/C, C is the risk of high myopia, and T is the risk of non-high myopia.

Specifically, the SNP1-SNP8 sites are all located in the chromosome 6p22.3 region, and the chromosome 6p22.3 region has a certain correlation with the high myopia risk.

A method for obtaining a combination of SNP markers for determining the risk of high myopia, comprising the steps of:

the sequencing data of the exome of 167 patients with high myopia and 259 local controls are analyzed, and the rare variation with low quality sites, tristate sites and low population frequency less than 0.05 is removed through data filtering, so that case-control correlation study is carried out on the obtained common variation data. For polymorphic sites with significant difference (p value is less than the threshold value of 1 e-6) in frequency distribution in a case group and a control group after multiple correction by a Benjamini-Hochberg method, considering that the sites have statistically significant correlation with the risk of high myopia;

and (3) sorting according to the size of the p value and the size of the OR value, removing the linkage sites with r2 more than 0.8 according to the linkage relation of the polymorphic sites in Chinese population in the genome of thousands of people, finally selecting the 8 polymorphic sites as the most obvious sites with the highest relative risk of diseases, and incorporating the sites into a next model for predicting the risk of high myopia.

According to another aspect of the present application, there is provided a primer probe combination comprising the following primer probe sets:

the primer probe set 1 is used for detecting SNP1 sites, and comprises a primer set 1 and a probe P1, wherein the primer set 1 comprises a primer F1 and a primer R1, and the primer F1 is a single-stranded DNA molecule shown in a sequence l of a sequence table; the primer R1 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table; the probe P1 is a single-stranded DNA molecule shown in sequence 3 of the sequence table; the 5 'end of the probe P1 is connected with a fluorescent group A1, and the 3' end is connected with a quenching group B1;

the primer probe set 2 is used for detecting SNP2 site, the primer probe set is composed of a primer set 2 and a probe P2, the primer set 2 is composed of a primer F2 and a primer R2, and the primer F2 is a single-stranded DNA molecule shown in sequence 4 of the sequence table; the primer R2 is a single-stranded DNA molecule shown in a sequence 5 in a sequence table; the probe P2 is a single-stranded DNA molecule shown in sequence 6 of the sequence table; the 5 'end of the probe P2 is connected with a fluorescent group A2, and the 3' end is connected with a quenching group B2;

the primer probe set 3 is used for detecting SNP3 site, the primer probe set is composed of a primer set 3 and a probe P3, the primer set 3 is composed of a primer F3 and a primer R3, and the primer F3 is a single-stranded DNA molecule shown in sequence 7 of the sequence table; the primer R3 is a single-stranded DNA molecule shown in a sequence 8 in a sequence table; the probe P3 is a single-stranded DNA molecule shown in a sequence 9 in a sequence table; the 5 'end of the probe P3 is connected with a fluorescent group A3, and the 3' end is connected with a quenching group B3;

the primer probe set 4 is used for detecting SNP4 sites, and comprises a primer set 4 and a probe P4, wherein the primer set 4 comprises a primer F4 and a primer R4, and the primer F4 is a single-stranded DNA molecule shown in a sequence l0 in a sequence table; the primer R4 is a single-stranded DNA molecule shown in a sequence 11 in a sequence table; the probe P4 is a single-stranded DNA molecule shown in a sequence 12 in a sequence table; the 5 'end of the probe P4 is connected with a fluorescent group A4, and the 3' end is connected with a quenching group B4;

the primer probe set 5 is used for detecting SNP5 site, the primer probe set is composed of a primer set 5 and a probe P5, the primer set 5 is composed of a primer F5 and a primer R5, and the primer F5 is a single-stranded DNA molecule shown in sequence 13 of a sequence table; the primer R5 is a single-stranded DNA molecule shown in a sequence 14 in a sequence table; the probe P5 is a single-stranded DNA molecule shown in a sequence 15 in a sequence table; the 5 'end of the probe P5 is connected with a fluorescent group A5, and the 3' end is connected with a quenching group B5;

the primer probe set 6 is used for detecting SNP6 site, the primer probe set is composed of a primer set 6 and a probe P6, the primer set 6 is composed of a primer F6 and a primer R6, and the primer F6 is a single-stranded DNA molecule shown in sequence 16 of the sequence table; the primer R6 is a single-stranded DNA molecule shown in a sequence 17 in a sequence table; the probe P6 is a single-stranded DNA molecule shown in a sequence 18 in a sequence table; the 5 'end of the probe P6 is connected with a fluorescent group A6, and the 3' end is connected with a quenching group B6;

the primer probe set 7 is used for detecting SNP7 site, the primer probe set is composed of a primer set 7 and a probe P7, the primer set 7 is composed of a primer F7 and a primer R7, and the primer F7 is a single-stranded DNA molecule shown in sequence 19 of a sequence table; the primer R7 is a single-stranded DNA molecule shown in a sequence 20 in a sequence table; the probe P7 is a single-stranded DNA molecule shown in a sequence 21 in a sequence table; the 5 'end of the probe P7 is connected with a fluorescent group A7, and the 3' end is connected with a quenching group B7; and

the primer probe set 8 is used for detecting SNP8 site, the primer probe set consists of a primer set 8 and a probe P8, the primer set 8 consists of a primer F8 and a primer R8, and the primer F8 is a single-stranded DNA molecule shown in sequence 22 of the sequence table; the primer R8 is a single-stranded DNA molecule shown in a sequence 23 in a sequence table; the probe P8 is a single-stranded DNA molecule shown in a sequence 24 in a sequence table; the 5 'end of the probe P8 is connected with a fluorescent group A8, and the 3' end is connected with a quenching group B8.

Optionally, the Primer probe combination further comprises a universal reverse Primer-R, wherein the universal Primer-R is a single-stranded DNA molecule shown as a sequence 25 in the sequence table.

Alternatively, the fluorophore A1-fluorophore A8 is independently selected from one of FAM, ROX, HEX, and CY 5;

the fluorescence quenching group B1-fluorescence quenching group B8 is respectively selected from one of BHQ1, BHQ2 and BHQ3, and can quench with the paired fluorescence groups. Specifically, for example, FAM can be mated with BHQ1 and BHQ2, and ROX can be mated with BHQ2, respectively.

Preferably, the fluorescent group A1-fluorescence quenching group B1, the fluorescent group A2-fluorescence quenching group B2, the fluorescent group A3-fluorescence quenching group B3 and the fluorescent group A4-fluorescence quenching group B4 are respectively selected from one of FAM-BHQ1, ROX-BHQ2, HEX-BHQ3 and CY5-BHQ 1; and

the fluorescent group A5-fluorescence quenching group B5, the fluorescent group A6-fluorescence quenching group B6, the fluorescent group A7-fluorescence quenching group B7 and the fluorescent group A8-fluorescence quenching group B8 are respectively selected from one of FAM-BHQ1, ROX-BHQ1, HEX-BHQ3 and CY5-BHQ 1.

More preferably, the fluorophore A1-fluorescence quenching group B1, the fluorophore A2-fluorescence quenching group B2, the fluorophore A3-fluorescence quenching group B3 and the fluorophore A4-fluorescence quenching group B4 are respectively FAM-BHQ1, ROX-BHQ2, HEX-BHQ3 and CY5-BHQ1 in sequence; and

the fluorescent group A5-fluorescence quenching group B5, the fluorescent group A6-fluorescence quenching group B6, the fluorescent group A7-fluorescence quenching group B7 and the fluorescent group A8-fluorescence quenching group B8 are respectively and sequentially FAM-BHQ1, ROX-BHQ1, HEX-BHQ3 and CY5-BHQ 1.

Specifically, the primer probe sets 1 to 8 have:

(1) the size of the amplicon is 100-500bp, the optimal size is 200bp +/-50 bp, and the range has the optimal amplification efficiency;

(2) the GC content of the primer is controlled to be 40-60 percent, the Tm value is 58-62 ℃, the excessive high or insufficient GC content of the primer is not beneficial to initiating reaction, and the exonuclease activity at the 5' end of the temperature range of the Tm value is the highest;

(3) the length of the probe is 19-25bp, and the quenching effect is poor when the length of the probe is too long;

(4) the SNP locus is in the middle position of the probe sequence, so that the sequences on both sides of the probe can be ensured to be complementarily matched with the template;

(5) the base of the SNP site of the probe is designed according to the wild type, but when the wild type site is G, the base at the site is designed based on the mutant type, the Tm value difference of the wild type probe and the mutant type probe is more than 3 ℃, and the phenomenon that two melting point peaks of a melting curve of a heterozygous sample are too close to each other to mislead the judgment of the genotype is prevented;

(6) the Tm value of each SNP site probe is at least 5 ℃ higher than that of the primer, and the probe is ensured to be combined with the template in a renaturation way before the primer;

(7) the difference of Tm values of probes marked by different fluorescent groups is more than 3 ℃, so that the fluorescent signal crosstalk between the fluorescent groups is prevented.

According to a further aspect of the present application, there is provided a kit for determining the risk of high myopia, comprising the primer probe composition described in any one of the above.

Preferably, the kit further comprises a universal reverse Primer-R, wherein the universal Primer-R is a single-stranded DNA molecule shown in a sequence 25 of a sequence table.

Optionally, the kit further comprises DNA polymerase, PCR buffer solution, MgCl₂And dNTPs. Specifically, the DNA polymerase may be Taq enzyme, and the PCR Buffer may be 10 XPCR Buffer.

According to still another aspect of the present application, there is provided a use of a primer probe composition for detecting a combination of SNP markers in the preparation of any one of the above-described kits for determining risk of high myopia,

the SNP marker combination is 8 SNP loci which are collected by individuals and are 1-8, and the method comprises the following steps:

1) typing said SNP site of DNA collected from a human individual;

2) determining the risk of high myopia of the individual according to the typing result obtained in the step 1);

wherein in the step 2), the risk is judged from high to low according to the sequence that the SNP1 is TT type, CT type and CC type;

in the step 2), judging the risk from high to low according to the sequence that the SNP2 is GG type, AG type and AA type;

in the step 2), judging the risk from high to low according to the sequence that the SNP3 is TT type, GT type and GG type;

in the step 2), judging the risk from high to low according to the sequence that the SNP4 is GG type, AG type and AA type;

in the step 2), judging the risk from high to low according to the sequence that the SNP5 is AA type, GA type and GG type;

in the step 2), judging the risk from high to low according to the sequence that the SNP6 is CC type, TC type and TT type;

in the step 2), judging the risk from high to low according to the sequence that the SNP7 is AA type, GA type and GG type; and

in the step 2), the risk is judged from high to low according to the sequence that the SNP8 is CC type, TC type and TT type.

Preferably, in the step 2), the SNP1 is judged as a high risk group when the SNP is TT type, and is judged as a low risk group when the SNP is CC type;

in the step 2), judging the SNP2 as a GG type to be a high risk group, and judging the SNP2 as an AA type to be a low risk group;

in the step 2), the SNP3 is judged as a high risk group in the TT type, and is judged as a low risk group in the GG type;

in the step 2), judging the SNP4 as a GG type to be a high risk group, and judging the SNP4 as an AA type to be a low risk group;

in the step 2), judging the SNP5 as an AA type to be a high risk group, and judging the SNP5 as a GG type to be a low risk group;

in the step 2), the SNP6 is judged to be a high risk group for CC type, and is judged to be a low risk group for TT type;

in the step 2), judging the SNP7 as an AA type to be a high risk group, and judging the SNP7 as a GG type to be a low risk group; and

in the step 2), the SNP8 is judged as a high risk group when the SNP is CC type, and is judged as a low risk group when the SNP is TT type.

Optionally, the step 1) of typing the SNP sites of DNA collected from a human individual specifically comprises:

a) extracting DNA of a sample to be detected, and carrying out multiplex fluorescence PCR by using any one of the primer probe compositions to detect at least one genotype of 8 SNP sites including SNP1-SNP 8;

b) and (3) determining whether the corresponding positive peak type exists in the melting curve peak of the amplification product or not by comparing, or determining the SNP site according to the Tm value of the melting curve peak.

Preferably, the primer probe sets 1 to 4 are added to a first reaction tube as a first reaction system, and the primer probe sets 5 to 8 are added to a second reaction tube as a second reaction system, to simultaneously or separately perform multiplex real-time fluorescent quantitative PCR reactions.

Optionally, the first reaction system comprises: 0.03-5 mu M of each primer group 1-4, 0.5 mu M of each probe P1-P4, 20-50ng of template genome DNA and water are filled in the reaction system to be 10-50 mu L;

the second reaction system comprises: 0.03-5. mu.M each of the primer sets 1-4, 0.5. mu.M each of the probes P1-P4, 20-50ng of template genomic DNA, and 10-50. mu.L of water in total to the reaction system.

Preferably, the first tube reaction system and the second reaction system further comprise: 10 XPCR Buffer, Taq enzyme, 2-4mM Mg²⁺And dNTPs.

Optionally, the real-time fluorescent quantitative PCR reaction conditions of the first reaction system and the second reaction system are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20 s, annealing at 65 ℃ for 30 s, extension at 72 ℃ for 40 s and a total of 50 cycles;

the melting curve was prepared under the following conditions: completely extending for 5min at 72 ℃; denaturation at 95 deg.C for 1 min; probe annealing and hybridizing at 40 ℃ for 1 min; the melting curve was recorded 5 times from 40 ℃ to 85 ℃ at 1 ℃ per liter.

Optionally, the step b) typing the SNP site according to the melting curve peak Tm value, or directly determining the high myopia risk of the individual according to the melting curve peak Tm value.

Preferably, the Tm values of the amplification products corresponding to the C/T polymorphism of the SNP1 detected by the primer probe set 1 are respectively 56 ℃/45 ℃;

the Tm values of amplification products corresponding to the A/G polymorphism of the SNP2 detected by the primer probe set 2 are 63 ℃/56 ℃ respectively;

the primer probe sets 3 respectively detect that the Tm values of amplification products corresponding to the G/T polymorphism of the SNP3 are respectively 55 ℃/65 ℃;

the primer probe sets 4 respectively detect that the Tm values of amplification products corresponding to the A/G polymorphism of the SNP4 are respectively 50 ℃/59 ℃;

the primer probe sets 5 respectively detect that the Tm values of amplification products corresponding to the G/A polymorphism of the SNP5 are respectively 55 ℃/63 ℃;

the primer probe sets 6 are used for respectively detecting the Tm values of amplification products corresponding to the C/T polymorphism of the SNP6 to be 55 ℃/67 ℃;

the Tm values of amplification products corresponding to G/A polymorphism of SNP7 detected by the primer probe set 7 are 66 ℃/58 ℃ respectively;

the Tm values of amplification products corresponding to the T/C polymorphism of the SNP8 detected by the primer probe sets 8 are 59 ℃/55 ℃.

Optionally, the step 2) calculates the risk probability of the high myopia phenotype according to the typing result and by using the prediction model.

Specifically, a multi-gene Risk Score (PRS) method is used to construct a model for predicting the Risk probability of the high myopia phenotype, and prspace software is used to calculate the Risk probability, wherein the formula for calculating the Risk probability of the phenotype by using the PRS value is as follows:

P（pheno）=-0.694+0.8805*PRSavg

the PRSavg value is calculated by the formula

。

Wherein, i refers to the number of SNP sites, n refers to the number of SNP sites, Gj refers to the genotype, the value is 0, 1 OR 2, and beta represents the OR value of the SNP sites;

PT represents the threshold value of P value in the analysis result of the association study (1 e-6), and SNP sites with P value smaller than the threshold value are selected and calculated.

As an implementation mode, samples of all genotype types capable of covering 1-88 SNP sites are selected for experiment, and the genotype of the selected sample is obtained through a whole exon sequencing method; then obtaining the Tm values of the melting point peaks of different SNP sites of each sample by a qPCR method; comparing with the known genotypes of different sites of the sample to obtain Tm values corresponding to different genotypes of the sites; and then the method is used for detecting the sample with unknown genotype, 8 SNP loci are obtained for typing, and the risk of suffering from high myopia is predicted.

According to still another aspect of the present application, there is provided a model for predicting the risk of high myopia based on the genotype of SNP sites of a SNP marker combination.

Specifically, the model for predicting the Risk of the high myopia adopts a multi-gene Risk Score (PRS) method to construct a model for predicting the phenotypic Risk probability of the high myopia, and is calculated by using prspace software, wherein a formula for calculating the phenotypic Risk probability by using a PRS value is as follows:

P（pheno）=-0.694+0.8805*PRSavg

the PRSavg value is calculated by the formula

PT represents the threshold value of P value in the analysis result of the association study (1 e-6), and SNP sites with P value smaller than the threshold value are selected and calculated. The model can rapidly and accurately detect the prediction model of the high myopia susceptibility gene aiming at the defects of the prior detection technology and the prior high myopia susceptibility prediction model.

Taking out a part of data of a patient with a high myopia phenotype as a verification data set, taking the other part of data as a training data set, performing statistical analysis after data quality control to screen SNP sites with significant difference in frequency distribution between the patient and a normal control, acquiring p values and OR values of the sites, performing weighted sum calculation on the sites by taking the OR values as weights to calculate the phenotype risk probability, and checking the accuracy and sensitivity of the phenotype risk probability method in the verification data set.

And constructing a phenotype risk probability model by screening SNP sites highly associated with the phenotype in the training data set, and checking the accuracy and the sensitivity of the model in the verification data set. On one hand, the method is not highly dependent on whole genome association analysis data, needs fewer sites, has the effects of simplicity, convenience and cost saving, and on the other hand, the method has good performance in verifying data concentration and proves that the risk scoring efficacy is not lost on the premise of saving cost.

Benefits of the present application include, but are not limited to:

1. the SNP marker combination has the advantages of predicting the high myopia risk based on the SNP marker combination, along with high accuracy, less required number of sites, simplicity, convenience and cost saving.

2. According to the primer probe combination and the kit, the multi-color probe melting curve analysis technology of combining multiple fluorescence PCR with the specific primer probe combination is adopted, the simultaneous detection of a plurality of target SNP sites is realized within two to three hours, the target gene typing is realized through standard comparison, and the high myopia susceptibility gene is subjected to gene typing. The detection flux is obviously improved, the detection cost is reduced, and the detection accuracy is improved; and accurately and quickly judging the genotype of each site of the sample according to the difference of the Tm values of all the detection channels.

3. The application of the primer probe composition for detecting the SNP marker combination in preparing the kit for judging the high myopia risk has the advantages of less required number of sites, simplicity, convenience and cost saving.

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 embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a FAM channel melting curve diagram of a first reaction system SNP1 site (rs35771565 site) of a sample QW-K8819 according to an embodiment of the present application, wherein a melting point peak Tm value of the FAM channel at the rs35771565 site is 56.3 ℃;

FIG. 2 is a ROX channel melting curve diagram of SNP2 site (rs2076486 site) of a first reaction system of QW-K8819 according to an example of the present application, wherein a peak Tm value of the ROX channel melting point at the rs2076486 site is 62.66 ℃.

FIG. 3 is a diagram of a melting curve of a HEX channel at SNP3 site (rs2301753 site) in a first reaction system QW-K8819 according to an example of the present application, wherein a Tm value of a melting point peak of the HEX channel at the rs2301753 site is 55 ℃/65 ℃.

FIG. 4 is a melting curve diagram of a CY5 channel at SNP4 site (rs3130573 site) of a first reaction system of QW-K8819 according to an example of the present application, wherein a Tm value of a CY5 channel melting point peak at rs3130573 site is 49.71 ℃/59 ℃.

FIG. 5 is a melting curve diagram of a FAM channel at SNP5 site (rs1065356 site) of a second reaction system of QW-K8819 according to an example of the present application, wherein a melting point peak Tm value of the FAM channel at the rs1065356 site is 55.41 ℃/62.86 ℃.

FIG. 6 is a melting curve diagram of a ROX channel at SNP6 site (rs3134605 site) of a second reaction system of QW-K8819 according to an example of the present application, wherein a melting point peak Tm value of the ROX channel at rs3134605 site is 67.35 ℃.

Fig. 7 is a graph of the HEX channel melting curve at SNP7 site (rs3131290 site) of the second reaction system of QW-K8819 according to the example of the present application, and the Tm value of the HEX channel melting point peak at rs3131290 site is 65.6 ℃.

FIG. 8 is a melting curve diagram of a CY5 channel at SNP8 site (rs2076523 site) of a sample QW-K8819 second reaction system according to an embodiment of the present application, wherein a Tm value of a CY5 channel melting point peak at rs2076523 site is 59.34 ℃.

Fig. 9 is a melting curve diagram of a FAM channel at SNP1 site (rs35771565 site) of a first reaction system of QW-K9017 according to an embodiment of the present application, where a Tm value of a FAM channel melting point peak at rs35771565 site is 56.3 ℃.

FIG. 10 is a melting curve diagram of a ROX channel at SNP2 site (rs2076486 site) of a sample QW-K9017 first reaction system according to an example of the present application, wherein a melting point peak Tm value of the ROX channel at the rs2076486 site is 62.76 ℃.

Fig. 11 is a melting curve diagram of an HEX channel at SNP3 site (rs2301753 site) of a first reaction system of QW-K9017 according to an example of the present application, where a Tm value of a HEX channel melting point peak at rs2301753 site is 55 ℃.

Fig. 12 is a melting curve diagram of a CY5 channel at a SNP4 site (rs3130573 site) of a first reaction system of a QW-K9017 according to an example of the present application, wherein a Tm value of a CY5 channel melting point peak at a rs3130573 site is 59.1 ℃.

Fig. 13 is a melting curve diagram of a FAM channel at SNP5 site (rs1065356 site) in a second reaction system QW-K9017 according to an example of the present application, where a Tm value of a FAM channel melting point peak at rs1065356 site is 55.31 ℃.

Fig. 14 is a melting curve diagram of a ROX channel at SNP6 site (rs3134605 site) of a second reaction system of QW-K9017 according to an example of the present application, and a melting point peak Tm value of a ROX channel at rs3134605 site is 55.63 ℃.

Fig. 15 is a graph of the melting curve of the HEX channel at SNP7 site (rs3131290 site) of the second reaction system QW-K9017 according to the example of the present application, and the Tm value of the HEX channel at rs3131290 site is 58.27 ℃.

FIG. 16 is a melting curve diagram of a CY5 channel at SNP8 site (rs2076523 site) of a sample QW-K9017 second reaction system according to an embodiment of the present application, wherein a Tm value of a CY5 channel melting point peak at rs2076523 site is 55.73 ℃.

FIG. 17 is a FAM channel melting curve diagram of SNP1 site (rs35771565 site) of the first reaction system of QW-K8949 according to the example of the present application, wherein Tm of FAM channel melting point peak at rs35771565 site is 45 ℃/56.5 ℃.

FIG. 18 is a melting curve diagram of a ROX channel at SNP2 site (rs2076486 site) of a sample QW-K8949 first reaction system according to an example of the present application, wherein a melting point peak Tm value of the ROX channel at the rs2076486 site is 56.13 ℃.

Fig. 19 is a melting curve diagram of an HEX channel at SNP3 site (rs2301753 site) of a first reaction system of QW-K8949 according to an example of the present application, wherein a Tm value of a HEX channel melting point peak at rs2301753 site is 65 ℃.

Fig. 20 is a melting curve diagram of a CY5 channel at a SNP4 site (rs3130573 site) of a first reaction system of a QW-K8949 according to an example of the present application, wherein a Tm value of a CY5 channel melting point peak at a rs3130573 site is 50.28 ℃.

Fig. 21 is a melting curve diagram of a FAM channel at SNP5 site (rs1065356 site) in a QW-K8949 reaction system according to an example of the present application, where a Tm value of a FAM channel melting point peak at the rs1065356 site is 62.8 ℃.

FIG. 22 is a melting curve diagram of a ROX channel at SNP6 site (rs3134605 site) of a sample QW-K8949 second reaction system according to an example of the present application, wherein a melting point peak Tm value of the ROX channel at rs3134605 site is 55.28 ℃/66.84 ℃.

FIG. 23 is a diagram of a melting curve of an SNP7 site (rs3131290 site) HEX channel of a second reaction system SNP 8949 according to an example of the present application, wherein a Tm value of an HEX channel melting point peak of the rs3131290 site is 57.5 ℃/65.5 ℃.

FIG. 24 is a melting curve diagram of a CY5 channel at SNP8 site (rs2076523 site) of a sample QW-K8949 second reaction system according to an example of the application, wherein a Tm value of a CY5 channel melting point peak at rs2076523 site is 55.01 ℃/59.53 ℃.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

The reagents used in the examples of the present application are all commercially available unless otherwise specified. The primer probe sequence was synthesized by Biotechnology engineering (Shanghai) Inc.

In the present invention, the SNP is preferably a SNP registered in the child public database, which can be identified from its reference number (referrence number). For example, there is a SNP identified by reference number rs of the SNP database (db SNP BUILD135) of NCBI (national center for Biotechnology information).

Collecting a sample of a subject, such as peripheral blood, saliva, buccal swab, etc., and extracting genomic DNA of the sample;

the extracted genome DNA is used as a template, high-fidelity and high-efficiency DNA polymerase is adopted, and the regions covering the locus rs35771565 of SNP1, the locus rs2076486 of SNP2, the locus rs2301753 of SNP3, the locus rs3130573 of SNP4, the locus rs1065356 of SNP5, the locus rs3134605 of SNP6, the locus rs3131290 of SNP7 and the locus rs2076523 of SNP8 are amplified by utilizing the optimized primer concentration, probe concentration, reaction temperature and reaction time.

The application of a primer probe composition for detecting SNP marker combination in preparing any one of the kits for determining the risk of high myopia is provided, which comprises the following steps:

1) typing said SNP sites of DNA collected from human individuals:

a) extracting DNA of a sample to be detected, and carrying out multiple fluorescence PCR by using 8 primer probe compositions to detect the genotypes of 8 SNP sites including SNP1-SNP 8;

b) determining whether a corresponding positive peak type exists in the melting curve peak of the amplification product or not by comparing, or typing the SNP locus according to the melting curve peak Tm value;

2) determining the high myopia risk of the individual according to the typing result obtained in the step 1) or according to the melting curve peak Tm value; or calculating the risk probability of the high myopia phenotype by using the obtained typing result and a prediction model.

Example 1

1. Extraction of genome DNA of sample to be detected

Human peripheral blood was collected, whole genome gDNA was extracted using a Tiangen blood extraction kit, DNA concentration was determined using an onedrop and Qubit3.0 apparatus, and the corresponding concentration and the ratio of 260/280 to 260/230 were recorded.

2. The combination of primers and probes for amplifying 8 kinds of sites in total, SNP1-SNP8, is shown in Table 1 (SEQ ID NOS: 1-25)

3. The extracted genome DNA is used as a template, a multiple fluorescent quantitative PCR system is respectively prepared according to the following tables 2 and 3, and the PCR reaction is carried out by adopting a Hangzhou Langzhou A200 PCR instrument.

Note: the first reaction system is the combination of four sites of SNP1 locus rs35771565, SNP2 locus rs2076486, SNP3 locus rs2301753 and SNP4 locus rs 3130573. F1, F2, F3 and F4 are corresponding 4 upstream primers, and R1, R2, R3 and R4 are corresponding 4 downstream primers. Probe P1 (Probe-1), Probe P2 (Probe-2), Probe P3 (Probe-3) and Probe P4 (Probe-4) correspond to 4 probes.

Note: the second reaction system is the combination of four sites of SNP5 site rs1065356, SNP6 site rs3134605, SNP7 site rs3131290 and SNP8 site rs 2076523. F5, F6, F7 and F8 are corresponding 4 upstream primers, and R5, R6, R7 and R8 are corresponding 4 downstream primers. Probe P5 (Probe-5), Probe P6 (Probe-6), Probe P7 (Probe-7) and Probe P8 (Probe-8) correspond to 4 probes.

4. The same amplification conditions were used for the tube reaction system, and the amplification conditions are shown in Table 4 below.

Fluorescence signals were collected at 65 ℃.

5. Analysis of PCR products by multicolor fluorescence dissolution curve

Setting the detection fluorescence channels as FAM, ROX, HEX and CY5, and setting the reaction conditions as follows: completely extending for 5min at 72 ℃; denaturation at 95 deg.C for 1 min; probe annealing and hybridizing at 40 ℃ for 1 min; collecting FAM, HEX, ROX and CY5 channel fluorescence signals for 5 times from 40-85 ℃ per liter at 1 ℃, determining a melting curve peak Tm value according to the collected fluorescence signals, and typing SNP sites according to the melting curve peak Tm value to judge the high myopia risk of the sample to be detected.

Example 2

The method of example 1 was used to test the polymorphisms at SNP1-SNP8 of sample QW-K8819, sample QW-K9017 and sample QW-K8949 of known genotypes and the corresponding melting curve peak Tm.

The genotypes of 8 SNP sites of SNP1-SNP8 of 3 samples are obtained by a whole exon sequencing method, and the sample genotypes are detailed in Table 5.

The statistics of Tm values of the sample genotype, detection channel and melting point peak for QW-K8819, QW-K9017 and sample QW-K8949 are shown in tables 6, 7 and 8, respectively.

Note: two replicates per sample were taken and the melting point peaks Tm averaged.

Melting curves of the sample QW-K8819, the sample QW-K9017 and the sample QW-K8949 in FAM, ROX, HEX and CY5 channels are shown in the attached drawings 1-24.

The Tm values corresponding to 8 different genotypes at 8 positions of 8 SNP1-SNP8 are shown in Table 9.

The information of 8 total loci of SNPs 1-SNP8 is shown in Table 10, wherein the 8 loci are located in the 6p22.3 region of chromosome.

Example 3

The method of example 1 and the corresponding relationship between the melting curve peak Tm and genotype obtained in example 2 were used to detect 5 samples of informed consenting volunteers with unknown phenotype of unknown genotype, samples 1-3 were highly myopic patients, samples 4-5 were non-highly myopic patients, and the genotypes of the 5 samples were obtained according to the Tm values of 8 SNP sites, as shown in Table 11.

The high myopia phenotype risk probabilities were calculated from the predictive models, and the high myopia phenotype risk probabilities for the 5 samples corresponded to their actual phenotypes as shown in table 12.

Specifically, taking sample 1 as an example, a calculation method of a probability model for predicting the risk of the high myopia phenotype is described as follows:

the calculation was performed using prsite software, where PRS values were calculated as follows:

P（pheno）=-0.694+0.8805*PRSavg

the PRSavg value is calculated by the formula

。

Gj refers to the genotype, the wild type value is 0, the heterozygous mutation is 1, the homozygous mutation is 2, beta represents the OR value of the SNP site (the OR values of 8 sites from SNP1 to SNP8 are 4.1845, 6.816, 4.332, 3.8005, 5.044, 3.1575, 4.216 and 3.609 in sequence), the data brought into the sample 1 are represented by the PRSavg formula: (1*4.184+1*6.816+1*4.332+1*3.8005+1*5.044+0*3.1575+0*4.216 +2*3.609)/16.

The high myopia phenotype risk probability is more than 0.5, the high myopia or the high myopia is determined, and the higher the value is, the higher the probability that the phenotype is the high myopia is; low myopia or emmetropia is defined as a high myopia phenotype risk probability of less than 0.5.

According to the content, other 50 high myopia volunteers are detected, and the detection results are consistent with the results of the actual detection standard equivalent sphere lens form, so that the kit and the prediction model can timely and accurately judge the high myopia risk.

In addition, a test method for predicting the sensitivity and specificity of high myopia includes:

the sensitivity is calculated as: sensitivity = true positive number/(true positive number + false negative number). 100%. The specific calculation formula is as follows: specificity = number of true negatives/(number of true negatives + number of false positives). 100%.

The model calculated by the training set obtains the results of sensitivity and specificity in the check set, and the sensitivity of the method for predicting the high myopia is calculated to be 82.14% and the specificity is calculated to be 50.69% according to the phenotype predicted by the phenotype risk probability of each sample and the actual phenotype through the test results in the check set of 249 high myopia samples and 49 normal non-myopia control samples.

The above description is only an example of the present application, and the protection scope of the present application is not limited by these specific examples, but is defined by the claims of the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong TCM university affiliated ophthalmic hospital

<120> SNP marker combination, primer probe combination, kit and application in preparation thereof

<130> JH-CNP210678YS

<160> 25

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> DNA

<213> Artificial sequence

<400> 1

cctgttgctc aaagtgtag 19

<210> 2

<211> 19

<212> DNA

<213> Artificial sequence

<400> 2

attttagtcc ctcgttctc 19

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<400> 3

cgtccttctt tccccctgag gacg 24

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence

<400> 4

ggtaacaagg tggacgact 19

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

aagtggaagc cctgaatcta 20

<210> 6

<211> 28

<212> DNA

<213> Artificial sequence

<400> 6

ggcgtgaaat atgatagaaa caaacgcc 28

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence

<400> 7

acctactcgg gaggctgat 19

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence

<400> 8

tggcattctt tggcttgt 18

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

<400> 9

tcagaaaaag aaaatggggt ctga 24

<210> 10

<211> 19

<212> DNA

<213> Artificial sequence

<400> 10

ccctccccac taggttcag 19

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<400> 11

cttggatgtg ccagtttct 19

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence

<400> 12

ccatgtaaca aaggactaat cttcg 25

<210> 13

<211> 19

<212> DNA

<213> Artificial sequence

<400> 13

gttcatgcca ttctgcttc 19

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence

<400> 14

tggacactca cagggacac 19

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence

<400> 15

agcttagtag aggctgcgtt tcaagct 27

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> 16

aagatggaaa ggaaataagg 20

<210> 17

<211> 19

<212> DNA

<213> Artificial sequence

<400> 17

aagacagaaa agggcagat 19

<210> 18

<211> 22

<212> DNA

<213> Artificial sequence

<400> 18

tcctcatggg gactgaggag ga 22

<210> 19

<211> 19

<212> DNA

<213> Artificial sequence

<400> 19

gcattactgg ttatccctt 19

<210> 20

<211> 19

<212> DNA

<213> Artificial sequence

<400> 20

gcagttgttt tgcttattt 19

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence

<400> 21

cgagtgaatg acaagatgat tgtccag 27

<210> 22

<211> 19

<212> DNA

<213> Artificial sequence

<400> 22

ggctgctctt tcttgttcc 19

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

tcttcctatt cctcctgcta 20

<210> 24

<211> 23

<212> DNA

<213> Artificial sequence

<400> 24

gccattcaag gtctttttat ggc 23

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence

<400> 25

cactactgat tccatcttgc tgccg 25

Claims

1. An SNP marker combination for judging the high myopia risk, which is characterized by comprising 8 SNP sites of SNP1-SNP8, wherein the SNP1-SNP8 sites are as follows:

the rs number of the SNP1 site is rs35771565, and the SNP is C/T polymorphism;

the rs number of the SNP2 locus is rs2076486, and the SNP is A/G polymorphism;

the rs number of the SNP3 site is rs2301753, and the SNP is G/T polymorphism;

the rs number of the SNP4 site is rs3130573, and the SNP is A/G polymorphism;

the rs number of the SNP5 site is rs1065356, and the SNP is G/A polymorphism;

the rs number of the SNP6 site is rs3134605, and the SNP is T/C polymorphism;

the rs number of the SNP7 site is rs3131290, and the SNP is G/A polymorphism;

the rs number of the SNP8 site is rs2076523, and the SNP is T/C polymorphism.

2. The primer probe combination for detecting the SNP marker combination according to claim 1, wherein the primer probe combination comprises the following primer probe sets:

3. The primer probe composition of claim 2, wherein the fluorophore A1-fluorophore A8 is selected from one of FAM, ROX, HEX and CY5, respectively;

the fluorescence quenching group B1-fluorescence quenching group B8 is respectively selected from one of BHQ1, BHQ2 and BHQ3, and can quench with the paired fluorescence groups.

4. The primer-probe composition of claim 2, wherein the fluorophore A1-fluorescence quencher B1, the fluorophore A2-fluorescence quencher B2, the fluorophore A3-fluorescence quencher B3, and the fluorophore A4-fluorescence quencher B4 are respectively selected from one of FAM-BHQ1, ROX-BHQ2, HEX-BHQ3, and CY5-BHQ 1; and

5. A kit for determining the risk of high myopia, comprising the primer probe composition of claim 3 or 4.

6. Use of a primer probe composition for detecting a combination of SNP markers for the preparation of a kit for determining the risk of high myopia according to claim 5,

the SNP marker combination is 8 SNP sites of the SNP1-SNP8 collected by individuals and described in claim 1, and comprises the following steps:

1) typing said SNP site of DNA collected from a human individual;

7. Use of a primer probe composition for detecting a combination of SNP markers in the preparation of a kit for determining the risk of high myopia according to claim 5 or 6, wherein said step 1) of typing said SNP sites of DNA collected from a human individual specifically comprises:

a) extracting DNA of a sample to be detected, and carrying out multiple fluorescence PCR by using the primer probe composition of claim 3 or 4 to detect the genotypes of 8 SNP sites including SNP1-SNP 8;

wherein the primer probe sets 1-4 are added into a first reaction tube to serve as a first reaction system, the primer probe sets 5-8 are added into a second reaction tube to serve as a second reaction system, and multiple real-time fluorescent quantitative PCR reactions are carried out synchronously or respectively.

8. Use of a primer probe composition for detecting a combination of SNP markers for the preparation of a kit for determining the risk of high myopia according to claim 7,

the first reaction system comprises: 0.03-5 mu M of each primer group 1-4, 0.5 mu M of each probe P1-P4, 20-50ng of template genome DNA and water are filled in the reaction system to be 10-50 mu L;

9. Use of a primer probe composition for detecting a combination of SNP markers for the preparation of a kit for determining the risk of high myopia according to claim 8,

the real-time fluorescent quantitative PCR reaction conditions of the first reaction system and the second reaction system are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20 s, annealing at 65 ℃ for 30 s, and extension at 72 ℃ for 40 s for 50 cycles;

10. Use of a primer probe composition for detecting a combination of SNP markers in the preparation of a kit for determining the risk of high myopia according to claim 7, wherein step b) is performed by typing the SNP sites according to the melting curve peak Tm values, or directly determining the risk of high myopia in the human individual according to the melting curve peak Tm values;

wherein, the Tm values of amplification products corresponding to the C/T polymorphism of the SNP1 detected by the primer probe set 1 are respectively 56 ℃/45 ℃;

the primer probe sets 6 are used for respectively detecting the Tm values of amplification products corresponding to the T/C polymorphism of the SNP6 to be 55 ℃/67 ℃;