一种眼科疾病相关snp位点引物组合物及应用

Ophthalmologic disease related SNP site primer composition and application

文档序号：3381 发布日期：2021-09-17 浏览：27次中文

1. An ophthalmic disease-associated SNP site primer composition, characterized in that: the SNP loci comprise: rs10034228, rs13382811, rs2730260, rs4395927, rs4455882, rs6469937, rs7839488, rs9318086, rs11911275, rs7615568, rs10483727, rs1063192, rs2487032, rs3213787, rs523096 and rs 7865618.

2. The primer composition of claim 1, wherein: the primer composition comprises:

3. the primer composition of claim 2, wherein: the ophthalmic diseases comprise one or more of myopia, age-related cataract or glaucoma.

4. Use of the primer composition of claim 1 for the preparation of a product for detecting and/or assessing ophthalmic diseases.

5. A product for detecting and/or assessing ophthalmic diseases, characterized in that: the product comprises the SNP site primer composition according to claim 1.

6. The product of claim 5, wherein: the product is an independent reagent or a kit.

7. Use of the primer composition of claim 1 or the product of claim 5 for detecting ophthalmic disease-associated SNP sites.

8. A method for detecting an SNP site associated with an ophthalmic disease, which is a non-disease diagnosis and treatment method, characterized in that: the method comprises detecting SNP sites in the genome of a sample to be detected by using the primer composition of claim 1 or the product of claim 5.

9. The method of claim 8, wherein: the method comprises the following steps:

(1) extracting genome DNA;

(2) 1, performing multiplex PCR reaction;

(3) magnetic bead purification of round 1;

(4) performing multiplex PCR reaction of 2 nd round;

(5) magnetic bead purification for round 2;

(6) quantifying the library;

(7) detecting the quality of the library;

(8) performing computer sequencing on the library to obtain a fastaq sequence file, and performing quality evaluation, comparison and SNP (single nucleotide polymorphism) calling to obtain the genotype information of the SNP of the capture region of the sample to be detected;

(9) assigning the genotype of each SNP to the OR value of the disease in the population;

(10) multiplying all OR values of SNPs involved in a disease in an individual by the absolute genetic risk of the individual for the disease;

(11) the absolute genetic risk is ranked in a thousand human genome/Chinese human genome queue, namely the relative genetic risk of the individual aiming at the disease is ranked within 5% as low risk, above 95% as high risk and between 5 and 95% as normal range.

10. The method of claim 9, wherein: the quality evaluation software in the step (8) is FASTQC software, the comparison software is BWA software, and the SNP calling software is GATK software.

Background

Ophthalmic diseases refer to diseases occurring in the ocular region, and common ophthalmic diseases include myopia, astigmatism, cataract, glaucoma, central serous retinopathy, dry eye, sympathetic ophthalmia, nyctalopia, amblyopia, trachoma, diabetic retinopathy, conjunctivitis, presbyopia, achromatopsia, retinitis pigmentosa, central retinal artery occlusion, retinal detachment, hypermetropia, pinkeye, snow blindness, aragonioma, muscae volitantes, and the like. The cataract is a common ophthalmic disease of the old, is the most common cause of blindness in the world, the incidence rate of the cataract of the old over 50 years old is 60 percent, and the incidence rate of the cataract of the old over 70 years old is increased to over 80 percent.

Besides the environmental effect, the genetic factors are important pathogenic factors of some ophthalmic diseases, and the evaluation of the genetic risk of the ophthalmic diseases has important significance for early screening and prevention of the ophthalmic diseases. As already known, age-related macular degeneration (AMD) is associated with genes such as CFH, ARMS2, HTRA1, etc. And the corneal curvature related genes MTOR, CMPK1, PDGFRA and RBP3 are related to high myopia.

A Single Nucleotide Polymorphism (SNP) is a polymorphism in a DNA sequence caused by a variation of a single nucleotide (including four forms of transition, transversion, deletion and insertion), and generally, only two forms of transition and transversion occur, and the ratio of the two forms is about 1: 2. SNPs are widely present in the human genome, and occur in about every five hundred to one thousand bases, accounting for more than 90% of all known genetic polymorphisms, and are the most common heritable variations in humans. Single nucleotide polymorphisms occur most often in CG sequences, with C-allelic switching to T-allelic being most common, and this may be related to the ease with which cytosine is methylated to form thymine. The single nucleotide polymorphism is used as a third generation DNA genetic marker which is explored, has the advantages of large quantity, large density, wide distribution, strong stability, easy detection and the like compared with the first two generations of markers, and is beneficial to the application and popularization of the single nucleotide polymorphism in the medical field.

Patent CN103201393B discloses a method for identifying individuals with advanced age-related macular degeneration (AMD) who have a greater risk of developing AMD, by using a multigene score calculated based on the results of genome-wide genetic association studies, requiring the use of thousands of SNPs. The relation between the high myopia and seven SNP sites of rs1656404, rs7829127, rs7084402, rs17648524, rs1254319, rs12205363 and rs3138144 was studied in the text of the correlation analysis of 7 single nucleotide polymorphism sites and the high myopia of Chinese Han population (Li Fang, Sichuan: university of electronic technology, 2017.). However, the above prior art provides only an evaluation method or related SNP sites for a certain ophthalmic disease, and does not provide a detection method or system for a plurality of ophthalmic diseases.

Therefore, it is highly desirable to construct a primer composition and related SNP sites capable of simultaneously targeting multiple ophthalmic diseases for prevention or treatment of ophthalmic diseases, so as to better maintain the health of human body.

Disclosure of Invention

In view of the above-mentioned disadvantages, the present invention provides an ophthalmic disease-related SNP site and a primer composition thereof. According to the invention, a group of SNP locus amplification primers are designed and developed by an NGS method, and the gene structure change related to the ophthalmic diseases in the individual body is detected by a bioinformatics analysis method, so that the individual disease risk is predicted, and the prevention and/or treatment effect of the ophthalmic diseases is improved. The primer composition and the detection method have the advantages of low detection cost, simplicity and convenience in operation, good sensitivity, high accuracy and good repeatability, and have great clinical application value.

In order to achieve the above object, the technical solution of the present invention is as follows:

in one aspect, the present invention provides an ophthalmic disease-related SNP site primer composition, where the SNP sites include: rs10034228, rs13382811, rs2730260, rs4395927, rs4455882, rs6469937, rs7839488, rs9318086, rs11911275, rs7615568, rs10483727, rs1063192, rs2487032, rs3213787, rs523096 and rs 7865618.

Specifically, the primer compositions are shown in table 1 below.

TABLE 1 primer compositions

Specifically, the ophthalmic diseases comprise myopia, age-related cataract, glaucoma and the like.

In another aspect, the invention provides the application of the SNP locus primer composition in the preparation of products for detecting and/or evaluating ophthalmic diseases.

In yet another aspect, the present invention provides a product for detecting and/or evaluating an ophthalmic disease, the product comprising the above-described SNP site primer composition.

Specifically, the product is an independent reagent or a kit.

In another aspect, the invention provides an application of the SNP site primer composition or the product in detecting ophthalmic disease related SNP sites.

In still another aspect, the present invention provides a method for detecting an SNP site associated with an ophthalmic disease, which is a non-disease diagnosis and treatment method, comprising detecting the SNP site in the genome of a sample to be tested using the above primer composition or product.

Specifically, the method comprises the following steps:

(1) extracting genome DNA;

(2) 1, performing multiplex PCR reaction;

(3) magnetic bead purification of round 1;

(4) performing multiplex PCR reaction of 2 nd round;

(5) magnetic bead purification for round 2;

(6) quantifying the library;

(7) detecting the quality of the library;

(9) assigning the genotype of each SNP to the OR value of the disease in the population;

(10) multiplying all OR values of SNPs involved in a disease in an individual by the absolute genetic risk of the individual for the disease;

Further specifically, the quality evaluation software in the step (8) is FASTQC software, the alignment software is BWA software, and the SNP calling software is GATK software.

Compared with the prior art, the invention has the advantages that:

1. the invention provides a primer composition for detecting ophthalmic disease SNP sites, which can simultaneously detect ophthalmic diseases such as myopia, age-related cataract, glaucoma and the like and evaluate the risks of diseases, and further improve the prevention and/or treatment effects of the ophthalmic diseases.

2. The SNP locus primer composition and the detection method provided by the invention can obtain enough genetic information from trace peripheral blood for genetic risk assessment, have good sensitivity, simultaneously, the efficient and specific amplification primer can accurately obtain the SNP locus information, avoid non-specific amplification, greatly improve the accuracy, reduce the experiment cost and have simple and convenient operation. The acquired SNP combination is subjected to calculation and prediction of the possible future disease risk of the individual by a specific analysis method, and the method has great clinical application value.

3. The SNP locus information contained in the invention covers various ophthalmic diseases, N genotypes of susceptible loci of the diseases are obtained for each disease, the genotype allele of each susceptible locus has an own OR value, the OR value corresponding to the genotype of the locus covered by a certain disease is obtained according to the genotype information of the locus obtained by a sequencing result, and the overall genetic risk value of the disease is further calculated. For risk assessment of a certain disease of an individual, the absolute genetic risk of the individual is put into a sufficiently large population for comparison, so that the risk of the disease of the individual can be represented more accurately. Therefore, the invention determines the genetic risk of the detected individual by taking the established natural population of China and the genome population of thousands of people as references. If 95% of the individuals are cut-off and the OR value of a certain disease exceeds 95% of the people, the genetic risk of the disease of the individual is considered to be higher.

Drawings

FIG. 1 is a graph of in-flight sequencing data.

FIG. 2 is a diagram of myopia genetic risk assessment.

Fig. 3 is a map of glaucoma genetic risk assessment.

Fig. 4 is a diagram for genetic risk assessment of age-related cataract.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

In certain embodiments, details of the reagents or instruments used are as follows:

anhydrous ethanol: the Beijing chemical industry is analytically pure;

AgencourtAMPure XP Kit：Beckman(A63881)；

QubitdsDNA HS Assay Kit：Life Technologies(Q32851)；

Nuclease-free water：Ambion(AM9930)；

High Sensitivity DNA Kit：Agilent(p/n 5067-4626)；

custom Panel: agutaikang biotechnology (beijing) ltd (IGMU062), including Box 1(-20 ℃ storage): IGT-I7 (5. mu.M), IGT-EM101 polymerase mix, Enhancer NB (1N) and Box 2(-20 ℃ storage): YF buffer B (stored at 4 ℃), IGT-I5(5μM)；

PCR instrument: ABI 9700 by Life corporation;

DynaMag-96 Side：Life Technologies(cat.no.12331D)；

Fluorometer：Thermo(Q33216)；

Agilent 2100Bioanalyzer system：Agilent(G2939AA)。

example 1 primer composition

The primer compositions described herein are specifically shown in table 2 below.

TABLE 2 primer compositions

Example 2 library construction and detection

1. Genomic DNA extraction

Saliva or blood genomic DNA (gDNA) extraction was performed according to commercial genomic DNA extraction kit procedures and quantified using the Qubit (Life technologies).

2. 1 st round of multiplex PCR reaction

1.1. The reaction system is shown in table 3 below.

TABLE 3 reaction System

Reagent	Volume(μL)
		ddH₂O	4-x
Enhancer NB(1N)	5
		Primer (Table 2)	2.4
gDNA	X(30ng)
		IGT-EM101polymerase mixture	3.6

Wherein the final concentration of each Primer in the Primer mixture is 0.5 pmol.

1.2. The reaction procedure is as follows: 30s at 95 deg.C for 3 min; 18 cycles at 98 ℃ for 20s and 60 ℃ for 8 min; 5min at 72 ℃.

3. Round 1 magnetic bead purification

3.1. Adding 15 mu L of AMPure XP magnetic beads which are balanced at room temperature into 15 mu L of PCR products, and sucking and uniformly mixing the products for a plurality of times by using a pipettor;

3.2. after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96 Side magnetic frame for 3 min;

3.3. thoroughly removing the supernatant, taking the PCR tube off the magnetic frame, adding 40 mu LYF buffer B into the tube, and sucking and mixing the mixture for several times by using a pipettor;

3.4. after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96 Side magnetic frame for 3 min;

3.5. removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, and standing for 30 s;

3.6. removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, standing for 30s, and completely removing the supernatant;

3.7. standing at room temperature for 10min to completely volatilize residual ethanol;

3.8. taking down the PCR tube from the magnetic frame, adding 22 mu L of nucleic-free water, gently sucking a pipette to beat the heavy suspension magnetic beads to avoid generating bubbles, and standing for 2min at room temperature;

3.9. placing the PCR tube on the magnetic frame again, and standing for 3 min;

3.10. pipette 9.4. mu.L of the supernatant, and transfer to a new 200. mu.L PCR tube, where the supernatant is the multiplex PCR product.

4. Multiplex PCR reaction round 2

4.1. The reaction system is shown in table 4 below.

TABLE 4 reaction System

Reagent	Volume(μL)
		Step 3.10 multiplex PCR reaction product mixture	9.4
IGT-I5(5μM)	1
		IGT-I7(5μM)	1
IGT-EM101polymerase mixture	3.6

4.2. The reaction procedure is as follows: 30s at 95 deg.C for 3 min; 20s at 98 ℃, 1min at 58 ℃, 30s at 72 ℃ and 8 cycles; 5min at 72 ℃.

5. Round 2 magnetic bead purification

5.1. Adding 13.5 mu L of AMPure XP magnetic beads which are balanced at room temperature into 15 mu L of PCR products, and sucking and uniformly mixing the mixture for a plurality of times by using a pipettor;

5.2. after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96 Side magnetic frame for 3 min;

5.3. thoroughly removing the supernatant, taking down the PCR tube from the magnetic frame, adding 40 mu L YF buffer B into the PCR tube, and sucking and mixing the mixture by a pipettor for several times;

5.4. after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96 Side magnetic frame for 3 min;

5.5. removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, and standing for 30 s;

5.6. removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, standing for 30s, and completely removing the supernatant;

5.7. standing at room temperature for 10min to completely volatilize residual ethanol;

5.8. taking down the PCR tube from the magnetic frame, adding 24 μ L of nucleic-free water or 1 × TE buffer (pH 8.0), gently pipetting the resuspended magnetic beads by a pipette to avoid generating bubbles, and standing at room temperature for 2 min;

5.9. placing the PCR tube on the magnetic frame again, and standing for 3 min;

5.10. pipette 20. mu.L of the supernatant, which is the prepared multiplex PCR library, into a new PCR tube.

6. Library quantification

2 μ L of library samples were usedFluorometer (QubitdsDNA HS Assay kit) performs library concentration determination and records library concentration.

7. Library quality detection

Using Agilent 2100Bioanalyzer system (High Sensitivity DNA Kit), 1 μ L of library sample was taken for library fragment length and purity measurement, the target fragment distribution interval of the normal library was between 300 and 400bp, and the main peak was around 360 bp.

8. And performing computer sequencing on the library to obtain a fastaq sequence file. And obtaining the genotype information of the SNP of the capture area of the sample to be detected through the steps of quality evaluation, comparison, SNP calling and the like, wherein FASTQC software is used for quality evaluation, BWA software is used for comparison, and GATK software is used for SNP calling.

9. The genotype of each SNP obtained is assigned to the OR value of the disease in the population study.

10. All OR values of SNPs involved in a disease in an individual are multiplied, i.e. the absolute genetic risk of the individual for the disease.

11. The absolute genetic risk is ranked in a thousand human genome/Chinese human genome queue, namely the relative genetic risk of the individual aiming at the disease is ranked within 5% as low risk, above 95% as high risk and between 5 and 95% as normal range.

Example 3

The correspondence between the ophthalmic diseases and the genes and the detection sites is shown in the following table 5.

TABLE 5

Experimental example 1 accuracy test

The 16 sites were tested using the assay method described herein and simultaneously verified by Sanger sequencing, the results of which are shown in Table 6 below.

TABLE 6 accuracy test results

Sample(s)	Site of the body	Genotype of the application	Sanger sequencing genotype
				1	rs10034228	TC	TC
2	rs13382811	CC	CC
				3	rs2730260	GT	GT
4	rs4395927	CT	CT
				5	rs4455882	AA	AA
6	rs6469937	GG	GG
				7	rs7839488	GG	GG
8	rs9318086	GG	GG
				9	rs11911275	AG	AG
10	rs7615568	TT	TT
				11	rs10483727	TT	TT
12	rs1063192	GG	GG
				13	rs2487032	AA	AA
14	rs3213787	AG	AG
				15	rs523096	AA	AA
16	rs7865618	GG	GG

As can be seen from Table 6, the accuracy of the primer composition and the detection method described in the present application was 100%.

Experimental example 2 repeatability test

1 of the samples A and 1 of the samples B were selected, and each sample was tested in a batch by repeating 3 times, and the test results are shown in Table 7 below.

TABLE 7 results of repeated measurements

Remarking: sample A-1 is sample A at the 1 st test.

As shown in the test results in Table 7, the repeatability of the primer composition and the test method disclosed in the present application is 100%.

Experimental example 3 detection of precision

1 of the samples A and 1 of the samples B were selected, and each sample was tested with three batches of the primer composition, and the test results are shown in Table 8 below.

TABLE 8 results of precision measurements

As is clear from the results in Table 8, the precision of the primer composition and the detection method described in the present application was 100%.

Experimental example 4

1 sample of peripherial blood A was selected and tested according to the methods of examples 1 and 2 of the present application and evaluated for genetic risk of ocular diseases, such as myopia, glaucoma and age-related cataract, and the genetic risk of this sample in the thousand population and the current self-test population is shown in FIGS. 2-4. Wherein the content of the first and second substances,the abscissa is the natural logarithm of the normalized relative risk OR value, and the ordinate is the probability density of the population on the corresponding OR value. The sample myopia OR ═ 0.5877, Rank_{Thousand human genomes}＝46.2％，Rank_Self-test35.2%; glaucoma OR 0.6241, Rank_{Thousand human genomes}＝99.7％，Rank_Self-test98.6 percent; age-related cataract OR ═ 1, Rank_{Thousand human genomes}＝0.5％，Rank_Self-test＝0.4％。

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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