一种复杂疾病相关snp位点引物组合物及应用

Complex disease related SNP site primer composition and application

文档序号：3382 发布日期：2021-09-17 浏览：26次中文

1. A complex disease related SNP site primer composition is characterized in that: the SNP loci comprise: rs10229583, rs1048886, rs10811661, rs10814916, rs10886471, rs10906115, rs1111875, rs1116357, rs11257655, rs117601636, rs11787792, rs12000501, rs12010175, rs12549902, rs13266634, rs1359790, rs1421085, rs1436955, rs1470579, rs147538848, rs 0315505500, rs163171, rs 16316361329, rs 29955379, rs17584499, rs 177982, rs 18018095, rs1845900, rs187060802, rs1965305, rs 202773617640, rs 4120796, rs2206734, rs2237892, rs2237895, rs 223782237897, rs 22835637799, rs 70779, rs 70777943779, rs 7077563756375637563756375648, rs 70779, rs 7037563756375637563756375637563756375637569, rs 70729, rs 7037563756375637563756375637563756375637563756375637567, rs 703756375637563756375637563756375637563756375637567, rs 70729, rs 7037563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, rs1745, rs 70729, rs 707245, rs1745, rs 7245, rs 70729, rs 729, rs 70729, rs 707245, rs 7072563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756300, rs 729, rs1745, rs 703756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756300, rs1745, rs 729, rs1745, rs 7037563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756300, rs1745, rs 703756375637563756375637563756375637563756375637563756375637563756375637563756375637, rs10883795, rs11038167, rs11038172, rs1635, rs16887244, rs2142731, rs835784, rs10821944, rs 11900600673, rs12379034, rs 12617617617617656, rs13192471, rs1854853, rs2075876, rs2233434, rs2841277, rs2867461, rs3093024, rs3124, rs3781913, rs 65707075, rs10865331, rs13210693, rs17095830, rs30187, rs4552569, rs10036748, rs10845606, rs10847697, rs 2208334, rs1167796, rs1234315, rs12494314, rs 1287707, rs 13113177851338574, rs 1388190, rs1913517, rs 220793, rs 2205756375637563756375639, rs 31779, rs 70575648, rs 31575648 5648, rs 704746, rs 725646, rs 725648, rs 445648, rs 44575646, rs 445646, rs 725648, rs 445648, rs 725648, rs 445648, rs 725648, rs 72563457435456345743545648, rs 725648, rs 445648, rs 725648, rs 445648, rs 725648, rs 43545648, rs 725648, rs 445648, rs 7256345743545648, rs 725648, rs 7256345743545648, rs 725648, rs 445634575648, rs 725648, rs 44563457435456345743545634575648, rs 445634575634575634575648, rs 445648, rs 7256345743545648, rs 445648, rs 435456345743545648, rs 445648, rs 725648, rs 445648, rs 725648, rs 43545648, rs 725648, rs 4456345743545648, rs 445648, rs 725648, rs 445648, rs 4354.

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

3. the primer composition of claim 2, wherein: the complex disease comprises one or more of type 2 diabetes, hashimoto's thyroiditis, Alzheimer's disease, allergic dermatitis, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, asthma or schizophrenia.

4. Use of the primer composition of claim 1 in the preparation of a product for detecting and/or assessing a complex disease.

5. A product for detecting and/or assessing a complex disease, 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 SNP sites associated with a complex disease.

8. A method for detecting SNP sites related to complex diseases, which is a non-disease diagnosis and treatment method and is 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

Complex Disease (Complex Disease) is a general name for a Disease with complicated etiology, hidden onset, long course of Disease and persistent Disease. In recent years, the incidence and mortality of chronic complex diseases have been on the rise with the progress of rapid township and aging of population. Epidemiological investigations have shown that the occurrence of chronic complex diseases is closely related to poor lifestyle and behavior. The monitoring of chronic complex diseases can find the health problems of the organism in time and provide correction measures aiming at the related problems. Early screening and early detection of chronic complex diseases are significant for reducing the harm of the chronic complex diseases, the prevention of the chronic complex diseases starts from the prevention and control of risk factors of the chronic complex diseases, and genetic background is one of the risk factors influencing the chronic complex diseases.

The complex diseases are caused by factors of complex regulation and control among multiple genes and environmental action, have obvious characteristics of genetic heterogeneity and phenotype complication, and are commonly seen as neoplastic diseases, cardiovascular diseases, autoimmune diseases, nervous system diseases and the like. Unlike the general monogenic diseases, which do not completely comply with Mendelian's law of inheritance, the development of complex diseases often involves multiple biological processes, such as the dysregulation of the network dynamics of interactions between multiple genes, proteins or related molecules, resulting in specific phenotypes and symptoms of various diseases. The early diagnosis of the complex disease is very difficult, the prognosis effect is poor, and the recurrence rate and the death rate are high, so the method has important practical significance for the research of the complex disease.

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.

The patent CN108334749B discloses a method and a system for detecting the epistasis of a complex disease based on a chromatin regulation loop, wherein the chromatin regulation loop based on gene interaction is established by utilizing data such as chromatin remote interaction, SNP interaction which can influence the phenotype of the complex disease in the regulation loop is calculated according to the chromatin regulation loop, the SNP interaction related to the complex disease can be rapidly and accurately explored, and a potential target is provided for subsequent drug involvement and the like. Patent CN105354444B discloses a method for screening complex disease susceptibility SNP combination based on susceptibility SNP, which can quickly find out susceptibility SNP combination of specific disease from a large amount of data, can be applied to screening of any complex disease susceptibility SNP combination and prediction of disease genetic risk, and provides a new method for disease risk evaluation. However, the above prior art only provides an evaluation method or system for a certain complex disease, and does not provide a detection method or system for a plurality of complex diseases.

Therefore, it is highly desirable to construct a primer composition for related SNP sites capable of simultaneously targeting multiple complex diseases and use thereof, so as to improve the effect of preventing or treating chronic complex diseases.

Disclosure of Invention

Aiming at the defects, the invention provides a complex disease related SNP locus primer composition and application thereof. According to the invention, a group of multiple SNP sites related to the complex disease and amplification primers thereof are designed and developed by an NGS method, and the change of the gene structure related to the complex disease in an 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 complex disease is improved. The evaluation system and the detection method have the advantages of low cost, simple and convenient 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 a complex disease-associated SNP site primer composition, comprising: rs10229583, rs1048886, rs10811661, rs10814916, rs10886471, rs10906115, rs1111875, rs1116357, rs11257655, rs117601636, rs11787792, rs12000501, rs12010175, rs12549902, rs13266634, rs1359790, rs1421085, rs1436955, rs1470579, rs147538848, rs 0315505500, rs163171, rs 16316361329, rs 29955379, rs17584499, rs 177982, rs 18018095, rs1845900, rs187060802, rs1965305, rs 202773617640, rs 4120796, rs2206734, rs2237892, rs2237895, rs 223782237897, rs 22835637799, rs 70779, rs 70777943779, rs 7077563756375637563756375648, rs 70779, rs 7037563756375637563756375637563756375637569, rs 70729, rs 7037563756375637563756375637563756375637563756375637567, rs 703756375637563756375637563756375637563756375637567, rs 70729, rs 7037563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, rs1745, rs 70729, rs 707245, rs1745, rs 7245, rs 70729, rs 729, rs 70729, rs 707245, rs 7072563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756300, rs 729, rs1745, rs 703756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756300, rs1745, rs 729, rs1745, rs 7037563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756300, rs1745, rs 703756375637563756375637563756375637563756375637563756375637563756375637563756375637, rs10883795, rs11038167, rs11038172, rs1635, rs16887244, rs2142731, rs835784, rs10821944, rs 11900600673, rs12379034, rs 12617617617617656, rs13192471, rs1854853, rs2075876, rs2233434, rs2841277, rs2867461, rs3093024, rs3124, rs3781913, rs 65707075, rs10865331, rs13210693, rs17095830, rs30187, rs4552569, rs10036748, rs10845606, rs10847697, rs 8334, rs1167796, rs1234315, rs12494314, rs 128569, rs 13177851338574, rs 1388190, rs1913517, rs 22057596409, rs 2200926, rs 09575637567746, rs 31779, rs 317746, rs 31575648 5648, rs 704746, rs 445646, rs 445648 5646, rs 445648, rs 445646, rs 725648, rs 445648, rs 44575646, rs 725648, rs 445648, rs 445646, rs 725648, rs 445648, rs 725648, rs 445648, rs 725648, rs 445648, rs 725648, rs 445648, rs 725648, rs 445648, rs 725648, rs 445648, rs 725648, rs 445648, rs 725648, rs 445648, rs 445634575648, rs 445648, rs 725634575648, rs 43545648, rs 4456345743545648, rs 445648, rs 725648, rs 445634575634575648, rs 445634575648, rs 445634575634575634575648, rs 445648, rs 445634579, rs 445648, rs 445634575648, rs 445634579, rs 445648, rs 445634575648, rs 445648.

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

TABLE 1 primer compositions

Specifically, the complex disease includes, but is not limited to, type 2 diabetes, hashimoto's thyroiditis, alzheimer's disease, allergic dermatitis, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, asthma, or schizophrenia.

On the other hand, the invention provides the application of the SNP locus primer composition in preparing products for detecting and/or evaluating complex diseases.

In yet another aspect, the present invention provides a product for detecting and/or evaluating a complex disease, which comprises 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 SNP sites related to complex diseases.

In still another aspect, the present invention provides a method for detecting a complex disease-associated SNP site, which is a non-disease diagnosis and treatment method, comprising detecting a SNP site in a test sample genome 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 an SNP locus related to complex diseases and a primer composition thereof, which can simultaneously detect and evaluate the risks of the complex diseases such as type 2 diabetes, hashimoto's thyroiditis, Alzheimer's disease, allergic dermatitis, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, asthma, schizophrenia and the like, thereby guiding people to prevent in advance from the aspects of diet, exercise, living habits and the like and prompting the long-term attention point of routine physical examination in hospitals in the future, and further improving the prevention and treatment effect of the chronic complex 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 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 showing the genetic risk assessment of Alzheimer's disease.

Fig. 3 is a diagram of the genetic risk assessment of adult asthma.

FIG. 4 is a chart of the genetic risk assessment of Classification disorders.

FIG. 5 is a graph of genetic risk assessment of type 2 diabetes.

FIG. 6 is a graph of genetic risk assessment of Hashimoto's thyroiditis.

FIG. 7 is a diagram of the genetic risk assessment of systemic lupus erythematosus.

FIG. 8 is a diagram of the genetic risk assessment of ankylosing spondylitis.

FIG. 9 is a diagram showing the genetic risk assessment of rheumatoid arthritis.

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;

Agencourt AMPure XP Kit：Beckman(A63881)；

Qubit dsDNA 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 (4 ℃ storage), IGT-I5 (5. mu.M);

PCR instrument: ABI 9700 by Life corporation;

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

3.0Fluorometer：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 used3.0fluorometer (qubit dsDNA HS Assay kit) to determine the concentration of the library, record the 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 complex disease and the gene and the detection site is shown in the following table 5.

TABLE 5

Experimental example 1 accuracy test

The 221 sites were tested using the assay method described herein and verified by Sanger sequencing, the results of which are shown in Table 6 below.

TABLE 6 accuracy test results

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 the peripheral blood A is selected to be detected according to the method of the application example 1 and example 2, and the genetic risk of the complex diseases is evaluated, wherein the genetic risk of the sample in the thousand human genome population and the current self-test genome population is shown in figures 2-9 by taking Alzheimer's disease, adult asthma, mental classification disease, type 2 diabetes, Hashimoto's thyroiditis, systemic lupus erythematosus, ankylosing spondylitis and rheumatoid arthritis as examples. Wherein, the abscissa is the natural logarithm of the normalized relative risk OR value, and the ordinate is the probability density of the crowd on the corresponding OR value. Alzheimer's disease OR ═ 1, Rank_{Thousand human genomes}＝20％，Rank_Self-test50 percent; adult asthma OR 1.1825, Rank_{Thousand human genomes}＝3.7％，Rank_Self-test13.6 percent; mental classification OR 1.4222 Rank_{Thousand human genomes}＝85.6％，Rank_Self-test94.97%; type 2 diabetes OR 0.4305, Rank_{Thousand human genomes}＝7％，Rank_Self-test23.6%; hashimoto's thyroiditis OR ═ 1, Rank_{Thousand human genomes}＝0.1％，Rank_Self-test0.2%; systemic lupus erythematosus OR 1.8085, Rank_{Thousand human genomes}＝76.3％，Rank_Self-test51.2 percent; ankylosing spondylitis OR ═ 1.26, Rank_{Thousand human genomes}＝6％，Rank_Self-test3.7 percent; rheumatoid arthritis OR ═ 1.11, Rank_{Thousand human genomes}＝1.9％，Rank_Self-test＝2％。

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

<110> Beijing institute of genomics (national center for bioinformatics)

<120> complex disease related SNP site primer composition and application

<130> 20210127

<160> 442

<170> SIPOSequenceListing 1.0

<210> 1

<211> 35

<212> DNA