阅读说明：本技术 用于同步检测apoe基因的两个snp位点的基因多态性的方法 (Method for synchronously detecting gene polymorphism of two SNP sites of APOE gene ) 是由 赵方圆 佟洪梅 智慧芳 倪君君 于 2020-04-30 设计创作，主要内容包括：本发明提供一种同步检测APOE基因两个SNP位点的引物对及检测方法,有益于检测通量的提高。本发明-提供用于同步检测APOE基因的两个SNP位点的基因多态性的引物对,包含一对引物,上游引物的核苷酸序列由SEQ ID NO.1所示,下游引物的核苷酸序列由SEQ ID NO.2所示。使用该引物对进行PCR扩增反应时,APOE基因的rs429358和rs7412位点同时进行扩增,即同时扩增包含2个SNP位点的基因片段。本发明通过一次反应可以对APOE基因的rs429358和rs7412位点的基因多态性进行检测。如此,可以同时检测90多例样本,不仅提高了检测效率,也很大程度的节约了成本费用。(The invention provides a primer pair and a detection method for synchronously detecting two SNP sites of an APOE gene, which are beneficial to the improvement of detection flux. The invention provides a primer pair for synchronously detecting gene polymorphism of two SNP sites of an APOE gene, which comprises a pair of primers, wherein the nucleotide sequence of an upstream primer is shown by SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown by SEQ ID NO. 2. When the primer pair is used for PCR amplification reaction, the rs429358 and rs7412 sites of the APOE gene are simultaneously amplified, namely, the gene fragment containing 2 SNP sites is simultaneously amplified. The invention can detect the gene polymorphism of the rs429358 and rs7412 sites of the APOE gene through one reaction. Therefore, more than 90 samples can be detected simultaneously, the detection efficiency is improved, and the cost is greatly saved.)

1. A primer pair for synchronously detecting gene polymorphism of two SNP sites of an APOE gene is characterized by comprising a pair of primers, wherein the nucleotide sequence of an upstream primer is shown by SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown by SEQ ID NO. 2.

2. The primer pair of claim 1, wherein the SNP sites are rs429358 and rs7412 sites of an APOE gene.

3. Use of the primer set according to claim 1 or 2 for the preparation of a kit for simultaneous detection of gene polymorphisms at two SNP sites of an APOE gene.

4. A kit for simultaneously detecting gene polymorphisms at two SNP sites of an APOE gene, which comprises the primer set according to claim 1 or 2.

5. The kit according to claim 4, further comprising a DNA polymerase, a PCR buffer, a mixture of 4 dNTPs, and ultrapure water.

6. The kit according to claim 5, wherein the DNA polymerase is used in an amount of 0.5 to 5U, each dNTP is used at a final concentration of 50 to 500. mu.M, and the primer is used at a final concentration of 20 to 300 nM.

7. The kit according to any one of claims 4 to 6, further comprising a test sample DNA extraction reagent or DNA extraction kit.

8. A method for detecting gene polymorphism of two SNP sites of an APOE gene simultaneously, which is characterized in that a primer pair according to claim 1 or 2 is used for carrying out PCR detection on a sample to be detected.

9. The detection method according to claim 8, characterized by comprising the steps of:

(1) extracting genome DNA from a sample to be detected as an amplification template;

(2) preparing a PCR reaction system containing the primer pair and the amplification template;

(3) carrying out PCR amplification reaction on the PCR reaction system to obtain a PCR product;

(4) and determining the gene polymorphism of two SNP sites of the APOE gene according to the PCR product.

10. The detection method according to claim 9, wherein the reaction conditions of the PCR amplification reaction are: 94 ℃ below zero: 1-10 min; 98 ℃ C: 5-20 s, and 58-68 ℃: 10-60 s, 68-72 ℃: 30 s-3 min, and 25-40 cycles in total; 68-72 ℃: 0-30 min.

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer pair and a detection method for synchronously detecting gene polymorphism of two SNP sites of APOE (apolipoprotein).

Background

The APOE (apolipoprotein) gene is located at 19q13.2, has a full length of 3.7kb and contains 4 exons, and generates APOE functional proteins which are ligands of low density lipoprotein, very low density lipoprotein and chylomicron receptor. The APOE gene is shown in polymorphism in normal population, and 6 different genotypes are formed by two SNP sites: E4/E4 type: rarely, it is associated with senile dementia, sudden death of coronary heart disease, intractable hypertension, cerebral ischemia, type IV hyperlipidemia, hypercholesterolemia and inheritance thereof; E4/E3: rarely, it is associated with senile dementia, hypercholesterolemia, myocardial infarction and high genetic susceptibility; E4/E2 type: rare, moderate frequency of cerebral recurrent bleeding infarct rate, and diabetic nephrotic syndrome incidence; E3/E3: frequently, heart and brain ischemia is low, but hemorrhage and death can recur, the damage of hypertension and viscera is late, and the senile dementia rate is low; E3/E2: the medicine is related to longevity, the senile dementia rate is low, and the patients possibly suffer from heart ischemia diseases, so that the total cholesterol can be increased by the female climacteric hormone replacement therapy; E2/E2 type: rarely, it is manifested as type III hyperlipidemia, early arteriosclerosis, but heart protection, and low incidence of diabetes. That is, there are different types of APOE in the population, and thus different pathogenic states are exhibited, resulting in significant variability in the types and risk frequencies of human disease.

PCR (Polymerase Chain Reaction) has been widely used in medicine, genetics, microbiology, and even throughout life sciences. At present, PCR detection tests can be respectively designed aiming at two polymorphic sites rs429358 and rs7412 of an APOE gene so as to detect the gene mutation condition. Because each PCR reaction only aims at one SNP site, the detection flux is low.

Disclosure of Invention

In order to solve the above-mentioned drawbacks, the present invention provides a primer set and a detection method for synchronously detecting two SNP sites of an APOE gene, which are beneficial to the improvement of detection throughput.

In order to achieve the purpose, the invention is realized by the following technical scheme:

firstly, a primer pair for synchronously detecting the gene polymorphism of two SNP sites of an APOE gene is provided, which comprises a pair of primers, wherein the nucleotide sequence of an upstream primer is shown by SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown by SEQ ID NO. 2. The primer pair is specially designed for specific amplification of the gene containing the rs429358 and rs7412 sites of APOE, can simultaneously, synchronously, efficiently, specifically and accurately amplify a specific gene fragment of the gene at the sites, and detect the gene polymorphism of the gene fragment by sequencing. When the primer pair is used for PCR amplification reaction, the rs429358 and rs7412 sites of the APOE gene are simultaneously amplified, namely, the gene fragment containing 2 SNP sites is simultaneously amplified. The detection flux can be improved to the maximum extent.

Multiple tests prove that the primer pair provided by the invention has better specificity and amplification accuracy, can be applied to the preparation of a kit for synchronously detecting the gene polymorphism of two SNP sites of an APOE gene, and can quickly and accurately obtain the result of the gene polymorphism of the APOE gene at the two sites after a sample is collected and tested by preparing a finished kit.

The kit mainly comprises the primer pair provided by the invention, wherein the final concentration of the primer is preferably 20-300 nM. Other PCR reagents may be selected according to conventional techniques, for example, a preferred embodiment further comprises a DNA polymerase, a PCR buffer, a mixture of 4 kinds of dNTPs (deoxyribose nucleotide triphosphates), and ultrapure water. Wherein the amount of DNA polymerase is 0.5-5U, and the final concentration of each dNTP is 50-500. mu.M.

The DNA polymerase can be Taq, KOD FX, etc., so that the PCR buffer solution is a concentrated buffer solution corresponding to the selected DNA polymerase, and the concentration degree can be 2X, 5X or 10X.

For example, when KOD FX is used as the DNA polymerase and 2 × concentrated buffer is used, the amounts of the components in the PCR system may be: 0.5-2 μ l of DNA polymerase, 18-30 μ l of PCR buffer solution, 1-10 μ l of mixture of various dNTPs, 1-5 μ l of each of upstream and downstream primers, 5-1000 ng of DNA, and an appropriate amount of ultrapure water to supplement water to 50 μ l. And may be other volume sizes formulated in the same proportions.

When the finished product kit is manufactured, a reagent for extracting the DNA of a sample or a professional DNA extraction kit can be selectively prepared according to actual needs; the DNA of the sample to be detected can be obtained more conveniently and quickly, and the convenience and the rapidity of the detection finished product kit are enhanced. The sample to be tested can be any blood, cell, tissue or buccal swab sample containing genomic DNA.

On the basis of the primer pair provided by the invention, the invention further provides a detection method for synchronously detecting the gene polymorphism of two SNP sites of the APOE gene, and the primer pair is adopted for PCR detection.

According to a preferred embodiment, the detection method comprises the following specific steps:

(1) extracting genome DNA from a sample to be detected as an amplification template;

(2) preparing a PCR reaction system containing the primer pair and the amplification template;

(3) carrying out PCR amplification reaction on the PCR reaction system to obtain a PCR product;

(4) and determining the gene polymorphism of two SNP sites of the APOE gene according to the PCR product.

Wherein the most preferable reaction conditions for the PCR amplification reaction are: 94 ℃ below zero: 1-10 min; 98 ℃ C: 5-20 s, and 58-68 ℃: 10-60 s, 68-72 ℃: 30 s-3 min, and 25-40 cycles in total; 68-72 ℃: 0-30 min.

On the basis of the method and the conditions, the detection method can quickly, effectively and conveniently synchronously obtain the gene polymorphism of two SNP loci of the APOE gene of the sample to be detected, most commonly rs429358 and rs7412 loci. May be used for non-diagnostic purposes. In general, when the rs429358 and rs7412 sites of the APOE gene are amplified by using the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2, the length of the corresponding amplified product is 728 bp. The DNA fragment was then recovered by cutting and sequenced.

According to a preferred embodiment of the present invention, the step (4) of determining gene polymorphism comprises: electrophoretically detecting the PCR product to verify the amplified fragment size of the PCR product; and after the verification is correct, performing sequence determination on the PCR product to obtain the gene polymorphism conditions of the rs429358 and rs7412 loci of the APOE gene of the sample to be detected. In detail, the PCR amplified fragment can be detected by agarose gel electrophoresis or polyacrylamide gel electrophoresis.

Compared with the prior art, the invention at least has the following beneficial effects: (1) and (3) improving the detection flux: generally, each PCR reaction only aims at one SNP site, but the invention can simultaneously detect 2 SNP sites of the APOE gene, and can detect the gene polymorphism of the rs429358 and rs7412 sites of the APOE gene through one reaction. Therefore, more than 90 samples can be detected simultaneously, the detection efficiency is improved, and the cost is greatly saved. (2) The cost is reduced: the invention can reduce the PCR reaction system from 2 systems/procedures to 1 system/procedure, thereby reducing the use amount of reagents and consumables such as DNA polymerase, dNTP and the like and greatly reducing the detection cost.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis detection according to an embodiment of the present invention;

FIG. 2 is a diagram showing a portion of a PCR product sequence determination result for a SNP site according to an embodiment of the present invention;

FIG. 3 shows a portion of the PCR product sequence determination result for another SNP site according to an embodiment of the present invention.

Detailed Description

To further illustrate the present invention, reference is made to the following examples. Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.