CN116356010A - Application of gene SNP detection kit in prediction of blood lipid metabolism level of type 2 diabetes patient - Google Patents

Application of gene SNP detection kit in prediction of blood lipid metabolism level of type 2 diabetes patient Download PDF

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CN116356010A
CN116356010A CN202211444148.5A CN202211444148A CN116356010A CN 116356010 A CN116356010 A CN 116356010A CN 202211444148 A CN202211444148 A CN 202211444148A CN 116356010 A CN116356010 A CN 116356010A
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李妍
李越
杨祉妍
杨蕊
董中华
王昕宇
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First Affiliated Hospital of Shandong First Medical University
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Abstract

The invention discloses an application of a gene SNP detection kit in predicting the blood lipid metabolism level of a type 2 diabetes patient, wherein the kit comprises a forward primer of rs3765467, a reverse primer of rs3765467, a forward primer of rs10305420 and a reverse primer of rs10305420, and the nucleotide sequences of the kit are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, and are the sequencing of the blood lipid metabolism coding genes of the type 2 diabetes patient in a non-disease diagnosis method. The invention detects GLP1R gene polymorphism sites related to blood lipid metabolism level of type 2 diabetes patients by utilizing Polymerase Chain Reaction (PCR) technology, and provides technical support for researching GLP1R gene polymorphism of type 2 diabetes people and scientifically predicting blood lipid metabolism level of T2DM patients.

Description

Application of gene SNP detection kit in prediction of blood lipid metabolism level of type 2 diabetes patient
Technical Field
The invention belongs to the technical field of blood lipid level prediction, and particularly relates to application of a gene SNP detection kit in predicting the blood lipid metabolism level of a type 2 diabetic patient.
Background
Type 2 diabetes mellitus (T2 DM) is an endocrine and metabolic disease characterized by hyperglycemia, and T2DM patients exhibit impaired insulin secretion or reduced function.
Glucagon-like peptide-1 (GLP-1) is an endogenous incretin secreted by intestinal L cells and plays an important role in maintaining blood glucose balance. GLP-1 promotes insulin synthesis and secretion, inhibits islet beta cell apoptosis, and promotes beta cell proliferation in a glucose-dependent manner by binding to and activating GLP-1 receptor (GLP-1R). GLP-1R belongs to one member of the G protein coupled receptor family, consists of 463 amino acids, contains 7 transmembrane domains, and is a therapeutic target of type 2 diabetes therapeutic drugs glucagon-like peptide-1 receptor agonists (GLP 1 RAs). GLP-1R is composed ofGLP-1RGene coding, the occurrence of which may affect the structural composition of GLP-1R and thus further affect receptor function, both sites rs10305420 and rs3765467 are located on chromosome 6 short arm (chr 6939048860 (GRCh38. P12)). The gene polymorphism at the locus rs10305420 causes proline at the locus 7 of the receptor to be changed into leucine, and the polymorphism at the locus rs3765467 causes arginine at the locus 132 of the receptor to be replaced by glutamine.
The function of GLP-1R has important reference value for predicting insulin secretion, pharmacological actions of GLP-1 and analogues thereof and the like.GLP-1RThe mutation sites are more, and the Single Nucleotide Polypeptide (SNP) site with the highest mutation frequency in the eastern Asian population is rs3765467 (G)>A) And rs10305420 (C)>T), the minimum gene frequencies were 22.7% and 13.7%, respectively. The research shows that after GLP1RAs treatment is received, rs3765467 wild type (GG type) patients can obtain better hypoglycemic and weight-reducing curative effects than rs3765467 mutant (GA/AA type) patients. Another study showed that rs10305420 (CT/TT type) is obese in Europe compared to rs10305420 (CC type)Reduced response to liraglutide (a GLP-RA) in humans and women with polycystic ovary syndrome is associated.
Currently, for the followingGLP-1RGene studies have focused mainly on blood glucose-related levels, butGLP-1RGenes exhibit a non-negligible effect on lipid metabolism. The increased risk of insulin resistance associated with obesity leads the adipose tissue of a patient with T2DM to show specific lipidomic characteristics, so that the control of T2DM not only needs to reduce blood sugar, but also needs to formulate reasonable comprehensive management targets of blood lipid reduction, blood pressure reduction, weight reduction and the like. For patients who have been diagnosed with T2DM, and for whom the presence or absence of lipid metabolism disorders has not been determined, it is necessary to scientifically predict future trends in their blood lipid levels.
Disclosure of Invention
Aiming at the problem that the blood lipid level of a type 2 diabetes patient lacks research and predictive factors in the prior art, the invention provides the application of a gene SNP detection kit in predicting the blood lipid metabolism level of the type 2 diabetes patient, and the blood lipid metabolism level of the type 2 diabetes patient is related by utilizing the Polymerase Chain Reaction (PCR) technologyGLP1RThe gene polymorphism sites are detected, so that the development trend of the blood lipid metabolism of a patient can be predicted, and guidance is provided for formulating a blood glucose and blood lipid management dosing scheme.
The invention is realized by the following technical scheme:
the application of a gene SNP detection kit in predicting the blood lipid metabolism level of a type 2 diabetic patient comprises a rs3765467 forward primer, a rs3765467 reverse primer, a rs10305420 forward primer and a rs10305420 reverse primer, wherein the nucleotide sequences of the kit are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4; the application is the sequencing of the blood lipid metabolism coding gene of the type 2 diabetes patients in a non-disease diagnosis method.
Further, the kit also comprises a detection reagent, a detection chip and a detection carrier.
Further, the detection reagent comprises Taq DNA polymerase, dNTP mixture, buffer DE-A, buffer DE-B, buffer W1, buffer W2, eluent, bigDye 2.5Xand BigDye Seq Buffer.
Further, the kit also comprises a resin for purification, a sample application and a palladium sheet for mass spectrometry detection and a human genome DNA extraction reagent.
Further, the using method of the kit comprises the following steps:
(1) Performing PCR amplification on a sample to be detected by using a primer group in the kit to obtain a first amplification product;
(2) Purifying the first amplification product;
(3) Re-amplifying the purified first amplified product to obtain a second amplified product;
(4) Sequencing the second amplification product.
Further, the PCR amplification system in the step (1) is as follows:
Figure 153591DEST_PATH_IMAGE001
。
further, the PCR amplification procedure in step (1) is:
Figure 133049DEST_PATH_IMAGE002
。
further, the PCR amplification system in the step (3) is as follows:
Figure 975103DEST_PATH_IMAGE003
。
further, the PCR amplification procedure in step (3) is:
Figure 432629DEST_PATH_IMAGE004
。
furthermore, the blood lipid metabolism level of the type 2 diabetes patient is predicted by using the sequencing result of the gene, which is favorable for scientifically predicting the lipid metabolism change trend of the T2DM patient, and an optimal scheme for blood lipid management is formulated in clinical practice.
Advantageous effects
The invention utilizes PCR technology for blood lipid metabolism level correlation of type 2 diabetes patientsGLP1RGene polymorphism site detection for researching type 2 diabetes patient groupGLP1RThe gene polymorphism and scientific prediction of the development trend of the blood lipid metabolism level of the T2DM patient provide technical support, are favorable for scientific prediction of the disease process of the T2DM patient, formulate an optimal scheme for blood lipid management in clinical practice, save medical cost, and provide guidance basis for the research and development of new drugs based on pharmacogenomics concept.
Drawings
FIG. 1 shows the two-locus polymorphism peak patterns obtained by detecting the amplified fragment of the rs10305420 (C > T) gene locus by Sanger dideoxy termination sequencing, wherein the two-locus polymorphism peak patterns are rs10305420 (heterozygote CT), rs10305420 (wild type CC) and rs10305420 (mutant homozygote TT) genotypes from left to right; FIG. 2 shows the two-locus polymorphism peak diagram obtained by detecting the amplified fragment of rs3765467 (G > A) gene locus through Sanger dideoxy termination sequencing; once from left to right, the rs3765467 (wild type GG), rs3765467 (heterozygote GA), rs3765467 (mutant homozygote AA) genotypes.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The gene SNP detection kit comprises specific primer sequences corresponding to rs3765467 and rs10305420 as shown in the following table 1, and comprises a rs3765467 forward primer, a rs3765467 reverse primer, a rs10305420 forward primer and a rs10305420 reverse primer, wherein the nucleotide sequences of the specific primer sequences are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
Table 1 Gene SNP detection kit specific primer sequence
Figure 43739DEST_PATH_IMAGE005
。
Example 2
Extraction of sample DNA
Collecting 2ml of peripheral venous blood of a type 2 diabetes patient, placing the peripheral venous blood in an EDTA anticoagulation tube, and immediately and reversely mixing the peripheral venous blood for 8 times after taking the blood so as to ensure that the blood and the anticoagulation agent are fully mixed; DNA extraction was performed according to blood genomic DNA extraction kit DP348 (China Tiangen Co., ltd.); DNA concentration determination was performed using a Siemens Nanodrop 4000 spectrophotometer. The extracted sample DNA concentration is diluted to 10 ng/. Mu.L by sterilized deionized water and used as a DNA template.
Example 3
Gene detection
The gene polymorphism of GLP1R rs3765467, rs10305420 was detected by using an ABI3730XL PCR sequencer according to the method of instruction.
3.1 one amplification
(1) The amplification system is shown in Table 2 below
TABLE 2 one-time amplification PCR amplification System
Figure 335043DEST_PATH_IMAGE006
；
(2) The amplification procedure is shown in Table 3 below
TABLE 3 PCR amplification procedure
Figure 664393DEST_PATH_IMAGE007
。
3.2 PCR product purification
(1) Cutting agarose gel containing target DNA (3.1 amplified DNA) in an ultra clean bench under irradiation of ultraviolet lamp with long wave of more than 300nm, then sucking the liquid on the surface of agarose gel with clean paper towel and cutting it up, calculating weight of agarose gel, and taking the gel with the weight as a gel volume (1 mg =1 μl);
(2) Adding 3 Buffer DE-A with gel volume, mixing Buffer DE-A with gel uniformly, heating at 75deg.C to melt gel block, intermittently oscillating and mixing uniformly to make gel block fully melt;
(3) Adding 0.5 Buffer DE-A volume of Buffer DE-B after the gel block is completely melted, and fully and uniformly mixing;
(4) Transferring the mixed solution prepared in the step 3 into a DNA preparation tube, placing the DNA preparation tube into a 2mL EP tube, placing the EP tube into a centrifuge for centrifugation at 12000r/min for 1min, and discarding the filtrate;
(5) The preparation tube was placed back into a 2mL EP tube, 500 μLBuffer W1 was added to the preparation tube, and then placed into a centrifuge for centrifugation at 12000r/min for 30s, and the filtrate was discarded;
(6) The preparation tube was placed back into a 2mL EP tube, 700. Mu.L Buffer W2 was added, and the mixture was centrifuged at 12000r/min for 30s, and the filtrate was decanted; adding 700 μL Buffer W2, centrifuging at 12000r/min for 1min, and discarding the filtrate;
(7) Placing the preparation tube back into the EP tube of 2mL, then placing the preparation tube into a centrifuge for centrifugation at 12000r/min for 1min, taking out the preparation tube, and discarding 2mL of the EP tube and the filtrate;
(8) Placing the preparation tube in a clean EP tube of 1.5 mL, dripping 30 mu L of Eluent or deionized water in the center of the preparation film, standing for 1min at room temperature, and centrifuging for 1min at 12000r/min in a centrifuge;
(9) The preparation tube was discarded to obtain purified DNA.
3.3 reamplification of the PCR products after purification
(1) The re-amplification system is shown in Table 4 below
TABLE 4 Secondary amplification PCR amplification System
Figure 925610DEST_PATH_IMAGE008
；
(2) The procedure for re-amplification PCR amplification is shown in Table 5 below
TABLE 5 re-amplification PCR amplification procedure
Figure 453543DEST_PATH_IMAGE009
。
3.4 Sequencing of PCR products
(1) Adding 30 mu LPCR re-amplified product into 96 well plates, sequentially adding 125mmol/L EDTA and 3mol/L NaAc 2 mu L each and 100% alcohol 50 mu L into each well of the 96 well plates, covering the 96 well plates, shaking for 4 times, and standing at room temperature for 15min;
(2) Putting the 96-well plate into a plate type centrifuge, centrifuging for 30min at 3000r/min at 4 ℃, immediately inverting the 96-well plate, centrifuging to 185r/min, closing the power supply of the centrifuge, and stopping centrifuging;
(3) Adding 70 mu L of 70% alcohol into a 96-well plate, centrifuging for 15min at 3000r/min at 4 ℃, immediately inverting the 96-well plate, centrifuging until 185r/min, turning off the power supply of a centrifuge, and stopping centrifuging; this step was repeated 1 time;
(4) Placing a 96-well plate at room temperature for a period of time, and adding 10 mu L of Hi-Di Formamide to dissolve DNA after alcohol naturally volatilizes cleanly;
(5) The pretreated sample was placed on a PCR instrument for denaturation: 4min at 95℃and 4min at 4 ℃;
(6) Placing the processed sample plate in an ABI3730XL sequencer;
(7) Opening the software of 3730 Data Collection v3.0 in the ABI3730XL sequencer and selecting a type Sequencing Analysis starting program;
(8) After electrophoresis, obtaining a sequencing result file;
(9) And analyzing and calibrating the sequencing result file.
(10) Amplified fragments containing the rs10305420 (C > T) and rs3765467 (G > A) gene loci were detected by Sanger dideoxy termination sequencing, and the two-locus polymorphism peak diagram results are shown in FIG. 1 and FIG. 2. Rs10305420 (heterozygote CT), rs10305420 (wild type CC), rs10305420 (mutant homozygote TT) genotypes are sequentially from left to right; FIG. 2 shows the two-locus polymorphism peak diagram obtained by detecting the amplified fragment of rs3765467 (G > A) gene locus through Sanger dideoxy termination sequencing; the genotypes of rs3765467 (wild GG), rs3765467 (heterozygote GA) and rs3765467 (mutant homozygote AA) are sequentially shown from left to right.
Genotyping was performed using the methods described in examples 1-3, and the results are shown in Table 6 below.
TABLE 6 genotyping detection results statistics
Figure 243645DEST_PATH_IMAGE010
；
The results of the detection of the index of blood lipid metabolism of the wild type rs10305420 and the wild type rs3765467 and the mutant type rs3765467 (heterozygous mutant type and pure and mutant type) at two gene loci are shown in the following table 7, and it is clear from the table that the gene polymorphism of rs10305420 is related to the HDL-C level of high density lipoprotein (HDL-C) and the HDL-C level of CC genotype patients is higher than that of T allele carriers; the gene polymorphism of rs3765467 correlated with Triacylglycerol (TG) levels in GG genotype patients with TG levels lower than a allele carriers, and this difference was only present in type 2 diabetics and was not found in healthy volunteers.
Table 7 blood lipid metabolism index of rs3765467 and rs10305420 wild type and mutant type 2 diabetes patients
Figure 732395DEST_PATH_IMAGE011
；
The invention provides technical support for predicting blood lipid metabolism of type 2 diabetes people in China and lays a genetic foundation for personalized pharmacogenomics.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art without affecting the spirit of the invention, within the scope of the claims.

Claims (10)

1. The application of the gene SNP detection kit in predicting the blood lipid metabolism level of a type 2 diabetic patient is characterized in that the kit comprises a forward primer of rs3765467, a reverse primer of rs3765467, a forward primer of rs10305420 and a reverse primer of rs10305420, and the nucleotide sequences of the kit are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4; the application is the sequencing of the blood lipid metabolism coding gene of the type 2 diabetes patients in a non-disease diagnosis method.
2. The use of claim 1, wherein the kit further comprises a detection reagent, a detection chip and a detection carrier.
3. The use according to claim 2, wherein the detection reagent comprises Taq DNA polymerase, dNTP mixture, buffer DE-A, buffer DE-B, buffer W1, buffer W2, eluent, bigDye 2.5.5X and BigDye Seq Buffer 5.5X.
4. The use according to claim 2, wherein the kit further comprises a resin for purification, a palladium chip for spotting and mass spectrometry, and a reagent for extraction of human genomic DNA.
5. The use according to claim 1, wherein the method of using the kit comprises the steps of:
(1) Performing PCR amplification on a sample to be detected by using a primer group in the kit to obtain a first amplification product;
(2) Purifying the first amplification product;
(3) Re-amplifying the purified first amplified product to obtain a second amplified product;
(4) Sequencing the second amplification product.
6. The use according to claim 5, wherein the PCR amplification system in step (1) is:
Figure 984661DEST_PATH_IMAGE001
。
7. the use according to claim 5, wherein the PCR amplification procedure in step (1) is:
Figure 495277DEST_PATH_IMAGE002
。
8. the use according to claim 5, wherein the PCR amplification system in step (3) is:
Figure 337331DEST_PATH_IMAGE003
。
9. the use according to claim 5, wherein the PCR amplification procedure in step (3) is:
Figure 591595DEST_PATH_IMAGE004
。
10. the use according to claim 1, wherein the sequencing result of the gene is used to predict the blood lipid metabolism level in a type 2 diabetic patient.
CN202211444148.5A 2022-11-18 2022-11-18 Application of gene SNP detection kit in prediction of blood lipid metabolism level of type 2 diabetes patient Pending CN116356010A (en)

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