A Kit and Detection Method for Detecting the Risk of Type 2 Diabetes

Abstract

The present invention belongs to the technical field of type 2 diabetes, and specifically relates to a type 2 diabetes risk detection kit and detection method, including PCR amplification primers SEQ IQ NO.1-4, SEQ IQ NO.7-10, SEQ IQ NO.13-16, SEQ IQ NO.19-22, SEQ IQ NO.25-28, SEQ IQ NO.31-34, SEQ IQ NO.37-40, SEQ IQ NO.43-46, SEQ IQ NO.49-52 and SEQ IQ NO.55-58 of SNP sites. The detection method includes: extracting and purifying a sample to obtain a gDNA sample to be detected; adding the gDNA sample to be detected into a PCR amplification system for amplification; mixing the amplified product with a hybridization reagent system for hybridization incubation; scanning the hybridized microarray chip with a scanner, collecting and recording the fluorescence signal. Through the design of specific primers and probes, the application of a high-density SNP microarray chip, and the construction of a weighted genetic risk model, the present invention realizes the high-sensitivity and high-specificity detection of type 2 diabetes-related SNP sites, and further accurately evaluates the risk of an individual suffering from type 2 diabetes, providing a strong basis for the early prevention and intervention of the disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of type 2 diabetes, and particularly relates to a kit and a detection method for detecting the risk of type 2 diabetes. Background Art

[0002] Type 2 diabetes (T2DM) is a chronic disease caused by insufficient insulin secretion or reduced efficiency, commonly found in adults, also known as adult-onset diabetes. This disease is caused by a combination of genetic and environmental factors such as lifestyle, overnutrition, and lack of physical activity. The initial symptoms are often mild, and many people are not diagnosed until they develop complications or during a routine physical examination.

[0003] Currently, there are already various kits for detecting type 2 diabetes on the market. However, many products have problems such as insufficient comprehensive detection sites, low sensitivity, and poor specificity, resulting in inaccurate and unreliable detection results, which in turn affect the effective assessment and prevention of the risk of type 2 diabetes. Summary of the Invention

[0004] In order to solve the problems existing in the prior art kits for detecting type 2 diabetes, such as complex operation, single detection site, incomplete effective sites, and over-detection of ineffective sites, the present invention provides a kit and a detection method for detecting the risk of type 2 diabetes. Through the design of specific primers and probes, the application of a high-density SNP microarray chip, and the construction of a weighted genetic risk model, high-sensitivity and high-specificity detection of type 2 diabetes-related SNP sites can be achieved, thereby accurately assessing the risk of an individual suffering from type 2 diabetes and providing a strong basis for early prevention and intervention of the disease.

[0005] The present invention solves its technical problems by adopting the following technical solutions:

[0006] The object of the present invention is to provide a kit for detecting the risk of type 2 diabetes, including PCR amplification primers SEQ IQ NO.1-4, SEQ IQ NO.7-10, SEQ IQ NO.13-16, SEQ IQ NO.19-22, SEQ IQ NO.25-28, SEQ IQ NO.31-34, SEQ IQ NO.37-40, SEQ IQ NO.43-46, SEQ IQ NO.49-52, and SEQ IQ NO.55-58 for SNP sites.

[0007] Further, it also includes probes SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.59 and SEQ ID NO.60.

[0008] Further, it also includes universal primer 1: SEQ ID NO.61, and universal primer 2: SEQ ID NO.62.

[0009] Further, the SNP sites highly susceptible to type 2 diabetes include rs4712523, rs2237892, rs1801282, rs4402960, rs13266634, rs10906115, rs2383208, rs8050136, rs1111875 and rs1436955.

[0010] A detection method for a type 2 diabetes risk detection kit includes the following steps:

[0011] S1. Treatment of the sample to be tested: Extract and purify the sample to obtain the gDNA sample to be tested;

[0012] S2. PCR amplification: Add the gDNA sample to be tested to the PCR amplification system for amplification;

[0013] S3. Hybridization incubation: Mix the amplified product with the hybridization reagent system for hybridization incubation;

[0014] S4. Scanner scanning: Use a scanner to scan the hybridized microarray chip, and collect and record the fluorescence signal.

[0015] Further, the PCR amplification includes the first-round PCR amplification and the second-round PCR amplification. The first-round PCR amplification system includes dNTPs with a final concentration of 200 μmol / L, MgCl2 with a final concentration of 1.5 mmol / L, Taq enzyme at 0.05 unit / μL, and a PCR amplification primer mixture, and the total reaction volume is 30 μL.

[0016] Further, the reaction conditions for the first round of PCR amplification are as follows: reacting at 95°C for 1 minute, then taking 94°C for 20 seconds and 65°C for 2 minutes as one cycle, repeating this cycle 25 times, and finally ending the reaction at 4°C.

[0017] Further, after the first round of PCR amplification reaction, the second round of PCR amplification reaction is carried out. The second-round PCR amplification system includes the product of the first-round PCR amplification reaction, universal primer 1, universal primer 2, dNTPs with a final concentration of 200 μmol / L, and Taq enzyme at 0.05 unit / μL. The total reaction volume is 30 μl. The reaction conditions for the second-round PCR amplification are as follows: reacting at 95°C for 1 minute, then taking 94°C for 10 seconds and 65°C for 30 seconds as one cycle, repeating this cycle 35 times, and finally ending the reaction at 4°C.

[0018] Further, the hybridization incubation includes: after mixing the hybridization buffer with the product of the second-round PCR amplification reaction, adding it to the microarray chip with the probe sequence immobilized on the surface for hybridization. The reaction conditions are: 45°C, and the hybridization reaction lasts for 6 hours.

[0019] Further, it also includes analyzing and processing the fluorescence signal: using data ratio analysis, if wild-type Cy3 / mutant Cy5 ≥ 2.0, then it is determined that this site is of the wild-type genotype; if wild-type Cy3 / mutant Cy5 ≤ 0.5, then it is determined that this site is of the mutant genotype; if 0.5 < wild-type Cy3 / mutant Cy5 < 2.0, then it is determined that this site is of the heterozygous genotype. Then, the genes at the detection sites for type 2 diabetes are combined, and a risk determination standard is established by screening the data.

[0020] Through screening a large amount of experimental data, the determination standard is as follows:

[0021] 。

[0022] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0023] 1. Traditional detection methods for type 2 diabetes can usually only detect single or a few gene loci, while the kit of the present invention can simultaneously detect 10 SNP loci related to type 2 diabetes through a high-density SNP microarray chip, greatly improving the comprehensiveness of detection.

[0024] 2. The design of specific primers and probes, as well as the strict control of experimental conditions, significantly improves the specificity of the detection results. When detecting a large number of samples, the specificity can be maintained above 98%, reducing the occurrence of false positive results.

[0025] 3. The detection kit of the present invention can detect SNP sites with high throughput, greatly reducing the detection cost, comprehensively assessing the risk of type 2 diabetes in the subjects, and timely and effectively avoiding the occurrence of the disease.

[0026] 4. The primer design method is not limited to the sites involved in the present invention. It can simultaneously detect different susceptible sites corresponding to the same disease, or simultaneously detect different sites of different diseases or even different detection sites of multiple diseases, and can more comprehensively and accurately detect the gene susceptibility of different diseases and different sites of the subjects.

[0027] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above content, its purpose, features and advantages of the present invention more obvious and understandable, the following specifically illustrates the specific embodiments of the present invention. Specific Embodiments

[0028] The following further details the technical solution of the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only for exemplarily illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0029] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.

[0030] The detection kit designed by the present invention mainly includes:

[0031] PCR amplification system: This system contains primer pairs composed of specific upstream and downstream primers for 10 target SNP sites for detecting type 2 diabetes to be tested. These primers can accurately recognize and bind to the target sequences in genomic DNA, and achieve efficient exponential specific amplification under suitable conditions. Tables 1-1, 1-2, 1-3, 1-4, and 1-5 show the specific primer and probe sequences corresponding to the 10 sites respectively. In order to achieve fluorescence labeling, universal primers are added after the amplification system to achieve the second-round amplification reaction. Different fluorescences are modified at the 5' end of the universal primer sequences (Universal Primer 1 - Cy3, Universal Primer 2 - Cy5, and other fluorescences or luminescent molecules such as biotin can also be modified and labeled) to adapt to the amplification of wild type and mutant types.

[0032] Hybridization reagent system: It is used for the purification of amplification products, and the purified amplification products interact with enzymes, buffers, etc. during the hybridization incubation process to ensure that the probes can stably and specifically bind to the targets.

[0033] High-density SNP microarray chip: As the core component of detection, its solid-phase support can be a silicon wafer, glass slide, ceramic chip, polystyrene sheet, nitrocellulose membrane, nylon membrane, PM membrane, etc. After surface group modification, probes can be immobilized, greatly improving the detection throughput and accuracy.

[0034] Table 1-1

[0035] ,

[0036] Table 1-2

[0037] Table 1-3 ,

[0038] Table 1-4

[0039] Table 1-5

[0040] The detection process of this kit is as follows:

[0041] Treatment of the sample to be tested: Extract and purify a biological sample containing genomic DNA. The biological sample includes, but is not limited to, human peripheral whole blood, sputum, nasopharyngeal swab, pleural effusion and other types of samples to obtain a genomic DNA (gDNA) sample to be detected.

[0042] PCR amplification: After adding the gDNA sample to be tested to the PCR amplification system, through specific amplification conditions, the primers can accurately bind to the target sequence in the DNA. Under the action of polymerases such as Taq enzyme, using dNTP (deoxyribonucleotide triphosphate) as raw material, a large number of target DNA fragments are synthesized according to the base complementary pairing principle (A pairs with T, C pairs with G). For the convenience of subsequent detection, in the second round of PCR amplification, a fluorescence labeling technique is adopted, that is, the universal primers are labeled with fluorescent groups so that the amplification products carry fluorescent signals.

[0043] Hybridization incubation: Mix the amplified product with the hybridization reagent system and carry out hybridization incubation under specific conditions such as temperature and time. In this process, the amplified product DNA fragments will specifically bind to the probes immobilized on the surface of the high-density SNP microarray chip through the base complementary pairing principle. The binding position and intensity determine the characteristics of the fluorescent signals detected by subsequent scanning.

[0044] Scanner Scanning: Use a professional scanner to scan the hybridized microarray chip, collect and record the fluorescence signals at different positions. By analyzing and processing information such as the intensity and color of the fluorescence signals, the genotypes of each SNP locus in the test sample can be determined.

[0045] Data Analysis and Risk Assessment: After obtaining the fluorescence data of the chip hybridization points, use data ratio analysis, and take the ratio of Cy3 / Cy5, also known as the R / G value. Generally, genes within the range of 0.5 - 2.0 do not show significant expression differences, and gene expression is considered to have changed significantly outside this range. Based on the above, if wild-type Cy3 / mutant Cy5 ≥ 2.0, then this locus is determined to be of the wild genotype; if wild-type Cy3 / mutant Cy5 ≤ 0.5, then this locus is determined to be of the mutant genotype; if 0.5 < wild-type Cy3 / mutant Cy5 < 2.0, then this locus is determined to be of the heterozygous genotype. Then, by combining the genes of the above 10 detection loci highly susceptible to type 2 diabetes, a risk model is established through a large number of experimental screening data. The analysis results of the model show that its predictive ability for the risk of type 2 diabetes reaches a relatively high level, and it can effectively screen out high-risk individuals.

[0046] Example 1: Risk Assessment Detection Process

[0047] 1. Treatment of Test Samples: Extract and purify a biological sample containing genomic DNA, such as a whole blood sample, to obtain a genomic DNA (gDNA) sample to be detected, that is:

[0048] Take the peripheral venous blood of the subject, perform genomic DNA extraction operation according to the nucleic acid extraction or purification reagent, and use the Xinbaoji nucleic acid extraction or purification reagent to obtain the extracted gDNA sample.

[0049] 2. Preparation of Amplification Kit

[0050] Mix Primers: Mix the specific primer pairs (100 μM) of 10 detection loci in a 1:1 ratio;

[0051] First-round PCR Amplification System: The total reaction volume is 30 μL. Take 5 μL of the primer mixture of the locus to be detected, add dNTPs (final concentration 200 μmol / L), MgCl2 (final concentration 1.5 mmol / L), Taq enzyme (0.05 unit / μL), and 2 μL of the gDNA (50 ng / μL) sample. The amplification reaction is carried out using the Tianlong automatic medical PCR analysis system (Gentier 96E). The reaction conditions for the first-round PCR amplification are: react at 95°C for 1 minute, then take 94°C for 20 seconds and 65°C for 2 minutes as a cycle, and repeat the cycle 25 times, and finally end the reaction at 4°C.

[0052] After the first round of PCR amplification reaction, the second round of PCR amplification reaction is carried out, introducing fluorescent labeling. The total reaction volume is 30 μl, including 15 μl of the product of the first round of PCR amplification reaction, 1 μl of universal primer (10 μM), adding dNTPs (final concentration 200 μmol / L), Taq enzyme (0.05 unit / μL); the amplification reaction is carried out using Tianlong automatic medical PCR analysis system (Gentier 96E). The conditions for the second round of PCR amplification reaction are: reacting at 95 °C for 1 minute, then taking 94 °C for 10 seconds and 65 °C for 30 seconds as a cycle, and repeating the cycle 35 times, and finally ending the reaction at 4 °C. The amplified product after the reaction is completed is stored at 4 °C for the chip hybridization step.

[0053] 3. Hybridization reagent system: After mixing 70 μl of hybridization buffer (5M tetramethylammonium chloride solution, 100 mM Tris solution) with the product of the second round of PCR amplification reaction, it is added to the chip with specific probe sequences immobilized on the surface for hybridization. The reaction conditions are: 45 °C, and the hybridization reaction is 6 hours. After hybridization, the chip is cleaned.

[0054] 4. Cleaning: Add an appropriate amount of cleaning solution (30% SSPE, 5% SDS) to the cleaning container and perform oscillating cleaning on the shaker for 5 minutes. After the cleaning is completed, an appropriate amount of re-washing solution (10% SSPE) can be added to the cleaning container again for re-washing for 5 minutes. After the cleaning is completed, a scanner equipped with 532 nm and 635 nm lasers (Biochip Luxscan 10KA) is used for scanning.

[0055] 5. Data analysis: The scanner scans and reads the fluorescence values of each point on the gene chip at two different laser wavelengths respectively, and uses ratio analysis for the ratio of Cy3 / Cy5, also known as the R / G value. Generally, genes within the range of 0.5 - 2.0 do not have significant expression differences, and gene expression is considered to have a significant change outside this range. Based on the above, if wild-type Cy3 / mutant Cy5 ≥ 2.0, then this site is judged as the wild genotype; if wild-type Cy3 / mutant Cy5 ≤ 0.5, then this site is judged as the mutant genotype; if 0.5 < wild-type Cy3 / mutant Cy5 < 2.0, then this site is judged as the heterozygous genotype.

[0056] 6. Result analysis: In the present invention, by combining the above 10 genes of detection sites highly susceptible to type 2 diabetes and screening the experimental data, the risk determination criteria are obtained as shown in Table 2 below:

[0057] Table 2

[0058] 。

[0059] Generate a risk assessment result for the example sample according to the risk judgment criteria.

[0060] Example 2: Composition of the kit and detection verification

[0061] 1. Select 3 Coriell standard DNA samples (NA12872, NA20764, NA19114) with known genotypes. The genotypes of 10 corresponding loci are as shown in Table 3 below:

[0062] Table 3

[0063] 。

[0064] After subjecting the DNA sample to the first-round amplification reaction and the second-round amplification reaction with the amplification reagent, perform a hybridization reaction. After the hybridization is completed and the washing step is finished, perform scanning and data analysis to determine the genotype result. The detection results are as shown in Table 4 below:

[0065] Table 4

[0066] 。

[0067] And perform 10 repeated detections on Sample 1 (NA12872) to achieve repeatability verification. After verification, the 10 repeated detection results of Sample 1 (NA12872) in this example are consistent with the inhibitory genotype result, confirming that the detection kit in the present invention has extremely high accuracy when applied to multiple SNP loci.

[0068] Example 3: Verification and evaluation of the prediction efficacy of the judgment criteria

[0069] 1. A verification group sample consisting of 200 subjects is formed. Genomic DNA extraction operations are performed on the peripheral venous blood of the 200 subjects respectively according to the nucleic acid extraction or purification reagent, and the new Baike nucleic acid extraction or purification reagent is used to obtain the extracted gDNA sample.

[0070] 2. After subjecting the DNA sample to the first-round amplification reaction and the second-round amplification reaction with the amplification reagent, perform a hybridization reaction. After the hybridization is completed and the washing step is finished, perform scanning and data analysis, and generate a risk assessment result according to the judgment criteria. The results are as shown in Table 5 below:

[0071] Table 5

[0072] 。

[0073] Track the disease status of the above verification group population, calculate the prevalence rate, prevalence rate = (actual number of patients ÷ sample size) × 100%, and the calculation results are as shown in Table 6 below:

[0074] Table 6

[0075] 。

[0076] In summary, a type 2 diabetes detection kit based on SNP microarray chips provided by the present invention designs specific primers and probes, provides ideas for multiplex amplification and fluorescence labeling in a very simple and convenient manner, detects 10 gene loci highly susceptible to type 2 diabetes, constructs a weighted genetic risk model, uses this weighted genetic risk model to evaluate the risk of samples, and evaluates the prediction efficacy of the model to determine the predictive ability of the SNP loci detected by this kit for the risk of type 2 diabetes. The present invention has high sensitivity and specificity for the detection of SNP loci related to type 2 diabetes, can accurately evaluate the risk of an individual having type 2 diabetes, and provides a strong basis for the early prevention and intervention of the disease.

[0077] The above describes the embodiments of the present invention, but the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. These all fall within the protection scope of the present invention.

Claims

1. A type 2 diabetes risk detection kit, characterized in that: PCR amplification primers including SNP sites SEQ ID NO.1-4, SEQ ID NO.7-10, SEQ ID NO.13-16, SEQ ID NO.19-22, SEQ ID NO.25-28, SEQ ID NO.31-34, SEQ ID NO.37-40, SEQ ID NO.43-46, SEQ ID NO.49-52 and SEQ ID NO.55-58; The SNP sites include rs4712523, rs2237892, rs1801282, rs4402960, rs13266634, rs10906115, rs2383208, rs8050136, rs1111875, and rs1436955; Also included are probes SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.59 and SEQ ID NO.60; Also included is universal primer 1: SEQ ID NO.61, and universal primer 2: SEQ ID NO.62.

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