Type 2 diabetes risk detection kit and detection method

Abstract

The invention belongs to the technical field of type 2 diabetes mellitus, and particularly relates to a type 2 diabetes mellitus risk detection kit and a detection method, the type 2 diabetes mellitus risk detection kit comprises PCR (polymerase chain reaction) amplification primers of SNP (single nucleotide polymorphism) sites: 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. The detection method comprises the following steps: extracting and purifying a sample to obtain a gDNA sample to be detected; in a PCR amplification system, adding a gDNA sample to be detected for amplification; mixing the amplified product with a hybridization reagent system, and carrying out hybridization incubation; scanning the hybridized microarray chip by adopting a scanner, and collecting and recording a fluorescence signal. Through the design of specific primers and probes, the application of the high-density SNP microarray chip and the construction of a weighted genetic risk model, the high-sensitivity and high-specificity detection of the type 2 diabetes related SNP loci is realized; further, the disease risk of the individual type 2 diabetes mellitus is accurately evaluated, and a powerful basis is provided for early prevention and intervention of diseases.

Description

Technical Field

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

[0002] Type 2 diabetes mellitus (T2DM) is a chronic disease caused by insufficient or inefficient insulin secretion, which is common in adults and is also called adult-onset diabetes. This disease is caused by genetics 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 complications occur or during routine physical examinations.

[0003] There are currently a variety of type 2 diabetes testing kits on the market, but many products have problems such as incomplete testing sites, low sensitivity, and poor specificity, which lead to inaccurate and unreliable test results, thus affecting the effective assessment and prevention of type 2 diabetes risks. Summary of the invention

[0004] In order to solve the problems of complex operation, single detection site, incomplete effective sites, excessive detection of invalid sites, etc. in the type 2 diabetes detection kit in the prior art, the present invention provides a type 2 diabetes risk detection kit and a detection method, which realizes high-sensitivity and high-specificity detection of SNP sites related to type 2 diabetes through the design of specific primers and probes, the application of high-density SNP microarray chips and the construction of a weighted genetic risk model, thereby accurately assessing the individual risk of type 2 diabetes, and providing a strong basis for early prevention and intervention of the disease.

[0005] The present invention solves the technical problem by adopting the following technical solutions: The object of the present invention is to provide a type 2 diabetes risk detection kit, comprising PCR amplification primers of SNP sites 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.

[0006] Furthermore, the probes include SEQ IQ NO.5, SEQ IQ NO.6, SEQ IQ NO.11, SEQ IQ NO.12, SEQ IQ NO.17, SEQ IQ NO.18, SEQ IQ NO.23, SEQ IQ NO.24, SEQ IQ NO.29, SEQ IQ NO.30, SEQ IQ NO.35, SEQ IQ NO.36, SEQ IQ NO.41, SEQ IQ NO.42, SEQ IQ NO.47, SEQ IQ NO.48, SEQ IQ NO.53, SEQ IQ NO.54, SEQ IQ NO.59 and SEQ IQ NO.60.

[0007] Furthermore, it also includes universal primer 1: SEQ IQ NO.61, and universal primer 2: SEQ IQ NO.62.

[0008] Furthermore, SNP sites with high susceptibility to type 2 diabetes include rs4712523, rs2237892, rs1801282, rs4402960, rs13266634, rs10906115, rs2383208, rs8050136, rs1111875 and rs1436955.

[0009] A detection method for a type 2 diabetes risk detection kit comprises the following steps: S1. Sample processing: extract and purify the sample to obtain the gDNA sample to be tested; S2. PCR amplification: Add the gDNA sample to be tested into the PCR amplification system for amplification; S3. Hybridization incubation: mixing the amplified product with the hybridization reagent system and performing hybridization incubation; S4. Scanning: Scan the hybridized microarray chip with a scanner to collect and record the fluorescence signal.

[0010] Furthermore, PCR amplification includes a first round of PCR amplification and a second round of PCR amplification, and the first round of PCR amplification system includes dNTPs with a final concentration of 200 μmol / L, MgCl2 with a final concentration of 1.5 mmol / L, 0.05 units / μL of Taq enzyme and PCR amplification primer mixture, and the total reaction volume is 30 μL.

[0011] Furthermore, the reaction conditions for the first round of PCR amplification were: 95°C for 1 minute, followed by 94°C for 20 seconds and 65°C for 2 minutes as one cycle, and the cycle was repeated 25 times, and the reaction was terminated at 4°C.

[0012] Furthermore, after the first round of PCR amplification reaction was completed, a second round of PCR amplification reaction was carried out. The second round of PCR amplification system included the first round of PCR amplification reaction product, universal primer 1, universal primer 2, dNTPs with a final concentration of 200 μmol / L and 0.05 unit / μL of Taq enzyme. The total reaction volume was 30 μl. The reaction conditions for the second round of PCR amplification were: 95°C for 1 minute, followed by a cycle of 94°C for 10 seconds and 65°C for 30 seconds, and the cycle was repeated 35 times, and the reaction was terminated at 4°C.

[0013] Furthermore, the hybridization incubation includes: mixing the hybridization buffer with the second round PCR amplification reaction product, adding it to the microarray chip with the probe sequence fixed on the surface for hybridization, and the reaction conditions are: 45° C., hybridization reaction for 6 hours.

[0014] Furthermore, it also includes analyzing and processing the fluorescence signal: using data ratio analysis, if wild-type Cy3 / mutant Cy5≥2.0, the site is judged to be a wild genotype; if wild-type Cy3 / mutant Cy5≤0.5, the site is judged to be a mutant genotype; if 0.5<wild-type Cy3 / mutant Cy5<2.0, the site is judged to be a heterozygous genotype, and then the genes of the type 2 diabetes detection site are combined, and the risk judgment standard is established by screening the data.

[0015] Through a large number of experimental screening data, the judgment criteria are: .

[0016] Compared with the prior art, the beneficial technical effects of the present invention are: 1. Traditional type 2 diabetes detection methods can usually only detect a single or a few gene sites, while the kit of the present invention can simultaneously detect 10 type 2 diabetes-related SNP sites through a high-density SNP microarray chip, greatly improving the comprehensiveness of the detection.

[0017] 2. The design of specific primers and probes, as well as strict control of experimental conditions, significantly improves the specificity of the test results. When testing a large number of samples, the specificity can be maintained at more than 98%, reducing the occurrence of false positive results.

[0018] 3. The detection kit of the present invention can detect SNP sites with high throughput, greatly reduce the detection cost, conduct a comprehensive risk assessment of type 2 diabetes in the subject, and avoid the occurrence of the disease in a timely and effective manner.

[0019] 4. The primer design method is not limited to the sites involved in the present invention. It can simultaneously detect different susceptibility sites corresponding to the same disease, or it can simultaneously detect different sites of different diseases or even different detection sites of multiple diseases, so as to more comprehensively and accurately detect the genetic susceptibility of the subject to different diseases and different sites.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0022] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0023] The detection kit designed by the present invention mainly comprises: PCR amplification system: This system contains primer pairs consisting of specific upstream and downstream primers for the 10 target SNP sites to be tested for type 2 diabetes. These primers can accurately identify and bind to the target sequence in the genomic DNA, and achieve efficient exponential specific amplification under appropriate conditions. Tables 1-1, 1-2, 1-3, 1-4, and 1-5 are the specific primers and probe sequences corresponding to the 10 sites. In order to achieve fluorescent labeling, universal primers are added after the amplification system to achieve the second round of amplification reaction. The 5' end of the universal primer sequence is modified with different fluorescence (universal primer 1-Cy3, universal primer 2-Cy5, and can also be modified to label other fluorescent, biotin and other fluorescent or luminescent molecules) to adapt to wild-type and mutant amplification.

[0024] Hybridization reagent system: used for purification of amplification products. The purified amplification products interact with enzymes, buffers, etc. during the hybridization incubation process to ensure that the probe and target can bind stably and specifically.

[0025] High-density SNP microarray chip: As the core component of the detection, its solid phase support can be silicon wafer, glass wafer, ceramic wafer, polystyrene wafer, nitrocellulose membrane, nylon membrane or PM membrane, etc. After surface group modification, the probe can be fixed, which greatly improves the throughput and accuracy of the detection.

[0026] Table 1-1 , Table 1-2 , Table 1-3 , Table 1-4 , Table 1-5 .

[0027] The detection process of this kit is as follows: Processing of samples to be tested: Extract and purify biological samples containing genomic DNA, including but not limited to: human peripheral whole blood, sputum, nasopharyngeal swab, pleural effusion and other types of samples, to obtain genomic DNA (gDNA) samples to be tested.

[0028] PCR amplification: After adding the gDNA sample to be tested to the PCR amplification system, the primers can be accurately combined with the target sequence in the DNA through specific amplification conditions. Under the action of polymerases such as Taq enzymes, dNTP (deoxyribonucleotide triphosphate) is used as raw materials to synthesize a large number of target DNA fragments according to the principle of base complementary pairing (A and T pairing, C and G pairing). In order to facilitate subsequent detection, fluorescent labeling technology is used in the second round of PCR amplification, that is, universal primers are labeled with fluorescent groups so that the amplified products have fluorescent signals.

[0029] Hybridization incubation: The amplified product is mixed with the hybridization reagent system and hybridized 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 principle of complementary base pairing. The location and intensity of the binding determine the characteristics of the fluorescent signal detected by the subsequent scan.

[0030] Scanning: Use a professional scanner to scan the hybridized microarray chip to collect and record the fluorescence signals at different positions. By analyzing and processing the intensity, color and other information of the fluorescence signal, the genotype of each SNP site in the test sample can be determined.

[0031] Data analysis and risk assessment: After obtaining the fluorescence data of the chip hybridization point, the ratio of Cy3 / Cy5, also known as the R / G value, is analyzed by data ratio analysis. Generally, there is no significant expression difference in genes within the range of 0.5-2.0, and it is considered that the expression of genes outside this range has changed significantly. According to the above, if wild-type Cy3 / mutant Cy5 ≥ 2.0, the site is judged to be a wild genotype, if wild-type Cy3 / mutant Cy5 ≤ 0.5, the site is judged to be a mutant genotype, and if 0.5 < wild-type Cy3 / mutant Cy5 < 2.0, the site is judged to be a heterozygous genotype. Then, by combining the above 10 detection site genes that are highly susceptible to type 2 diabetes, a risk model is established through a large amount of experimental screening data. The model analysis results show that its ability to predict the risk of type 2 diabetes has reached a high level, and it can effectively screen out high-risk individuals.

[0032] Example 1: Risk Assessment and Testing Process 1. Sample processing: Extract and purify biological samples containing genomic DNA, such as whole blood samples, to obtain genomic DNA (gDNA) samples to be tested, that is: Peripheral venous blood is obtained from the subject, and genomic DNA is extracted using nucleic acid extraction or purification reagents, using Xinbaiji nucleic acid extraction or purification reagents to obtain extracted gDNA samples.

[0033] 2. Preparation of amplification kit Mixed primers: Mix the specific primer pairs (100 μM) of the 10 detection sites in a 1:1 ratio; First round PCR amplification system: The total reaction volume is 30μL. Take 5μL of the primer mixture of the test site, add dNTPs (final concentration 200μmol / L), MgCl2 (final concentration 1.5mmol / L), Taq enzyme (0.05 units / μL), and 2μl of gDNA (50ng / μL) sample. The amplification reaction is carried out using the Tianlong fully automatic medical PCR analysis system (Gentier 96E). The first round of PCR amplification reaction conditions are: 95℃ for 1 minute, then 94℃, 20 seconds, 65℃, 2 minutes as a cycle, and repeat the cycle 25 times, and finally end the reaction at 4℃.

[0034] After the first round of PCR amplification reaction, the second round of PCR amplification reaction was carried out to introduce fluorescent markers. The total reaction volume was 30 μl, including 15 μl of the first round of PCR amplification reaction product, 1 μl of universal primers (10 μM), dNTPs (final concentration 200 μmol / L), and Taq enzyme (0.05 units / μL); the amplification reaction was carried out using the Tianlong fully automatic medical PCR analysis system (Gentier 96E). The second round of PCR amplification reaction conditions were: 95°C for 1 minute, followed by 94°C for 10 seconds and 65°C for 30 seconds as a cycle, and repeated 35 times, and finally ended the reaction at 4°C. The completed amplification product was stored at 4°C for the chip hybridization step.

[0035] 3. Hybridization reagent system: 70μl hybridization buffer (5M tetramethylammonium chloride solution, 100mM Tris solution) is mixed with the second round of PCR amplification reaction products and added to the chip with specific probe sequences fixed on the surface for hybridization. The reaction conditions are: 45℃, hybridization reaction for 6 hours. After hybridization, the chip is cleaned.

[0036] 4. Cleaning: Add an appropriate amount of cleaning solution (30% SSPE, 5% SDS) to the cleaning container and perform shaking cleaning on an oscillator for 5 minutes. After cleaning, add an appropriate amount of re-washing solution (10% SSPE) to the cleaning container and re-wash for another 5 minutes. After cleaning, use a scanner equipped with 532nm and 635nm lasers (Bio Luxscan 10KA) for scanning.

[0037] 5. Data analysis: The scanner scans and reads the fluorescence value of each point on the gene chip at two different laser wavelengths, and uses ratio analysis to calculate the ratio of Cy3 / Cy5, also known as R / G value. Generally, there is no significant expression difference in genes within the range of 0.5-2.0, and it is considered that the expression of genes outside this range has changed significantly. Based on the above, if wild-type Cy3 / mutant Cy5 ≥ 2.0, the site is judged to be a wild genotype, if wild-type Cy3 / mutant Cy5 ≤ 0.5, the site is judged to be a mutant genotype, and if 0.5 < wild-type Cy3 / mutant Cy5 < 2.0, the site is judged to be a heterozygous genotype.

[0038] 6. Result analysis: The present invention combines the above 10 detection site genes that are highly susceptible to type 2 diabetes and obtains the risk determination criteria as shown in Table 2 by screening the experimental data: Table 2 .

[0039] Generate risk assessment results for the implementation example samples according to the risk determination criteria.

[0040] Example 2: Kit composition and detection verification 1. Select three Coriell standard DNA samples with known genotypes (NA12872, NA20764, NA19114). The genotypes of the 10 corresponding loci are shown in Table 3: Table 3 .

[0041] The DNA sample was subjected to the first round of amplification reaction and the second round of amplification reaction by the amplification reagent, and then a hybridization reaction was performed. After the hybridization and the cleaning step, the sample was scanned and the data was analyzed to determine the genotype result. The test results are shown in Table 4: Table 4 .

[0042] Sample 1 (NA12872) was tested 10 times to verify repeatability. It was verified that the results of the 10 repeated tests of sample 1 (NA12872) in this embodiment were consistent with the suppressed genotype results, confirming that the detection kit of the present invention has extremely high accuracy when applied to multiple SNP sites.

[0043] Example 3: Validation and evaluation of the predictive effectiveness of the judgment criteria 1. The validation group samples consisted of 200 subjects. The peripheral venous blood of the 200 subjects was subjected to genomic DNA extraction operation using nucleic acid extraction or purification reagents, and the extracted gDNA samples were obtained using Xinbaiji nucleic acid extraction or purification reagents.

[0044] 2. The DNA sample is subjected to the first round of amplification reaction and the second round of amplification reaction by the amplification reagent, and then a hybridization reaction is performed. After the hybridization and the cleaning step are completed, the sample is scanned and the data is analyzed. The risk assessment results are generated according to the judgment criteria. The results are shown in Table 5: Table 5 .

[0045] The disease status of the above validation group was tracked and the prevalence was calculated. Prevalence = (actual prevalence ÷ sample size) × 100%. The calculation results are shown in Table 6: Table 6 .

[0046] In summary, the present invention provides a type 2 diabetes detection kit based on SNP microarray chip, designs specific primers and probes, provides ideas for multiple amplification and fluorescence labeling in an extremely simple and convenient way, constructs a weighted genetic risk model by detecting 10 gene sites that are highly susceptible to type 2 diabetes, uses the weighted genetic risk model to evaluate the risk of samples, and evaluates the predictive efficiency of the model to determine the predictive ability of the SNP sites detected by the kit for the risk of type 2 diabetes. The present invention has high sensitivity and specificity for the detection of SNP sites related to type 2 diabetes, can accurately evaluate the risk of individual type 2 diabetes, and provides a strong basis for early prevention and intervention of the disease.

[0047] The embodiments of the present invention are described above, but the present invention is not limited to the above-mentioned specific implementation modes. The above-mentioned specific implementation modes are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which are all within the protection of the present invention.

Claims

1. A type 2 diabetes risk detection kit, characterized in that: PCR amplification primers including SNP sites: 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; The SNP sites include rs4712523, rs2237892, rs1801282, rs4402960, rs13266634, rs10906115, rs2383208, rs8050136, rs1111875 and rs1436955.

2. A type 2 diabetes risk detection kit as claimed in claim 1, characterized in that: Also included are probes SEQ IQ NO.5, SEQ IQ NO.6, SEQ IQ NO.11, SEQ IQ NO.12, SEQ IQ NO.17, SEQ IQ NO.18, SEQ IQ NO.23, SEQ IQ NO.24, SEQ IQ NO.29, SEQ IQ NO.30, SEQ IQ NO.35, SEQ IQ NO.36, SEQ IQNO.41, SEQIQNO.42, SEQIQNO.47, SEQIQNO.48, SEQIQNO.53, SEQIQNO.54, SEQIQNO.59 and SEQIQNO.

60.

3. A type 2 diabetes risk detection kit as claimed in claim 1, characterized in that: It also includes universal primer 1: SEQ IQ NO.61, and universal primer 2: SEQ IQ NO.

62.

4. The detection method of a type 2 diabetes risk detection kit according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Sample processing: extract and purify the sample to obtain the gDNA sample to be tested; S2. PCR amplification: Add the gDNA sample to be tested into the PCR amplification system for the first round of PCR amplification and the second round of PCR amplification; S3. Hybridization incubation: mixing the amplified product with the hybridization reagent system and performing hybridization incubation; S4. Scanning: Scan the hybridized microarray chip with a scanner to collect and record the fluorescence signal.

5. The detection method of the type 2 diabetes risk detection kit according to claim 4, characterized in that: The first round of PCR amplification system included dNTPs with a final concentration of 200 μmol / L, MgCl2 with a final concentration of 1.5 mmol / L, 0.05 units / μL of Taq enzyme and PCR amplification primer mixture, and the total reaction volume was 30 μL.

6. The detection method of the type 2 diabetes risk detection kit according to claim 5, characterized in that: The first round of PCR amplification reaction conditions were: 95°C for 1 minute, followed by 94°C for 20 seconds and 65°C for 2 minutes as one cycle, and the cycle was repeated 25 times, and the reaction was terminated at 4°C.

7. The detection method of the type 2 diabetes risk detection kit according to claim 6, characterized in that: After the first round of PCR amplification reaction, a second round of PCR amplification reaction was carried out. The second round of PCR amplification system included the first round of PCR amplification reaction product, universal primer 1, universal primer 2, dNTPs with a final concentration of 200 μmol / L and 0.05 unit / μL Taq enzyme. The total reaction volume was 30 μl. The reaction conditions for the second round of PCR amplification were: 95°C for 1 minute, followed by a cycle of 94°C for 10 seconds and 65°C for 30 seconds, and the cycle was repeated 35 times, and the reaction was terminated at 4°C.

8. The detection method of the type 2 diabetes risk detection kit according to claim 4, characterized in that: The hybridization incubation includes: mixing the hybridization buffer with the second round PCR amplification reaction product, adding it to the microarray chip with the probe sequence fixed on the surface for hybridization, and the reaction conditions are: 45° C., hybridization reaction for 6 hours.

9. The detection method of the type 2 diabetes risk detection kit according to claim 4, characterized in that: It also includes analysis and processing of fluorescence signals: using data ratio analysis, if wild-type Cy3 / mutant Cy5≥2.0, the site is judged to be a wild genotype; if wild-type Cy3 / mutant Cy5≤0.5, the site is judged to be a mutant genotype; if 0.5<wild-type Cy3 / mutant Cy5<2.0, the site is judged to be a heterozygous genotype, and then the genes of type 2 diabetes detection sites are combined, and risk judgment criteria are established by screening data.