Reagent kit for detecting diabetes

A diabetes and kit technology, applied in the field of detection, can solve the problems of less research on pyruvate dehydrogenase phosphatase and unclear functional localization, etc.

Active Publication Date: 2019-04-02
广东创晟控股集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there are few studies on pyruvate dehydroge

Method used

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  • Reagent kit for detecting diabetes
  • Reagent kit for detecting diabetes
  • Reagent kit for detecting diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Sample Selection and DNA Extraction

[0022] 1.1 Object

[0023] From January 2016 to December 2017, 120 patients with type 2 diabetes treated in Yichun People's Hospital were collected as the experimental group; 120 normal people who underwent physical examination during the same period were selected as the control group. The subjects in the two groups were all Chinese Han nationality, male and female Half, and there was no statistically significant difference between ages (P>0.05). All subjects had no family history of other genetic diseases and tumor history. There was no genetic relationship among the subjects, and all subjects gave informed consent.

[0024] 1.2 Specimen source

[0025] 2 mL of peripheral blood was collected from the two groups of subjects, anticoagulated with sodium citrate and stored in a low-temperature refrigerator at -70 °C.

[0026] Genomic DNA is extracted from whole blood, and the extraction method and steps of genomic DNA are a...

Embodiment 2

[0033] Embodiment 2PCR amplification detection

[0034] 1.1 Design and synthesis of primers

[0035] Primers are specific primers and probes that can detect the SNP site through numerous designs and optimizations:

[0036] Upstream primer sequence: 5'-atacattttttagcctaa-3',

[0037] Downstream primer sequence: 5'-gatctccttatttaaacta-3',

[0038] Probe: 5'-gtaattgaaagattttctatttt-3'; the 3' end of the probe is blocked to avoid extension during the reaction, and the primers are synthesized by Shanghai Shenggong Company.

[0039] 1.2 HRM typing based on quantitative PCR

[0040] In the reaction system, the concentration ratio of upstream and downstream primers is 1:10, that is, in a 10 μL reaction system, the final concentration of upstream primers is 0.3 pmol,

[0041] The final concentration of downstream primers is 3 pmol, and the concentration of probe is 3 pmol. The amplification length is 300 bp, and the probe is specific and complementary to G. The total volume of PCR re...

Embodiment 3

[0055] Embodiment 3 kit preparation and actual detection

[0056] The probes and primers of the present invention are prepared into a kit according to the conventional storage concentrations of primers and probes in the art. Take 5 blood samples from diabetics and 6 blood samples from normal people, and carry out DNA extraction and PCR detection according to the same method as before. The curves of 11 samples are as follows: figure 1 As shown in the dissolution curve diagram of the test sample; there are 6 SNPs in the test samples with GG phenotype, 1 SNP with CC phenotype in total, and 4 cases with SNP in samples with CG phenotype, that is to say, CC phenotype plus CG A total of 5 cases were phenotyped, and the PCR test results also corresponded to the results of the samples being diabetic, indicating that the detection method has good accuracy.

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PUM

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Abstract

The invention provides a reagent kit for detecting diabetes. The reagent kit contains an SNP site relevant to properties of type II diabetes, the site is the 166th of PDP1 of which the nucleotide sequence is SEQID NO:1, and a basic group is G or C. The invention also provides a probe and typing primer for detecting the SNP site. The site polymorphism is detected in human diabetes potential groupsthrough a high-resolution melting curve technique, and relevance of the genotype frequency of the site and the properties of the diabetes is analyzed. The site can be used as a fast detection index tobe used for large-scale potential patient screening.

Description

technical field [0001] The invention belongs to the field of detection, in particular to a detection kit for diabetes. Background technique [0002] Pyruvate dehydrogenase kinase (also known as pyruvate dehydrogenase complex kinase, PDC kinase, or PDHK) is a kinase that inactivates pyruvate dehydrogenase by using ATP to phosphorylate it. [0003] The pyruvate dehydrogenase complex (pyruvate dehydrogenase Complex, PDC) in the mitochondria can catalyze the oxidative decarboxylation of pyruvate to generate NADH and acetyl-CoA, and closely links glycolysis with the tricarboxylic acid cycle and the generation of ATP. This important response is critical for tissues that require large amounts of ATP (eg, brain, muscle). Acetyl-CoA produced by PDC is also very important for fat-producing tissues such as liver and fat, because acetyl-CoA in mitochondria can enter the synthesis process of fatty acids through the formation of citric acid (the precursor of cellular acetyl-CoA) . It i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 巫满香张玲
Owner 广东创晟控股集团有限公司
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