Nucleic acid sequence for detecting diabetes risk site, kit and detection method thereof

A nucleic acid sequence and nucleotide sequence technology, applied in the field of nucleic acid sequence detection of diabetes risk sites, can solve the problems of restricted development and application, easy pollution, complicated operation, etc., to avoid non-specific amplification problems, and to achieve a high degree of automation , the effect of simplifying the operation process

Inactive Publication Date: 2018-08-03
PRIMBIO GENES BIOTECH WUHAN CO LTD
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Sanger sequencing method is the gold standard for mutation analysis, which can find known and unknown mutation sites, but the instrument is expensive, the sensitivity is low, the operation is complicated, the cycle is long, and it is easy to contaminate; the ordinary PCR-RFLP method is simple in technology, cheap in price, and suitable for a small amount of samples laboratory testing, but it can only detect mutations with enzyme cleavage sites, and cannot be detected without enzy...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Nucleic acid sequence for detecting diabetes risk site, kit and detection method thereof
  • Nucleic acid sequence for detecting diabetes risk site, kit and detection method thereof
  • Nucleic acid sequence for detecting diabetes risk site, kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Design of primers and verification templates for 5 polymorphic sites in diabetes

[0045] The ARMS primers selected in the present invention are designed for five polymorphic sites of diabetes (rs228648, rs114202595, rs7754840, rs7756992 and rs7574865). The selected ARMS primers are a set of primer combinations artificially added with mismatched mutation sites at the 3' end of a sequence and its vicinity. By screening primers capable of distinguishing alleles at a single site, the accuracy of PCR amplification is ensured. Highly specific.

[0046] After experimental verification, the specific detection primers for the 5 polymorphic sites of diabetes were finally determined, and the nucleotide sequences were as follows:

[0047] UST2 gene rs228648 site wild-type upstream primer (DM1-F1), mutant upstream primer (DM1-F2) and common downstream primer (DM1-R1)

[0048] DM1-F1 (SEQ ID NO: 1): 5`-tgctcggactcataaatcctt-3`

[0049] DM1-F2 (SEQ ID NO:2): 5`-tgctcgga...

Embodiment 2

[0093] Embodiment 2: Preparation of 5 polymorphism kits of diabetes

[0094] For the five polymorphic sites, the wild-type upstream primers, mutant upstream primers and common downstream primers designed in Example 1 were used for synthesis, and SYBR Green I dye was used for staining, which is a dye that binds to dsDNA The dye with green excitation wavelength in the minor groove region of the double helix, in the free state, SYBR Green I emits weak fluorescence, but once combined with double-stranded DNA, the fluorescence is greatly enhanced, so the fluorescence intensity of SYBR Green I is related to the amount of double-stranded DNA Correlation, so the amount of double-stranded DNA present in the PCR system can be detected based on the fluorescent signal.

[0095] A kit for detecting diabetes risk loci, including 10 PCR reaction solutions, positive controls, negative controls, and chromogenic reagents.

[0096] There are 10 kinds of PCR reaction liquids among the present in...

Embodiment 3

[0107] Embodiment 3: Detect 5 polymorphic sites of diabetes with the kit prepared by the present invention

[0108]For the determination of genomic DNA, almost any biological sample that contains genomic DNA (eg, impure red blood cells) can be used. For example, and without limitation, genomic DNA may be conveniently obtained from blood, semen, saliva, tears, urine, feces, sweat, buccal cells, skin or hair. In order to utilize DNA assays, the target nucleic acid must be obtained from a cell or tissue expressing the target sequence.

[0109] The sample to be tested in this embodiment is DNA extracted from 10 cases of oral swabs collected by Wuhan Liangpei Gene Biotechnology Co., Ltd. and quantified as a template for PCR detection. For the main operation steps of DNA extraction, refer to Tiange Company’s buccal swab DNA extraction kit to extract genomic DNA from samples. The purity and concentration of the extracted genomic DNA were calculated by an ultraviolet spectrophotomet...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a nucleic acid sequence for detecting a diabetes risk site, a kit and a detection method thereof. A nucleic acid sequence combination disclosed by the invention is proposed specific to five polymorphic sites (rs228648, rs114202595, rs7754840, rs7756992 and rs7574865) of diabetes, and comprises a wild upstream primer, a mutant upstream primer and a universal downstream primer of each site. The kit for rapidly detecting the diabetes risk site and prepared from the nucleic acid sequence combination has the characteristics of convenience in use, easiness and convenience inoperation, high automation degree, lower pollution in an operation process, good detection effect, high sensitivity, high specificity, high accuracy and high precision. The detection method disclosedby the invention is convenient and rapid to use through adoption of a complete closed-tube operation; a detection result is obtained by directly exploring a fluorescent signal value in a PCR (Polymerase Chain Reaction) process, so that PCR posttreatment or electrophoresis detection is not required, the technical problems of great probability of pollution and false positive value generation in theconventional PCR technology are solved, and non-specific amplification can be effectively avoided; the detection method is suitable for large-scale sample detection.

Description

technical field [0001] The invention relates to the fields of medicine and biotechnology, in particular to a nucleic acid sequence for detecting diabetes risk sites, a kit and a detection method thereof. Background technique [0002] Diabetes mellitus is a polygenic disease in which both genetic and environmental factors participate in the interaction. Both type 1 and type 2 diabetes have obvious genetic heterogeneity. Type 1 diabetes mellitus (T1DM) is an immunomodulatory disease, which is caused by the production of inflammatory cytokines by immune dysregulation, which induces inflammation in pancreatic islets. Type 2 diabetes mellitus (T2DM) is a complex polygenic disease that results in hyperglycemia due to insulin resistance and β-cell secretion defects. More than 90% of diabetic patients have type 2 diabetes. [0003] UST2 (rs228648) affects intracellular Ca after binding to the G protein-coupled receptor GPR14 2+ Therefore, the serum UTS2 of T2DM patients is signifi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 周艳琳
Owner PRIMBIO GENES BIOTECH WUHAN CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap