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Primer group for detection of vitamin E metabolism gene TTPA (tocopherol (Alpha) transfer protein) and multiple PCR (polymerase chain reaction) detection method

A vitamin and primer set technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of low detection throughput, improve detection efficiency, reduce detection cost, and save costs Effect

Active Publication Date: 2019-09-20
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Low detection throughput since each PCR reaction targets only one exon

Method used

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  • Primer group for detection of vitamin E metabolism gene TTPA (tocopherol (Alpha) transfer protein) and multiple PCR (polymerase chain reaction) detection method
  • Primer group for detection of vitamin E metabolism gene TTPA (tocopherol (Alpha) transfer protein) and multiple PCR (polymerase chain reaction) detection method
  • Primer group for detection of vitamin E metabolism gene TTPA (tocopherol (Alpha) transfer protein) and multiple PCR (polymerase chain reaction) detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Design and synthesize primer sets, including the following steps:

[0040] Step 1.1: Based on the 25 mutation sites of exons 1-5 of the TTPA gene, design upstream and downstream primers for specifically amplifying the gene mutation site region.

[0041] For the design of primers, Primer Quest and Primer Premier 5.0 were used to design primers and analyze dimers and stem-loop mismatches. Primers were designed at both ends of the SNP (Single Nucleotide Polymorphism) site, and the annealing temperature of the five pairs of primers was basically maintained. unanimous.

[0042] The primer set provided in this example covers exons 1-5 of the TTPA gene and the pathogenic sites therein. Since small sequence changes will lead to a significant decrease in primer amplification efficiency and poor specificity, multiple PCR primer sets were designed for different sites / exons, and after pre-experimental screening, the length and position of the product fragments were integrated. Ac...

Embodiment 2

[0047] Genomic DNA is extracted from the sample to be tested, comprising the following steps:

[0048] Step 2.1: Collect oral exfoliated cells or fresh peripheral blood samples with oral swabs.

[0049] In this embodiment, the sample source is a normal human body, that is, a non-AVED patient.

[0050] Step 2.2: Use Tiangen Oral Swab Genomic DNA Extraction Kit (DP322), or, use Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304) to extract genomic DNA from the sample, and use NP80-touch (Germany IMPLEN ) to determine the concentration and purity of DNA, and to preserve genomic DNA.

Embodiment 3

[0052] A multiplex PCR detection method for synchronous detection of 5 exons of the vitamin E metabolism gene TTPA, comprising the following steps:

[0053] Step 3.1: Use the genomic DNA obtained in step 2.2 as an amplification template, and use the primer set synthesized in step 1.2 to configure a multiplex PCR reaction system.

[0054] In this example, the DNA polymerase and buffer in the KOD FX enzyme system (article number: KFX-101) of TOYOBO Co., Ltd. were used as the basic raw materials. concentration, buffer concentration, and enzyme dosage to prepare a multiplex PCR amplification system. The specific composition of this reaction system is shown in Table 3 below. Of course, the proportional amplification / reduction of the reaction system is within the protection scope of the embodiment of the present invention; the purpose of amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate ratio.

[0055] table 3

[0056] ...

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Abstract

The invention provides a primer group for detection of vitamin E metabolism gene TTPA (tocopherol (Alpha) transfer protein) and a multiple PCR (polymerase chain reaction) detection method. The primer group includes at least two of five primer pairs for detecting first to five exons of vitamin E metabolism gene TTPA respectively; nucleotide sequences of upstream and downstream primers to the five primer pairs are shown as SEQ ID NO. 1 to 10 respectively. When applied to multiple PCR detection, the primer group is suitable for detecting gene mutations of at least two exons through a single reaction, so that the detection flux is increased.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer set and multiplex PCR detection method for detecting vitamin E metabolism gene TTPA. Background technique [0002] Vitamin E can be divided into 8 types due to their different structures: α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol Enols and δ-tocotrienols, among which α-tocopherols have physiological functions. [0003] Vitamin E exists in the membranous structure of cells and lipid droplets and lipoproteins of adipocytes, and achieves antioxidation by blocking the chain oxidation reaction between peroxide free radicals and unsaturated fatty acids in biofilms. Moreover, vitamin E can assist sulfhydryl groups to scavenge free radicals. Some studies believe that vitamin E can also protect the central and peripheral nerves by regulating the neurotoxicity of glutamic acid. [0004] Vitamin E transport in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/16C12Q2600/156
Inventor 赵方圆佟洪梅智慧芳倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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