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Primer group, kit and method for detecting vitamin D metabolic gene mutation

A vitamin and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as activation failure, calcium and phosphorus imbalance in the body, and weakened affinity

Active Publication Date: 2020-07-03
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gene mutation can weaken the zinc finger structure stability of VDR receptor, so that VDR and 1,25(OH) 2 D. 3 The affinity is weakened, or can not be with 1,25 (OH) 2 D. 3 Specific binding, resulting in the failure of the promoter activation of the gene regulated by the VDR receptor, which in turn leads to an imbalance of calcium and phosphorus in the body

Method used

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  • Primer group, kit and method for detecting vitamin D metabolic gene mutation
  • Primer group, kit and method for detecting vitamin D metabolic gene mutation
  • Primer group, kit and method for detecting vitamin D metabolic gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Design and synthesize primer sets, including the following steps:

[0102] Step 1.1: According to a total of 22 high-frequency mutation sites and their upstream and downstream sequences corresponding to the three target genes of vitamin D metabolism genes CYP24A1, CYP27B1 and VDR, design and specifically amplify the locations of high-frequency mutation sites of vitamin D metabolism genes Upstream and downstream primers for exonic or intronic regions.

[0103] Among them, the 22 high-frequency mutation sites are shown in Table 1 above.

[0104] 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 mutation site, and the annealing temperature of each pair of primers was basically the same.

[0105] Since small sequence changes lead to a significant decrease in primer amplification efficiency and poor specificity, the embodiments of the present inven...

Embodiment 2

[0115] DNA is extracted from the sample to be tested, including the following steps:

[0116] Step 2.1: The sample to be tested can be: oral exfoliated cells collected from the human body with a buccal swab, or biological samples containing human DNA such as fresh peripheral blood collected from the human body.

[0117] Step 2.2: Specifically, use the Tiangen Oral Swab DNA Extraction Kit (DP322), or use the Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304) to extract DNA from the sample, and use the NP80-touch (Germany IMPLEN) measure the concentration and purity of DNA, and preserve the DNA.

Embodiment 3

[0119] A method for amplifying a mutation in a vitamin D metabolism gene, comprising the following steps:

[0120] Step 3.1: Use the DNA obtained in step 2.2 as an amplification template, and use the amplification primer set synthesized in step 1.2 to prepare a multiplex PCR reaction system.

[0121] The embodiment of the present invention uses the DNA polymerase and buffer in the KOD FX enzyme system (article number: KFX-101) of TOYOBO Company as the basic raw materials, and adjusts the concentration of the primers on the basis of the amplification system in the instruction manual of the enzyme system. , dNTP concentration, buffer concentration, and enzyme dosage to prepare a multiplex PCR amplification system. The specific composition of this reaction system is shown in Table 4 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 a...

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Abstract

The invention provides a primer group, a kit and a method for detecting vitamin D metabolic gene mutation. The primer group comprises ten primer pairs of which the nucleotide sequences of upstream anddownstream primers are shown as SEQ ID NO.1-20 respectively. When the primer group is used for amplifying vitamin D metabolic genes; a plurality of exon and intron regions of the vitamin D metabolicgenes CYP24A1, CYP27B1 and VDR can be simultaneously amplified through one-time reaction, so that a plurality of high-frequency mutation sites of the vitamin D metabolic genes CYP24A1, CYP27B1 and VDRcan be simultaneously detected.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer set, a kit and a method for detecting mutations in vitamin D metabolism genes. Background technique [0002] Vitamin D (Vitamin D) is a kind of fat-soluble vitamin and a multifunctional hormone that can maintain the health of the body. It plays an important role in controlling the level of calcium and phosphorus in the body and bone calcification, and it is also important for the extraskeletal system, including the cardiovascular system. , immune system, nervous system, glucose metabolism, cell proliferation and differentiation, etc., all play a very important role. [0003] Vitamin D can be obtained through diet and sunlight exposure. Vitamin D itself does not have an active form. It needs two steps of hydroxylation to become a biologically active substance, which can regulate blood calcium and blood phosphorus in the human body. The absorption, transport...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/16C12Q2600/156C12Q2531/113C12Q2537/143C12Q2565/125
Inventor 李晓娇赵方圆智慧芳倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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