Primer set for detecting vitamin B12 metabolism gene mutation and application method thereof

A technology for B12 and vitamins, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of low detection throughput

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

[0004] However, because the conventional PCR reaction only targets one or a few mutation sites of a vitamin B12 metabolism gene, the detection throughput is low

Method used

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  • Primer set for detecting vitamin B12 metabolism gene mutation and application method thereof
  • Primer set for detecting vitamin B12 metabolism gene mutation and application method thereof
  • Primer set for detecting vitamin B12 metabolism gene mutation and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Design and synthesize primer sets

[0086] Step 1.1: According to the hotspot mutation sites of TCN1 gene, GIF gene, CUBN gene, AMN gene, TCN2 gene and CD320 gene related to vitamin B12 metabolism, design upstream and downstream primers for specific amplification gene sequence.

[0087] For the design of primers, according to the hotspot mutation sites of vitamin B12 metabolism genes, Primer Quest and Primer Premier 5.0 were used to design primers and analyze dimers and stem-loop mismatches, and design primers at both ends of the mutation sites. Among them, The annealing temperatures of the 22 pairs of primers were basically consistent.

[0088] The primer set composed of 22 primer pairs provided in this example covers 49 pathogenic gene mutation sites of TCN1 gene, GIF gene, CUBN gene, AMN gene, TCN2 gene and CD320 gene related to vitamin B12 metabolism. Multiple PCR primer sets were designed for different sites / exons, and after pre-experimental screening, the primers...

Embodiment 2

[0096] Genomic DNA is extracted from the sample to be tested as an amplification template

[0097] Step 2.1: The samples to be tested are: EDTA whole blood, buccal swab or dried blood paper sample.

[0098] Step 2.2: Specifically, 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 (IMPLEN, Germany) measure the concentration and purity of DNA, and preserve genomic DNA.

Embodiment 3

[0100] Prepare PCR reaction system

[0101] Step 3.1: Using the genomic DNA obtained in step 2.2 as an amplification template, and using the primer set synthesized in step 1.2, prepare a multiplex PCR reaction system.

[0102] 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.

[0103] It can be understood that 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.

[0104] table 3

[0105] Reagent components Volume / Amount 2×PCR buffer for KODFX 30μl 2mM dNTPs...

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PUM

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Abstract

The invention provides a primer set for detecting a vitamin B12 metabolism gene mutation and an application method thereof. The primer set comprises at least two primer pairs in 22 primer pairs shownin SEQ ID NO.1 to SEQ ID NO.44. According to the primer set, the detection throughput of vitamin B12 metabolism gene detection can be improved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a primer set for detecting mutations in vitamin B12 metabolism genes and an application method thereof. Background technique [0002] Vitamin B12 is an essential nutrient for all human tissues and is key to releasing energy from food. The absorption and metabolism process of vitamin B12 is very complicated, and there are many mutation sites that affect the absorption of vitamin B12. At present, the pathogenic mutations in TCN1 gene, GIF gene, CUBN gene, AMN gene, TCN2 gene and CD320 gene related to vitamin B12 metabolism There are mainly 49 loci. [0003] Currently, polymerase chain reaction (Polymerase Chain Reaction, PCR) detection experiments can be designed for the hotspot mutation sites of vitamin B12 metabolism genes to detect gene mutations. [0004] However, because the conventional PCR reaction only targets one or a few mutation sites of a vitamin B12 metabolism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/125
Inventor 任静静赵方圆智慧芳倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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