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Primer, kit and method for detecting PIEZO1 gene mutation

A technology for sequencing primers and genes, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as easy missed or misdiagnosed, strong phenotypic heterogeneity, and limited diagnostic methods. Achieve the effect of reducing cost and difficulty, difficult detection and high cost

Pending Publication Date: 2021-03-16
CHANGSHA ADICON CLINICAL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The heterogeneity of Hst clinical phenotype is strong and the diagnostic methods are limited. It is easy to miss or misdiagnose, so that inappropriate splenectomy is performed, which will increase the risk of serious thrombosis

Method used

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  • Primer, kit and method for detecting PIEZO1 gene mutation
  • Primer, kit and method for detecting PIEZO1 gene mutation
  • Primer, kit and method for detecting PIEZO1 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Primers for detecting PIEZO1 gene mutation sites, which are designed for the amplification of PIEZO1 exons, including:

[0053] The primers for amplifying the whole exon sequence of PIEZO1 gene, its base sequence is:

[0054]

[0055]

[0056]

[0057] Kits for detecting PIEZO1 gene mutations, including:

[0058] (i) Blood DNA extraction reagents;

[0059] (ii) detection system PCR reaction solution;

[0060] (iii) Sequencing system reagents;

[0061] Among them, the tissue DNA extraction reagent can be purchased from commercial reagents such as Tiangen DNA extraction kit.

[0062] The detection system PCR amplification reaction solution includes: 2×PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U / μl); the concentration of the upstream and downstream primers of the exon sequence of the PIEZO1 gene is 10μM.

[0063] Sequencing system reagents include: sequencing purification solution (ExoI: 0.6U, CIP: 1.2U), EDTA (125mM), absolute ethanol, 75% ethanol, HIDI ...

Embodiment 2

[0065] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):

[0066] (1) Extract tissue DNA from blood: 1) Extract 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inverting, and place at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 12,000rpm for 1min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed. 2) Add 20 μl proteinase K solution and mix well. 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap. 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap. 5) Add the solution and flocculent precipitate obtained in the previous step ...

Embodiment 3

[0093] One case of clinical samples was taken to extract genome, prepare reagents, amplify and sequence according to the reagents and methods of Examples 1 and 2. Add 1 μl of sample to each detection system PCR reaction solution. Electrophoresis results such as figure 2 , 3 , 4 shows that the primers PIEZO1-1F / R, PIEZO1-2F / R, PIEZO1-3-4F / R, PIEZO1-5F / R, PIEZO1-6F / R, PIEZO1-7F / R, PIEZO1 of the present invention -8-10F / R, PIZEO1-11-12F / R, PIEZO1-13-15F / R, PIEZO1-16F / R, PIEZO1-17F / R, PIEZO1-18-19F / R, PIEZO1-20-21F / R , PIEZO1-23F / R, PIEZO1-24-25F / R, PIEZO1-26-27F / R, PIEZO1-28-29F / R, PIEZO1-30F / R, PIEZO1-31F / R, PIEZO1-32-33F / R, PIEZO1-35-36F / R, PIEZO1-37F / R, PIEZO1-38F / R, PIEZO1-39F / R, PIEZO1-40-42F / R, PIEZO1-42-43F / R, PIEZO1-44F / R, PIEZO1-45-47F / R, PIEZO1-47-48F / R, PIEZO1-49-50F / R, PIEZO1-51F / R can effectively amplify blood samples with bright bands.

[0094] The sequencing results of this sample showed that there were 11 mutation sites, and each mutation site is shown in t...

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Abstract

The invention relates to a primer and method for detecting PIEZO1 gene mutation. The primer comprises a primer for amplifying an exon sequence of a PIEZO1 gene, and a Sanger sequencing technology anda sequencing primer are adopted. According to the invention, the mutation of an exon of the PIEZO1 gene can be rapidly detected. The primer, the kit and the method provided by the invention are accurate in detection result, can assist in diagnosing hereditary mouth polycythemia, and have important reference significance for disease diagnosis.

Description

technical field [0001] The invention belongs to the field of molecular detection, in particular to a primer, a kit and a detection method for detecting PIEZO1 gene mutation. Background technique [0002] Hereditary stomatocytosis (HSt) is a group of rare hereditary hemolytic anemia characterized by abnormal ion permeability of red blood cell membrane and morphological changes of mature red blood cells. Different proportions of stomatocytosis appear in the blood. Erythrocytes are red blood cells characterized by wide transverse clefts or mouth shapes, which are found in various acquired genetic diseases. In hereditary stomatocytosis, the proportion of stomatocytosis is greater than 5% (normally less than 4%), up to 10% to 50%. [0003] Hereditary stomatocytosis includes dehydrating HSt, also known as hereditary xerocytosis (Hx), and hydrating HSt, and Hx is the most common subtype of HSt. The diagnosis of Hx is mainly based on clinical features and peripheral red blood cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/6883C12Q1/686C12Q2600/156
Inventor 刘赵玲王淑一
Owner CHANGSHA ADICON CLINICAL LAB
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