rhd-t163p mutant and its detection

A rhd-t163p, 1.rhd-t163p technology, applied in the field of molecular biology, can solve problems such as inability to obtain correct results and difficult judgment of results, and achieve the effect of extensive scientific research application value, high sensitivity and high precision detection

Active Publication Date: 2022-07-05
WUXI NO 5 PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, serological techniques have certain limitations
Due to the influence of disease or other factors, it is difficult to judge the results of red blood cells in some individuals during serological typing; the serological results of patients with chronic long-term blood transfusion sometimes show the phenomenon of "mixed vision"; when red blood cells cannot be obtained or the red blood cell samples are insufficient, Such as fetal blood type identification, forensic identification of remnants, etc., serological testing can not obtain correct results

Method used

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  • rhd-t163p mutant and its detection

Examples

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Effect test

Embodiment 1

[0034] Example 1 Preparation of DNA Template

[0035] Whole blood genomic DNA was extracted using a purchased kit. Specific steps are as follows:

[0036] (1) Take a sterile 2.0 mL centrifuge tube and add 1 mL of cell lysis solution.

[0037] (2) Gently shake the EDTA anticoagulated whole blood sample until thoroughly mixed; then pipette 500 μL of blood sample into the above centrifuge tube containing cell lysate, and gently pour the centrifuge tube 5-6 times to mix.

[0038] (3) Incubate at room temperature for 10 minutes (invert the centrifuge tube 2-3 times to mix evenly).

[0039] (4) Centrifuge at 12000 rpm for 5 minutes at room temperature.

[0040] (5) Use a pipette to slowly remove the supernatant as much as possible, being careful not to aspirate the white matter at the junction of the two phases.

[0041] (6) Mix vigorously with a vortex shaker (Votex) until the leukocytes are resuspended (10-15 seconds).

[0042] (7) Add 300 μL of nuclear lysate to the resuspen...

Embodiment 2

[0052] Embodiment 2RHD487A>C allele detection:

[0053] Instruments: Veriti 96 PCR instrument, BIO-RAD Gel Doc XR+ gel imager (Bole Corporation, USA), gel electrophoresis instrument (Beijing Liuyi Corporation).

[0054] Reagents: QIAamp DNA extraction kit (Qiagen Company, Germany); DNA Isolation Kit extraction kit (Beijing PELFREEZ Company); PCR buffer, dNTP, Taq enzyme (ABI Company, USA); primers were synthesized by Shanghai Sangon Biological Co., Ltd.

[0055] (1) RHD802A>G allele amplification: total reaction volume: 50 μL, containing PCR 5× buffer 10 μL, DNA template 5.0 μL, Taq polymerase (1U / μL) 1.0 μL, MgCl 2 The final concentration is 2.0 mmol / L, the final concentration of dNTP is 200 nmol / L, and the final concentration of specific upper and lower primers is 200 nmol / L, and the total volume is 50 μL by adding sterile double-distilled water.

[0056] Reaction conditions: pre-denaturation at 95°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, followed b...

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Abstract

The invention discloses RhD-T163P mutant and its detection. The invention provides a RhD blood group antigen RhD-T163P mutant RHD487A>C allele site g.25300946A>C mutation. Primer sequences for gene mutation sites were designed, and RHD487A>C allele detection was performed on the region containing gene mutation by gene amplification method.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, in particular to RhD-T163P mutants and their detection. Background technique [0002] Rh blood group is the most complex and polymorphic system in the human red blood cell blood group system, and it is also the main red blood cell blood group that causes clinical transfusion reactions and severe neonatal hemolytic disease. At present, more than 50 kinds of Rh blood group antigens have been found, among which RhD antigen has strong immunogenicity, which is encoded by RHD gene and is the focus of blood group research. Clinically, Rh blood group antigens are divided into RhD positive and RhD negative according to whether D antigen is detected on the surface of red blood cell membrane. [0003] At present, the routine detection method of Rh blood group D antigen is to use serological saline method and indirect anti-human globulin test and absorption and emission test for identifica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12Q1/6881C12Q1/6858C12N15/11
CPCC07K14/47C12Q1/6881C12Q1/6858C12Q2600/156C12Q2600/172C12Q2531/113C12Q2565/125Y02A50/30
Inventor 顾娟邵超鹏王学东王保龙王玥苹姬艳丽马静
Owner WUXI NO 5 PEOPLES HOSPITAL
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