RhD-T163P mutant and detection thereof

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

Active Publication Date: 2021-06-11
WUXI NO 5 PEOPLES HOSPITAL
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  • Summary
  • 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 detection thereof
  • RhD-T163P mutant and detection thereof
  • RhD-T163P mutant and detection thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation of embodiment 1 DNA template

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

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

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

[0038] (3) Incubate at room temperature for 10 minutes (during this period, invert the centrifuge tube 2-3 times to mix well).

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

[0040] (5) Use a pipette to slowly remove the supernatant as much as possible, and be careful not to suck out the white substance at the junction of the two phases.

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

[0042] ...

Embodiment 2

[0052] Example 2 RHD487A>C allele detection:

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

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

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

[0056] Reaction conditions: pre-denaturation at 95°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, followed by annealing at 61°C for 40 seconds, an...

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Abstract

The invention discloses an RhD-T163P mutant and detection thereof. The invention provides an RhD blood type antigen RhD-T163P mutant RHD487A>C allele locus g.25300946>C mutation. The method comprises the following steps: designing a primer sequence aiming at a gene mutation site, and carrying out RHD487A>C allele locus detection on a region containing gene mutation by virtue of a gene amplification method.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(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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