RH blood type DEL-type RHD93T>A allele and detection method thereof

A technology of alleles and detection methods, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve problems such as cumbersome operations, inability to obtain correct results, and unsuitability for large-scale clinical applications, and achieve high sensitivity And the effect of high-precision detection and extensive scientific research application value

Inactive Publication Date: 2014-08-06
WUXI NO 5 PEOPLES HOSPITAL
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AI Technical Summary

Problems solved by technology

This method is not only cumbersome to operate, but also unstable
In addition, 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"; Sometimes, such as fetal blood group identification, forensic identification of leftovers, etc., serological tests cannot obtain correct results
[0005] The detection of DEL blood type by immunoserological method depends on the specificity and operation method of the anti-D antibody used, the reliability is low, and it is not suitable for large-scale clinical application due to cumbersome steps. The reliable method is to apply gene Detection method to detect DEL blood type

Method used

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  • RH blood type DEL-type RHD93T>A allele and detection method thereof
  • RH blood type DEL-type RHD93T>A allele and detection method thereof
  • RH blood type DEL-type RHD93T>A allele and detection method thereof

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Experimental program
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Embodiment 1

[0025] The preparation of embodiment 1 DNA template

[0026] The preparation process of the DNA template adopts the purchased kit to extract whole blood genomic DNA, and the specific steps are as follows:

[0027] (1) Take a sterile 2.0mL centrifuge tube and add 1mL of cell lysate.

[0028] (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.

[0029] (3) Incubate at room temperature for 10 minutes (during this period, invert the centrifuge tube 2-3 times to mix well).

[0030] (4) Centrifuge at room temperature for 5 minutes at 12000 rpm.

[0031] (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.

[0032] (6) Vigorously mix with a vortex shaker (Votex) until the le...

Embodiment 2

[0043] Example 2 RHD93T>A allele detection technical scheme:

[0044] Instruments: Veriti96 PCR instrument, BIO-RAD Gel Doc XR+ gel imager (Bio-Rad, USA), gel electrophoresis (Beijing Liuyi Company).

[0045] Reagents: QIAamp DNA Extraction Kit (Qiagen, Germany); DNA Isolation Kit Extraction Kit (Beijing PELFREEZ Company); PCR buffer, dNTP, Taq enzyme (ABI Company, USA); primers and probes were purchased from Shanghai Sangon Biotechnology Co., Ltd. synthesis.

[0046] (1) Primer design: According to the RHD gene (serial number: BN000065) recorded in GenBank of the National Center for Biological Information (NCBI) and the sequences of various RHD alleles in Chinese, primers were designed by Oligo6.0 primer software, and finally 1 For specific oligonucleotide primer sequences (RHD93A-F, GCTCTCATTC TCCTCTTCTA TTTTTTA; RHD93A-R, CCTGCTATTT GCTCCTGTGA CCACTT), the length of the amplified product fragment is 125bp; and a pair of internal reference primer sequences were introduced a...

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Abstract

The invention relates to an RH blood type DEL-type RHD93T>A allele and a detection method thereof, and belongs to the technical field of molecular biology analysis and detection. According to the detection method, primer sequences capable of amplifying areas including target sites with mutant gene sequences are designed through a PCR-specific primer technology; PCR amplification is selectively conducted on the areas of the target sites including the mutant genes through a gene amplification method so as to specially detect specific RHD93T>A allele. The detection method adopts a molecular biology method for gene-level detection, so that the detection sensitivity and specificity are improved, and the detection method is simple, convenient and fast. Due to differences in RHD gene of different ethnic groups, the detection method is designed specially for RHD93T>A allele on the basis of correlational research and according to the Chinese RHD gene molecule background. The RH blood type DEL-type RHD93T>A allele and the detection method thereof not only are of important clinical practical significance in making up for deficiencies of the serological technique, but also has a wide application value on scientific research.

Description

technical field [0001] The invention relates to a RHD93T>A allele and a detection method thereof, in particular to a method for doping a known mutant gene in a wild-type gene cluster, and belongs to the technical field of molecular biology analysis and 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] In 1984, Japanese scholar Okubo et al....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12Q1/68
Inventor 王学东顾娟邵超鹏王俊潘兆麟黄利华裴豪陆雁
Owner WUXI NO 5 PEOPLES HOSPITAL
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