Single-nucleotide polymorphism locus of ABO blood variant inducing hemolytic transfusion reaction
A technology of ABO blood type and blood transfusion reaction, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/testing, etc., to achieve rapid detection and avoid the effect of acute hemolytic transfusion reaction
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Embodiment 1
[0026] Example 1: Confirmation of the mutation site of type A variant from the gene detection of the proband of ABO blood type A variant gene.
[0027] 1. Extraction of peripheral blood genomic DNA:
[0028] In line with the relevant national policies and regulations, and on the basis of the consent of the sampling subjects, draw 2-5ml of peripheral venous blood from members of the ABO blood type A variant gene family and put it into EDTA-Na 2 In anticoagulant tubes, freeze at -80°C for later use; DNA extraction uses the DNA extraction kit from LIFE Company, and the specific operation process is as follows:
[0029] After thawing the frozen EDTA anticoagulated blood at room temperature, take 500ul and put it in a centrifuge tube, add an equal volume of erythrocyte lysate (pH8.0), vortex and mix for 5 minutes, centrifuge at 12000rpm for 5 minutes, and discard the supernatant. Add 500ul of red blood cell lysate repeatedly, vortex and mix for 5 minutes, centrifuge at 12000rpm fo...
Embodiment 2
[0041] Verification of Example 2 SNP site
[0042] 3. Perform haplotype sequencing on exon 7 of the ABO gene to determine the mutation site of the A variant gene in the proband
[0043] Primers for PCR amplification of target fragments: ABO-E7F: 5'-ATCCGCCTGCCTTGCAGATA-3'; ABO-E7R: 5'-AGCCTAGGCTTCAGTTACTCACAACA-3'.
[0044]The PCR amplification primers may also select other primer pairs capable of amplifying the above-mentioned SNP sites.
[0045] Reaction conditions and reaction system for PCR amplification of target fragments:
[0046] (1) PCR reaction conditions: pre-denaturation at 95°C for 5 minutes; 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 50 seconds; extension at 72°C for 5 minutes, and incubation of the amplified product at 10°C.
[0047] (2) Reaction system: (TAKARA LA-Taq polymerase)
[0048]
[0049] The reaction system was used to carry out the amplification reaction between the genomic DNA template of each family member and the pri...
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