Kit for simultaneously detecting thalassemia mutant type and deletion type and application thereof
A thalassemia and deletion type technology, which is applied in the determination/testing of microorganisms, chemical libraries, combinatorial chemistry, etc., can solve the problems of missed detection and improvement of thalassemia detection technology, and achieves significant advantages, small detection range and low cost Effect
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Embodiment 1
[0089] The inventors designed PCR primers for the α-globin gene and β-globin gene of thalassemia, and then performed multiple PCR combination screening on the obtained PCR primers, and designed matching primer tag sequences. details as follows:
[0090] 1. Primer design
[0091] (1) Design of mutant primers
[0092] First, bioinformatics analysis was performed on the sequences of HBA1, HBA2, and HBB genes (two α-globin genes and one β-globin gene, respectively), and the pseudogenes ψα1, ψα2, and β-globin with high sequence similarity to HBA genes were analyzed. Other genes in the protein gene cluster were included in the scope of bioinformatics analysis to find out the conserved and specific sequences of HBA1, HBA2, and HBB genes, and then design primers. The designed primer sequences were subjected to genome-wide comparison (blast) analysis. Except for the accurate alignment to the target HBA1, HBA2, and HBB genes, the primers without accurate alignment at other positions i...
Embodiment 2
[0144] Using the kit with 96 sets of index primers obtained in Example 1, referring to the above-mentioned "general method", the results after sanger sequencing are known (including 7 deletion types, no mutations, common mutations, rare mutations and newly discovered mutations ) of 95 samples were tested. details as follows:
[0145] 1. Sample extraction
[0146] DNA was extracted from 95 blood samples using a KingFisher automatic extractor, and each blood sample was from Chenzhou People's Hospital. The main extraction steps are as follows: Take out 3 deep-well plates and 1 shallow-well plate matched with the Kingfisher automatic extractor, add a certain amount of matching reagents according to the instructions and mark them, and place all the well-plates with reagents in the corresponding Select the program "Bioeasy_200ul Blood DNA_KF.msz" and press "star" to execute the program for nucleic acid extraction. After the program is finished, about 100ul of the eluted product i...
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