Primer for rapidly detecting HLA-B*1502 allele, kit and detection method
A technology of HLA-B and reagent kit, which is applied in the field of genetic engineering, can solve the problems of prone to false positives, failure to meet high specificity detection, and few genotypes, so as to save operation time and workload and prevent false positives The effect of producing and experimenting with low cost
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[0024] Example 1: Primer design for rapid detection of HLA-B*1502 gene
[0025] All HLA allele data required for PCR primer design comes from IMGT / HLADatabase (Release3.15.0, 2014-01-17), the specific website is: http: / / www.ebi.nc.uk / ipd / imgt / hla / . The primers were designed manually, and the designed primers were compared in the IMGT database to confirm that the primers can specifically bind to the HLA-B*1502 allele. In the primer design process, the key is to make the designed primers able to specifically amplify the HLA-B*1502 gene in a specific PCR buffer system environment, that is, the primers are a kind of "sequence-specific SSP".
[0026] In order to use specific PCR-SSP to screen the HLA-B*1502 gene, specific primers EPF01 (SEQ ID NO. 1) and EPR01 (SEQ ID NO. ID NO.2), primer EPF02 (SEQ ID NO.3) and primer EPR02 (SEQ ID NO.4), 4 primers are combined into two groups of P1 and P2 respectively, to prevent the missed detection of HLA-B*1502 gene, The P1 group is EPF01-...
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[0034] Example 2: Preparation of a kit for rapid detection of HLA-B*1502 gene
[0035]1. Synthesis of primers
[0036] The primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd., the primer sequences are shown in SEQ ID NO.1-6 respectively, and the specific sequences are shown in Table 1.
[0037] 2. Preparation of PCR reaction mixture
[0038] As shown in Table 1, primer sets P1 and P2 were prepared, respectively, and each primer shown in the P1 and P2 groups was mixed with the internal control primers, dNTPs, the dye cresol red and PCR buffer, respectively. The concentrations of detection primers SEQ ID NO.1-4 were 0.5 μM, the concentrations of internal reference primers SEQ ID NO.5-6 were 0.2 μM, the concentration of dye cresol red was 0.01%, and the concentration of dNTPs was 0.2 mM. The PCR buffers were: Tris-HCl concentration of 10 mM, potassium chloride concentration of 50 mM, magnesium chloride concentration of 1.5 mM, and gelatin concentration of 0.001...
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[0039] Example 3: Typing detection of HLA-B*1502 gene in samples
[0040] Eight DNA samples with known HLA-B gene results were selected. Add to two PCR tubes containing the P1 and P2 primer sets, respectively, while adding Taq enzyme to each tube. After the addition of samples, the reaction mixture was mixed well, centrifuged briefly, and the PCR reaction was performed.
[0041] The conditions of the PCR reaction are: 94°C for 5 min; then run 30 cycles according to the following procedure, 94°C for 1 min, 65°C for 2 min, 72°C for 1 min; finally, 72°C for 10 min, cooling to 15°C for gel electrophoresis detection.
[0042] Prepare a 2% agarose gel using 0.5×TBE buffer. Take 3ul PCR product and directly spot it on the gel well, electrophoresis at 150V for 40 minutes, and then take pictures and record under ultraviolet light. For the specific gel electrophoresis diagram, see the attached file. figure 1 .
[0043] Interpretation of results: There are two possibilities for the d...
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