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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

Active Publication Date: 2014-09-17
SHANGHAI TISSUEBANK BIOTECH +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although, generally speaking, PCR-SSP is a relatively simple, convenient, and highly specific method, and there are currently kits for detecting HLA-B*1502 with single-specific primers based on the PCR-SSP method, but currently on the market The kit covers fewer genotypes and is prone to false positives, which is far from meeting the needs of fast, simple and highly specific detection of HLA-B*1502 gene for drug safety

Method used

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  • Primer for rapidly detecting HLA-B*1502 allele, kit and detection method
  • Primer for rapidly detecting HLA-B*1502 allele, kit and detection method
  • Primer for rapidly detecting HLA-B*1502 allele, kit and detection method

Examples

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Effect test

Embodiment 1

[0024] Example 1: Design of primers for rapid detection of HLA-B*1502 gene

[0025] The data of all HLA alleles 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 could 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 primer is a "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 primer EPR01 (SEQ ID NO. ID NO.2), primer EPF02 (SEQ ID NO.3) and primer EPR02 (SEQ ID NO.4), the four primers were combined into two groups, P1 and P2, to prevent the missed detection of the HLA-B*1502 gene, The P1 group is...

Embodiment 2

[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., and the sequences of the primers are respectively shown in SEQ ID NO.1-6, and the specific sequences are shown in Table 1.

[0037] 2. Preparation of PCR reaction mixture

[0038] Prepare primer sets P1 and P2 respectively as shown in Table 1, and mix each primer shown in P1 set and P2 set with internal control primers, dNTPs, dye cresyl red, and PCR buffer respectively. The concentrations of detection primers SEQ ID NO.1-4 are all 0.5 μM, the concentrations of internal reference primers SEQ ID NO.5-6 are respectively 0.2 μM, the concentration of dye cresyl red is 0.01%, and the concentration of dNTPs is 0.2 mM. The PCR buffer solution is: Tris-HCL at a concentration of 10 mM, potassium chloride at a concentration of 50 mM, magnesium chloride at a concentration of 1.5 mM, and gelatin a...

Embodiment 3

[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, and add Taq enzyme to each tube at the same time. After adding the sample, mix the reaction mixture evenly, centrifuge briefly, and carry out the PCR reaction.

[0041] The conditions of the PCR reaction were: 94°C for 5 minutes; followed by 30 cycles of 94°C for 1 minute, 65°C for 2 minutes, and 72°C for 1 minute; finally, 72°C for 10 minutes, then cooled to 15°C for gel electrophoresis detection.

[0042] Use 0.5×TBE buffer to prepare 2% agarose gel. Take 3ul of PCR products and directly spot on the gel well, run electrophoresis at 150V for 40 minutes, and then take pictures and record under ultraviolet light. For specific gel electrophoresis pictures, please refer to the attached figure 1 .

[0043] Interpretation of the results: When the internal control bands app...

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Abstract

The invention belongs to the technical field of genetic engineering and discloses a primer for rapidly detecting an HLA-B*1502 gene, a kit comprising the primer and a method for rapidly detecting the HLA-B*1502 gene by the primer and kit. According to the invention, by virtue of the HLA-B*1502 gene-specific primer and the combinational design of multiplex primers, an SNP site of the HLA-B*1502 gene is completely covered, the specific binding is enhanced and the false positive generation is more effectively prevented. In addition, by pre-mixing the specific primer, dNTPs, PCR buffer and dyes, the operating time and workload are significantly saved, the method is fast, simple, accurate and intuitive, the entire gene screening and classification experiment can be completed within three hours and the problem of safe medication instruction of the HLA-B*1502 gene in antiepileptic drugs is solved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, a kit and a method for rapidly detecting HLA-B*1502 alleles. Background technique [0002] Epilepsy is an ancient common disease of the nervous system, which seriously endangers human health. There are more than 9 million epilepsy patients in my country, of which 6 million patients still have seizures every year, and there will be 600,000 new cases every year. Carbamazepine (CBZ), phenytoin (PHT), lamotrigine (LTG), oxcarbazepine (OXC) and phenobarbital (PB) are common aromatic antiepileptic drugs (AEDs). These drugs are widely used clinically and can effectively control seizures and neuralgia, but they can also cause cutaneous adverse drug reactions (cADRs). [0003] With the continuous deepening of human genomics and pharmacogenomics research, the relationship between severe skin reactions caused by some drugs and human leukocyte antigen HLA gene has been...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113
Inventor 郑仲征潘捷谢志聪
Owner SHANGHAI TISSUEBANK BIOTECH
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