Primer, kit and method for quickly detecting HLA-B*5801 allele

A HLA-B, 58PF01 technology, applied in the field of genetic engineering, can solve the problems of missed detection, inability to meet high specificity detection, incomplete genotypes, etc. Effect

Active Publication Date: 2014-09-03
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*5801 with single-specific primers based on the PCR-SSP method, but currently available on the market The genotypes covered by the kit are incomplete, and it is easy to miss detection, which is far from meeting the needs of fast, simple and highly specific detection of HLA-B*5801 gene for drug safety

Method used

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  • Primer, kit and method for quickly detecting HLA-B*5801 allele
  • Primer, kit and method for quickly detecting HLA-B*5801 allele
  • Primer, kit and method for quickly detecting HLA-B*5801 allele

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Design of primers for rapid detection of HLA-B*5801 gene

[0023] The data of all HLA alleles required for PCR primer design comes from IMGT / HLA Database (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*5801 allele. In the primer design process, the key is to make the designed primers specifically amplify the HLA-B*5801 gene in a specific PCR buffer system environment, that is, the primers are a kind of "sequence-specific SSP".

[0024] In order to screen the HLA-B*5801 gene using specific PCR-SSP, specific primers 58PF01 (SEQ ID NO.1) and 58PR01 (SEQ ID NO. ID NO.2), primer 58PF02 (SEQ ID NO.3), primer 58PR02 (SEQ ID NO.4), primer 58PF03 (SEQ ID NO.5) and 58PR03 (SEQ ID NO.6), 6 primers were used for multiplex Combining, merging into one specific reaction....

Embodiment 2

[0032] Example 2: Preparation of a kit for rapid detection of HLA-B*5801 gene

[0033] 1. Synthesis of primers

[0034] The primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and the sequences of the primers are respectively shown in SEQ ID NO.1-8, and the specific sequences are shown in Table 1.

[0035] 2. Preparation of PCR reaction mixture

[0036]Mix primers shown in SEQ ID NO.1-8, dNTPs, dye cresyl red, and Taq enzyme buffer. The concentration of detection primers SEQ ID NO.1-6 is 0.5 μM, the concentration of internal reference primers SEQ ID NO.6-7 is 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 at a concentration of 0.001%. The above-mentioned PCR reaction mixture and Taq enzyme are subpackaged and packaged to form a kit.

Embodiment 3

[0037] Example 3: Typing detection of HLA-B*5801 gene in samples

[0038] 23 DNA samples with known HLA-B site results were selected. Add to PCR tubes separately, 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.

[0039] The conditions of the PCR reaction are: 96°C for 2 minutes; then run 35 cycles in sequence according to the following procedures, 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec; finally, 72°C for 10 min, then cool down to 15°C for gel electrophoresis detection.

[0040] Use 0.5×TBE buffer to prepare 1.5% agarose gel. Take 3ul PCR products and directly spot on the gel well, electrophoresis for 20 minutes, and then take pictures and record under ultraviolet light. For specific gel electrophoresis pictures, see the attached figure 1 .

[0041] Interpretation of the results: When the internal control bands appear normally, there are two possibi...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses a primer for quickly detecting HLA-B*5801 allele, a kit containing the primers and a method for quickly detecting HLA-B*5801 allele by using the primers and kit. By using the HLA-B*5801 gene specific primer and adopting the multiplex primer combination design, the SNP (single-nucleotide polymorphism) site of the HLA-B*5801 gene is completely covered, the specific combination is enhanced, and the generation of the false positive is prevented more effectively. Besides, the specific primer, dNTPs (deoxyribonucleotide triphosphates), PCR (polymerase chain reaction) buffer solution and dyes are mixed previously, thereby greatly saving the operation time and workload; and the method is quick, simple, accurate and visual, and can the screening and typing experiment of the whole gene within 3 hours, thereby solving the problem of safe application instructions of the HLA-B*5801 gene in drugs for treating gout and the like.

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*5801 alleles. Background technique [0002] Gout is a metabolic disease caused by abnormal purine metabolism leading to increased uric acid synthesis. Gout is related to the metabolic imbalance of the human body and is closely related to the increase of blood uric acid level. In my country, the prevalence of the disease is increasing year by year, and the incidence of hyperuricemia in some areas has reached 13.3%, and it often coexists with hypertension, diabetes, hyperlipidemia, and obesity. In my country, allopurinol is the drug of choice for the treatment of gout. However, the use of allopurinol will cause certain adverse drug reactions. In recent years, many studies have shown that the severe drug eruption associated with allopurinol is closely related to HLA-B*5801, which has been affirmed. As early as 2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2537/143C12Q2531/113C12Q2545/101
Inventor 郑仲征潘捷谢志聪
Owner SHANGHAI TISSUEBANK BIOTECH
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