Human HLA-B*5801 gene polymorphism detection kit

A technology of genetic polymorphism and detection kit, which is applied in the fields of biotechnology and medicine, can solve the problems that whole blood samples cannot be directly detected, and the detection effect of low-concentration genomic DNA is not ideal, so as to achieve true and reliable results and avoid false positives and false negative results, time-consuming effects

Inactive Publication Date: 2017-07-07
WUHAN YZY MEDICAL SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a detection kit for HLA-B*5801 gene polymorphism, so as to solve the problem that the whole blood sample cannot be directly detected in the existing gene polymorphism detection technology, and the detection effect of low-concentration genomic DNA is not good. ideal question

Method used

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  • Human HLA-B*5801 gene polymorphism detection kit
  • Human HLA-B*5801 gene polymorphism detection kit
  • Human HLA-B*5801 gene polymorphism detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Prepare the human HLA-B*5801 gene detection kit of the present invention, comprising the following steps:

[0093] 1. Primer and probe synthesis:

[0094] Design and synthesize specific primers SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, specific probes SEQ ID NO:4 and SEQ ID NO:5, and at the 5' end of SEQ ID NO:4 Label the FAM fluorescent group, label the NFQ-MGB non-luminescent quenching group at the 3' end, label the VIC fluorescent group at the 5' end of SEQ ID NO:5, and label the NFQ-MGB non-luminescent quenching group at the 3' end. The primers and probes were prepared into 100 μM stock solutions for storage.

[0095]2. Prepare the internal standard system: design and synthesize a pair of internal standard primers for the human gene GAPDH, the primer pair sequences are SEQ ID NO:6 and SEQ ID NO:7; design and synthesize internal standard probes, the The probe is SEQ ID NO:8. The ROX fluorescent group is labeled at the 5' end of SEQ ID NO:8, and the BHQ2 non-luminesce...

Embodiment 2

[0105] The human HLA-B*5801 gene detection kit prepared in Example 1 was used to detect the samples to be tested.

[0106] In this example, 10 samples of EDTA anticoagulated whole blood were collected, treated with a blood treatment agent, and then tested with the human HLA-B*5801 gene detection kit prepared in Example 1.

[0107] 1. Blood Sample Processing

[0108] Take 10 μl of whole blood, add 90 μl of blood treatment agent, shake and mix for 1 min.

[0109] 2. Fluorescent quantitative PCR detection of samples

[0110] Add 2 μl of the sample in step 1 to 23 μl of the HLA-B*5801 detection reaction system of the kit in Example 1, so that the total volume of the reaction system is 25 μl, and put it into a fluorescent quantitative PCR instrument, and set it as follows Amplification reaction after PCR reaction procedure:

[0111] 37℃10min;

[0112] 95°C for 5 minutes;

[0113] 95°C for 15s, 60°C for 60s, 40 cycles; after each cycle, the fluorescence signals of FAM, VIC and ...

Embodiment 3

[0118] Take the remaining whole blood sample whose test result is heterozygous mutation in Example 2, extract genomic DNA, measure the DNA concentration, dilute the DNA to 10ng / μl, 1ng / μl and 0.1ng / μl in sequence, and evaluate the detection performance of the kit .

[0119] 1. Genomic DNA Extraction

[0120] Take 300 μl of whole blood, add 900 μl of cell lysate CL, mix by inverting, and let stand for 5 minutes;

[0121] Centrifuge at 10,000rpm (11,500×g) for 1min, suck off the supernatant, leave the cell pellet, add 200μl buffer GS to the cell pellet collected by centrifugation, shake until thoroughly mixed;

[0122] Add 20μl Proteinase K solution and mix well;

[0123] Add 200μl buffer GB, mix well by inversion, place at 56°C for 10min, invert and mix several times during this time, the solution should become clear (if the solution is not completely clear, please prolong the lysis time until the solution is clear);

[0124] Add 200 μl of absolute ethanol, and mix thoroughl...

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Abstract

The invention relates to a human HLA-B*5801 gene polymorphism detection kit. The human HLA-B*5801 gene polymorphism detection kit comprises PCR damping liquid, a specific primer, a specific probe, an interior label system, a Taq enzyme, a UNG enzyme, a weakly-positive control group and a blank control group, and also comprises a blood treating agent; a blood sample is simply treated and can be directly subjected to PCR amplification, the DNA extracting process is omitted, and operating time is saved. According to the human HLA-B*5801 gene polymorphism detection kit, the SNP probe is used in cooperation with the technology of the ARMS primer, and it is achieved that two different gene types are detected in one pipe; meanwhile, the interior label system is designed and used for monitoring the quality of the sample, and the weakly-positive control group and the blank control group are designed and used for monitoring the quality of the kit. The human HLA-B*5801 gene polymorphism detection kit for detecting HLA-B*5801 alleles has the advantages of being high in specificity and sensitivity, rapid and easy to operate, safe, objective in result interpretation and the like when being used for detecting HLA-B*5801 alleles.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to a human HLA-B*5801 gene detection kit. Background technique [0002] Allopurinol is the first-line drug for the treatment of gout. It is currently the only xanthine oxidase inhibitor. It is mainly used for the treatment of gout and the adjuvant treatment of gouty nephropathy, secondary hyperuricemia and severe epilepsy. Its metabolite, oxypurinol, inhibits the activity of xanthine oxidase (the latter can convert hypoxanthine into xanthine, and then convert xanthine into uric acid), so that the production of uric acid is reduced, and the uric acid content in blood and urine is reduced. To the level below the solubility, so as to prevent the deposition of uric acid stones, contribute to the re-dissolution of gouty nodules and uric acid crystals. But allopurinol can cause serious side effects, including Stevens-John syndrome (SJS) and toxic epidermal necrosis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/686C12Q2600/106C12Q2600/156C12Q2535/137C12Q2561/101C12Q2563/107C12Q2545/101C12Q2545/114
Inventor 蔡从利张喆彭盼周鹏飞
Owner WUHAN YZY MEDICAL SCI & TECH
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