Primer pair and kit for detecting polymorphism of Etanercept-medication-related genes

A technology of gene polymorphism and etanercept, which is applied in the field of primer pairs and kits for detecting etanercept drug-related gene polymorphisms, can solve the problems that cannot meet the needs of typing

Pending Publication Date: 2021-04-20
南昌豪仕医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In the existing publicly reported literature, there are only genotyping tests for SLCO1C1 and STAT4 genes alone (the purpose is not for etanercept medication), but when they are used alone, they often

Method used

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  • Primer pair and kit for detecting polymorphism of Etanercept-medication-related genes
  • Primer pair and kit for detecting polymorphism of Etanercept-medication-related genes
  • Primer pair and kit for detecting polymorphism of Etanercept-medication-related genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1: the preparation of kit (30 tests / box)

[0114] 1. Design and synthesis of primers and probes

[0115] For human SLCO1C1 gene, PTPRC gene, STAT4 gene, HLA-E gene, TRAF1 gene and KLRD1 gene, select specific mutation sites rs3794271, rs10919563, rs7574865, rs1264457, rs3761847 and rs2302489, and select primers and probes designed at the mutation sites Points and nearby conserved regions, avoiding SNPs in the primer-binding region (retrieve the SNP of the target gene sequence through the online NCBI website), perform PrimerBlast through the online NCBI website, design allele-specific PCR amplification primers, and confirm the primers For the specific amplification of the PCR primer, when there is a mismatch between the base at the 3' end of the PCR primer and its template DNA, the amplification efficiency will drop sharply, and the PCR amplification signal will only appear when the 3' base of the primer is paired with the template. The probe is located in the...

Embodiment 2

[0174] Embodiment 2: the use of kit

[0175] 1. Sample testing

[0176] Prepare the system according to the number of templates: take the PCR reaction tube, add the corresponding primer solution, PCR premix solution, sterilized purified water, add sample DNA, sterilized purified or positive control substance as template to form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0177] There are two kinds of reaction solutions for each site: wild (WT) and mutant (MT), and there are 12 kinds of reaction solutions for 6 sites. Each reaction solution was prepared as follows:

[0178] Table 18. Preparation composition of each reaction solution

[0179]

[0180] The system reaction procedure is as follows:

[0181] Table 19. PCR reaction program

[0182]

[0183] 2. ABI7500 fluorescent quantitative PCR

[0184] Press the power button on the right to start ABI 7500. After starting up, the "power" indicator on the left end of the m...

Embodiment 3

[0219] Embodiment 3: Large sample verification of the kit

[0220] 1. According to the preparation method shown in Example 1, the relevant components of the kit were prepared and stored at -20°C for later use.

[0221] 2. Take 30 cases of known whole blood samples, use "nucleic acid extraction or purification reagents" (record number: Xiangchang Machinery Equipment 20160167) to extract sample DNA, and use a nucleic acid protein analyzer to detect DNA sample concentration, 30 samples A260 / 280 All are between 1.6 and 2.0.

[0222] 3. According to the steps shown in Example 2, add DNA samples and perform detection on an ABI 7500 fluorescent quantitative PCR instrument.

[0223] 4. According to the interpretation standard shown in embodiment 2, the results are interpreted and counted (the test result coincidence rate statistics), the sample coincidence rate is 100%; the test result specific information is as follows:

[0224]

[0225]

[0226]

[0227]

[0228]

...

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Abstract

The invention relates to a primer pair and kit for detecting polymorphism of Etanercept-medication-related genes and belongs to the technical field of in-vitro nucleic acid examination. The primer pair comprises amplification primers aiming at allelic genes, i.e., SLCO1C1rs3794271, PTPRCrs10919563, STAT4rs7574865, HLA-Ers1264457, TRAF1rs3761847 and KLRD1rs2302489 and an internal reference gene GAPDH separately; and the kit comprises a primer solution containing the amplification primers. According to the kit provided by the invention, the sensitivity is high and can reach one-ten-thousandth, the minimum detection limit is only 1-2 copies, and thus, the kit is particularly applicable to detection on low-content mutation samples; compared with a sequencing method, the primer pair and the kit have the advantages that a detection result can be observed in real time, a product has no need of gel electrophoresis detection, complete-closed tube operation is carried out, and thus, the risk of PCR product contamination is effectively lowered; and furthermore, the primer pair and the kit further have the advantages of being high in detection speed and applicable to high-pass sample detection.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a primer pair and a kit for detecting etanercept drug-related gene polymorphisms. Background technique [0002] Rheumatic diseases refer to a type of disease involving joints and surrounding soft tissues, including muscles, ligaments, bursae and fascia. In addition to pain, joint lesions are often accompanied by swelling and movement disorders, and some patients may also experience joint and visceral dysfunction. damage, seriously affecting the quality of life of patients. The etiology and pathogenesis of rheumatic diseases are complex, but mostly autoimmunity is the main pathogenesis and inflammatory response is the main pathological basis. Tumor necrosis factor (TNF)-α, as a pro-inflammatory factor, plays an important role in the pathogenesis of rheumatic diseases. TNF-α inhibitors can inhibit this cytokine to exert anti-inflammatory and immunosuppressi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCY02A50/30
Inventor 滕祥云廖敏
Owner 南昌豪仕医学检验实验室有限公司
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