Primer pair and kit for detecting related gene polymorphism of adalimumab medication

A technology of adalimumab and gene polymorphism, applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. problems, to achieve the effect of fast sequencing speed, easy operation, and short reaction time

Active Publication Date: 2021-04-27
南昌豪仕医学检验实验室有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, the record in China in 2018 was less than 200 million yuan. Compared with the 15 indications approved globally for Humira, it only has 3 indications for rheumatoid arthritis, ankylosing spondylitis and psoriasis in China. Approved, the target population is small; Humira is not included in the medical insurance, so patients are mainly at their own expense, and the price is too high
[0015] In the existing publicly reported literature, only the genotyping detection of TNF, KLRC1, FCGR2A, PTPRC, HLA-E, TRAF1 and KLRD1 genes (the purpose is not for adalimumab), but when used alone, often It cannot meet the needs of genotyping related to clinical adalimumab drug use, and needs to be used in combination with seven gene tests. There are many operational problems and system crossover problems in clinical practice;

Method used

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  • Primer pair and kit for detecting related gene polymorphism of adalimumab medication
  • Primer pair and kit for detecting related gene polymorphism of adalimumab medication
  • Primer pair and kit for detecting related gene polymorphism of adalimumab medication

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Embodiment 1: the preparation of kit (30 tests / box)

[0133] 1. Design and synthesis of primers and probes

[0134] For human TNF gene, KLRC1 gene, FCGR2A gene, PTPRC gene, HLA-E gene, TRAF1 gene and KLRD1 gene, select specific mutation sites rs1800629, rs7301582, rs1801274, rs10919563, rs1264457, rs3761847 and rs2302489, the selected primers and probes The needle is designed at the mutation site and the nearby conserved region to avoid SNPs in the primer binding region (search the SNP of the target gene sequence through the online NCBI website), perform Primer Blast through the online NCBI website, and design allele-specific PCR Amplify the primers to confirm the specific amplification of the primer pair. When there is a mismatch between the 3' terminal base of the PCR primer and its template DNA, the amplification efficiency will drop sharply. Only when the 3' base of the primer is paired with the template can it be A PCR amplification signal appears. The probe is l...

Embodiment 2

[0201] Embodiment 2: the use of kit

[0202] 1. Sample testing

[0203] Prepare the system according to the number of templates: take a PCR reaction tube, add the corresponding primer solution, PCR premix solution, sterilized purified water, add sample DNA, sterilized purified water or positive control as a template to form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0204] There are two kinds of reaction solutions for each site: wild (WT) and mutant (MT), and there are 14 kinds of reaction solutions for 7 sites. Each reaction solution was prepared as follows:

[0205] Table 18. Preparation composition of each reaction solution

[0206]

[0207]

[0208] The system reaction procedure is as follows:

[0209] Table 19. PCR reaction program

[0210]

[0211] 2. ABI7500 fluorescent quantitative PCR

[0212] Press the power button on the right to start ABI 7500. After starting up, the "power" indicator on the left end ...

Embodiment 3

[0249] Embodiment 3: Large sample verification of the kit

[0250] 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.

[0251] 2. Take 30 cases of whole blood samples with known genotypes, 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 the concentration of DNA samples, 30 cases of samples A260 / 280 are all between 1.6 and 2.0.

[0252] 3. According to the steps shown in Example 2, add DNA samples and perform detection on an ABI 7500 fluorescent quantitative PCR instrument.

[0253] 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:

[0254]

[0255]

[02...

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Abstract

The invention relates to a primer pair and a kit for detecting related gene polymorphism of adalimumab medication, and belongs to the technical field of in-vitro nucleic acid detection. The primer pair comprises amplification primers aiming at TNFrs1800629, KLRC1rs7301582, FCGR2Ars1801274, PTPRCrs10919563, HLA-Ers1264457, TRAF1rs3761847 and KLRD1rs2302489 allelic genes and GAPDH reference genes respectively. The kit comprises a primer solution containing the amplification primers. The kit provided by the invention is high in sensitivity which can reach one ten- thousandth, and the lowest detection limit is only 1-2copies, so that the kit is particularly suitable for detecting low-content mutation samples; and compared with a sequencing method, a detection result can be observed in real time, a product does not need gel electrophoresis detection, tube closing operation is completely carried out, and the risk of PCR product pollution is effectively reduced; and in addition, the kit has the advantages of being high in detection speed and suitable for high-throughput 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 adalimumab drug-related gene polymorphisms. Background technique [0002] Adalimumab (Humira) has been approved by the China Food and Drug Administration (CFDA) for 3 indications, namely rheumatoid arthritis, ankylosing spondylitis, and psoriasis. The stock of patients with the three diseases is 5 million, totaling 15 million. [0003] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by symmetry, multiple joints, and small joints. It is known as "undead cancer". Pain, swelling, and stiffness produce symptoms of fatigue and weakness, and in advanced stages can lead to stiffness and deformity, severe impairment of function, and eventually loss of joint function. Humira is used in combination with methotrexate for the treatment of adults with moderately to severely active rheumatoid arthritis who ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2600/166C12Q2531/113C12Q2563/107C12Q2537/143C12Q2535/137
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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