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Gene polymorphism detection kit for predicting efficacy of tumor necrosis factor alpha inhibitor as well as detection method and application of gene polymorphism detection kit

A technology of tumor necrosis factor and gene polymorphism, which is applied in biochemical equipment and methods, microbiological measurement/testing, DNA/RNA fragments, etc., can solve the problems of cumbersome operation and time-consuming, and achieve simplified sequencing process and the effect of time

Inactive Publication Date: 2021-11-19
上海普然生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the methods of obtaining blood DNA mainly include traditional column extraction and magnetic bead extraction, both of which are time-consuming and cumbersome to operate

Method used

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  • Gene polymorphism detection kit for predicting efficacy of tumor necrosis factor alpha inhibitor as well as detection method and application of gene polymorphism detection kit
  • Gene polymorphism detection kit for predicting efficacy of tumor necrosis factor alpha inhibitor as well as detection method and application of gene polymorphism detection kit
  • Gene polymorphism detection kit for predicting efficacy of tumor necrosis factor alpha inhibitor as well as detection method and application of gene polymorphism detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the preparation of kit

[0042] (1) Specific primer design

[0043] The kit of the present invention designs specific amplification primers and sequencing primers for the TNFα (G308A) gene polymorphism, and is used for pyrophosphate PCR detection. The gene polymorphism sequence is subject to the public sequence in Genebank, and the primer sequences are shown in Table 1 below.

[0044] Table 1 Primer sequence list

[0045]

[0046] (2) Kit composition

[0047] The detection kit includes the components shown in Table 2 below:

[0048] Table 2 Kit component list

[0049]

[0050]

[0051] (3) The configuration system of the sample treatment solution is as follows:

[0052] Sample treatment solution, including 0.4% lithium dodecyl sulfate, 0.3% Triton X-100, 15mg / mL sodium hydroxide, 10mM betaine, 10% trehalose, 5mM BSA, 50mM Tris-HCl, 100mM NaCl, pH=9.

[0053] (4) The detection kit amplification reagent 1 single serving configuration system o...

Embodiment 2

[0060] Embodiment 2, kit detection steps

[0061] The instruments adopted in the present invention are as follows: thermostat, pyrosequencer (Wuhan First Biotechnology Co., Ltd.).

[0062] 1) Take 30 μL EDTA anticoagulated whole blood sample in a PCR amplification tube;

[0063] 2) Add 100 μL of sample treatment solution and 4ul of magnetic beads, and let stand for 5 minutes;

[0064] 3) Place the PCR amplification tube on the magnetic stand, and suck out the mixed solution from the opposite side of the magnetic beads after all the magnetic beads are adsorbed to one side;

[0065] 4) Add amplification reagent 1 into the dry powder of amplification reagent 2, fully dissolve and mix;

[0066] 5) Add 25ul of the prepared PCR reaction solution into the PCR amplification tube obtained in step 4), fully mix the magnetic beads and the PCR reaction solution, centrifuge, and perform constant temperature reaction.

[0067] 6) Amplify using a PCR instrument, the reaction system is 25 ...

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Abstract

The invention discloses a gene polymorphism detection kit for predicting the efficacy of a tumor necrosis factor alpha inhibitor as well as a detection method and application of the gene polymorphism detection kit. The efficacy of the tumor necrosis factor alpha inhibitor reflects the metabolic capability by detecting the polymorphism of a TNF alpha G308A gene; according to the kit, a specific amplification primer and a sequencing primer are designed aiming at the polymorphism of TNF alpha G308A, and the kit comprises the following components: a sample treatment solution, magnetic beads, an amplification reagent 1, an amplification reagent 2, the TNF alpha G308A sequencing primer and a positive control. On one hand, a mode of extracting and amplifying in the same tube is adopted, so that the risk of nucleic acid loss due to repeated tube transfer during extraction is avoided; on the other hand, rapid isothermal amplification is carried out by adding an anti-inhibitor; the invention aims to detect the gene polymorphism of the efficacy of the tumor necrosis factor alpha inhibitor by combining constant-temperature PCR (Polymerase Chain Reaction) and pyrophosphoric acid detection, and provides suggestions from the gene perspective for clinical personalized medication.

Description

technical field [0001] The invention relates to a gene polymorphism detection kit for predicting drug efficacy of a tumor necrosis factor alpha inhibitor, a detection method and an application thereof, and belongs to the field of gene detection. Background technique [0002] Tumor necrosis factor (TNF-α) inhibitors are divided into macromolecular inhibitors and active small molecule inhibitors, and macromolecular inhibitors are mainly composed of monoclonal antibodies and other proteins. Monoclonal antibodies include Infliximab (Infliximab, INF), Adalimumab (Adalimumab, ADA), Certolizumab (Certolizumab, CDP) and Golimumab (Golimumab, ONTO-148), other proteins There are etanercept (ETA), progranulin (PGRN); active small molecule inhibitors mainly include TACE inhibitors and P38 mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) inhibition agent. Tumor necrosis factor (TNF-α) inhibitors are currently the most widely used biological agents for the treat...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12Q1/6806C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q1/6806C12Q2600/106C12Q2600/156C12Q2600/166C12Q2531/119C12Q2537/1376C12Q2522/101C12Q2521/507C12Q2535/122C12Q2545/113C12Q2527/125C12Q2563/143C12Q2563/149
Inventor 孙悦周虹桥刘丹
Owner 上海普然生物科技有限公司
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