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Detection kit for budesonide metabolism marker and detection method and application thereof

A technology for detection kits and metabolic markers, used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome operation and time-consuming, and simplify the sequencing process and time Effect

Pending Publication Date: 2021-11-19
菲思特(上海)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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  • Detection kit for budesonide metabolism marker and detection method and application thereof
  • Detection kit for budesonide metabolism marker and detection method and application thereof
  • Detection kit for budesonide metabolism marker and detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, preparation and use of kit

[0043] (1) Specific primer design

[0044] The kit of the present invention designs specific amplification primers and sequencing primers for the polymorphism of the GLCCI1 (A-1106G) gene, 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.

[0045] Table 1

[0046]

[0047] (2) Kit composition

[0048] The detection kit includes the components shown in Table 2 below:

[0049] Table 2 Kit component list

[0050]

[0051]

[0052] (3) The configuration system of the sample treatment solution is as follows:

[0053] 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.

[0054] (4) The detection kit amplification reagent 1 single serving configuration system of the ...

Embodiment 2

[0061] Embodiment 2, kit detection steps

[0062] The instruments adopted in the present invention are as follows: thermostat, pyrosequencer (Wuhan First Biotechnology Co., Ltd.).

[0063] 1) Take 30 μL EDTA anticoagulated whole blood sample in a PCR amplification tube;

[0064] 2) Add 100 μL of sample treatment solution and 4ul of magnetic beads, and let stand for 5 minutes;

[0065] 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;

[0066] 4) Add amplification reagent 1 into the dry powder of amplification reagent 2, fully dissolve and mix;

[0067] 5) Add 25ul of the prepared PCR reaction solution to the PCR amplification tube obtained in step 4), fully mix the magnetic beads with the PCR reaction solution, centrifuge, and perform a constant temperature reaction;

[0068] 6) Amplify using a PCR instrument, the reaction system is 25...

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Abstract

The invention discloses a detection kit for a budesonide metabolism marker and a detection method and application thereof. The kit designs a specific amplification primer and a sequencing primer aiming at the polymorphism of GLCCI1A-1106G, and the kit comprises the following components: a sample treating fluid, magnetic beads, an amplification reagent 1, an amplification reagent 2, a GLCCI1A-1106G sequencing primer and a positive control. The detection kit is mainly optimized from two aspects: 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 curative effect of budesonide by combining rapid DNA (deoxyribonucleic acid) preparation, constant-temperature PCR (polymerase chain reaction) amplification and pyrosequencing technologies, and provides suggestions from the gene perspective for clinical personalized medication.

Description

technical field [0001] The invention relates to a detection kit for budesonide metabolic markers and a detection method and application thereof, belonging to the field of gene detection. Background technique [0002] Budesonide is a glucocorticoid with highly effective local anti-inflammatory effects, which can enhance the stability of endothelial cells, smooth muscle cells and lysosomal membranes, inhibit immune reactions and reduce antibody synthesis, thereby preventing the production of allergic active media such as histamine. The release and activity are reduced, and it can alleviate the enzymatic process stimulated by the combination of antigen and antibody, inhibit the synthesis and release of bronchoconstriction substances, and reduce the contraction response of smooth muscle. It is clinically used in patients with glucocorticoid-dependent or non-dependent bronchial asthma and asthmatic chronic bronchitis. Refractory asthma refers to asthma that cannot be well contro...

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 陈立波刘丹孙悦
Owner 菲思特(上海)生物科技有限公司
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