A kind of endonuclease mus81 detection kit and its detection method and application

A detection kit and endonuclease technology, applied in the field of malignant tumor detection, can solve the problems of malignant tumors such as easy recurrence, metastasis, rapid progress, and poor prognosis, and achieve good clinical application prospects and highly targeted effects

Active Publication Date: 2018-12-25
海博泰(上海)医疗科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Malignant tumors are prone to recurrence, metastasis, rapid progression, and poor prognosis

Method used

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  • A kind of endonuclease mus81 detection kit and its detection method and application
  • A kind of endonuclease mus81 detection kit and its detection method and application
  • A kind of endonuclease mus81 detection kit and its detection method and application

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Experimental program
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Embodiment 1

[0035] This embodiment provides a detection kit for endonuclease MUS81, including the following reagents:

[0036] Digestion substrate: plasmid DNA.

[0037] Lysis solution: a system for lysing tumor cells, including: 0.01mol / L phosphate solution PBS (pH 7.4), 4% CHAPS, RNase inhibitor (0.2U / μl).

[0038] Digestion solution: a medium for the determination of endonuclease MUS81 activity, including: 0.01mol / L phosphate solution PBS (pH 7.4), 4% CHAPS, RNase inhibitor (0.2U / μl), 1mmol / L MgCl 2 , 0.1mmol / L DTT.

Embodiment 2

[0040] (1) Sample preparation:

[0041] 1. Collect cells: (1) Tumor tissue specimens: place the tissue in 1ml 1×PBS solution, crush the tissue with a high-speed homogenizer, filter it with an autoclaved copper wire mesh, and wash it with PBS to obtain a cell suspension ; (2) Peripheral blood samples: remove plasma, add 1:1 normal saline (NS) to the blood cell pellet according to the volume ratio, after mixing, gently add it to the ficoll separation solution (volume ratio 1:1), 1500rpm Centrifuge for 20 minutes, collect the white blood cell layer, add NS according to the volume ratio (1:4), mix well, and centrifuge at 1800rpm for 10min; remove the lower layer, add NS according to the volume ratio (1:4), mix well, and centrifuge at 1800rpm for 10min, collect a single nucleus cell. Then, use erythrocyte lysate (8.29g NH4CL+1g KHCO 3 +200ul 0.5M EDTA dissolved in 1L distilled water) to remove the red blood cells in the collected cells. (3) Isolation of purely cultured primary t...

Embodiment 3

[0054] (1) Reagent preparation:

[0055] LB solid medium containing Amp: tryptone 10g + yeast extract (yeast extract) 5g + sodium chloride 10g, add distilled water to 1L. The pH was adjusted to 7.0 with 1M NaOH, and finally autoclaved. Take the above-prepared LB culture solution and add it to agar (15g / L), after autoclaving, cool to about 60°C, add ampicillin (100mg / L) to make the final concentration 50ug / ml, shake well and spread the plate; Prepare LB plates containing ampicillin.

[0056] (2) Sample preparation:

[0057] 1. Collect cells: (1) Tumor tissue specimens: place the tissue in 1ml 1×PBS solution, crush the tissue with a high-speed homogenizer, filter it with an autoclaved copper wire mesh, and wash it with PBS to obtain a cell suspension ; (2) Peripheral blood samples: remove the plasma, add 1:1 normal saline (NS) to the blood cell pellet according to the volume ratio and mix well, then gently add it to the ficoll separation solution (1:1 volume ratio), and centr...

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Abstract

The invention relates to an endonuclease MUS81 detection kit, and a detecting method and the application of the endonuclease MUS81 detection kit. The endonuclease MUS81 detection kit comprises following reagents of a digestion substrate, a lysis solution and a digestion solution. The detecting method comprises the step of determining the activity of endonuclease MUS81 through an electrophoresis method or a germ clone forming method. According to the endonuclease MUS81 detection kit, the detecting method and the application, disclosed by the invention, the activity of the endonuclease MUS81 in peripheral blood of a malignant tumor patient can be detected, and the needs of clinic diagnosis, treatment monitoring and prognosis judgment are met; the specificity of activity change of the endonuclease MUS81 in the peripheral blood of the malignant tumor patient is completely considered, the pertinence is strong, and a better clinic application prospect is obtained.

Description

technical field [0001] The invention belongs to the field of malignant tumor detection, in particular to a detection kit for endonuclease MUS81, a detection method and application thereof. Background technique [0002] In recent years, due to changes in the environment and lifestyle, the incidence of malignant tumors in my country has increased significantly, especially lung cancer, intestinal cancer, and ovarian cancer. Studies have confirmed that genome instability plays an important role in the occurrence and development of tumors; endonuclease is an enzyme that plays a key role in maintaining genome integrity, so changes in its activity are closely related to the occurrence and development of tumors. [0003] Enzymes that can degrade DNA molecules are usually called ribozymes, which are divided into two types: enzymes that can hydrolyze nucleic acids from the end of polynucleotide chains are called exonucleases; and enzymes that hydrolyze nucleic acids from the middle of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/44
Inventor 卢仁泉郭林高翔
Owner 海博泰(上海)医疗科技有限责任公司
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