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Method for optimizing ctDNA detection accuracy

An accurate, silica technology, applied in the field of tumor DNA detection, can solve the problems of high cost of gene chip detection, low detection sensitivity, non-specific signals, etc., to enhance thermal cycle stability, good dispersion, and prevent blood hanging. wall effect

Active Publication Date: 2022-04-08
武汉承启医学检验实验室有限公司
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  • Application Information

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Problems solved by technology

[0003] At present, the detection methods of ctDNA mainly include enzyme-free PCR reaction, DNA sequencing, gene chip and enzyme-assisted PCR amplification, but there are still some shortcomings in these methods. False positives, while the instruments and equipment used in the DNA sequencing method are relatively expensive, and the detection time is long, the detection cost of the gene chip is expensive, the detection sensitivity is low, and the repeatability is poor. expensive

Method used

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  • Method for optimizing ctDNA detection accuracy
  • Method for optimizing ctDNA detection accuracy
  • Method for optimizing ctDNA detection accuracy

Examples

Experimental program
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preparation example 1-5

[0043] In the preparation example 1-5, the isocyanate silane coupling agent is selected from Nanjing Quanxi Chemical Co., Ltd., the article number is QX-225; the nano-lanthanum oxide is selected from Zhengzhou Chengao Chemical Products Co., Ltd., the article number is 526-98-70, and the particle size is 40nm.

preparation example 1

[0044] Preparation Example 1: Dissolve 40g of heparin sodium in 100g of sodium citrate buffer solution with a pH value of 4.5, add 40kg of 1-ethyl-3(3-dimethylpropylamine), stir well, and place at 2°C 5h, the sodium heparin solution was added to 100g concentration of chitosan in the acetic acid solution of 3%, under nitrogen protection, stirred evenly at room temperature, after centrifugation and drying, added 300g nano lanthanum oxide treated with isocyanate silane coupling agent, After mixing evenly, dry in vacuum. The method for treating nano-lanthanum oxide with isocyanate-silane coupling agent is: put 10g of isocyanate-silane coupling agent in 200g of deionized water, add 100g of nano-lanthanum oxide, ultrasonically disperse at room temperature for 20min, filter, and dry.

preparation example 2

[0045] Preparation Example 2: Dissolve 80g of heparin sodium in 200g of sodium citrate buffer solution with a pH value of 4.7, add 80g of 1-ethyl-3(3-dimethylpropylamine), stir well, and place at 6°C 5h, the sodium heparin solution was added to 200g concentration of 4% chitosan in the acetic acid solution, under nitrogen protection, stirred evenly at room temperature, after centrifugation and drying, added 300g nano lanthanum oxide treated with isocyanate silane coupling agent, After mixing evenly, dry in vacuum. The method for treating nano-lanthanum oxide with isocyanate-silane coupling agent is: put 10g of isocyanate-silane coupling agent in 200g of deionized water, add 100g of nano-lanthanum oxide, ultrasonically disperse at room temperature for 20min, filter, and dry.

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Abstract

The invention relates to the field of tumor DNA detection, and particularly discloses a method for optimizing ctDNA detection accuracy, which comprises the following steps: S1, blood sample treatment: carrying out the following operations on a blood sample within 2 hours of blood sampling: centrifuging the blood sample, and taking upper-layer plasma; s2, extraction of ctDNA: adding a lysate, a protein enzymolysis agent and silicon dioxide into the plasma, uniformly mixing, centrifuging, taking a lower-layer precipitate, adding an EB buffer solution, carrying out water bath, taking out, centrifuging at room temperature, taking a supernatant as a ctDNA sample, and storing; s3, intermediate treatment of the ctDNA sample: heating the ctDNA sample to 1-10 DEG C, and carrying out ultrasonic treatment; and S4, ctDNA content detection: carrying out PCR amplification on the ctDNA sample, mixing the amplified ctDNA with a biosensor, carrying out a hybridization reaction, and detecting the fluorescence intensity. The ctDNA detection method has the advantages of high plasma extraction rate, short extraction time, high protein hydrolysis degree, high accuracy and high sensitivity.

Description

technical field [0001] This application relates to the field of tumor DNA detection, more specifically, it relates to a method for optimizing the accuracy of ctDNA detection. Background technique [0002] ctDNA is circulating tumor DNA, which refers to the DNA fragments that are secreted into the blood by tumor cells through necrosis, apoptosis or normal secretion. It carries information about gene variation related to cancer and is a new type of tumor biomarker. [0003] At present, the detection methods of ctDNA mainly include enzyme-free PCR reaction, DNA sequencing, gene chip and enzyme-assisted PCR amplification, but there are still some shortcomings in these methods. False positives, while the instruments and equipment used in the DNA sequencing method are relatively expensive, and the detection time is long, the detection cost of the gene chip is expensive, the detection sensitivity is low, and the repeatability is poor. expensive. [0004] Because ctDNA only accoun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6806C12Q1/6886G01N27/30
Inventor 刘涛王鑫
Owner 武汉承启医学检验实验室有限公司
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