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Detection of mutation site of PIK3CA gene in ctDNA in urine

A urine sample and mixed solution technology, applied in the biological field, can solve problems such as ctDNA to be developed

Inactive Publication Date: 2018-07-24
索真(北京)医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, the method of obtaining ctDNA and its application are still to be developed

Method used

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  • Detection of mutation site of PIK3CA gene in ctDNA in urine
  • Detection of mutation site of PIK3CA gene in ctDNA in urine
  • Detection of mutation site of PIK3CA gene in ctDNA in urine

Examples

Experimental program
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Effect test

Embodiment 1

[0038] In this embodiment, the following method is used to determine whether the ctDNA in the urine sample carries a predetermined mutation site:

[0039] 1. Isolation of ctDNA from urine

[0040] The patients with lung cancer were treated with gefitinib, and the urine samples were continuously sampled before and every 21 days after the treatment, and the fasting morning urine of the patients was collected. Use a 120ml collection cup to collect 110ml of urine, add EDTA to make the final concentration 10mmol / L, to inhibit the activity of nuclease. Centrifuge the mixture at a speed of 1800g and a temperature of 18°C ​​to 23°C for 10min, collect the supernatant, concentrate the supernatant to 4mL through an ultrafiltration tube, mix the concentrated sample with 750μl of Q Sepharose anion exchange resin, place in Incubate at room temperature on a rotator, and use the Micro Bio-Spin to collect the precipitate TM (BioRad) chromatographic column was washed and eluted, and the elute...

Embodiment 2

[0060] In this example, the accuracy of using urine ctDNA as the detection object was verified.

[0061] Specifically, three control groups were set up:

[0062] Control group 1: Drugs for lung cancer patients (gefitinib), collected tissue biopsy samples every 21 days before the drug and after the drug, embedded the collected samples in paraffin, estimated the tumor cell content after hematoxylin and eosin staining, Samples containing tumor tissue were available for subsequent analysis, and cancer tissue DNA was extracted using a commercial kit, QIAamp DNA FFPE Tissue Kit (Qiagen).

[0063] Control group 2: Drugs (gefitinib) were administered to patients with lung cancer, samples were taken every 21 days before and after the medication, and peripheral blood was collected from the patient's fasting vein on the same day. Blood samples were collected with EDTA anticoagulated vacuum blood collection tubes from Streck Company. Plasma was separated within 30 minutes after blood col...

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Abstract

The invention relates to detection of a mutation site of a PIK3CA gene in the ctDNA in urine, and particularly provides a method for separating the ctDNA from a urine sample, a method for building a sequencing library based on the urine sample, a method for performing nucleic acid sequencing based on the urine sample according to the sequencing library, equipment for separating the ctDNA from theurine sample and a system for determining the sensitivity of an individual drug. The method for separating the ctDNA from the urine sample comprises the following steps: adding EDTA into the urine sample to obtain a first mixed solution; centrifugating the mixed solution, and collecting supernate; concentrating the supernate to obtain a concentrated solution; mixing the concentrated solution withanion exchange resin to obtain a second mixed solution; performing chromatographic separation treatment on the second mixed solution to obtain eluent; purifying the eluent to obtain the ctDNA. According to the detection disclosed by the invention, a large quantity of ctDNAs can be extracted quickly from a large quantity of urine samples, and can be subjected to nucleic acid sequencing, and the sensitivity of the individual drug can be determined, so that the application prospect of the ctDNA in the urine is enlarged.

Description

technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to the detection of PIK3CA gene mutation sites in urine ctDNA. Background technique [0002] Circulating tumor DNA (ctDNA) is a small genome fragment released by tumor cells into the circulatory system such as the blood. It is derived from all tumor sites in the body and has extremely low content, large inter-individual differences, short half-life, and homogeneity. , Carrying characteristics such as tumor-specific genetic / epigenetic variation. These DNA fragments from the tumor genome will carry certain characteristics, including mutations, deletions, insertions, rearrangements, copy number abnormalities, and methylation. [0003] However, the method of obtaining ctDNA and its application are still to be developed. Contents of the invention [0004] The present invention aims to solve at least one of the technical problems existing in the prior art ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6886C40B50/06C12M1/00C12M1/34
CPCC12N15/101C12N15/1093C12Q1/6806C12Q1/6886C12Q2600/106C12Q2600/156C40B50/06C12Q2525/191C12Q2531/113
Inventor 陈谦张建民李新新贾海雪刘芳周瑜
Owner 索真(北京)医学科技有限公司
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