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Method for separating and identifying biliary tract cancer peripheral blood CTC (circulating tumor cells) and detecting downstream genes by HSPG (high-throughput sequencing assay)

A technology of peripheral blood and biliary tract, applied in the field of biological and medical detection, can solve the problems of short survival period, low degree of cell differentiation, poor response to chemotherapy, etc.

Pending Publication Date: 2020-11-24
中国人民解放军海军军医大学第三附属医院
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  • Description
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Problems solved by technology

The content of HSPG increases in poorly differentiated adenocarcinoma, and the sulfation level of HSPG in GBC tissues with low degree of cell differentiation, local metastasis, and advanced clinical stage increases, and patients with positive sulfated HSPG have poorer chemotherapy response and shorter survival time , but there are few studies on the overall expression of HSPG in biliary tract cancer

Method used

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  • Method for separating and identifying biliary tract cancer peripheral blood CTC (circulating tumor cells) and detecting downstream genes by HSPG (high-throughput sequencing assay)
  • Method for separating and identifying biliary tract cancer peripheral blood CTC (circulating tumor cells) and detecting downstream genes by HSPG (high-throughput sequencing assay)
  • Method for separating and identifying biliary tract cancer peripheral blood CTC (circulating tumor cells) and detecting downstream genes by HSPG (high-throughput sequencing assay)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] A method for separating and identifying CTCs in peripheral blood of biliary tract cancer by HSPG, comprising the steps of:

[0082] (1) Sampling: Take 7.5 mL of peripheral blood from patients with biliary tract cancer, place it in an anticoagulant tube and mix the whole blood sample;

[0083] (2) Remove plasma protein and plasma (clear) nucleic acid from the blood sample collected in step (1), add buffer solution to the whole blood, remove the supernatant by layered centrifugation, and gently shake the centrifuge tube to mix and precipitate the cells;

[0084] (3) Removal of red blood cells: After mixing, add the lysate to the centrifuge tube, place the centrifuge tube in a vertical mixer to mix, centrifuge to discard the supernatant, shake the centrifuge tube to mix and settle the cells, and then add buffer;

[0085] (4) Layered centrifugation: Add the layered liquid into a new centrifuge tube, superimpose all the liquid in step (3) on the top layer of the layered liqu...

Embodiment 2

[0122] A HSPG method for downstream gene detection of peripheral blood CTCs of biliary tract cancer, using a high-throughput sequencing method to perform genome-wide amplification of nucleic acids derived from blood circulating tumor cells of biliary tract cancer, the specific steps are as follows:

[0123] (1) Thaw and lyse the frozen CTC PCR plate on ice, and add 0.4M HCl to each well after lysing;

[0124] (2) Multiple replacement amplification was used for whole genome amplification, and the reaction mixture was prepared. For each reaction, sterilized water, 10× reaction buffer, BSA, DTT, dNTPs in the replication genome amplification kit, random six After the aggregates are mixed and acted according to the amount, they are then incubated on a thermal cycler at 30°C;

[0125] (3) The paramagnetic beads in the nucleic acid purification kit are used to terminate the subsequent MDA reaction, add 100 μL of the paramagnetic beads in the nucleic acid purification kit to each reac...

Embodiment 3

[0160] (1) Determine the sample size:

[0161] (a) 100 patients with biliary tract cancer were enrolled in the early stage, the detection system and indicators were evaluated, and the follow-up sample size was calculated.

[0162] (b) According to the previous research results, statisticians will preliminarily calculate and count the number of CTCs identified by HSPG antibody sorting specific for biliary tract cancer, and compare the sorting efficiency and differences among individual individuals, and count the sensitivity and specificity of markers , to further calculate the total sample size required, it is expected to enroll about 500 patients.

[0163] (c) According to the previous research results, the number and frequency of gene mutations derived from CTC nucleic acid are further counted, and the total sample size required is calculated. It is estimated that about 500 patients will be enrolled.

[0164] (2) Inclusion criteria:

[0165] The subjects of the study were p...

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Abstract

The invention provides a method for separating and identifying biliary tract cancer peripheral blood CTC (circulating tumor cells) and detecting downstream genes by HSPG (high-throughput sequencing assay). The method includes capturing tumor cells in the peripheral blood CTC to separate and identify the tumor cells; extracting DNA (deoxyribonucleic acid) from samples by the aid of DNA extraction kits; carrying out whole-genome amplification, library construction, sequencing data filtering and quality evaluation, sequencing depth coverage statistics, variation detection, key variation identification and the like on biliary tract cancer blood circulating tumor cell-derived nucleic acid by the aid of high-throughput sequencing processes; building a complete set of high-throughput sequencing analysis system; acquiring tumor specific molecular information for improving the specificity; capturing the tumor cells in blood by the aid of specific sorting antibodies; and acquiring spider silk tracks of genetic information carried by the released DNA and detecting and analyzing the spider silk tracks. The method has the advantage that the DNA can be analyzed and detected by the aid of gene expression level change of CTC-derived nucleic acid and gene expression level change of tissue-derived nucleic acid.

Description

technical field [0001] The invention relates to the technical field of biological and medical detection, in particular to a HSPG separation and identification method for peripheral blood CTC of biliary tract cancer and a downstream gene detection method. Background technique [0002] Circulating Tumor Cells (CTCs) in human peripheral blood refer to tumor cells that disseminate from tumor lesions into the peripheral blood circulation, and can develop into tumor metastatic lesions under certain conditions. Since more than 90% of cancer deaths are caused by metastasis, and CTCs are the direct source of tumor metastasis, the isolation of CTCs from blood and their molecular detection have attracted more and more attention. The components of peripheral blood are complex, among which the red blood cell count is 5.0-6.0×1012 / mL, the white blood cell count is 4.0-10.0×106 / mL, and there may be only 0-100 circulating tumor cells. At present, the detection of CTC has expanded from simp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/6869C12Q1/6886G16B20/00G16B30/10G16B35/00G16B40/00
CPCC12N5/0693C12N2509/00C12Q1/6869C12Q1/6886C12Q2600/106C12Q2600/156G16B20/00G16B30/10G16B35/00G16B40/00C12Q2535/122
Inventor 易滨袁磊吴英俊姜小清
Owner 中国人民解放军海军军医大学第三附属医院
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