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Circulating gastric tumor cell identifying kit

A technology for tumor cells and gastric cancer cells, applied in the field of molecular biology, can solve the problems of missed treatment opportunities, contaminated blood samples, insufficient operation rate, etc., and achieve the effect of improving accuracy and reliability and improving accuracy.

Inactive Publication Date: 2017-06-20
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of specific clinical symptoms and the limitations of clinical diagnosis methods in most gastric cancer patients, patients are usually in the advanced stage of the disease when they are diagnosed, and the best opportunity for treatment is missed. The operation rate is less than 10%, and the survival rate of patients after surgery shorter period
At present, the identification and typing of CTCs in patients with various cancers mainly depends on the detection of the expression of common CTCs epithelial and mesenchymal marker molecules. Contamination of blood samples by epithelial cells, coupled with differential expression of some epithelial and mesenchymal markers in different cancer types, can lead to some false positive and false negative test results

Method used

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  • Circulating gastric tumor cell identifying kit
  • Circulating gastric tumor cell identifying kit
  • Circulating gastric tumor cell identifying kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] There are two types of gastric cancer circulating tumor cell identification kits described in this example, those with labeled probes and those without labeled probes.

[0042] Among them, the gastric cancer circulating tumor cell identification kit A with labeled probes mainly includes:

[0043] 1. Capture probe

[0044] The capture probe consists of three parts, from the 5' end to the 3' sequence is the sequence P1 that is complementary to the mRNA of the corresponding marker gene, the spacer sequence, and the P2 sequence that is complementary to the corresponding amplification probe P3 sequence, the same category The P2 sequences in the capture probes of the marker genes are the same. The spacer is used to space the capture probe P2 sequence from the target mRNA, and by setting a spacer sequence of appropriate length inside the probe, it can reduce steric hindrance, improve the efficiency of the hybridization reaction and the specificity of the hybridization reactio...

Embodiment 2

[0073] Example 2 Using kit A in Example 1 to detect circulating tumor cells in peripheral blood of patients with gastric cancer

[0074] The formula of described various solutions is as follows:

[0075]

[0076]

[0077] The probe mixture, amplification mixture, and chromogenic mixture in this example all use all the probes in the corresponding gene list in Example 1 (including all marker genes being CEA, HER2, KRT7, KRT8, KRT17, KRT18, KRT19, KRT20, AKT2, HIF-1A, TWIST1, VIMENTIN, CD45).

[0078] 1. Sample pretreatment, filtering gastric cancer CTCs onto the filter membrane

[0079] 1. Preserve the blood sample in the sample preservation tube with preservation solution, centrifuge at 600×g for 5 minutes, discard the supernatant, and remove the red blood cells.

[0080] 2. Add 4ml of PBS and 1ml of fixative, vortex to mix, and let stand at room temperature for 8min.

[0081] 3. Sample filtration: transfer the liquid in the sample storage tube to the filter, turn on t...

Embodiment 3

[0140] Example 3 Using the kit A in Example 1 to detect gastric cancer cell lines

[0141] 1. Selection of cell lines

[0142] The gastric cancer cell marker genes of the kit of the present invention are selected from: one or more of CEA and HER2; epithelial cell marker genes are selected from: one or more of KRT7, KRT8, KRT17, KRT18, KRT19, KRT20; mesenchymal cell markers The gene is selected from: one or more of AKT2, HIF-1A, TWIST1, VIMENTIN; the leukocyte marker gene is CD45. The various marker genes in gastric cancer cell marker genes, epithelial cell marker genes, mesenchymal cell marker genes and leukocyte marker genes selected in the present invention are the genes specifically expressed on gastric cancer CTCs obtained by the inventor through a large number of experiments and statistical analysis and screening. , has good specificity and accuracy in the detection of gastric cancer cells.

[0143] In this example, the mixed epithelial-mesenchymal gastric cancer cell l...

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Abstract

The invention relates to a circulating gastric tumor cell identifying kit. The identifying kit, in accordance with each marker gene mRNA, comprises a capture probe, an amplification probe and a marking probe, wherein the marker gene mRNAs include three varieties: gastric tumor cell marker gene mRNA selected from at least one of CEA and HER2, epithelial cell marker gene selected from at least one of KRT7, KRT8, KRT17, KRT18, KRT19 and KRT20, and mesenchymal cell marker gene selected from at least one of AKT2, HIF-1A, TWIST1 and VIMENTIN. The identifying kit provided by the invention, which adds detection on gastric tumor cell marker gene on the basis of CTCs epithelial cell marker genes and mesenchymal cell marker genes which are common in various tumors, can avoid a false positive result caused by such factors as non-tumor epithelial cells in blood, the introduction of normal epithelial cells to a sampling process and the like, so that the detected epithelial cell marker gene and / or mesenchymal cell marker gene cells are circulating gastric tumor cells; and the accuracy and reliability of a detection result can be further enhanced.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a gastric cancer circulating tumor cell identification kit. Background technique [0002] Gastric cancer (gastric cancer) is a malignant tumor derived from gastric mucosal epithelium, accounting for the third of all malignant tumors and the first of digestive tract malignant tumors. It is one of the most common and deadly malignant tumors in the world. The second most common cause of death from cancer. At present, in order to improve the curative effect of gastric cancer and reduce the mortality rate, the key is to do a good job in the following three aspects: early detection, early diagnosis and early treatment. However, due to the lack of specific clinical symptoms and the limitations of clinical diagnosis methods in most gastric cancer patients, patients are usually in the advanced stage of the disease when they are diagnosed, and the be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q1/6841C12Q2600/158C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 刘苏燕吴诗扬董艳
Owner SUREXAM BIO TECH
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