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Osteosarcoma circulating tumor cell identification kit

A technology for tumor cells and osteosarcoma, applied in the field of molecular biology, can solve problems affecting the sensitivity and specificity of detection

Inactive Publication Date: 2017-12-15
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The patent application with the application number 201510919145.6 we filed earlier, due to the relatively small number of markers screened in the technical solution, has affected the sensitivity and specificity of the detection to a certain extent

Method used

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  • Osteosarcoma circulating tumor cell identification kit
  • Osteosarcoma circulating tumor cell identification kit
  • Osteosarcoma circulating tumor cell identification kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The osteosarcoma circulating tumor cell identification kit described in this embodiment has two types, one with a labeled probe and one without a labeled probe.

[0043] Among them, the osteosarcoma circulating tumor cell identification kit A with labeled probes mainly includes:

[0044] One, capture probe

[0045] The capture probe is composed of three parts, from 5'to 3'are 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 sequence in the capture probe is the same as the marker gene of. The spacer is used to separate the P2 sequence of the capture probe from the target mRNA. By setting a spacer sequence of an appropriate length inside the probe, steric hindrance can be reduced, and the efficiency of the hybridization reaction and the specificity of the hybridization reaction can be improved. . The s...

Embodiment 2

[0072] Example 2 Using kit A in Example 1 to detect circulating tumor cells in the peripheral blood of patients with osteosarcoma

[0073] The formulations of the various solutions are as follows:

[0074]

[0075]

[0076] The probe mixture, amplification mixture, and color-developing mixture in this example all use all the probes in the corresponding gene list of kit A in Example 1.

[0077] 1. Sample pretreatment, filtering osteosarcoma CTCs onto the filter membrane

[0078] 1. Use the preservation solution to store the blood sample in the sample preservation tube, centrifuge at 600×g for 5 min, discard the supernatant, and remove the red blood cells.

[0079] 2. Add 4ml PBS and 1ml fixative, vortex to mix, and let stand for 8min at room temperature.

[0080] 3. Sample filtration: transfer the liquid in the sample preservation tube to the filter, turn on the vacuum filtration pump to exhaust the liquid; add 4ml PBS to the preservation tube, wash the wall of the tube and filter the liq...

Embodiment 3

[0139] Example 3 Using kit A in Example 1 to detect osteosarcoma cell lines

[0140] 1. Selection of cell lines

[0141] The epithelial cell marker gene of the kit of the present invention is selected from two or more of EpCAM, CDH1, KRT8, KRT16, KRT17, KRT18, KRT19, and KRT20, and the osteosarcoma metastasis-related mesenchymal cell marker gene is selected from: MMP2, MMP9 Two or more of VIM, CDH2, FN1, HIF-1a, SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL, and the white blood cell marker gene is CD45. The epithelial cell marker genes, osteosarcoma metastasis-related mesenchymal cell marker genes and various marker genes in leukocytes of the present invention are genes specifically expressed on osteosarcoma CTCs that the inventors have obtained through a large number of comparative experiments and statistical analysis. At the same time, the classification and metastasis evaluation of osteosarcoma CTCs derived from mesenchymal cells have good sensitivity, specificity and accuracy.

[014...

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PUM

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Abstract

The invention discloses an osteosarcoma circulating tumor cell identification kit. The kit comprises capture probes for marker gene mRNAs and a signal amplification system. The marker genes comprise at least two epithelial cell marker genes selected from EpCAM, CDH1, KRT8, KRT16, KRT17, KRT18, KRT19 and KRT20 and at least two osteosarcoma metastasis-related interstitial cell marker genes selected from MMP2, MMP9, VIM, CDH2, FN1, HIF-1a, SERPINE1, SURVIVIN, TWIST1, AKT2 and SNAIL. The kit can completely detect osteosarcoma circulating tumor cells, evaluate their metastatic ability to avoid the possibility of the false negative result caused by different expression levels of CTCs in different types of cancers and further improve the accuracy of detection.

Description

Technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and specifically relates to an osteosarcoma circulating tumor cell identification kit. Background technique [0002] Osteosarcoma (osteosarcoma), also called osteosarcoma, is a more common malignant bone tumor that occurs in adolescents or children under the age of 20, accounting for about 60% of primary malignant bone tumors in adolescents. Osteosarcoma develops from a mesenchymal cell line. It has a high degree of malignancy, rapid growth, and high frequency of distant metastasis. The rapid growth of tumors is due to the formation of tumor bone-like tissue and bone directly or indirectly through the cartilage stage. organization. At present, osteosarcoma is still a disease with high mortality of malignant tumors in children and adolescents. Its malignant degree is very high and the prognosis is extremely poor. It can appear lung metastasis within a few months,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q1/6841C12Q1/6886C12Q2600/158C12Q2563/107
Inventor 刘苏燕吴诗扬董艳
Owner SUREXAM BIO TECH
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