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Colon cancer nucleic acid aptamer obtained by rapid screening of tissue samples and application of colon cancer nucleic acid aptamer to detection preparation

A nucleic acid aptamer and colon cancer technology, which is applied in the preparation of colon cancer clinical diagnosis and treatment reagents, can solve the problems of low patient acceptance, unsatisfactory sensitivity and specificity, and the risk of bleeding and perforation

Pending Publication Date: 2020-11-17
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, colonoscopy is considered to be the gold standard for the diagnosis of colon cancer, but it is an invasive test that requires intestinal preparation, and there is a risk of complications such as bleeding and perforation during the test, which is not well accepted by patients and costs a lot
Although the fecal occult blood test is a non-invasive test, the test results are easily affected by factors such as diet
Stool DNA mutation detection and stool RNA-specific gene detection are non-invasive detection methods with good patient adaptability, but the sensitivity and specificity are not ideal for screening colon cancer

Method used

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  • Colon cancer nucleic acid aptamer obtained by rapid screening of tissue samples and application of colon cancer nucleic acid aptamer to detection preparation
  • Colon cancer nucleic acid aptamer obtained by rapid screening of tissue samples and application of colon cancer nucleic acid aptamer to detection preparation
  • Colon cancer nucleic acid aptamer obtained by rapid screening of tissue samples and application of colon cancer nucleic acid aptamer to detection preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Nucleic acid aptamer screening of clinical colon cancer tissue

[0021] Based on the Tissue-SELEX technology, clinical colon cancer tissues were used as positive screening samples, and colon cancer paracancerous tissues were used as negative screening samples for screening. Firstly, the X-Aptamer library with affinity group modification was synthesized, and then the initial library was incubated with colon cancer paracancerous tissue, and the nucleic acid sequence combined with the paracancerous tissue was retained and isolated after incubation. Simultaneously, the nonbinding nucleic acid sequences were incubated with clinical colon cancer tissue. Nucleic acid sequences bound to colon cancer tissue were retained and isolated after incubation. Then, in the second round of screening, the positive screening and negative screening libraries retained in the previous round were divided into three groups, respectively, and sterilized water (blank control group), co...

Embodiment 2

[0025] Example 2: Determining the sequence with the strongest specific binding ability to colon cancer cell line SW480

[0026] First, the 8 candidate sequences and the reference library strands were preprocessed. The candidate sequence and the control library were diluted with sterilized water to a final concentration of 250 nM, then denatured in a 95°C metal bath for 5 minutes, refolded on ice for 10 minutes, and then placed at room temperature. Second, colon cancer cells SW480 were pretreated. Wash the cells 2-3 times with DPBS; then digest the adherent colon cancer cells SW480 from the culture dish with 0.2% EDTA, and then pass through the washing buffer (Washing buffer, containing 0.45% glucose, 5 mM magnesium chloride) Cells were pipetted and collected into centrifuge tubes. Finally, candidate sequences and binding buffer (Binding Buffer: DPBS, 0.45% glucose, 5 mM magnesium chloride, 100mg / LtRNA, 1g / L BSA) were added to the treated cells. After shaking and mixing, pla...

Embodiment 3

[0029] Example 3: Determining the sequence with the strongest specific binding ability to colon cancer cell HCT116

[0030] First, the 8 candidate sequences and the reference library strands were preprocessed. The candidate sequence and control library were diluted to a final concentration of 250 nM, then denatured in a 95°C metal bath for 5 minutes, refolded on ice for 10 minutes, and then placed at room temperature. Second, colon cancer cells HCT116 were pretreated. The cells were washed 2-3 times with DPBS; the colon cancer cells HCT116 in the adherent state were digested from the culture dish with 0.2% EDTA, and then the cells were collected by blowing through the washing buffer. Finally, the prepared nucleic acid sequence and binding buffer are added to the treated cells. After shaking and mixing, place on a shaker at 4°C and incubate for 1 h. After the incubation was completed, centrifuge and wash 3 times with washing buffer, and then perform fluorescence detection by...

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Abstract

The invention discloses a colon cancer nucleic acid aptamer obtained by rapid screening of tissue samples and an application of the colon cancer nucleic acid aptamer to a detection preparation. The sequence of the nucleic acid aptamer is selected from any one of sequences shown in SEQ ID NO.1 to SEQ ID NO.8, or the sequence of the nucleic acid aptamer is marked and modified. The nucleic acid aptamer has the characteristics similar to or even superior to those of a biological antibody, and is mainly reflected in that the nucleic acid aptamer has specificity, high substantivity, low toxicity andlow immunogenicity, can be synthesized in vitro, and is convenient to store and transport. When the nucleic acid aptamer provided by the invention is used for early colon cancer tissue detection after being labeled by a fluorophore, the operation is simple and feasible, and the detection result is more intuitive. Besides, the nucleic acid aptamer disclosed by the invention is a short-chain nucleic acid sequence, and the in-vitro synthesis cost of the nucleic acid aptamer is relatively low.

Description

technical field [0001] The present invention relates to a nucleic acid aptamer and its application, in particular to a nucleic acid aptamer which can be used for the detection of colon cancer cells and clinical colon cancer sample tissue and its application in the preparation of reagents for clinical diagnosis and treatment of colon cancer. Background technique [0002] Colon cancer is the second leading malignancy in cancer-related mortality. About 95% of colon cancers are adenocarcinomas, and other rarer types include lymphomas, adenosquamous and squamous cell carcinomas. With the change of people's lifestyle and dietary structure, the incidence and mortality of colon cancer are increasing year by year. Colorectal cancer is also one of the most common malignant tumors in the United States, but from 2000 to 2013, the incidence rate of adults over the age of 50 decreased by 32%, and the incidence rate of distal colon cancer in people ≥65 years old decreased the most, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/574
CPCC12N15/115C12N15/1048G01N33/57419C12N2310/16
Inventor 胡小晓谭蔚泓马艺菡彭程李娟
Owner HUNAN UNIV
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