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Recognition probe, detection method and application of cancer cells

A technology of tumor cells and detection methods, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve the problems of low detection sensitivity, achieve high detection sensitivity, high detection efficiency, and chemical stability good sex effect

Active Publication Date: 2013-04-24
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In the prior art, nucleic acid aptamers are used to detect tumor cells by cross-linking nucleic acid aptamers on glass slides and other substrates, and the detection sensitivity is low.

Method used

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  • Recognition probe, detection method and application of cancer cells
  • Recognition probe, detection method and application of cancer cells
  • Recognition probe, detection method and application of cancer cells

Examples

Experimental program
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Embodiment 1

[0054] Human lung cancer cells were used as tumor cells to be tested, and a recognition probe (recognition probe 1) whose sequence was SEQ ID No.2 was designed based on the nucleic acid aptamer S11e (SEQ ID No.1) of human lung cancer A549 cells.

[0055] The length of S11e is 45bp, and a 32bp extended sequence is added to the 5' end of S11e to form probe 1.

[0056] Specific binding experiment of probe 1 to human lung cancer A549 cells

[0057] Specific steps are as follows:

[0058] Label the 5' end of probe 1 with fluorescein FAM to form FAM-probe 1;

[0059] Human lung adenocarcinoma A549 cells were cultured in laser confocal dishes;

[0060] Wash human lung adenocarcinoma A549 cells 3 times with phosphate buffered solution PBS;

[0061] Dilute FAM-probe 1 with phosphate buffer;

[0062] Mix the phosphate buffer solution of FAM-probe 1 with human lung cancer A549 cells, and incubate at room temperature for 20 minutes; use the lung cancer A549 cells without FAM-probe 1...

Embodiment 2

[0084] Human lung cancer cells were used as tumor cells to be tested, and a recognition probe (recognition probe 2) whose sequence was SEQ ID No.5 was designed based on the nucleic acid aptamer S11e (SEQ ID No.1) of human lung cancer A549 cells.

[0085] In order to eliminate primer-dimer interference during real-time fluorescent quantitative PCR, the sequence of the recognition probe was increased. The length of S11e was 45 bp, and a 55 bp extended sequence was added to the 5' end of S11e to form probe 2.

[0086] PCR amplification experiment using probe 2 as template

[0087] According to the sequence of probe 2, an upstream primer whose sequence is shown in SEQ ID No.6 and a downstream primer shown in SEQ ID No.7 were designed. The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0088] After PCR, the PCR products were electrophoresed with 2% agarose gel, and the results were as follows: Figure 4 As shown, the 100bp band i...

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Abstract

The invention discloses a recognition probe of cancer cells. The length of the recognition probe of the cancer cells is larger or equal to 70bp, and the recognition probe of the cancer cell comprises an nucleic acid aptamer which is combined with the cancer cells in a specific binding mode, and an extending sequence which is arranged at a 5' end or a 3' end of the nucleic acid aptamer, wherein the extending sequence is a single stranded nucleotide sequence which is not enabled to form a secondary structure by itself. The invention further provides a detection method of the cancer cells by using the recognition probe and a kit. The recognition probe of the cancer cells is simple in manufacture, free from immunogenicity, free from toxicity, and is good in chemical stability. The detection method of the cancer cells combines the recognition probe based on nucleic acid aptamer design with a nucleic acid amplification technology, is good in detection sensitivity, and is good in detection efficiency.

Description

technical field [0001] The present invention relates to a recognition probe for detecting tumor cells; a method for detecting tumor cells using the recognition probe; and a kit for the detection method. Background technique [0002] Due to environmental pollution, unhealthy lifestyle and other factors, the incidence of tumors is getting higher and higher, and it is gradually showing a younger trend, which seriously threatens the safety and health of human life. Molecular biology and clinical studies have shown that tumor invasion and micrometastasis have appeared in the early stages of tumorigenesis. [0003] At present, traditional tumor diagnosis methods mainly rely on the morphological characteristics of tumor tissue and tumor cells for judgment. It is necessary to sample and detect tumor tissue with a certain volume, and it is impossible to detect the existence of tumor cells when the number of tumor cells is small, that is, it is impossible to realize the tumor. Early ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/68C12Q1/06G01N21/64
Inventor 蔡林涛龚萍石碧华杨月婷王碧张鹏飞郑明彬胡德红盛宗海刘朋高笃阳郑翠芳
Owner SHENZHEN INST OF ADVANCED TECH
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