Switch mode aptamer probe and application thereof in tumor living cell and vital detection

A nucleic acid aptamer and tumor cell technology, which can be used in preparations for in vivo experiments, measuring devices, microbial determination/testing, etc., can solve the problem of inability to meet the analysis requirements of high specificity and high sensitivity of tumor cells and limit the early diagnosis of tumors To achieve the effect of shortening the detection time, improving the sensitivity and specificity, and expanding the detection system

Inactive Publication Date: 2010-08-25
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the inevitable non-specific adsorption between nucleic acid aptamers and non-target cells, this type of method cannot meet the requirements of convenient, rapid, high-specificity and high-sensitivity analysis of tumor cells in complex mixed systems, which limits the clinical detection of tumor cells. Realization of early diagnosis and development of relevant treatment

Method used

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  • Switch mode aptamer probe and application thereof in tumor living cell and vital detection
  • Switch mode aptamer probe and application thereof in tumor living cell and vital detection
  • Switch mode aptamer probe and application thereof in tumor living cell and vital detection

Examples

Experimental program
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Effect test

Embodiment 1

[0053] (Schematic diagram of the detection principle of the switch-type nucleic acid aptamer probe designed for CCRF-CEM tumor cells)

[0054] A switch-type nucleic acid aptamer probe of the present invention as shown in Figure 1, the nucleic acid aptamer probe includes a probe body 1 and a fluorescence generating unit 2 and a fluorescence extinguishing unit 3 respectively connected to the two ends of the probe body 1 , the probe body 1 includes a nucleic acid aptamer fragment 11 having a specific recognition function for target tumor cells and a nucleic acid fragment 13 complementary to the partial sequence of the nucleic acid aptamer fragment 11, and the nucleic acid aptamer fragment 11 and the nucleic acid fragment 13 are connected by a Fragments 12 are linked to form a hairpin structure. The ability of the nucleic acid fragment 13 to competitively hybridize to the nucleic acid aptamer fragment 11 is weaker than that of the target tumor cell. The fluorescence generating un...

Embodiment 2

[0065] Fluorescence Spectral Characterization of Switched Nucleic Aptamer Probes in Buffer

[0066] In four 200μL buffer (Dulbecco's PBS, 4.5g / L glucose, 5mM MgCl 2 , 1mg / mL BSA) samples were added 100nM of the switch type nucleic acid aptamer probe, switch type control nucleic acid probe, single fluorescently labeled nucleic acid aptamer probe and single fluorescently labeled control nucleic acid probe of the above-mentioned embodiment 1 , the nucleotide sequences of the four probes are as follows:

[0067] Switch-type aptamer probe: [5'-(Cy5)-CTA ACC GT TTT TTT TTT TTT TTT TTT TT ATC TAA CTGCTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-(BHQ2)-3'];

[0068]Switch control nucleic acid probe: [5'-(Cy5)-ACG GTT AG TTT TTT TTT TTT TTT TT ATA CGG TGACTG CGC CGC CGG GAA AAT ACT GTC TAA CCG TA-(BHQ2)-3'];

[0069] Single fluorescently labeled aptamer probe: [5'-(Cy5)-ATC TAA CTG CTG CGC CGC CGG GAA AATACT GTA CGG TTA GA-3'];

[0070] Single fluorescently labeled control nucleic acid...

Embodiment 3

[0075] Highly Specific Detection of Tumor Cells in Buffer with Switched Nucleic Aptamer Probes

[0076] Disperse 2 x 10 in 200 µL 5 Add 25 nM of the switch-type nucleic acid aptamer probe and the switch-type control nucleic acid probe prepared in Example 2 to two buffer samples of CCRF-CEM tumor cells respectively, mix them evenly, and incubate at room temperature in the dark for 15 minutes, then immediately The fluorescence signal of the cells was detected with a FACSCalibur flow cytometer (Becton-Dickinson, USA). In addition, the buffer containing negative control cells such as Ramos cells, human multiple myeloma cells (abbreviated as U266 cells), African marmoset B lymphocytes (abbreviated as B95-8 cells) and other negative control cells were tested, and the operation was as described above. The detection results of buffers containing the aforementioned four different tumor cells are shown in FIG. 5 . It can be seen from Fig. 5 that both the switch-type nucleic acid aptam...

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Abstract

The invention belongs to the nucleic acid detection technical field, in particular discloses a switch mode aptamer probe and application thereof. The aptamer probe comprises a probe body as well as a fluorescence generating unit and a fluorescence extinguishing unit which are respectively connected at the two ends of the probe body, the probe body contains an aptamer segment with specificity identification function to target tumor cell and a nucleic acid segment forming complementary sequence with the aptamer segment, and the aptamer segment and the nucleic acid segment are connected to form a hairpin type structure by a connection segment with the length of 7-15nm; and the capacity of the nucleic acid segment competitive hybridization aptamer segment is weaken than that of the target tumor cell. The application of the probe of the invention can be at least one of specificity detection of tumor living cell in buffer solution, effective detection on tumor living cell in serum and real-time imaging and vital diction of tumor in moving organism. The probe of the invention has higher stability, specificity and sensibility, application operation thereof is simple, fast, sensible and specific, and cost is low.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a nucleic acid aptamer probe and its application in tumor detection. Background technique [0002] Tumor is a common disease that threatens human health. Accurate immunophenotyping of tumor cells is conducive to early diagnosis and treatment of tumors, thereby improving the cure rate of tumors. Antigen-antibody reaction is the basis of immunological diagnosis. The rapid development of antibody technology in the past few decades has made great contributions to the research on tumor diagnosis and treatment. However, the emergence of nucleic acid aptamers in recent years has brought unprecedented challenges to antibody technology. [0003] Nucleic acid aptamers (also known as Aptamers) are artificially synthesized oligonucleotides designed using the strict recognition ability and affinity between nucleotides, and through the systematic evolution of ligand...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11A61K49/00G01N21/64
CPCG01N21/6428G01N21/6456C12Q1/6886A61K49/0054C12Q1/6818G01N2021/6432C12Q2525/301C12Q2525/205A61K48/0058
Inventor 王柯敏石慧何晓晓叶晓生伍旭郭秋平周冰
Owner HUNAN UNIV
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