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H4 nucleic acid aptamer and detection kit for detecting GBM cells and application of H4 nucleic acid aptamer and detection kit

A nucleic acid aptamer and detection kit technology, applied in the field of analysis and detection, can solve the problems of insufficient targeting and inability to accurately navigate the border of glioma, achieve good targeting, avoid interference from false positive signals, Good biocompatibility effect

Pending Publication Date: 2022-05-13
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the lack of targeting in the application of existing clinical fluorescent agents and the inability to accurately navigate to the border of glioma, there is an urgent need for a fast and sensitive method to detect GBM

Method used

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  • H4 nucleic acid aptamer and detection kit for detecting GBM cells and application of H4 nucleic acid aptamer and detection kit
  • H4 nucleic acid aptamer and detection kit for detecting GBM cells and application of H4 nucleic acid aptamer and detection kit
  • H4 nucleic acid aptamer and detection kit for detecting GBM cells and application of H4 nucleic acid aptamer and detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Detection of SVG(P12) glial cells

[0045] Select the SEQ ID NO.10 sequence of the 5' end and the 3' end to connect the fluorescent group Cy5 and the quenching group BHQ2 respectively (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.), and the obtained powdery DNA H4-10 (SEQ ID NO.10) was centrifuged at 4000rpm for 60s, and 10μL of deionized water was added to make a stock solution DNA with a final concentration of 100μM, and then 90μL of buffer solution (5mM Tris-HCl, 250mM NaCl, 50mM MgCl 2 ) was further diluted to a final concentration of 10 μM stock solution DNA, fully shaken and mixed, heated to 95 ° C in a metal bath, kept for 5 min, and then slowly cooled to room temperature, and DNA with a hairpin structure was obtained through the above annealing steps. Such as figure 1 In (A) when the hairpin structure is not formed, the concentration of the aptamer ([aptamer]) and 666nm (F 666nm The fluorescence intensity at ) is positively correlated, and the func...

Embodiment 2

[0051] Detection of U87 glioma cells

[0052] Using the synthetic method of Example 1, the obtained powdered H4-10 (SEQ ID NO.10) DNA was centrifuged at 4000rpm for 60s, and 10 μL of deionized water was added to prepare a stock solution DNA with a final concentration of 100 μM, and then buffer solution (5 mM Tris -HCl, 250mM NaCl, 50mM MgCl 2 ) was further diluted to a final concentration of 10 μM stock solution DNA, fully shaken and mixed, heated to 95 ° C in a metal bath, kept for 5 min, and then slowly cooled to room temperature, and DNA with a hairpin structure was obtained through the above annealing steps.

[0053] Take 10 μL of the DNA solution of the above-mentioned hairpin structure and dissolve it in 900 μL DMEM, and co-incubate with U87 cells in a confocal glass-bottom dish at 37°C for 20 min.

[0054] Discard the supernatant, wash twice with PBS, fix with 4% paraformaldehyde for 20 min, wash away the 4% paraformaldehyde with PBS, and add 50 μL of cell nucleus dye ...

Embodiment 3

[0057] Detection of LN229 glioma cells

[0058] Using the synthetic method of Example 1, the obtained powdered H4-10 (SEQ ID NO.10) DNA was centrifuged at 4000rpm for 60s, and 10 μL of deionized water was added to prepare a stock solution DNA with a final concentration of 100 μM, and then a buffer solution (5 mM Tris-HCl, 250mM NaCl, 50mM MgCl 2 ) was further diluted to a final concentration of 10 μM stock solution DNA, fully shaken and mixed, heated to 95 ° C in a metal bath, kept for 5 min, and then slowly cooled to room temperature, and DNA with a hairpin structure was obtained through the above annealing steps.

[0059] Take 10 μL of the DNA solution of the above-mentioned hairpin structure and dissolve it in 900 μL DMEM, and co-incubate with LN229 cells in a confocal glass-bottom dish at 37°C for 20 min.

[0060] Discard the supernatant, wash twice with PBS, fix with 4% paraformaldehyde for 20 min, wash away the 4% paraformaldehyde with PBS, and add 50 μL of cell nucleus...

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Abstract

The invention discloses an H4 nucleic acid aptamer and a detection kit for detecting glioblastoma multiforme and application of the H4 nucleic acid aptamer and the detection kit. The nucleotide sequence of the H4 nucleic acid aptamer is shown as any one of SEQ ID NO.1-10. According to the present invention, the H4 nucleic acid aptamer is adopted to specifically recognize the proteins of the tumor markers Ku70 and Ku80, the fluorescence imaging technology is combined, the 5'end and the 3 'end of the H4 nucleic acid aptamer are respectively modified with the complementary bases, the fluorophore and the quenching group thereof, the hairpin structure is formed in the base complementary pairing manner, and the kit comprising the H4 nucleic acid aptamer is used for rapid detection of GBM-related diseases, the detection method is simple and high in feasibility, has potential clinical value, and is expected to realize intraoperative navigation of brain glioma resection surgery in the future.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to an H4 nucleic acid aptamer for detecting GBM cells, a detection kit and applications thereof. Background technique [0002] Glioblastoma multiforme (GBM) is one of the deadliest primary central nervous system tumors in both adults and children, accounting for 52% of all primary brain tumors. Several studies have shown that the most important factor affecting the survival of glioma patients is the degree of tumor resection. Achieving complete surgical resection of the tumor is limited by the location of the tumor itself (close to active regions of the brain) and the difficulty of identifying tumor tissue at the margin of resection. 8-70% of surgical cases will have tumor false-positive resection margins, which leads to a five-year survival rate of glioma patients of only about 9.8%, with an average survival rate of only 14.6 months. [0003] Fluorescen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/569G01N33/574
CPCC12N15/115G01N33/56966G01N33/57407C12N2310/16
Inventor 王兆寅尤永平胡席席戴志晖张军霞
Owner NANJING NORMAL UNIVERSITY
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