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Method for detecting multiple targets based on multiple positioning of green solvent and programmable oligonucleotide probe

A technology of oligonucleotide probes and green solvents, which is applied in the field of multiple positioning detection of multiple targets based on green solvents and programmable oligonucleotide probes, can solve the problem of no signal amplification process or insufficient signal amplification, difficult to low Effective detection of abundant molecular targets, unfavorable personnel and environmental protection and other issues, to achieve the effect of short detection time and amplified detection of target signals

Pending Publication Date: 2021-11-23
CHINA PHARM UNIV
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Problems solved by technology

Compared with the method of batch antibody staining, this method improves the speed of multi-target detection, but it needs to repeatedly rely on toxic reagents for the dissociation of fluorescent probes, which is not conducive to the protection of personnel and the environment in real practice; The method has no signal amplification process or insufficient signal amplification, and it is difficult to effectively detect low-abundance molecular targets, especially when there are common fluorescence scattering and fluorescence background effects in the sample to be tested

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  • Method for detecting multiple targets based on multiple positioning of green solvent and programmable oligonucleotide probe
  • Method for detecting multiple targets based on multiple positioning of green solvent and programmable oligonucleotide probe
  • Method for detecting multiple targets based on multiple positioning of green solvent and programmable oligonucleotide probe

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Embodiment 1

[0050] (1) Experimental materials and reagents:

[0051] Streptavidin (purchased from Beijing Boaosen); human breast cancer cell line MDA-MB 231 (purchased from Shanghai ATCC cell bank); citrate buffer, phosphate buffered saline (1×PBS solution, pH 7.4 ) (purchased from Gibco); DMEM medium (containing penicillin-streptomycin double antibody) (purchased from KGI); sterile fetal bovine serum (FBS) (purchased from Natocor-Industria Biológica); bovine serum albumin ( Bovine serum albumin, BSA) was purchased from (Amresco); cell culture dish (purchased from Wuxi Neisi); biotin, formamide, polyethylene glycol tert-octyl phenyl ether (Triton X-100) (purchased from Sigma- Aldrich); 4', 6-diamidino-2-phenylindole (DAPI) (purchased from Wuhan Boster); all probes, oligonucleotides, and template molecules were prepared by Shanghai Sangon Biotechnology Co., Ltd.; dNTPs (purchased from Beijing Saibaisheng); RNaseA, salmon sperm DNA solution (SalmonSperm DNA Solution) (purchased from Thermo...

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Abstract

The invention discloses a method for detecting multiple targets (such as proteins or other targets in cells) based on multiple positioning of a green solvent and a programmable oligonucleotide probe. The method comprises the steps of: directly or indirectly connecting target-specific oligonucleotides with the targets; and specifically connecting the oligonucleotides connected with one or more targets with corresponding fluorescent probes, and implementing positioning analysis of the corresponding targets through positioning detection of the fluorescent probes. In order to implement more multi-target positioning analysis, according to the method, the green solvent is used for removing the detected fluorescent probes, then the oligonucleotides on other unanalyzed targets are specifically connected to the corresponding fluorescent probes so as to implement positioning analysis of other corresponding targets through positioning detection on the fluorescent probes. Therefore, the method cannot be influenced by signal crossing of different specific fluorescent probes, also cannot be hindered by a limited number of detection channels of fluorescence detection equipment, and implement multi-target positioning analysis.

Description

technical field [0001] The invention belongs to the field of multiple target positioning detection in samples to be tested, in particular to a method for multiple positioning detection of multiple targets based on green solvents and programmable oligonucleotide probes. Background technique [0002] Cells are the basic unit of organisms; all physiological functions of organisms are carried out through communication between various types of cells and their mutual cooperation. Therefore, it is very important to elucidate the molecular mechanism of the biological functions of each cell in the organs and tissues of the body. Therefore, in the context of the in situ biological environment, characterizing the molecular composition of individual cells and tissue samples is crucial for in-depth exploration of life activities and disease mechanisms. [0003] Immunofluorescence imaging is an ideal tool for the characterization of molecular targets in biological samples (such as cells ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6841G01N33/68G01N33/58
CPCC12Q1/6841G01N33/68G01N33/582C12Q2563/107C12Q2527/125C12Q2565/601
Inventor 王琛邹秉杰宋沁馨
Owner CHINA PHARM UNIV
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