Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Composite locked nucleic acid magnetic bead probe for detecting miRNA marker, construction method and diagnostic reagent containing probe

A technology of locked nucleic acid probe and construction method, applied in the field of composite locked nucleic acid magnetic bead probe and diagnostic reagent, can solve the problems of low expression amount, low sensitivity, large sample volume requirement, etc., achieve strong binding force and reduce background signal Effect

Pending Publication Date: 2022-03-01
CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They have their own advantages and disadvantages: the method without amplification is easy to operate, but requires a large sample size and low sensitivity; while the method based on amplification has high sensitivity, but it is limited by the inability to directly detect the level of miRNAs and non-specific heterosexual expansion
The reason is that the sequence of miRNAs is very short, and these amplification methods and corresponding probe design are very delicate and complicated processes
Most traditional PCR techniques make indirect judgments by examining their precursors, which cannot reflect the real level of miRNAs
Moreover, the expression of miRNAs in body fluids is much lower than that in tissues, so the conventional techniques for detecting miRNAs in tissues are not necessarily applicable to the detection of miRNAs in body fluids.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Composite locked nucleic acid magnetic bead probe for detecting miRNA marker, construction method and diagnostic reagent containing probe
  • Composite locked nucleic acid magnetic bead probe for detecting miRNA marker, construction method and diagnostic reagent containing probe
  • Composite locked nucleic acid magnetic bead probe for detecting miRNA marker, construction method and diagnostic reagent containing probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Selection of the optimal incubation temperature

[0044] 1. DNA probe preparation

[0045] According to the molecular base sequence of the marker miRNA486-5p to be detected, the corresponding locked nucleic acid (LNA) was synthesized as the capture probe LNA probe-486-5p, and the FAM fluorophore and biotin and other molecules were modified at both ends respectively. .

[0046] 2. DNA probe molecules immobilized on magnetic beads

[0047] The superparamagnetic microspheres are covalently combined with high-purity streptavidin to form streptavidin magnetic beads (streptavidinMBs, SMBs). Filter off the supernatant and redisperse into 2×B&W buffer. An appropriate amount of LNA probe molecules were preheated at 95°C for 5 minutes to eliminate the cross-hybridization phenomenon, and then mixed with the SMBs suspension, and the concentration of each LNA in the prepared test solution was 1 μM. Slightly shake the test solution and incubate at 37°C for 20 minutes to...

Embodiment 2

[0055] Example 2 Minimum detection concentration test

[0056] 1. DNA probe preparation

[0057] According to the molecular base sequence of the marker miRNA486-5p to be detected, the corresponding locked nucleic acid (LNA) was synthesized as the capture probe LNA probe-486-5p, and the FAM fluorophore and biotin and other molecules were modified at both ends respectively. .

[0058] 2. DNA probe molecules immobilized on magnetic beads

[0059] The superparamagnetic microspheres are covalently combined with high-purity streptavidin to form streptavidin magnetic beads (streptavidinMBs, SMBs). Filter off the supernatant and redisperse into 2×B&W buffer. An appropriate amount of LNA probe molecules were preheated at 95°C for 5 minutes to eliminate the cross-hybridization phenomenon, and then mixed with the SMBs suspension, and the concentration of each LNA in the prepared test solution was 1 μM. Slightly shake the test solution and incubate at 37°C for 20 minutes to better cou...

Embodiment 3

[0070] Example 3 Cross-reaction degree test

[0071] 1. DNA probe preparation

[0072] According to the molecular base sequence of the marker miRNA486-5p to be detected, the corresponding locked nucleic acid was synthesized as the capture probe LNAprobe-486-5p, and the FAM fluorophore and biotin and other molecules were modified at both ends.

[0073] 2. DNA probe molecules immobilized on magnetic beads

[0074]Superparamagnetic microspheres are covalently combined with high-purity streptavidin to form streptavidin magnetic beads (streptavidinMBs, SMBs). At room temperature, take a certain amount of SMBs and wash them with 1×B&W buffer. Filter off the supernatant, and then disperse into 2×B&W buffer. Take an appropriate amount of LNA detection molecules and preheat them at 95°C for 5 minutes to clear the cross-hybridization phenomenon, and then mix them with the SMBs suspension. The concentration of each LNA in the prepared test solution is 1 μM. Shake the test solution sli...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a composite locked nucleic acid magnetic bead probe for detecting a miRNA marker, a construction method of the composite locked nucleic acid magnetic bead probe and a diagnostic reagent containing the composite probe. The composite locked nucleic acid magnetic bead probe comprises three probes in the same magnetic bead, wherein the three probes comprise an FAM fluorophore-labeled LNA probe-486-5p probe, a Cy5 fluorophore-labeled LNA probe-21 probe and a TAMRA fluorophore-labeled LNA probe-210 probe. According to the invention, a DNA probe molecule modified with a fluorescence label is fixed on a magnetic bead through biotin-streptavidin connection. A DNA probe sequence and target miRNA are matched and hybridized to form double strands, under the action of DSN enzyme, the DNA probe in the hybrid double strands is hydrolyzed into fragments, fluorophore molecules are suspended in reaction liquid, but miRNA is kept complete and hybridized with the DNA probe again, and DSN enzyme digestion reaction is carried out. The steps are repeated, and the number of fluorophores of the final system is remarkably amplified. The sensitive detection of the concentration of the target miRNA can be realized by testing the signal intensity of the fluorophore in the supernatant. According to the invention, three different lung cancer marker miRNAs in body fluid can be conveniently and ultrasensitively detected at the same time, and the lung cancer can be accurately diagnosed.

Description

technical field [0001] The invention relates to the fields of molecular biology and nucleic acid chemistry, in particular to a composite locked nucleic acid magnetic bead probe for detecting miRNA markers, a construction method and a diagnostic reagent containing the probe. Background technique [0002] Among the many causes of death of residents in large and medium cities in my country, cancer is the first. According to the latest authoritative conclusion of the World Health Organization, if cancer patients can be detected early, the cure rate can reach more than 80%. Therefore, early diagnosis of cancer is very necessary. Early detection and treatment of cancer can reduce the pain, mental and economic burden of patients. [0003] Among cancer patients, lung cancer accounts for more than 20%. Traditional early detection techniques for lung cancer include chest radiography, low-dose computed tomography (LDCT), and lung biopsy. However, these techniques have several limit...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6834C12Q1/6806C12N15/11
CPCC12Q1/6886C12Q1/6834C12Q1/6806C12Q2600/158C12Q2600/178C12Q2563/107C12Q2563/143C12Q2563/149C12Q2563/131C12Q2521/301C12Q2527/127
Inventor 何露·德杰比查克·特里里周大明穆罕默德·巴里孔祥东石彪田荣王德强
Owner CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com