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Marking and identifying method of micro-particle carrier and application of marking and identifying method

A microparticle and carrier technology, applied in the field of labeling and identification of microparticle carriers, can solve the problems of poor quantitative specificity, expensive instruments and signal analysis software, low sensitivity, etc., achieve good stability, improve stability and distinguishability, and facilitate Qualitative and quantitative detection of effects

Pending Publication Date: 2018-09-14
杜权
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are low sensitivity, poor quantitative specificity, expensive instruments and signal analysis software, and considerable technical difficulties in probe preparation and hybridization.

Method used

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  • Marking and identifying method of micro-particle carrier and application of marking and identifying method
  • Marking and identifying method of micro-particle carrier and application of marking and identifying method
  • Marking and identifying method of micro-particle carrier and application of marking and identifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Experimental process of nucleic acid detection scheme

[0087] The nucleic acid detection scheme provided by the present invention includes six operation steps: fluorescent labeling of particle carriers, capture probe loading, nucleic acid sample extraction, fluorescent labeling of nucleic acid samples, liquid phase hybridization and hybridization signal analysis. Now take the detection of the expression level of microRNA-146b-5p (5'-ugagaacugaauuccauaggcu, nucleotide sequence shown in SEQ ID NO: 1) in 3T3-L1 cells as an example to illustrate the technical solution provided by the present invention.

[0088] 1. Fluorescent labeling of microparticle carriers: the microparticle carriers used in this example are agarose microspheres (Streptavidin High Performance, 17-5113-01), the surface of the microspheres is coupled with streptavidin molecules that can be combined with biotin. The marker molecule used for fluorescent labeling (fluorescence coding) agarose m...

Embodiment 2

[0102] Example 2: Technical optimization of nucleic acid detection scheme

[0103] In order to establish a stable liquid-phase hybridization scheme, the present invention systematically optimizes the hybridization conditions. The details of the experimental materials and experimental procedures are as follows.

[0104] A. Microsphere carrier: Streptavidin from GE Healthcare Bio-Sciences High Performance Agarose Microsphere Carrier.

[0105] B. Microsphere labeling molecules:

[0106] Marker molecule 1: 5'-Alexa 488-AAAAAAAAAA-biotin

[0107] Marker 2: 5'-Texas Red-AAAAAAAAAA-biotin

[0108] C. Detection of nucleic acid samples: chemically synthesized four microRNA analogues as detection samples, the RNA sequences of which are shown in SEQ ID NO: 6-SEQ ID NO: 9.

[0109] mmu-miR-146b-5p: 5'-ugagaacugaauuccauaggcu

[0110] mmu-miR-222-3p:5'-agcuacaucuggcuacugggu

[0111] mmu-miR-302a-3p:5'-uaagugcuuccauguuuugguga

[0112] mmu-miR-let-7d-5p:5'-agagguaguagguugcauaguu

[01...

Embodiment 3

[0144] Example 3: Technical verification of nucleic acid detection scheme

[0145] In order to verify the sensitivity and specificity of the technical solution of the present invention, the present invention carried out technical verification research on the nucleic acid detection solution. The details of the experimental materials and experimental procedures are as follows.

[0146] A. Microsphere carrier: Streptavidin from GE Healthcare Bio-Sciences High Performance Agarose Microsphere Carrier.

[0147] B. Microsphere labeling molecules:

[0148] Marker molecule 1: 5'-Alexa 488-AAAAAAAAAA-biotin

[0149] Marker 2: 5'-Texas Red-AAAAAAAAAA-biotin

[0150] C. Detection of nucleic acid samples: chemically synthesized four microRNA analogues as detection samples, the RNA sequences of which are shown in SEQ ID NO: 6-SEQ ID NO: 9.

[0151] mmu-miR-146b-5p: 5'-UGAGAACUGAAUUCCAUAGGCU

[0152] mmu-miR-222-3p: 5'-AGCUACAUCUGGCUACUGGGU

[0153] mmu-miR-302a-3p: 5'-UAAGUGCUUCCAUG...

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Abstract

The invention discloses a marking and identifying method of a micro-particle carrier and application of the marking and identifying method. Under a certain coupling condition, a certain amount of at least one detectable marking molecule and a certain amount of the micro-particle carrier are in contact and are combined so that the micro-particle carrier is specifically marked through the marking molecule. By qualitatively and quantitatively detecting the marking molecule coupled with the micro-particle carrier, the type of the marked micro-particle carrier is identified. According to the marking and identifying method disclosed by the invention, the marking molecule which is artificially synthesized and has a certain amount of detectable groups is utilized to realize accurate and quantitative marking of the micro-particle carrier, and the stability and distinguishable degree of micro-particle carrier markers with the same type are remarkably improved. Based on the marking and identifying method of the micro-particle carrier, provided by the invention, the invention further provides a detection method of nucleic acid molecules and a detection method of microorganisms, and rapid and convenient qualitative and quantitative detection can be carried out on dozens of the known nucleic acid molecules and dozens of the known microorganisms; the marking and identifying method has the advantages of high flux, high precision and good stability.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection, in particular to a method for labeling and identifying microparticle carriers and its application. Background technique [0002] As a carrier of genes and expressed genes, nucleic acid detection is an important aspect in biomedical research and applications. At present, the mainstream nucleic acid detection technologies include Northern blot, RT-PCR, real-time fluorescent quantitative PCR, gene chip and next-generation sequencing technology. [0003] Northern blot is used to detect specific RNA and is the most classic gene expression detection method. It can detect gene expression level qualitatively or quantitatively, but the detection throughput is low, and the operation steps are cumbersome and technical requirements are high. [0004] RT-PCR technology is roughly divided into two types: qualitative RT-PCR and quantitative real-time fluorescent quantitative RT-PCR. It is usuall...

Claims

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Application Information

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IPC IPC(8): C12Q1/6816C12Q1/06C12Q1/04
CPCC12Q1/6816C12Q2563/131
Inventor 杜权王华全
Owner 杜权
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