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Micro-RNA (Ribonucleic Acid) colorimetric detection method based on rolling circle amplification

A technology of rolling circle amplification and detection method, which is applied in the detection field of biological macromolecules, can solve the problems of complex equipment and high requirements for experimental conditions, and achieve the effect of reducing detection costs

Active Publication Date: 2013-08-07
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the reaction relies on complex equipment, and the temperature needs to be continuously and accurately controlled during the reaction process, which requires high experimental conditions.

Method used

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Embodiment

[0028] figure 1 The overall process of the present invention is shown. The gold nanoparticles used in this embodiment have a particle size of 13nm, can absorb green light, and can produce a strong absorption peak in the visible spectrum at 520nm, showing wine red. The detection instrument of the colorimetric method used in this embodiment is an ultraviolet spectrophotometer. The base sequences of the target microRNAs used in this embodiment, linear template DNA and DNA probes modified by sulfhydryl groups are shown in Table 1:

[0029] Table I

[0030]

[0031] 1.1. Preparation of long-chain products containing multiple target microRNA fragments

[0032] a. Mix 10 μL target microRNA with 10 μL linear template DNA (1 μM, 2 mM PBS, pH 7.4), and place at 37° C. for 30 min.

[0033] b. Add 1 μL T4RNA ligase 2 to the above system for ligation, react at 37°C for 30 minutes, and then heat at 70°C for 10 minutes to inactivate it.

[0034] c. Add phi29 polymerase (final concentr...

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Abstract

The invention discloses a micro-RNA (Ribonucleic Acid) colorimetric detection method based on rolling circle amplification. The method is characterized by comprising the following steps of: amplifying the target micro-RNA into a long-chain product comprising a plurality of target micro-RNA segments using a rolling circle amplification technology, fixing a gold nanoparticle DNA (Deoxyribonucleic Acid) probe to the long-chain product comprising the plurality of target micro-RNA segments by means of hybridization reaction between nucleic acids, detecting the color change reaction of the gold nanometer particle of the gold nanometer particle DNA probe fixed on the long-chain product comprising the plurality of target micro-RNA segments, and detecting the long-chain product comprising the plurality of target micro-RNA segments so as to achieve the purpose of detecting the target micro-RNA. The detection method disclosed by the invention can be used for preventing the application of expensive instruments such as a PCR (Polymerase Chain Reaction) instrument and reducing the detection cost, and is conductive to the early diagnosis of a relevant disease.

Description

technical field [0001] The invention relates to a microRNA colorimetric detection method based on rolling circle amplification, which belongs to the detection field of biological macromolecules. Background technique [0002] MicroRNA (microRNA, miRNA) is a class of endogenous, non-coding RNA molecules, encoded by the genome of higher eukaryotes, with a length of 18-25 nucleotides. miRNAs can regulate the expression of other genes. Through specific binding to target messenger ribonucleic acid (mRNA), RNA-induced silencing complex (RNA Induced Silence ComplexX, RISC) degrades mRNA or hinders its translation, thereby inhibiting post-transcriptional gene expression. miRNA plays an important role in regulating gene expression, cell cycle, organism development timing, immune response and so on. And participate in tumorigenesis and viral infection. Most miRNAs are highly conserved, temporally and tissue-specific. Currently, only a small number of biological functions of miRNAs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 缪鹏韩坤刘涛白鹏利尹焕
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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