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Method for detecting microRNA by combining CRISPR/Cas13a with electrochemical luminescence system and application thereof

A luminescent system, electrochemical technology, applied in biochemical equipment and methods, chemiluminescence/bioluminescence, analysis by making materials undergo chemical reactions, etc., can solve the problems that have not yet been detected such as microRNA

Active Publication Date: 2020-04-10
SOUTH CHINA NORMAL UNIVERSITY
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Problems solved by technology

However, so far, there is no report on the combination of CRISPR / Cas system and electrochemiluminescence system for the detection of microRNA

Method used

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Experimental program
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Effect test

Embodiment 1

[0072] 1. Expression and purification of Cas13a protein

[0073] The amino acid sequence of the Cas13a protein (ie LbuCas13a protein) is shown in SEQ ID NO.1, and the gene sequence encoding the Cas13a protein is shown in SEQ ID NO.2. The plasmid used in the present invention is pET-Sumo-LbuCas13a (Liu, L.; Li, X.Y.; Ma, J.; Li, Z.Q.; You, L.L.; Wang, J.Y.; Wang, M.; Zhang, X.Z.; Wang, Y.L. The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a. Cell 2017, 170, 714-726.). First transform the plasmid into Escherichia coli Rosetta2(DE3) (Novagen) in TB medium (OXOID) (containing 34 μg / mL chloramphenicol (Sangon) and 50 μg / mL kanamycin (Sangon)) medium Incubate at 37°C. When the cell number of Escherichia coli OD 600 =0.6, protein expression was induced by 0.1 mM isopropyl-1-thio-b-d-galactoside (IPTG, sigma) for 12 hours at 16°C. Escherichia coli Rosetta (DE3) (Novagen) cells were collected and sonicated in a buffer containing, 20 mM Tris-HCl (pH 7.5), 1 M NaCl, 20 ...

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Abstract

The invention discloses a method for detecting microRNA by combining CRISPR / Cas13a with an electrochemical luminescence system and application thereof. The method comprises the following steps that aCas13a / crRNA complex is used for recognizing Target RNA and the activity of a Cas13a non-specific shearing RNA probe is triggered to shear pre-trigger; dephosphorylation treatment is performed on theobtained product through a T4PNK enzyme to obtain a mature-trigger, and then EXPAR reaction is made to obtain a dsDNA product; and an EXPAR amplification product is mixed with a solution containing 'photoswitch 'molecules and TPrA and then pBPE-ECL detection is performed, and miRNAs detection is performed based on an obtained effective signal value.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method and application of CRISPR / Cas13a combined with an electrochemiluminescence system for detecting microRNA. Background technique [0002] Mature microRNAs (miRNAs) are a class of non-coding small RNAs with regulatory functions in eukaryotes, with a length of about 18-25 nt. Current studies have shown that the occurrence of many cancers is closely related to the abnormal expression of miRNAs, and miRNAs are expected to become potential markers for early cancer diagnosis. Therefore, rapid and sensitive detection of miRNAs is of great significance for early diagnosis and treatment of cancer. [0003] Due to the low expression level of miRNAs in cells, short sequences and unstable hybridization with probes, the detection of miRNAs is very challenging. At present, traditional miRNAs detection methods include northern blotting, real-time quantitative PCR, microarr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/6886G01N21/76G01N27/327
CPCC12Q1/682C12Q1/6886G01N27/3275G01N21/76C12Q2600/178C12Q2600/158Y02A50/30
Inventor 邢达黄茹周婷黄梦琪
Owner SOUTH CHINA NORMAL UNIVERSITY
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