Method for detecting miRNA (ribonucleic acid)

A step-by-step, self-supporting technology that can be used in biochemical equipment and methods, measuring devices, and microbial determination/inspection, etc., and can solve the problems of long detection time, complicated operation and high cost.

Inactive Publication Date: 2014-04-02
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a general-purpose, high-sensitivity, convenient and fast detection and low-cost miRNAs detection technology that generally has defects such as low sensitivity, long detection time, complicated operation and high cost. Quantitative detection method of miRNAs

Method used

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  • Method for detecting miRNA (ribonucleic acid)
  • Method for detecting miRNA (ribonucleic acid)
  • Method for detecting miRNA (ribonucleic acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Reagents and materials

[0042]For the convenience of description, the four ssDNA single strands whose sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 are called Tetra-A, Tetra-B, respectively. Tetra-C and Tetra-D, the tetrahedral DNA nanostructure probes are formed by self-assembly of the four ssDNA single strands. The specific information is: Tetra-A (75bp, molecular weight 23028.0, ssDNA), Tetra-B (55bp, molecular weight 17018.0, ssDNA modified with thiol at the 5' end), Tetra-C (55bp, molecular weight 16898.0, ssDNA modified with thiol at the 5' end ) and Tetra-D (55bp, molecular weight 16877.0, ssDNA modified with thiol at the 5' end), the four ssDNA single strands were purchased from Dalian Takara Bioengineering Co., Ltd. The four DNAs that constitute the tetrahedral structure probe contain three structural domains, each of which is complementary to the corresponding domains of the other three single-stranded DNAs (17 pairs of base...

Embodiment 2

[0076] Embodiment 2 sensitivity test

[0077] Different concentrations of hsa-miR-141 (1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM) were detected.

[0078] Other steps of detection are the same as in Example 1, and the results are shown in Figure 3(A) and Figure 3(B), as can be seen from the results in the figure, the electrochemical signal increases with the concentration of target miRNA hsa-miR-141 (~10aM ) increases monotonously and changes linearly in the range from 1aM to 10pM. Using this curve, quantitative analysis of the target miRNA can be achieved. The results also show that when detecting hsa-miR-141 as low as 1aM, the electrochemical signal can reach 78nA, which is still much higher (>3SD) than the background signal (SD represents the standard deviation), indicating that the detection limit of the present invention can be lower to 1aM.

Embodiment 3

[0079] Embodiment 3 specificity test

[0080] The helper-miR-141 chain among the example 1 is changed into helper-let-7a, helper-let-7d respectively, for let-7 family miRNAs (let-7a, let-7b, let-7c, let-7d , let-7e, let-7f, let-7g, let-7i, miR-98) were detected to verify the specificity of the present invention, and hsa-miR-141 was used as a negative control. The sequences are as follows:

[0081] helper-let-7a: 5'-AGG CAA GAT GAA CTA TAC AAC CTA CTA CCT CAT GCG ACC T-3' (SEQ ID NO.7);

[0082] helper-let-7d: 5'-AGG CAA GAT GAA CTA TGC AAC CTA CTA CCT CTT GCG ACC T-3' (SEQ ID NO.8);

[0083] The biotin-modified DNA signal probe is the same as in Example 1.

[0084] Target miRNAs:

[0085] hsa-let-7a: 5'-UGA GGU AGU AGU AGG UUG UAU AGU U-3' (SEQ ID NO.9);

[0086] hsa-let-7b: 5'-UGA GGU AGU AGG UUG UGU GGU U-3' (SEQ ID NO.10);

[0087] hsa-let-7c: 5'-UGA GGU AGU AGG UUG UAU GGU U-3' (SEQ ID NO.11);

[0088] hsa-let-7d: 5'-AGA GGU AGU AGU AGG UUG CAU AGU U-3' (SEQ ID NO.1...

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Abstract

The invention discloses a method for detecting miRNA (ribonucleic acid). The method comprises the steps: (1) synthesizing a DNA (deoxyribonucleic acid) tetrahedron probe; (2) connecting three peak points of the DNA tetrahedron probe to the surface of a working electrode of an electrochemical device to obtain a working electrode with a capture probe; (3) adding a target miRNAs to be detected, a signal probe and an auxiliary chain into a reaction system to implement hybridization reaction to form a composite body I, and immersing the working electrode into the reaction system to implement hybridization reaction to form a composite body II; (4) generating reaction between the composite body II and enzyme capable of generating catalytic oxidation reduction reaction; (5) adding a primer produced by the enzyme catalytic oxidation to realize electrochemical detection analysis. The target miRNAs can be directly detected by the method disclosed by the invention without being marked and subjected to pre-PCR (polymerase chain reaction) multiplication; the method is easy to operate, so that the experiment cost is greatly reduced, and the experiment efficiency is improved.

Description

technical field [0001] The invention belongs to the field of nucleic acid hybridization detection, and relates to a method for detecting miRNA. Specifically, the invention relates to a method for detecting miRNA based on a self-supporting interface of a three-dimensional nanostructure of DNA and adopting an electrochemical gap analysis method. Background technique [0002] MicroRNAs (miRNAs) are a newly discovered class of endogenous non-coding single-stranded small RNAs with a length of 19-23 nucleotides, which widely exist in different organs of various organisms, mainly through specific recognition of targets. Messenger RNA (mRNA) regulates the expression of mRNA. Studies on the human genome have shown that miRNAs not only play an important regulatory role in a series of processes in cell development, but also that the expression of miRNAs is related to the occurrence and development of diseases, and it is particularly noteworthy that it is directly related to various typ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/327C12Q1/68
Inventor 刘刚樊春海闻艳丽左小磊林美华许丽徐勤李兰英梁文李妍
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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