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MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe

A technology of constant temperature amplification and fluorescent nanotechnology, which is applied in the field of detection and analysis, to achieve the effects of improving amplification efficiency, good selectivity, and adjustable luminous color

Inactive Publication Date: 2014-12-24
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is still a challenge to develop simple and direct analytical techniques with high sensitivity, high specificity, and accurate detection of microRNAs.

Method used

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  • MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe
  • MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe
  • MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe

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Embodiment 1

[0047] An analysis method for the quantitative detection of miR-141 (5'-UAACACUGUCUGGUAAAGAUGG-3') synthesized by a fluorescent nano-silver cluster probe using a constant temperature amplification reaction, the detection principle is as follows figure 1 shown. use (exo-)DNA polymerase and Nt.BstNBI endonuclease. The sequence arrangement on the DNA amplification template is the target miR-141 binding sequence (5'-CCATCTTTACCAGACAGTGTTA-3'), the Nt.BstNBI restriction sequence (5'-TCTTGACTC-3'), the miR-141 binding sequence (5' -CCATCTTTACCAGACAGTGTTA-3'), Nt.BstNBI restriction sequence (5'-TCTTGACTC-3'), and the DNA complementary sequence for the synthesis of fluorescent nanosilver clusters (5'-TTGGGCGGGTGGGTGGG-3'), phosphorylated at the 3' end.

[0048] The following specific implementation of miR-141 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions...

Embodiment 2

[0052] An analysis method for the quantitative detection of miR-21 (5'-UAGCUUAUCAGACUGAUGUUGA-3') synthesized by a fluorescent nano-silver cluster probe using a constant temperature amplification reaction, the detection principle is as follows Figure 4 shown. use (exo-)DNA polymerase and Nt.BstNBI endonuclease. The sequence arrangement on the DNA amplification template is the target miR-21 binding sequence (5'-TCAACATCAGTCTGATAAGCTA-3'), the Nt.BstNBI restriction sequence (5'-ACGTGACTC-3'), the miR-21 binding sequence (5' -TCAACATCAGTCTGATAAGCTA-3'), Nt.BstNBI restriction sequence (5'-ACGTGACTC-3'), and the DNA complementary sequence for the synthesis of fluorescent nanosilver clusters (5'-TTGGGCGGGTGGGTGGG-3'), phosphorylated at the 3' end.

[0053] The following specific implementation of miR-21 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or...

Embodiment 3

[0056] An analysis method for the quantitative detection of let-7a (5'-UGAGGUAGUAGGUUGUAUAGUU-3') synthesized by a fluorescent nano-silver cluster probe using a constant temperature amplification reaction, the detection principle is as follows Figure 5 shown. Using phi29 DNA polymerase and Nb.BbvCI endonuclease. The sequence arrangement on the DNA amplification template is the target let-7a binding sequence (5'-AACTATACAACCTACTACCTCA-3'), the Nb.BbvCI restriction sequence (5'-GCTGAGG-3'), the let-7a binding sequence (5' -AACTATACAACCTACTACCTCA-3'), Nb.BbvCI cleavage sequence (5'-GCTGAGG-3'), and the DNA complementary sequence of synthetic fluorescent nanosilver clusters (5'-TTGGGCGGGTGGGTGGG-3'), phosphorylated at the 3' end.

[0057] The following specific implementation of let-7a detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions su...

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Abstract

The invention provides a microRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize a fluorescent nano silver cluster probe. The method is as below: a DNA amplification template containing three kinds of functional sequences is designed: a sequence binding with a target microRNA, a nicking endonuclease enzyme sequence and a DNA complementary sequence for synthesis of fluorescent nano cluster; when the target microRNA and the DNA amplification template are combined, isothermal amplification reaction and specific enzyme reaction of nicking endonuclease are induced to obtain a large number of single-stranded DNA products; the DNA sequence for synthesis of fluorescent nano silver cluster and a silver nitrate solution are employed to prepare the fluorescent nano silver cluster probe under the reduction of sodium borohydride; fluorescence signal of the reaction system are determined, and a fluorescence change value is calculated; the value is compared with a standard working curve to calculate the concentration of the target microRNA. The method has the advantages of high sensitivity, strong specificity, wide linear detection range, low background signal and simple operation, and can be widely applied to microRNA detection of biological samples such as tissue, blood or cells.

Description

technical field [0001] The invention relates to an analysis method for detecting microRNA with a fluorescent nano-silver cluster probe, belonging to the field of detection and analysis. Background technique [0002] MicroRNA is a kind of endogenous non-coding RNA with regulatory function found in eukaryotes, and its size is about 18-25 nucleotides in length. It is involved in a wide variety of regulatory pathways, including development, viral defense, hematopoietic processes, organ formation, cell proliferation and apoptosis, fat metabolism, etc. Recent studies have found that microRNA plays a vital role in the process of tumorigenesis and has become an ideal tumor marker. Therefore, quantitative detection of microRNA is of great significance for understanding its biological function, early diagnosis of cancer and development of new drugs. Compared with traditional nucleic acid detection, the unique characteristics of microRNA, such as short sequence, high family sequence ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 叶邦策尹斌成刘玉强
Owner EAST CHINA UNIV OF SCI & TECH
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