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Method for detecting biomolecules based on label-free fluorochrome and nucleic acid aptamers

A technology of nucleic acid aptamers and biomolecules, applied in the field of bioengineering, can solve the problems of low sensitivity and specificity and inability to detect, and achieve the effects of improving detection sensitivity and specificity, wide application, and shortening the detection time

Inactive Publication Date: 2015-03-25
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the instrumental analysis method, other various methods, although the detection method is fast, have low sensitivity and specificity
PCR and RT-PCR methods based on nucleic acid amplification polymerase chain reaction technology have high detection sensitivity, but for non-microbial samples, PCR or RT-PCR detection cannot be performed

Method used

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  • Method for detecting biomolecules based on label-free fluorochrome and nucleic acid aptamers
  • Method for detecting biomolecules based on label-free fluorochrome and nucleic acid aptamers
  • Method for detecting biomolecules based on label-free fluorochrome and nucleic acid aptamers

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Rapid detection of toxins

[0025] The following program takes germantoxin OTA and OTA aptamer (sequence 5'-GCATCTGATCGGGTGTGGGTGGCGTAAAGG-3') as an example to illustrate the rapid detection method based on PicoGreen technology:

[0026] Draw the standard curve:

[0027] Take 10 microliters of 10nmol / L OTA nucleic acid aptamer and 10 microliters of OTA solutions with final concentrations of 0, 0.1, 1, 2, 4, 8, and 10ng / mL, respectively, and mix and react in a 96-well plate. The reaction buffer is: (10mmol / L Tris, 120mmol / L NaCl, 5mmol / L KCl, 20mmol / L CaCl 2 , pH8.5), the reaction volume is 50 microliters, and the reaction time is 5-10 minutes. Add 10 microliters of 10nmol / L single-stranded oligonucleotide complementary to the OTA aptamer (sequence: 5'-CCTTTACGCCACCCACACCCGATCAGATGC-3'), react for 5-10 minutes, and then add 10 microliters of PicoGreen (final concentration: 1 pmol / L), mix well. After reacting for 15 minutes, use a microplate reader or a fl...

Embodiment 2

[0044] Embodiment 2: the detection of antibiotics

[0045] The following program takes ampicillin Amp and Amp aptamer (sequence 5'-GCGGGCGGTTGTATAGCGG-3') as an example to illustrate the rapid detection method based on PicoGreen technology:

[0046] Draw the standard curve:

[0047] Take 10 microliters of 10nmol / L Amp nucleic acid aptamer and 10 microliters of Amp solutions with final concentrations of 0, 0.1, 1, 2, 4, 8, and 10ng / mL, respectively, and mix and react in a 96-well plate. The reaction buffer is: (10mmol / L Tris, 120mmol / L NaCl, 5mmol / L KCl, 20mmol / L CaCl 2 , pH8.5), the reaction volume is 50 microliters, and the reaction time is 5-10 minutes. Add 10 microliters of 10nmol / L single-stranded oligonucleotide complementary to the Amp aptamer (sequence: 5'-CCGCTATACAACCGCCCGC-3'), react for 5-10 minutes, then add 10 microliters of PicoGreen (final concentration is 1 pmol / L), mix well. After reacting for 15 minutes, use a microplate reader or a fluorescence spectrop...

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Abstract

The invention relates to the field of bioengineering and aims to provide a method for detecting biomolecules based on label-free fluorochrome and nucleic acid aptamers. The method comprises the following steps: the nucleic acid aptamers are mixed with to-be-detected biomolecules, oligonucleotide chains complementary with the nucleic acid aptamers are added, the nucleic acid aptamers and the to-be-detected biomolecules are subjected to specific binding, redundant nucleic acid aptamers not subjected to specific binding are hybridized with the complementary oligonucleotide chains to generate double-stranded DNAs, the double-stranded DNAs are bonded with the label-free fluorochrome Pico Green to release fluorescence, when the to-be-detected biomolecules with different concentrations are bonded with the nucleic acid aptamers with a fixed concentration, the fluorescence strengths in a reaction solution are different, and rapid and quantitative detection on the biomolecules can be realized through detection of the fluorescence strengths. According to the invention, the detection time is shortened to be 25-40 minutes while the detection sensitivity and the specificity are improved; the detection sensitivity of a to-be-detected substance can reach the pg-ng level; the adopted nucleic acid aptamers can be conveniently synthetized and are low in cost.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a rapid detection method for detecting biomolecules based on a non-labeled fluorescent dye PicoGreen and a nucleic acid aptamer. Background technique [0002] The detection of microorganisms, proteins, polysaccharides, peptides, toxins, chemical drugs and other biomolecules can be divided into different detection principles: enzyme-linked immunosorbent assay based on antigen-antireaction technology, polymerase chain reaction technology based on nucleic acid amplification PCR and RT-PCR method, colloidal gold method based on lateral flow chromatography, mass spectrometry and chromatography based on instrumental analysis technology, etc. In addition to the instrumental analysis method, other various methods have low sensitivity and specificity although the detection method is fast. Although the PCR and RT-PCR methods based on nucleic acid amplification polymerase chain re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64C12Q1/04
Inventor 宋厚辉金庆日杨梦华邵春艳程昌勇王晓杜杨杨杨永春卫芳芳桂海娈张亚军方维焕
Owner ZHEJIANG FORESTRY UNIVERSITY
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