Bisphenol A detecting method and detecting kit based on dual-quenching-group nucleic acid self-assembling technology

A detection kit and self-assembly technology, which is applied in the field of environmental analytical chemistry, can solve the problems of low sensitivity, time-consuming and labor-consuming, high detection cost, etc., and achieve the effects of improving detection sensitivity, reducing fluorescent background, and good selectivity

Active Publication Date: 2015-10-07
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional bisphenol A detection techniques mainly include high performance liquid chromatography, gas chromatography, mass spectrometry, and enzyme-linked immunosorbent assay. These techniques require cumbersome sample pretreatment and expensive instruments. Widely promoted
Moreover, the traditional fluorescence detection method has a high background value and low sensitivity, making it difficult to detect trace pollutants

Method used

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  • Bisphenol A detecting method and detecting kit based on dual-quenching-group nucleic acid self-assembling technology
  • Bisphenol A detecting method and detecting kit based on dual-quenching-group nucleic acid self-assembling technology
  • Bisphenol A detecting method and detecting kit based on dual-quenching-group nucleic acid self-assembling technology

Examples

Experimental program
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Embodiment 1

[0042] A bisphenol A detection kit based on double quencher nucleic acid self-assembly technology, comprising the following components:

[0043] (1) DNA1, the 5' end or 3' end of the nucleic acid aptamer of bisphenol A is extended to form DNA1, which includes b* region, a* region and bisphenol A nucleic acid aptamer region, the sequence is as follows:

[0044] Nucleic acid aptamer of b*a*bisphenol A

[0045] 5'-AGTCTAGGATTCGGCGTG--GGTTAA--CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGTATGGCCCAGCGCATCACGGGTTCGCACCA-3' (SEQ ID NO. 1);

[0046] (2) DNA2, DNA2 is complementary to the a* region of DNA1 and part of the bisphenol A nucleic acid aptamer region, the sequence is as follows:

[0047] a

[0048] 5'-CTGACCACCCCACCGG--TTAACC-3' (SEQ ID NO.2);

[0049] (3) DNA H1, the 24th base at the 5' end T-modifies the fluorescent group FAM, the sequence is as follows:

[0050] a b c b*

[0051] 5'-TTAACC--CACGCCGAATCCTAGACT--CAAAGT--AGTCTAGGATTCGGCGTG-3' (SEQ ID NO. 3);

[0052] It includes a,...

Embodiment 2

[0063] A method for detecting bisphenol A based on double quencher nucleic acid self-assembly technology, comprising the steps of:

[0064] (1) All DNAs were dissolved in 20 mM Tric-HCl buffer, and 100 nM DNA1 and 400 nM DNA2 were hybridized in Tric-HCl buffer for 20 minutes to form a DNA1-DNA2 complex;

[0065] (2) Add the sample to be tested to the DNA1-DNA2 complex and react at room temperature for 45 minutes;

[0066] (3) Add 1mM DNA H1 and 1mM DNA H2, react at room temperature for 60 minutes, then record the fluorescence intensity of the system, the excitation light is 490nm, the emission peak is 530nm, calculate the bisphenol A in the sample to be tested according to the established standard curve concentration.

Embodiment 3

[0068] Detection of different concentrations of bisphenol A:

[0069] Prepare bisphenol A standard solutions with concentrations of 1pM, 10pM, 100pM, 1nM, 10nM and 100nM, respectively, and store them at room temperature.

[0070] Add bisphenol A solutions of different concentrations to the reaction system described in Example 1 respectively, and detect the fluorescence intensity after sufficient reaction, as shown in figure 2 As shown, as the concentration of bisphenol A increases, the fluorescence intensity decreases, and when the concentration of bisphenol A exceeds 10 nM, it gradually reaches saturation. Taking the logarithm of bisphenol A concentration (lgC) as the abscissa, the fluorescence intensity ratio (F 0 / F) is the ordinate, draw the standard curve, the two have a good linear relationship, the linear range is from 1pM to 10nM, the linear equation is: F 0 / F=1.49lgC+0.54 (R=0.945), (F 0 : initial fluorescence intensity; F: fluorescence intensity corresponding to...

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Abstract

The invention discloses a bisphenol A detecting method and detecting kit based on the dual-quenching-group nucleic acid self-assembling technology. Two DNAs of stem loop structures are designed, wherein one DNA of the stem loop structure is used for modifying a fluorophore, and the other DNA of the stem loop structure is used for modifying two quenching groups; and under the condition that bisphenol A exists, loop opening hybridization can be continuously carried out on the two DNAs of the stem loop structures, so that the fluorophore and the quenching groups get close to each other, fluorescence energy transferring happens, and fluorescence is accordingly quenched. According to the detecting method, signal amplification is sourced from continuous loop opening complementation of the two DNAs of the stem loop structures, and no protease is needed to be used, so that operation is easy, a reaction can be completed at the room temperature, the sensitivity is high, and the selectively is good. The two quenching groups exist on one DNA of the stem loop structure at the same time, and the fluorophore on the other DNA of the stem loop structure can be quenched more effectively, so that the fluorescence background is effectively reduced, the detection sensitivity is greatly improved, and the detection limit is 0.4 pM.

Description

technical field [0001] The invention belongs to the field of environmental analytical chemistry, and relates to a bisphenol A detection method and a detection kit based on double quenching group nucleic acid self-assembly technology. Background technique [0002] Bisphenol A (Bisphenol A, BPA) is widely used in the synthesis of cyclotrophic resins, polycarbonate and other polymer materials. With the rapid development of the modern packaging industry, a large amount of bisphenol A enters the ecological environment through various channels, seriously Affect environmental safety and human health. Bisphenol A is enriched and difficult to degrade. Even a very low concentration will have a series of adverse effects on the endocrine system, immune system, and reproductive system, and is closely related to the occurrence of a series of cancers. Therefore, the detection of bisphenol A is of great significance. Traditional bisphenol A detection techniques mainly include high perform...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/115C12N15/11
CPCC12Q1/6818C12Q2525/205C12Q2525/301C12Q2565/101
Inventor 陈俊华周顺桂
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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