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Nucleic acid aptamer sequence and detection method for quantitative and rapid detection of mercury ions

A nucleic acid aptamer and mercury ion technology, which is applied in the field of nucleic acid sequences for mercury ion detection, can solve the problems of low sensitivity, troublesome and time-consuming operation, and poor specificity, and achieve the effects of low detection cost, convenient operation, and simple structure

Inactive Publication Date: 2014-12-17
HENAN ACAD OF AGRI SCI
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Problems solved by technology

[0005] The technical problem to be solved in the present invention is to provide a nucleic acid aptamer sequence and its detection method that can be used for quantitative and rapid detection of mercury ions in view of the problems of low sensitivity, poor specificity, troublesome and time-consuming operation in the detection of metal ions in the prior art, The method has the advantages of high sensitivity and selectivity, low detection cost, rapid detection and easy operation.

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  • Nucleic acid aptamer sequence and detection method for quantitative and rapid detection of mercury ions

Examples

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example 1

[0027] Example 1: The nucleic acid aptamer sequence used for quantitative and rapid detection of mercury ions contains a stem-loop structure of a specific length of thymine T, and a fluorescent group is connected to the 5' end of the single strand of the stem-loop structure, and a corresponding The fluorescence quenching group forms a self-assembled detection system; when mercury ions exist, the stem-loop structure is closed, and the fluorescence intensity of the reaction system decreases, and the fluorescence intensity is inversely proportional to the concentration of mercury ions.

[0028] The specific structure of the nucleic acid aptamer sequence is: fluorescent group-T （m） -N （n） -T （m） -Fluorescence quenching group; N represents one or more of the nitrogenous bases A, G and C in the nucleic acid, but A and G should be avoided at the same time; wherein m is the number of thymine, and n is the nucleic acid containing The number of nitrogen bases. Nucleic acid aptamers...

example 2

[0032] Example 2: Screening of Standard Curve for Mercury Ion Detection

[0033] (1) According to Example 1, design nucleic acid aptamer sequences of different thymine lengths (see Table 1), couple the FAM fluorescent group to its 5' end, and couple the Dabcyl fluorescence quencher to its 3' end, the sequence Synthesized by a commercial primer company.

[0034] (2) Add mercury ion standard solutions with concentrations of 0, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 1024ng / ml to the corresponding microplate (for fluorescence) respectively. In the well, 50 μl / well, each sample was set to repeat at least once;

[0035] (3) Dilute nucleic acid aptamer sequences of different thymine lengths into a 50nM solution with PBS solution, add to the corresponding microwell of the microplate, 50μl per well, and mix well on a shaker, at 37°C Incubate in the dark for 20 minutes;

[0036] (3) Place the microplate plate on a multi-functional microplate reader for reading.

[0037] Experimenta...

example 3

[0043] Example 3: Method for Concentration Detection of Mercury Ions in Water Using Nucleic Aptamers

[0044] (1) Dilute the T6 sequence in Example 2 with PBS solution to form a nucleic acid aptamer solution with a concentration of 50nM; filter the water sample to be tested with filter paper to remove impurities;

[0045] (2) Add the mercury ion standard solution with the concentration of 0, 2, 4, 8, 16, 32, 64ng / ml and the sample to be tested into the corresponding microwells of the microplate (for fluorescence) (50μl / Wells), each sample was set to repeat at least once;

[0046] (3) Add the diluted nucleic acid aptamer solution T6 (50nM) into the corresponding microwell, 50μl / well, mix well on a shaker, and incubate at 37°C in the dark for 20min;

[0047] (4) Place the microplate plate on a multi-functional microplate reader to read the value, and obtain the fluorescence value of the standard substance and the sample to be tested.

[0048] Experimental result processing:...

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Abstract

The invention relates to a nucleic acid aptamer sequence and detection method for quantitative and rapid detection of mercury ions. The nucleic acid aptamer sequence contains a stem-loop structure of thymine T with a specific length, a fluorophore is connected to a 5' terminal of a single strand, and a corresponding fluorescence quenching group is connected to a 3' terminal of the single strand, thus a self-assembling detection system is formed; when mercury ions exist, the stem-loop structure is closed, the fluorescence intensity of a reaction system is reduced, and the fluorescence intensity and the concentration of the mercury ions are in an inverse proportion. The nucleic acid aptamer sequence disclosed by the invention can be applied to the detection of the mercury ions, is relatively high in sensitivity and selectivity, and is relatively low in detection cost; a to-be-tested sample is directly added in the reaction system during detection, no other reagents need to be added in the whole detection process, and a detection result can be obtained in one step only; and the detection method is high in detection speed and is simple and convenient to operate.

Description

technical field [0001] The invention relates to a nucleic acid sequence for detecting mercury ions, in particular to a nucleic acid aptamer sequence for quantitatively detecting mercury ions in a solution system and a detection method thereof. Background technique [0002] There are many analysis methods for metal ions. According to different detection principles, commonly used analysis methods include chemical methods, physical methods, and biological analysis methods. Among them, the chemical method has low detection sensitivity, poor specificity, troublesome and time-consuming operation, and cannot meet the requirements of determination. Physical methods mainly rely on high-precision instruments and equipment. The current analysis methods mainly include: spectrophotometry, atomic absorption spectroscopy, mass spectrometry, neutron activation analysis and electrochemical analysis. [0003] Atomic absorption analysis is an important method for heavy metal analysis at prese...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N21/64
Inventor 邓瑞广王方雨胡骁飞邢广旭杨继飞张改平
Owner HENAN ACAD OF AGRI SCI
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