Mercury ion detection method based on nucleic acid probe head-to-tail complementation strategy, and mercury ion detection kit based on nucleic acid probe head-to-tail complementary strategy

A detection kit and detection method technology, which can be used in material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of low detection sensitivity, troublesome operation, increase experimental cost, etc., and achieve the effects of high sensitivity, simple operation and good specificity

Active Publication Date: 2015-10-14
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the potential disadvantage is that the detection sensitivity is low, and it is difficult to meet the needs of environmental sample detection, so it is necessary to improve the detection sensitivity through signal amplification
At present, tool enzymes (exonuclease, endonuclease, DNA polymerase, etc.) are often used to amplify the detection signal, but the use of these proteases not only increases the cost of the experiment, but also is troublesome to operate, and the enzyme is easily affected by the reaction system and environmental factors. effect, not suitable for rapid detection

Method used

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  • Mercury ion detection method based on nucleic acid probe head-to-tail complementation strategy, and mercury ion detection kit based on nucleic acid probe head-to-tail complementary strategy
  • Mercury ion detection method based on nucleic acid probe head-to-tail complementation strategy, and mercury ion detection kit based on nucleic acid probe head-to-tail complementary strategy
  • Mercury ion detection method based on nucleic acid probe head-to-tail complementation strategy, and mercury ion detection kit based on nucleic acid probe head-to-tail complementary strategy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A detection kit for mercury ions based on the nucleic acid probe head-to-tail complementary strategy, including the following components:

[0041] (1) Streptavidin-modified magnetic beads;

[0042] (2) Biotin-modified DNA1, the sequence is as follows:

[0043] 5'-CAGTTTGGTTTTCTCTTGC-Biotin-3' (SEQ ID NO. 1)

[0044] (3) DNA2, the sequence is as follows:

[0045] 5'-GCTTGAGATTTTTCCATTCTGACTACTAGGGTCTGAGGG-3' (SEQ ID NO. 2)

[0046] (4) DNA3 and DNA4, the sequences are as follows:

[0047] DNA3: 5'-TACTCCCCCAGGTGCCCCTCAGACCCTAGTAGT-3' (SEQ ID NO. 3);

[0048] DNA4: 5'-GCACCTGGGGGAGTAACTACTAGGGTCTGAGGG-3' (SEQ ID NO. 4).

[0049] The 5' end of DNA3 is complementary to the 5' end of DNA4, the 3' end of DNA3 is complementary to the 3' end of DNA4, and the 3' end of DNA3 is complementary to the 3' end of DNA2;

[0050] (5) 20 mM Tris-acetate buffer (pH 7.5, containing 50 mM sodium acetate).

[0051] (6) Sybr Green I solution.

[0052] The working principle of this kit...

Embodiment 2

[0058] The method for detecting mercury ions based on the head-to-tail complementary strategy of nucleic acid probes is carried out according to the following steps:

[0059](1) Add 1 mM biotin-modified DNA1 to the streptavidin-modified magnetic bead solution, mix well, react at room temperature for 30 minutes, separate the magnetic beads, and remove excess DNA1.

[0060] (2) The magnetic bead-DNA1 mixture was resuspended with 20 mM Tris-acetate buffer (pH 7.5, containing 50 mM sodium acetate), and then 1 mM DNA2 was added, as well as Hg 2+ , mix well, react at room temperature for 30 minutes, separate with magnetic beads, and remove excess DNA2.

[0061] (3) The above mixture was resuspended with 20 mM Tris-acetic acid buffer (pH 7.5, containing 50 mM sodium acetate), then added 2 mM DNA3 and 2 mM DNA4, reacted at room temperature for 60 minutes, and separated with magnetic beads to remove excess DNA3 and DNA4.

[0062] (4) The above mixture was resuspended in 20 mM Tris-ace...

Embodiment 3

[0064] For different concentrations of Hg 2+ detection of:

[0065] Prepare Hg 2+ Standard solutions with concentrations of 10pM, 100 pM, 1 nM, 10 nM, 100 nM and 500 nM were stored at room temperature.

[0066] Different concentrations of Hg 2+ Solution is added in the reaction system described in embodiment 1 respectively, detects fluorescence intensity after sufficient reaction, as figure 2 As shown, with Hg 2+ As the concentration increases, the corresponding fluorescence intensity also increases, when Hg 2+ When the concentration exceeds 100 nM, it gradually reaches saturation. in Hg 2+ Logarithm of concentration (lg 汞离子浓度 ) is the abscissa, the fluorescence intensity is the ordinate, draw the standard curve, the two have a good linear relationship, the linear range is from 10pM to 100 nM, the linear equation is: F = 191.8 lg C + 18.4 (R=0.989), according to the standard of 3 times signal-to-noise ratio (3S / N), the detection limit is 2 pM.

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Abstract

The present invention discloses a mercury ion detection method based on nucleic acid probe head-to-tail complementation strategy, and a mercury ion detection kit based on nucleic acid probe head-to-tail complementation strategy. According to the present invention, T base-rich nucleic acid is designed as a molecule recognition element, and is combined with a capture probe through T-Hg<2+>-T pairing so as to be immobilized on magnetic beads, two nucleic acid probes having head-to-tail complementation are added after the magnetic beads are separated, the long double-stranded DNA is formed through continuous hybridization complementation, a fluorescent intercalator Sybr Green I is added after the magnetic beads are separated, the fluorescent intercalator Sybr Green I has strong fluorescence property after being combined with the double-stranded DNA, and the fluorescence intensity and the mercury ion concentration have the good correlation so as to achieve the purpose of mercuric ion detection; the detection method and the detection kit have high sensitivity, the detection limit on Hg<2+> is 2 pM, the good specificity is provided, and other common interference ions do not affect the detection; and the signal amplification process is derived from the continuous head-to-tail complementation of the DNA probe without protease, the operation is simple, and the cost is low.

Description

technical field [0001] The invention belongs to the field of heavy metal ion detection, and relates to a mercury ion detection technology, in particular to a mercury ion detection method based on a nucleic acid probe head-to-tail complementary strategy and a detection kit. Background technique [0002] Mercury ions (Hg 2+ ) is a carcinogenic, teratogenic, and mutagenic environmental pollution source, which is a serious hazard to the ecological environment and human safety, and is an important indicator for environmental testing. Exposure to enrichment of mercury ions can lead to kidney failure, brain damage, nervous system and immune system damage, therefore, the effect on Hg 2+ Detection is important. The United States Environmental Protection Agency stipulates that the maximum allowable level of mercury ions in drinking water shall not exceed 10 nM. At present, the routine detection methods of mercury ions mainly include atomic absorption method, atomic fluorescence spe...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 陈俊华周顺桂
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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