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Difunctional aptamer detection kit and detection method

A technology for detection kits and detection methods, which is applied in the fields of environmental analysis and food safety, and can solve problems such as inability to simultaneously detect metal ions

Inactive Publication Date: 2014-03-26
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are limited to the detection of only one metal ion (Hg 2+ or Ag + ), Hg cannot be carried out at the same time 2+ and Ag + Detection of two metal ions

Method used

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  • Difunctional aptamer detection kit and detection method
  • Difunctional aptamer detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Reagent preparation: Prepare reagents at the following concentrations: 1 micromole per liter of the first template DNA, 1 micromole per liter of the second template DNA, 2.5 millimole per liter of dNTPs (a mixture of four deoxyribonucleotides) , 10× First Reaction Buffer (0.2 M Trisacetic Acid, pH 7.9, 0.1 M Magnesium Acetate, 0.5 M Potassium Acetate, 0.01 M Dithiothreitol) , 10× reaction buffer 2 (0.1 mol per liter of Trisaminomethane hydrochloride, pH 7.9, 0.1 mol per liter of magnesium chloride, 0.5 mol per liter of sodium chloride, 0.01 mol per liter of dithiothreitol), 10 units per microliter of DNA cutting enzymes Nb.BbvCI, Nb.BtsI and Nt.AlwI, 5 units per microliter of DNA polymerase Klenow fragment (3'-5'exo-), 100×BSA (bovine serum albumin, 10mg / mL), 100 micromole per liter of the first fluorophore quencher probe (QF probe-1) and the second fluorophore quencher probe (QF probe-2), sterile water, and different concentrations of mercury ions and silver ions.

...

Embodiment 2

[0087] Oligonucleotide strand displacement amplification reaction validation experiment..

[0088] The oligonucleotide strand displacement amplification reaction was verified by non-denaturing polyacrylamide gel electrophoresis analysis.

[0089] The result is as image 3As shown, in the absence of Hg 2+ and Ag + In the case of the control tube electrophoresis lane (control lane), no amplified band was found; in the presence of only Hg 2+ or Ag + In the case of , obvious oligonucleotide target bands can be found in the 20bp down of the electrophoresis lane, which indicates that the strand displacement amplification reaction has been successfully realized. in the presence of Hg 2+ and Ag + In the case of oligonucleotide target bands can also be clearly observed, and the bands are obviously brighter than the presence of a single metal ion, which shows that in the presence of two metal ions at the same time, successful amplification two different oligonucleotides. In conc...

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Abstract

The invention relates to a difunctional aptamer detection kit and a detection method capable of simultaneously detecting two metal ions of Hg<2+> and Ag<+1>. According to the difunctional aptamer detection method, in the simultaneous presence of the two metal ions of Hg<2+> and Ag<+1>, T-Hg<2+>-T and C-Ag<+1>-C structures are formed to respectively connect mismatch of thymine and mismatch of cytosine on the 3' end of a first template DNA and the 3' end of a second template DNA, so that the hybridized first template DNA and the second template DNA can be amplified, a first oligonucleotide and a second oligonucleotide formed by cutting of a first DNA nickase are respectively hybridized with a first probe sequence and a second probe sequence, so that the first probe sequence and the second probe sequence are respectively cut by a second DNA nickase and a third DNA nickase, a first fluorophore and a second fluorophore are exposed finally, and the two metal ions of Hg<2+> and Ag<+1> are simultaneously detected by fluorescence detection.

Description

technical field [0001] The invention relates to the fields of environmental analysis and food safety, in particular to a method capable of simultaneous Hg 2+ and Ag + A dual-function aptamer detection kit and detection method for the detection of two metal ions. Background technique [0002] Mercury and silver are two very common heavy metals that are widely used in the pharmaceutical, battery and photographic industries. However, when these products are by-products or are discarded after use, some heavy metals will eventually enter the environment in the form of ions, causing serious pollution to water bodies and soil. Mercury ions have a great impact on animal, plant and human health. Long-term exposure to the environment containing mercury ions can cause permanent damage to the central nervous system and other organs. The direct toxicity of silver ions is relatively small, but it can produce a strong enrichment effect in aquatic organisms, and thus enter the human body...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
CPCC12Q1/6844C12Q2521/101C12Q2521/301C12Q2563/107
Inventor 张春阳朱桂池
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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