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Preparation method of up-conversion fluorescence recognition probe as well as product and application of up-conversion fluorescence recognition probe

A fluorescent recognition and probe technology, which is applied in the field of preparation of up-conversion fluorescent recognition probes, can solve problems such as no related reports, and achieve the effects of wide detection range, good specificity and good versatility

Pending Publication Date: 2022-02-18
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no relevant reports on the molecular detection methods of pathogenic bacteria based on fluorescent nanomaterials and DNA nanotechnology.

Method used

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  • Preparation method of up-conversion fluorescence recognition probe as well as product and application of up-conversion fluorescence recognition probe
  • Preparation method of up-conversion fluorescence recognition probe as well as product and application of up-conversion fluorescence recognition probe
  • Preparation method of up-conversion fluorescence recognition probe as well as product and application of up-conversion fluorescence recognition probe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Step 1, preparing graphene oxide quantum dots (GOQDs): using an organic synthesis method to obtain a dispersion with a size of about 15 nm;

[0048] Step 2, preparation of water-soluble fluorescent nanomaterials: ① Preparation of oil-soluble up-conversion fluorescent nanomaterials (UCNPs-OA): UCNPs-OA was obtained by high-temperature thermal decomposition method, as follows: ErCl 3 ·6H 2 O (0.03mmol), YbCl 3 ·6H 2 O (0.17mmol) and YCl 3 ·6H 2 O (0.8mmol) was ultrasonically dissolved in 5mL of methanol, 7mL of 1-octadecene and 3mL of oleic acid were added, then transferred to a flask and mixed evenly. Introduce nitrogen and heat up to exhaust the air and water, and stop heating until the solution is stirred until it becomes transparent. Add a mixture of 2.5mmol NaOH and 4mmol ammonium fluoride dissolved in methanol dropwise and continue magnetic stirring, then raise the temperature to remove methanol and air in the device, then continue to raise the temperature to 3...

Embodiment 2

[0057] Step one, two are with embodiment 1;

[0058] Step 3, preparation of Escherichia coli O157:H7-specific DNA nanoprobe: select Escherichia coli O157:H7 claudhesin eaeA gene as the detection object, design nanoprobe ssDNA, and synthesize 5'-NH 2 -(CH 2 ) 6 -ATGCTAACGGTAAGGCAACCGTAACGTTGA-3';

[0059] Step 4, synthesis of up-conversion fluorescent recognition probes: using EDC / NHS amide condensation reaction to connect aminated DNA nanoprobes to UCNPs-COOH to obtain UCNPs-ssDNA;

[0060] Step five, construction of fluorescent biosensing system: 600 μL GOQDs (1 mg mL -1 ) and 200 μ LUCNPs-ssDNA synthesized in step 4 (0.5 mg·mL -1 ) mixing, which is the specific sensing and detection system constructed;

[0061] Step 6, the establishment of the molecular detection method for Escherichia coli O157:H7: add 200 μL of the eaeA target sequence (5'-TCAACGTTACGGTTGCCTTACCGTTAGCAT-3') of Escherichia coli O157:H7 at different concentrations into the specific sensing detection sys...

Embodiment 3

[0067] Step one, two are with embodiment 1;

[0068] Step 3, preparation of Listeria monocytogenes-specific DNA nanoprobes: select the inlA gene unique to Listeria monocytogenes as the detection object, design nanoprobe ssDNA, and synthesize 5'- NH 2 -(CH 2 ) 6 -GGAACACACCGCCTACAACA-3';

[0069] Step four, five are with embodiment 2;

[0070] Step 6, the establishment of a molecular detection method for Listeria monocytogenes: 200 μL of Listeria monocytogenes inlA target sequence (5'-AGTGACAGGTTGGCTAAAGGTATAGCTTACTTCAT-3') of different concentrations were added to the specific sensing detection In the system, the up-conversion fluorescence spectrum is collected, and a quantitative analysis model between the fluorescence intensity at the characteristic peak and the concentration of the inlA target sequence is established.

[0071] Step 7, detection of Listeria monocytogenes in aquatic products: Take marine fish as an example, weigh 5g of marine fish sample, wash it several...

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Abstract

The invention discloses a preparation method of an up-conversion fluorescence recognition probe as well as a product and application of the up-conversion fluorescence recognition probe, and relates to the technical field of food safety detection. The preparation method comprises the following steps: preparing UCNPs-OA from hydrated rare earth chloride, 1-octadecene, oleic acid, NaOH, ammonium fluoride and the like, and then modifying the UCNPs-OA to obtain water-soluble UCNPs-COOH; selecting a specific gene according to the type of pathogenic bacteria, designing a nanoprobe ssDNA, and carrying out amino modification on the nanoprobe to obtain an amino-functionalized DNA nanoprobe; and connecting the water-soluble UCNPs-COOH with the aminated DNA nanoprobe through a condensation reaction so as to obtain the up-conversion fluorescence recognition probe. According to the invention, the stable luminescence characteristic of UCNPs, the specific accurate recognition function of the DNA nanoprobe and the excellent fluorescence quenching performance of GOQDs are combined, so detection sensitivity and specificity are improved.

Description

technical field [0001] The invention relates to the technical field of food safety detection, in particular to a preparation method of an up-conversion fluorescent recognition probe and its product and application. Background technique [0002] Food safety incidents caused by food-borne pathogenic bacteria contamination occur frequently, seriously affecting public health and global economic development. The main pathogenic bacteria that contaminate food are Staphylococcus aureus, Salmonella, Escherichia coli, Listeria monocytogenes, etc. Although the commonly used traditional culture detection method is reliable and accurate, it is cumbersome and time-consuming (average 2 - 4 days to get preliminary results, 7-10 days to confirm the results), which cannot meet the needs of rapid detection of pathogenic bacteria contamination in the food industry; compared with traditional culture detection methods, immunological detection of pathogenic bacteria takes less time, but the cost ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6811C12N15/11C12N15/10C12R1/445C12R1/19C12R1/42C12R1/01
CPCC12Q1/689C12Q1/6811C12Q2563/107Y02A50/30
Inventor 刘蕊陈全胜许婧许艺张运莲李欢欢
Owner JIANGSU UNIV
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