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Method for detecting food-borne enteropathogenic bacteria O157: H7 based on nucleic acid aptamers, nanoparticles and quantum dot markers

A nucleic acid aptamer and nanoparticle technology, applied in biochemical equipment and methods, nanotechnology for materials and surface science, nanotechnology, etc., can solve the problems of low sensitivity and specificity, complicated operation process, and time-consuming Long and other problems, to achieve the effect of wide range of target molecules, high sensitivity and good stability

Active Publication Date: 2021-12-31
南京海关动植物与食品检测中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are currently the most basic and common detection methods, they have their own shortcomings, such as complicated operation process, long time-consuming, low sensitivity and specificity, poor repeatability, etc. Therefore, it is necessary to establish an efficient and rapid detection method. The necessary basis for effective prevention of colibacillosis and ensuring public health safety

Method used

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  • Method for detecting food-borne enteropathogenic bacteria O157: H7 based on nucleic acid aptamers, nanoparticles and quantum dot markers
  • Method for detecting food-borne enteropathogenic bacteria O157: H7 based on nucleic acid aptamers, nanoparticles and quantum dot markers
  • Method for detecting food-borne enteropathogenic bacteria O157: H7 based on nucleic acid aptamers, nanoparticles and quantum dot markers

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1. Construction and screening of nucleic acid aptamers of Escherichia coli O157:H7

[0036] 1. Coarse screening of nucleic acid aptamers of Escherichia coli O157:H7

[0037] The specific screening steps are as follows:

[0038] (1) For the first round of screening, take 200 pmol single-stranded random DNA oligonucleotide templates, dilute to 500 μL with binding buffer (pH=7.6), heat denature at 95°C for 10 minutes, and quickly place in ice water at 0°C Ice-bathed for 10 minutes, then placed at room temperature for 30 minutes.

[0039] (2) Mix the single-stranded random DNA oligonucleotide template after ice bathing with 200 μL of Escherichia coli O157:H7, and shake at room temperature for 60 minutes. Centrifuge at 6000rmp for 5min and discard the supernatant. Wash three times with 400 μL wash buffer. The purpose is to wash away the single-stranded random DNA oligonucleotide template that does not bind E. coli O157:H7.

[0040] (3) Take the precipitate obtai...

Embodiment 2

[0076] Example 2. Preparation of magnetic nanoparticles and functionalization of nucleic acid aptamers

[0077] In this paper, sodium hydroxide solution was used to hydrolyze ferric chloride hexahydrate (FeCl 3 ·6H 2 O) and ferrous sulfate heptahydrate (FeSO 4 ·7H 2 O) mixed solution to prepare Fe 3 o 4 Nanoparticles, also known as co-precipitation method to prepare MNP, its chemical principle is 2Fe 3+ +Fe 2+ +8OH - → Fe 3 o 4 ↓+4H 2 O, iron ions Fe3+ and ferrous ions Fe should be strictly controlled 2+ The ratio is 2:1.

[0078] Synthesis of Fe by Co-precipitation 3 o 4 The steps of magnetic nanoparticles are as follows: the concentration is 0.12mol / L FeSO 4 ·7H 2 O and concentration is 0.2mol / L FeCl 3 ·6H 2 O was dissolved in deionized aqueous solution, mechanically stirred and mixed, and nitrogen gas was passed through to fully remove the dissolved oxygen in the solution, and then a sodium hydroxide solution with a concentration of 2.5mol / L was added dropw...

Embodiment 3

[0081] Example 3. Preparation of avidinized nano-magnetic beads and functionalized quantum dots

[0082] The specific method of preparing avidinated amino magnetic beads based on the glutaraldehyde method refers to the method of Wu et al., with slight changes, as follows: Weigh 5 mg of aminated magnetic beads and dissolve them in 5 mL of 10 mM phosphate buffer for 20 min; Add 1.25mL of 25% glutaraldehyde, shake slowly at room temperature for 1h, enrich and separate Fe under the action of magnet 3 o 4 And washed 3 times with PBS to remove the physically adsorbed glutaraldehyde; 3 o 4 Add 500ul 1mg / mL streptavidin to the magnetic particles, shake slowly at room temperature for 6h; enrich and separate the avidinized amino magnet under the action of a magnet, discard the supernatant containing free avidin, and wash repeatedly with PBS; Add 5mL of 10mg / mL BSA to the primed amino magnetic beads and shake slowly at room temperature for 6h to block unreacted and non-specific bindin...

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Abstract

The invention discloses a method for detecting food-borne enteropathogenic bacteria O157: H7 based on nucleic acid aptamers, nanoparticles and quantum dot markers, which comprises the following steps: taking nucleic acid aptamer functionalized nano-enriched magnetic beads as a capture probe, capturing target bacteria existing in a reaction system, incubating, and recovering magnetic bead aptamer-target bacteria through a magnetic separator; then, adding nucleic acid aptamer functionalized quantum dots; and enriching magnetic beads under the action of a magnetic field, resuspending the precipitate in a buffer solution, determining the fluorescence intensity by using a fluorescence chemical analyzer, and determining the total bacterial count of the food-borne enteropathogenic bacteria O157: H7 according to the fluorescence intensity. According to the invention, the functionalized quantum dots are applied to detection of E.coli O157: H7 in food, and an efficient and rapid detection method of E.coli O157: H7 is finally established in combination with a nucleic acid aptamer functionalized magnetic bead enrichment method.

Description

technical field [0001] The invention relates to a method for detecting food-borne intestinal pathogenic bacteria O157: H7 based on nucleic acid aptamers, nanoparticles and quantum dot markers, belonging to the technical field of biological detection. Background technique [0002] Escherichia coli (Escherichia coli, E.coli), also known as Escherichia coli, is the most important and most numerous bacteria in the intestinal tract of humans and animals, and belongs to Gram-negative bacteria. The antigen composition of Escherichia coli is complex, which can be divided into bacterial antigen (O), flagellar antigen (H) and surface antigen (K), the latter has the ability of resisting body phagocytosis and anti-complement. According to the different bacterial antigens, Escherichia coli can be divided into more than 150 types, the vast majority of Escherichia coli are not pathogenic, while a small part of Escherichia coli can cause diseases in the gastrointestinal tract, urinary syste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543G01N33/58C12N15/115C01G49/08B82Y30/00B82Y40/00
CPCG01N33/56916G01N33/54326G01N33/54346G01N33/582G01N33/588C12N15/115C01G49/08B82Y30/00B82Y40/00G01N2333/245C12N2310/16Y02A50/30
Inventor 蒋鲁岩王毅谦朱振华曾德新高渊杨天宇封振徐振东张娜
Owner 南京海关动植物与食品检测中心
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