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A rapid on-site detection method for food-borne pathogenic bacteria

A food-borne pathogenic bacteria and detection method technology, applied in the direction of measuring devices, fluorescence/phosphorescence, instruments, etc., can solve the problems of high detection microenvironment requirements, difficult antibody preparation, complex processing, etc., achieve low cost and improve detection Sensitivity and the effect of improving the detection signal-to-noise ratio

Active Publication Date: 2021-06-29
SHAOXING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these techniques have disadvantages such as complex sample pretreatment, high requirements for the detection microenvironment, difficulty in antibody preparation, and easy failure.

Method used

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  • A rapid on-site detection method for food-borne pathogenic bacteria
  • A rapid on-site detection method for food-borne pathogenic bacteria
  • A rapid on-site detection method for food-borne pathogenic bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: Using LiLuF 4 :Yb, Er-Aptamer / cDNA-Alexa Fluor 546 FRET nano-FRET probe for detection of Escherichia coli E. coli O157:H7

[0024] First, Escherichia coli was screened using SELEX technology E. coli The aptamer of O157:H7 simultaneously synthesizes a single-strand complementary oligonucleotide corresponding to the aptamer. Synthesis of LiLuF with particle size less than 10 nm by high temperature co-precipitation method 4 :Yb,Er upconversion fluorescent nanoparticles, and through the ligand exchange method in LiLuF 4 : Yb, Er surface modified amino group. Using EDC and Sulfo-NHS as coupling agents, Aptamer-UCNPs nanoprobes and AF546-cDNA were obtained respectively.

[0025] Second, configure a known concentration of Escherichia coli E. coli O157:H7 standard solution, diluted in proportion to 1-10 5 cfu / mL different concentrations of bacterial liquid. The prepared equivalent amount of LiLuF 4 : Yb, Er-Aptamer / cDNA-Alexa Fluor546 nanometer FRE...

Embodiment 2

[0027] Embodiment 2: Using NaGdF 4 :Yb, Er-Aptamer / cDNA-Alexa Fluor 546 nanometer FRET probe for detection of Salmonella

[0028] First, the aptamer of Salmonella was screened by SELEX technology, and the complementary oligonucleotide single strand corresponding to the aptamer was synthesized at the same time. Synthesis of NaGdF with Particle Size Less than 10 nm by High Temperature Co-precipitation Method 4 :Yb,Er upconversion fluorescent nanoparticles, and through the ligand exchange method in NaGdF 4 : Yb, Er surface modified amino group. Using EDC and Sulfo-NHS as coupling agents, Aptamer-UCNPs nanoprobes and AF546-cDNA were obtained respectively.

[0029] Secondly, configure a Salmonella standard solution of known concentration and dilute it proportionally to 1-10 6 cfu / mL different concentrations of bacterial liquid. Prepared equal amount of NaGdF 4 :Yb, Er-Aptamer / cDNA-Alexa Fluor 546nm FRET probes were added to different concentrations of the bacteria solution t...

Embodiment 3

[0031] Example 3: Using LiYF 4 :Yb, Er-Aptamer / cDNA-Alexa Fluor 546 nanometer FRET probe for detection of Vibrio parahaemolyticus

[0032] First, the aptamer of Vibrio parahaemolyticus was screened by using SELEX technology, and the complementary oligonucleotide single strand corresponding to the aptamer was synthesized at the same time. Synthesis of LiYF with particle size less than 10 nm by high temperature co-precipitation method 4 :Yb,Er upconversion fluorescent nanoparticles, and through the ligand exchange method in LiYF 4 : Yb, Er surface modified amino group. Using EDC and Sulfo-NHS as coupling agents, Aptamer-LiYF was obtained respectively 4 : Yb, Er nanoprobes and AF546-cDNA.

[0033] Secondly, configure Vibrio parahaemolyticus standard solution of known concentration, and dilute it proportionally to 1-10 7 cfu / mL different concentrations of bacterial liquid. The prepared equivalent amount of LiYF 4 : Yb, Er-Aptamer / cDNA-Alexa Fluor 546 nanometer FRET probes ...

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Abstract

The invention relates to a rapid on-site detection method for food-borne pathogenic bacteria, synthesis of nano-FRET probes: synthesis of up-conversion fluorescent nanoparticles AReF by high-temperature co-precipitation method 4 : Yb, Er, utilize coupler EDC, sulfo-NHS to AReF 4 : Yb, Er and the aptamer of the food-borne pathogenic bacteria to be detected, the complementary oligonucleotide single-stranded cDNA of the aptamer and Alexa Fluor 546 fluorescent dye were respectively covalently coupled, and the two coupling products were covalently coupled. Incubation results in nano-FRET probes. The invention constructs AReF 4 : Yb, Er‑Aptamer / cDNA‑Alexa Fluor 546 nanometer FRET probe has a simple process, the established qualitative detection method is fast and simple, and the quantitative detection method is fast, sensitive and specific. The invention is applicable to the field of on-site rapid detection of food-borne pathogenic bacteria, and has positive significance for the control of food safety.

Description

technical field [0001] The invention relates to the technical field of detection of food-borne pathogenic bacteria, in particular to a rapid, high-sensitivity and high-specificity food-borne pathogenic bacteria detection method based on Aptamer / up-conversion fluorescent nanoprobes to realize fluorescence resonance energy transfer . Background technique [0002] In recent years, food poisoning incidents caused by food-borne pathogenic bacteria have occurred continuously, which has greatly affected people's health and life, and at the same time caused huge economic losses. Food safety problems caused by pathogenic microorganisms have been widely concerned by countries all over the world. . Common pathogenic bacteria of bacterial food poisoning mainly include Vibrio parahaemolyticus, Salmonella, Proteus, Staphylococcus aureus, Bacillus cereus, and diarrhea-causing Escherichia coli. Therefore, establishing a rapid, accurate, convenient, high-sensitivity, and specificity detect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N21/78
CPCG01N21/6486G01N21/78
Inventor 俞樟森钱沁清张建华孙爱静张衡方剑张小娟
Owner SHAOXING UNIVERSITY