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A method for rapid detection of pathogenic bacteria in food

A technology for pathogenic bacteria and food, applied in the field of rapid detection of pathogenic bacteria in food, can solve the problems of high cost, inability to use food sample detection, and inability to detect multiple pathogenic bacteria at the same time, and achieve a rapid detection method. Effect

Active Publication Date: 2014-10-22
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have not been applied to the detection of actual food samples so far.
[0005] As another option, some researchers have tried to directly use magnetic nucleic acid probes to isolate and concentrate pathogen DNA from food samples instead of the pathogen itself, but there are also some problems. For example, a capture magnetic probe can only For one pathogenic bacteria, it is impossible to detect multiple pathogenic bacteria at the same time, and the cost is high

Method used

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  • A method for rapid detection of pathogenic bacteria in food
  • A method for rapid detection of pathogenic bacteria in food
  • A method for rapid detection of pathogenic bacteria in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Amino magnetic beads adsorb the genomic DNA of Salmonella in artificially contaminated milk and PCR for rapid detection of Salmonella in raw milk

[0054] Fe 3 O 4 Preparation of magnetic nanoparticles: Fe 3 O 4 Magnetic nanoparticles are prepared by co-precipitation method. 0.02mol FeCl 3 ·6H 2 O and 0.01mol FeSO 4 ·7H 2 O was dissolved in 150ml of deionized water, ammonia was added dropwise to pH 11, heated to 85°C under nitrogen protection, and stirred continuously for a heating duration of 25min, and then cooled to room temperature. The black precipitate was washed repeatedly with ethanol and deionized water, and finally vacuum dried.

[0055] Silicon coated Fe 3 O 4 Preparation of magnetic nanoparticles: Preparation of silicon-coated Fe by inverse microemulsion method 3 O 4 Magnetic nanoparticles, 32.0ml Triton 100, 128.0ml cyclohexane, 8.0ml deionized water, 32.0ml n-hexanol, 280mg Fe 3 O 4 The magnetic nanoparticles were mixed and ultrasonicated to form a r...

Embodiment 2

[0060] Example 2: Absorption of genomic DNA of Listeria monocytogenes in artificially contaminated raw milk by amino magnetic beads combined with PCR for rapid detection of Listeria monocytogenes in raw milk

[0061] Fe 3 O 4 The preparation of magnetic nanoparticles is the same as in Example 1.

[0062] Silicon coated Fe 3 O 4 Preparation of magnetic nanoparticles: mix 25ml ethanol, 6.5ml water, and 1ml ammonia water, this is liquid A; mix 25ml ethanol with 0.5ml ethyl orthosilicate, and add 0.1g Fe 3 O 4 Magnetic nanoparticles, this is liquid B. Mix liquid A and liquid B and stir for 12h at room temperature. Wash the ethanol and water several times, and then soak in 1mol / L hydrochloric acid solution for 24h.

[0063] The amination modification procedure is the same as in Example 1.

[0064] Take 10ml of raw milk contaminated with artificial gradient of Listeria monocytogenes (GIM1.229), centrifuge at 6000r / min for 20min, add 450μl of TE buffer solution containing 0.5% Triton 100, a...

Embodiment 3

[0067] Example 3: Amino magnetic beads adsorb the genomic DNA of pathogenic bacteria in artificially contaminated raw milk combined with PCR to quickly detect Salmonella and Listeria monocytogenes in raw milk

[0068] Fe 3 O 4 Preparation of Nanoparticles and Silicon Coated Fe 3 O 4 The preparation of the nanoparticles is the same as in Example 1.

[0069] Fe on silicon 3 O 4 Nanoparticles are coated twice: mix 25ml of ethanol, 6.5ml of water, and 1ml of ammonia, this is liquid A; mix 25ml of ethanol with 0.5ml of ethyl orthosilicate, and add 0.1g of coated silicon Magnetic nanoparticles, this is liquid B. Mix liquid A and liquid B and stir for 12h at room temperature.

[0070] The amination modification procedure is the same as in Example 1.

[0071] Take 10ml of raw milk contaminated with artificial gradient of Salmonella and Listeria monocytogenes, centrifuge at 6000r / min for 20min, add 450μl of TE buffer solution containing 0.5% Triton 100, add 40μl of 20μg / μl of proteinase K, 40...

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Abstract

The invention relates to the field of food safety, in particular to a method for rapidly detecting pathogenic bacteria in food, wherein the method is on the basis that non-marking amination nano magnetic beads capture trace desoxyribonucleic acid (DNA) of genome, and a polymerase chain reaction (PCR) is combined to rapidly detect the pathogenic bacteria in the food. The defects of a method for rapidly detecting pathogenic bacteria in food in the prior art include that a national standard cultivation method needs bacteria to be added, detection time is long, an immunoassay method needs high-quality immune bodies, one immune body is only corresponding to one kind of pathogenic bacterium, a simple PCR detecting technique also needs bacteria to be added in advance, and the detection time of the PCR technology in the prior art is long. The method for rapidly detecting the pathogenic bacteria in the food includes the steps of preparing amino magnetic beads which are suitable for being added into a PCR system, capturing the DNA in food samples which are processed in a simple mode, and eventually directly carrying out PCR detection by a combination object. The method for rapidly detecting the pathogenic bacteria in the food has the advantages of being rapid in detection, low in cost, high in sensitivity, capable of aiming at different pathogenic bacteria to carry out detection, capable of detecting multiple kinds of pathogenic bacteria simultaneously, and capable of carrying out semi-quantitative detection and quantitative determination.

Description

Technical field [0001] The invention relates to the field of food safety, in particular to a detection method capable of rapidly detecting pathogenic bacteria in food. Background technique [0002] Pathogenic bacteria are an important biological factor that affects food safety and have received widespread attention from consumers and industrial production. Compared with traditional culture-based methods, PCR is a landmark discovery for detecting living substances. PCR technology can amplify several or even one target sequence to millions of copies in a short time. In this way, a reliable, rapid, and specific detection method is provided for the detection of pathogenic bacteria. In theory, as long as there is a target sequence of the pathogenic bacteria, the PCR method can detect it. However, when PCR is applied to the detection of food samples (such as milk), fat, protein and other food ingredients will strongly inhibit the PCR reaction. Although the PCR reaction has a strong ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04
Inventor 史贤明白亚龙施春雷宋明辉王大鹏崔妍
Owner SHANGHAI JIAOTONG UNIV