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A functionalized magnetic nanoparticle for efficiently enriching microorganisms and its preparation and application

A technology of magnetic nanoparticles and microorganisms, which can be used in microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc., and can solve the problems of high antibody price, inability to detect bacteria, and inconvenient storage and transportation.

Active Publication Date: 2017-10-13
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now there are a variety of enrichment methods for bacteria, mainly based on antibodies, aptamers, antibiotics, chemical groups, etc., which can specifically bind to bacteria, but these methods also have some problems, such as antibody The price is high and can only identify a single bacterium, and it is inconvenient to store and transport; antibiotics such as vancomycin modified nano-magnetic beads will change the properties of the surface of the magnetic beads due to the modification effect and weaken the dispersibility in the liquid, which is harmful to bacteria The capture efficiency decreases; chemical groups such as amino groups often bind negatively charged bacteria on the surface through charge, which leads to the inability to effectively separate and purify the genomes of captured bacteria, resulting in the inability to detect the captured bacteria at the nucleic acid level
These defects have led to the fact that the magnetic beads currently used for enrichment cannot be effectively applied in actual life and production.

Method used

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  • A functionalized magnetic nanoparticle for efficiently enriching microorganisms and its preparation and application
  • A functionalized magnetic nanoparticle for efficiently enriching microorganisms and its preparation and application
  • A functionalized magnetic nanoparticle for efficiently enriching microorganisms and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Functionalization of Amino Magnetic Beads by Vancomycin and PEG

[0063] 1. Take 4.14mL amino magnetic beads, perform magnetic enrichment on a magnetic separation rack, add 30mM / pH6 MES buffer to wash three times and resuspend in 1mL 30mM / pH6 MES buffer.

[0064] 2. Weigh 0.03g of vancomycin hydrochloride, 0.06g of N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide on an electronic balance Hydrochloride (EDC·HCl) 0.08g (the above medicines are purchased from Shanghai Sigma-Aldrich Company);

[0065] 3. Add the amino magnetic beads in step 1) and the drug in step 2) into a 15mL centrifuge tube, and add 30mM / pH6 MES buffer to make up the reaction system to 10mL, and react in a shaking table at a temperature of 25°C. Rotational speed 180rpm, time 6h.

[0066] 4. After the reaction is completed, magnetically separate the magnetic beads, wash with 30mM / pH6 MES buffer three times, and resuspend in 10mL 30mM / pH6 MES buffer to obtain the nano-...

Embodiment 2

[0069] Example 2 Functionalized magnetic beads carry out magnetic enrichment of microorganisms in food

[0070] The microorganisms in the food are pathogenic bacteria: Listeria monocytogenes (Listeria monocytogenes) (the strain number is CMCC54007, purchased from the Guangzhou Institute of Microbiology), the bacteria that were cultured overnight in 10mL LB medium, the OD600 was 1, the concentration About 1×10 8 cfu / mL, take 1mL of the above bacterial solution at 10000rpm, centrifuge at 4°C for 2min, collect the precipitate, resuspend in 1mL of 30mM MES / 0.1M NaCl pH6 buffer, and perform 10-fold serial dilution to obtain 1×10 6 cfu / mL to 1×10 0 cfu / mL dilution bacteria solution 1mL each.

[0071] Add the above 1×10 to 15mL centrifuge tube 6 cfu / mL to 1×10 0 cfu / mL bacterium solution 1mL, add sterilized LB culture medium to it, supplement the volume to 10mL, then add 1mL of double-labeled magnetic beads prepared in Example 1 to it (magnetic separation first, only add solid C...

Embodiment 3

[0073] Example 3 Bacteria of different concentrations are detected by the platform of this scheme

[0074] The magnetic beads / bacteria complex obtained in Example 2 was magnetically separated, 110 μL of TE buffer solution was added thereto, mixed evenly, and then 70 μL of lysozyme (purchased from Tiangen Biochemical Technology Co., Ltd.) was added, reacted in a water bath, and the temperature The temperature is 37°C, and the time is 1h. After the reaction was completed, 20 μL of proteinase K (purchased from Tiangen Biochemical Technology Co., Ltd.) was added, mixed evenly, and reacted in a water bath at a temperature of 56 °C for 30 min. After the reaction was completed, magnetic separation was performed, and the aqueous phase was transferred to 1.5 In a centrifuge tube, add 220 μL of pre-cooled absolute ethanol, vortex to mix, and briefly centrifuge.

[0075] The above product was isolated and purified with a bacterial genome DNA extraction kit (purchased from Tiangen Bioche...

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Abstract

The present invention discloses functionalized magnetic nanoparticles for efficiently enriching microorganisms, preparation and applications thereof. The method for carrying out efficient enrichment and nucleic acid detection on microorganisms in food mainly comprises: (1) modifying and functionalizing magnetic nanoparticles; (2) carrying out magnetic enrichment on microorganisms in food in a liquid environment; (3) carrying out genome isolation and purification on the enriched bacteria; and (4) carrying out PCR-based nucleic acid detection on the isolated and purified bacterial genome. With the technical scheme, the pathogenic bacteria in the food can be efficiently enriched within a short time, and the high quality genome template is isolated and purified from the enriched bacteria and then is subjected to nucleic acid detection. Compared with the traditional method, the method of the present invention has characteristics of rapidity, manpower saving, material resource saving, efficient detection of the low concentration pathogenic bacteria in the food, and reliable detection result, and is easily generalized and applied in the fields of production, life, scientific research and the like.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a functionalized magnetic nanoparticle for efficiently enriching microorganisms, as well as its preparation and application. Background technique [0002] With the progress of society, people pay more and more attention to food safety, especially the pathogenic bacteria in food pose a serious threat to people's health. At present, the main detection method is to detect microorganisms through culture medium enrichment and observation of plate colony morphology. This method is relatively reliable, but it also has this obvious deficiency. It often takes more than 5 days for culture medium enrichment, single colony division and identification, which can no longer meet the needs of high circulation and timeliness in the current market, and cannot effectively prevent the infection of food pathogens. At the same time, detection based on colony morphology is eve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/686C12Q2563/143C12Q2563/149
Inventor 邢达周小明朱敏君
Owner SOUTH CHINA NORMAL UNIVERSITY