Functionalized magnetic nanoparticles for efficiently enriching microorganisms, preparation and applications thereof
A magnetic nanoparticle and microbial technology, applied in the direction of microbial-based methods, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of inability to effectively separate and purify bacterial genomes, weakened dispersibility, and high antibody prices
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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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