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Method for evaluating biosafety by using mouse intestinal flora
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A technology of biosafety and intestinal flora, applied in biochemical equipment and methods, microbial measurement/testing, biological testing, etc., can solve problems such as unclear impact on human health
Inactive Publication Date: 2020-10-30
ZHEJIANG UNIV +1
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Embodiment 1
[0045] (1) Isolation of bacteriophages from mid-Pacific ridge sediments:
[0046] 1. Weigh 10g of the mid-Pacific ridgesediment sample (see Table 1-1 for sample information) in a 50mL centrifuge tube, add 5mL of SM solution (25mM Tris-HCl, 200mM NaCl, 20mM MgCl 2 ,pH=7.5), shake and resuspend for 20min;
[0047] 2. Centrifuge at 5000×g for 10 minutes at 4°C;
[0048] 3. Continue to add SM solution to the precipitate to resuspend, repeat 5 times;
[0049] 4. Combine the supernatants collected five times, first pass through a 0.45 μm filter membrane, and then pass through a 0.22 μm filter membrane to filter and sterilize;
[0050] 5. Add PEG-6000 with a final concentration of 10% to the solution, and precipitate overnight;
[0051] 6. Centrifuge at 40000×g for 2 hours at 4°C;
[0052] 7. Resuspend the precipitate with an appropriate amount of sterile water;
[0053] 8. Take 10 μL of the virus solution, stain it with tungstenphosphate, and observe it with a transmission el...
Embodiment 2
[0107] (1) Isolation of phages from seamount sediments:
[0108] 1. Weigh 10g of seamount sediment samples in a 50mL centrifuge tube, add 5mL of SM solution (25mM Tris-HCl, 200mM NaCl, 20mM MgCl 2 ,pH=7.5), shake and resuspend for 20min;
[0109] 2. Centrifuge at 5000×g for 10 minutes at 4°C;
[0110] 3. Continue to add SM solution to the precipitate to resuspend, repeat 5 times;
[0111] 4. Combine the supernatants collected five times, first pass through a 0.45 μm filter membrane, and then pass through a 0.22 μm filter membrane to filter and sterilize;
[0112] 5. Add PEG-6000 with a final concentration of 10% to the solution, and precipitate overnight;
[0113] 6. Centrifuge at 40000×g for 2 hours at 4°C;
[0114] 7. Resuspend the precipitate with sterile water;
[0116] Table 2-1 Seamount sediment sample information
[0117] ...
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Abstract
The invention discloses a method for evaluating biosafety by using mouse intestinal flora. According to the method, mouse intestinal flora is used as a biological model, mixed bacteriophage separatedfrom deep sea sediments is added into the biological model, and continuous treatment is performed for 3 days. After one week, an excrement sample of the mouse is taken to perform 16S rRNA gene sequencing, physiological indexes of the mouse are measured and compared with a control sample to determine the influence of the deep-sea bacteriophage on the intestinal flora structure of the mouse and thehealth of the mouse. Compared with a control group, if significant bacterial abundance changes and mouse physiological index changes exist in the three repetitions in the treatment group, it is provedthat the group of deep seabacteriophage has the biosafety problem. The invention provides a method for evaluating the biological safety of the deep sea bacteriophage.
Description
technical field [0001] The invention belongs to the field of biological safety evaluation, in particular to a method for evaluating the biological safety of deep-sea bacteriophages by using the intestinal flora of mice. Background technique [0002] Viruses are ubiquitous, most abundant, and genetically highly diverse in marine ecosystems. The depth of virus-host interactions in the global ocean is great, and viruses may infect a variety of organisms, ranging from simple green algae to large eukaryotes. Marine viruses have a major impact on nutrient and energy cycling, biogeochemistry, population dynamics, genetic exchange, and evolution in the marine environment. Although most marine viruses are nonculturable, most of them can be computationally associated with dominant, ecologically relevant microbial hosts, i.e. most are bacteriophages (bacteriophages). Due to their broad host range, deep-sea phages may have the ability to infect the human gut microbiota. [0003] The ...
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