Method for quantitively detecting methane-oxidizing bacterium
A technology for methane-oxidizing bacteria and detection methods, which is applied in the measurement/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., and can solve the problems that methane-oxidizing bacteria cannot be cultured and survived, the experimental workload is large, genome extraction and fluorescence The problem of high cost of quantitative PCR determination can achieve the effect of easy observation and subsequent analysis, reducing experimental error and no cross-contamination
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[0023] Example 1:
[0024] 1. Collect 50 g of paddy field soil with a sampling depth of 50 cm.
[0025] 2. Put the sample in a 500 mL small-necked Erlenmeyer flask, then add 200 mL of PBS solution, place the small-necked Erlenmeyer flask on a shaker, shake at 200 rpm for 30 minutes, and let it settle naturally for 15 minutes.
[0026] 3. Use a double-layer 96-well filter membrane with an upper pore size of 5 μm and a lower pore size of 0.2 μm to filter the supernatant; take 5 known methanooxidizing bacteria with different concentrations as positive controls, and make 3 for each concentration Parallel test; set a negative control and do 3 parallel tests.
[0027] 4. Remove the upper microporous filter membrane plate, and add 100 μL of 100 mg / mL FITC-labeled pMMO monoclonal antibody to each well of the lower filter membrane plate, and moisturize at 37°C for 40 min.
[0028] 5. Add 100 μL of 0.01 mol / L pH 7.4 PBST solution to each well of the lower filter membrane plate and perform suctio...
Example Embodiment
[0030] Example 2:
[0031] 1. Collect 50 g of the bottom mud of the pond with a sampling depth of 30 cm.
[0032] 2. Put the sample into a 500 mL small-necked Erlenmeyer flask, then add 200 mL of PBS solution, place the small-necked Erlenmeyer flask on a shaker, shake at 200 rpm for 30 minutes, add 12.5 g of NaCl, and let it settle naturally for 15 minutes.
[0033] 3. Use a double-layer 96-well filter membrane with an upper pore size of 5 μm and a lower pore size of 0.2 μm to filter the supernatant; take 5 known methanooxidizing bacteria with different concentrations as positive controls, and make 3 for each concentration Parallel test; set 1 negative control and do 3 parallel tests.
[0034] 4. Remove the upper microporous filter membrane plate, and add 100 μL of 100 mg / mL FITC-labeled pMMO monoclonal antibody to each well of the lower filter membrane plate, and moisturize at 37°C for 40 min.
[0035] 5. Add 100 μL of 0.01 mol / L pH 7.4 PBST solution to each well of the lower filter ...
Example Embodiment
[0037] Example 3:
[0038] 1. Collect 50 g of paddy field soil with a sampling depth of 50 cm.
[0039] 2. Put the sample in a 500 mL small-necked Erlenmeyer flask, then add 200 mL of PBS solution, place the small-necked Erlenmeyer flask on a shaker, shake at 200 rpm for 30 minutes, and let it settle naturally for 15 minutes.
[0040] 3. Use a double-layer 384-hole filter membrane with an upper pore size of 5 μm and a lower pore size of 0.2 μm to filter the supernatant; take 5 known methanooxidizing bacteria with different concentrations as a positive control, and 3 for each concentration Parallel test; set 1 negative control and do 3 parallel tests.
[0041] 4. Remove the upper microporous filter membrane plate, and add 100 μL of 100 mg / mL FITC-labeled pMMO monoclonal antibody to each well of the lower filter membrane plate, and moisturize at 37°C for 40 min.
[0042] 5. Add 100 μL of 0.01 mol / L pH 7.4 PBST solution to each well of the lower filter membrane plate and perform suction ...
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