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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

Active Publication Date: 2011-08-17
GUANGZHOU ENENTA CHEM SCI & TECH
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Since the cultivation of methane-oxidizing bacteria requires a continuous supply of methane, the samples need to be treated at low temperature or dried, and the indoor culture conditions have changed greatly from the living conditions of the methane-oxidizing bacteria in the original sample, so most of the methane-oxidizing bacteria in the sample Cannot be cultured to survive, which will artificially cause large counting errors
In addition, if a large number of samples are to be cultured indoors to count viable bacteria, the experimental workload is very heavy, and rapid quantitative detection of a large number of samples cannot be achieved.
[0007](3) Compared with the chemical method and culture method, the molecular biology method has better accuracy and greatly reduces the workload, but the cost of genome extraction and fluorescent quantitative PCR measurement Higher, requires specialized technicians and large instruments and equipment as support, and the average person can only process about 15 samples per day, which greatly limits the popularization and application of this method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Collect 50 g of rice field soil, and the sampling depth is 50 cm.

[0025] 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 it at 200 rpm for 30 minutes, and let it settle naturally for 15 minutes.

[0026] 3. Use a double-layer 96-well filter plate with a pore size of 5 μm in the upper layer and a 96-well filter plate in the lower layer with a pore size of 0.2 μm to filter the supernatant; take 5 methane-oxidizing bacteria with known different concentrations as positive controls, and make 3 samples for each concentration Parallel experiments; set up a negative control and do 3 parallel experiments.

[0027] 4. Remove the upper microporous membrane filter plate, and add 100 μL of 100 mg / mL FITC-labeled pMMO monoclonal antibody to each well of the lower filter membrane plate, and keep it moist at 37°C for 40 min.

[0028] 5. Add 100 μL of 0.01 mol / L pH 7.4 PBST s...

Embodiment 2

[0031] 1. Collect 50 g of pond bottom mud, and the sampling depth is 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 min, add 12.5 g of NaCl, and allow it to settle naturally for 15 min.

[0033] 3. Use a double-layer 96-well filter plate with a pore size of 5 μm in the upper layer and a 96-well filter plate in the lower layer with a pore size of 0.2 μm to filter the supernatant; take 5 methane-oxidizing bacteria with known different concentrations as positive controls, and make 3 samples for each concentration Parallel experiments; set up a negative control and do 3 parallel experiments.

[0034] 4. Remove the upper microporous membrane filter plate, and add 100 μL of 100 mg / mL FITC-labeled pMMO monoclonal antibody to each well of the lower filter membrane plate, and keep it moist at 37°C for 40 min.

[0035] 5. Add 100 μL of 0.01 mol / L...

Embodiment 3

[0038] 1. Collect 50 g of rice field soil, and the sampling depth is 50 cm.

[0039] 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 it at 200 rpm for 30 minutes, and let it settle naturally for 15 minutes.

[0040] 3. Use a double-layer 384-well filter plate with an upper pore size of 5 μm and a lower pore size of 0.2 μm to filter the supernatant; take 5 methane-oxidizing bacteria with known different concentrations as positive controls, and make 3 samples for each concentration Parallel experiments; set up a negative control and do 3 parallel experiments.

[0041] 4. Remove the upper microporous membrane filter plate, and add 100 μL of 100 mg / mL FITC-labeled pMMO monoclonal antibody to each well of the lower filter membrane plate, and keep it moist 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 plate and pe...

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Abstract

The invention discloses a method for quantitively detecting methane-oxidizing bacterium. The method comprises the following steps: sampling; washing bacterium with PBS (Phosphate Buffer Solution) by shaking samples; designing and selecting a double-layer microfiltration membrane plate with specific pores according to the number of the samples; and filtering the bacteria, passing the membrane and fixing; and conducting fluorescent dyeing and result analysis. According to the fluorescent strength of samples on the microfiltration membrane plate, the number of the methane-oxidizing bacterium in soil samples or sediment samples can be quantitively calculated, so as to provide technical support for microorganism oil-gas exploration and microorganism oil-gas reservoir characteristics.

Description

technical field [0001] The invention relates to the technical field of quantitative detection of microorganisms, in particular to a high-throughput quantitative detection method for methanotrophic bacteria. Background technique [0002] Methane-oxidizing bacteria are a member of the obligate hydrocarbon-oxidizing bacteria group, which is a highly specific bacterial group that uses methane as the only carbon source. The survival of methanotrophs depends on a continuous supply of methane. Therefore, the abnormal generation of methane-oxidizing bacteria in surface soil or sediment is often related to the continuous micro-leakage of deep methane, and the abnormality of methane-oxidizing bacteria is an important indicator for microbial oil and gas exploration and microbial oil and gas reservoir characterization. [0003] Determination of methanotroph abnormalities requires high-throughput enumeration of methanotroph populations. The conventional method for counting methane-oxid...

Claims

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Application Information

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IPC IPC(8): C12Q1/06G01N21/64
Inventor 王江海吴酬飞袁建平许红吕宝凤郑贵洲郑新宁燕腾鹏刘权徐小明彭娟徐小燕
Owner GUANGZHOU ENENTA CHEM SCI & TECH
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