Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample

A sewage biological treatment and microbial technology, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of extracting microbial intracellular and extracellular DNA at the same time, avoiding loss, good lysis effect and high yield. Effect

Inactive Publication Date: 2015-09-16
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, there is no method suitable for simultaneous extraction of microbia

Method used

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  • Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample
  • Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] When using a membrane bioreactor (membrane bioreactor, MBR) to treat domestic sewage, analyze the intracellular and extracellular DNA content of the water sample inside the membrane bioreactor.

[0034] Using 8L submerged hollow fiber membrane MBR, continuous aeration of 200L / h, HRT of 24h, sludge load of 0.24kg COD / kg VSS·d, stable operation at room temperature. Take 10mL of MBR internal mixed liquid for DNA extraction operation, the mixed liquid sample contains more activated sludge, and the color is light brown yellow.

[0035] Add 10mL NaH to the MBR mixture 2 PO 4 The solution (0.12M, pH 8.0) and 1g of polyvinylpyrrolidone (Polyvinylpolypyrrolidone, PVPP) were placed on a shaker (250rpm) at room temperature for 10 minutes to shake and mix well.

[0036] Then the sample was centrifuged (10000g, 4°C, 10min), and the centrifuged supernatant was further filtered with a sterile filter membrane (pore size 0.22μm), centrifuged and filtered to obtain the precipitate with...

Embodiment 2

[0040] When using a membrane bioreactor (membrane bioreactor, MBR) to treat domestic sewage, analyze the intracellular and extracellular DNA content of the membrane bioreactor effluent sample.

[0041] The difference between this example and Example 1 is that the sewage sample comes from the effluent of the reactor, the color is clearer, and the microbial content is less, so the sediment is obtained by centrifugation and filtration with NaH 2 PO 4 (0.12M, pH 8.0) after resuspension, repeat the operation of centrifugation and filtration only once, and use the freeze-thaw method to disrupt the cells, that is, alternately place the mixed solution in liquid nitrogen (1min) and 60°C water bath (20min) only once, other The operation was the same as in Example 1, and finally the content of intracellular DNA in the water sample was 0.091±0.013 μg / mL, the content of extracellular DNA was 0.064±0.032 μg / mL, and the ratios of absorbance at 260 nm to absorbance at 280 nm were 1.45 and 1.5...

Embodiment 3

[0043] Using the pure water sample as a blank control, the same steps as in Example 1 were used to extract the contents of intracellular and extracellular DNA to be 0.

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Abstract

The invention relates to a method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in a sewage biological treatment water sample, belonging to the technical field of sewage treatment. The method comprises the following steps: pretreating; and separating intracellular and extracellular DNAs, and respectively extracting and purifying the intracellular DNA and extracellular DNA. The method can simultaneously extract the intracellular and extracellular DNAs from a biological treatment sewage sample of an activated sludge process or the like and perform accurate quantification, has the advantages of high yield, high purity and low thallus cracking cross contamination, is simple and easy to implement, is very suitable for performing contrast experiments and scientific research on the sewage sample containing abundant microbes, and has application prospects for developing and processing business analysis kits.

Description

technical field [0001] The invention belongs to the technical field of sewage treatment, and relates to a method for extracting microbial intracellular and extracellular DNA, in particular to a method for simultaneously extracting microbial intracellular and extracellular DNA in sewage biological treatment water samples. Background technique [0002] Degradation of soluble and suspended organic pollutants by microorganisms is the core process of sewage treatment plants, and the most representative one is the activated sludge process. As long as a suitable growth environment is provided for microorganisms and the microorganisms grow sufficiently, organic pollutants can be removed to the greatest extent and the quality of effluent water can be improved. In other words, the number and type of microorganisms directly determine the effect of sewage biological treatment. However, the microbial community structure in sewage is complex, and the traditional purebred culture method o...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 沈亮图姆斯基·斯巴图·海伯特马里安袁霞卢英华王远鹏何宁
Owner XIAMEN UNIV
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