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Method for extracting and detecting free-state DNA in sewage

A technology of extraction method and quantitative detection method, which is applied in the field of extraction and detection of free DNA in sewage, can solve the optimization research on the separation parameters of free DNA in sewage, interfere with the efficient separation and accurate quantification of free DNA, and affect the free state in sewage. DNA separation efficiency and other issues, to achieve the effect of improving recovery rate and purity, shielding interference, and simple process operation

Inactive Publication Date: 2020-07-10
NANHUA UNIV
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Problems solved by technology

[0004] The composition of sewage is complex, containing many impurities such as salts and organic matter, which may interfere with the efficient separation and accurate quantification of free DNA
In addition, the selection of separation reagents, separation system volume, separation temperature and other parameters will also seriously affect the separation efficiency of free DNA in sewage, but there is no optimization study on the separation parameters of free DNA in sewage

Method used

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  • Method for extracting and detecting free-state DNA in sewage
  • Method for extracting and detecting free-state DNA in sewage
  • Method for extracting and detecting free-state DNA in sewage

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Embodiment 1

[0059] The extraction of embodiment 1 sewage free state DNA

[0060] 1. Pretreatment of sewage samples

[0061] Sewage samples were taken from the inflow and outflow points of seven actual sewage plants in Wuxi City, Jiangsu Province, placed in sterilized brown glass bottles, and transported to the laboratory in ice boxes. The sewage was filtered with a 0.22 μm filter membrane, and the filtrate was stored in a -20°C refrigerator.

[0062] 2. Isolation of free DNA

[0063] (1) Take 8 mL of the filtrate in step 1 and place it in a 50 mL centrifuge tube. Add 4mL of 7.5M ammonium acetate and mix by inversion. Add 24 mL of 4°C pre-cooled absolute ethanol, invert and mix again, and let stand on ice for 1 h;

[0064] (2) After centrifugation at 4° C., 14000 g, for 30 min, carefully remove the supernatant. Add 0.8mL 4°C precooled 70vol% ethanol to the centrifuge tube. After slight mixing, transfer the ethanol / precipitation mixture to a new 2mL centrifuge tube;

[0065] (3) Afte...

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Abstract

The invention relates to the technical field of sewage treatment, in particular to a method for extracting and detecting free-state DNA in sewage. According to the extraction method and a quantitativedetection method for free-state DNA in the sewage provided by the invention, an ethanol-ammonium acetate precipitation method is adopted in the extraction process for concentrating and separating free-state DNA in a filtrate, and the whole extraction process only needs one time of precipitation and two times of washing, so that the process is simple to operate, and the yield and purity of the DNAcan be improved; and in the detection step, the concentration of free-state DNA is detected by using a high-sensitivity Picogreen fluorescent dye. The method can effectively shield the interference of pollutants such as common salts and organic matters and the like in sewage, thereby providing a sensitive and reliable analysis means for the research of trace free-state DNA in sewage.

Description

technical field [0001] The invention relates to the technical field of sewage treatment, in particular to a method for extracting and detecting free DNA in sewage. Background technique [0002] Cell-free DNA is considered as one of the important sources of nutrition for microorganisms in the water environment because it is rich in elements such as nitrogen and phosphorus. A large number of studies have reported the existence of free DNA in natural water bodies such as seawater, lake water, river water, and pond water, and its concentration ranges from pg / mL to ng / mL. In addition to the widespread detection of free DNA in various water bodies, studies have also proved that free DNA in water environments can be naturally transformed by bacteria, which shows that free DNA is also a potential carrier of horizontal gene transfer in water environments. More importantly, the latest research has found that free DNA in natural water bodies (including river water, well water, drinkin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6851G01N21/64
CPCC12N15/1003C12Q1/6851G01N21/6486
Inventor 周帅熊聪高媛媛唐振平刘迎九
Owner NANHUA UNIV
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