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Qualitative and/or quantitative extraction method of microbial DNA on the surface of large particle matrix of constructed wetland

A technology of constructed wetlands and extraction methods, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, and treatment of granular microorganism carriers, etc. question

Active Publication Date: 2020-06-16
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The methods disclosed in the above patents are limited to the direct extraction of environmental samples with extremely fine particles, and do not mention the rough and uneven surface of large-grained matrix samples, especially how to quantitatively extract the microbial DNA on the surface of large-grained matrix samples
In constructed wetlands, large-grained gravel, volcanic rock, zeolite and other substrates are often used as bed fillers to prevent system blockage. The single-grain weight of these large-grained substrates often exceeds 0.5g, and it is impossible to use trace amounts of soil like soil, sediment and silt. DNA extraction kits are used to extract DNA, and the kits are expensive, which is not economically feasible for the extraction of large quantities of samples
[0004] Therefore, it is necessary to develop an efficient, fast, economical and batch-scale method suitable for the extraction of microbial DNA on the surface of large-grained substrates, so as to solve the problem of direct extraction of microbial DNA on the surface of large-grained substrate samples by conventional methods, and the presence of humus and other PCR inhibitors in the extracted DNA. And the problems of inaccurate quantification and low DNA yield

Method used

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  • Qualitative and/or quantitative extraction method of microbial DNA on the surface of large particle matrix of constructed wetland
  • Qualitative and/or quantitative extraction method of microbial DNA on the surface of large particle matrix of constructed wetland
  • Qualitative and/or quantitative extraction method of microbial DNA on the surface of large particle matrix of constructed wetland

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Extraction of microbial DNA from gravel surfaces in constructed wetlands

[0063] Constructed wetland gravel sample collection:

[0064] In this embodiment, gravel samples are collected from a group of horizontal subsurface flow constructed wetlands for treating domestic sewage. The wetland device is 80 cm long, 60 cm wide, and 80 cm high. The wetland matrix is ​​gravel (particle size 1-2cm), and the filling height is 60cm. The no-plant group, the canna group and the Zailihua group are respectively set. The hydraulic load of the device is 2.5m / d, and it operates continuously throughout the day. The matrix samples are collected near the water inlet and near the water outlet respectively. The matrix at the surface 0-10cm is mixed as the surface sample, and the matrix at the bottom 30-40cm is mixed as the bottom sample. There are 6 samples in three devices, 3 of which are surface samples and 3 are bottom samples. About 200g of matrix is ​​collected and put into...

Embodiment 2

[0085] Example 2: Extraction of microbial DNA from gravel surfaces in constructed wetlands

[0086] Constructed wetland gravel sample collection:

[0087] In this example, gravel samples are collected from a group of horizontal subsurface flow artificial wetlands for domestic sewage treatment. The wetland device is 80 cm long, 60 cm wide, and 80 cm high. The wetland matrix is ​​gravel (particle size 1-2 cm), and the filling height is 60 cm. group, Canna group and Zailihua group. The hydraulic load of the device is 2.5m / d, and it operates continuously throughout the day. The matrix samples are collected near the water inlet and near the water outlet respectively. The matrix at the surface 0-10cm is mixed as the surface sample, and the matrix at the bottom 30-40cm is mixed as the bottom sample. There are 6 samples in three devices, 3 of which are surface samples and 3 are bottom samples. About 200g of matrix is ​​collected and put into sterile sealed bags for later use. Each sa...

Embodiment 3

[0108] Embodiment 3 comparative test

[0109]In order to compare the present invention (embodiment 1 and embodiment 2, claim the present invention in the following data and samples) and the extraction effect of existing non-corruption treatment method and kit method, the applicant has carried out utilizing non-corrosion treatment simultaneously Method and kit method experiments, specific operations are as follows:

[0110] (1) This method does not carry out calcium chloride and sodium oxalate treatment, and other steps are identical with embodiment 1 or embodiment 2, and the formula of DNA extraction liquid is (100mM Tris-HCl, 100mM EDTA, 200mM NaCl, 2%PVPP, 3% CTAB, pH 8.0).

[0111] (2) use DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) was used to extract genomic DNA, and all the liquid and grinding beads in the PowerBead Tubes were transferred to a 2mL centrifuge tube containing 0.5g of precipitate, and the rest of the steps were carried out according t...

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Abstract

The invention discloses a qualitative and / or quantitative extraction method of total DNA of large-grain substrate surface microorganisms in constructed wetlands. The method comprises the steps that large-grain substrates used for constructed wetlands filling serve as materials, biological membranes on the surfaces of the large-grain substances are eluted through a shaking table oscillation method, centrifugation is conducted to obtain precipitates, the precipitates are subjected to humic acid removing treatment with a humic acid removing buffer solution and a calcium chloride solution, cells are subjected to pyrolysis through an SDS-lysozyme method, chloroform extraction is conducted, and isopropanol precipitating is conducted to obtain DNA. Compared with an existing kit extraction method without pretreatment, the extracted total DNA of the substrate surface microorganisms in the constructed wetlands is high in concentration and purity and good in integrity, a small amount of information of microbial diversity is lost, and the method can be directly used for PCR, real-time quantitative PCR and DGGE experiment analysis. The method has the advantages of being low in extraction cost, short in consumed time and the like, the contamination rate in the sample treatment process is decreased, the DNA loss caused by freeze thawing is reduced, and the problems that when the total DNA of the large-grain substrate surface microorganisms in the constructed wetlands are extracted directly, the yield ratio is low, the purity is low and a small amount of the diversity information is obtained are solved.

Description

technical field [0001] The invention relates to the technical field of microbial molecular ecology, more specifically, to a method for qualitatively and / or quantitatively extracting microbial DNA from gravel surfaces in constructed wetlands. Background technique [0002] Biomass and community structure are two important characteristics of constructed wetlands, and their distribution characteristics in wetlands are closely related to the degradation behavior of pollutants in wetlands. With the development of molecular biology and its application in ecology, especially the establishment and development of metagenomics methods in the direction of ecology, the cultivation methods are far from meeting the needs of large-flux species information in constructed wetlands. Therefore, the research points to the entire environmental genomic DNA, including cultured and non-cultured microorganisms, through high-throughput sequencing and qPCR and other molecular biology techniques to obta...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6888C12Q1/686C12Q1/6869C12Q1/04C12Q1/06C02F3/32C02F3/34
CPCC02F3/327C02F3/34C02F2003/001C12N15/1003C12Q1/6806C12Q1/686C12Q1/6869C12Q1/6888C12Q2527/125C12Q2565/125C12Q2535/122C12Q2531/113Y02W10/40
Inventor 杨扬黄文达陶然郭菁菁满滢
Owner JINAN UNIVERSITY
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