Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for extracting metagenome DNA of compost and application thereof in identifying denitrifying bacteria

A technology of denitrifying bacteria and metagenomics, applied in the direction of recombinant DNA technology, DNA preparation, etc., can solve the problems of limiting the research and utilization of soil microbial resources and their genetic resources, time-consuming, poor accuracy, etc., to achieve short and efficient extraction , the effect of high application value

Inactive Publication Date: 2012-07-04
CHINA AGRI UNIV
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional screening method for denitrifying bacteria is to use medium for culture and inoculation, and the single culture cycle takes 14 days. The entire screening process generally takes more than two weeks, which takes too long and is not very accurate.
Moreover, 99% of the microorganisms in the environment are not cultivable, and traditional screening methods greatly limit the research and utilization of soil microbial resources and their genetic resources.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for extracting metagenome DNA of compost and application thereof in identifying denitrifying bacteria
  • Method for extracting metagenome DNA of compost and application thereof in identifying denitrifying bacteria
  • Method for extracting metagenome DNA of compost and application thereof in identifying denitrifying bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Extraction of compost metagenomic DNA and its application in identifying denitrifying bacteria

[0053] 1. Production of chicken manure compost

[0054] 25kg of chicken manure, 12.3kg of sawdust and 13kg of water are mixed, the C / N of the mixture is 25-30, and the water content is 55-60%, and the mixture is fermented at room temperature (about 23° C.) for 20 days.

[0055] Chicken manure was obtained from the chicken farm of China Agricultural University, and sawdust was obtained from Yongfeng Woodware Factory, Haidian District, Beijing. The properties of the materials are shown in Table 1.

[0056] Table 1 Basic properties of compost raw materials

[0057]

[0058] Samples were taken on the 1st, 4th, 7th, 11th, 16th, and 20th days of fermentation for subsequent experiments.

[0059] 2. Extraction of compost metagenomic DNA

[0060] 1. Weigh about 4g of chicken manure compost prepared in step 1 and place it in a 50ml centrifuge tube.

[0061] 2. Add 13...

Embodiment 2

[0084] Example 2. Extraction of compost metagenomic DNA and its application in identifying denitrifying bacteria

[0085] 1. Production of chicken manure compost

[0086] Same as step 1 in Example 1.

[0087] 2. Extraction of compost metagenomic DNA

[0088] 1. Weigh about 4g of chicken manure compost prepared in step 1 and place it in a 50ml centrifuge tube.

[0089] 2. Add 10ml of DNA extraction solution to the centrifuge tube in step 1, mix well and then perform physical lysis (repeat the following operation twice: liquid nitrogen treatment for 10 minutes, thawing in a 63°C water bath).

[0090] 3. After the liquid in the centrifuge tube in step 2 is slightly cooled, add 20 μl lysozyme (80 mg / ml), bathe in 36°C water for 25 minutes, and mix it upside down every once in a while (about 5 minutes).

[0091] 4. Add 100 μl of proteinase K (8 mg / ml) to the centrifuge tube in step 3, bathe in water at 36°C for 25 minutes, and mix by inverting up and down every once in a while (...

Embodiment 3

[0105] Example 3. Extraction of compost metagenomic DNA and its application in identifying denitrifying bacteria

[0106] 1. Production of pig manure compost

[0107] 25kg of pig manure and 3.1kg of sawdust are mixed, the C / N of the mixture is 25-30, and the water content is 55-60%, and the mixture is fermented at room temperature (about 23° C.) for 20 days.

[0108] Fresh pig manure was obtained from Shangzhuang pig farm in Beijing, and sawdust was obtained from Yongfeng Woodware Factory in Haidian District, Beijing. The properties of the materials are shown in Table 2.

[0109] Table 2 Basic properties of compost raw materials

[0110]

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for extracting metagenome DNA of compost and application thereof in identifying denitrifying bacteria. The method comprises the following steps: (1), a compost sample is subjected to physical splitting decomposition in DNA extraction solution; (2), lysozyme is added into the mixture; (3), protease K is added into the mixture; (4), SDS water solution is added into the mixture; (5), liquid supernatant is collected by centrifugation; (6), DNA extraction liquid and SDS water solution are added into sediment obtained in the step (5), then liquid supernatant is collected by centrifugation; (7), the liquid supernatant obtained in the step (5) and the liquid supernatant obtained in the step (7) are mixed; (8), phenol-chloroform-isoamyl alcohol is extracted; and (9) the liquid supernatant obtained in the step (8) and isopropyl alcohol are blended, then sediment, which is metagenome DNA of compost, is collected. The method for extracting metagenome DNA of compost and the application thereof in identifying denitrifying bacteria are of great application value for the research on the varieties of bacteria (especially the variety of denitrifying bacteria) in the compost samples produced by different fermentation times. And effective technical support is provided for the research and the application of the compost as well as the research on molecular microbial ecology.

Description

technical field [0001] The invention relates to a method for extracting compost metagenomic DNA and its application in identifying denitrifying bacteria. Background technique [0002] Compost itself contains many inhibitory factors, and a large amount of humic acid is produced during the composting process. These substances will have a strong impact on the extraction of total DNA (metagenomic DNA), and it is difficult to extract total DNA that can be used as a template for PCR amplification. [0003] Denitrifying bacteria are very important bacteria in the research of nitrification and denitrification. The traditional screening method for denitrifying bacteria is to use medium for culture and inoculation, and the single culture period takes 14 days. The entire screening process generally takes more than two weeks, which is too time-consuming and has poor accuracy. Moreover, 99% of microorganisms in the environment are not cultivable, and traditional screening methods have g...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/10C12Q1/68
Inventor 李季霍培书陈雅娟
Owner CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com