Method for extracting microbial metagenome DNA from intestinal tract content

A technology of metagenomic and content, applied in the field of extraction of microbial metagenomic DNA, can solve problems such as cracking and inability to obtain species abundance, and achieve the effects of reducing degradation, reducing species preference, and reducing pollution

Active Publication Date: 2018-02-02
AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The methods often used in kits often have a preference for cleavage, and the real species abundance cannot be obtained.

Method used

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  • Method for extracting microbial metagenome DNA from intestinal tract content
  • Method for extracting microbial metagenome DNA from intestinal tract content
  • Method for extracting microbial metagenome DNA from intestinal tract content

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0045] Experimental example 1. Extracting microbial metagenomic DNA from chicken intestinal contents

[0046] 1. Collection of intestinal contents of chicken intestine duodenum, jejunum, ileum, cecum, colorectal

[0047] Various fresh chicken intestinal contents were obtained by dissection, and 2 times volume of the chicken intestinal contents were added with 4°C pre-cooled normal saline containing 0.1% Tween80, and the samples were shaken and mixed thoroughly to obtain contents samples.

[0048] Take about 5-10g samples of duodenal contents, about 10-20g samples of jejunum contents, and about 10-20g samples of ileum contents into centrifuge tubes for subsequent steps; take about 0.5-1.0g samples of cecal contents, about 0.5 -1.0g sample of colorectal contents to centrifuge tube for subsequent steps.

[0049] 2. Extract microbial metagenomic DNA from chicken intestinal contents

[0050] 1. Differential centrifugation to wash and enrich intestinal microbial cells

[0051] 1) Centrifuge a...

Embodiment 2

[0094] Example 2: Different lysis and fragmentation methods and analysis of microorganisms belonging to metagenomic DNA obtained

[0095] In this embodiment, a fresh chicken feces sample is used as a sample.

[0096] 1. Extract metagenomic DNA

[0097] 1. Differential centrifugation to wash and enrich microbial cells

[0098] Take 2 g of fresh chicken feces sample, wash and enrich the microbial cells according to the differential centrifugation in 1 of Experimental Example 12 to obtain microbial cells.

[0099] 2. The lysis of microbial cells

[0100] Divide the microbial cells obtained in the above 1 into 4 groups and lyse and break them according to the following 4 methods, specifically, they are divided into 4 2mL centrifuge tubes:

[0101] Method 1: Use 800uL cell lysate (QIAGEN company ASL buffer), lyse at 70℃ for 15min, centrifuge at 19000g for 5min, and collect the supernatant;

[0102] Method 2: Use 800uL cell lysate (QIAGEN ASL buffer), lyse at 95℃ for 15min, centrifuge at 19000g ...

experiment example 3

[0113] Experimental example 3: Exploring the importance of differential centrifugal washing steps

[0114] 1. Extract metagenomic DNA

[0115] 1. Collect the washing solution of the ileal contents that have not been washed by differential centrifugation

[0116] Take 0.5g sample of chicken intestinal ileum content, add 1mL of 4℃ pre-cooled normal saline (containing 0.1% Tween80), shake well to mix the sample, centrifuge at 4℃ and 19000g for 15min at high speed, sediment the chyme and Microbial cells are collected from the washing liquid of the ileal contents.

[0117] 2. The metagenomic DNA sample from the cecal contents obtained in Experimental Example 1 was diluted to a concentration of 50ng / uL to become the DNA sample d1.

[0118] The DNA molecular weight standard (Tiangen Biochemical Technology Co., Ltd., D15000) with a concentration of 50ng / uL is the DNA sample e1. Take 5uL DNA samples d1 and e1, mix them with 5uL physiological saline (containing 0.1% Tween80), and react at room ...

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Abstract

The invention discloses a method for extracting a microbial metagenome DNA from intestinal tract content. Themethod for extracting the microbial metagenome DNA from the intestinal tract content comprises the following steps that 1, the intestinal tract content or excrement of an animal is subjected to differential centrifugation; 2, microbial cells are combined and are broken by using a liquid nitrogen repeated freezing and thawing cracking method, a glass bead grinding method and a lysate high-temperature cracking method to obtain broken cells; 3, genomes DNA of the broken cells are firstly extracted and then are purified. By adding the steps of washing and enriching microbial cells, the pollution of host and food DNA is reduced. By combining the multiple microbial cell cracking and breaking methods, and the speciespreference of the metagenome DNA is reduced. By adding a purifying step, the purity of the DNA is ensured, and the DNA requirements for establishing second generation of high-throughput sequencing libraries can be met.

Description

Technical field [0001] The invention relates to the field of metagenomics, in particular to a method for extracting microbial metagenomic DNA from intestinal contents. The obtained metagenomic DNA meets the requirements for constructing a second-generation high-throughput sequencing library. Background technique [0002] Metagenomics is the sum of the genetic material of all microorganisms in the biological environment, that is, the genome of the microbial population. At present, it mainly refers to the sum of the genomes of bacteria, archaea and fungi. Metagenomics takes the microbial population genome as the research object to study the genetic material composition of microorganisms and the function of microbial community. In recent years, with the development of high-throughput sequencing technology and the reduction of sequencing costs, metagenomic sequencing technology has gradually penetrated into many fields, and people have gained a new understanding of microbial flora, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006C12Q2563/149
Inventor 张艳江帆樊伟刘博李书曲王恒超刘聪辉任钰为尹丽娟
Owner AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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