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Pretreatment method for extracting metagenome DNA of intestinal contents

A technology of metagenomic and content, which is applied in the field of pretreatment for the extraction of metagenomic DNA from intestinal content, and achieves the effect of strong practicability

Active Publication Date: 2017-12-15
广东美格基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to: overcome the problems that the existing method is difficult to extract metagenomic DNA in the intestinal substance of corrosive fish, and provide a pretreatment method for extracting the metagenomic DNA of intestinal contents. After conventional DNA extraction steps, the complete metagenomic DNA of intestinal contents can be extracted efficiently

Method used

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  • Pretreatment method for extracting metagenome DNA of intestinal contents
  • Pretreatment method for extracting metagenome DNA of intestinal contents
  • Pretreatment method for extracting metagenome DNA of intestinal contents

Examples

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Effect test

Embodiment 1

[0030] The method of the present invention extracts the metagenomic DNA of the intestinal contents of bighead carp:

[0031] (1) Take 0.25g of fresh bighead carp intestinal content sample into a 15ml centrifuge tube, add a decongestant, fully vortex, and centrifuge at 11,000g for 10min (can be washed twice).

[0032] (2) Discard the supernatant, add a mixture of detergent + 0.5mM EDTA, and vortex well.

[0033] (3) Freeze at -80°C for 15 minutes, and heat at 65°C for 5 minutes (repeated 3 times).

[0034] (4) Add lysozyme, 37°C, 25min (then 95°C, 5-10min).

[0035] (5) Add proteinase K, 10 molar mass % SDS at 65° C., 1 h (upside down every 15 min).

[0036] (6) RNase was added at 37° C. for 15 min (to a final concentration of 20 mg / ml).

[0037] (7) Centrifuge at 11000rpm for 10min at 4°C.

[0038] (8) Pipette the supernatant into a new 50ml centrifuge tube, add equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and extract twice (the protein layer can be seen). ...

Embodiment 2

[0043] The method of the present invention extracts the metagenomic DNA of the intestinal contents of grass carp:

[0044] (1) Take 0.25g of fresh grass carp intestinal content sample into a 15ml centrifuge tube, add a decontamination agent, fully vortex, and centrifuge at 11,000g for 10min (can be washed twice).

[0045] (2) Discard the supernatant, add a mixture of detergent + 1 mM EDTA, and vortex well.

[0046] (3) Freeze at -80°C for 15 minutes, and heat at 65°C for 5 minutes (repeated 3 times).

[0047] (4) Add lysozyme, 37°C, 25min (then 95°C, 5-10min).

[0048](5) Add proteinase K, 10 molar mass % SDS at 65° C., 1 h (upside down every 15 min).

[0049] (6) RNase was added at 37° C. for 15 min (to a final concentration of 20 mg / ml).

[0050] (7) Centrifuge at 11000rpm for 10min at 4°C.

[0051] (8) Pipette the supernatant into a new 50ml centrifuge tube, add equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and extract twice (the protein layer can be seen)...

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Abstract

The invention discloses a pretreatment method for extracting metagenome DNA of intestinal contents. The pretreatment method comprises the following steps of: adding a necrotic-tissue removing agent into the intestinal contents to wash for twice, then adding nuclease eliminating solution for washing, cracking cells by adopting a freezing-thawing method and a cracking method, fully releasing DNA in the cells, finally adding protease K, SDS and RNA enzyme to remove protein, fiber and RNA in the cells, and treating products by adopting a conventional method to obtain the metagenome DNA of the intestinal contents. The pretreatment method disclosed by the invention has the advantages that the effect for extracting the DNA of the metagenome DNA of the intestinal contents of omnivorous animals is obvious, and both the extracting purity and the extracting amount can meet downstream experiments such as PCR augmentation and high-throughput sequencing and the like, so that the practicability is very strong.

Description

technical field [0001] The invention belongs to the technical field of gene extraction, and more particularly relates to a pretreatment method for the extraction of metagenomic DNA from intestinal contents. Background technique [0002] Fish gut microbes play a very important role in fish development and health. In 1998, the American Academy of Sciences first proposed the concept of metagenomics. By analyzing the metagenome of fish gut contents, the usual dietary habits of fish can be known. The composition of probiotics / harmful bacteria and other foreign invading bacteria in the gut, so as to better understand the balance of the intestinal environment of fish and the composition of bacteria. However, due to the extremely complex environment of fish in water and many food sources for fish, serious degradation or failure to extract occurs during the extraction process. Extraction is very difficult. [0003] At present, the methods of extracting fish gut metagenomes include ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2521/537C12Q2521/301
Inventor 张杰豪束文圣周集中叶脉徐卓菲刘玉莲
Owner 广东美格基因科技有限公司
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