Kit for extracting aquatic animal fecal like bacteria DNA and method thereof

A technology of aquatic animals and a kit, which is applied in the field of extracting bacterial DNA from fecal samples, can solve the problems of extracting DNA with many impurities and incomplete extraction, achieve comprehensive information, rapid extraction methods, and reduce physical health damage

Inactive Publication Date: 2008-02-13
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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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 provide a kit capable of rapidly and efficiently extracting bacterial genome DNA from aquatic animal excrement samples and the extraction method of bacterial genome DNA, so as to solve the problem of traditional method extraction when using molecular means to study the intestinal microbial community of aquatic animals. DNA impurities, incomplete extraction, PCR inhibitors and other defects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Make the bacterial DNA extraction kit of aquatic animal feces samples according to the following formula:

[0035] Reagent A: Contains 70% (V / V) ethanol and 0.5% (V / V) Triton X-100.

[0036] Reagent B: Contains 50 mM Tris-Cl (pH 8.0), 50 mM EDTA, 50 mM NaCl, 2% (W / V) of PVP, 1% (W / V) of CTAB and 3% (W / V) of SDS.

[0037] Reagent C: RNAase concentration is 20mg / ml.

[0038] Reagent D: Contains 6.5M GuSCN (guanidine isothiocyanate) and 100mM Tris, adjust pH to 6.

[0039] Reagent E: prepared by mixing 10mM Tris-Cl (pH7.5) and absolute ethanol at a ratio of 1:4.

[0040] Reagent F: 10 mM Tris-Cl (pH 8.0).

[0041] DNA adsorption column: silica gel column

[0042] Follow the procedure below to extract bacterial DNA from aquatic animal fecal samples:

[0043] 1. Sample pretreatment

[0044] Take 200 mg of fish feces sample, add 400 μl of reagent A, place on ice for 30 min, centrifuge at 12000 g for 5 min.

[0045]2. Bacterial Lysis

[0046] Discard the supernatant, a...

Embodiment 2

[0054] Prepare various reagents according to the following formula:

[0055] Reagent A: Contains 75% (V / V) ethanol and 0.1% (V / V) Triton X-100.

[0056] Reagent B: Contains 200 mM Tris-Cl (pH 8.0), 100 mM EDTA, 100 mM NaCl, 1% (W / V) of PVP, 0.5% (W / V) of CTAB and 2% (W / V) of SDS.

[0057] Reagent C: RNAase concentration is 10 mg / ml.

[0058] Reagent D: Contains 6.5M GuSCN (guanidine isothiocyanate) and 50mM Tris, adjust pH to 6.

[0059] Reagent E: prepared by mixing 10 mM Tris-Cl (pH 7.5) and absolute ethanol 1:4.

[0060] Reagent F: ddH 2 O.

[0061] DNA adsorption column: silica gel column

[0062] Follow the procedure below to extract bacterial DNA from aquatic animal fecal samples:

[0063] 1. Sample pretreatment

[0064] Take 100mg of prawn feces, add 200μl reagent A, place on ice for 20min, centrifuge at 12000g for 5min.

[0065] 2. Bacterial Lysis

[0066] Discard the supernatant, add 200 μl of reagent B and 4 μl of reagent C to the pellet, stir well with a pi...

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Abstract

This invention provides an aquatic dung bacteria DNA distilling kit and its method. In this invention, the dung is disposed by ethanol and TritonX-100 to increase sensitivity of the bacteria to cracking liquid. Then cell is cracked by cracking liquid with SDS, the liquid also has PVP that with absorption to PCR restrictor. Then a high concentration GuSCN, low pH environment is provided for further denaturation of protein in the solution, and the water solubility is reinforced, and the DNA can be binding on silica gel column. Finally, DNA is eluted from the column in low concentration buffer solution or water. The goal of the maximum diversities information, high quality and higher output DNA of aquatic dung bacteria community can be reached by using this kit and its distilling method. The DNA got can be used to molecular ecology, system evolution and other researches.

Description

technical field [0001] The invention relates to a kit for extracting bacterial DNA from feces samples of aquatic animals and a method for extracting bacterial DNA from feces samples using the kit. Background technique [0002] The intestinal bacterial community of aquatic animals is closely related to its growth, immunity, and disease occurrence, and has gradually received attention in recent years. Previous studies of bacteria in the environment, including the guts of animals, have been based on traditional culture methods. This method first needs to isolate and cultivate a large number of strains from the sample, and then perform biochemical identification separately, so it is time-consuming and laborious. And because in the natural environment, it is reported that the bacteria that can be cultured account for less than 1% of the existing bacterial species, and the analysis of the intestinal bacterial community based on culture often cannot reflect its true composition. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C07H21/04C07H1/08
Inventor 罗鹏胡超群张吕平
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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