High-temperature combined method for extracting genomic DNA of fish enteric microorganisms

Abstract

The invention discloses a high-temperature combined method for extracting genomic DNA of fish enteric microorganisms. The high-temperature combined method includes the steps that firstly, the walls of various fish enteric microorganisms are properly broken through a lysozyme method and an ultrasonic method at the same time, wherein the ultrasonic physical wall breaking condition ranges from 150 w to 250 w, ultrasonic treatment is carried out for 2-3 s and then carried out again after an interval of 5 s, and the process is repeated 50-60 times; secondly, the sample obtained after ultrasonic treatment and lysozyme are jointly incubated for 30 min in a water-bath constant-temperature oscillator at the temperature of 55-65 DEG C, and the mixture and RNase A are jointly incubated at the temperature of 27-32 DEG C. According to the method, the walls of the various microorganisms are properly broken under the proper and mild ultrasonic condition, and the genomic DNA is prevented from being broken and degraded. As the specific temperature range for incubating the lysozyme and the sample and the specific temperature range for incubating the RNase A and the sample are set, the splitting decomposition effect of the lysozyme can be improved, and it can be avoided that the genomic DNA is degraded by DNA enzymes in tissues and cells. Operation is easy, the extracted genomic DNA is complete and low in degradation rate, and the fish enteric microorganism structure can be truly reflected by the types of the microorganisms identified according to the extracted DNA.

Description

technical field [0001] The invention relates to a method for extracting the genome of fish gut microbes, in particular to a high-temperature composite method for extracting the genome DNA of fish gut microbes. Background technique [0002] Methods for extracting microbial genomic DNA include kit method, cetyltrimethylammonium bromide (CTAB) method, lysozyme method, physical method (such as ultrasonic method, liquid nitrogen grinding method, bead milling method) and the like. There are many types of kits for extracting microbial genomic DNA on the market, most of which are aimed at mammals, soil environments or direct extraction of single bacterial species genomic DNA, which are poorly specific to fish intestinal microorganisms; the obtained DNA fragments are small and the yield is low, and The price is very expensive. [0003] The CTAB method has a good function of removing humic acid and other environmental impurities, but it only uses chemical methods to break the wall, a...

AI Technical Summary

Problems solved by technology

There are many types of kits for extracting microbial genomic DNA on the market, most of which are aimed at mammals, soil environments or direct extraction of single bacterial species genomic DNA, which are poorly specific for fish intestinal microorganisms; the obtained DNA fragments are small and the yield is low, and very expensive

[0003] The CTAB method has a good function of removing humic acid and other environmental impurities, but it only uses chemical methods to break the wall, and there are concerns about whether the wall is broken completely; lysozyme can effectively hydrolyze the peptidoglycan of the bacterial cell wall, and is effective for Gram-positive bacteria. Bacteria lysis is relatively complete; while only the inner wall layer of Gram-negative bacteria is peptidoglycan, therefore, lysozyme cannot completely lyse the cell wall of Gram-negative bacteria

Although the physical method of breaking the wall is efficient, it needs to explore various conditions, including power, frequency, time, etc. If the strength is low, the wall cannot be completely broken; if the strength is high, the DNA will be broken or even degraded.

At present, the methods for extracting the genomic DNA of animal intestinal microorganisms are relatively simple, and do not take into account Gram-negative bacteria, Gram-positive bacteria and other types of microorganisms.

In addition, in the existing test methods, both lysozyme and RNaseA work at a temperature of 37°C, while lyases such as DNase in tissues and cells are most active at this temperature, which will cause a large amount of DNA degradation

Due to the particularity of the living environment of fish, the composition of intestinal microorganisms is complex and easily affected by external environmental factors. Composition of fish gut microbes

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