KR101760726B1 - Method for isolation of metagenomic DNA from animal food with detaching of bacteria and phenol-chloroform - Google Patents

Abstract

The present invention relates to a method for separating metagenomic DNA from animal food using microorganism elimination and phenol-chloroform, and can separate methogenomic DNA of bacteria from animal food in a high yield compared with the conventional commercial kit. Therefore, when the present invention is used, bacteria that have been infected with animal food can be accurately detected through PCR or the like.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating metagenomic DNA from an animal food using microorganism elimination and phenol-chloroform,

The present invention relates to a method for separating meta genomic DNA from animal food, and more particularly, to a method for separating meta genomic DNA from an animal food using microbial elimination and phenol-chloroform. The present invention can effectively isolate bacteria from animal foods and efficiently isolate and purify metagenomic DNA. The existing methods of desalting and DNA purification are improved to separate and purify metagenomic DNA from food without loss .

Metagenomics is a science that analyzes and studies the total genome of various bacteria in the environment. It is mainly used to identify constitutional information of bacterial communities in food and other environments. do.

Meta-genomics has the advantage of studying not only strains that can be cultured, but also strains that have been previously studied, since the total DNA is isolated and purified without culturing the strains. As a result, it has attracted a lot of attention in recent years as a next generation genome analysis method.

Due to recent advances in sequencing technology, there has been a vigorous study of bacterial community analysis using meta-genomics in many areas. In particular, in the case of food, metagenomics can be used to analyze changes in bacterial communities in food according to various conditions. This has enabled us to gain a deeper understanding of the characteristics of food and to promote technological development in the fields of production, processing, storage and transport.

However, there are various difficulties in separating and purifying bacterial DNA from food, compared to separating and purifying DNA from a pure bacterial culture. When isolating DNA using conventional commercial kits, a variety of factors present in the food act as inhibitors to lower the DNA isolation yield, or to obtain a large amount of high purity DNA by mixing with impurities and lowering the DNA purity There was a limit.

 Accordingly, a new technique for cleaving bacterial DNA efficiently from food and purifying it at high purity is required.

Korean Patent Laid-open No. 10-2002-0034087 (published on May 05, 2002) discloses a process for preparing an aqueous suspension of (a) an aqueous suspension of a sample; (b) forming an extract mixture by adding an appropriate organic solvent to the aqueous suspension under conditions suitable to remove undesirable material from the suspension while retaining some or all of the high molecular weight DNA; (c) separating the extraction mixture into an aqueous phase and an organic phase containing DNA; (d) precipitating DNA from an aqueous solution, wherein the high molecular weight DNA is separated from a plurality of species in the sample. Korean Patent Publication No. 10-2006-0061494 (published on June 26, 2006) discloses a functionalized silica magnetic nanoparticle for purification and separation of nucleic acid and a method for producing the same, wherein the surface of the silica-coated magnetic nanoparticle is coated with an amine amino group or / and a chloro group, and a method for producing the functional silica magnetic nanoparticle. Korean Patent Laid-Open No. 10-2003-0006505 (published on Jan. 23, 2003) discloses a method for producing a polysaccharide by adding Tris-Cl, EDTA, a detergent, a salt of acetic acid, And a DNA extraction method using the same.

The present invention seeks to develop and provide a novel method for isolating bacterial metagenomic DNA from animal foods without loss of DNA.

The present invention comprises the steps of (1) putting a buffered peptone water (BPW) and an animal food sample into a filter-bag and then using a spindle or stomacher machine to remove the microorganism; After step (1), a step (2) of applying sterilized gauze to a sterilized beaker and pouring the BPW containing the microorganisms desorbed in the step (2); A step (3) of performing primary centrifugation using the centrifugal separator after the step (2); A step (4) of discarding the supernatant after the primary centrifugation, releasing the pellet with TES buffer, followed by secondary centrifugation; After the secondary centrifugation, discard the supernatant and release the pellet into TE buffer (step 5); After step (5), lysozyme is mixed and reacted to produce a first reaction product (6); A step (7) of freezing and thawing the reactant in the step (6); (8) after the thawing, adding a proteinase K and then reacting to produce a second reaction product; Adding a mixed solution of phenol, chloroform and isoamyl alcohol to the second reactant, inverting and mixing the mixture until the layer is invisible (step 9); After step (9), centrifugal separation is carried out, and the upper layer of the two separated layers is separated and transferred to a new tube (10); After step (10), RNase A is added to a tube and reacted to produce a tertiary reaction product (11); After the step (11), NaOAc is added to the tertiary reactant, the mixture is inverted and added with ice-cold ethanol (12); A step (13) of performing quadratic centrifugation after the step (12); After the quaternary centrifugation, removing the supernatant and washing the pellet with ice-cold ethanol (14); A step (15) of the fifth centrifugation after the step (14); After the fifth centrifugation, discard the supernatant and allow the pellet to dry at room temperature (16); And (17) adding the TE buffer and dissolving the pellet after the step (16).

On the other hand, in the method of separating the metagenomic DNA from the animal food of the present invention, in the step (a), the spindle is preferably applied to an animal food which is likely to be broken or separated from food during the desorption process, It is preferable to apply stomacher to animal food.

On the other hand, in the method for separating meta genomic DNA from the animal food of the present invention, it is preferable that the primary centrifugation is carried out preferably at 4 DEG C and 9000 rpm for 15 minutes, and the secondary centrifugation is preferably performed at 4 DEG C, The centrifugation is preferably performed at 4 ° C and 5000 rpm for 5 minutes. The quaternary centrifugation is preferably performed at 4 ° C and 13200 rpm for 20 minutes. The fifth centrifugation is preferably carried out at 4 ° C and 13200 rpm for 5 minutes. On the other hand, in the method for separating meta genomic DNA from the animal food of the present invention, the TES buffer of step (4) preferably comprises 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and 0.1 M NaCl It is preferable to use TE buffers of step (5) and step (16) which are composed of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA.

On the other hand, in the method for separating meta genomic DNA from the animal food of the present invention, the reaction of step (6) is preferably performed by adding lysozyme and reacting at 37 DEG C for 1 hour.

On the other hand, in the method for separating meta genomic DNA from the animal food of the present invention, the step (7) is preferably such that the reaction product of step (6) is frozen at -80 ° C for 10 minutes and then thawed at 37 ° C for 10 minutes good.

On the other hand, in the method for separating metagenomic DNA from the animal food of the present invention, the proteinase K of step (8) is preferably a proteinase K mixture composed of EDTA, proteinase K and SDS, (8) is preferably carried out at 37 DEG C for 1 hour.

Meanwhile, in the method for separating metagenomic DNA from the animal food of the present invention, the solution of phenol, chloroform and isoamyl alcohol in step (9) is preferably a mixture of phenol: chloroform: isoamyl alcohol in a ratio of 25: 24: 1, and it is preferable to add the same amount as the second reaction product.

On the other hand, in the method for separating the metagenomic DNA from the animal food of the present invention, it is preferable that the steps (9) to (10) are repeatedly carried out until no impurities appear.

In step (12), preferably, 3 M NaOAc (pH 5.2) is added to the tertiary reaction product, followed by inverting and mixing. Then, 100% ice- It is advisable to add ice-cold ethanol.

On the other hand, in the method for separating meta genomic DNA from the animal food of the present invention, the ice-cold ethanol in step (14) is preferably 70% ice-cold ethanol.

On the other hand, in the method for separating metagenomic DNA from the animal food of the present invention, preferably, the step (17) is carried out by adding a TE buffer and dissolving the pellet at 55 ° C for 1 hour.

The present invention can separate the meta-genomic DNA of bacteria from animal foods at a high yield compared to the case of using a conventional commercialization kit. Therefore, when the present invention is used, bacteria that have been infected with animal food can be accurately detected through PCR or the like.

FIG. 1 shows the results of detection of Vibrio vulnificus using gyrB -specific PCR from metagenomic DNA isolated from oyster samples.

The conventional commercialized DNA purification kit has a merit that the DNA is bound to the column, and then the impurities are removed in turn and the bound DNA is recovered again, so that the DNA can be conveniently used. However, since the column plays a major role in the purification of DNA, when the metagenomic DNA is separated and purified from the food, the column is clogged by foodstuffs or impurities, or an impurity or an inhibitor derived from the food reacts with the DNA on the column There is a problem that the yield of the DNA is remarkably reduced due to damage to the DNA.

In order to solve such a problem, in the present invention, a method of separating and purifying DNA by applying phenol-chloroform after first eliminating bacteria from food has been developed.

As a result of the experiment, it was confirmed that when methanomic DNA was separated from food using phenol - chloroform, DNA could be separated and purified at a higher yield than the commercial kit.

Details of the microorganism elimination and DNA separation and purification method described in the present invention are as follows.

1. Add 225 ml of BPW (buffered peptone water, sold by BD Difco) and 25 g of food sample to the filter-bag, and then remove the microorganism using a desalting machine.

1-1. For food products that are broken or separated during the desalination process, use a spindle as a tearing machine and desorb for 2 minutes.

1-2. If it does not correspond to 1-1 above, it is desorbed for 2 minutes using a stomacher.

2. Pour sterilized gauze into sterilized beakers in 2 layers, and pour off BPW in 1.

3. Centrifuge at 4 ° C and 9000 rpm for 15 minutes using a centrifuge.

4. After discarding the supernatant, discard the pellet with 10 ml TES buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 M NaCl) and centrifuge for 10 min at 4 ° C at 6000 rpm.

5. Discard the supernatant and dissolve the pellet in 400 μl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA).

6. Add 50 μl of lysozyme (100 mg / ml) to 5 samples and incubate at 37 ° C for 1 hour.

7. Samples of sample 6 are frozen at -80 ° C for 10 minutes and then dissolved at 37 ° C for 10 minutes.

8. Add Proteinase K mixture (140 μl of 0.5 M EDTA, 20 μl of Proteinase K (20 mg / ml) and 40 μl of 10% SDS) and incubate at 37 ° C for 1 hour .

9. Add the same amount of Phenol: Chloroform: Isoamylalcohol = 25: 24: 1 as the sample and invert until the layer is invisible.

10. Centrifuge at 5000 rpm for 5 minutes at 4 ° C. Separate the upper layer of the two separate layers and place in a new tube.

11. Add 10 μl of RNase A (30 mg / ml) to the tube and incubate at 37 ° C for 1 hour.

12. Add 80 μl of 3 M NaOAc (pH 5.2), mix by inverting, and add 1 ml of 100% ice-cold ethanol.

13. Centrifuge the sample at 4 ° C and 13200 rpm for 20 minutes.

14. Remove the supernatant and wash the pellet with 1 ml of 70% ice-cold ethanol.

15. Centrifuge the sample for 5 minutes at 4 ° C and 13200 rpm.

16. Discard the supernatant and allow the pellet to dry well at room temperature.

17. Add 100 μl of TE buffer and dissolve the pellet at 55 ° C for 1 hour.

On the other hand, it is preferable to carry out the above steps 9 to 10 repeatedly until no impurities appear.

In the DNA isolation method of the present invention, nucleic acid including DNA has hydrophilicity due to a charge due to a phosphate group present in the template, while impurities such as protein have a relatively low hydrophilicity and hydrophobic property.

It is possible to separate DNA from various impurities by dissolving the bacterial extract in a mixed solvent of an inorganic solvent such as water and an organic solvent such as phenol or chloroform and then separating only the aqueous solution layer in which DNA alone exists.

In particular, by separating the bacteria from the food first using a talli machine, the contamination and damage of the bacterial meta-genomic DNA by the food tissue or the food inhibitor can be minimized.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments and experimental examples, and includes modifications of equivalent technical ideas.

[ Example  : Bacteria in oyster samples to which the present invention is applied Metagenomic  DNA isolation and purification]

In order to obtain the meta-genomic DNA of the bacteria from the oyster sample, the bacteria were desorbed using a stomacher, and the method of isolating and purifying DNA using the phenol-chloroform introduced in the present invention and the QIAamp DNA Stool Mini Kit (2001), Microbiology and Biotechnology, 22 (3): 219-222). In this study, we used two methods DNA was isolated, and the concentration and purity of DNA isolated from each method were compared. Three repetition experiments were performed for each method, and the results were as follows.

Refining method Target Food Concentration (ng / μl) Purity (A260 / A280) Using Phenol-chloroform oyster 1,813 1.912 2,175 1.916 2,048 1.919 Using commercialization kit oyster 8.8 1.76 8.3 1.287 6.4 2.228

As a result of comparing the method using the phenol-chloroform method of the present invention with the method using the existing commercialization kit, it has been found that when methanomic DNA is separated using phenol-chloroform of the present invention, there was.

Further, when Vibrio vulnificus was detected by PCR using the DNA, Vibrio vulnificus was detected more sensitively when phenol-chloroform of the present invention was used.

FIG. 1 shows the results of detection of Vibrio vulnificus using gyrB -specific PCR from metagenomic DNA isolated from oyster samples.

Claims (12)

A step (1) of putting a buffered peptone water (BPW) and an animal food sample in a filter-bag and then using a spindle or stomacher machine to remove the microorganism;
After step (1), a step (2) of applying sterilized gauze to a sterilized beaker and pouring the BPW containing the microorganisms desorbed in the step (2);
A step (3) of performing primary centrifugation using the centrifugal separator after the step (2);
A step (4) of discarding the supernatant after the primary centrifugation, releasing the pellet with TES buffer, followed by secondary centrifugation;
After the secondary centrifugation, discard the supernatant and release the pellet into TE buffer (step 5);
After step (5), lysozyme is mixed and reacted to produce a first reaction product (6);
A step (7) of freezing and thawing the reactant in the step (6);
After the thawing, a step (8) of adding a proteinase K mixture composed of EDTA, proteinase K and SDS, followed by performing the reaction at 37 ° C for 1 hour to produce a second reaction product;
A solution of phenol, chloroform and isoamyl alcohol in a weight ratio of 25: 24: 1 was added to the second reaction product in an amount equivalent to that of the second reaction product, and the solution was inverted until no layer was visible. Step (9);
After step (9), centrifugal separation is carried out, and the upper layer of the two separated layers is separated and transferred to a new tube (10);
After step (10), RNase A is added to a tube and reacted to produce a tertiary reaction product (11);
After the step (11), NaOAc is added to the tertiary reactant, and the mixture is inverted and added with ice-cold ethanol (12);
A step (13) of performing quadratic centrifugation after the step (12);
After the quaternary centrifugation, removing the supernatant and washing the pellet with ice-cold ethanol (14);
A step (15) of the fifth centrifugation after the step (14);
After the fifth centrifugation, discard the supernatant and allow the pellet to dry at room temperature (16);
And (17) adding the TE buffer and dissolving the pellet after the step (16).
The method according to claim 1,
In the step (1), the spindle is applied to an animal food in which the foodstructure is broken or is likely to be separated during the elimination process, and the stomarker is applied to the animal food that is not likely to be separated. .
The method according to claim 1,
The primary centrifugation was carried out at 4 DEG C and 9000 rpm for 15 minutes,
The secondary centrifugation was carried out at 4 DEG C at 6000 rpm for 10 minutes,
The tertiary centrifugation was carried out at 4 DEG C and 5000 rpm for 5 minutes,
The quaternary centrifugation was carried out at 4 ° C at 13200 rpm for 20 minutes,
Wherein the 5 < th > centrifugation is carried out at 4 DEG C and 13200 rpm for 5 minutes.
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