CN115011588A

CN115011588A - Bacterial genome DNA extraction kit and use method thereof

Info

Publication number: CN115011588A
Application number: CN202210610398.5A
Authority: CN
Inventors: 肖晓文; 王文朋; 李妍; 东昱汝
Original assignee: Tsingke Biotechnology Co Ltd
Current assignee: Tsingke Biotechnology Co Ltd
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-09-06
Anticipated expiration: 2042-05-31
Also published as: CN115011588B

Abstract

The invention belongs to the technical field of biology, and discloses a bacterial genome DNA extraction kit and a bacterial genome DNA extraction method. The kit provided by the invention adopts a unique buffer solution system to match with a centrifugal column for specifically adsorbing DNA, can rapidly and efficiently extract the genomic DNA of various gram-negative/positive bacteria within 1h, and can remove impurity proteins and other metabolic substances in cells to the maximum extent.

Description

Bacterial genome DNA extraction kit and use method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a kit for extracting bacterial genome DNA and a using method thereof.

Background

With the development of biochemistry and molecular biology, such as molecular cloning technology, PCR technology, genetic engineering, and whole-gene DNA sequencing technology, the research on biological DNA is becoming more and more extensive and intensive. The extraction of genomic DNA is often the first step of research on biological DNA and is also the basis of the research, and the quality of DNA extraction directly determines the success or failure of downstream experiments.

Bacteria belong to prokaryotes, and have a relatively simple cell structure and a tough cell wall composed of peptidoglycan. The first step of extracting bacterial DNA needs to destroy the cell wall to release the contents such as nucleic acid in the cell, and the current methods for extracting bacterial genome DNA mainly comprise: cell disruption and DNA extraction are carried out by mechanical disruption, enzymatic method, chemical method, and the like. Wherein the mechanical method mainly comprises the steps of crushing bacterial cell walls by using mechanical acting force such as ultrasonic crushing, repeated freeze thawing, micro-bead oscillation and the like; the enzyme method mainly dissolves and breaks the bacterial cell wall through biological enzyme such as lysozyme and the like; the chemical method extracts protein impurities by combining chemical reagents such as strong base NaOH, surfactant SDS (sodium dodecyl sulfate), CTAB and the like to crack bacteria, denature proteins and the like with organic solvents, thereby obtaining DNA with higher purity.

The research on the genome of bacteria is becoming more extensive and important, so the high-efficiency and high-quality extraction of the genome is also important. The existing various bacterial genome extraction methods have different defects, such as long time consumption, use of toxic reagents, low product purity and the like. Therefore, a kit for extracting bacterial genomes needs to be developed, and the corresponding problem of extracting bacterial genomes is effectively solved.

Disclosure of Invention

The invention aims to provide a kit for extracting bacterial genome DNA, which effectively solves the corresponding problem of bacterial genome extraction.

Accordingly, in a first aspect, the present invention provides a bacterial genomic DNA extraction kit comprising a suspension, a binding solution, a deproteinizing solution, a rinsing solution and an eluting solution,

said suspension comprising 1-2%, preferably 1.5%, of sarcosyl in a first Tris-EDTA buffer;

the binding solution comprises 2-5M, preferably 2.5M GuHCl and 20-60mM, preferably 40mM urea in sodium citrate buffer;

the deproteinizing solution comprises 2-3M, preferably 2.5M GuHCl in a first Tris-HCl buffer solution, and ethanol with a final concentration of 30-60%, preferably 55%, by volume;

the rinsing liquid comprises a second Tris-HCl buffer solution, and ethanol with the final concentration of 75-80%, preferably 80% by volume is added into the second Tris-HCl buffer solution;

the eluent comprises a second Tris-EDTA buffer.

In one embodiment, the kit further comprises a DNA adsorption column and proteinase K.

In one embodiment, the DNA adsorption column is a silicon substrate membrane adsorption column.

In one embodiment, the first Tris-EDTA buffer has a pH of 7.5 to 8.5, preferably 8, and comprises 20 to 100mM, preferably 25 to 50mM Tris-HCl and 10 to 20mM EDTA.

In one embodiment, the pH of the sodium citrate buffer is between 5.0 and 6.0, preferably between 5.0 and 5.5, more preferably between 5.0 and 5.05, at a concentration of 20 to 60mM, preferably 50 mM.

In one embodiment, the first Tris-HCl buffer has a pH of 6.8-7.5, preferably 7.0, at a concentration of 20-50mM, preferably 30 mM.

In one embodiment, the second Tris-HCl buffer has a pH of 6.8-7.5, preferably 7.0, and a concentration of 10-20mM, preferably 15 mM.

In one embodiment, the second Tris-EDTA buffer has a pH of 7.5 to 8.5, preferably 8, and comprises 5 to 20mM, preferably 10mM Tris-HCl and 0.1 to 10mM, preferably 1mM, EDTA.

In a second aspect, the present invention provides a method for extracting bacterial genomic DNA, comprising:

1) breaking bacterial cells, degrading proteins and releasing bacterial genome DNA of a bacterial sample;

2) adding the binding solution of the invention for incubation;

3) precipitating DNA with ethanol;

4) transferring the liquid for precipitating the DNA into an adsorption column for centrifugation, and removing a liquid phase;

5) adding the deproteinized liquid and anhydrous ethanol mixture into the adsorption column, centrifuging, and removing a liquid phase;

6) adding a mixture of the rinsing liquid and the absolute ethyl alcohol into the adsorption column, centrifuging, removing a liquid phase, preferably repeating for 1-2 times, and then airing;

7) eluting DNA from the adsorption column.

In one embodiment, in 1), the cell walls of gram-negative bacteria are disrupted with the suspension of the invention and the cell walls of gram-positive bacteria are disrupted with lysozyme.

In one embodiment, in 1), the protein is degraded with proteinase K.

In one embodiment, in 2), incubating at 56 ℃ for 10-15 min.

In one embodiment, in 4) -6), centrifugation is performed at 12,000rpm (about 13,400 Xg) for 1 min.

In one embodiment, in 7), the elution is carried out with TE Buffer or sterile water, preferably at a temperature of 65 ℃.

The components in the kit are cooperatively matched, so that the excellent DNA extraction effect is realized. In the development process, the inventor finds that not all the same reagents can achieve good effects, for example, in suspension, if SLS is replaced by SDS, Tween20, triton X-100 or other reagents such as guanidine hydrochloride, the problem of high RNA residue or poor amplification inhibition can be caused, or the effect of SDS and salts such as sodium chloride can be reduced. For example, if GuSCN, sodium acetate, NP-40, PEG-8000 or Tris-HCl, TritonX-100, AEO-15 are added into the binding solution, the solution may have the problems of flocculent precipitate, poor extraction effect or unstable effect. The deproteinizing solution mainly has the functions of creating conditions of high salt and low pH value, keeping nucleic acid adsorbed on the silicon substrate membrane and simultaneously promoting protein to be dissolved in the deproteinizing solution and eluted from the silicon substrate membrane; however, improper selection of deproteinizing solution causes problems such as salt residue and PCR inhibition; in the system of the invention, the selection of the salt of the deproteinizing solution comprehensively considers the components of the suspension and the binding solution, and the ideal impurity removing effect can be achieved only by mutual matching, so that the purity of the product is improved; for example, the results are worse with sodium chloride or GuSCN or with guanidine hydrochloride, or with the addition of auxiliary ingredients such as urea, TritonX-100. In addition, the components may also affect each other, for example, the respective components of the suspension and the binding solution need to be matched to achieve a balanced effect to effectively lyse the bacterial cells, and if SDS is used in the suspension, the solution may be turbid, and the column may be blocked after the binding solution is added. In addition, the amount and pH of each reagent will also influence the result. For example, when the concentration of guanidine hydrochloride is too high, the amount of residual RNA is significantly increased; guanidine hydrochloride affects the pH measurement and requires the pH of the binding solution to be adjusted by the pH of the sodium citrate buffer.

The kit and the method for extracting the bacterial genome DNA by using the DNA adsorption column can be used for quickly extracting the genomic DNA of various gram-negative bacteria and gram-positive bacteria, and ensure that the obtained genomic DNA has higher purity and is compatible with downstream application.

Drawings

FIG. 1 shows gel electrophoresis of bacterial genomic DNA extracted from different formulations of suspensions and binding solutions;

FIG. 2 shows a gel electrophoresis of bacterial genomic DNA extracted from suspension A with different binding solutions; wherein, the picture (A) is a gel electrophoresis picture of the extracted escherichia coli genome DNA, and the picture (B) is a gel electrophoresis picture of the extracted agrobacterium genome DNA;

FIG. 3 shows gel electrophoresis of bacterial genomic DNA extracted using deproteinised solutions of different formulations; wherein, the picture (A) is a gel electrophoresis picture of the extracted Escherichia coli genome DNA, and the picture (B) is a gel electrophoresis picture after PCR amplification by using different primers;

FIG. 4 shows gel electrophoresis of bacterial genomic DNA extracted using deproteinizing solutions of different formulations; wherein, the picture (A) is a gel electrophoresis picture of the extracted Escherichia coli genome DNA, and the picture (B) is a gel electrophoresis picture after PCR amplification by using different primers;

FIG. 5 shows a gel electrophoresis image of bacterial genomic DNA extracted after adjusting the buffer in the deproteinizing solution;

FIG. 6 shows gel electrophoresis images of genomic DNAs extracted from different bacteria in the kit and the commercially available kit.

Detailed Description

The present invention is described in detail below. It is to be understood that the following description is intended to illustrate the present invention by way of example only and is not intended to limit the scope of the invention, which is defined by the appended claims. Also, those skilled in the art will appreciate that modifications can be made to the disclosed embodiments without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

Where a range of values is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the subject matter described. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.

The kit provided by the invention adopts a unique buffer solution system to match with a centrifugal column for specifically adsorbing DNA, can rapidly and efficiently extract the genomic DNA of various gram-negative/positive bacteria within 1h, and can remove impurity proteins and other metabolic substances in cells to the maximum extent. The kit does not need to use phenol/chloroform for extraction, does not need a time-consuming isopropanol or ethanol precipitation process, and is safe to operate. The genome DNA obtained by using the kit has good integrity and high purity, and can be directly used for experiments such as PCR, enzyme digestion, hybridization and the like.

Without wishing to be bound by any theory, the inventors believe that the kit of the present invention achieves better bacterial DNA extraction, i.e. better bacterial genomic DNA integrity and higher purity, than the prior art conventional methods by accurate reagent component loading sequence and precise pH and concentration.

Examples are given.

Example 1 reagent components and concentrations of kit.

Formula 1:

suspension: Tris-HCl (pH8), 50 mM; EDTA (pH8), 10 mM; sarcosyl (SLS), 1.5%.

Binding liquid: sodium citrate (pH5.0-5.05), 50 mM; GuHCl, 2.5M; urea, 40 mM.

Protein removing liquid: Tris-HCl (pH7), 30 mM; GuHCl, 2.5M; 55 percent of ethanol.

Rinsing liquid: Tris-HCl (pH7), 15 mM; 80 percent of ethanol.

Eluent (TE pH 8): Tris-HCl (pH8), 10 mM; EDTA (pH8), 1 mM.

And (2) formula:

suspension: Tris-HCl (pH8), 25 mM; EDTA (pH8), 10 mM; sarcosyl (SLS), 1.5%.

Binding liquid: sodium citrate (ph5.0), 50 mM; GuHCl, 2.5M; urea, 40 mM.

Rinsing liquid: Tris-HCl (pH7), 15 mM; 80 percent of ethanol.

Eluent (TE pH 8): Tris-HCl (pH8), 10 mM; EDTA (pH8), 1 mM.

And (3) formula:

suspension: Tris-HCl (pH8), 25 mM; EDTA (pH8), 10 mM; sarcosyl (SLS), 1.5%.

Binding liquid: sodium citrate (ph5.2), 50 mM; GuHCl, 2.0M; urea, 20 mM.

Protein removing liquid: Tris-HCl (pH7.5), 50 mM; GuHCl, 3M; 60 percent of ethanol.

Rinsing liquid: Tris-HCl (pH7.5), 10 mM; 80 percent of ethanol.

Eluent (TE pH 8): Tris-HCl (pH8), 10 mM; EDTA (pH8), 1 mM.

And (4) formula:

suspension: Tris-HCl (pH8), 50 mM; EDTA (pH8), 10 mM; sarcosyl (SLS), 1.5%.

Binding liquid: sodium citrate (ph5.03), 50 mM; GuHCl, 2.5M; urea, 40 mM.

Protein removing liquid: Tris-HCl (pH6.8), 20 mM; GuHCl, 2.5M; 45 percent of ethanol.

Rinsing liquid: Tris-HCl (pH6.8), 20 mM; 75 percent of ethanol.

Eluent (TE pH 8): Tris-HCl (pH8), 10 mM; EDTA (pH8), 1 mM.

And (5) formula:

suspension: Tris-HCl (pH8), 50 mM; EDTA (pH8), 20 mM; sarcosyl (SLS), 1.0%.

Binding liquid: sodium citrate (ph5.5), 40 mM; GuHCl, 2.5M; urea, 30 mM.

Rinsing liquid: Tris-HCl (pH7), 15 mM; 80 percent of ethanol.

Eluent (TE pH 8): Tris-HCl (pH8), 10 mM; EDTA (pH8), 1 mM.

Example 2, bacterial genomic DNA extraction method steps.

1. And (4) sample preparation.

1.1 gram-negative bacteria.

1) Taking 1-5 mL (not more than 1 × 10) of bacterial culture ⁹ Individual cells) were placed in a centrifuge tube and centrifuged at 12,000rpm (-13,400 Xg) for 1min, with the supernatant being aspirated as clean as possible.

2) 200. mu.L of the suspension of the present invention was added thereto, and the mixture was shaken until the cells were completely suspended.

3) Optionally adding 5 mu L of RNase A, oscillating for 15s, and standing at room temperature for 5-15 min.

4) Add 20. mu.L proteinase K solution to the tube and mix well.

1.2 gram-positive bacteria.

2) Adding 180. mu.L lysozyme, shaking until the thallus is completely suspended, and incubating at 37 ℃ for 30min or more. The concentration of the bacterial enzyme is 20mg/mL, and the preparation method of the lysozyme buffer solution comprises the following steps: 20mM Tris, pH8.0; 2mM Na ₂ -EDTA；1.2％Triton X-100。

4) Add 20. mu.L proteinase K solution to the tube and mix well.

2. Adding 220 mu L of the binding solution, oscillating, mixing uniformly, and incubating for 10-15 min at 56 ℃.

3. Add 220. mu.L of absolute ethanol and mix by shaking, at which time a flocculent precipitate may appear.

4. Transferring the mixed solution obtained in the previous step into an adsorption column filled in a collecting pipe, centrifuging for 1min at 12,000rpm (13,400 Xg), pouring waste liquid, and returning the adsorption column into the collecting pipe. The adsorption column can be a silicon substrate film adsorption column, and has the advantages that: the operation is simple, the nucleic acid separation and purification process is changed into simple filtration operation, the operation requirement is low, and the repeatability is good; the purification effect is good, and the yield is high; high safety and no need of contacting large amount of toxic reagent.

5. Anhydrous ethanol is added into 500 mu L deproteinized liquid, the volume final concentration of the ethanol is 55 percent, centrifugation is carried out for 1min at 12,000rpm (13,400 Xg), and waste liquid is discarded.

6. Anhydrous ethanol was added to 600. mu.L of the rinse solution at a final concentration of 55% by volume, centrifuged at 12,000rpm (. about.13,400 Xg) for 1min, and the waste solution was discarded, preferably repeated once.

7. The adsorption column was returned to the collection tube, centrifuged at 12,000rpm (13,400 Xg) for 2min, the waste liquid was decanted, and the column was left open at room temperature for several minutes to allow for full air drying.

8. Taking out the adsorption column, putting the adsorption column into a clean 1.5mL centrifuge tube, suspending and dripping 50-100 mu L TE Buffer or sterilized water into the center of the adsorption membrane for elution (preferably 65 ℃), standing the adsorption membrane at room temperature for 2-5 min, and centrifuging the adsorption membrane at 12,000rpm (13,400 Xg) for 2 min. The solution obtained by centrifugation is preferably added to the adsorption column again, left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) for 2 min.

Example 3 comparison of different reagent components and concentrations of the kit.

The reagent components and the concentration of the kit are matched with each other, and the steps of the method for extracting the bacterial genome DNA can quickly and efficiently extract the genome DNA of various gram-negative/positive bacteria within 1 hour, and remove impurity proteins and other metabolic substances in cells to the maximum extent.

1. A suspension and a binding solution.

1) Coli and agrobacterium genomic DNA extraction was tested with suspensions and binding solutions adjusted to the following ingredients. When searching the components of the suspension and the binding solution, the deproteinizing solution is Buffer PW in a promoter plant gene DNA extraction kit (TSP101-50), the rinsing solution is corresponding Wash Buffer, and the eluent is corresponding TE Buffer. The ingredients and concentrations are shown in tables 1 and 2.

TABLE 1

TABLE 2

Table 3 and fig. 1 show the effect of the suspension in combination with the binding liquid. 260 represents the absorbance of the nucleic acid at a wavelength of 260nm of the highest absorption peak; 280 most reflects protein concentration (protein has multiple absorption peaks, 280 uses more); 230 are most reactive to the level of impurities such as carbohydrates, salts (e.g., guanidinium salts), etc. 260/230 and 260/230 may reflect the purity of the nucleic acid to some extent.

For SLS and SDS, the inventors found in experiments that suspensions using SDS did not clear after lysis, there was a potential for column blockage, while treatments with SLS appeared to be more stable, and in other experiments also demonstrated that SLS alone was superior to a mixture of SDS and SLS. From 260/230 in Table 3, it can be seen that the value of the self-prepared B suspension is 1.27 to 1.876, while empirically, a value of about 2.0 indicates a good purity, and a value that is too low may result in an excessively high impurity content. As can also be seen from fig. 1, the band brightness of suspension a is overall better than that of suspension B. The method proves that the overall yield of the product extracted by using the sodium dodecyl sarcosinate is higher and the effect is stable.

For the binding solution, 260/280, except that the value of the suspension B + the Tiangen GB is lower, the other values are in a reasonable range; 260/230, the whole suspension B group is lower, the suspension A group is lower, the binding solution 2 is lower, and the electrophoresis chart of figure 1 shows that the binding solutions 1 and 3 have better effects, and probably because sodium acetate solution is selected as a buffer solution component in the binding solution 2, the solution after cracking cannot become clear, and a large amount of flocculent precipitates are generated to block the columns after ethanol is added; the binding solution 4 is prepared according to the detection result, the binding solution 5 is added with 1% of AEO-15 on the basis of the binding solution 1, and according to the experimental result, the extraction effect cannot be improved by adding the AEO-15.

In addition, it can be seen that, when the suspensions a and B are used with several different binding solutions, the variation trends are not consistent, which indicates that the suspensions and the binding solutions are mutually cooperated and matched.

TABLE 3

2) On the basis, different buffers or no buffer are added to prepare a binding solution to compare the effect of extracting the Escherichia coli genome DNA. Consistent with the previous experimental results, the addition of AEO-15 increases the residual amount of RNA in the sample, resulting in a preference for measurement and a higher purity measurement. The effect is better without adding buffer solution or by using Tris-HCl and sodium citrate. The inventors further compared the use effect of the sodium citrate buffer and the Tris-HCl buffer, and found that the effect of using the sodium citrate buffer is better. The use effect of the sodium citrate buffer solution and the Tris-HCl buffer solution is further compared by using escherichia coli and agrobacterium, and the effect of the sodium citrate buffer solution is better. The ingredients and concentrations are shown in table 4. The effect of extraction of E.coli/Agrobacterium genomic DNA is shown in Table 5 and FIG. 2 (wherein (A) is E.coli and (B) is Agrobacterium).

TABLE 4

TABLE 5

In addition to the above experimental results for discussing the combination of different components, the inventors also performed experiments on the conditions of the amount of each component, the pH value, etc., and determined that the optimal mixture ratio of the suspension and the binding solution is: suspension: Tris-HCl (pH8), 50 mM; EDTA (pH8), 10 mM; sarcosyl (SLS), 1.5%. Binding liquid: sodium citrate (pH5.0-5.05), 50 mM; GuHCl, 2.5M; urea, 40 mM. Wherein the dosage of the suspension is 200 mu L, the dosage of the binding solution is 220 mu L, namely the volume dosage ratio of the suspension to the binding solution is 1: 1.1.

2. The components and content of deproteinized liquid.

The reagent components of the cracking system in the early stage have certain influence on the subsequent purification process of the adsorption column, so that the type and the content of salt in the binding solution are matched with the type and the content of salt used in the protein removing solution in the later stage, the ideal impurity removing effect can be achieved, and the purity of the product is improved. Coli genomic DNA extraction was tested with protein solutions adjusted to the following composition. Further comparative tests were performed below for the more effective treatments in the above several schemes.

1) One set of deproteinized fluid and results on E.coli. The components and concentrations are shown in Table 6, and the results are shown in Table 7 and FIG. 3 (wherein the graph (A) is a gel electrophoresis of the extracted E.coli genomic DNA, and the graph (B) is a gel electrophoresis of the E.coli genomic DNA amplified with different primers using gold MIX). Obviously, the addition of TritonX-100 did not provide a better result.

TABLE 6

TABLE 7

2) The combined effect of adding NaCl or GuHCl + NaCl is further discussed. The components and concentrations are shown in Table 8, and the results are shown in Table 9 and FIG. 4 (in which the graph (A) is a gel electrophoresis of the extracted E.coli genomic DNA and the graph (B) is a gel electrophoresis of the DNA amplified with different primers using gold MIX). It can be seen that the purity is obviously affected after the sodium chloride is added, and in addition, the protein removing liquid prepared by mixing NaCl and GuHCl has an extraction effect similar to that of GuHCl only, but partial impurity residues may affect PCR.

TABLE 8

TABLE 9

On the basis of this, the addition of urea to the deproteinizing solution was attempted, and this treatment resulted in an increase in product yield, but significantly affected the 260/230 ratio and possibly the PCR. An experiment that GuSCN replaces GuHCl or is mixed with GuHCl is also tried, and GuSCN is found to have residue and influence the subsequent PCR; therefore GuHCl is the chosen deproteinizing solution of the present invention.

3. Buffer concentration and pH in deproteinizing solution.

The effect of extraction of genomic DNA from Agrobacterium is shown in Table 10 and FIG. 5.

Watch 10

Comparing the influence of the concentration and the pH of the Tris-HCl buffer solution in the deproteinized solution on the extraction effect, wherein the effect is better when the concentration is 30 mM; the difference of pH 6.8/7.0 was not significant, and for the sake of later production simplification, the same pH as Tris-HCl buffer in the rinse was used, 7.0.

The main components in the deproteinizing solution are determined to be the following three types: salts, buffers, alcohols. 2.5M GuHCl is used for manufacturing a high-salt environment, and the high-salt environment is matched with the salt concentration in the binding solution to effectively remove protein impurities; Tris-HCl is selected as a buffer, the pH value is 7.0, and the concentration is 30 mM; in addition, about 55% ethanol was used.

4. Comparison with commercially available products.

The kit of the invention (formulation 1, product number TSP701) was compared with a similar kit available on the market.

Several gram-negative bacteria and gram-positive bacteria were selected and compared with the same type of commercially available kit using the kit of the present invention (formulation 1), and the results are shown in FIG. 6. As can be seen, the kit has similar effect with the commercial kit for model bacteria (Escherichia coli, Bacillus subtilis and Staphylococcus aureus); for some common functional bacteria, such as agrobacterium, bacillus thuringiensis, acetic acid bacteria, lactococcus lactis and the like, the effect of the kit is superior to that of a product sold in the market; for other strains, such as pseudomonas aeruginosa, xanthomonas flavipes, salmonella typhimurium and enterobacter aerogenes, the kit is obviously superior to T company, such as staphylococcus epidermidis, streptococcus agalactiae, bacillus licheniformis and rhodococcus, and the kit is obviously superior to T company and O company. Generally, the kit has good universality and has good extraction effect on different types of bacteria.

While the invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention, and it is intended that the following claims be interpreted to include all such embodiments and equivalent variations. In addition, the contents of all references cited herein are hereby incorporated by reference.

Claims

1. A kit for extracting bacterial genome DNA comprises a suspension, a binding solution, a deproteinizing solution, a rinsing solution and an eluent,

the suspension comprises 1-2% of sodium dodecyl sarcosinate in a first Tris-EDTA buffer solution;

the binding solution comprises 2-5M GuHCl and 20-60mM urea in sodium citrate buffer solution;

the deproteinizing solution comprises 2-3M GuHCl in a first Tris-HCl buffer solution, and ethanol with the final concentration of 30-60% by volume is added;

the rinsing liquid comprises a second Tris-HCl buffer solution, and ethanol with the final concentration of 75-80% by volume is added into the second Tris-HCl buffer solution;

the eluent comprises a second Tris-EDTA buffer.

2. The kit of claim 1, further comprising a DNA adsorption column and proteinase K.

3. The kit according to claim 1 or 2, wherein the DNA adsorption column is a silicon substrate membrane adsorption column.

4. Kit according to claim 1 or 2, the first Tris-EDTA buffer having a pH of 7.5 to 8.5, preferably 8, comprising 20 to 100mM, preferably 25 to 50mM Tris-HCl and 10 to 20mM EDTA.

5. The kit according to claim 1 or 2, the pH of the sodium citrate buffer being between 5.0 and 6.0, preferably between 5.0 and 5.5, at a concentration of between 20 and 60mM, preferably 50 mM.

6. Kit according to claim 1 or 2, the second Tris-EDTA buffer having a pH comprised between 7.5 and 8.5, preferably 8, comprising between 5 and 20mM, preferably 10mM Tris-HCl and between 0.1 and 10mM, preferably 1mM EDTA.

7. A method for extracting bacterial genomic DNA using the kit of any one of claims 1 to 6, comprising:

2) adding the binding solution for incubation;

3) precipitating DNA with ethanol;

5) adding the deproteinized liquid and absolute ethyl alcohol mixture into the adsorption column, centrifuging, and removing a liquid phase;

6) adding the rinsing liquid and absolute ethyl alcohol mixture into the adsorption column, centrifuging, removing a liquid phase, preferably repeating for 1-2 times, and then drying;

7) eluting DNA from the adsorption column.

8. The method according to claim 7, wherein in 1), the cell wall of gram-negative bacteria is disrupted by the suspension, and the cell wall of gram-positive bacteria is disrupted by lysozyme.

9. The method according to claim 7 or 8, wherein in 1), the protein is degraded with proteinase K.

10. The method according to claim 7 or 8, wherein in 2), the incubation is carried out at 56 ℃ for 10-15 min.