CN115369111A - Extraction kit and extraction method for soil microorganism DNA - Google Patents

Abstract

The invention discloses a soil microorganism DNA extraction kit, which comprises a grinding material, a lysis buffer solution, a binding solution, a rinsing solution a, a rinsing solution b and an eluent; wherein the grinding material consists of grinding beads and steel balls; the lysis buffer solution comprises CTAB, SDS, naCl, tris-HCl and EDTA; the binding solution comprises a magnetic bead solution, PEG6000 and NaCl; the rinsing liquid a comprises guanidinium isothiocyanate, guanidinium hydrochloride, ethanol and Tris-HCl; the rinsing liquid b comprises ethanol and Tris-HCl; the eluent is Tris-HCl buffer solution. The invention adopts a unique grinding system, which is more beneficial to cell lysis; in addition, toxic and harmful chemical reagents such as chloroform, phenol and the like are not needed, and a good impurity removal effect can be achieved by virtue of the binding liquid and the rinsing liquid; in addition, the magnetic bead purification is adopted, and the magnetic bead purification is combined with related equipment, so that the extraction efficiency of the DNA can be effectively improved.

Description

Extraction kit and extraction method for soil microorganism DNA

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an extraction kit and an extraction method for soil microorganism DNA.

Background

Most of microorganisms such as bacteria and fungi in soil are not culturable, but with the rapid development of NGS (high throughput sequencing) technology in recent years, particularly, techniques such as metagenome sequencing and amplicon sequencing which are established on the NGS technology, analysis related to microbial community structures can be realized without isolated culture of soil microorganisms.

Due to the complex type of soil samples, especially the difficult effective removal of humic acid substances in soil during the extraction process, humus has similar physicochemical properties to nucleic acid. Thus, most of the humus is co-separated from the DNA together with adsorbed organic molecules. The humic acid has great inhibition effect on subsequent analysis with participation of biological enzymes, such as PCR amplification, endonuclease digestion, high-throughput sequencing and the like. In most metagenomic studies, it is a great challenge to isolate high-purity metagenomic DNA free of humus from soil samples from various sources. The NGS technology is based on the extraction of DNA, and the existing extraction process of soil microorganism DNA is complicated and has low efficiency, so that the DNA extraction efficiency and the DNA extraction quality cannot be considered at the same time.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide an extraction kit and an extraction method for soil microorganism DNA.

In order to achieve the purpose, the invention adopts the following technical means:

the invention provides a soil microorganism DNA extraction kit, which comprises a grinding material, a lysis buffer solution, a binding solution, a rinsing solution a, a rinsing solution b and an eluent;

wherein the grinding material consists of 1mm grinding beads and 4mm steel beads; the lysis buffer solution comprises CTAB, SDS, naCl, tris-HCl and EDTA; the binding liquid comprises magnetic beads, PEG6000 and NaCl; the rinsing liquid a comprises guanidinium isothiocyanate, guanidinium hydrochloride, ethanol and Tris-HCl; the rinsing liquid b comprises ethanol and Tris-HCl; the eluent is Tris-HCl buffer solution.

Preferably, the abrasive material consists of 0.1-0.3g of 1mm abrasive beads and 1-3 4mm steel beads for disrupting cells, making the cells more easily lysed;

preferably, in the solution buffer solution, the concentration of CTAB is 0.1-1 wt%, the concentration of SDS is 0.1-1 wt%, the concentration of NaCl is 2-6M, the concentration of Tris-HCl is 0.1-0.3M, and the concentration of EDTA is 0.1-0.3; the lysis buffer is used for lysing cells, releasing nucleic acid and complexing some impurities;

preferably, the concentration of PEG6000 in the binding solution is 25wt% -35wt%, and the concentration of NaCl is 1-3M; the magnetic beads are silicon-based magnetic beads and are used for directionally adsorbing nucleic acid;

preferably, in the rinsing liquid a, the concentration of the guanidinium isothiocyanate is 1-3M, the concentration of the guanidinium hydrochloride is 2-4M, the volume fraction of the ethanol is 25% -35%, and the concentration of the Tris-HCl is 0.3-0.7M. The rinsing liquid a is used for removing residual salt substances;

preferably, in the rinsing liquid b, the volume fraction of the ethanol is 65-75%, and the concentration of Tris-HCl is 0.1-0.3M; the rinsing liquid b is used for further removing residual salt substances;

preferably, the eluent is Tris-HCl buffer solution with the concentration of 8-12mM, and is used for eluting the nucleic acid adsorbed by the magnetic beads.

The second aspect of the present invention provides the use of the kit according to the first aspect of the present invention in soil DNA extraction.

The third aspect of the invention provides an extraction method of the kit of the first aspect of the invention for soil DNA extraction, which comprises the following specific steps:

s1, adding a grinding material into a container a, then adding a soil sample, then adding a lysis buffer solution, grinding for 10-15min, and then carrying out water bath for 5-15min in water at the temperature of 60-70 ℃;

s2, cooling the container a to room temperature, and then centrifuging at the rotating speed of 11000-13000rpm for 2-5min; and sucking the supernatant liquid into the container 2;

s3, adding the binding liquid into the container b, then placing the container b on a mixing instrument, and oscillating and mixing the mixture for 1-2min at room temperature;

s4, placing the container b uniformly mixed in the step S3 on a special magnetic frame, standing, and discarding supernatant after the magnetic beads are completely adsorbed;

s5, adding the rinsing liquid a into the container b with the supernatant discarded in the step S4, transferring the container b to a blending instrument, oscillating and blending, placing the container b on a special magnetic frame for standing, and discarding the supernatant after the magnetic beads are completely adsorbed;

s6, adding a rinsing liquid b into the container b with the supernatant discarded in the step S5, transferring the container b to a blending instrument, oscillating and blending, placing the container b on a special magnetic frame for standing, and discarding the supernatant after the magnetic beads are completely adsorbed;

s7, airing the container b at room temperature; adding eluent, transferring to a mixing instrument, shaking at constant temperature of 60-70 deg.C, mixing for 1-3min, standing container b on special magnetic frame, and transferring supernatant to container c after magnetic beads are completely adsorbed; wherein the supernatant in the container c contains a soil microorganism DNA sample.

Preferably, the soil sample in step S1 is selected from one of rhizosphere soil of paddy field, soil around compost, garden soil, mountain forest soil, river sludge and anaerobic sludge.

Preferably, the container a, the container b and the container c are an EP tube a, an EP tube b and an EP tube c, respectively.

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts a unique grinding system, which is more beneficial to cell lysis; in addition, toxic and harmful chemical reagents such as chloroform, phenol and the like are not needed, and a good impurity removal effect can be achieved by virtue of the binding liquid and the rinsing liquid; in addition, the magnetic bead purification is adopted and is combined with related equipment, so that the extraction efficiency of the DNA can be effectively improved.

Drawings

FIG. 1 is a graph showing the result of electrophoresis on soil DNA extraction in example 3 of the present invention.

FIG. 2 is a graph showing the results of electrophoresis on soil DNA extraction in example 4 of the present invention and comparative example 1.

Detailed Description

Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. Where applicable, the contents of any patent, patent application, or publication referred to in this application are incorporated herein by reference in their entirety, and the equivalent family of patents is also incorporated by reference, in particular for the definitions set forth in these documents regarding synthetic techniques, product and process designs, polymers, comonomers, initiators or catalysts, and the like, in the art. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.

The numerical ranges in this application are approximations, and thus may include values outside of the ranges unless otherwise specified. A numerical range includes all numbers from the lower value to the upper value, in increments of 1 unit, provided that there is a separation of at least 2 units between any lower value and any higher value. For example, if a compositional, physical, or other property (e.g., molecular weight, melt index, etc.) is recited as 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing a numerical value less than 1 or containing a fraction greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is considered to be 0.0001,0.001,0.01, or 0.1, as appropriate. For ranges containing single digit numbers less than 10 (e.g., 1 to 5), 1 unit is typically considered 0.1. These are merely specific examples of what is intended to be presented, and all possible combinations of numerical values between the lowest value and the highest value enumerated, are to be considered to be expressly stated in this application.

When used with respect to chemical compounds, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively) unless expressly specified otherwise. In addition, unless explicitly stated otherwise, the use of the terms "a", "an" or "the" are intended to include the plural forms as well.

The terms "comprising," "including," "having," and derivatives thereof do not exclude the presence of any other component, step or procedure, and are not relevant to whether such other component, step or procedure is disclosed herein. To the exclusion of any doubt, all compositions herein using the terms "comprising", "including", or "having" may include any additional additive, adjuvant, or compound, unless explicitly stated otherwise. Rather, the term "consisting essentially of 8230 \8230; \8230composition" excludes any other components, steps or processes from the scope of any of the terms hereinafter recited, insofar as they are necessary for performance. The term "consisting of 823070 \8230composition" does not include any components, steps or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clear, the present invention is further described in detail below with reference to the embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

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 this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Example 1

The embodiment provides an extraction kit of soil microorganism DNA, which comprises a grinding material, a lysis buffer solution, a binding solution, a rinsing solution a, a rinsing solution b and an eluent;

a grinding material consisting of 0.2g of 1mm grinding beads and 1 4mm steel balls;

lysis buffer (1L): weighing 1g CTAB,1g SDS,234g NaCl,0.292g EDTA and 100mL1M Tris-HCl, and adding sterile water to 1L.

Binding solution (100 mL): 1mL of the magnetic bead solution, 30g of PEG6000 and 11.7g of NaCl are taken, and sterile water is added to the solution to reach 100mL.

Rinse liquid a (1L): 236.3g of guanidinium isothiocyanate, 286.6g of guanidinium hydrochloride, 300mL of absolute ethanol, 500mL1M Tris-HCl and sterile water are weighed to 1L.

Rinse liquid b (1L): taking 700mL of absolute ethyl alcohol, 100mL of 1M Tris-HCl, and supplementing sterile water to 1L.

Eluent (1L): 1L of 10mL of 1M Tris-HCl was added with sterile water.

Example 2

This example provides the use of the kit of example 1 for soil DNA extraction.

S1, adding grinding beads and steel balls into an EP (EP) tube a, then adding 0.2g of soil sample, then adding 1mL of lysis buffer, grinding for 15min (50 Hz), and then carrying out water bath for 10min in 65 ℃;

s2, cooling the EP pipe a to room temperature, and then centrifuging for 2min at the rotating speed of 12000 rpm; and the supernatant was aspirated into EP tube 2

S3, sucking the supernatant into an EP tube b, and adding binding liquid with the volume of 3/4 of that of the supernatant. And (4) placing the EP pipe b on a blending instrument, and shaking and blending for 1min at room temperature.

S4, placing the EP tube b uniformly mixed in the step S3 on a special magnetic frame, standing for 1min, and discarding supernatant after the magnetic beads are completely adsorbed;

s5, adding 800 mu L of rinse liquid a into the EP tube b with the supernatant discarded in the step S4, transferring the EP tube b to a blending instrument, oscillating and blending for 1min, then placing the EP tube b on a special magnetic frame for standing for 30S, and discarding the supernatant after the magnetic beads are completely adsorbed;

s6, adding 800 mu L of rinsing liquid b into the EP tube b with the supernatant discarded in the step S5, transferring the EP tube b to a blending instrument, oscillating and blending for 1min, placing the EP tube b on a special magnetic frame for standing for 30S, and discarding the supernatant after the magnetic beads are completely adsorbed;

s7, airing the EP tube b for 5min at room temperature; then adding 100 mu L of eluent, transferring the eluent to a blending instrument, oscillating and blending the eluent at the constant temperature of 65 ℃ for 1min, placing the EP tube b on a special magnetic frame for standing for 2min, and transferring the supernatant to the EP tube c after the magnetic beads are completely adsorbed; wherein, the supernatant in the EP tube c is the DNA product.

Example 3

This example used the kit of example 1 to extract DNA from soils 1 to 6 according to the method of example 2, wherein 1 is paddy rhizosphere soil, 2 is compost surrounding soil, 3 is garden soil, 4 and 5 are sandy soil, and 6 is pond sludge; the electrophoresis results are shown in FIG. 1. Wherein M is 15000bp marker,1-6 are different types of soil samples in 6.

As can be seen from FIG. 1, the DNA of the soil microorganism extracted by the kit of example 1 has clear main bands, no degradation, and no miscellaneous bands such as protein and RNA. The method shows that the quality of the soil microorganism DNA extracted by the method is higher.

Example 4

This example uses the kit of example 1 and performs DNA extraction on the soil around the humic acid-rich compost as described in example 2, and the results of electrophoresis are shown in the left 1-3 of FIG. 2, where M is 15000bp Marker, and are repeated three times.

Comparative example 1

Comparative example 1 DNA extraction was carried out three times using the kit of example 1 on the soil around compost containing a large amount of humic acid according to the method described in patent CN107228787B, and the results of electrophoresis are shown in the right side 1-3 of FIG. 2.

As can be seen from FIG. 2, the DNA of the soil microorganism extracted by the method of example 2 using the kit of example 1 was clearly banded, without degradation, whereas the DNA extracted by the method described in patent CN107228787B was degraded and banded more.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (9)

1. An extraction kit of soil microorganism DNA is characterized by comprising a grinding material, a lysis buffer solution, a binding solution, a rinsing solution a, a rinsing solution b and an eluent; wherein the grinding material consists of grinding beads and steel balls; the lysis buffer solution comprises CTAB, SDS, naCl, tris-HCl and EDTA; the binding liquid comprises magnetic beads, PEG6000 and NaCl; the rinsing liquid a comprises guanidinium isothiocyanate, guanidinium hydrochloride, ethanol and Tris-HCl; the rinsing liquid b comprises ethanol and Tris-HCl; the eluent is Tris-HCl buffer solution.

2. The kit for extracting DNA from soil microorganisms according to claim 1, wherein the grinding material comprises 0.1-0.3g of 1mm grinding beads and 1-3 beads of 4 mm.

3. The kit for extracting soil microorganism DNA according to claim 1, characterized in that in the buffering solution, CTAB is 0.1-1 wt%, SDS is 0.1-1 wt%, naCl is 2-6M, tris-HCl is 0.1-0.3M, and EDTA is 0.1-0.3M.

4. The soil microorganism DNA extraction kit of claim 1, wherein the concentration of PEG6000 in the binding solution is 25wt% -35wt%, and the concentration of NaCl is 1-3M; the magnetic beads are silicon-based magnetic beads.

5. The soil microorganism DNA extraction kit of claim 1, wherein the rinsing solution a contains 1-3M guanidinium isothiocyanate, 2-4M guanidinium hydrochloride, 25-35% ethanol by volume and 0.3-0.7M Tris-HCl.

6. The soil microorganism DNA extraction kit as claimed in claim 1, wherein the rinsing solution b contains 65-75% by volume of ethanol and 0.1-0.3M concentration of Tris-HCl.

7. The soil microorganism DNA extraction kit as claimed in claim 1, wherein the eluent is Tris-HCl buffer solution with concentration of 8-12 mM.

8. A method for extracting DNA from soil using the kit for extracting DNA from soil microorganisms according to any one of claims 1 to 7, comprising the steps of:

s1, adding a grinding material into a container a, then adding a soil sample, then adding a lysis buffer solution, grinding for 10-15min, and then carrying out water bath for 5-15min in water at the temperature of 60-70 ℃;

s2, cooling the container a to room temperature, and then centrifuging at the rotating speed of 11000-13000rpm for 2-5min; and sucking the supernatant into a container b;

s3, adding the binding liquid into the container b2, then placing the container b on a mixing instrument, and oscillating and mixing uniformly for 1-2min at room temperature;

s4, placing the container b uniformly mixed in the step S3 on a magnetic frame, standing, and discarding supernatant after the magnetic beads are completely adsorbed;

s5, adding the rinsing liquid a into the container 2 with the supernatant discarded in the step S4, transferring the container b to a mixing instrument, oscillating and mixing uniformly, placing the container b on a magnetic frame for standing, and discarding the supernatant after the magnetic beads are completely adsorbed;

s6, adding a rinsing liquid b into the container b with the supernatant discarded in the step S5, transferring the container b to a blending instrument, oscillating and blending, placing the container b on a magnetic frame for standing, and discarding the supernatant after the magnetic beads are completely adsorbed;

s7, airing the container b at room temperature; adding eluent, transferring to a mixing instrument, shaking at constant temperature of 60-70 deg.C, mixing for 1-3min, placing container b on magnetic frame, standing, and transferring supernatant to container c after magnetic beads are completely adsorbed; wherein, the supernatant in the container c contains a soil microorganism DNA sample which is a DNA product.

9. The extraction method according to claim 8, wherein the soil sample in step S1 is selected from one of rice field rhizosphere soil, compost surrounding soil, garden soil, mountain forest soil, river sludge and anaerobic sludge.

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