A soil microorganism DNA extraction kit and extraction method

Abstract

The application discloses a soil microorganism DNA extraction kit, which comprises grinding materials, a lysis buffer, a binding liquid, a rinsing liquid a, a rinsing liquid b and an eluent; wherein the grinding materials are composed of grinding beads and steel beads; the lysis buffer comprises CTAB, SDS, NaCl, Tris-HCl and EDTA; the binding liquid comprises a magnetic bead solution, PEG6000 and NaCl; the rinsing liquid a comprises guanidine isothiocyanate, guanidine hydrochloride, ethanol and Tris-HCl; the rinsing liquid b comprises ethanol and Tris-HCl; and the eluent is a Tris-HCl buffer solution. The unique grinding system of the application is more helpful for cell lysis; and the application does not need to use toxic and harmful chemical reagents such as chloroform and phenol, and can achieve a good impurity removal effect by means of the binding liquid and the rinsing liquid; furthermore, the application adopts magnetic bead purification, and can effectively improve the DNA extraction efficiency in combination with related equipment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a soil microorganism DNA extraction kit and an extraction method. BACKGROUND

[0002] Most of the microorganisms such as bacteria and fungi in soil are not cultivable, but with the rapid development of NGS (high-throughput sequencing) technology in recent years, especially the macro-genome sequencing and amplicon sequencing technologies based on NGS technology, the analysis of microorganism community structure can be realized without the separation and cultivation of soil microorganisms.

[0003] Due to the complex types of soil samples, especially the humic acid substances in soil, which are difficult to effectively remove in the extraction process, humus has physical and chemical properties similar to nucleic acids. Therefore, most of the humus is separated together with the adsorbed organic molecules and the DNA. Humic acid has a great inhibitory effect on subsequent biological enzyme involved analysis, such as PCR amplification, endonuclease digestion, high-throughput sequencing, etc. In most macro-genome studies, it is a great challenge to separate high-purity macro-genome DNA without humus from soil samples of various sources. The basis of NGS technology is the extraction of DNA, and the existing soil microorganism DNA extraction process is relatively cumbersome and low in efficiency, so it cannot balance the DNA extraction efficiency and DNA extraction quality. SUMMARY

[0004] In order to solve the problems in the prior art, the purpose of the present application is to provide a soil microorganism DNA extraction kit and an extraction method.

[0005] In order to achieve the above purpose, the present application adopts the following technical means:

[0006] The first aspect of the present application is to provide a soil microorganism DNA extraction kit, which comprises grinding material, lysis buffer, binding liquid, rinse liquid a, rinse liquid b and eluent;

[0007] The grinding material is composed of 1mm grinding beads and 4mm steel beads; the lysis buffer comprises CTAB, SDS, NaCl, Tris-HCl and EDTA; the binding liquid comprises magnetic beads, PEG6000 and NaCl; the rinse liquid a comprises guanidine isothiocyanate, guanidine hydrochloride, ethanol and Tris-HCl; the rinse liquid b comprises ethanol and Tris-HCl; and the eluent is Tris-HCl buffer.

[0008] Preferably, the grinding material is composed of 0.1-0.3g of 1mm grinding beads and 1-3 of 4mm steel beads, which is used to break the cells, so that the cells are more easily lysed;

[0009] Preferably, in the lysis buffer, the concentration of CTAB is 0.1wt%-1wt%, the concentration of SDS is 0.1wt%-1wt%, the concentration of NaCl is 2-6M, the concentration of Tris-HCl is 0.1-0.3M, and the concentration of EDTA is 0.1-0.3M; the lysis buffer is used for lysing cells to release nucleic acids and complex some impurities;

[0010] Preferably, in the binding liquid, the concentration of PEG6000 is 25wt%-35wt%, and the concentration of NaCl is 1-3M; the magnetic beads are silica-based magnetic beads and are used for directional adsorption of nucleic acids;

[0011] Preferably, in the rinsing liquid a, the concentration of guanidine isothiocyanate is 1-3M, the concentration of guanidine hydrochloride is 2-4M, the volume fraction of ethanol is 25%-35%, and the concentration of Tris-HCl is 0.3-0.7M; the rinsing liquid a is used for removing residual salt substances;

[0012] Preferably, in the rinsing liquid b, the volume fraction of 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;

[0013] Preferably, the elution liquid is a Tris-HCl buffer with a concentration of 8-12mM and is used for eluting nucleic acids adsorbed by the magnetic beads.

[0014] The second aspect of the present application provides application of the kit of the first aspect of the present application in soil DNA extraction.

[0015] The third aspect of the present application provides an extraction method for soil DNA extraction using the kit of the first aspect of the present application, and the specific steps include:

[0016] S1, grinding material is added to container a, then soil sample is added, and then lysis buffer is added, and grinding is performed for 10-15min, and then water bath is performed in water at 60-70℃ for 5-15min;

[0017] S2, container a is cooled to room temperature, and then centrifugation is performed at a speed of 11000-13000rpm for 2-5min; and supernatant is sucked into container 2;

[0018] S3, binding liquid is added to container b, and then container b is placed on a mixing instrument, and mixing is performed at room temperature for 1-2min;

[0019] S4, container b after mixing in step S3 is placed on a special magnetic stand, and is left to stand, and after the magnetic beads are completely adsorbed, supernatant is discarded;

[0020] S5, adding the rinsing liquid a into the container b in which the supernatant is discarded in step S4, then transferring the container b to a mixing instrument, mixing and shaking, then placing the container b on a special magnetic stand, and discarding the supernatant after the magnetic beads are completely adsorbed;

[0021] S6, adding the rinsing liquid b into the container b in which the supernatant is discarded in step S5, then transferring the container b to a mixing instrument, mixing and shaking, then placing the container b on a special magnetic stand, and discarding the supernatant after the magnetic beads are completely adsorbed;

[0022] S7, drying the container b at room temperature, then adding the elution liquid and transferring to a mixing instrument, mixing and shaking at a constant temperature of 60-70℃ for 1-3 min, then placing the container b on a special magnetic stand, and transferring the supernatant to the container c after the magnetic beads are completely adsorbed; wherein the supernatant in the container c contains the soil microbial DNA sample.

[0023] Preferably, the soil sample in step S1 is selected from one of paddy rhizosphere soil, compost surrounding soil, garden soil, mountain forest soil, river channel silt and anaerobic sludge.

[0024] Preferably, the container a, the container b and the container c are EP tube a, EP tube b and EP tube c respectively.

[0025] Advantages of the present application

[0026] Compared with the prior art, the present application has the following advantages:

[0027] The unique grinding system adopted in the present application is more conducive to cell lysis; and the present application does not need to use toxic and harmful chemical reagents such as chloroform and phenol, but relies on binding liquid and rinsing liquid to achieve good impurity removal effect; furthermore, the magnetic bead purification adopted in the present application can effectively improve the extraction efficiency of DNA when combined with related equipment. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 An electrophoresis result diagram of soil DNA extraction by the embodiment 3 of the present application is shown.

[0029] Figure 2 An electrophoresis result diagram of soil DNA extraction by the embodiment 4 and the comparative example 1 of the present application is shown. DETAILED DESCRIPTION

[0030] Unless otherwise indicated, all parts and percentages in the present application are on a weight basis, and all tests and measurements are made in accordance with standard techniques synchronous with the date of the present application. To the extent that any patent, patent application, or publication is cited in this application, the contents of all such citations are incorporated by reference into this application, and equivalents thereof are also incorporated by reference, as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that any definition or usage provided by any document incorporated by reference into the present application contradicts or conflicts with the definition or usage of a term or phrase contained in this application, the definition or usage of the term or phrase contained in this application shall control.

[0031] Numerical ranges in the present application are approximations, and thus the endpoints of ranges are not to be understood as being significantly bound by the recited starting and ending points. Unless otherwise indicated, all numerical ranges are inclusive of the recited endpoints. A numerical range includes all values from and including the lower and the upper values, in increments of one unit. In addition, unless otherwise indicated, the various numerical ranges are inclusive of the endpoints, and also include all sub-ranges falling within the given ranges. In other words, unless specifically noted, the numerical ranges are continuous ranges, thereby including all possible sub-ranges. For numerical ranges that include less than one, or more than one, unit, the ranges are inclusive of the recited values. For example, "room temperature to 30 °C" is intended to indicate a range of room temperature (approximately 20 °C to 25 °C) to 30 °C inclusive of all increments of temperature within this range. Similarly, "less than or equal to 5" is intended to indicate a range of "less than or equal to 5" inclusive of all increments of less than or equal to 5.

[0032] As used in reference to chemical compounds, the singular includes all isomeric forms, unless specifically indicated otherwise (e.g., "hexane" alone or in combination, includes all isomeric forms of hexane). Additionally, the use of "a" or "an" or "the" when referring to a grammatical object, is intended to include one or more of the object, unless specifically indicated otherwise.

[0033] The terms "comprising," "including," "containing," and "having," and their derivatives, are not intended to exclude any component, step or procedure not specified, and are used to mean "comprising" unless otherwise indicated. To the extent that any term in the present application, including the claims, is dependent head "comprising," "including," "containing", or "having," for the purposes of United States patent law, such term is intended to be construed in the alternative, as "consisting of. For the purposes of United States patent law, the term "consisting essentially of means "consisting of" with the proviso that the claimed subject matter does not exclude any additional components, steps or procedures. The term "consisting of means "consisting of" with the proviso that the claimed subject matter does not include any additional components, steps or procedures.

[0034] In order to make the technical problems solved by the present application, the technical solutions and the beneficial effects clearer, the present application will be further explained in details in combination with the embodiments.

[0035] Embodiment

[0036] The following examples are presented to demonstrate preferred embodiments of the present application. Those skilled in the art will appreciate that the technology disclosed in the following examples represents the best of the inventor's knowledge of how to practice the present application and, as such, can be considered to be preferred embodiments of the present application. However, those skilled in the art will appreciate from the present disclosure that various modifications can be made to the specific embodiments disclosed herein, without departing from the spirit or scope of the application.

[0037] Unless otherwise defined, 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 application belongs. The materials disclosed herein are cited for their disclosure prior to the filing date of the present application. The citation of a reference herewith is made solely for purposed of providing additional disclosure.

[0038] Those skilled in the art will appreciate that numerous equivalents to the specific procedures described herein will suggest themselves to the skilled person and will be within the spirit and scope of the application. Such equivalents are considered to be within the scope of the application.

[0039] Embodiment 1

[0040] The present embodiment provides a soil microbial DNA extraction kit, comprising grinding material, lysis buffer, binding liquid, rinse liquid a, rinse liquid b and elution liquid;

[0041] The grinding material consists of 0.2 g of 1 mm grinding beads and 1 steel ball of 4 mm;

[0042] Lysis buffer (1 L): weigh 1 g CTAB, 1 g SDS, 234 g NaCl, 0.292 g EDTA and 100 mL 1 M Tris-HCl, make up to 1 L with sterile water.

[0043] Binding buffer (100 mL): take 10 mL of magnetic beads solution, 30 g PEG6000, 11.7 g NaCl, make up to 100 mL with sterile water.

[0044] Rinse buffer a (1 L): weigh 236.3 g guanidine isothiocyanate, 286.6 g guanidine hydrochloride, 300 mL absolute ethanol, 500 mL 1 M Tris-HCl, make up to 1 L with sterile water.

[0045] Rinse buffer b (1 L): take 700 mL absolute ethanol, 100 mL 1 M Tris-HCl, make up to 1 L with sterile water.

[0046] Elution buffer (1 L): take 10 mL 1 M Tris-HCl, make up to 1 L with sterile water.

[0047] Example 2

[0048] This example provides the application of the kit of example 1 in soil DNA extraction.

[0049] S1, add grinding beads and steel beads to EP tube a, then add 0.2 g soil sample, then add 1 mL lysis buffer, grind for 15 min (50 Hz), then water bath at 65 °C for 10 min;

[0050] S2, cool EP tube a to room temperature, then centrifuge at 12000 rpm for 2 min; and aspirate the supernatant into EP tube 2

[0051] S3, aspirate the supernatant into EP tube b, add binding buffer to 3 / 4 volume of the supernatant. Place EP tube b on a vortex mixer, vortex for 1 min at room temperature.

[0052] S4, place the vortexed EP tube b of step S3 in a dedicated magnetic stand, stand for 1 min, after the magnetic beads are completely adsorbed, discard the supernatant;

[0053] S5, add 800 μL of rinse buffer a to the EP tube b of step S4 after discarding the supernatant, transfer the EP tube b to a vortex mixer, vortex for 1 min, then place the EP tube b in a dedicated magnetic stand for 30 s, after the magnetic beads are completely adsorbed, discard the supernatant;

[0054] S6, add 800 μL of the rinsing solution b to the EP tube b in which the supernatant is discarded in step S5, transfer the EP tube b to a mixer, shake and mix for 1 min, then place the EP tube b on a special magnetic stand and stand for 30 s, after the magnetic beads are completely adsorbed, discard the supernatant;

[0055] S7, dry the EP tube b at room temperature for 5 min, then add 100 μL of the elution solution and transfer to a mixer, shake and mix at 65℃ for 1 min, then place the EP tube b on a special magnetic stand and stand for 2 min, after the magnetic beads are completely adsorbed, transfer the supernatant to an EP tube c; wherein the supernatant in the EP tube c is the DNA product.

[0056] Example 3

[0057] In this example, the kit of Example 1 is used to extract DNA from soil 1-6 according to the method of Example 2, wherein 1 is a paddy field rhizosphere soil, 2 is a soil around compost, 3 is a garden soil, 4 and 5 are sandy soil, and 6 is a pond sludge; the electrophoresis result is shown in Figure 1 , wherein M is 15000bp Marker, and 1-6 are 6 different types of soil samples.

[0058] As can be seen from Figure 1 , the main band of the soil microbial DNA extracted by the kit of Example 1 is clear, without degradation, and without protein, RNA and other impurity bands. It shows that the soil microbial DNA extracted by the method has high quality.

[0059] Example 4

[0060] In this example, the kit described in Example 1 is used to extract DNA from soil around compost containing more humic acid according to the method described in Example 2, which is repeated three times, and the electrophoresis result is shown in Figure 2 1-3 on the left, wherein M is 15000bp Marker.

[0061] Comparative Example 1

[0062] Comparative Example 1 uses the kit of Example 1 to extract DNA from soil around compost containing more humic acid according to the method described in patent CN107228787B, which is repeated three times, and the electrophoresis result is shown in Figure 2 1-3 on the right.

[0063] As can be seen from Figure 2 , the main band of the soil microbial DNA extracted by the kit of Example 1 according to the method of Example 2 is clear, without degradation, while the DNA extracted by the method described in patent CN107228787B is degraded, and has more impurity bands.

[0064] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the disclosure and the appended claims.

Claims

1. A soil microorganism DNA extraction kit characterized by comprising: Consist of grinding material, lysis buffer, binding liquid, rinse liquid a, rinse liquid b and eluent; wherein, The grinding material consists of 0.2g of 1mm grinding beads and 1 piece of 4mm steel beads; The lysis buffer consists of CTAB, SDS, NaCl, Tris-HCl and EDTA, the concentration of CTAB is 1g / L, the concentration of SDS is 1g / L, the concentration of NaCl is 4M, the concentration of Tris-HCl is 0.1M, and the concentration of EDTA is 0.292g / L; The binding liquid consists of magnetic beads, PEG6000 and NaCl, the concentration of PEG6000 is 30wt%, and the concentration of NaCl is 2M; the magnetic beads are silicon-based magnetic beads; The rinse liquid a consists of guanidine isothiocyanate, guanidine hydrochloride, ethanol and Tris-HCl, the concentration of guanidine isothiocyanate is 2M, the concentration of guanidine hydrochloride is 3M, the volume fraction of ethanol is 30%, and the concentration of Tris-HCl is 0.5M; The rinse liquid b consists of ethanol and Tris-HCl, the volume fraction of ethanol is 70%, and the concentration of Tris-HCl is 0.1M; The eluent is 10mM Tris-HCl buffer.

2. An extraction method for extracting soil DNA using the soil microorganism DNA extraction kit according to claim 1, characterized by, Comprise the following steps: S1, add grinding material to container a, then add soil sample, then add lysis buffer, grind for 10-15min, then water bath in water at 60-70℃ for 5-15min; S2, cool container a to room temperature, then centrifuge at a speed of 11000-13000rpm for 2-5min; and aspirate the supernatant into container b; S3, add binding liquid to container b, then place container b on a vortex mixer, and vortex mix at room temperature for 1-2min; S4, place container b after vortex mixing in step S3 on a magnetic stand, stand still, and after the magnetic beads are completely adsorbed, discard the supernatant; S5, add rinse liquid a to container b after discarding the supernatant in step S4, then transfer container b to a vortex mixer, vortex mix, then place container b on a magnetic stand, stand still, and after the magnetic beads are completely adsorbed, discard the supernatant; S6, add rinse liquid b to container b after discarding the supernatant in step S5, then transfer container b to a vortex mixer, vortex mix, then place container b on a magnetic stand, stand still, and after the magnetic beads are completely adsorbed, discard the supernatant; S7, dry container b at room temperature; then add eluent and transfer to a vortex mixer, vortex mix at a constant temperature of 60-70℃ for 1-3min, then place container b on a magnetic stand, stand still, and after the magnetic beads are completely adsorbed, transfer the supernatant to container c; wherein the supernatant in container c contains a soil microbial DNA sample, which is a DNA product.

3. The extraction method according to claim 2, characterized in that, The soil sample in step S1 is selected from one of paddy rhizosphere soil, compost surrounding soil, garden soil, mountain forest soil, river channel silt and anaerobic sludge.

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