CN114292841A - Nucleic acid extraction reagent and nucleic acid extraction method - Google Patents

Abstract

The invention belongs to the field of nucleic acid extraction, and relates to a nucleic acid extraction reagent and a nucleic acid extraction method. Specifically, the present invention relates to a cleavage reagent comprising trihydroxymethylaminomethane, lithium chloride, NaSCN, glycerol and lithium dodecyl sulfate, inhibitor removal reagents comprising potassium aluminum sulfate, sodium acetate and acetic acid and Corresponding buffers prepared therefrom and their application in DNA extraction from environmental samples. The present invention also relates to a nucleic acid extraction method using the lysis reagent and/or inhibitor removal reagent. The nucleic acid extraction reagent provided by the invention is simple and fast to operate, and can achieve high-quality extraction of microbial DNA in different environmental samples.

Description

Nucleic acid extraction reagent and nucleic acid extraction method

technical field

The present invention relates to the field of nucleic acid extraction reagents, in particular to extraction or detection reagents for extracting nucleic acids from biological samples in the environment, especially samples containing microorganisms.

Background technique

The study of microbial communities in environmental samples requires the extraction of nucleic acids from biological samples. Nucleic acid extraction and analysis of such samples are extremely challenging, mainly due to the following reasons. 1) Environmental samples such as soil, feces, compost, sludge, wastewater and other samples contain a variety of PCR inhibitors, such as heavy metals, polysaccharides, polyphenols, bile salts, humic acids, etc. Inhibits the PCR reaction. For example, soil samples are rich in humic acid, fulvic acid and humin, and the DNA solution of the conventional method for extracting soil microorganisms is yellow-brown, which is the pollution of the above substances. 2) Microorganisms in environmental samples such as soil, feces and wastewater are more difficult to lyse than simply cultured microorganisms. Conventional extraction methods obtain a low lysis rate of microbial cells, so the obtained DNA cannot reflect the species abundance in the original sample.

Therefore, efficient and practical basic research tools and reagents are highly needed to ensure accurate characterization of the composition of microbial communities, study interdependent functions within microbial communities, and allow manipulation of interactions within microbial communities and between microbial communities and their hosts. Next-generation tools, such as isolation and purification kits for microbial nucleic acids, are urgently needed.

DNA Isolation Kit，Power

DNA IsolationKit和

DNA Isolation kit，Qiagen公司提供的

ProDNA Kit、

Fast DNA Stool Mini Kit、

Pro Kit和

Kit，但是其操作非常繁琐，需要反复离心，且售价极为昂贵，单个样本的提取为30-50元RMB，操作不同的样本需要适用不同的提取试剂盒。Kits currently on the market for DNA isolation from environmental and biological samples include MO BIO Laboratories

DNA Isolation Kit, Power

DNA IsolationKit and

DNA Isolation kit, supplied by Qiagen

ProDNA Kit,

Fast DNA Stool Mini Kit,

Pro Kit and

However, the operation is very cumbersome, requires repeated centrifugation, and the price is extremely expensive. The extraction of a single sample is 30-50 RMB, and different extraction kits are required for different samples.

Chinese invention patent CN107475249 A proposes a method for extracting total microbial DNA from soil with low biomass and rich in humus, through liquid nitrogen grinding and proteinase K digestion, but its operation still requires Qiagen's soil DNA extraction kit To further purify the crudely extracted DNA, the essence of the invention is to provide a pretreatment method for soil samples and obtain crude DNA extracts, rather than to provide a complete solution.

To sum up, although the prior art cannot provide a universal, simple and convenient kit to solve the high-quality extraction of microbial genomic DNA from a variety of complex environmental samples, that is, to obtain good purity, good integrity and high yield. microbiome DNA.

SUMMARY OF THE INVENTION

The purpose of the embodiments of the present invention is to provide a nucleic acid extraction reagent. The nucleic acid extraction reagent provided by the present invention is simple and fast to operate, and can achieve high-quality extraction of microbial DNA in different environmental samples.

In order to achieve the above object of the present invention, the present invention provides a cleavage reagent in the first aspect, the cleavage reagent includes the following components: trihydroxymethylaminomethane, lithium chloride, NaSCN, glycerol and dodecyl sulfate lithium.

The present invention provides, in a second aspect, a lysis buffer prepared from the lysis reagent described in the first aspect of the present invention.

Preferably, the lysis buffer contains 50-500mM tris, 100-500mM lithium chloride, 1-5M NaSCN, 0.5-20% glycerol, 0.1-5% of lithium dodecyl sulfate and a pH of 8.0-12.5.

More preferably, the lysis buffer contains 100-200 mM tris, 200-300 mM lithium chloride, 2-3 M NaSCN, 1-5% glycerol, 0.5-2 % lithium dodecyl sulfate and pH 8.5-11.5.

Further preferably, the lysis buffer contains 200 mM tris(hydroxymethyl)aminomethane, 200 mM lithium chloride, 3 M NaSCN, 5% glycerol, 2% lithium dodecyl sulfate in a mass-to-volume ratio, and the pH is 11.0.

In a third aspect, the present invention provides an inhibitor removal reagent, the inhibitor removal reagent includes the following components: aluminum potassium sulfate, sodium acetate and acetic acid.

The present invention provides, in a fourth aspect, an inhibitor removal buffer, which is prepared from the inhibitor removal reagent described in the third aspect of the present invention.

Preferably, the inhibitor removal buffer contains 0.1-2M potassium aluminum sulfate, 0.5-5M sodium acetate, and the pH is adjusted to pH 3.5-7.5 using acetic acid.

More preferably, the inhibitor removal buffer contains 0.2-1M potassium aluminum sulfate, 0.5-2M sodium acetate, and the pH is adjusted to pH 4-6 using acetic acid.

It is further preferred that the inhibitor removal buffer contains 0.5M potassium aluminum sulfate, 2M sodium acetate, and the pH is adjusted to pH 4 using acetic acid.

The present invention provides a nucleic acid extraction method in a fifth aspect, the method comprising the steps of:

S1: mechanically lysing the environmental sample with grinding beads, and chemically lysing the environmental sample with a lysis buffer, thereby obtaining a crude lysate;

S2: centrifuge the crude lysate to obtain a supernatant, add inhibitor removal buffer to the supernatant to remove PCR inhibitors contained in the environmental sample, and centrifuge to obtain a crude DNA product;

S3: use magnetic microspheres and a binding buffer to bind the DNA in the crude DNA product to obtain a bound DNA product;

S4: washing with a washing solution to obtain a washed DNA product;

S5: Use TE buffer to heat and elute the washed DNA product to obtain the final DNA.

The present invention has no special requirements on the rotation speed and time of centrifugation. In the context of the application, unless otherwise specified, the centrifugation involved in each step of the method of the present invention adopts the centrifugation conditions of a rotating speed of 6000-12000 rpm and a time of 1-2 min. Preferably, in step S1, the grinding beads are 0.1-3mm zirconia beads, 0.1-5mm glass beads, 0.2-1.5mm garnet, 0.2-0.5mm silicon carbide, 0.3-4.5mm Metal balls and/or combinations thereof. Preferably, the grinding beads are 0.1-3mm zirconia beads, 0.1-2mm glass beads, 0.3-1.0mm garnet and/or combinations thereof. More preferably, the grinding beads are 0.1-3 mm zirconia beads, 0.1-1 mm glass beads and/or combinations thereof.

Also preferably, the lysis buffer is prepared from the lysis reagent described in the first aspect of the present invention or is the lysis buffer described in the second aspect of the present invention.

Preferably, in step S2, the inhibitor removal buffer is prepared from the inhibitor removal buffer reagent described in the third aspect of the present invention, or the inhibitor removal buffer described in the fourth aspect of the present invention.

Preferably, in step S3, the magnetic microspheres are silicon hydroxyl magnetic beads, glass magnetic beads, carboxyl magnetic beads, amino magnetic beads, tosyl magnetic beads, epoxy magnetic beads and/or combinations thereof. More preferably, the magnetic microspheres are silicon hydroxyl magnetic beads, glass magnetic beads, carboxyl magnetic beads and/or combinations thereof; further preferably, the magnetic microspheres are magnetic beads produced by Suzhou Cretaceous Biotechnology Co., Ltd. Bead series products, such as silicon hydroxyl magnetic beads MagH1N Silica (Cat#BMD007511), silicon hydroxyl magnetic beads MagH1Silica (Cat#BMD003511), glass magnetic beads Mag2000 Silica (Cat#BMD004511).

Also preferably, in step S3, the binding buffer is a mixture of chaotropic salt and alcohol. More preferably, the chaotropic salt is guanidine hydrochloride, potassium thiocyanate, guanidine isothiocyanate, sodium thiocyanate, potassium thiocyanate, sodium iodide, sodium perchlorate and/or combinations thereof. It is also preferred that the alcohol is ethanol or isopropanol. It is further preferred that the binding buffer contains 1-5M guanidine cyanogen isosulphate, 0.2-2M sodium iodide and isopropanol in a volume ratio of 30-70%. Still further preferably, the binding buffer contains 2-3M guanidine cyanogen isosulphate, 0.5-2M sodium iodide and isopropanol in a volume ratio of 30-60%.

Preferably, in step S4, the cleaning is performed with a first washing solution and a second washing solution in sequence, and the first washing solution contains 0.5-4 cyanoguanidine isosulphate, 0.5-3M lithium chloride, 0.1 -0.5M trishydroxymethylaminomethane, 30-70% ethanol by volume, and pH 5.0-8.0, the second washing solution is 0.1-1M sodium chloride, 0.1-0.2M trishydroxymethylaminomethane The sum-to-product ratio is 50-90% ethanol;

Preferably, the first washing solution contains 1-2M cyanoguanidine isosulphate, 1-2M lithium chloride, 0.1-0.2M trihydroxymethylaminomethane and 50-60% ethanol by volume, and has a pH of 5.0-8.0, the second washing solution is 0.5M sodium chloride, 0.1M trihydroxymethylaminomethane and the volume ratio is 70-80% ethanol.

Preferably, in step S5, the TE buffer contains 10 mM Tris-HCl and 1 mM EDTA, and the pH is 8.5. More preferably, the heating temperature of the heating elution is 55°C to 65°C (eg, 60°C). It is further preferred that the step of removing alcohol by volatilization is also carried out before the heating and elution.

In some more specific embodiments, the method includes the steps of:

S1: Provide a lysis buffer and matching grinding beads, and use a tissue grinder to simultaneously chemically and mechanically lyse microorganisms in environmental samples to generate crude lysates;

S2: Centrifuge the crude lysate (take the supernatant, add IRS buffer (Inhibitor RemovalSolution), vortex and shake to remove PCR inhibitors in the environmental sample, and obtain a crude lysate substantially free of inhibitors after centrifugation. DNA product;

S3: use magnetic microspheres and a binding buffer to bind the crude DNA product to obtain a bound DNA product;

S4: use the first washing solution to wash once, and use the second washing solution to wash once;

S5: After volatilizing and removing alcohol, use TE Buffer to heat and elute to obtain the final (pure) DNA.

The present invention has no particular requirements on the conditions of vortex oscillation. In the context of the present application, unless otherwise stated, the vortexing conditions involved in each step of the method of the present invention are vortexing at a speed of 1000 rpm and a time of 1 min.

In the step S1, the lysis buffer adopts a specific combination of reagents, a specific ratio and a suitable pH, which can dissolve impurities and release nucleic acids in the sample. The NaSCN and glycerol described therein are used to dissolve PCR inhibitors in environmental samples such as humic acid and bile acid.

In other more specific embodiments, the method includes the following steps:

S1: Provide a lysis buffer (100-200mM tris, 200-300mM lithium chloride, 2-3M NaSCN, 1-5% glycerol, 0.5-2% dodecyl sulfate Lithium, pH 8.5-11.5.) and accompanying grinding beads (0.1-3 mm zirconia beads, 0.1-1 mm glass beads, and/or combinations thereof) for simultaneous chemical and Mechanical lysis to produce crude lysate;

S2: Centrifuge the crude lysate to take the supernatant, add IRS buffer (100-250 mM potassium aluminum sulfate, 200-500 mM sodium acetate, and use acetic acid to adjust the pH to pH 4-6), vortex Then, the PCR inhibitors in the environmental samples were removed, and after centrifugation, a crude DNA product substantially free of inhibitors was obtained;

S3: Use magnetic microspheres (silicone magnetic beads, glass magnetic beads, carboxyl magnetic beads and/or combinations thereof) and a binding buffer (2-3M guanidine cyanosulphate, 1-2M sodium iodide and a volume ratio of 30- 60% isopropanol) to bind the above DNA to obtain a bound DNA product;

S4: Use the first washing solution (1-2M guanidine cyanogen isosulphate, 1-2M lithium chloride, 0.1-0.2M trihydroxymethylaminomethane, 50-60% ethanol by volume, pH 5.0-8.0) for cleaning 1 time, the second washing solution (0.2M sodium chloride, 0.1M trihydroxymethylaminomethane, the volume ratio is 70-80% ethanol) was washed once to obtain the cleaned DNA product;

S5: After volatilizing and removing alcohol, use TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.5) to perform heating and elution to obtain pure DNA.

In other more specific embodiments, the method includes the following steps in sequence:

1) Weigh 0.05-0.5 g of environmental samples (the environmental samples can be selected from one or more samples from soil, feces, filtration wastewater membranes, biofilms, etc.), add 0.4-1 mL of lysis buffer and 0.25- 2g of grinding beads, use a bead mill for mechanical cracking, the recommended grinding speed is 4-6m/s, and the grinding time is 1-3 minutes.

2) Centrifuge the above crude lysate to take the supernatant, add 0.1-0.4 mL of IRS buffer, vortex, remove PCR inhibitors by centrifugation, and transfer the supernatant to a clean centrifuge tube.

3) Add 1-2 mg of magnetic microspheres and an equal volume of binding buffer, and vortex for 5 min for nucleic acid binding.

4) Magnetic separation, remove the supernatant, add 0.5-1 mL of the first washing solution, and vortex for 1 min to remove macromolecular impurities.

5) Magnetic separation, remove the supernatant, add 0.5-1 mL of the second washing solution, and vortex for 1 min to remove inorganic salt impurities.

6) Magnetic separation, remove the supernatant, evaporate the alcohol at room temperature, use 50-200 μL TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.5) to heat and elute at 60 degrees for 5 min, after magnetic separation, suck the supernatant solution to obtain pure DNA.

The present invention provides, in a sixth aspect, a kit for extracting nucleic acid from an environmental sample, the kit comprising the lysis reagent described in the first aspect of the present invention and/or the inhibitor described in the third aspect of the present invention Remove reagents. Optionally or additionally preferably, the kit further includes a reagent for preparing the first washing solution according to the fifth aspect of the present invention. Optionally or additionally preferably, the kit further includes reagents for preparing the second washing solution according to the fifth aspect of the present invention. Optionally or additionally preferably, the kit further includes the grinding beads described in the fifth aspect of the present invention. Optionally or additionally preferably, the kit further includes the magnetic microspheres according to the fifth aspect of the present invention. Optionally or additionally preferably, the kit further comprises reagents for preparing the binding buffer according to the fifth aspect of the present invention (eg chaotropic salts and alcohols, the chaotropic salts and alcohols are preferably as described above. ). Optionally or additionally preferably, the kit further includes reagents (eg Tris-HCl and EDTA) for preparing the TE buffer according to the fifth aspect of the present invention. The reagents mentioned in the sixth aspect of the present invention may be as described in the first to fifth aspects of the present invention, which will not be repeated here.

In the seventh aspect, the present invention provides the lysis reagent according to the first aspect of the present invention, the lysis buffer according to the second aspect of the present invention, and the third aspect of the present invention included in the kit according to the sixth aspect of the present invention. Application of one or more combinations of the inhibitor removal reagent, the inhibitor removal buffer described in the fourth aspect of the present invention, and the grinding beads in DNA extraction or detection of environmental samples; or, the present invention The kit according to the sixth aspect includes the lysis reagent according to the first aspect of the present invention, the lysis buffer according to the second aspect of the present invention, the inhibitor removal reagent according to the third aspect of the present invention, the present invention One or more of the inhibitor removal buffer and the grinding beads described in the fourth aspect are further combined with the binding buffer (or the reagent for preparing the binding buffer), the TE buffer (or the reagent used to formulate the TE buffer), the first wash solution (or the reagent used to formulate the first wash solution), the second wash solution (or the reagent used to formulate the second wash solution) The application of a combination of reagents for washing solutions) in DNA extraction or detection of environmental samples.

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

1) The present invention provides a nucleic acid extraction reagent for microbial DNA of an environmental sample, which can achieve good effects on microbial DNA in samples such as soil, feces, compost, and sludge.

2) The method of the present invention does not require complicated operations, the extraction time is short, and high-quality DNA can be extracted within 30 minutes.

3) The present invention provides a nucleic acid extraction reagent that can almost completely remove PCR inhibitors in environmental samples, so that downstream PCR and NGS experiments can be successfully completed.

Description of drawings

Figure 1 shows the data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents in Example 3 of the present invention for extracting microbial genomic DNA from 6 different types of soil samples and detecting the DNA concentration with Nanodrop.

Figure 2 shows the data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents in Example 3 of the present invention for extracting microbial genomic DNA from 6 different types of soil samples and detecting the DNA concentration with Qubit.

Figure 3 shows the results of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents in Example 3 of the present invention for extracting microbial genomic DNA from 6 different types of soil samples and detecting their DNA purity (A260/280) with Nanodrop data.

Figure 4 shows the results of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents in Example 3 of the present invention for extracting microbial genomic DNA from 6 different types of soil samples and detecting their DNA purity (A260/230) with Nanodrop data.

Figure 5 shows the data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents in Example 4 of the present invention for extracting microbial genomic DNA from stool samples of 6 volunteers and detecting the DNA concentration with Nanodrop.

Figure 6 shows the data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents in Example 4 of the present invention for extracting microbial genomic DNA from stool samples of 6 volunteers and detecting the DNA concentration with Qubit.

Figure 7 shows that the nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents in Example 4 of the present invention extract the microbial genomic DNA from the stool samples of 6 volunteers and use Nanodrop to detect the DNA purity (A260/280) The data.

Figure 8 shows that nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents in Example 4 of the present invention extract microbial genomic DNA from stool samples of 6 volunteers and use Nanodrop to detect the DNA purity (A260/230) The data.

Fig. 9 shows the gel of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagent in Example 4 of the present invention for extracting microbial genomic DNA from stool samples of 6 volunteers and performing 16S rDNA PCR amplification on the DNA picture.

Detailed ways

The present application will be further introduced below with reference to the embodiments.

In order to describe the embodiments of the present invention or the technical solutions in the prior art more clearly, in the following description, different "an embodiment" or "embodiments" do not necessarily refer to the same embodiment. Different embodiments can be replaced or combined, and for those skilled in the art, other implementations can also be obtained according to these embodiments without creative efforts.

definition

Before the present teachings are described in detail, it is to be understood that this disclosure is not limited to particular compositions or process steps, as these may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

When ranges of values are provided herein, unless expressly stated otherwise, the range is intended to include starting and ending values, as well as values or ranges of values therebetween. For example, "from 0.2-0.5" means 0.2, 0.3, 0.4, 0.5; ranges therebetween such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments therebetween such as 0.25, 0.35, 0.220, 0.325, 0.49; Increment range such as 0.26-0.39 and so on.

The term "sample" or "sample" as used herein will be understood to mean any sample that may contain nucleic acid material of interest, the term "sample" or "sample" or may include solutions such as aqueous solutions, cells, tissues, biopsies , powder, or a combination of one or more of them.

It is to be understood that temperatures, masses, weights, volume ratios, concentrations, times, etc. discussed in this disclosure are preceded by the implied "about" such that slight and insubstantial deviations are within the scope of the teachings herein. Generally, the term "about" refers to an insubstantial change in the amounts of the components of the composition that does not have any significant effect on the efficacy or stability of the composition. Also, the use of "comprising", "containing", and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the present teachings. To the extent that any material incorporated by reference is inconsistent with the presentation of the present disclosure, the presentation shall control.

Unless otherwise specified, embodiments in the specification described as "comprising" various components also mean "consisting of" or "consisting essentially of said components in addition to containing the recited components. ” means; an embodiment in the specification described as “consisting of various components” means “comprising” or “consisting essentially of said components” in addition to being expressed only by the recited components .

In this application, "extracting," "separating," or "purifying" means that one or more components of a sample are removed or separated from other sample components. Sample components contain target nucleic acids, often in a generally aqueous solution phase, which may also contain cellular fragments, proteins, carbohydrates, lipids, salt ions, metal ions, and other nucleic acids. "Extraction", "isolation" or "purification" do not imply any degree of purification. Typically, isolation or purification removes at least 70% or at least 80% or at least 90% of the target nucleic acid from other sample components.

For the methods herein, the collected sample is mixed thoroughly to produce a homogeneous sample before a portion is weighed. Use 02 g ± 0.05 g of animal stool samples or environmental samples for use in 2 mL bead tubes. For fecal samples from small animals, an amount of 0.1 ± 0.05 g was used. These sample volumes can be adjusted accordingly depending on the volume of the tubes used in the methods herein.

Purity analysis: In addition to affecting lysis efficiency, the purity of the nucleic acid can also affect downstream analysis, as some methods retain more and different interferents than others. Assays used to characterize DNA or RNA recovered from environmental and biological samples containing microbial populations using the protocols and reagents described herein. A (NanoDrop Spectrophotometer (NanoDrop Technologies Inc) and a Qubit 4.0 Fluorometer (Invitrogen Inc) were used. It was observed that the DNA concentration measured by the spectrophotometer was generally slightly higher than that measured by the Qubit 4.0 Fluorometer. The Qubit 4.0 assay that is specific to DNA is believed to provide a more reliable concentration readout. The A260/280nm ratio as well as the A260/230nm ratio is a measure of purity, pure DNA has an A260280nm ratio of 1.8-1.86, while pure RNA has a ratio of about 2.0 A260/280 ratio. The A260/230nm ratio is a measure of contaminants absorbed at 230nm; the expected A260/230nm ratio is 2.0-2.5 for each of DNA and RNA.

Example

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with the examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the materials can be obtained from commercial sources unless otherwise specified.

Example 1: Preparation of the following reagents (the sum of the reagent components used is collectively referred to as nucleic acid extraction kit A)

This embodiment prepares the following nucleic acid extraction kit A, which comprises the following components:

Lysis buffer: 200 mM tris, 200 mM lithium chloride, 3 M NaSCN, 5% glycerol, 2% lithium dodecyl sulfate, pH 11.0.

Grinding beads: composite grinding beads of 0.25 g of 0.7 mm zirconia beads and 0.25 g of 0.1-0.2 mm glass beads.

IRS buffer: 0.5M potassium aluminum sulfate, 2M sodium acetate and pH adjusted to pH 4 using acetic acid.

Magnetic microspheres: 1.5 mg glass magnetic beads Mag2000 Silica (Cat#BMD00451, 100 mg/mL).

Binding buffer: 3M guanidine cyanogen isosulphate, 0.5M sodium iodide, and 60% isopropanol by volume.

The first washing solution: 2M guanidine cyanate isosulphate, 1M lithium chloride, 0.1M trishydroxymethylaminomethane, 50% ethanol by volume, pH 6.0.

The second washing solution: 0.5M sodium chloride, 0.1M trihydroxymethylaminomethane, the volume ratio is 70% ethanol.

TE Buffer: 10mM Tris-HCl, 1mM EDTA, pH8.5

Example 2: Preparation of the following kit (the sum of the reagent components used is collectively referred to as nucleic acid extraction kit B)

This embodiment provides the following nucleic acid extraction kit B, which comprises the following components:

Lysis buffer: 100 mM tris, 300 mM lithium chloride, 2 M NaSCN, 1% glycerol by volume, 0.5% lithium dodecyl sulfate, pH 9.5.

Grinding beads: composite grinding beads of 0.25g 1mm zirconia beads, 0.25g 0.5mm zirconia beads and 0.5g glass beads of 0.1-0.2.

IRS buffer: 0.2M potassium aluminum sulfate, 0.5M sodium acetate and pH adjusted to pH 6 using acetic acid.

Magnetic Microspheres: 2mg MagH1N Silica (Cat#BMD007511, 60mg/mL)

Binding buffer: 1M guanidine cyanogen isosulphate, 1M sodium iodide, and 30% by volume isopropanol.

The first washing solution: 1M guanidine cyanate isosulphate, 2M lithium chloride, 0.2M trishydroxymethylaminomethane, 60% ethanol by volume, pH 5.0.

The second washing solution: 0.5M sodium chloride, 0.1M trihydroxymethylaminomethane, the volume ratio is 80% ethanol.

TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.5

Example 3: Using nucleic acid extraction kits A and B to extract microbial genomic DNA from 6 different types of soil samples and compare with commercial reagents.

The operation of the nucleic acid extraction kit A in Example 1 is as follows:

(MP Biomedicals)匀浆仪时，速度为6.0m/s，时间为1分钟。1) Weigh 0.25g soil sample, add 0.6mL lysis buffer and grinding beads provided in Example 1, use FastPrep-

(MP Biomedicals) homogenizer with a speed of 6.0 m/s and a time of 1 minute.

2) Centrifuge the above crude lysate at 12,000 rpm for 1 min to take the supernatant, add 0.2 mL of IRS buffer, vortex, centrifuge at 12,000 rpm for 1 min to remove PCR inhibitors, and transfer the supernatant to a clean centrifuge tube middle.

3) Add 1.5 mg of glass magnetic beads Mag2000 Silica and an equal volume of binding buffer, and vortex for 5 min for nucleic acid binding.

4) Use a magnetic stand for magnetic separation, remove the supernatant, add 0.7 mL of the first washing solution, and vortex for 1 min to remove macromolecular impurities.

5) Magnetic separation, remove the supernatant, add 0.7 mL of the second washing solution, and vortex for 1 min to remove inorganic salt impurities.

6) Magnetic separation, remove the supernatant, evaporate the alcohol at room temperature, use 100 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.5) to heat and elute at 60 degrees for 5 min, after magnetic separation, aspirate the supernatant solution to obtain pure DNA.

The operation of the nucleic acid extraction kit B in Example 2 is as follows:

(MP Biomedicals)匀浆仪时，速度为6.0m/s，时间为1分钟。1) Weigh 0.25g soil sample, add 0.5mL lysis buffer and grinding beads provided in Example 1, use FastPrep-

(MP Biomedicals) homogenizer with a speed of 6.0 m/s and a time of 1 minute.

2) Centrifuge the above crude lysate at 12,000 rpm for 1 min to take the supernatant, add 0.25 mL of IRS buffer, vortex, centrifuge at 12,000 rpm for 1 min to remove PCR inhibitors, and transfer the supernatant to a clean centrifuge tube middle.

3) Add 2 mg of MagH1N Silica and an equal volume of binding buffer, and vortex for 5 min for nucleic acid binding.

4) Use a magnetic stand for magnetic separation, remove the supernatant, add 0.5 mL of the first washing solution, and vortex for 1 min to remove macromolecular impurities.

5) Magnetic separation, remove the supernatant, add 0.5 mL of the second washing solution, and vortex for 1 min to remove inorganic salt impurities.

6) Magnetic separation, remove the supernatant, evaporate the alcohol at room temperature, use 100 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.5) to heat and elute at 60 degrees for 5 min, after magnetic separation, aspirate the supernatant solution to obtain pure DNA.

Pro Kit(Qiagen,Cat#47014)，称取同样质量的土壤样本并按照试剂盒提供的说明书(Protocol)进行核酸提取。The commercial control nucleic acid extraction reagents are:

Pro Kit (Qiagen, Cat#47014), weigh soil samples of the same quality and perform nucleic acid extraction according to the instructions (Protocol) provided by the kit.

Pro Kit明显要高，尤其是对于高腐殖酸和高杂质的森林土和蚯蚓土，说明本发明的核酸提取试剂对于复杂的土壤样本处理能力更强。从图3和图4的Nanodrop纯度比较，我们发现核酸提取试剂盒A和核酸提取试剂盒B对于森林土和蚯蚓土的纯度要高于商品化试剂，其余的4个样本与商品化试剂接近。As a result, the comparison of Nanodrop and Qubit concentration data in Figure 1 and Figure 2 shows that the nucleic acid yield of nucleic acid extraction kit A and nucleic acid extraction kit B is relatively commercialized

The Pro Kit is obviously higher, especially for forest soil and earthworm soil with high humic acid and high impurities, indicating that the nucleic acid extraction reagent of the present invention is more capable of processing complex soil samples. From the comparison of Nanodrop purity in Figure 3 and Figure 4, we found that the purity of nucleic acid extraction kit A and nucleic acid extraction kit B for forest soil and earthworm soil is higher than that of commercial reagents, and the remaining 4 samples are close to commercial reagents.

Example 4: Using nucleic acid extraction kits A and B to extract microbial genomic DNA from 6 different stool samples (6 volunteers) and compare with commercial reagents.

Fast DNA Stool Mini Kit(Qiagen,Cat#51604)，称取同样质量的粪便样本并按照试剂盒的说明书(Protocol)进行核酸提取。The operation process of nucleic acid extraction kits A and B for fecal samples is the same as that of Example 3 for soil samples, and the commercial extraction reagents are

Fast DNA Stool Mini Kit (Qiagen, Cat#51604), weigh stool samples of the same quality and perform nucleic acid extraction according to the kit's instructions (Protocol).

Fast DNA Stool Mini Kit高出1倍多。从图7和图8的Nanodrop纯度比较，我们发现核酸提取试剂盒A和核酸提取试剂盒B对于粪便样本的纯度要高于商品化试剂。我们进一步通过16S rDNA PCR扩增进行验证，使用16S通用引物，商品化2x PCR Master Mix，在20μL体系中我们加入4μL DNA模板产物，如图9所述，发现核酸提取试剂盒A和B都可以很好进行扩增，而商品化试剂提取的DNA模板明显有含有PCR抑制剂，样本3和4的DNA产物中PCR抑制剂的含量过高导致无法进行有效的扩增。As a result, the comparison of Nanodrop and Qubit concentration data in Figure 5 and Figure 6 shows that the nucleic acid yield of nucleic acid extraction kit A and nucleic acid extraction kit B is relatively commercialized

Fast DNA Stool Mini Kit is more than 1x higher. From the comparison of Nanodrop purity in Figure 7 and Figure 8, we found that the purity of nucleic acid extraction kit A and nucleic acid extraction kit B for stool samples was higher than that of commercial reagents. We further verified by 16S rDNA PCR amplification, using 16S universal primers, commercial 2x PCR Master Mix, and adding 4 μL DNA template product to the 20 μL system, as described in Figure 9, and found that both nucleic acid extraction kits A and B can Amplification is very good, but the DNA templates extracted by commercial reagents obviously contain PCR inhibitors. The content of PCR inhibitors in the DNA products of samples 3 and 4 is too high, so that effective amplification cannot be performed.

The descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The above description of the disclosed embodiments enables any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

It should be noted that the above embodiments can be freely combined as required. The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. a cleavage reagent, is characterized in that, described cleavage reagent comprises the following components: trihydroxymethylaminomethane, lithium chloride, NaSCN, glycerol and lithium dodecyl sulfate. 2. a lysis buffer, is characterized in that: The lysis buffer is prepared by the reagent of claim 1; Preferably, the lysis buffer contains 50-500mM tris, 100-500mM lithium chloride, 1-5M NaSCN, 0.5-20% glycerol, 0.1-5% lithium dodecyl sulfate, and the pH is 8.0-12.5; More preferably, the lysis buffer contains 100-200 mM tris, 200-300 mM lithium chloride, 2-3 M NaSCN, 1-5% glycerol, 0.5-2 % lithium dodecyl sulfate, and the pH is 8.5-11.5; Further preferably, the lysis buffer contains 200 mM tris(hydroxymethyl)aminomethane, 200 mM lithium chloride, 3M NaSCN, 5% glycerol, 2% lithium dodecyl sulfate in a mass-to-volume ratio, and the pH is 11.0. 3. An inhibitor removal reagent, characterized in that, the inhibitor removal comprises the following components: aluminum potassium sulfate, sodium acetate and acetic acid. 4. an inhibitor removal buffer, is characterized in that: The inhibitor removal buffer is prepared from the inhibitor removal reagent of claim 3; Preferably, the inhibitor removal buffer contains 0.1-2M potassium aluminum sulfate, 0.5-5M sodium acetate, and the pH is adjusted to pH 3.5-7.5 using acetic acid; More preferably, the inhibitor removal buffer contains 0.2-1M potassium aluminum sulfate, 0.5-2M sodium acetate, and the pH is adjusted to pH 4-6 using acetic acid; It is further preferred that the inhibitor removal buffer contains 0.5M potassium aluminum sulfate, 2M sodium acetate, and the pH is adjusted to pH 4 using acetic acid. 5. a nucleic acid extraction method, is characterized in that, described method comprises the steps: S1: mechanically lysing the environmental sample with grinding beads, and chemically lysing the environmental sample with a lysis buffer, thereby obtaining a crude lysate; S2: centrifuge the crude lysate to obtain a supernatant, add inhibitor removal buffer to the supernatant to remove PCR inhibitors contained in the environmental sample, and centrifuge to obtain a crude DNA product; S3: use magnetic microspheres and a binding buffer to bind the DNA in the crude DNA product to obtain a bound DNA product; S4: washing with washing solution to obtain washing DNA product; S5: Use TE buffer to heat and elute the washed DNA product to obtain the final DNA. 6. method according to claim 5, is characterized in that, in step S1: The grinding beads are zirconia beads of 0.1-3mm, glass beads of 0.1-5mm, garnets of 0.2-1.5mm, silicon carbide of 0.2-0.5mm, metal balls of 0.3-4.5mm and/or combinations thereof; preferably Preferably, the grinding beads are 0.1-3mm zirconia beads, 0.1-2mm glass beads, 0.3-1.0mm garnet and/or combinations thereof; more preferably, the grinding beads are 0.1-3mm oxide beads Zirconium beads, 0.1-1mm glass beads and/or combinations thereof; Also preferably, the lysis buffer is prepared from the lysis reagent described in claim 1 or the lysis buffer described in claim 2 . 7. The method according to claim 5 or 6, characterized in that, in step S2: The inhibitor removal buffer is prepared from the inhibitor removal reagent of claim 3, or the inhibitor removal buffer of claim 4. 8. The method according to any one of claims 5 to 7, wherein in step S3: The magnetic microspheres are silicon hydroxyl magnetic beads, glass magnetic beads, carboxyl magnetic beads, amino magnetic beads, tosyl magnetic beads, epoxy magnetic beads and/or combinations thereof; preferably, the magnetic microspheres are Silanol magnetic beads, glass magnetic beads, carboxyl magnetic beads and/or combinations thereof; more preferably, the magnetic microspheres are magnetic beads series products produced by Suzhou Cretaceous Biotechnology Co., Ltd., such as Silanol Magnetic Beads MagH1N Silica(Cat#BMD007511), Silica MagH1 Silica(Cat#BMD003511), Glass Mag2000 Silica(Cat#BMD004511); Also preferably, the binding buffer is a mixture of chaotropic salt and alcohol; more preferably, the chaotropic salt is guanidine hydrochloride, potassium thiocyanate, guanidine isothiocyanate, sodium thiocyanate, sulfur Potassium cyanate, sodium iodide, sodium perchlorate and/or a combination thereof, and/or the alcohol is ethanol or isopropanol; further preferably, the binding buffer contains 1-5M guanidine cyanogen isosulphate, 0.2-2M sodium iodide and a volume ratio of 30-70% isopropanol; it is further preferred that the binding buffer contains 2-3M cyanoguanidine isosulphate, 0.5-2M sodium iodide and a volume ratio of 30 -60% isopropyl alcohol. 9. The method according to any one of claims 5 to 8, wherein in step S4: The cleaning is performed by using a first washing solution and a second washing solution in sequence, and the first washing solution contains 0.5-4 cyanoguanidine isosulphate, 0.5-3M lithium chloride, 0.1-0.5M trihydroxymethylaminomethane , the volume ratio is 30-70% ethanol, and the pH is 5.0-8.0, the second washing solution is 0.1-1M sodium chloride, 0.1-0.2M trihydroxymethylaminomethane and the volume ratio is 50-90% ethanol ; Preferably, the first washing solution contains 1-2M cyanoguanidine isosulphate, 1-2M lithium chloride, 0.1-0.2M trihydroxymethylaminomethane and 50-60% ethanol by volume, and has a pH of 5.0-8.0, the second washing solution is 0.5M sodium chloride, 0.1M trihydroxymethylaminomethane and the volume ratio is 70-80% ethanol. 10. A kit for extracting nucleic acid in environmental samples, characterized in that the kit comprises: the lysis reagent according to claim 1; and/the inhibitor removal reagent according to claim 3; Preferably, the kit further includes one or more of the following reagents: (1) a reagent for preparing the first washing solution according to claim 9; (2) a reagent for preparing the first washing solution according to claim 9; (3) the grinding beads described in claim 6; (4) the magnetic microspheres described in claim 8; (5) used to prepare the binding buffer described in claim 8 (6) a reagent for preparing the TE buffer of claim 10.

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