CN114292841A - Nucleic acid extraction reagent and nucleic acid extraction method - Google Patents
Nucleic acid extraction reagent and nucleic acid extraction method Download PDFInfo
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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 invention relates to a cracking reagent containing trihydroxymethyl aminomethane, lithium chloride, NaSCN, glycerol and lithium dodecyl sulfate, an inhibitor removing reagent containing aluminum potassium sulfate, sodium acetate and acetic acid, a corresponding buffer solution prepared from the inhibitor removing reagent, and application of the corresponding buffer solution in extraction of DNA of an environmental sample. The invention also relates to a nucleic acid extraction method using the lysis reagent and/or the inhibitor removal reagent. The nucleic acid extraction reagent provided by the invention is simple and rapid to operate, and can realize high-quality extraction of microbial DNA in different environmental samples.
Description
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 from the environment, in particular samples containing microorganisms.
Background
The research of microbial community in environmental samples requires the extraction of nucleic acid from biological samples, and the extraction and analysis of nucleic acid from such samples are very challenging, mainly for several reasons. 1) Environmental samples such as soil, manure, compost, sludge, wastewater and the like contain various PCR inhibitors, for example: the PCR reaction is strongly inhibited by the extremely small amount of heavy metals, polysaccharides, polyphenols, cholate, humic acid, etc. For example, soil samples are rich in humic acid, fulvic acid and humins, and the DNA solution obtained by the conventional method for extracting soil microorganisms is yellowish brown and is polluted by the substances. 2) The microbial organisms in environmental samples such as soil, excrement, wastewater and the like are more difficult to crack than the microorganisms which are simply cultured, and the conventional extraction method has low microbial cell cracking rate, so the obtained DNA cannot reflect the species abundance in the original sample.
Therefore, there is a high need for efficient and practical basic research tools and reagents to ensure accurate characterization of the composition of a microbial community, to study the function of interdependencies within a microbial community, and to allow manipulation of interactions within a microbial community and between a microbial community and its host. Next-generation tools, such as isolation and purification kits for microbial nucleic acids, are urgently needed.
The kits currently available on the market for isolating DNA from environmental and biological samples include those provided by MO BIO Laboratories Inc
DNA Isolation Kit，Power
DNA Isolation Kit and
DNA Isolation kit, Qiagen company provides
Pro DNA Kit、
Fast DNA Stool Mini Kit、
Pro Kit and
kit, however, is very cumbersome to operate, requires repeated centrifugation, and is very expensive to sell, and the extraction of a single sample is 30-50 RMB, and different extraction kits are required to be adapted to different samples to operate.
The invention of Chinese patent application CN 107475249A proposes a method for extracting microorganism total DNA from soil with low biomass and rich humus by liquid nitrogen grinding and proteinase K digestion, but the operation still needs the soil DNA extraction kit of Qiagen company to further purify the roughly extracted DNA.
In summary, although the prior art cannot provide a universal, simple and convenient kit to solve the problem of high-quality extraction of microbial genome DNA in various complex environmental samples, the method can obtain high-purity, high-integrity and high-quality microbial genome DNA.
Disclosure of Invention
The nucleic acid extraction reagent provided by the invention is simple and quick to operate, and can realize 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 in a first aspect a lysis reagent comprising the following components: trihydroxymethyl aminomethane, lithium chloride, NaSCN, glycerol and lithium dodecyl sulfate.
In a second aspect, the present invention provides a lysis buffer formulated from a lysis reagent according to the first aspect of the present invention.
Preferably, the lysis buffer comprises 50-500mM of trihydroxymethyl aminomethane, 100-500mM of lithium chloride, 1-5M of NaSCN, 0.5-20% by mass volume of glycerol, 0.1-5% of lithium dodecyl sulfate, and a pH of 8.0-12.5.
More preferably, the lysis buffer comprises 100-200mM of trihydroxymethylaminomethane, 200-300mM of lithium chloride, 2-3M of NaSCN, 1-5% by mass/volume of glycerol, 0.5-2% of lithium dodecyl sulfate, and a pH of 8.5-11.5.
It is further preferred that the lysis buffer comprises 200mM tris, 200mM lithium chloride, 3M NaSCN, 5% glycerol by mass/volume, 2% lithium dodecyl sulfate, and a pH of 11.0.
The present invention provides, in a third aspect, an inhibitor-removing agent comprising the following components: potassium aluminum sulfate, sodium acetate and acetic acid.
In a fourth aspect, the present invention provides an inhibitor-removing buffer prepared from the inhibitor-removing reagent according to the third aspect of the present invention.
Preferably, the inhibitor removal buffer comprises 0.1-2M potassium aluminum sulfate, 0.5-5M sodium acetate, and acetic acid is used to adjust the pH to 3.5-7.5.
More preferably, the inhibitor removal buffer comprises 0.2-1M aluminum potassium sulfate, 0.5-2M sodium acetate, and acetic acid is used to adjust the pH to pH 4-6.
It is further preferred that the inhibitor removal buffer comprises 0.5M potassium aluminum sulfate, 2M sodium acetate and acetic acid is used to adjust the pH to pH 4.
The present invention provides, in a fifth aspect, a method for nucleic acid extraction, the method comprising the steps of:
s1: mechanically lysing an environmental sample using grinding beads, and chemically lysing the environmental sample using a lysis buffer, thereby obtaining a crude lysate;
s2: centrifuging the crude lysate to obtain a supernatant, adding an inhibitor removal buffer to the supernatant to remove PCR inhibitors contained in the environmental sample, and centrifuging to obtain a crude DNA product;
s3: binding DNA in the crude DNA product by using magnetic microspheres and a binding buffer solution to obtain a bound DNA product;
s4: washing with a washing solution to obtain a washed DNA product;
s5: and (3) heating and eluting the washing DNA product by using TE buffer solution to obtain the final DNA.
The invention has no special requirement on the rotating speed and time of the centrifugation. In the context of the application, the centrifugation involved in the steps of the method of the invention is carried out at 6000-12000rpm for a period of 1-2min, unless otherwise stated. Preferably, in step S1, the grinding beads are zirconia beads of 0.1 to 3mm, glass beads of 0.1 to 5mm, garnet of 0.2 to 1.5mm, silicon carbide of 0.2 to 0.5mm, metal balls of 0.3 to 4.5mm and/or combinations thereof. Preferably, the grinding beads are zirconia beads of 0.1 to 3mm, glass beads of 0.1 to 2mm, garnet of 0.3 to 1.0mm and/or combinations thereof. More preferably, the grinding beads are zirconia beads of 0.1 to 3mm, glass beads of 0.1 to 1mm and/or combinations thereof.
It is further preferred that the lysis buffer is prepared from a lysis reagent according to the first aspect of the invention or is a lysis buffer according to the second aspect of the invention.
Preferably, in step S2, the inhibitor removal buffer is prepared from the inhibitor removal buffer reagent according to the third aspect of the present invention, or is the inhibitor removal buffer according to 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 a combination thereof; further preferably, the magnetic microspheres are a series of magnetic beads produced by Suzhou Chalkbrook Biotechnology Co., Ltd, such as Silica hydroxyl magnetic bead MagH1N Silica (Cat # BMD007511), Silica hydroxyl magnetic bead MagH1Silica (Cat # BMD003511), and glass magnetic bead Mag2000 Silica (Cat # BMD 004511).
It is also preferred that, in step S3, the binding buffer is a mixture of a chaotropic salt and an 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 comprises 1-5M cyanoguanidine iso-sulfate, 0.2-2M sodium iodide and 30-70% by volume isopropanol. It is still further preferred that the binding buffer comprises 2-3M cyanoguanidine isothiocyanate, 0.5-2M sodium iodide and 30-60% by volume isopropanol.
Preferably, in step S4, the cleaning is performed by using a first cleaning solution and a second cleaning solution in sequence, and the first cleaning solution comprises 0.5-4% of cyanoguanidine iso-sulfate, 0.5-3M of lithium chloride, 0.1-0.5M of trihydroxy methyl aminomethane, 30-70% of ethanol by volume ratio, and has a pH of 5.0-8.0, and the second cleaning solution comprises 0.1-1M of sodium chloride, 0.1-0.2M of trihydroxy methyl aminomethane, and 50-90% of ethanol by volume ratio;
preferably, the first wash solution comprises 1-2M cyanoguanidine iso-sulfate, 1-2M lithium chloride, 0.1-0.2M trihydroxymethylaminomethane and 50-60% by volume ethanol, and has a pH of 5.0-8.0, and the second wash solution comprises 0.5M sodium chloride, 0.1M trihydroxymethylaminomethane and 70-80% by volume ethanol.
Preferably, in step S5, the TE buffer contains 10mM Tris-HCl and 1mM EDTA, and has a pH of 8.5. More preferably, the heating temperature for the heated elution is 55 ℃ to 65 ℃ (e.g., 60 ℃). It is further preferred that the heating elution is further preceded by a step of volatilizing the alcohol.
In some more specific embodiments, the method comprises the steps of:
s1: providing a lysis buffer and milling beads for use therewith, and simultaneously chemically and mechanically lysing microorganisms in an environmental sample using a tissue milling apparatus to produce a crude lysate;
s2: centrifuging the crude lysate (taking supernatant, adding IRS buffer Solution (Inhibitor Removal Solution), removing PCR Inhibitor in an environmental sample after vortex oscillation, and centrifuging to obtain a crude DNA product which does not contain Inhibitor basically;
s3: binding the crude DNA product by using magnetic microspheres and a binding buffer solution to obtain a bound DNA product;
s4: washing with the first washing solution for 1 time, and washing with the second washing solution for 1 time;
s5: after the alcohol was evaporated, the elution was carried out by heating using TE Buffer to obtain the final (pure) DNA.
The present invention has no particular requirement on the condition of vortex oscillation. In the context of the present application, unless otherwise indicated, the vortex conditions at a speed of 1000rpm for a period of 1min are used for the vortex oscillations involved in the steps of the process of the present invention.
In step S1, the lysis buffer can dissolve impurities and release nucleic acids in the sample by using a specific reagent combination and a specific ratio and a suitable pH. Wherein the NaSCN and the glycerol are used for dissolving PCR inhibitors in environmental samples such as humic acid, bile acid and the like.
In other more specific embodiments, the method comprises the steps of:
s1: providing a lysis buffer (100-;
s2: centrifuging the crude lysate to obtain a supernatant, adding an IRS buffer solution (100 plus 250mM aluminum potassium sulfate, 200 plus 500mM sodium acetate and adjusting the pH to 4-6 by using acetic acid), removing the PCR inhibitor in the environmental sample after vortex oscillation, and centrifuging to obtain a crude DNA product which does not contain the inhibitor basically;
s3: binding the DNA by using magnetic microspheres (silicon hydroxyl magnetic beads, glass magnetic beads, carboxyl magnetic beads and/or combinations thereof) and binding buffer solution (2-3M of guanidine isocyanate sulfate, 1-2M of sodium iodide and 30-60% of isopropanol by volume) to obtain a bound DNA product;
s4: washing with a first washing solution (1-2M cyanoguanidine isothiocyanate, 1-2M lithium chloride, 0.1-0.2M trihydroxymethyl aminomethane, 50-60% ethanol by volume, pH 5.0-8.0) for 1 time, and washing with a second washing solution (0.2M sodium chloride, 0.1M trihydroxymethyl aminomethane, 70-80% ethanol by volume) for 1 time to obtain a washed DNA product;
s5: after the alcohol was evaporated, TE Buffer (10mM Tris-HCl, 1mM EDTA, pH8.5) was used for heat elution to obtain pure DNA.
In other more specific embodiments, the method comprises the following steps in sequence:
1) weighing 0.05-0.5g of environmental sample (the environmental sample can be selected from one or more samples of soil, feces, filtered wastewater membrane, biofilm, etc.), adding 0.4-1mL of lysis buffer and 0.25-2g of grinding beads, and performing mechanical lysis by using a bead mill, wherein the grinding speed is 4-6m/s, and the grinding time is 1-3 minutes.
2) And centrifuging the crude lysate to obtain a supernatant, adding 0.1-0.4mL of IRS buffer solution, performing vortex oscillation, centrifuging to remove the PCR inhibitor, and transferring the supernatant into 1 clean centrifuge tube.
3) Adding 1-2mg magnetic microspheres and an equal volume of binding buffer solution, and performing vortex oscillation for 5min to perform nucleic acid binding.
4) Performing magnetic separation, removing supernatant, adding 0.5-1mL of first washing solution, and performing vortex oscillation for 1min to remove macromolecular impurities.
5) Performing magnetic separation, removing supernatant, adding 0.5-1mL of second washing solution, and performing vortex oscillation for 1min to remove inorganic salt impurities.
6) Magnetic separation, removing supernatant, volatilizing at room temperature to remove alcohol, eluting with 50-200 μ L TE Buffer (10mM Tris-HCl, 1mM EDTA, pH8.5) at 60 deg.C for 5min, and magnetically separating, and sucking supernatant 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 a lysis reagent according to the first aspect of the invention and/or an inhibitor removal reagent according to the third aspect of the invention. Alternatively or additionally preferably, the kit further comprises reagents for formulating the first wash solution according to the fifth aspect of the invention. Alternatively or additionally preferably, the kit further comprises reagents for formulating a second wash solution according to the fifth aspect of the invention. Alternatively or additionally preferably, the kit further comprises a grinding bead according to the fifth aspect of the invention. Alternatively or additionally preferably, the kit further comprises magnetic microspheres according to the fifth aspect of the invention. Alternatively or additionally preferably, the kit further comprises reagents for formulating a binding buffer according to the fifth aspect of the invention (e.g. chaotropic salts and alcohols, preferably as described above). Alternatively or additionally preferably, the kit further comprises reagents (e.g. Tris-HCl and EDTA) for formulating the TE buffer according to the fifth aspect of the 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, and will not be described herein again.
In a seventh aspect, the present invention provides the use of one or more of the lysis reagent of the first aspect of the present invention, the lysis buffer of the second aspect of the present invention, the inhibitor-removing reagent of the third aspect of the present invention, the inhibitor-removing buffer of the fourth aspect of the present invention, and the grinding beads contained in the kit of the sixth aspect of the present invention in the extraction or detection of DNA from an environmental sample; alternatively, the kit of the sixth aspect of the present invention comprises one or more of the lysis reagent of the first aspect of the present invention, the lysis buffer of the second aspect of the present invention, the inhibitor removal reagent of the third aspect of the present invention, the inhibitor removal buffer of the fourth aspect of the present invention, and the grinding beads in combination, further comprising a binding buffer (or a reagent for preparing the binding buffer), a TE buffer (or a reagent for preparing the TE buffer), a first wash solution (or a reagent for preparing the first wash solution), and a second wash solution (or a reagent for preparing the second wash solution) for use in DNA extraction or detection of an environmental sample.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention provides a nucleic acid extraction reagent for microbial DNA of an environmental sample, which can achieve better effect on microbial DNA in samples such as soil, excrement, compost, sludge and the like.
2) The method does not need complicated operation, has short extraction time, and can extract high-quality DNA within 30 minutes.
3) The invention provides a nucleic acid extraction reagent which can almost completely remove a PCR inhibitor in an environmental sample, so that downstream PCR and NGS experiments are successfully completed.
Drawings
FIG. 1 shows data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents according to example 3 of the present invention, in which genomic DNA of microorganisms in 6 different types of soil samples was extracted and DNA concentration thereof was detected by Nanodrop.
FIG. 2 shows data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents according to example 3 of the present invention, in which genomic DNAs of microorganisms were extracted from 6 different types of soil samples and their DNA concentrations were measured by using a Qubit.
FIG. 3 shows data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents according to example 3 of the present invention, in which genomic DNA of microorganisms was extracted from 6 different types of soil samples and DNA purity thereof was measured using Nanodrop (A260/280).
FIG. 4 shows data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents according to example 3 of the present invention, in which genomic DNA of microorganisms was extracted from 6 different types of soil samples and DNA purity thereof was measured by Nanodrop (A260/230).
FIG. 5 shows data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents according to example 4 of the present invention, in which genomic DNA of microorganisms in 6 human fecal samples was extracted and DNA concentration thereof was measured by Nanodrop.
FIG. 6 shows data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents according to example 4 of the present invention, in which genomic DNA of microorganisms in feces samples of 6 volunteers was extracted and DNA concentration thereof was detected by using a Qubit.
FIG. 7 shows data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents according to example 4 of the present invention, in which genomic DNA of microorganisms in feces samples of 6 volunteers was extracted and DNA purity (A260/280) thereof was measured by Nanodrop.
FIG. 8 shows data of nucleic acid extraction kit A, nucleic acid extraction kit B and commercial reagents according to example 4 of the present invention, in which genomic DNA of microorganisms in feces samples of 6 volunteers was extracted and DNA purity thereof was measured by Nanodrop (A260/230).
FIG. 9 shows a gel diagram of the extraction of microbial genomic DNA and 16S rDNA PCR amplification of DNA from 6 human volunteers feces samples using the nucleic acid extraction kit A, the nucleic acid extraction kit B and the commercial reagents of example 4 of the present invention.
Detailed Description
The present application is further described below with reference to examples.
In the following description, different "one embodiment" or "an embodiment" may not necessarily refer to the same embodiment, in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art. Various embodiments may be replaced or combined, and other embodiments may be obtained according to the embodiments without creative efforts for those skilled in the art.
Definition of
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, the ranges are intended to include the starting and ending values, as well as the values or ranges of values therebetween, unless expressly stated otherwise. For example, "from 0.2 to 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 therein such as 0.25, 0.35, 0.220, 0.325, 0.49; and increments therein ranging from 0.26-0.39 and the like.
The term "sample" or "specimen" as used herein will be understood to mean any such sample: the term "sample" or "specimen" may include a sample that may contain the nucleic acid species of interest, or may include a solution, such as an aqueous solution, cells, tissue, biopsy, powder, or a combination of one or more thereof.
It is understood that there is an implied "about" before the temperatures, masses, weights, volume ratios, concentrations, times, etc. discussed in this disclosure such that slight and insubstantial deviations are within the scope of the teachings herein. Generally, the term "about" refers to insubstantial changes in the amounts of the components of the composition, which do not have any significant effect on the effectiveness or stability of the composition. Also, the use of "including," "containing," and "including" is 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 does not conform to the teachings of the present disclosure, that description controls.
Unless otherwise indicated, embodiments in the specification that are described as "comprising" various components mean "consisting of or" consisting essentially of the recited components in addition to the recited components; embodiments in the specification that are described as "consisting of various components" mean "including" or "consisting essentially of" the recited components in addition to the recited components alone.
In this application, "extraction," "isolation," or "purification" refers to the removal of one or more components of a sample or the separation from other sample components. The sample components comprise target nucleic acids, often in a generally aqueous solution phase, which may also comprise cell fragments, proteins, carbohydrates, lipids, salt ions, metal ions, and other nucleic acids. "extraction", "isolation" or "purification" does not imply any degree of purification. Typically, the 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 well to produce a homogenous sample before weighing a portion. 02 g. + -. 0.05g of stool or environmental samples from the animals were used for use in 2mL beads. For the faecal samples of the small animals, an amount of 0.1. + -. 0.05g was used. These sample volumes can be adjusted accordingly based on the volume of the tube used in the methods herein.
Purity analysis: in addition to affecting the efficiency of lysis, the purity of the nucleic acids can also affect downstream analysis, as some methods retain more and different interferents than others. Assays used to characterize DNA or RNA include those that are recovered from environmental and biological samples containing a collection of microorganisms using the protocols and reagents described herein. Using (NanoDrop spectrophotometers (NanoDrop Technologies Inc) and Qubit 4.0 fluorometers (Invitrogen Inc) it has been observed that the DNA concentration measured by the spectrophotometer is typically slightly higher than that measured by the Qubit 4.0 fluorometer the Qubit 4.0 assay specific to DNA is believed to provide a more reliable concentration readout the a260/280nm ratio and 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 2.0a260/280, the a260/230nm ratio is a measure of the contaminant absorbed at 230nm, the expected a260/230nm ratio for each of DNA and RNA is 2.0-2.5.
Examples
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the materials are commercially available, unless otherwise specified.
Example 1: the following reagents were prepared (wherein the total sum of the reagent components used is referred to as a nucleic acid extraction kit A)
This example formulated the following nucleic acid extraction kit a, which contained the following components:
lysis buffer: 200mM trihydroxymethyl aminomethane, 200mM lithium chloride, 3M NaSCN, 5% glycerol by mass and volume, 2% lithium dodecyl sulfate, pH 11.0.
Grinding beads: 0.25g of 0.7mm zirconia beads and 0.25g of 0.1-0.2mm glass beads.
IRS buffer solution: 0.5M aluminum potassium sulfate, 2M sodium acetate and acetic acid was used to adjust the pH to 4.
Magnetic microspheres: 1.5mg of glass magnetic beads Mag2000 Silica (Cat # BMD00451, 100 mg/mL).
Binding buffer: 3M cyanoguanidine iso-sulfate, 0.5M sodium iodide and 60% by volume isopropanol.
A first washing solution: 2M cyanoguanidine iso-sulfate, 1M lithium chloride, 0.1M trihydroxymethyl aminomethane, 50% ethanol by volume, pH 6.0.
A second washing solution: 0.5M sodium chloride, 0.1M trihydroxymethyl aminomethane, volume ratio is 70% ethanol.
TE Buffer：10mM Tris-HCl，1mM EDTA，pH8.5
Example 2: the following kit (wherein the total sum of the reagent components used is referred to as nucleic acid extraction kit B)
This example provides a nucleic acid extraction kit B comprising the following components:
lysis buffer: 100mM trihydroxymethyl aminomethane, 300mM lithium chloride, 2M NaSCN, 1% glycerol by mass and volume, 0.5% lithium dodecyl sulfate, pH 9.5.
Grinding beads: 0.25g of 1mm zirconia beads, 0.25g of 0.5mm zirconia beads and 0.5g of 0.1 to 0.2 glass beads.
IRS buffer solution: 0.2M aluminum potassium sulfate, 0.5M sodium acetate and acetic acid was used to adjust the pH to pH6.
Magnetic microspheres: 2mg MagH1N Silica (Cat # BMD007511,60mg/mL)
Binding buffer: 1M cyanoguanidine iso-sulfate, 1M sodium iodide and 30% by volume isopropanol.
A first washing solution: 1M of cyanoguanidine iso-sulfate, 2M of lithium chloride, 0.2M of trihydroxymethyl aminomethane, 60% by volume of ethanol, pH 5.0.
A second washing solution: 0.5M sodium chloride, 0.1M trihydroxymethyl aminomethane, 80% ethanol by volume.
TE buffer solution: 10mM Tris-HCl, 1mM EDTA, pH8.5
Example 3: the microbial genomic DNA from 6 different types of soil samples was extracted using nucleic acid extraction kits A and B and compared with commercial reagents.
The operation of the nucleic acid extraction kit a in example 1 is as follows:
1) 0.25g of soil sample was weighed, 0.6mL of lysis buffer and the milling beads provided in example 1 were added, FastPrep-
(MP Biomedicals) homogenizer speed 6.0m/s for 1 minute.
2) Centrifuging the crude lysate at 12000rpm for 1min, collecting the supernatant, adding 0.2mL IRS buffer solution, vortexing, centrifuging at 12000rpm for 1min to remove PCR inhibitor, and transferring the supernatant to 1 clean centrifuge tube.
3) 1.5mg of glass magnetic beads Mag2000 Silica and an equal volume of binding buffer were added and nucleic acid binding was performed by vortexing and vortexing for 5 min.
4) And (3) magnetically separating by using a magnetic frame, removing supernatant, adding 0.7mL of first washing liquid, and performing vortex oscillation for 1min to remove macromolecular impurities.
5) And magnetically separating, removing supernatant, adding 0.7mL of second washing solution, and performing vortex oscillation for 1min to remove inorganic salt impurities.
6) Magnetic separation, removing supernatant, volatilizing alcohol at room temperature, heating and eluting with 100 μ L TE buffer (10mM Tris-HCl, 1mM EDTA, pH8.5) at 60 deg.C for 5min, and magnetic separation, and sucking supernatant to obtain pure DNA.
The operation of the nucleic acid extraction kit B in example 2 is as follows:
1) 0.25g of soil sample was weighed, 0.5mL of lysis buffer and the milling beads provided in example 1 were added, FastPrep-
(MP Biomedicals) homogenizer speed 6.0m/s for 1 minute.
2) Centrifuging the crude lysate for 1min at 12000rpm, taking supernatant, adding 0.25mL IRS buffer solution, performing vortex oscillation, centrifuging at 12000rpm for 1min, removing PCR inhibitor, and transferring the supernatant to 1 clean centrifuge tube.
3) Nucleic acid binding was performed by adding 2mg of MagH1N Silica and an equal volume of binding buffer and vortexing for 5 min.
4) And (3) magnetically separating by using a magnetic frame, removing supernatant, adding 0.5mL of first washing liquid, and performing vortex oscillation for 1min to remove macromolecular impurities.
5) And magnetically separating, removing supernatant, adding 0.5mL of second washing solution, and performing vortex oscillation for 1min to remove inorganic salt impurities.
6) Magnetic separation, removing supernatant, volatilizing alcohol at room temperature, heating and eluting with 100 μ L TE buffer (10mM Tris-HCl, 1mM EDTA, pH8.5) at 60 deg.C for 5min, and magnetic separation, and sucking supernatant to obtain pure DNA.
Commercial control nucleic acid extraction reagents were:
pro Kit (Qiagen, Cat #47014), soil samples of the same mass were weighed and subjected to nucleic acid extraction according to the instructions (Protocol) provided in the Kit.
As a result, as shown in the comparison of the Nanodrop and the Qubit concentration data in FIGS. 1 and 2, the nucleic acid yield of the nucleic acid extraction kit A and the nucleic acid extraction kit B was relatively commercialized
The Pro Kit is obviously higher, especially for forest soil and earthworm soil with high humic acid and high impurities, which shows that the nucleic acid extraction reagent has stronger processing capability on complex soil samples. From the Nanodrop purity comparison of fig. 3 and 4, we found that the purity of the nucleic acid extraction kit a and the nucleic acid extraction kit B was higher than that of the commercial reagents for forest soil and earthworm soil, and the remaining 4 samples were close to those of the commercial reagents.
Example 4: microbial genomic DNA was extracted from 6 different stool samples (6 volunteers) using nucleic acid extraction kits a and B and compared to commercial reagents.
The operation flow of the nucleic acid extraction kit A and B on the fecal samples is the same as that of the soil samples in example 3, and the commercial extraction reagent is
Fast DNA Stool Mini Kit (Qiagen, Cat #51604), Stool samples of the same mass were weighed and subjected to nucleic acid extraction according to the Kit's instructions (Protocol).
As a result, as shown in comparison of the Nanodrop and the Qubit concentration data in FIGS. 5 and 6, the nucleic acid yield of the nucleic acid extraction kit A and the nucleic acid extraction kit B was relatively commercialized
Fast DNA pool Mini Kit was more than 1-fold higher. From the Nanodrop purity comparison of fig. 7 and 8, we found that the purity of the nucleic acid extraction kit a and the nucleic acid extraction kit B was higher for the stool samples than for the commercial reagents. We further validated by 16S rDNA PCR amplification using 16S universal primers, a commercial product2 XPCR Master Mix was formulated, and we added 4 uL DNA template product to a 20 uL system, as shown in FIG. 9, it was found that both nucleic acid extraction kits A and B could amplify well, while the DNA template extracted with commercial reagents apparently contained PCR inhibitors, and the PCR inhibitors in the DNA products of samples 3 and 4 were too high to amplify effectively.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The previous description of the disclosed embodiments is provided to enable 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 applied to 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 necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A lysis reagent comprising the following components: trihydroxymethyl aminomethane, lithium chloride, NaSCN, glycerol and lithium dodecyl sulfate.
2. A lysis buffer characterized by:
the lysis buffer is prepared from the reagent of claim 1;
preferably, the lysis buffer comprises 50-500mM of trihydroxymethyl aminomethane, 100-500mM of lithium chloride, 1-5M of NaSCN, 0.5-20% by mass volume of glycerol, 0.1-5% of lithium dodecyl sulfate, and a pH of 8.0-12.5;
more preferably, the lysis buffer comprises 100-200mM of trihydroxymethyl aminomethane, 200-300mM of lithium chloride, 2-3M of NaSCN, 1-5% by mass/volume of glycerol, 0.5-2% of lithium dodecyl sulfate, and a pH of 8.5-11.5;
it is further preferred that the lysis buffer comprises 200mM tris, 200mM lithium chloride, 3M NaSCN, 5% glycerol by mass/volume, 2% lithium dodecyl sulfate, and a pH of 11.0.
3. An inhibitor removal reagent, wherein the inhibitor removal reagent comprises the following components: potassium aluminum sulfate, sodium acetate and acetic acid.
4. An inhibitor removal buffer, characterized in that:
the inhibitor removal buffer is formulated from the inhibitor removal reagent of claim 3;
preferably, the inhibitor removal buffer comprises 0.1-2M aluminum potassium sulfate, 0.5-5M sodium acetate, and acetic acid is used to adjust the pH to 3.5-7.5;
more preferably, the inhibitor removal buffer comprises 0.2-1M aluminum potassium sulfate, 0.5-2M sodium acetate, and acetic acid is used to adjust the pH to pH 4-6;
it is further preferred that the inhibitor removal buffer comprises 0.5M potassium aluminum sulfate, 2M sodium acetate, and acetic acid is used to adjust the pH to pH 4.
5. A method for extracting nucleic acid, 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: centrifuging the crude lysate to obtain a supernatant, adding an inhibitor removal buffer to the supernatant to remove PCR inhibitors contained in the environmental sample, and centrifuging to obtain a crude DNA product;
s3: binding DNA in the crude DNA product by using magnetic microspheres and a binding buffer solution to obtain a bound DNA product;
s4: washing with a washing solution to obtain a washed DNA product;
s5: and (3) heating and eluting the washing DNA product by using TE buffer solution to obtain the final DNA.
6. The method according to claim 5, characterized in that in step S1:
the grinding beads are zirconia beads of 0.1-3mm, glass beads of 0.1-5mm, garnet of 0.2-1.5mm, silicon carbide of 0.2-0.5mm, metal balls of 0.3-4.5mm and/or combinations thereof; preferably, the grinding beads are zirconia beads of 0.1 to 3mm, glass beads of 0.1 to 2mm, garnet of 0.3 to 1.0mm and/or combinations thereof; more preferably, the grinding beads are zirconia beads of 0.1 to 3mm, glass beads of 0.1 to 1mm and/or combinations thereof;
further preferably, the lysis buffer is prepared from the lysis reagent of claim 1 or is the lysis buffer of claim 2.
7. Method according to claim 5 or 6, characterized in that in step S2:
the inhibitor removal buffer is formulated 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 silicon hydroxyl magnetic beads, glass magnetic beads, carboxyl magnetic beads and/or a combination thereof; more preferably, the magnetic microspheres are a series of magnetic beads manufactured by Suzhou Chalkbrook Biotechnology Co., Ltd, such as Silica hydroxyl magnetic beads MagH1N Silica (Cat # BMD007511), Silica hydroxyl magnetic beads MagH1Silica (Cat # BMD003511), and glass magnetic beads Mag2000 Silica (Cat # BMD 004511);
it is further preferred that the binding buffer is a mixture of a chaotropic salt and an 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, and/or the alcohol is ethanol or isopropanol; it is further preferred that the binding buffer comprises 1-5M cyanoguanidine iso-sulfate, 0.2-2M sodium iodide and 30-70% by volume isopropanol; it is still further preferred that the binding buffer comprises 2-3M cyanoguanidine isothiocyanate, 0.5-2M sodium iodide and 30-60% by volume isopropanol.
9. The method according to any one of claims 5 to 8, wherein in step S4:
the cleaning is carried out by adopting a first cleaning solution and a second cleaning solution in sequence, the first cleaning solution comprises 0.5-4% of cyanoguanidine iso-sulfate, 0.5-3M of lithium chloride, 0.1-0.5M of trihydroxy methyl aminomethane, 30-70% of ethanol in volume ratio and has the pH of 5.0-8.0, and the second cleaning solution comprises 0.1-1M of sodium chloride, 0.1-0.2M of trihydroxy methyl aminomethane and 50-90% of ethanol in volume ratio;
preferably, the first wash solution comprises 1-2M cyanoguanidine iso-sulfate, 1-2M lithium chloride, 0.1-0.2M trihydroxymethylaminomethane and 50-60% by volume ethanol, and has a pH of 5.0-8.0, and the second wash solution comprises 0.5M sodium chloride, 0.1M trihydroxymethylaminomethane and 70-80% by volume ethanol.
10. A kit for extracting nucleic acid from an environmental sample, the kit comprising: the lysis reagent of claim 1; and/or the inhibitor-removing agent of claim 3; preferably, the kit further comprises one or more of the following reagents: (1) reagents for formulating the first wash liquor of claim 9; (2) reagents for formulating a second wash liquor according to claim 9; (3) the abrasive bead of claim 6; (4) the magnetic microspheres of claim 8; (5) reagents for formulating the binding buffer of claim 8; (6) a reagent for preparing TE buffer according to claim 10.
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