CN118147272A - Environment DNA extraction kit and method suitable for species diversity analysis of eDNA macro-bar code technology - Google Patents
Environment DNA extraction kit and method suitable for species diversity analysis of eDNA macro-bar code technology Download PDFInfo
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- 238000007400 DNA extraction Methods 0.000 title claims abstract description 26
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- 239000000243 solution Substances 0.000 claims abstract description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
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- 238000005406 washing Methods 0.000 claims abstract description 28
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- 238000001179 sorption measurement Methods 0.000 claims abstract description 20
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 18
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 18
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 18
- QCWXUUIWCKQGHC-UHFFFAOYSA-N Zirconium Chemical compound [Zr] QCWXUUIWCKQGHC-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052726 zirconium Inorganic materials 0.000 claims abstract description 16
- 239000011324 bead Substances 0.000 claims abstract description 15
- 238000005336 cracking Methods 0.000 claims abstract description 12
- 239000003480 eluent Substances 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000007983 Tris buffer Substances 0.000 claims abstract description 9
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000005695 Ammonium acetate Substances 0.000 claims abstract description 8
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 8
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 8
- 229960000789 guanidine hydrochloride Drugs 0.000 claims abstract description 8
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims abstract description 8
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims abstract description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 7
- WZUKKIPWIPZMAS-UHFFFAOYSA-K Ammonium alum Chemical compound [NH4+].O.O.O.O.O.O.O.O.O.O.O.O.[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O WZUKKIPWIPZMAS-UHFFFAOYSA-K 0.000 claims abstract description 7
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims abstract description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000741 silica gel Substances 0.000 claims abstract description 7
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 7
- 239000002904 solvent Substances 0.000 claims abstract description 6
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims abstract description 5
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims abstract description 5
- 239000000706 filtrate Substances 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- 238000000227 grinding Methods 0.000 claims description 13
- 239000006166 lysate Substances 0.000 claims description 11
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- 238000000197 pyrolysis Methods 0.000 claims description 3
- 102000053602 DNA Human genes 0.000 description 46
- 108020004414 DNA Proteins 0.000 description 46
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- 239000000523 sample Substances 0.000 description 24
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- 229910052782 aluminium Inorganic materials 0.000 description 2
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- 239000012472 biological sample Substances 0.000 description 2
- 239000003093 cationic surfactant Substances 0.000 description 2
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- 230000001954 sterilising effect Effects 0.000 description 2
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- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses an environment DNA extraction kit suitable for species diversity analysis of eDNA macro-bar code technology, wherein a cracking medium is pure zirconium beads, a cracking liquid comprises 100-200mM guanidine isothiocyanate and 100-300mM disodium hydrogen phosphate, and a solvent is water; the components of the impurity removing liquid comprise 200-400mM ammonium acetate and 30-100mM ammonium aluminum sulfate dodecahydrate; the binding solution comprises 2-10M guanidine hydrochloride; the nucleic acid adsorption column is a silica gel membrane adsorption column, and the washing solution I is 20-200mM Tris-HCl with pH of 7.5; the washing liquid II is ethanol solution with the volume ratio of 75%; the components of the eluent comprise 5-15mM Tris, pH8.0 and 0.2-1mM disodium ethylenediamine tetraacetate, pH 8.0. The environmental sample DNA extracted by the kit can meet the high requirements of the environmental DNA macro-bar code technology on the integrity and diversity of the environmental sample nucleic acid.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an environment DNA extraction kit and method suitable for species diversity analysis of an eDNA macro-bar code technology.
Background
Environmental DNA refers to free DNA released into the environment by way of skin shed, residue, body fluid, gametes, feces, secretions, etc., while the sample carrying these eDNA may be water, soil, sediment, feces, air, etc. Environmental DNA macro-barcode technology refers to the high throughput of multiple species identification using DNA isolated in environmental samples (e.g., soil, sediment, water, etc.). The traditional sampling method is used for carrying out ecological research, the sample collection of some species is generally difficult, time and labor are consumed, environmental DNA allows non-invasive sampling from soil, water or animal hair, sloughed cells, feces, urine, blood, sperms, ova, roots, leaves, fruits and the like contained in the air, so that genetic detection is successfully carried out, the method has lower cost, the sample collection is less influenced by climatic conditions, and a larger number of sample collection can be carried out
The environmental DNA macro-barcode technology has high requirements on the integrity and diversity of the nucleic acid of the environmental sample, so an extraction method capable of obtaining comprehensive and complete DNA is needed to extract the DNA of the environmental sample for species diversity analysis by the eDNA macro-barcode technology.
Disclosure of Invention
In order to solve at least one of the problems, the invention provides an environment DNA extraction kit and a method suitable for species diversity analysis of eDNA macro-bar code technology.
In order to achieve the above purpose, the invention adopts the following technical means:
The first aspect of the invention provides an environmental DNA extraction kit suitable for species diversity analysis of eDNA macro-bar code technology, which comprises a lysis medium, a lysis solution, a impurity removing solution, a binding solution, a nucleic acid adsorption column, a washing solution I, a washing solution II and an eluent;
The cracking medium is pure zirconium beads;
The components of the lysate comprise 100-200mM guanidine isothiocyanate and 100-300mM disodium hydrogen phosphate, and the solvent is water;
the components of the impurity removing liquid comprise 200-400mM ammonium acetate and 30-100mM ammonium aluminum sulfate dodecahydrate;
the binding solution comprises 2-10M guanidine hydrochloride;
the washing solution I is 20-200mM Tris-HCl with pH of 7.5;
the washing liquid II is ethanol solution with the volume ratio of 75%;
The components of the eluent comprise 5-15mM Tris with pH8.0 and 0.2-1mM disodium ethylenediamine tetraacetate with pH 8.0.
In some embodiments of the invention, the lysis medium is a mixture of 0.1mm of 95% pure zirconium beads, 0.5mm of 95% pure zirconium beads, and 3mm of 95% pure zirconium beads.
In some embodiments of the invention, the nucleic acid adsorption column is a silica gel membrane adsorption column.
In some embodiments of the invention, the composition of the lysate comprises 150-250mM guanidine isothiocyanate, 150-250mM disodium hydrogen phosphate, and the solvent is water. Preferably, the components of the lysate include 200mM guanidine isothiocyanate, 200mM disodium hydrogen phosphate, and the solvent is water.
In some embodiments of the invention, the components of the impurity removal solution include 300-400mM ammonium acetate, 80-100mM aluminum ammonium sulfate dodecahydrate. Preferably, the components of the impurity removal solution include 350mM ammonium acetate, 100mM aluminum ammonium sulfate dodecahydrate.
In some embodiments of the invention, the binding solution comprises 5-8M guanidine hydrochloride. Preferably, the binding solution comprises 6M guanidine hydrochloride.
In some embodiments of the invention, the wash solution I is 50-80mM Tris-HCl pH 7.5. Preferably, the wash I is 50mM Tris-HCl pH 7.5.
In some embodiments of the invention, the components of the eluate include 8-12mM Tris, pH8.0, 0.3-0.8mM disodium edetate, pH 8.0. Preferably, the components of the eluate include 10mM Tris, pH8.0, 0.5mM disodium edetate, pH 8.0.
The environmental DNA extraction kit can be applied to the species diversity analysis of the eDNA macro-bar code technology.
The second aspect of the invention provides an environmental DNA extraction method suitable for species diversity analysis of an eDNA macro barcode technology, comprising the steps of:
S1, adding a proper amount of environmental sample into a grinding tube filled with a cracking medium for full grinding;
s2, adding the lysate, uniformly mixing, performing water bath pyrolysis at 55 ℃ to release DNA in cells, and centrifuging to obtain supernatant;
S3, adding impurity removing liquid into the supernatant, incubating at 4 ℃, centrifuging at room temperature, and removing impurities in a sample;
S4, adding a binding solution into the supernatant, and uniformly mixing by vortex to fully bind sample nucleic acid;
s5, the mixed solution passes through a nucleic acid adsorption column, filtrate is discarded after centrifugation,
S6, adding the washing liquid I, centrifuging, and discarding filtrate;
s7, adding the washing liquid II, centrifuging, and discarding filtrate;
S8, adding eluent, incubating at 65 ℃, and centrifuging to obtain purified DNA.
In some embodiments of the invention, the centrifugation speed in each step is 10000-12000rpm for a period of 1-3min.
Preferably: the method for extracting the environment DNA comprises the following steps:
S1, adding a proper amount of environmental sample into a grinding tube filled with a cracking medium for full grinding;
S2, adding a lysate, mixing by vortex for 15S, performing water bath at 55 ℃ for 10min for lysis to release DNA in cells, regulating a vortex instrument to a maximum rotation speed, mixing for 5min, and centrifuging at 12000rpm for 3min at room temperature to obtain a supernatant;
S3, adding impurity removing liquid into the supernatant, incubating for 5min at 4 ℃, and centrifuging for 3min at 12000rpm at room temperature to remove impurities in the sample;
S4, adding a binding solution into the supernatant, and uniformly mixing by vortex to fully bind sample nucleic acid;
s5, passing the mixed solution through a nucleic acid adsorption column, centrifuging at 12000rpm for 1min, discarding the filtrate,
S6, adding the washing liquid I, centrifuging at 12000rpm for 1min, and discarding the filtrate;
S7, adding the washing liquid II, centrifuging at 12000rpm for 1min, and discarding the filtrate;
S8, adding eluent, incubating for 2min at 65 ℃, and centrifuging for 1min at 12000rpm to obtain purified DNA.
The beneficial effects of the invention are that
Compared with the prior art, the invention has the following beneficial effects: in the scheme of the invention, the collision force of pure zirconium beads with different sizes is adopted to realize sample cracking, wherein cationic surfactant and salt environment are favorable for cracking biological samples through the change of osmotic pressure, so that nucleic acid contained in the biological samples is released, and meanwhile, the addition of the cationic surfactant is favorable for the specific combination of the nucleic acid with negative electricity and a conjugate; aluminum sulfate is dissolved in water and ionized to form sulfate radical and aluminum ion, and the sulfate radical and the aluminum ion are hydrolyzed to form aluminum hydroxide colloid, wherein the colloid has a large surface area, can adsorb suspended matters in water, and can effectively remove impurities in mixed liquid; by adopting a silica gel membrane nucleic acid adsorption column, the DNA skeleton based on negative charge has high affinity to the positively charged filter membrane, the silica gel membrane can selectively adsorb DNA under the environment of high salt and low PH, and other pollutants, mainly proteins, are removed by membrane washing. A low salt concentration at high pH releases DNA. The purpose of purifying DNA can be achieved by utilizing this characteristic of the silica gel film.
The environmental DNA extracted by the scheme of the invention has good integrity and diversity, and meanwhile, the extracted DNA is amplified by adopting universal primers of different species, so that good amplification results can be obtained, and the analysis of species diversity by using the eDNA macro-bar code technology is facilitated.
Drawings
FIG. 1 is an electrophoretogram of genomic DNA extracted from water, soil and sediment in examples 1-3 and examples 5-7, with numbers 1-12 for the extraction results using the marine animal tissue DNA extraction kit of Tiangen, and 13-24 for the extraction results of the method of the present invention, wherein numbers 1-3 and 13-15 for the water filtered using the acetate filter; the numbers 4-6 and 18-18 are water bodies filtered by the glass cellulose filter membrane; the numbers 7-9 and 19-21 are soil samples; deposit extraction numbers 10-12 and 22-24, M is marker;
FIG. 2 is a diagram of an electrophoresis analysis of the obtained DNA after PCR amplification with primers of different species, wherein the number 1-12 is the result of extraction using a marine animal tissue DNA extraction kit, the number 13-24 is the result of extraction using the method, and the number 1-3 and the number 13-15 are the water bodies filtered by using a cellulose acetate filter membrane; the numbers 4-6 and 18-18 are water bodies filtered by the glass cellulose filter membrane; the numbers 7-9 and 19-21 are soil samples; deposit extraction numbers 10-12 and 22-24, M is marker;
Wherein 12S-10 and 12S-12 are fish, 18S rRNA V9-7 and COI-10 are zooplankton, rbcL-5 is phytoplankton, and M is marker;
FIG. 3 shows that the method of the invention extracts genomic DNA of environmental samples, the obtained DNA is amplified by PCR by using universal primers for amplifying different species, the obtained PCR product selection part is subjected to second generation sequencing, and the sequencing data is subjected to quality control, effective sequence splicing, OTU clustering and OTU species annotation; carrying out species richness analysis result graphs according to OTU species annotation;
FIG. 4 shows that the method of the invention extracts genomic DNA of environmental samples, the obtained DNA is amplified by PCR by using universal primers for amplifying different species, the obtained PCR product selection part is subjected to second generation sequencing, and the sequencing data is subjected to quality control, effective sequence splicing, OTU clustering and OTU species annotation; the graph of the results of the species diversity analysis was performed according to OTU species annotation.
Detailed Description
The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
The technical scheme of the patent is further described in detail below with reference to the specific embodiments.
Example 1
Respectively placing a 0.45 mu m acetate fiber filter membrane and a glass fiber filter membrane in a filter head interlayer, screwing up a filter head device, and filtering and collecting water sample microorganisms on the filter membranes by using a needle cylinder or a suction filtration pump, wherein the number is: HSD-1-1, HSD-5-1, HSD-14-1, SZ4# -2, SZ44# -2, DJ3#. Two sets of sterilizing forceps are used for picking up the white filter membrane at the opposite edges, rolling the filter membrane into a cylinder, leading the top surface of the cylinder to be inward, and the cylinder is arranged in a grinding pipe;
The extraction number of the marine animal tissue DNA extraction kit is as follows: the operation steps of the microbial DNA of the water samples of HSD-1-1, HSD-5-1, HSD-14-1, SZ4# -2, SZ44# -2 and DJ3# are strictly carried out according to the instruction of the kit, and the concentration of the extracted products is detected by electrophoresis analysis and Nanodrop and Qubit, and the results are shown in FIG. 1a and Table 1.
Universal primers using the eDNA technique respectively: 12S-10, 12S-12, 18S V9-7, COI-10, rbcL-5 pairs numbered: the results of PCR amplification and electrophoretic analysis of the PCR products of the water sample microorganism DNA of HSD-1-1, HSD-5-1, HSD-14-1, SZ4# -2, SZ44# -2 and DJ3# -are shown in figure 2.
Example 2
0.25-0.5G of soil sample is taken and added into a grinding tube, and the extraction number of the marine animal tissue DNA extraction kit is as follows: SXMT, SGNW, SDM the DNA in the soil sample, the operation steps are strictly carried out according to the instruction of the kit, the concentration of the extracted products is detected by electrophoresis analysis, nanodrop and Qubit, and the results are shown in FIG. 1a and Table 1.
Universal primers using the eDNA technique respectively: 12S-10, 12S-12, 18S V9-7, COI-10, rbcL-5 pairs numbered: SXMT, SGNW, SDM, performing PCR amplification on DNA in the soil sample, and performing electrophoresis analysis on the PCR product, wherein the result is shown in FIG. 2.
Example 3
Adding 0.25-0.5g of sediment into a grinding tube, and extracting the DNA (deoxyribonucleic acid) of the marine animal tissue with the extraction number of: the procedure of DNA in the sediment samples of M-H-2, MLB- ② and L-L-1 is strictly carried out according to the instruction of the kit, and the concentration of the extracted products is detected by electrophoresis analysis and Nanodrop and Qubit, and the results are shown in FIG. 1a and Table 1.
Universal primers using the eDNA technique respectively: 12S-10, 12S-12, 18S V9-7, COI-10, rbcL-5 pairs numbered: the DNA in the sediment samples of M-H-2, MLB- ② and L-L-1 was amplified by PCR, and the PCR products were analyzed by electrophoresis, and the results are shown in FIG. 2.
Example 4
An environment DNA extraction kit suitable for species diversity analysis of eDNA macro-bar code technology comprises a lysis medium, a lysis solution, an impurity removal solution, a binding solution, a nucleic acid adsorption column, a washing solution I, a washing solution II and an eluent;
wherein the lysis medium comprises 0.1mm of 95% pure zirconium beads, 0.5mm of 95% pure zirconium beads, and 3mm of 95% pure zirconium beads. The specific configuration method comprises the following steps: 1.56g of 0.1mm of 95% pure zirconium beads, 1.44g of 0.5mm of 95% pure zirconium beads and 3mm of 95% pure zirconium were weighed accurately into a 2ml freezer.
The lysate included 200mM guanidine isothiocyanate and 200mM disodium hydrogen phosphate. The specific configuration method comprises the following steps: 23.6g of guanidine isothiocyanate and 28.4g of disodium hydrogen phosphate are weighed, and a proper amount of pure water is added for dissolution, and then the volume is fixed to 1L.
The impurity removal solution included 350mM ammonium acetate and 100mM aluminum ammonium sulfate dodecahydrate. The specific configuration method comprises the following steps: 28.9g of ammonium acetate and 43.5g of ammonium aluminum sulfate dodecanol were weighed, and after adding a proper amount of pure water for dissolution, the volume was fixed to 1L.
The binding solution is 6M guanidine hydrochloride, and the specific preparation method comprises the following steps: 573.2g of guanidine hydrochloride was weighed, and after adding a proper amount of pure water for dissolution, the volume was set to 1L.
The nucleic acid adsorption column is a silica gel membrane adsorption column.
Washing solution I was tris-HCl with pH 7.5 at 50mM, and the specific preparation method was: 6g of tris was weighed into a beaker, dissolved in a suitable amount of pure water, and then adjusted to pH 7.5 with HCl, and then fixed to volume to 1L with pure water.
The washing liquid II is ethanol solution with the volume ratio of 75%, and the specific preparation method comprises the following steps: 750ml of absolute ethanol and 250ml of pure water were weighed and mixed.
The eluent comprises 10mM Tris of pH8.0 and 0.5mM disodium ethylenediamine tetraacetate of pH8.0, and the specific preparation method comprises: 1.214g of Tris and 0.1866g of EDTA.2NA are weighed into a beaker, dissolved in a proper amount of pure water, adjusted to pH8.0 with HCl, and then fixed to volume to 1L with pure water.
Example 5
The environmental DNA extraction kit of example 1 was used to extract DNA from a water sample, as follows:
(1) Respectively placing a 0.45 mu m acetate fiber filter membrane and a glass fiber filter membrane in a filter head interlayer, screwing up a filter head device, and filtering and collecting water sample microorganisms on the filter membranes by using a needle cylinder or a suction filtration pump, wherein the number is: HSD-1-1, HSD-5-1, HSD-14-1, SZ4# -2, SZ44# -2, DJ3#. Two sets of sterilizing forceps are used for picking up the white filter membrane at the opposite edges, rolling the filter membrane into a cylinder, leading the top surface of the cylinder to be inward, and the cylinder is arranged in a grinding pipe;
(2) Adding the lysate into a grinding tube containing the filter membrane, mixing by vortex for 15s, and carrying out water bath at 55 ℃ for 10min;
(3) Mixing the vortex instrument to the maximum rotation speed, and centrifuging at 12000rpm for 3min at room temperature for 5 min;
(4) Transferring the supernatant to a new centrifuge tube, adding impurity removing liquid, incubating for 5min at 4 ℃, and centrifuging for 3min at 12000rpm at room temperature to remove impurities in a sample;
(5) Transferring the supernatant to a new centrifuge tube, adding the binding liquid into the centrifuge tube, and mixing uniformly by vortex;
(6) Sleeving the adsorption column in a collecting pipe, passing the mixed solution through the adsorption column, centrifuging at 12000rpm for 1min, and discarding the filtrate;
(7) Adding the washing solution I, centrifuging at 12000rpm for 1min, and discarding the filtrate;
(8) Adding the washing liquid II, centrifuging at 12000rpm for 1min, and discarding the filtrate;
(9) Adding eluent, incubating for 2min at 65 ℃, centrifuging for 1min at 12000rpm to obtain purified DNA,
The concentration was measured by electrophoresis analysis, nanodrop and Qubit, and the results are shown in FIG. 1b and Table 1.
(10) The purified DNA was extracted by PCR amplification using the universal primer of eDNA technology, and the PCR product was analyzed by electrophoresis, and the result was shown in FIG. 2.
Example 6
DNA was extracted from soil samples using the environmental DNA extraction kit of example 1, as follows:
(1) 0.25-0.5g of soil sample was added to the grind tube, numbered: SXMT, SGNW, SDM, adding a lysate into a grinding tube, uniformly mixing for 15s by vortex, and carrying out water bath at 55 ℃ for 10min;
(2) Mixing the vortex instrument to the maximum rotation speed, and centrifuging at 12000rpm for 3min at room temperature for 5 min;
(3) Transferring the supernatant to a new centrifuge tube, adding impurity removing liquid, incubating for 5min at 4 ℃, and centrifuging for 3min at 12000rpm at room temperature to remove impurities in a sample;
(4) Transferring the supernatant to a new centrifuge tube, adding the binding liquid into the centrifuge tube, and mixing uniformly by vortex;
(5) The mixed solution passes through an adsorption column, is centrifuged at 12000rpm for 1min, and the filtrate is discarded;
(6) Adding the washing solution I, centrifuging at 12000rpm for 1min, and discarding the filtrate;
(7) Adding the washing liquid II, centrifuging at 12000rpm for 1min, and discarding the filtrate;
(8) The eluate was added, incubated at 65℃for 2min, centrifuged at 12000rpm for 1min to obtain purified DNA, and the concentration was measured by electrophoresis, nanodrop and Qubit, and the results are shown in FIG. 1b and Table 1.
(9) The purified DNA was extracted by PCR amplification using the universal primer of eDNA technology, and the PCR product was analyzed by electrophoresis, and the result was shown in FIG. 2.
Example 7
DNA was extracted from sediment samples using the environmental DNA extraction kit of example 1, as follows:
(1) 0.25-0.5g of the deposit was added to a milling tube, numbered: adding the pyrolysis liquid into a grinding tube, mixing the mixture for 15s by vortex, and carrying out water bath at 55 ℃ for 10min;
(2) Mixing the vortex instrument to the maximum rotation speed, and centrifuging at 12000rpm for 3min at room temperature for 5 min;
(3) Transferring the supernatant to a new centrifuge tube, adding impurity removing liquid, incubating for 5min at 4 ℃, and centrifuging for 3min at 12000rpm at room temperature to remove impurities in a sample;
(4) Transferring the supernatant to a new centrifuge tube, adding the binding liquid into the centrifuge tube, and mixing uniformly by vortex;
(5) Sleeving the adsorption column in a collecting pipe, passing the mixed solution through the adsorption column, centrifuging at 12000rpm for 1min, and discarding the filtrate;
(6) Adding the washing solution I, centrifuging at 12000rpm for 1min, and discarding the filtrate;
(7) Adding the washing liquid II, centrifuging at 12000rpm for 1min, and discarding the filtrate;
(8) The eluate was added, incubated at 65℃for 2min, centrifuged at 12000rpm for 1min to obtain purified DNA, and the concentration was measured by electrophoresis, nanodrop and Qubit, and the results are shown in FIG. 1b and Table 1.
(9) The purified DNA was extracted by PCR amplification using the universal primer of eDNA technology, and the PCR product was analyzed by electrophoresis, and the result was shown in FIG. 2.
Performing second generation sequencing on the product selected part of the DNA extracted in the embodiment 5-7 after PCR amplification, and performing quality control, effective sequence splicing, OTU clustering and OTU species annotation on the sequencing data; species abundance analysis was performed according to OTU species annotation, with detailed results in fig. 3; the results of species diversity analysis are detailed in FIG. 4.
TABLE 1 detection results of the concentration of the extraction products Nanodrop and Qubit in different extraction modes
The results show that: the DNA extracted by the invention shows that the band is brighter than the band extracted by the marine animal tissue DNA extraction kit through agarose gel electrophoresis detection, and the qubit concentration of most samples is higher than that of control experiments. After amplification by using each universal primer, the samples successfully amplified by the method are found to be more than those of a control experiment.
The environmental DNA extracted by the scheme of the invention has good integrity and diversity, and meanwhile, the extracted DNA is amplified by adopting universal primers of different species, so that good amplification results can be obtained, and the analysis of species diversity by using the eDNA macro-bar code technology is facilitated.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be understood that various changes and modifications may be made by those skilled in the art after reading the above teachings of the application, and such equivalents are intended to fall within the scope of the application as defined by the claims.
Claims (10)
1. An environment DNA extraction kit suitable for species diversity analysis of eDNA macro-bar code technology, which is characterized in that: comprises a cracking medium, a cracking liquid, an impurity removing liquid, a binding liquid, a nucleic acid adsorption column, a washing liquid I, a washing liquid II and an eluent;
The cracking medium is pure zirconium beads;
The components of the lysate comprise 100-200mM guanidine isothiocyanate and 100-300mM disodium hydrogen phosphate, and the solvent is water;
the components of the impurity removing liquid comprise 200-400mM ammonium acetate and 30-100mM ammonium aluminum sulfate dodecahydrate;
the binding solution comprises 2-10M guanidine hydrochloride;
the washing solution I is 20-200mM Tris-HCl with pH of 7.5;
the washing liquid II is ethanol solution with the volume ratio of 75%;
The components of the eluent comprise 5-15mM Tris with pH8.0 and 0.2-1mM disodium ethylenediamine tetraacetate with pH 8.0.
2. An environmental DNA extraction kit suitable for analysis of species diversity by the eDNA macro barcode technology according to claim 1, characterized in that: the cracking medium was a mixture of 0.1mm of 95% pure zirconium beads, 0.5mm of 95% pure zirconium beads, and 3mm of 95% pure zirconium beads.
3. An environmental DNA extraction kit suitable for analysis of species diversity by the eDNA macro barcode technology according to claim 1, characterized in that: the nucleic acid adsorption column is a silica gel membrane adsorption column.
4. An environmental DNA extraction kit suitable for analysis of species diversity by the eDNA macro barcode technology according to claim 1, characterized in that: the components of the lysate comprise 150-250mM guanidine isothiocyanate and 150-250mM disodium hydrogen phosphate, and the solvent is water.
5. An environmental DNA extraction kit suitable for analysis of species diversity by the eDNA macro barcode technology according to claim 1, characterized in that: the components of the impurity removing liquid comprise 300-400mM ammonium acetate and 80-100mM ammonium aluminum sulfate dodecahydrate.
6. An environmental DNA extraction kit suitable for analysis of species diversity by the eDNA macro barcode technology according to claim 1, characterized in that: the binding solution comprises 5-8M guanidine hydrochloride.
7. An environmental DNA extraction kit suitable for analysis of species diversity by the eDNA macro barcode technology according to claim 1, characterized in that: the washing solution I is 50-80mM Tris-HCl with pH 7.5.
8. An environmental DNA extraction kit suitable for analysis of species diversity by the eDNA macro barcode technology according to claim 1, characterized in that: the components of the eluent comprise 8-12mM Tris with pH8.0 and 0.3-0.8mM disodium ethylenediamine tetraacetate with pH 8.0.
9. An environmental DNA extraction method suitable for species diversity analysis of an eDNA macro barcode technology is characterized by comprising the following steps:
S1, adding a proper amount of environmental sample into a grinding tube filled with a cracking medium for full grinding;
s2, adding the lysate, uniformly mixing, performing water bath pyrolysis at 55 ℃ to release DNA in cells, and centrifuging to obtain supernatant;
S3, adding impurity removing liquid into the supernatant, incubating at 4 ℃, centrifuging at room temperature, and removing impurities in a sample;
S4, adding a binding solution into the supernatant, and uniformly mixing by vortex to fully bind sample nucleic acid;
s5, the mixed solution passes through a nucleic acid adsorption column, filtrate is discarded after centrifugation,
S6, adding the washing liquid I, centrifuging, and discarding filtrate;
s7, adding the washing liquid II, centrifuging, and discarding filtrate;
S8, adding eluent, incubating at 65 ℃, and centrifuging to obtain purified DNA.
10. The method for extracting environmental DNA suitable for species diversity analysis of eDNA macro-bar code technology according to claim 9, wherein the centrifugal speed in each step is 10000-12000rpm, and the duration is 1-3min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935647A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit and method for extracting microbial DNA |
| CN111876410A (en) * | 2020-08-13 | 2020-11-03 | 武汉生之源生物科技股份有限公司 | Genomic column extraction kit and extraction method |
| CN116083420A (en) * | 2023-03-02 | 2023-05-09 | 上海美吉生物医药科技有限公司 | Method for efficiently extracting metagenome high-quality DNA in activated sludge and feces |
| CN116286794A (en) * | 2023-02-21 | 2023-06-23 | 江苏康为世纪生物科技股份有限公司 | Extraction reagent combination, extraction kit and extraction method for microbial genome DNA in soil |
-
2024
- 2024-01-23 CN CN202410095518.1A patent/CN118147272A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935647A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit and method for extracting microbial DNA |
| CN111876410A (en) * | 2020-08-13 | 2020-11-03 | 武汉生之源生物科技股份有限公司 | Genomic column extraction kit and extraction method |
| CN116286794A (en) * | 2023-02-21 | 2023-06-23 | 江苏康为世纪生物科技股份有限公司 | Extraction reagent combination, extraction kit and extraction method for microbial genome DNA in soil |
| CN116083420A (en) * | 2023-03-02 | 2023-05-09 | 上海美吉生物医药科技有限公司 | Method for efficiently extracting metagenome high-quality DNA in activated sludge and feces |
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