CN113088516A - DNA extraction kit and extraction method for fecal microorganism genome - Google Patents
DNA extraction kit and extraction method for fecal microorganism genome Download PDFInfo
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- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
Abstract
The invention discloses a DNA extraction kit and an extraction method for a microbial genome of excrement, belonging to the field of nucleic acid extraction in molecular biology. Correspondingly, the extraction method of the DNA extraction kit for the fecal microorganism genome is sequentially executed according to the impurity removal step, the cracking step, the rinsing step and the elution step. The DNA extraction kit and the extraction method for the microbial genome of the excrement disclosed by the invention can effectively remove PCR inhibitors such as humic acid, polysaccharide, polyphenol, pigment and the like in the excrement, and can conveniently, economically and quickly obtain DNA with high purity, high recovery rate and good integrity.
Description
Technical Field
The invention relates to the field of nucleic acid extraction in molecular biology, in particular to a DNA extraction kit and an extraction method for a fecal microorganism genome.
Background
Recent studies on intestinal flora have been emerging, and the role of intestinal flora in human health has also been receiving more and more attention. The intestinal flora is normal microorganisms in human intestinal tracts, and is various in types and numerous in number. According to estimation, the total weight of intestinal bacteria in a normal adult can reach 1-1.5 kg. They can affect body weight and digestive ability, protect against the risk of infection and autoimmune diseases, and in addition, have close relationship with the occurrence of some endocrine metabolic diseases, kidney diseases, neurological diseases, cardiovascular diseases, respiratory diseases and cancers.
Therefore, it is particularly important to study the intestinal flora response, and by looking at the state of the intestinal flora of the host, the health condition or the relationship with diseases of the host can be judged, and an improvement or treatment scheme can be provided according to the condition. At present, the distribution condition or change condition of the intestinal flora is mainly researched through 16S rDNA or metagenome, so that the acquisition of the nucleic acid DNA of the intestinal flora is the basis of subsequent research. However, the traditional nucleic acid extraction method is complicated in steps and time-consuming at present, and the commercial fecal microorganism genome DNA extraction kit is high in price, so that the kit which is simple and easy to operate, low in cost and good in extraction effect can be provided, and the research range of intestinal flora can be greatly expanded.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a DNA extraction kit for microbial genomes of excrement, which can effectively remove PCR inhibitors such as humic acid, polysaccharide, polyphenol, pigment and the like in excrement, so that medical workers can conveniently and efficiently extract microbial genome DNA of excrement through the DNA extraction kit, and the kit is helpful for the medical workers to research intestinal flora and provide quality assurance for subsequent PCR or high-throughput library establishment.
Accordingly, another technical problem to be solved by the present invention is to provide a method for extracting DNA from microbial genome of feces, which comprises the steps of removing impurities, cracking, rinsing and eluting, and thus, the medical workers can extract nucleic acid simply and conveniently, and the method is simple and easy to operate, and has reliable and excellent extraction effect.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a DNA extraction kit for a microbial genome of excrement, which comprises an impurity removal liquid A, a lysis liquid B, a rinsing liquid C, a rinsing liquid D and an eluent E, wherein the impurity removal liquid A, the lysis liquid B, the rinsing liquid C, the rinsing liquid D and the eluent E are sequentially added into a test tube to react with the microbial genome of the excrement so as to extract DNA, and the impurity removal liquid A comprises the following substances: CTAB, EDTA, PVP30, NaCl, TritonX-100, Tween-20, citric acid and sodium citrate; lysate B included the following: guanidine hydrochloride, CTAB, PVP30, citric acid and sodium citrate.
The preferable technical scheme of the invention is that in the impurity removing liquid A, the mass ratio range of CTAB is configured to be 1-3% M/M, the volume molar concentration range of EDTA is configured to be 20-30 mM, the mass ratio range of PVP30 is configured to be 1-3% M/M, the volume molar concentration range of NaCl is configured to be 1.5-2.0M, the volume ratio range of TritonX-100 is configured to be 1-3% v/v, the volume ratio range of Tween-20 is configured to be 0.5-1% v/v, the volume molar concentration range of citric acid is configured to be 0.1-0.5M, the volume molar concentration range of sodium citrate is configured to be 0.2-1M, and the pH range of the impurity removing liquid A is configured to be 4.0-4.5.
The preferable technical scheme of the invention is that in the lysate B, the volume molar concentration range of guanidine hydrochloride is configured to be 4-6M, the mass ratio range of CTAB is configured to be 0.5-1.5% M/M, the mass ratio range of PVP30 is configured to be 0.5-1.5% M/M, the volume molar concentration range of citric acid is configured to be 0.1-0.5M, the volume molar concentration range of sodium citrate is configured to be 0.2-1M, and the pH range of the lysate B is configured to be 4.0-4.5.
The preferable technical scheme of the invention is that the rinsing liquid C comprises the following substances: guanidine hydrochloride, NaCl and absolute ethyl alcohol.
The preferable technical scheme of the invention is that the rinsing liquid D comprises the following substances: tris and absolute ethyl alcohol.
The preferred technical scheme of the invention is that the eluent E comprises the following substances: tris and EDTA.
The preferable technical scheme of the invention is that in the rinsing liquid C, the volume molar concentration range of guanidine hydrochloride is configured to be 2-4M, the volume molar concentration of NaCl is configured to be 0.15M, and the volume ratio of absolute ethyl alcohol is configured to be 56% v/v.
The preferable technical scheme of the invention is that in the rinsing liquid D, the volume molar concentration of Tris is configured to be 25mM, the volume ratio of absolute ethyl alcohol is configured to be 70% v/v, and the pH range of the rinsing liquid D is configured to be 7-7.5.
In a preferred embodiment of the present invention, in the eluent E, the molar concentration of Tris is set to 10mM, the molar concentration of EDTA is set to 0.5mM, and the pH of the eluent E is set to 8.0.
The extraction method of the DNA extraction kit for the fecal microorganism genome provided by the invention is sequentially executed according to the impurity removal step, the cracking step, the rinsing step and the elution step,
the impurity removing step specifically comprises the following steps:
1) weighing fresh excrement and placing the fresh excrement in a sterilized centrifugal tube;
2) adding impurity removing solution A into the centrifuge tube, and homogenizing in vortex oscillator for 5 min;
3) the homogenized samples were incubated in a metal bath at 70 ℃ for 5min, during which the mixture was mixed 1 time by turning upside down every 2 min:
4) after the incubation is finished, putting the sample in a centrifuge for centrifugation at 14000rpm for 1 min;
wherein, 1ml of impurity removing liquid A is added into every 200mg of feces;
the cracking step specifically comprises the following steps:
5) taking the supernatant obtained in the step 4) into a new sterilized centrifugal tube;
6) adding lysis solution B and proteinase K;
7) mixing by turning upside down for several times, incubating in a metal bath at 70 deg.C for 10min, and mixing by turning upside down for 1 time every 3 min:
8) after the incubation is finished, adding absolute ethyl alcohol;
9) slightly reversing the upper part and the lower part, uniformly mixing, sucking the sterilized centrifuge tube solution, passing the sterilized centrifuge tube solution through a nucleic acid purification adsorption column, and placing the nucleic acid purification adsorption column in a centrifuge for 30s at 12000 rpm;
10) discarding the waste liquid;
11) repeating the steps 9) to 10) until all the lysate passes through the column;
the rinsing step specifically comprises the following steps:
12) adding rinsing liquid C into the adsorption column, and centrifuging at 12000rpm for 30s in a centrifuge;
13) discarding the waste liquid;
14) adding rinsing liquid D into the adsorption column, and centrifuging at 12000rpm for 30s in a centrifuge;
15) discarding the waste liquid;
16) putting the adsorption column into the centrifuge again, and standing at 12000rpm for 3 min;
the elution step specifically comprises the following steps:
17) transferring the adsorption column to a sterile centrifuge tube, and adding an eluent E;
18) incubating at room temperature for 1min, centrifuging at 12000rpm in a centrifuge for 2min, and recovering eluate;
wherein, the impurity removal solution A, the supernatant fluid, the lysis solution B and the protease K in the step 4), the absolute ethyl alcohol, the rinsing solution C, the rinsing solution D and the eluent E in the step 8) are sequentially mixed according to the ratio of 1000: 600: 600: 25: 600: 600: 600: the proportion of 60 is added.
The invention has the beneficial effects that:
the invention provides a DNA extraction kit and an extraction method for fecal microbial genomes, wherein medical workers can conveniently remove impurities such as protein, polysaccharide, phenols and the like after adding an impurity removing solution A into a fecal sample so as to separate fecal microbial genome nucleic acid, then add a lysis solution B which can destroy cell membranes and nuclear membranes and separate protein from DNA, inhibit the activity of Dnase in cells so as to crack host cell tissues or microorganisms such as bacteria, fungi, viruses and the like, then add a rinsing solution C and a rinsing solution D to further rinse and filter the impurities such as protein, polysaccharide, phenols and the like so as to improve the purity of the extracted DNA, and finally add an eluent E to elute, purify and separate the fecal microbial genome DNA. . Compared with a commercially available fecal DNA extraction kit, the DNA extraction kit has lower cost on the premise of ensuring that the DNA extraction effect meets the requirement. Through above-mentioned process and material, PCR inhibitors such as humic acid, polysaccharide, polyphenol, pigment in not only can getting rid of the excrement effectively, can make things convenient for, economy, swift obtain the DNA that the purity is high, the rate of recovery is high, the integrality is good moreover, make medical worker can extract excrement and urine microorganism genome DNA through the DNA extraction kit of this application conveniently high-efficiently, help medical worker to study the intestinal fungus crowd, build the storehouse for follow-up PCR or high flux and provide the quality assurance.
Drawings
FIG. 1 is a schematic diagram of the integrity of gDNA obtained by genomic DNA agarose gel electrophoresis quality inspection provided in an embodiment of the present invention;
FIG. 2 is a schematic diagram showing the distribution comparison between the DNA extraction kit for fecal microorganism genome and the foreign one-market kit flora after performing the library construction sequencing analysis respectively according to the embodiment of the present invention.
Detailed Description
The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings.
Example one
In order to make medical personnel can conveniently extract excrement and urine microorganism genome DNA through the DNA extraction kit of this application high-efficiently, help medical personnel to study intestinal flora, build the storehouse for follow-up PCR or high flux and provide quality assurance, furthermore, the DNA extraction kit for excrement and urine microorganism genome that provides in this embodiment, including edulcoration liquid A, lysate B, rinsing liquid C, rinsing liquid D and eluant E, edulcoration liquid A, lysate B, rinsing liquid C, rinsing liquid D and eluant E add in proper order in the test tube and react with excrement and urine microorganism biology, in order to extract DNA, edulcoration liquid A includes following substance: CTAB, EDTA, PVP30, NaCl, TritonX-100, Tween-20, citric acid and sodium citrate; lysate B included the following: guanidine hydrochloride, CTAB, PVP30,Citric acid and sodium citrate. Preferably, in the impurity removing liquid A, the mass ratio range of CTAB is configured to be 1-3% M/M, the volume molar concentration range of EDTA is configured to be 20-30 mM, the mass ratio range of PVP30 is configured to be 1-3% M/M, the volume molar concentration range of NaCl is configured to be 1.5-2.0M, the volume ratio range of TritonX-100 is configured to be 1-3% v/v, the volume ratio range of Tween-20 is configured to be 0.5-1% v/v, the volume molar concentration range of citric acid is configured to be 0.1-0.5M, the volume molar concentration range of sodium citrate is configured to be 0.2-1M, and the pH range of the impurity removing liquid A is configured to be 4.0-4.5. Preferably, in the lysate B, the volume molar concentration range of guanidine hydrochloride is configured to be 4-6M, the mass ratio range of CTAB is configured to be 0.5-1.5% M/M, the mass ratio range of PVP30 is configured to be 0.5-1.5% M/M, the volume molar concentration range of citric acid is configured to be 0.1-0.5M, the volume molar concentration range of sodium citrate is configured to be 0.2-1M, and the pH range of the lysate B is configured to be 4.0-4.5. The medical care worker can conveniently remove impurities such as protein, polysaccharide, phenols and the like after adding the impurity removing liquid A into the excrement sample so as to separate the excrement microorganism genome nucleic acid, then add the lysis liquid B so as to destroy cell membranes and nuclear membranes, separate the protein from DNA, inhibit the activity of Dnase in cells so as to crack host cell tissues or microorganisms such as bacteria, fungi and viruses, then add the rinsing liquid C and the rinsing liquid D so as to further rinse and filter the impurities such as protein, polysaccharide, phenols and the like, improve the purity of extracted DNA, and finally add the eluent E so as to elute, purify and separate the excrement microorganism genome DNA. In which NaCl is used to provide a high salt environment to dissolve DNA sufficiently in a liquid phase, and high salt concentration is always added when precipitating DNA, and the purpose of NaCl is to destroy two factors that can stabilize protein, and to separate and precipitate protein and DNA. At this time, the cation of the salt can be combined with DNA to form DNA-cation salt, and the DNA-cation salt can be precipitated by using absolute ethyl alcohol. CTAB is a surfactant that binds nucleic acids to form soluble, stable complexes at high salt concentrations and precipitates nucleic acids with acidic polysaccharides at low ionic strength (0.1-0.5M NaCl), while proteins and neutral polysaccharides remain in solution. In the presence of high ionsIn solution of strength: (>0.7mol/L NaCl), CTAB forms a complex with proteins and polysaccharides, but nucleic acids cannot be precipitated. Through organic solvent extraction, protein, polysaccharide, phenol and other impurities are removed, and then ethanol is added for precipitation, so that the nucleic acid can be separated out. EDTA is an enzyme inhibitor, and can chelate divalent metals and inhibit metal-dependent enzymes. Thus, by adding EDTA, Mg can be chelated2+Or Mn2+Ion, inhibiting the activity of DNase enzyme in cell, degrading DNA, EDTA as bivalent ion mixture capable of decocting Mg2+And Ca2+Divalent cations are needed, and the activity of various RNases is required to depend on the divalent cations, so that the EDTA can inhibit the activity of the RNases by decocting the divalent cations so as to ensure the integrity of the extracted microbial genome DNA. PVP30 can reduce the influence of phenols, quinones, and tannins, has antioxidant effect, and can complex polyphenols and terpenoids, prevent polyphenols from browning and oxidizing, and remove polysaccharides and pigment, which is a strong PCR inhibitor and must be removed. Triton X-100 is a milder detergent used as an additive to stabilize proteins, especially membrane proteins, so that proteinase K remains active in a 1% Triton X-100 solution, allowing the subsequent addition of proteinase K to degrade proteins into small peptides or amino acids, allowing the DNA molecules to be completely separated. And the TritonX-100 has no killing effect on microorganisms such as bacteria and the like so as to ensure the integrity of the extracted microorganism DNA. Tween-20 is a nonionic surfactant used for eluting unbound protein and reducing nonspecific binding. Some fusion proteins did not bind efficiently in the presence of 0.2% Triton X-100 or 0.25% Tween-20, while others were unaffected. Citric acid is used to maintain the reagents in an acidic environment. The sodium citrate can control the ionic strength of a reaction system, and simultaneously has a certain buffering effect, so that the relative stable state of the PH of the system is ensured. Guanidine hydrochloride is a strong inhibitor of nucleases, it is not a strong enough denaturant to allow extraction of intact RNA from RNase-rich tissues. Compared with a commercially available fecal DNA extraction kit, the DNA extraction kit has lower cost on the premise of ensuring that the DNA extraction effect meets the requirement. Through the upper partThe process and the substances can effectively remove PCR inhibitors such as humic acid, polysaccharide, polyphenol, pigment and the like in excrement, and can conveniently, economically and quickly obtain DNA with high purity, high recovery rate and good integrity, so that medical workers can conveniently, efficiently extract the genome DNA of excrement microorganisms through the DNA extraction kit, the research on intestinal flora is facilitated for the medical workers, and the quality assurance is provided for subsequent PCR or high-throughput library building.
Preferably, the rinsing liquid C comprises the following: guanidine hydrochloride, NaCl and absolute ethyl alcohol. In the rinsing liquid C, the volume molar concentration range of guanidine hydrochloride is 2-4M, the volume molar concentration of NaCl is 0.15M, and the volume ratio of absolute ethyl alcohol is 56% v/v. Guanidine hydrochloride is a strong inhibitor of nucleases, but is not a sufficiently strong denaturant to allow intact RNA to be extracted from RNase-rich tissues, the guanidine hydrochloride solution solubilizes proteins, resulting in cellular structural damage, nuclear protein secondary structural damage, dissociation from nucleic acids, and the RNase enzyme can be inactivated by reducing agents such as guanidine hydrochloride. The ethanol is mixed and dissolved with absolute ethanol and water to prepare 56% v/v ethanol, the DNA is precipitated by the 56% v/v ethanol, and the ethanol does not have any chemical reaction with nucleic acid, so the DNA is safe. Nucleic acid and acidic polysaccharide can be precipitated by proportioning low-concentration 0.15M NaCl. By the above substances, residual macromolecular substances such as proteins can be removed, and DNA can be sufficiently precipitated, so that complete DNA can be extracted.
Preferably, the rinsing liquid D comprises the following: tris and absolute ethyl alcohol. In the rinsing liquid D, the volume molar concentration of Tris is configured to be 25mM, the volume ratio of absolute ethyl alcohol is configured to be 70% v/v, and the pH range of the rinsing liquid D is configured to be 7-7.5. Since DNA is insoluble in 70% v/v ethanol, but salt ions are soluble, the DNA precipitate can be washed with 70% ethanol, and the precipitation effect of ethanol on proteins and nucleic acids increases with increasing molecular weight. The DNA is rinsed by proportioning 70% v/v ethanol, so as to remove residual salts and remove impurities such as excessive SDS, phenol and the like, and the SDS is kept in a dissolved state in the 70% v/v ethanol and does not coprecipitate with the DNA, so that the detergent is removed by discarding supernatant fluid, and the influence on the subsequent PCR reaction is avoided. By adding Tris, the solution has a certain buffering effect, and the relatively stable state of the PH of the system is ensured.
Preferably, eluent E comprises the following: tris and EDTA. In eluent E, the molar concentration of Tris was set to 10mM, the molar concentration of EDTA was set to 0.5mM, and the pH of eluent E was set to 8.0. EDTA is proportioned, so that the EDTA is decocted into divalent cations to inhibit the activities of RNA enzyme and DNase enzyme, thereby ensuring the integrity of the extracted microbial genome DNA. By proportioning Tris, the solution has a certain buffering effect, and the relatively stable state of the system PH is ensured.
Example two
As shown in fig. 1 and fig. 2, in order to enable medical workers to simply and conveniently extract nucleic acid by the method, the operation is simple and easy, and the extraction effect is reliable and excellent, further, the extraction method of the DNA extraction kit for fecal microorganism genome provided in this embodiment is performed by the following steps of removing impurities, lysing, rinsing, and eluting:
the impurity removing step specifically comprises the following steps:
1) weighing fresh excrement and placing the fresh excrement in a sterilized centrifugal tube;
2) adding impurity removing solution A into the centrifuge tube, and homogenizing in vortex oscillator for 5 min;
3) the homogenized samples were incubated in a metal bath at 70 ℃ for 5min, during which the mixture was mixed 1 time by turning upside down every 2 min:
4) after the incubation is finished, putting the sample in a centrifuge for centrifugation at 14000rpm for 1 min;
wherein, 1ml of impurity removing liquid A is added into every 200mg of feces;
secondly, the cracking step specifically comprises the following steps:
5) taking the supernatant obtained in the step 4) into a new sterilized centrifugal tube;
6) adding lysis solution B and proteinase K;
7) mixing by turning upside down for several times, incubating in a metal bath at 70 deg.C for 10min, and mixing by turning upside down for 1 time every 3 min:
8) after the incubation is finished, adding absolute ethyl alcohol;
9) slightly reversing the upper part and the lower part, uniformly mixing, sucking the sterilized centrifuge tube solution, passing the sterilized centrifuge tube solution through a nucleic acid purification adsorption column, and placing the nucleic acid purification adsorption column in a centrifuge for 30s at 12000 rpm;
10) discarding the waste liquid;
11) repeating the steps 9) to 10) until all the lysate passes through the column;
the rinsing step specifically comprises the following steps:
12) adding rinsing liquid C into the adsorption column, and centrifuging at 12000rpm for 30s in a centrifuge;
13) discarding the waste liquid;
14) adding rinsing liquid D into the adsorption column, and centrifuging at 12000rpm for 30s in a centrifuge;
15) discarding the waste liquid;
16) putting the adsorption column into the centrifuge again, and standing at 12000rpm for 3 min;
fourthly, the elution step specifically comprises the following steps:
17) transferring the adsorption column to a sterile centrifuge tube, and adding an eluent E;
18) incubating at room temperature for 1min, centrifuging at 12000rpm in a centrifuge for 2min, and recovering eluate;
wherein, the impurity removal solution A, the supernatant fluid, the lysis solution B and the protease K in the step 4), the absolute ethyl alcohol, the rinsing solution C, the rinsing solution D and the eluent E in the step 8) are sequentially mixed according to the ratio of 1000: 600: 600: 25: 600: 600: 600: the proportion of 60 is added.
The analysis will be specifically described below with reference to the experimental group and the control group.
First, main reagent and instrument
(1) Reagent
Experimental groups: CTAB, EDTA, PVP30, NaCl, TritonX-100, Tween-20, Tris, guanidine hydrochloride, citric acid and sodium citrate supplied by MACKLIN; absolute ethanol is produced by the national medicine group;
control group: QIAamp Fast DNA pool Mini Kit (commercially available fecal microorganism genomic DNA extraction Kit)
(2) Instrument for measuring the position of a moving object
MultiskanTMThe GO microplate reader is from ThermoFisher Scientific; PCR 2720 instrument from ThermoFisher Scientific; desktop centrifuge 5430 is from Eppendorf; VORTEX oscillator VORTEX-6 from its Linebel; gel imaging System KS-680D was from Shanghai culture broth;
3.0Fluorometer from ThermoFisher Scientific.
Second, sample source
The fecal sample for verifying the effect of the DNA extraction kit on microbial genome DNA extraction is derived from freshly collected human feces.
Third, formula of experimental group
Removing impurity solution A: 2% (M/M) CTAB, 25mM EDTA, 1% (M/M) PVP30, 1.5M NaCl, 2% (v/v) TritonX-100, 0.5% (v/v) Tween-20, 0.5M citric acid, 1M sodium citrate, pH 4.2.
Lysis solution B: 4M guanidine hydrochloride, 1% (M/M) CTAB, 1% (M/M) PVP30, 0.5M citric acid, 1M sodium citrate, pH 4.0-4.5.
Rinsing liquid C: 2M guanidine hydrochloride, 0.15M NaCl, 56% (v/v) absolute ethanol.
Rinsing liquid D: 25mM Tris, 70% (v/v) absolute ethanol, and pH 7-7.5.
Eluent E: 10mM Tris, 0.5mM EDTA, pH 8.0.
Fourthly, concrete operation steps of DNA extraction and DNA concentration and purity determination
(1) Experimental group
1. Weighing 200mg of fresh excrement and placing the fresh excrement into a 2ml sterilized centrifugal tube;
2. adding 1ml of impurity removing solution A into the centrifuge tube, and homogenizing in a vortex oscillator for 5 min;
3. the homogenized samples were incubated in a metal bath at 70 ℃ for 5min, during which the mixture was mixed 1 time by turning upside down every 2 min:
4. after the incubation is finished, putting the sample in a centrifuge for centrifugation at 14000rpm for 1 min;
5. taking 600ul of the supernatant fluid to a new 2ml sterilized centrifuge tube;
6. adding 600ul of lysate B and 25ul of proteinase K;
7. mixing by turning upside down for 15 times, incubating in a metal bath at 70 deg.C for 10min, and mixing by turning upside down for 1 time every 3 min:
8. after the incubation, 600ul of absolute ethyl alcohol was added;
9. gently mixing by turning upside down, sucking 600ul of the mixture through a nucleic acid purification adsorption column, and centrifuging the mixture in a centrifuge at 12000rpm for 30 s;
10. discarding the waste liquid;
11. repeating for 9-10 times until all the lysate passes through the column;
12. adding 600ul of rinsing liquid C into the adsorption column, and centrifuging for 30s at 12000rpm in a centrifuge;
13. discarding the waste liquid;
14. adding 600ul of rinsing liquid D into the adsorption column, and centrifuging for 30s at 12000rpm in a centrifuge;
15. discarding the waste liquid;
16. putting the adsorption column into the centrifuge again, and performing air separation in the centrifuge at 12000rpm for 3 min;
17. transferring the adsorption column to a 1.5ml sterile centrifuge tube, and adding 60ul of eluent E;
18. incubating at room temperature for 1min, and centrifuging at 12000rpm for 2 min;
19. detecting the concentration and purity of the genome DNA by using an enzyme-labeling instrument and a Qubit3.0;
the integrity of the genomic DNA was checked by means of 20.1.5% agarose gel electrophoresis.
(2) Control group
1. Using a commercial QIAamp Fast DNA Stool Mini Kit to extract fecal DNA according to the operation instruction;
2. detecting the concentration and purity of the genome DNA by using an enzyme-labeling instrument and a Qubit3.0;
3.1.5% agarose gel electrophoresis to check the integrity of genomic DNA.
Fifth, experimental results
(1) Determination of the concentration and purity of gDNA solutions
Human feces were used as samples for experimental comparison with the QIAamp Fast DNA pool Mini Kit commercially available from the present patent, and the quality control was as follows in Table 1:
TABLE 1 determination of concentration and purity
Remarking: in order to ensure the accuracy of the experimental results, the experimental group and the control group respectively select three same samples from the same batch of kits to carry out parallel experiments, wherein QI1, QI2 and QI3 are 3 parallel experiments of a QIAamp Fast DNA Stool Mini Kit, and CG1, CGI2 and CG3 are 3 parallel experiments of the Kit.
(2) The 6 nucleic acids generated in the above experiment were subjected to 1.5% agarose gel nucleic acid electrophoresis quality check to see the integrity of the gDNA as shown in FIG. 1.
(3) QI1 and CG2 were used for sequencing of the bacterial 16S V4 library, and the distribution of the flora was shown in FIG. 2, and the primer sequences, amplification reaction systems and reaction conditions for bacterial 16S V4 library construction are shown in tables 2, 3 and 4 below.
TABLE 2 primer sequences
| Primer F | 5’-GTGYCAGCMGCCGCGGTAA-3’ |
| Primer R | 5’-GGACTACNVGGGTWTCTAAT-3’ |
TABLE 3 amplification reaction System
| KAPA 2×Mix | 10ul |
| Primer F/R | 1ul |
| Template | 2ul |
| NF·H2O | 7ul |
TABLE 4 reaction conditions
(4) Analysis of results
From the nucleic acid quality inspection, the invention proves that the extraction effect on the genomic DNA of the fecal microorganisms is better, the purity is equivalent to that of QIAamp Fast DNASOL Mini Kit, the integrity is good, and the recovery rate is enough to meet the downstream experiment although the concentration is low;
from the view of the relative abundance distribution of the microbial flora, the invention has no influence on the relative abundance distribution of the microbial flora in the sample, and the feasibility and the advantages of the invention are proved again.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. The present invention is not to be limited by the specific embodiments disclosed herein, and other embodiments that fall within the scope of the claims of the present application are intended to be within the scope of the present invention.
Claims (10)
1. A DNA extraction kit for fecal microorganism genome comprises an impurity removal solution A, a lysis solution B, a rinsing solution C, a rinsing solution D and an eluent E, wherein the impurity removal solution A, the lysis solution B, the rinsing solution C, the rinsing solution D and the eluent E are sequentially added into a test tube to react with fecal microorganism to extract DNA, and the kit is characterized in that:
the impurity removing liquid A comprises the following substances: CTAB, EDTA, PVP30, NaCl, TritonX-100, Tween-20, citric acid and sodium citrate;
the lysis solution B comprises the following substances: guanidine hydrochloride, CTAB, PVP30, citric acid and sodium citrate.
2. The DNA extraction kit for fecal microbial genome according to claim 1 characterized by:
in the impurity removing liquid A, the mass ratio range of CTAB is configured to be 1-3% M/M, the volume molar concentration range of EDTA is configured to be 20-30 mM, the mass ratio range of PVP30 is configured to be 1-3% M/M, the volume molar concentration range of NaCl is configured to be 1.5-2.0M, the volume ratio range of TritonX-100 is configured to be 1-3% v/v, the volume ratio range of Tween-20 is configured to be 0.5-1% v/v, the volume molar concentration range of citric acid is configured to be 0.1-0.5M, the volume molar concentration range of sodium citrate is configured to be 0.2-1M, and the pH range of the impurity removing liquid A is configured to be 4.0-4.5.
3. The DNA extraction kit for fecal microbial genome according to claim 1 characterized by:
in the lysis solution B, the volume molar concentration range of guanidine hydrochloride is configured to be 4-6M, the mass ratio range of CTAB is configured to be 0.5-1.5% M/M, the mass ratio range of PVP30 is configured to be 0.5-1.5% M/M, the volume molar concentration range of citric acid is configured to be 0.1-0.5M, the volume molar concentration range of sodium citrate is configured to be 0.2-1M, and the pH range of the lysis solution B is configured to be 4.0-4.5.
4. The DNA extraction kit for fecal microbial genome according to claim 1 characterized by:
the rinsing liquid C comprises the following substances: guanidine hydrochloride, NaCl and absolute ethyl alcohol.
5. The DNA extraction kit for fecal microbial genome according to claim 1 characterized by:
the rinsing liquid D comprises the following substances: tris and absolute ethyl alcohol.
6. The DNA extraction kit for fecal microbial genome according to claim 1 characterized by:
the eluent E comprises the following substances: tris and EDTA.
7. The DNA extraction kit for fecal microbial genome according to claim 4 characterized by:
in the rinsing liquid C, the volume molar concentration range of guanidine hydrochloride is 2-4M, the volume molar concentration of NaCl is 0.15M, and the volume ratio of absolute ethyl alcohol is 56% v/v.
8. The DNA extraction kit for fecal microbial genome according to claim 5 characterized by:
in the rinsing liquid D, the volume molar concentration of Tris is configured to be 25mM, the volume ratio of absolute ethyl alcohol is configured to be 70% v/v, and the pH range of the rinsing liquid D is configured to be 7-7.5.
9. The DNA extraction kit for fecal microbial genome according to claim 6 characterized by:
in the eluent E, the volume molar concentration of Tris is configured to be 10mM, the volume molar concentration of EDTA is configured to be 0.5mM, and the pH of the eluent E is configured to be 8.0.
10. An extraction method for the DNA extraction kit for fecal microbial genome according to any of claims 1 to 9, performed in order of the steps of decontamination, lysis, rinsing and elution, characterized in that:
the impurity removing step specifically comprises the following steps:
1) weighing fresh excrement and placing the fresh excrement in a sterilized centrifugal tube;
2) adding the impurity removing liquid A into the centrifuge tube, and homogenizing for 5min in a vortex oscillator;
3) the homogenized samples were incubated in a metal bath at 70 ℃ for 5min, during which the mixture was mixed 1 time by turning upside down every 2 min:
4) after the incubation is finished, putting the sample in a centrifuge for centrifugation at 14000rpm for 1 min;
wherein 1ml of the impurity removal liquid A is added into every 200mg of excrement;
the cracking step specifically comprises the following steps:
5) taking the supernatant obtained in the step 4) into a new sterilized centrifugal tube;
6) adding the lysis solution B and protease K;
7) mixing by turning upside down for several times, incubating in a metal bath at 70 deg.C for 10min, and mixing by turning upside down for 1 time every 3 min:
8) after the incubation is finished, adding absolute ethyl alcohol;
9) slightly reversing the upper part and the lower part, uniformly mixing, sucking the sterilized centrifuge tube solution, passing the sterilized centrifuge tube solution through a nucleic acid purification adsorption column, and placing the nucleic acid purification adsorption column in a centrifuge for 30s at 12000 rpm;
10) discarding the waste liquid;
11) repeating the steps 9) to 10) until all the lysate passes through the column;
the rinsing step specifically comprises the following steps:
12) adding the rinsing liquid C into an adsorption column, and placing the adsorption column in a centrifuge for centrifugation at 12000rpm for 30 s;
13) discarding the waste liquid;
14) adding the rinsing liquid D into an adsorption column, and placing the adsorption column in a centrifuge for centrifugation at 12000rpm for 30 s;
15) discarding the waste liquid;
16) putting the adsorption column into the centrifuge again, and standing at 12000rpm for 3 min;
the elution step specifically comprises the steps of:
17) transferring the adsorption column to a sterile centrifuge tube, and adding the eluent E;
18) incubating at room temperature for 1min, centrifuging at 12000rpm in a centrifuge for 2min, and recovering eluate;
wherein, the impurity removing solution A, the supernatant in the step 4), the lysis solution B, the protease K, the absolute ethyl alcohol in the step 8), the rinsing solution C, the rinsing solution D and the eluent E are sequentially mixed according to the ratio of 1000: 600: 600: 25: 600: 600: 600: the proportion of 60 is added.
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| CN115532242A (en) * | 2022-10-18 | 2022-12-30 | 广州美基生物科技有限公司 | Humic acid adsorbent and preparation method thereof soil DNA extraction kit |
| CN116287106A (en) * | 2022-12-30 | 2023-06-23 | 上海碧云天生物技术有限公司 | Method and reagent for enhancing firefly luciferase ATP bioluminescence detection performance |
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