CN113186250B - Impurity removal reagent for extracting microbial genome DNA of excrement - Google Patents
Impurity removal reagent for extracting microbial genome DNA of excrement Download PDFInfo
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- CN113186250B CN113186250B CN202110523659.5A CN202110523659A CN113186250B CN 113186250 B CN113186250 B CN 113186250B CN 202110523659 A CN202110523659 A CN 202110523659A CN 113186250 B CN113186250 B CN 113186250B
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Abstract
The invention discloses an impurity removal reagent for extracting microbial genome DNA of excrement, belonging to the field of nucleic acid extraction in molecular biology, and the impurity removal reagent for extracting the microbial genome DNA of excrement comprises the following substances: CTAB, EDTA, PVP30, NaCl, TritonX-100, citric acid and sodium citrate. The impurity removal reagent for extracting the microbial genome DNA of the excrement can effectively remove PCR inhibitors such as humic acid, polysaccharide, polyphenol, pigment and the like in the excrement, and can be used for obtaining gDNA with high purity, high recovery rate and good integrity by matching with lysate Buffer AL, rinse AW1 and Buffer AW2 of QIAGEN company.
Description
Technical Field
The invention relates to the field of nucleic acid extraction in molecular biology, in particular to an impurity removal reagent for extracting microbial genome DNA of excrement.
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.
The intestinal tract contains a large number of intestinal flora with rich varieties, and the intestinal flora and a host form a mutual-benefit symbiotic relationship, so that the intestinal flora is the second set of genome of human beings and has direct or indirect influence on the health of the host. Therefore, the research on the host intestinal flora is particularly important, the health condition or the relation with diseases of the host can be judged by looking at the state of the host intestinal flora, and an improvement or treatment scheme can be provided according to the judgment. The research on the intestinal flora relates to the extraction of the nucleic acid of the intestinal flora, so that the acquisition of high-quality gDNA of the intestinal flora is particularly important, and the subsequent PCR and high-throughput sequencing can be influenced. However, the conventional method for removing the inhibitor is complex to operate, and the commercial fecal microorganism genome impurity removal reagent is high in price, so that the provision of the impurity removal reagent which is simple and easy to operate and low in cost is increasingly important.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an impurity removal reagent for extracting microbial genome DNA 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 by using the impurity removal reagent, the research on intestinal flora by the medical workers is facilitated, and the quality assurance is provided for subsequent PCR or high-throughput library establishment.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an impurity removal reagent for extracting microbial genome DNA of excrement, which comprises the following substances: CTAB, EDTA, PVP30, NaCl, TritonX-100, citric acid and sodium citrate.
The preferable technical scheme of the invention is that the mass ratio range of CTAB is configured to be 1-3% m/m.
The preferable technical scheme of the invention is that the volume molar concentration range of the EDTA is configured to be 20-30 mM.
The preferable technical scheme of the invention is that the mass ratio range of PVP30 is configured to be 1-3% m/m.
The preferable technical scheme of the invention is that the volume molar concentration range of NaCl is configured to be 1.5-2.0M.
The preferable technical scheme of the invention is that the volume ratio range of TritonX-100 is 1-3% v/v.
The preferable technical scheme of the invention is that the volume molar concentration range of the citric acid is configured to be 0.1-0.5M.
The preferable technical scheme of the invention is that the volume molar concentration range of the sodium citrate is configured to be 0.2-1M.
The preferable technical scheme of the invention is that the pH range of the impurity removal reagent for extracting the genomic DNA of the fecal microorganisms is configured to be 4.0-4.5.
The invention has the beneficial effects that:
the impurity removal reagent for extracting the microbial genome DNA of the excrement can effectively remove PCR inhibitors such as humic acid, polysaccharide, polyphenol, pigment and the like in the excrement, can obtain gDNA with high purity, high recovery rate and good integrity by matching with lysate Buffer AL, rinse AW1 and Buffer AW2 of QIAGEN company, provides quality guarantee for subsequent PCR or high-flux library construction, shows the same effect compared with a kit sold in the market abroad, does not generate difference influence on the abundance distribution of flora, and has the advantages of simple operation, low cost, effective removal of the PCR inhibitors and the like. Through the process, impurities can be conveniently and efficiently removed by medical workers through the impurity removal reagent, so that fecal microorganism genome DNA is extracted, the research on intestinal flora is facilitated for the medical workers, and the quality assurance is provided for subsequent PCR or high-flux library establishment.
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 comparison of flora distribution after performing library construction sequencing analysis on the impurity removal reagent for extracting genomic DNA of fecal microorganisms and a foreign commercially available kit 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.
As shown in fig. 1 and fig. 2, in order to enable medical workers to conveniently and efficiently remove impurities through the impurity removal reagent of the present application so as to extract fecal microbial genomic DNA, which is helpful for the medical workers to study intestinal flora and provide quality assurance for subsequent PCR or high-throughput library establishment, further, the impurity removal reagent for fecal microbial genomic DNA extraction provided in this embodiment includes the following substances: CTAB, EDTA, PVP30, NaCl, TritonX-100, citric acid and sodium citrate. After medical care worker adds the edulcoration reagent of this application in to the excrement and urine sample, can conveniently get rid of impurity such as protein, humic acid, polysaccharide, polyphenol, pigment to isolate excrement and urine microorganism genome nucleic acid.
Preferably, the mass ratio of CTAB is configured to be 1-3% m/m. 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 a solution of high ionic strength (>0.7mol/L NaCl), CTAB forms complexes with proteins and polysaccharides, except that nucleic acids cannot be precipitated. Extracting with organic solvent to remove protein, polysaccharide, phenols and other impurities, and precipitating with ethanol to separate nucleic acid.
Preferably, the volume mol concentration range of EDTA is configured to be 20-30 mM. EDTA is an enzyme inhibitor, and can chelate divalent metals and inhibit metal-dependent enzymes. Thus, by adding EDTA, Mg can be chelated 2+ Or Mn 2+ Ion, inhibiting the activity of DNase enzyme in cell, degrading DNA, EDTA as bivalent ion mixture capable of decocting Mg 2+ And Ca 2+ 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.
Preferably, the mass ratio of the PVP30 is configured to be 1-3% m/m. 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.
Preferably, the volume molar concentration range of NaCl is configured to be 1.5-2.0M. NaCl is used to provide a high salt environment to fully dissolve DNA in the liquid phase, and high concentrations of salt are 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.
Preferably, the volume ratio range of TritonX-100 is configured to be 1-3% v/v. 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.
Preferably, the volume molar concentration range of the citric acid is configured to be 0.1-0.5M. Citric acid is used to maintain the reagents in an acidic environment.
Preferably, the volume molar concentration range of the sodium citrate is configured to be 0.2-1M. The sodium citrate can control the ionic strength of a reaction system, and has a certain buffering effect to ensure the relatively stable state of the PH of the system.
Preferably, the pH range of the impurity removal reagent for extracting the genomic DNA of the fecal microorganisms is 4.0-4.5. The pH value of the impurity removal reagent can ensure an excellent impurity removal effect in an acidic environment of 4.0-4.5.
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, citric acid and sodium citrate supplied by MACKLIN; the absolute ethyl alcohol is produced by national medicine group; buffer AL, Buffer AW1, Buffer AW2, ATE from QIAamp Fast DNA pool Mini Kit.
Control group: QIAamp Fast DNA pool Mini Kit (commercially available fecal microorganism genomic DNA extraction Kit, including Buffer A, Buffer AL, Buffer AW1, Buffer AW2, ATE)
(2) Instrument
Multiskan TM The 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
And the excrement sample for verifying the extraction effect of the impurity removal reagent on the microbial genome DNA is derived from freshly collected human excrement.
Third, formula
(1) Experimental group
Removing impurities from the reagent: 2% (M/M) CTAB, 25mM EDTA, 3% (M/M) PVP30, 1.5M NaCl, 1% (v/v) TritonX-100, 0.5M citric acid, 1M sodium citrate, pH 4.2;
lysis solution: buffer AL;
rinsing liquid: buffer AW1, Buffer AW 2;
eluent: and ATE.
(2) Control group
Removing impurities from the reagent: buffer A;
lysis solution: buffer AL;
rinsing liquid: buffer AW1, Buffer AW 2;
eluent: and ATE.
Fourthly, concrete operation steps of DNA extraction and DNA concentration and purity determination
The experimental group and the control group are respectively carried out according to the following steps:
1. weighing 200mg of fresh excrement and placing the fresh excrement into a 2ml sterile centrifuge tube;
2. adding 1ml of impurity removing reagent into the centrifuge tube, and homogenizing for 5min in a vortex oscillator;
3. incubating the homogenized sample in a metal bath at 70 deg.C for 5min at intervals of 2min
Mix 1 time by turning upside down the mixture in minutes:
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 lysate AL 600ul and protease K25 ul of QIAGEN company;
7. mixing by turning upside down for 15 times, incubating in 70 deg.C metal bath for 10min, 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. 600ul of rinsing Buffer AW1 was added to the adsorption column and centrifuged at 12000rpm for 30s in a centrifuge;
13. discarding the waste liquid;
14. 600ul of rinsing Buffer AW2 was added to the adsorption column and centrifuged at 12000rpm for 30s 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 ATE;
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.
Fifth, experimental results
(1) Determination of the concentration and purity of gDNA solutions
The human feces are used as samples, the impurity removal reagent of the invention patent and the impurity removal reagent of the commercial QIAamp Fast DNA Stool Mini Kit are used for experimental comparison, and the quality inspection is as the following table 1:
TABLE 1 determination of concentration and purity
Remarking: in order to ensure the accuracy of the experimental results, three identical samples of the reagents of the experimental group and the control group are respectively selected from the same batch of reagents to carry out parallel experiments, wherein QI1, QI2 and QI3 are 3 parallel experiments of QIAamp Fast DNA pool Mini Kit, and CG1, CGI2 and CG3 are 3 parallel experiments of the impurity removal reagent.
(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
| |
10ul |
| Primer F/R | 1ul |
| Template | 2ul |
| NF·H 2 O | 7ul |
TABLE 4 reaction conditions
(4) Analysis of results
From the aspect of nucleic acid quality inspection, the invention proves that the extraction effect on the genomic DNA of the fecal microorganisms is better, the purity of the genomic DNA is equivalent to that of a foreign famous Kit QIAamp Fast DNA stock Mini Kit, the integrity of the genomic DNA 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 (1)
1. An impurity removal reagent for extracting microbial genome DNA of excrement is characterized in that:
it consists of the following substances: 1-3% M/M CTAB, 20-30 mM EDTA, 1-3% M/mVP 30, 1.5-2.0M NaCl, 1-3% v/v TritonX-100, 0.1-0.5M citric acid and 0.2-1M sodium citrate;
the pH range of the impurity removal reagent for extracting the fecal microorganism genome DNA is configured to be 4.0-4.5.
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| CN104450685A (en) * | 2014-12-29 | 2015-03-25 | 福建师范大学 | Kit and extraction method for rapidly extracting RNA of animal fecal microorganism |
| CN104498477A (en) * | 2014-12-29 | 2015-04-08 | 福建师范大学 | Kit for extracting animal fecal microbial genomes by CTAB method, and extraction method of kit |
| CN104531680A (en) * | 2014-12-29 | 2015-04-22 | 福建师范大学 | Kit and extraction method for quickly extracting microbial genome DNA from animal fecal microorganisms |
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