CN114231526B - Method for extracting genome DNA of high-abundance fecal microorganisms - Google Patents
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Abstract
The invention provides a method for extracting genome DNA of a high-abundance fecal microorganism, which is realized based on a magnetic bead method and comprises buffer solution RB1, buffer solution RB2, lysis solution, buffer solution P, buffer solution M, cleaning solution 1, cleaning solution 2 and eluent, wherein the specific process comprises the following steps: and (3) pretreating the excrement sample, and then cracking, combining, washing and eluting to obtain the excrement microbial genome DNA. After the sample is pretreated by buffer solutions RB1 and RB2, impurities such as protein, humic acid, polysaccharide, lipid and the like in the sample can be effectively removed, preliminary cracking effect on the sample is realized, and high-purity and high-quality sample DNA is finally obtained through subsequent further digestion and cracking, so that high-quality guarantee is provided for detection of downstream PCR and the like. The invention also improves the abundance of the microorganisms in the extracted excrement sample, and can be combined with the magnetic bead method for extraction, thereby realizing automation and high flux.
Description
Technical Field
The invention relates to the field of nucleic acid extraction, in particular to a method for extracting genomic DNA of high-abundance fecal microorganisms.
Background
The human gastrointestinal tract is populated by a wide variety of microorganisms, of about thousands of different species, known as gut microbiota, and such a community of microorganisms living in the body's gut is collectively known as the gut microbiota. Recent evidence of more and more studies shows that the intestinal flora is directly related to the occurrence of various human diseases, and among them, various top academic journals including Nature and Science successively published the latest research results of the relationship between intestinal microorganisms and human health. Scientists have discovered that the intestinal flora is associated with a variety of diseases such as autism, alzheimer's disease, parkinson's disease, hypertension, type 2 diabetes, inflammatory bowel disease, colorectal cancer, and even gradually freezing. Meanwhile, the intestinal microbiota is also an important part of the human body, and comprises the functions of helping the body digest food, producing vitamins, reducing bile acid, regulating the body immunity and even treating cancers.
However, in order to study microorganisms in human intestinal tracts, the intestinal flora needs to be separated and identified firstly, but many microorganisms in nature cannot be separated and identified by the existing culture method, and most of the intestinal flora needs an anaerobic environment for growth, and meanwhile, the bacterial strain cannot be accurately identified only by morphological and physiological biochemical detection, which causes limitation of the culture method. With the development and popularization of molecular biology in recent years, researchers often combine culture methods with molecular biology methods to study the flora of interest. Genes of all microorganisms in the intestinal tract, also called as a microbial group, are 100-150 times of the human genome, and the intestinal microorganisms are sometimes called as 'forgotten organs'. The gut microorganisms are therefore considered to be the second genome of the human body. For the research of the intestinal microbial genome, the microbial genome DNA with higher concentration and purity needs to be extracted, and further for the research of the intestinal microbial diversity, the microbes with more types and higher abundance need to be extracted.
For the extraction of the genomic DNA of the fecal microorganisms, the common extraction methods at present comprise a phenol/chloroform extraction method, a column chromatography method and a magnetic bead method.
Wherein, the phenol/chloroform extraction method has a more complicated flow, and toxic reagents such as chloroform and the like are used, which seriously affects the body health of operators; the multi-step centrifugal operation is adopted in the extraction by the column-passing method, and the process is time-consuming and labor-consuming; the existing magnetic bead method mainly aims at optimizing the extraction process or effectively removing inhibitors in a sample, and few methods are used for extracting microorganism DNA with more types and higher abundance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a method for extracting genomic DNA of high-abundance fecal microorganisms, which realizes automatic and rapid separation and purification of DNA.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for extracting genome DNA of high-abundance fecal microorganisms is realized based on a magnetic bead method, and comprises the following specific processes:
the fecal sample is pretreated and then subjected to cracking, combination, washing and elution to obtain fecal microorganism genome DNA, wherein the cracking process comprises the following steps: adding 200-;
the lysis solution comprises a mixture of 20-200mmol/L sodium chloride, 1-10mmol/L disodium ethylene diamine tetraacetate, 10-100mmol/L Tris-HCl, 2-5mol/L guanidine thiocyanate, 30-40% isopropanol and 1-10% polyethylene glycol p-isooctyl phenyl ether, and the pH value of the Tris-HCl is 8.0; the buffer solution P is 20-50mg/mL proteinase K.
Further, the combination process is specifically as follows: after the lysis is finished, adding 200-500 mu L of anhydrous ethanol and 15 mu L of buffer solution M, and shaking and combining for 10min at room temperature; after magnetic separation, the supernatant was removed completely;
the buffer solution M is superparamagnetic silicon hydroxyl nano magnetic microspheres, the particle size is 50-500nm, and the concentration is 50-200 mg/mL.
Further, the washing process is specifically as follows: adding 500-1000 mu L of cleaning solution into the centrifugal tube, carrying out vortex oscillation for 1min, placing the centrifugal tube on a magnetic frame, standing until the magnetic beads are completely adsorbed, and completely removing the supernatant to finish the cleaning process.
Further, the cleaning liquid comprises a cleaning liquid 1 and a cleaning liquid 2,
the cleaning solution 1 comprises 6-8mol/L guanidine hydrochloride, 5-50mmol/L citric acid, 5-50mmol/L sodium citrate and 60-80% ethanol solution, and the pH value of the cleaning solution 1 is between 4 and 6;
the cleaning solution 2 is 80% ethanol.
Further, the cleaning process is sequentially completed by the cleaning solution 1 and the cleaning solution 2.
Further, adding 50-100 μ L of eluent into the centrifuge tube during the elution process, uniformly mixing for 20s by vortex, and oscillating at the constant temperature of 1300rpm for 10min at 60 ℃; the eluent is 5-10mmol/L Tris-HCl, and the pH value of the eluent is 8.0.
Further, alcohol removal is performed before the elution, and the alcohol removal process is to place the centrifugal tube on a magnetic frame and air-dry the centrifugal tube in a fume hood for 5 min.
Further, the pretreatment process comprises sample treatment and inhibitor removal;
the sample processing process is specifically as follows: taking a 50-200mg fecal sample into a 2 mL centrifuge tube, adding 400-800 muL buffer solution RB1, fully whirling, shaking and mixing uniformly, heating, shaking and cracking at 1500rpm for 15min at 70 ℃, then centrifuging at 12,000 rpm for 2 min, and transferring about 300-600 muL supernatant to a new 2 mL centrifuge tube;
the inhibitor removal process is specifically as follows: adding 100 plus 400 mu L buffer solution RB2, vortex, shaking and mixing uniformly, placing for 5min at 4 ℃, centrifuging for 2 min at 12,000 rpm, and transferring 200 plus 500 mu L supernatant to a new 2 mL centrifuge tube.
Further, the buffer solution RB1 comprises 20-200mmol/L sodium chloride, 10-20mmol/L disodium ethylene diamine tetraacetate, 10-100mmol/L Tris-HCl, 20-200mmol/L guanidine thiocyanate, 0.5-2% sodium dodecyl sulfate, 0.5-5% polyethylene glycol p-isooctyl phenyl ether, and the pH value of the Tris-HCl is 8.0;
the buffer RB2 comprises 0.5-2mol/L potassium acetate, 2-5mol/L guanidine hydrochloride, 0.1-10mmol/L polyaluminum chloride, and the pH value of the buffer RB2 is 5-6.
Compared with the prior art, the invention has the beneficial effects that: the types of the extracted fecal microorganisms are increased, and automation and high flux can be realized by combining the extraction with the magnetic bead method.
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The disclosure of the present invention is illustrated with reference to the accompanying drawings. It is to be understood that the drawings are designed solely for the purposes of illustration and not as a definition of the limits of the invention. In the drawings, like reference numerals are used to refer to like elements throughout. Wherein:
FIG. 1 shows schematically agarose gel electrophoresis of DNA from 3 different stool samples extracted using the present invention; wherein M represents DL 15000 DNA Marker; 1 represents that the fecal sample 1 is used for extracting genome DNA; 2, extracting genome DNA from the fecal sample 2; 3, extracting genome DNA from the fecal sample 3;
FIG. 2 schematically shows species profiling histograms at the level of classification of samples by the column method as well as the magnetic bead method.
Detailed Description
It is easily understood that according to the technical solution of the present invention, a person skilled in the art can propose various alternative structures and implementation ways without changing the spirit of the present invention. Therefore, the following detailed description and the accompanying drawings are merely illustrative of the technical aspects of the present invention, and should not be construed as all of the present invention or as limitations or limitations on the technical aspects of the present invention.
A method for extracting genome DNA of high-abundance fecal microorganisms comprises the following steps:
s1 sample processing: taking a 50-200mg fecal sample, adding 400-800 muL buffer solution RB1 into a 2 mL centrifuge tube, fully swirling, shaking and mixing uniformly, and heating, shaking and cracking at 1500rpm for 15min at 70 ℃. Then centrifuged at 12,000 rpm for 2 min, transferring approximately 300 and 600 μ L of supernatant to a new 2 mL centrifuge tube.
S2. inhibitor removal: adding 100-400-mu L buffer solution RB2, vortexing, shaking and mixing uniformly, and standing at 4 ℃ for 5 min. Centrifuging at 12,000 rpm for 2 min, and transferring 200 and 500 mu L of supernatant to a new 2 mL centrifuge tube.
S3 nucleic acid cleavage: adding 200-plus 500 muL lysis solution and 20 muL buffer solution P, shaking and mixing uniformly, and heating and cracking at 70 ℃ at 1500rpm for 10 min.
S4 nucleic acid binding: after the cracking is completed, 200-fold 500 muL of anhydrous ethanol and 15 muL of buffer solution M are added, and the room temperature is vibrated and combined for 10 min. Magnetic separation, thoroughly remove the supernatant, during which avoid contact with magnetic beads.
S5, cleaning 1: 500-. And (4) placing the centrifuge tube on a magnetic frame, standing until the magnetic beads are completely adsorbed, and completely removing the supernatant.
S6, cleaning 2: 500-. And (4) placing the centrifuge tube on a magnetic frame, standing until the magnetic beads are completely adsorbed, and completely removing the supernatant. Repeat step 1 time.
S7, removing alcohol: placing the centrifuge tube on a magnetic frame, placing in a fume hood, and air drying for 5 min.
S8, elution: adding 50-100 μ L of eluent, mixing by vortex for 20s, and shaking at 60 deg.C at 1300rpm for 10 min.
S9, nucleic acid transfer: and (3) placing the centrifugal tube on a magnetic frame, standing for 1min, and transferring the supernatant into a new centrifugal tube after the magnetic beads are completely adsorbed to obtain the fecal microorganism genome DNA.
Wherein the buffer solution RB1 comprises 20-200mmol/L sodium chloride, 10-20mmol/L disodium ethylene diamine tetraacetate, 10-100mmol/L Tris-HCl (pH 8.0), 20-200mmol/L guanidine thiocyanate, 0.5-2% sodium dodecyl sulfate, 0.5-5% poly (ethylene diamine tetraacetic acid)Ethylene glycol p-isooctyl phenyl ether. The buffer RB2 comprises 0.5-2mol/L potassium acetate, 2-5mol/L guanidine hydrochloride, 0.1-10mmol/L polyaluminum chloride, and pH 5-6. The preferable buffer solutions RB1 and RB2 can effectively remove impurities such as protein, humic acid, polysaccharide, lipid and the like in the sample, thereby obtaining the sample DNA with higher purity and providing high-quality assurance for detection of downstream PCR and the like. Disodium edetate is a compound capable of reacting with Mg 2+ 、Ca 2+ 、Mn 2+ These divalent metal ion-binding chelators, since most nucleases act in the presence of Mg2+, such as the enzyme Dnase, are often used as nuclease inhibitors to protect DNA from denaturation or degradation. Guanidine thiocyanate is a powerful protein denaturant, and can dissolve proteins, and the main function is to crack cells and depolymerize nucleic acid substances in the cells to release the nucleic acid substances. Sodium dodecyl sulfate is a known detergent capable of denaturing proteins, solubilizing lipids and proteins on cell membranes to disrupt the cell membranes, and at high temperatures to disrupt the binding of proteins to nucleic acids to facilitate nucleic acid release. Polyethylene glycol p-isooctyl phenyl ether is a common nonionic surfactant, one of the common components of cell lysate, and can improve the permeability of cell membranes. Potassium ions in potassium acetate react with sodium dodecyl sulfate to form a precipitate, while sodium dodecyl sulfate can be combined with protein, on average, one sodium dodecyl sulfate molecule can be combined on two amino acids, and the precipitate generated by potassium sodium ion displacement can precipitate most of protein. Polyaluminum chloride is often used as a flocculating agent, and humic acid in a sample can form coagulation with aluminum salt, so that the content of humic acid in the sample is effectively reduced, and the purity of DNA is improved. Guanidine hydrochloride with higher concentration is additionally added into the buffer solution, which can dissolve protein, destroy the cell structure, destroy the secondary structure of nucleoprotein, release pure DNA, realize the effect of primary cell lysis and lay the foundation for further cell lysis combination.
The lysis solution comprises 20-200mmol/L sodium chloride, 1-10mmol/L disodium ethylene diamine tetraacetate, 10-100mmol/L Tris-HCl (pH 8.0), 2-5mol/L guanidine thiocyanate, 30-40% isopropanol and 1-10% polyethylene glycol p-isooctyl phenyl ether. The buffer solution P is 20-50mg/mL proteinase K. The addition of the preferred lysis solution and buffer solution P further realizes the sufficient lysis of the DNA sample, thereby obtaining the DNA sample with higher concentration. Tris-HCl is a common buffer, providing a relatively suitable environment for lysis and nucleic acids. Proteinase K is a powerful proteolytic enzyme, and has the main function of digesting histone combined with nucleic acid to make DNA free in solution. Isopropanol is a strongly hydrophilic solvent, and when added to a solution, the aqueous solution becomes saturated and the DNA molecules exhibit a tendency to escape from the solution and thus be adsorbed onto the surface of the solid support.
The buffer solution M is superparamagnetic silicon hydroxyl nano magnetic microsphere with the particle size of 50-500nm and the concentration of 50-200 mg/mL. The preferred magnetic microspheres in buffer M are one of the most widely used magnetic beads, which can adsorb DNA under high ion concentration salt and release DNA under low ion salt.
The cleaning solution 1 comprises 6-8mol/L guanidine hydrochloride, 5-50mmol/L citric acid, 5-50mmol/L sodium citrate, 60-80% ethanol solution with pH of 4-6. Cleaning solution 2 is 80% ethanol. Preferably, the washing solutions 1 and 2 can remove impurities such as proteins and salts in the sample DNA, and finally obtain high-purity DNA. Guanidine hydrochloride not only rapidly destroys cell membranes, denaturing proteins, causing them to denature and precipitate, thus allowing nucleic acids to break free of protein entanglement, it is also a strong inhibitor of nucleases. The sodium citrate can control the ionic strength in the reaction system, and also has a certain buffering effect, so that the relative stability of the pH value in the buffer solution is ensured. The 80% ethanol DNA is insoluble, and the salt ion is soluble, so that the DNA by the solution washing, can effectively remove the residual salt.
The eluent is 5-10mmol/L Tris-HCl, pH 8.0. The optimized eluent adopts a Tris-HCl buffer system, so that the relative stability of pH in the solution is ensured, and the elution efficiency of the solution on DNA is higher than that of sterilized water.
The technical effects of the present invention will be specifically described below with reference to examples.
Example 1 selection of 3 different stool samples for extraction using the method
1. Sample treatment: 100mg of the fecal sample is taken and placed in a 2 mL centrifuge tube, 700 mu L of buffer solution RB1 is added, and after full vortex shaking and uniform mixing, heating shaking cracking is carried out at 70 ℃ and 1500rpm for 15 min. Then centrifuged at 12,000 rpm for 2 min, transferring about 500 μ L of supernatant to a new 2 mL centrifuge tube.
2. Inhibitor removal: adding 200 mu L buffer solution RB2, vortex, shake and mix evenly, and placing for 5min at 4 ℃. Centrifuge at 12,000 rpm for 2 min, transfer 400 μ L of supernatant to a new 2 mL centrifuge tube.
3. Nucleic acid cleavage: adding 400 mu L of lysis solution and 20 mu L of buffer solution P, shaking and uniformly mixing, and heating and cracking at 70 ℃ and 1500rpm for 10 min.
4. Nucleic acid binding: and after the cracking is finished, adding 400 mu L of absolute ethyl alcohol and 15 mu L of buffer solution M, and oscillating and combining for 10min at room temperature. Magnetic separation, thoroughly remove the supernatant, during which avoid contact with magnetic beads.
5. Cleaning 1: add 800. mu.L of Wash 1 to the centrifuge tube and vortex for 1 min. And (4) placing the centrifuge tube on a magnetic frame, standing until the magnetic beads are completely adsorbed, and completely removing the supernatant.
6. And (3) cleaning 2: add 800. mu.L of Wash 2 to the tube and vortex for 1 min. And (4) placing the centrifuge tube on a magnetic frame, standing until the magnetic beads are completely adsorbed, and completely removing the supernatant. Repeat step 1 time.
7. Removing alcohol: placing the centrifuge tube on a magnetic frame, placing in a fume hood, and air drying for 5 min.
8. And (3) elution: adding 80 μ L of eluent, mixing by vortex for 20s, and shaking at 60 deg.C 1300rpm for 10 min.
9. Nucleic acid transfer: and (3) placing the centrifugal tube on a magnetic frame, standing for 1min, and transferring the supernatant into a new centrifugal tube after the magnetic beads are completely adsorbed to obtain the fecal microorganism genome DNA.
After completion of the extraction, the content and purity of DNA in the eluate were measured using a micro ultraviolet spectrophotometer (NanoDrop) as shown in Table 1, and the quality of DNA was checked by agarose gel electrophoresis as shown in FIG. 1. According to the following results, it can be seen from the table that the fecal microorganism genomic DNA extracted by the invention has high concentration and high purity, and from the electrophoresis chart, the genomic DNA extracted by the invention has clear main band, basically no impurity band and better integrity.
TABLE 1 NanoDrop determination of DNA content and purity in the eluate of example 1
Example 2 comparison of the extraction Effect of the invention with that of the existing column-type fecal genomic DNA extraction kit
3 different fecal samples were selected, two 100mg samples were taken from each sample, and placed in 2 mL centrifuge tubes, and extracted using the method and the conventional fecal genomic DNA extraction Kit of column method (QIAamp Fast DNA pool Mini Kit, cat # 51604), respectively, the extraction method being performed according to the instruction of the purchased Kit. After extraction, the fecal genomic DNA extracted by the above two methods was subjected to DNA content and purity determination in the eluate using a micro ultraviolet spectrophotometer (NanoDrop) as shown in table 2, and simultaneously subjected to downstream 16s library-building sequencing, followed by analysis of the sequencing data as shown in table 3 and fig. 2.
According to the following results, the extraction amount of the fecal genome DNA extracted by the invention is obviously higher than that of the existing column method kit; meanwhile, sequencing result analysis shows that the OTU abundance and the aroma index of the extracted sample are higher than those of the conventional column method, and the histogram also shows that the extracted sample is rich in species, so that the diversity of the extracted sample is higher, and a favorable extraction method is provided for the subsequent intestinal flora research.
TABLE 2 NanoDrop determination of DNA content and purity in the eluate of example 2
TABLE 3 DNA 16s sequencing of the sample DNA by the two methods mentioned above and Shannon index analysis
Note: OTUs, an abbreviation of Operational taxomic Units, artificially sets the same mark for a certain classification unit (strain, genus, species, group, etc.) in phylogenetic or population genetics research for the convenience of analysis. Theoretically one OTU represents one species of microorganism.
Shannon, the flavor intensity index, is one of the indices of flora diversity, and a higher Shannon value indicates a higher flora diversity.
The technical scope of the present invention is not limited to the above description, and those skilled in the art can make various changes and modifications to the above-described embodiments without departing from the technical spirit of the present invention, and such changes and modifications should fall within the protective scope of the present invention.
Claims (4)
1. A method for extracting genome DNA of high-abundance fecal microorganisms is characterized by being realized based on a magnetic bead method, and the specific process is as follows:
the fecal sample is pretreated and then subjected to cracking, combination, washing and elution to obtain fecal microorganism genome DNA, wherein the cracking process comprises the following steps: adding 200-;
the lysis solution is a mixture of 20-200mmol/L sodium chloride, 1-10mmol/L disodium ethylene diamine tetraacetate, 10-100mmol/L Tris-HCl, 2-5mol/L guanidine thiocyanate, 30-40% isopropanol and 1-10% polyethylene glycol p-isooctyl phenyl ether, and the pH value of the Tris-HCl is 8.0; the buffer solution P is 20-50mg/mL proteinase K;
the pretreatment process comprises sample treatment and inhibitor removal;
the sample processing process is specifically as follows: taking 50-200mg of a fecal sample, adding 400-fold 800-microliter buffer RB1 into a 2 mL centrifuge tube, fully whirling, vibrating and uniformly mixing, heating, vibrating and cracking at 1500rpm at 70 ℃ for 15min, then centrifuging at 12,000 rpm for 2 min, and transferring 300-fold 600-microliter supernatant to a new 2 mL centrifuge tube;
the inhibitor removal process is specifically as follows: adding 100 plus 400 mu L buffer solution RB2, uniformly mixing by vortex oscillation, placing for 5min at 4 ℃, centrifuging for 2 min at 12,000 rpm, and transferring 200 plus 500 mu L supernatant to a new 2 mL centrifuge tube;
the buffer solution RB1 is 20-200mmol/L sodium chloride, 10-20mmol/L disodium ethylene diamine tetraacetate, 10-100mmol/L Tris-HCl, 20-200mmol/L guanidine thiocyanate, 0.5-2% sodium dodecyl sulfate and 0.5-5% polyethylene glycol p-isooctyl phenyl ether, and the pH value of the Tris-HCl is 8.0;
the buffer RB2 is 0.5-2mol/L potassium acetate, 2-5mol/L guanidine hydrochloride and 0.1-10mmol/L polyaluminum chloride, and the pH value of the buffer RB2 is 5-6;
the binding process is specifically as follows: after the lysis is finished, adding 200-500 mu L of anhydrous ethanol and 15 mu L of buffer solution M, and shaking and combining for 10min at room temperature; after magnetic separation, the supernatant was removed completely;
the buffer solution M is superparamagnetic silicon hydroxyl nano magnetic microspheres, the particle size is 50-500nm, and the concentration is 50-200mg/mL;
the cleaning liquid comprises a cleaning liquid 1 and a cleaning liquid 2,
the cleaning solution 1 is 6-8mol/L guanidine hydrochloride, 5-50mmol/L citric acid, 5-50mmol/L sodium citrate, an ethanol solution with the concentration of 60-80%, and the pH value of the cleaning solution 1 is 4-6;
the cleaning solution 2 is 80% ethanol;
the cleaning process is completed by cleaning solution 1 and cleaning solution 2 in sequence.
2. The method for extracting genomic DNA of abundant fecal microorganisms according to claim 1, wherein the washing process is specifically as follows: adding 500-1000 mu L of cleaning solution into the centrifuge tube, carrying out vortex oscillation for 1min, placing the centrifuge tube on a magnetic frame, standing until magnetic beads are completely adsorbed, and completely removing the supernatant to finish the cleaning process.
3. The method for extracting genomic DNA of high-abundance fecal microorganisms according to claim 1, wherein in the elution process, 50-100 μ L of eluent is added into a centrifuge tube, vortexed and mixed for 20s, and shaken at a constant temperature of 1300rpm for 10min at 60 ℃; the eluent is 5-10mmol/L Tris-HCl, and the pH value of the eluent is 8.0.
4. The method for extracting genomic DNA of abundant fecal microorganisms according to claim 1, wherein the elution is preceded by alcohol removal by placing the centrifuge tube on a magnetic rack and air drying in a fume hood for 5 min.
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| CN113604464A (en) * | 2021-07-30 | 2021-11-05 | 中国科学院合肥物质科学研究院 | Kit for extracting and purifying DNA (deoxyribonucleic acid) of trace complex material to be detected and extraction method |
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