CN110592076A - Reagent and method for extracting microbial community DNA of fermented bean curd - Google Patents
Reagent and method for extracting microbial community DNA of fermented bean curd Download PDFInfo
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- CN110592076A CN110592076A CN201911022260.8A CN201911022260A CN110592076A CN 110592076 A CN110592076 A CN 110592076A CN 201911022260 A CN201911022260 A CN 201911022260A CN 110592076 A CN110592076 A CN 110592076A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- 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
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Abstract
The invention discloses a reagent and a method for extracting microbial community DNA of fermented bean curd. According to the method, starting from the characteristics of high soybean residue content and abundance of organic acids and amino acids of a fermented bean curd sample, processes of microorganism recovery, cell lysis, removal of PCR reaction substances and high-efficiency DNA recovery of fermented bean curd are designed. In the aspect of removing PCR inhibiting substances, a method combining ammonium sulfate, guanidine hydrochloride and CTAB is applied, and polysaccharide, polyphenol and interfering protein are removed simultaneously. Thereby obtaining high-quality DNA for subsequent molecular biology experiments. The method can simultaneously extract the DNA of the bacteria and the fungi of the fermented bean curd, and the extracted DNA is successfully applied to the PCR reaction and the high-throughput sequencing of 16S rRNA and ITS genes and also successfully applied to the metagenome sequencing and analysis.
Description
Technical Field
The invention belongs to the technical field of genome extraction, and relates to a reagent and a method for extracting microbial community DNA of fermented bean curd, in particular to the following steps: 1) a method for separating and recovering microorganisms in the fermented bean curd; 2) breaking microbial cells of the fermented bean curd, and removing polysaccharide, polyphenol and foreign protein precipitates; 3) a method for lysis of microbial cells and recovery of DNA; 4) the reagent formula used in the extraction process.
Background
The fermented bean curd has delicious taste and unique flavor, and is one of Chinese characteristic fermented soybean foods. The traditional fermented bean curd fermentation is divided into two stages, namely a front stage and a rear stage, and is a complex biochemical process. After microbial fermentation, the protein contained in the bean curd embryo is gradually hydrolyzed under the action of microbial protease to generate small molecular substances such as peptides, amino acids and the like; the starch is converted into monosaccharide and oligosaccharide, so that the starch is easily absorbed by a human body, and the bioavailability is maximized.
Because the fermented bean curd fermentation environment is open, the fermentation period is long, and a heating sterilization process is not needed, a large amount of important microorganisms are inevitably accumulated. In addition to the fermentation species, there is therefore the possibility of the entry of various microorganisms from the environment and from the raw materials, with the consequent formation of complex microbial communities, which play a decisive role in the quality of the fermented bean curd.
The research on the microorganisms of the preserved beancurd mainly focuses on the separation, identification and screening of the basic traditional culture strains in the early period, however, the recent research shows that the microorganisms such as exogenous bacteria, fungi and the like have a vital role on the quality of the preserved beancurd and the generation of flavor substances. Most of these exogenous microorganisms belong to the non-culturable type, and their colony composition characteristics need to be analyzed by molecular biological means.
At present, the community structure and species composition analysis of environmental bacteria and fungi is usually carried out by taking 16S rRNA and ITS genes as typical markers and combining a clone library or a high-throughput sequencing method for analysis. The application of the molecular biology method is premised on obtaining high-quality microbial DNA, however, most of fermented bean curd samples are soybean dregs and contain a large amount of organic acid, amino acid and other substances, and the existence of the substances makes the conventional microbial single-bacterium extraction method not suitable for fermented bean curd microbial communities. In addition, polysaccharides, polyphenols, polypeptides and the like contained in a large amount in the fermented bean curd belong to PCR reaction inhibiting substances, and a DNA extraction method for specifically removing the substances is a precondition for applying a molecular biological method to the research of microbial communities of the fermented bean curd. Meanwhile, the bacteria and fungus community in the fermented bean curd are greatly different in composition and function, and the bacteria and the fungus have great difference in composition such as cell walls, so that a method for simultaneously and efficiently extracting the DNA of the bacteria and fungus community is urgently needed.
Disclosure of Invention
Aiming at the problem that reagents for efficiently removing PCR reaction inhibiting substances such as polysaccharide, polyphenol, polypeptide and the like are lacked in the prior art, the invention mainly aims to provide a group of fermented bean curd microorganism DNA extraction reagents.
Another purpose of the invention is to provide a method for extracting the microbial community DNA of the fermented bean curd.
The purpose of the invention is realized by the following technical scheme:
a group of reagents for extracting microorganism DNA of fermented bean curd comprises cell separation solution (SFS buffer), cell washing solution (SFW buffer), cell lysis solution (SFLB buffer), PCR inhibiting substance precipitation solution (PIMP buffer) and cleaning solution (SFWbuffer);
the cell separation solution contains 10-20 mM potassium dihydrogen phosphate (KH)2PO4) 10 to 20mM disodium hydrogen phosphate (Na)2HPO4·12H2O), 100-200 mM sodium chloride (NaCl), 1-2 mM potassium chloride (KCl), pH 7-8;
the cell washing solution contains 100-200 mM potassium dihydrogen phosphate (KH)2PO4) 100 to 200mM disodium hydrogen phosphate (Na)2HPO4·12H2O), 0.05-0.10% Tween 20, and the pH value is 7-8; wherein, the Tween 20 is used as a surfactant to help remove the fermented bean curd vegetable protein adsorbed on the cell surface;
the cell lysate contains 0.5-1.0% SDS (sodium dodecyl sulfate), 0.5-1.0% polyethylene glycol octyl phenyl ether (Triton x-100), 100-200 mM Tris-HCl, 0.5-1.0 mM EDTA, 50-100 mM KCl, and the pH value is 7-8; wherein SDS is used as a detergent, Triton x-100 is used as a surfactant, and cell lysis and DNA release are facilitated; SDS can assist the precipitation of cell debris, polysaccharide and foreign protein at the same time;
the PCR inhibitor precipitation solution contains 100-200 mM potassium dihydrogen phosphate (KH)2PO4) 100 to 200mM disodium hydrogen phosphate (Na)2HPO4·12H2O), 50 to 100mM ammonium sulfate [ (NH)4)2SO4]200 to 500mM guanidine hydrochloride (CH)6ClN3) 1-2% CTAB (cetyl trimethyl ammonium bromide) and a pH value of 7-8; wherein ammonium sulfate is used as salting-out precipitator, which is helpful for precipitating polysaccharide, polypeptide and foreign protein; guanidine hydrochloride as a denaturant aids in the precipitation of heteroproteins; CTAB is helpful for removing polysaccharide and polyphenol;
the cleaning solution contains 75-85% of ethanol, 100-200 mM NaCl, 10-20 mM Tris and the pH value is 7-8; the cleaning solution containing ethanol as the main component can wash away high-salt components remained in the previous step, and is helpful for the combination of DNA and the silica gel filter membrane.
A method for extracting microbial community DNA of fermented bean curd adopts the extraction reagent for extraction, and comprises the following steps:
(1) mashing a fermented bean curd sample, adding tungsten carbide beads and a cell separation solution, shaking and fully mixing, standing for several minutes, centrifuging, and collecting precipitates;
the step of mashing the sample is to disperse the sample, increase the specific surface area of the sample and improve the contact between the reagent and the sample in the subsequent step to the maximum extent;
the diameter of the tungsten carbide beads is preferably 10-30 mm, and the large-diameter tungsten carbide beads are beneficial to separation of microbial cells from soybean particles in the fermented bean curd;
preferably, the vibration in the steps (1) and (2) is performed for 5-20 minutes on a shaking bed at room temperature of 100-150 revolutions per minute;
the centrifugation in the steps (1), (2), (4), (5) and (6) is preferably (12000-13000) x g centrifugation for 1-5 minutes;
(2) adding a cell flushing fluid into the precipitate obtained in the step (1), shaking for sufficiently mixing, standing for several minutes, centrifuging, and collecting the precipitate; removing the vegetable protein of the preserved beancurd adsorbed on the cell surface;
(3) adding cell lysate into the precipitate obtained in the step (2), adding garnet with the diameters of 0.7mm and 0.15mm respectively, and shaking for full mixing; adding a lysozyme reaction solution, carrying out water bath at 37 ℃ for 1-2 hours, adding proteinase K and 20% SDS, shaking for 0.5-1 hour in a shaking table at 37 ℃, then putting into a water bath at 65 ℃, taking out and shaking once every 10 minutes, and continuing for 1.0-1.5 hours;
the physical method is adopted to break the cells, and the surfaces of garnet particles are rough and uneven, so that the cell walls are easy to break; but the hardness is relatively low, and the damage to DNA is small under the condition of grinding and striking of a vortex instrument; the matching of the garnets with two different diameters is beneficial to breaking the walls of microorganisms (gram-positive bacteria and gram-negative bacteria, bacteria and fungi) with different cell wall structures, and the release efficiency of DNA is obviously improved; meanwhile, under the action of proteinase K and SDS, cell wall decomposition of biochemical method is carried out, which is helpful for DNA release;
in the step (3), 0.5-1.0 g of garnet with the diameter of 0.7mm and 1-2 g of garnet with the diameter of 0.15mm are added into each g of sediment;
oscillating for 5-10 minutes by adopting a vortex instrument in the oscillating steps (3) and (4);
in the step (3), 1-2 ml of proteinase K (20mg/m L) and 1-2 ml of 20% SDS are added into each 10ml of cell lysate;
(4) shaking the solution finally obtained in the step (3), fully mixing, then freezing and centrifuging, and sucking supernatant fluid to transfer into a new centrifugal tube; adding a PCR inhibiting substance precipitation solution into the supernatant, uniformly mixing, carrying out warm bath at 37 ℃ for 5-10 minutes, centrifuging, and transferring the supernatant into a new centrifugal tube;
this step consists in separating the insoluble microbial cell debris and other organic-inorganic components from the DNA; then adding PCR inhibiting substance precipitation solution, wherein ammonium sulfate is used as salting-out precipitator, which is helpful for precipitating polysaccharide, polypeptide and foreign protein; guanidine hydrochloride as a denaturant aids in the precipitation of heteroproteins; CTAB is helpful for removing polysaccharide and polyphenol;
the freezing centrifugation in the steps (4) and (5) is preferably carried out at 4-6 ℃ (12000-13000) x g for 15-20 minutes;
the addition amount of the PCR inhibiting substance precipitation solution is preferably 1:1 in volume ratio to the supernatant;
(5) adding a phenol-chloroform-isoamylol mixed solution into the supernatant, uniformly mixing, performing refrigerated centrifugation, and sucking the supernatant and transferring the supernatant into a new centrifuge tube; adding anhydrous ethanol, mixing, adsorbing to silica gel membrane centrifugal column, centrifuging, and discarding filtrate; in the step, ethanol is added to precipitate DNA particles, so that the DNA is favorably adsorbed to a silica gel filter membrane and the elution of soluble polysaccharide, polyphenol and foreign protein is favorably realized;
in the mixed solution of phenol, chloroform and isoamylol, the volume ratio of phenol, chloroform and isoamylol is 25:24: 1; the volume ratio of the phenol-chloroform-isoamyl alcohol mixed solution to the supernatant is preferably 1: 1;
the volume ratio of the absolute ethyl alcohol to the supernatant is preferably 1: 1;
(6) adding a cleaning solution into the silica gel membrane centrifugal column, centrifuging, discarding the filtrate, repeating the process for 2-3 times, and washing off impurities such as protein adsorbed on the silica gel membrane; then centrifuging the silica gel membrane centrifugal column to spin dry the ethanol on the silica gel membrane (the ethanol influences the final elution of DNA); finally adding preheated nuclease-free sterilized water at 65 ℃, incubating the centrifugal column filter membrane for several minutes at room temperature, centrifuging, and collecting DNA;
the silica gel membrane centrifugal column is centrifuged for 3-5 minutes by placing the centrifugal column at 4-8 ℃ (12000-13000) x g.
Compared with the prior art, the invention has the following advantages and effects:
according to the method, starting from the characteristics of high soybean residue content and abundance of organic acids and amino acids of a fermented bean curd sample, processes of microorganism recovery, cell lysis, removal of PCR reaction substances and high-efficiency DNA recovery of fermented bean curd are designed. In the aspect of removing PCR inhibiting substances, a method combining ammonium sulfate, guanidine hydrochloride and CTAB is applied, and polysaccharide, polyphenol and interfering protein are removed simultaneously. Thereby obtaining high-quality DNA (high purity and less protein) for subsequent molecular biology experiments. The method can simultaneously extract the DNA of the bacteria and the fungi of the fermented bean curd, and the extracted DNA is successfully applied to the PCR reaction and the high-throughput sequencing of 16S rRNA and ITS genes and also successfully applied to the metagenome sequencing and analysis.
Drawings
FIG. 1 shows the colony composition of red and white fermented bean curd.
FIG. 2 shows the species composition of the fungal community of red and white fermented bean curd.
FIG. 3 shows the functional composition of microbial communities of red and white fermented bean curds.
FIG. 4 shows the carbohydrate metabolism function composition of the microbial communities of red and white fermented bean curds; wherein the carbohydrate metabolism function is shown as ko00010, glycolysis/gluconeogenesis; ko00020, citric acid cycle; ko00030, the pentose acid pathway; ko00040, pentose and glucuronate transformations; ko00051, fructose and mannose metabolism; ko00052, galactose metabolism; ko00053, ascorbic acid metabolism; ko00500, starch and sucrose metabolism; ko00520, amino sugar and nucleotide sugar metabolism; ko00620, pyruvate metabolism; ko00630, glyoxylic acid and dicarboxylic acid metabolism; ko00640, propionic acid metabolism; ko00650, methyl butyrate metabolism; ko00660, c 5-branched diacid metabolism; ko00562, inositol phosphate metabolism.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
A set of reagents for extracting microorganism DNA of fermented bean curd comprises cell separating liquid, cell flushing liquid, cell lysate, PCR inhibiting substance precipitation liquid and cleaning liquid.
Preparation of cell separation solution: 1.36g KH was weighed out2PO4，3.58g Na2HPO4·12H2O, 5.84g of NaCl and 0.07g of KCl, and adding 800ml of deionized water, stirring uniformly by using a glass rod, adjusting the pH value to 7.5, and adding deionized water to a constant volume of 1L.
Preparation of cell washing liquid: 1.36g KH was weighed out2PO4，3.58g Na2HPO4·12H2O, 1ml of Tween 20, 800ml of deionized water is added, the mixture is stirred uniformly by using a glass rod, the pH value is adjusted to 7.5, and the deionized water is added to the mixture until the volume is 1L.
Preparation of cell lysate: 24.2g Tris (Tris hydroxymethyl aminomethane), 74.5g KCl and 0.3g EDTA are weighed, 800ml deionized water is added, after uniform stirring by using a glass rod, 5ml SDS and 5ml Triton x-100 are added, the pH value is adjusted to 7.5 by 100% HCl, and deionized water is added to the solution to reach the constant volume of 1L.
Preparing a PCR inhibition substance precipitation solution: 1.36g KH was weighed out2PO4，3.58g Na2HPO4·12H2O，13.2g(NH4)2SO4，19.1CH6ClN3And 800ml of deionized water was added thereto, and after stirring with a glass rod, 10ml of CTA was added theretoAnd (4) adjusting the pH value of the solution B to 7.5, and adding deionized water to the solution B until the volume is 1L.
Preparing a cleaning solution: weighing 5.8g NaCl and 1.2g Tris, adding 100ml deionized water, stirring uniformly by using a glass rod, adjusting the pH value to 7.5, and adding 100% ethanol to fix the volume to 1L.
Example 2
Commercial fermented bean curd microorganism DNAs of different manufacturers are extracted, the extraction reagent provided in example 1 is adopted for extraction, and 12 fermented bean curd samples are obtained in total, and the steps are as follows:
(1) a10 g sample of fresh fermented bean curd was mashed with a Mixer Mill MM 400 stirring homogenizer from RETSCH company;
(2) weighing 10g of mashed fermented bean curd sample, adding 1g of tungsten carbide beads with the diameter of 16mm, adding 50ml of cell separation solution SFS buffer, and shaking on a shaking table at room temperature of 120 revolutions per minute for 15 minutes;
(3) standing the sample at room temperature for 5 minutes, centrifuging at 13000 Xg for 1 minute, collecting the precipitate, pouring out all supernatant, and weighing about 5g of precipitate for the next step;
(4) adding 25mL of SFW buffer of cell washing liquid, shaking for 10 minutes on a shaking table at room temperature of 120 rpm, standing for 5 minutes at room temperature, centrifuging for 1 minute at 13000 Xg, collecting precipitate, pouring out all supernate, and recording the weight of the precipitate;
(5) adding 15ml of cell lysate SFLB buffer, and strongly shaking for 5 minutes on a vortex instrument according to the weight of 2.5g of garnet with the diameter of 0.7mm and 5g of garnet with the diameter of 0.15 mm;
(6) adding 1.5mL of lysozyme reaction solution (50mg/mL), carrying out water bath at 37 ℃ for 1-2 hours, adding 1.5mL of proteinase K (20mg/m L) and 1.5mL of 20% SDS, continuing shaking for 0.5 hour in a shaking table at 37 ℃, then putting the mixture into a water bath kettle at 65 ℃, taking out the mixture every 10 minutes, and shaking for 1 hour;
(7) after the solution is strongly shaken on a vortex instrument for 5 minutes, the temperature is 4 ℃, 13000 Xg is carried out, after centrifugation for 15 minutes, 10mL of supernatant is sucked and transferred into a new centrifuge tube;
(8) adding 10mL of PCR inhibiting substance precipitation liquid PIMP buffer, reversing the mixture up and down for 5 times, carrying out warm bath at 37 ℃ for 5 minutes, centrifuging at 13000 Xg for 3 minutes, and transferring supernatant into a new centrifuge tube;
(9) adding 10mL of phenol-chloroform-isoamyl alcohol (volume ratio is 25:24:1), turning upside down, shaking for 30 seconds by a vortex instrument, centrifuging for 15 minutes at 4 ℃, and sucking supernatant and transferring to a new centrifuge tube;
(10) adding 20mL of 100% ethanol, completely mixing, sucking into a silica gel membrane centrifugal column, centrifuging at room temperature at 13000 Xg for 2 minutes, and discarding the filtrate;
(11) adding 750ul of SFW buffer on a silica gel membrane centrifugal column, centrifuging for 2 minutes at room temperature of 13000 Xg, discarding filtrate, and repeating the process for 3 times;
(12) centrifuging the column at 4 deg.C and 13000 Xg for 5min, and spin-drying ethanol on silica gel film;
(13) adding 100ul of nuclease-free sterilized water preheated at 65 ℃, incubating the filter membrane of the centrifugal column for 5 minutes at room temperature, then, 13000 Xg, centrifuging for 2 minutes, and collecting DNA.
(14) DNA Nanodrop measures the mass and concentration of DNA, as shown in Table 1.
TABLE 1. microbial DNA concentration of fermented bean curd
| Sample(s) | Concentration (ng/. mu.l) | 260/280 | 260/230 | Volume (μ l) | Total amount of μ g |
| P2-1-1 | 421.7 | 1.84 | 1.13 | 40 | 16.868 |
| P2-1-2 | 532.5 | 1.88 | 1.58 | 40 | 21.3 |
| P3-1-1 | 161.9 | 1.63 | 0.49 | 40 | 6.476 |
| P3-1-2 | 186.6 | 1.78 | 1.01 | 40 | 7.464 |
| P4-1-1 | 104.8 | 1.72 | 0.65 | 40 | 4.192 |
| P4-1-2 | 147.7 | 1.66 | 0.63 | 40 | 5.908 |
| N1-1-1 | 91.5 | 1.79 | 0.59 | 40 | 3.66 |
| N1-1-2 | 85.3 | 1.71 | 1.19 | 40 | 3.412 |
| FR14-1-1 | 32.1 | 1.81 | 0.45 | 40 | 1.284 |
| FR14-1-2 | 81.1 | 1.86 | 0.98 | 40 | 3.244 |
| FR15-1-1 | 31.4 | 1.67 | 0.3 | 40 | 1.256 |
| FR15-1-2 | 42.4 | 2.08 | 0.3 | 40 | 1.696 |
As can be seen from Table 1, the concentration of the extracted DNA of the fermented bean curd is 31.4-532.5 ng/. mu.l, the total amount is 1.26-21.3. mu.g, 260/280 is 1.63-2.08, and the value of 260/230 is 0.3-1.58, which indicates that the extracted DNA has high purity and contains little protein. The extracted DNA meets the requirements of amplicon and metagenome sequencing.
Example 3
6 commercial fermented red beancurd and 6 commercial fermented white beancurd are collected, DNA of the fermented red beancurd and 6S rRNA genes are extracted, and the bacterial community composition of the fermented beancurd sample is identified by PCR amplification and high-throughput sequencing. The extraction reagent provided in example 1 was used for extraction, and 12 fermented bean curd samples in total were obtained by the following steps:
(1) the DNA extraction method was the same as in example 2, followed by PCR amplification of the 16S rRNA gene and high throughput sequencing of the amplicon and bioinformatic analysis;
(2) the V4 region of the 16S rRNA gene was amplified using 1. mu.l of DNA as a template. 515F (5 '-GTG CCA GCM GCC GCG GTA A-3') +806R (5 '-GGA CTA CHV GGG TWT CTA AT-3') is used as a primer for PCR amplification. The total volume of the PCR amplification system is 50 mul, and the reaction system comprises: mu.l each of the upstream and downstream primers (concentration: 10. mu.M), 2. mu.l of 10 XExtag reaction solution (Shanghai Dalbao Bio Inc.), 1. mu.l (0.5U) of Extag enzyme (Shanghai Dalbao Bio Inc.), 2. mu.l of dNTPs (2.5mM), and 5. mu.l of sterilized water. The PCR program comprises pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at 60 ℃ for 30 sec, extension at 72 ℃ for 30 sec, 28 cycles of repeated denaturation, annealing and extension, and then extension at 72 ℃ for 5 min.
(3) The PCR product of the 16S rRNA gene is purified by a Cycle Pure Kit (Omega company) and then sent to Shenzhen Yikeji Biotech limited for high-throughput sequencing, and the used instrument is a HiSeq2500 sequencer of Ilumina company.
(4) The obtained 16S rRNA sequences were subjected to original quality control, and then the species composition information of the microorganisms was analyzed in QIIME software. The bacterial community composition of fermented bean curd is shown in figure 1.
The results show that the composition diversity of the two types of fermented bean curd bacteria, namely red (RHB sample) and white (WXN sample) is high, and meanwhile, the relative abundance of Lactococcus in red fermented bean curd is higher than that of white fermented bean curd.
The results show that the method can effectively extract the DNA of the fermented bean curd, the extracted DNA is used for PCR of 16S rRNA gene and subsequent amplicon sequencing, and the bacterial colony species composition of the two fermented bean curds is successfully identified.
Example 4
6 commercial fermented red bean curds and 6 commercial fermented white bean curds are collected, DNA of the fermented red bean curds and the commercial fermented white bean curds is extracted, ITS genes are amplified by PCR, and the bacterial community composition of the fermented bean curds sample is identified by high-throughput sequencing. The extraction reagent provided in example 1 was used for extraction, and 12 fermented bean curd samples in total were obtained by the following steps:
(1) the DNA extraction method was the same as in example 2, followed by PCR amplification of ITS genes and high throughput sequencing of amplicons and bioinformatic analysis;
(2) mu.l of DNA was used as a template to amplify the ITS2 region of the ITS gene. ITS3f (5'-GCA TCG ATG AAG AAC GCA GC-3') + ITS4R (5'-TCC TCC GCT TAT TGA TAT GC-3') are primers for PCR amplification. The total volume of the PCR amplification system is 50 mul, and the reaction system comprises: mu.l each of the upstream and downstream primers (concentration: 10. mu.M), 2. mu.l of 10 XExtag reaction solution (Shanghai Dalbao Bio Inc.), 1. mu.l (0.5U) of Extag enzyme (Shanghai Dalbao Bio Inc.), 2. mu.l of dNTPs (2.5mM), and 5. mu.l of sterilized water. The PCR program comprises pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 30 sec, 28 cycles of repeated denaturation, annealing and extension, and then extension at 72 ℃ for 5 min.
(3) The PCR product of ITS gene is purified by Cycle Pure Kit (Omega company), and then sent to Shenzhen Yikeji Biotech limited for high-throughput sequencing, and the used instrument is HiSeq2500 sequencer of Ilumina company.
(4) The obtained ITS sequences were subjected to original quality control, and then the species composition information of the microorganisms was analyzed in QIIME software. The bacterial community composition of fermented bean curd is shown in FIG. 2.
The results show that the composition diversity of the two types of fermented bean curd fungi, namely red (RHB sample) and white (WXN sample) is high, and the relative abundance of Monascus (Monascus) in red fermented bean curd is higher than that of white fermented bean curd.
The results show that the method can effectively extract the DNA of the fermented bean curd, and the extracted DNA is used for PCR of ITS genes and subsequent amplicon sequencing, so that the fungal community species composition of the fermented bean curd is successfully identified.
Example 5
6 commercial fermented red beancurd and white beancurd are collected, DNA of the fermented red beancurd and white beancurd is extracted, high-throughput metagenome sequencing is carried out by using a Hiseq sequencer of Illumina company, and the functional composition of microorganisms of fermented beancurd samples is identified by sequencing. The extraction reagent provided in example 1 was used for extraction, and 12 fermented bean curd samples in total were obtained by the following steps:
(1) the DNA extraction method was the same as in example 2, followed by high throughput metagenomic sequencing and bioinformation analysis methods;
(2) fragmenting DNA of the fermented bean curd, constructing a sequencing library, and then performing double-end sequencing on PE150 by using a Hiseq sequencing platform of Illumina company;
(3) performing quality control on the original sequencing sequence by using FASTX-Toolkit software, and removing a low-quality sequence;
(4) high quality metagenomic sequences KEGG database aligned with Diamond softwarehttps:// www.genome.jp/kegg/Acquiring the functional composition condition of the system;
(5) extracting a functional composition profile associated with carbohydrate metabolism from the KEGG results;
(6) and calculating the functional components of the microorganisms with significant difference in the red and white fermented bean curds by a t-test statistical method in SPSS software.
The results show that membrane transport, carbohydrate metabolism and amino acid metabolism are the main functional metabolic components of the fermented bean curd microorganisms (fig. 3). The functions of red and white fermented bean curd with significant differences include energy metabolism, lipid metabolism, cell mobility, etc. Amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism and pyruvate metabolism are the main functional components of the microorganisms of fermented bean curd in terms of carbohydrate functional composition (fig. 4), wherein pyruvate metabolism, c 5-branched diacid metabolism and fructose and mannose metabolic processes have significant differences in the red and white fermented bean curd.
The results show that the extracted microbial DNA of the fermented bean curd can be successfully applied to metagenome sequencing, and the functional components with significant differences in red and white fermented bean curds are identified.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A group of reagents for extracting microorganism DNA of fermented bean curd is characterized by comprising cell separation liquid, cell flushing liquid, cell lysate, PCR inhibiting substance precipitation liquid and cleaning liquid;
the cell separation solution contains 10-20 mM potassium dihydrogen phosphate, 10-20 mM disodium hydrogen phosphate, 100-200 mM sodium chloride, 1-2 mM potassium chloride and pH value of 7-8;
the cell washing solution contains 100-200 mM potassium dihydrogen phosphate, 100-200 mM disodium hydrogen phosphate, 0.05-0.10% Tween 20 and a pH value of 7-8;
the cell lysate contains 0.5-1.0% SDS, 0.5-1.0% polyethylene glycol octyl phenyl ether, 100-200 mM Tris-HCl, 0.5-1.0 mM EDTA, 50-100 mM KCl, and the pH value is 7-8;
the PCR inhibitor precipitation solution contains 100-200 mM potassium dihydrogen phosphate, 100-200 mM disodium hydrogen phosphate, 50-100 mM ammonium sulfate, 200-500 mM guanidine hydrochloride, 1-2% CTAB, and has a pH value of 7-8;
the cleaning solution contains 75-85% of ethanol, 100-200 mM NaCl, 10-20 mM Tris and pH value of 7-8.
2. A method for extracting microorganism community DNA of fermented bean curd, which is characterized in that the reagent of claim 1 is used for extraction, and the steps are as follows:
(1) mashing a fermented bean curd sample, adding tungsten carbide beads and a cell separation solution, shaking and fully mixing, standing for several minutes, centrifuging, and collecting precipitates;
(2) adding a cell flushing fluid into the precipitate obtained in the step (1), shaking for sufficiently mixing, standing for several minutes, centrifuging, and collecting the precipitate;
(3) adding cell lysate into the precipitate obtained in the step (2), adding garnet with the diameters of 0.7mm and 0.15mm respectively, and shaking for full mixing; adding a lysozyme reaction solution, carrying out water bath at 37 ℃ for 1-2 hours, adding proteinase K and 20% SDS, shaking for 0.5-1 hour in a shaking table at 37 ℃, then putting into a water bath at 65 ℃, taking out and shaking once every 10 minutes, and continuing for 1.0-1.5 hours;
(4) shaking the solution finally obtained in the step (3), fully mixing, then freezing and centrifuging, and sucking supernatant fluid to transfer into a new centrifugal tube; adding a PCR inhibiting substance precipitation solution into the supernatant, uniformly mixing, carrying out warm bath at 37 ℃ for 5-10 minutes, centrifuging, and transferring the supernatant into a new centrifugal tube;
(5) adding a phenol-chloroform-isoamylol mixed solution into the supernatant, uniformly mixing, performing refrigerated centrifugation, and sucking the supernatant and transferring the supernatant into a new centrifuge tube; adding anhydrous ethanol, mixing, adsorbing to silica gel membrane centrifugal column, centrifuging, and discarding filtrate;
(6) adding a cleaning solution into the silica gel membrane centrifugal column, centrifuging, discarding the filtrate, and repeating the process for 2-3 times; then, the silica gel membrane centrifugal column is centrifuged, finally, nuclease-free sterilized water preheated at 65 ℃ is added, the centrifugal column filter membrane is incubated for a few minutes at room temperature, and the DNA is collected after centrifugation.
3. The method of claim 2, wherein: in the step (3), 0.5-1.0 g of garnet with the diameter of 0.7mm and 1-2 g of garnet with the diameter of 0.15mm are added into each g of sediment.
4. The method of claim 2, wherein: in the step (3), 1-2 ml of 20mg/m L concentration proteinase K and 1-2 ml of 20% SDS are added into every 10ml of cell lysate.
5. The method of claim 2, wherein: the diameter of the tungsten carbide bead in the step (1) is 10-30 mm.
6. The method of claim 2, wherein: the centrifugation in the steps (1), (2), (4), (5) and (6) is (12000-13000) x g centrifugation for 1-5 minutes.
7. The method of claim 2, wherein: the freezing centrifugation in the steps (4) and (5) is performed at 4-6 ℃ and (12000-13000) Xg for 15-20 minutes.
8. The method of claim 2, wherein: and (4) the volume ratio of the PCR inhibiting substance precipitation solution to the supernatant is 1: 1.
9. The method of claim 2, wherein: in the mixed solution of phenol, chloroform and isoamylol in the step (5), the volume ratio of phenol, chloroform and isoamylol is 25:24: 1; the volume ratio of the phenol-chloroform-isoamyl alcohol mixed solution to the supernatant is 1: 1.
10. The method of claim 2, wherein: the volume ratio of the absolute ethyl alcohol to the supernatant in the step (5) is 1: 1.
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