CN106497914B - Qualitative and/or quantitative extraction method for microbial DNA on large-particle substrate surface of constructed wetland - Google Patents
Qualitative and/or quantitative extraction method for microbial DNA on large-particle substrate surface of constructed wetland Download PDFInfo
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Abstract
The invention discloses a qualitative and/or quantitative extraction method of microbial total DNA on the surface of a large-particle substrate of an artificial wetland, which is characterized in that the large-particle substrate filled in the artificial wetland is taken as a material, a shaking table oscillation method is adopted to elute a biological membrane on the surface of the large-particle substrate, the biological membrane is centrifuged to obtain a precipitate, the precipitate is subjected to de-corrosion treatment by a de-corrosion buffer solution and a calcium chloride solution, cells are cracked by an SDS-lysozyme method, chloroform extraction is carried out, and isopropanol precipitation is carried out to obtain DNA. Compared with the existing kit extraction method without pretreatment, the constructed wetland substrate biofilm extracted by the invention has high total DNA concentration, high purity, good integrity and small loss of microbial diversity information quantity, and can be directly used for PCR, real-time quantitative PCR and DGGE experimental analysis. The method has the advantages of low extraction cost, short time consumption and the like, reduces the pollution probability in the sample treatment process and the DNA loss caused by freeze thawing, and solves the problems of low total DNA yield, low purity and less diversity information amount in the process of directly extracting the large-particle substrate of the artificial wetland.
Description
Technical Field
The invention relates to the technical field of microbial molecular ecology, in particular to a qualitative and/or quantitative extraction method of microbial DNA on the gravel surface of an artificial wetland.
Background
The biomass and community structure are two important characteristics of the artificial wetland, and the distribution characteristics of the biomass and community structure in the wetland are closely related to the degradation behavior of pollutants in the wetland. With the development of molecular biology and the application thereof in ecology, particularly the establishment and development of metagenomic methods in the direction of ecology, the culture means can not meet the requirement of species information with large flux for the artificial wetland. Thus, research has been directed to obtaining information covering the entire microbial community in a more accurate manner by molecular biology techniques such as high throughput sequencing and qPCR on genomic DNA of the entire environment including cultured and non-cultured microorganisms. The precondition for obtaining such information is to provide sufficient quality, purity and complete genomic DNA, such as the requirement of metagenomic sequencing of Huada gene GmbH: the total amount is more than or equal to 1.5 mug, OD260/280Between 1.8 and 2.0, OD260/280Is more than 1.5, and the electrophoresis detection strip has no obvious degradation. When qPCR is required to quantify bacteria, archaea, ammonia oxidizing bacteria and the like, it is required that genomic DNA can be subjected to PCR reaction, and that genomic DNA be quantitatively extracted, and data repeatability is good so as to accurately quantify the content of microorganisms in a substrate.
Environmental samples such as soil, sediment, silt and the like contain a large amount of humus, which is one of main factors causing the reduction of the extraction efficiency and the quality of sample DNA, so various DNA extraction technologies capable of effectively removing the rot gradually appear in China, and the high-quality DNA is expected to be effectively obtained from the humus-rich samples. Chinese patent application with application number 201010573017.8 discloses a method for extracting total DNA of freshwater sediment, Chinese patent application with application number 201110090363.5 discloses a method for extracting total DNA of soil and sediment and an extraction kit, Chinese patent application with application number 201110178774.X discloses a method for extracting total DNA of an environment sample for efficiently removing humus, Chinese patent application with application number 201210137580.X discloses a method for extracting and purifying total DNA from a lake sediment sample, Chinese patent application with application number 201410581883.X discloses a kit and a method for extracting soil microorganism DNA based on a paramagnetic particle method, and the like. The method disclosed in the above patent is limited to directly extracting environment sample with extremely fine particles, and does not mention coarse and uneven surface of large particle matrix sample, especially how to quantitatively extract microorganism DNA on the surface of large particle matrix sample. In the constructed wetland, in order to prevent the system from being blocked, matrixes such as large-particle gravels, volcanic rocks and zeolites are often used as bed fillers, the single particle weight of the large-particle matrixes is often more than 0.5g, the DNA cannot be extracted by using a trace soil DNA extraction kit like soil, sediments and silt, the kit is expensive, and the extraction of a large amount of samples is not economically feasible.
Therefore, a method which is suitable for extracting the microbial DNA on the surface of the large-particle matrix and is efficient, rapid, economical and capable of being carried out in batches needs to be developed, and the problems that the conventional method is difficult to directly extract the microbial DNA on the surface of the large-particle matrix sample, PCR inhibitors such as humus exist in the extracted DNA, the quantification is inaccurate, and the DNA yield is low are solved.
Disclosure of Invention
Aiming at the defect that the existing DNA extraction technology is difficult to directly obtain the microbial DNA on the surface of the large-particle substrate, the invention establishes a method for extracting the microbial DNA on the surface of the large-particle substrate of the artificial wetland, which is low in cost, rapid and quantitative, and can realize qualitative and quantitative detection and analysis. The DNA obtained by the method can be directly used as a PCR template for gene quantitative detection and high-throughput sequencing, and the biomass and community structure analysis of the constructed wetland substrate biofilm can be carried out.
The purpose of the invention is realized by the following technical scheme:
providing an artificial wetA method for qualitatively and/or quantitatively extracting the total DNA of microbes on the surface of large granular matrix includes such steps as using the large granular matrix filled in artificial wet land as raw material, shaking by table to elute the biomembrane on the surface of gravel, centrifugal separation to obtain deposit, removing the deposit by use of the buffer solution and calcium chloride solution, cracking cells by SDS-lysozyme method, extracting by chloroform, and depositing by isopropanol to obtain DNA. DNA obtained by the method, OD thereof260/280And OD260/280Are all around 1.8. The DNA obtained by the method of the invention can be directly used for PCR reaction without dilution and purification.
The large-particle matrix is made of materials including gravel, volcanic rock or zeolite.
The extraction method comprises the steps of matrix biomembrane elution, decay removal pretreatment and DNA extraction.
Specifically, the method comprises the following specific steps:
s1, elution and corrosion removal pretreatment of a matrix biological membrane:
s11, collecting the large-particle matrix, adding sterile normal saline, and oscillating to elute the biological membrane on the surface of the large-particle matrix to obtain turbid liquid;
s12, sucking the suspension obtained in the step S11 into a sterile centrifuge tube with a proper volume, centrifuging, removing the supernatant, continuously adding the suspension, centrifuging again, and storing the centrifuge tube containing the precipitate in a refrigerator at-20 ℃ for later use after the precipitate in the tube reaches the preset precipitate quality;
s13, taking out the sterile centrifuge tube with the preserved precipitate, standing at normal temperature for thawing, adding sterile quartz sand into the precipitate, adding a decay-removing buffer solution into the precipitate, uniformly mixing by vortex, centrifuging, removing supernatant, and carrying out the next step of DNA extraction on the obtained precipitate. The composition of the de-corrosion buffer solution is 100mmol/L Tris-HCl (pH8.0) and 100mmol/L Na2P2O7、100mmol/L Na2EDTA (pH8.0), 1% PVP, 100mmol/L NaCl, 0.05% Triton X-100; the pH value of the de-corrosion buffer solution is 8.0.
S2, DNA extraction:
s21, adding 0.5mol/L calcium chloride into the precipitate obtained in the step S13, uniformly mixing in a vortex manner, centrifuging, and then removing supernatant to obtain precipitate;
s22, adding 0.05mol/L sodium oxalate into the precipitate obtained in the step S21, uniformly mixing in a vortex mode, carrying out centrifugal treatment, and then removing supernatant to obtain the precipitate;
s23, adding the DNA extracting solution and lysozyme into the precipitate obtained in the step S22, uniformly mixing by vortex, and carrying out shaking table treatment;
the DNA extracting solution consists of 100mM Tris-HCl, 1.5mol/L NaCl and 1% CTAB, and the pH value is 8.0; the concentration of the lysozyme is 100 mg/mL;
s24, adding 100uL of 20% SDS after short-time centrifugation, uniformly mixing by vortex, treating in a water bath, and shaking up for several times by reversing at intervals;
s25, after centrifugal treatment, collecting an intermediate liquid phase layer, and repeatedly washing and precipitating once;
s26, adding mixed liquor of chloroform and isoamylol with the same volume into the collected supernatant fluid for extraction, and collecting an upper liquid phase layer after centrifugal treatment; the mixing volume ratio of the chloroform to the isoamyl alcohol is 24: 1;
s27, adding 0.1 time volume of 3mol/L sodium acetate and 0.6 time volume of isopropanol into the collected liquid phase layer, and standing at-20 ℃ for 20 min;
s28, centrifuging and then discarding the supernatant; adding 1mL of 70% ethanol into the precipitate for washing, centrifuging again, discarding the supernatant, and repeatedly washing once to obtain the precipitate;
s29, drying and dissolving the precipitate obtained in the step S28 to obtain the DNA solution.
Preferably, the concentration of the physiological saline in the step S11 is 0.85%.
Preferably, the mass-to-volume ratio of the large granular matrix and the sterile physiological saline in the step S11 is 1 g: 1 mL.
Preferably, the shaking elution in step S11 is performed in a sterile polyethylene bottle.
Preferably, the shaking in step S11 is performed at 15 ℃ and 225rpm after shaking for 3h, so as to sufficiently elute the biofilm on the surface of the large-particle substrate (gravel).
Preferably, the centrifugation in step S12 is performed at 12000rp at 4 deg.C for 10 min.
Preferably, the grain size of the sterile quartz sand of step S13 is about 1 mm.
Preferably, the centrifugation process of step S13 is performed at 12000rp for 5min at room temperature.
Preferably, the de-corrosion buffer of step S13 is prepared by the following steps: 100mmol/L Tris-HCl (pH8.0), 100mmol/L Na2P2O7、100mmol/L Na2EDTA (pH8.0), 1% PVP, 100mmol/L NaCl, 0.05% TritonX-100, 4% skimmed milk powder, pH8.0, filtering with 0.44 μm cellulose membrane, and filtering with 0.22 μm sterile filter head.
Preferably, the centrifugation treatment in step S21 is centrifugation at 12000rpm for 5min at room temperature.
Preferably, the centrifugation treatment in step S22 is centrifugation at 12000rpm for 5min at room temperature.
Preferably, the shaking treatment conditions in step S23 are 37 ℃ and 225rpm for 30 min;
preferably, the temperature of the water bath treatment in the step S24 is 65 ℃, and the water bath treatment time is 1 h.
Preferably, the centrifugation treatment in step S25 is centrifugation at 12000rpm for 5min at room temperature.
Preferably, the centrifugation treatment in step S26 is centrifugation at 12000rpm for 10min at room temperature.
Preferably, the extraction time of step S26 is 10 min.
Preferably, the pH of the sodium acetate in step S27 is 5.2.
Preferably, the centrifugation treatment in step S28 is centrifugation at 12000rpm for 10min at room temperature; the second centrifugation treatment is centrifugation for 5min at 12000rpm at room temperature;
preferably, the step of collecting the intermediate liquid phase layer and repeatedly washing once in step S26 is performed according to the following steps: sucking 900 mu L of the intermediate liquid phase layer to a new centrifuge tube (2mL) by a liquid transfer machine; adding 800 μ L of DNA extract and 100 μ L of 20% SDS into the precipitate again, mixing uniformly by vortexing, then bathing at 65 ℃ for 10min, shaking up for several times by inversing the intervals, centrifuging at 12000rpm for 5min at room temperature, and collecting 800 μ L of intermediate liquid phase layer to a new centrifuge tube (2 mL).
Preferably, the drying and DNA dissolution of step S29 is performed according to the following steps: oven-drying at 60 deg.C for 5min, adding 50 μ L10mmol/L Tris-HCl (pH8.0), flicking with finger to dissolve precipitate, and combining the same sample DNA of two tubes to 100 μ L.
OD of the DNA solution obtained by the method of the present invention260/280And OD260/280All are about 1.8, can be directly used for PCR reaction without dilution and purification, and can realize qualitative and/or quantitative detection through electrophoresis and quality calculation.
If the quantitative detection is needed, on the basis of the steps, the method also comprises the following steps:
step S11 further includes the step of recording the mass M of the large granular matrix and the volume V of sterile saline; step S12 includes recording the total volume V of the suspension added1A step of; calculating the mass M of the large-particle matrix corresponding to the precipitate1Is composed of
30g of the same large-particle matrix are weighed in addition, dried and weighed, and the mass is recorded as m2. Thus, m2/(30m1-30m0) I.e. the dry weight of the matrix of the sediment, the total DNA extracted from the sediment in the tube reaching the sediment quality set by the detection is marked as mgDNA。
Calculating the formula: the matrix DNA content (ng/g) is 30mgDNA(m1-m0)/m2。
The weighing after drying is to place the large-particle matrix in an oven to be dried for 48 hours at 70 ℃.
The extraction method is combined with a PCR (polymerase chain reaction) and an electrophoresis method and is applied to the qualitative and/or quantitative detection aspect of the microbial total DNA on the surface of the large-particle substrate of the constructed wetland.
The invention has the beneficial effects that:
the invention provides a qualitative and/or quantitative extraction method of microbial total DNA on the surface of a large-particle substrate of an artificial wetland, which successfully elutes a biological membrane on the surface of gravel by adopting a shaking table oscillation method aiming at the large-particle substrate filled in the artificial wetlandCentrifuging to obtain precipitate, reasonably removing the precipitate with a de-corrosive buffer solution and a calcium chloride solution, cracking cells by an SDS-lysozyme method, extracting by chloroform, and precipitating by isopropanol to obtain DNA. DNA obtained by the method, OD thereof260/280And OD260/280Are all around 1.8. The DNA obtained by the method of the invention can be directly used for PCR reaction without dilution and purification. The method for extracting the microbial DNA on the large-particle substrate surface of the constructed wetland is low in cost, rapid and quantitative, the DNA obtained by the method can be directly used as a PCR template, dilution and purification are not needed, and the method can be well used for gene quantitative detection and high-throughput sequencing and carrying out constructed wetland substrate biofilm biomass and community structure analysis.
Based on the method, the total DNA of the microorganisms on the surface of the large-particle substrate of the constructed wetland can be successfully and quantitatively detected by recording the related quality step by step.
Drawings
FIG. 1 is an electrophoretic image of DNA extracted directly without being subjected to a depassivation treatment. The numbers 1-6 in the figure are S-HF + UP, B-HF + UP, S-HF + TD, B-HF + TD, S-HF + CI and B-HF + CI in sequence, and the sample application amount is 2 uL. The sample loading amount of lambda DNA as a quantitative standard (Beijing ancient Changsheng biotechnology limited) is 10ng, 30ng and 50ng from left to right in sequence.
FIG. 2 is an electrophoresis test chart of DNA extracted by the kit method. The samples numbered 1-6 in the figure are the same as figure 1, the sample application amount is 2uL, and the samples are extracted three times respectively to be used as parallels. Lambda DNA as a quantitative standard was loaded with 10ng, 30ng, 50ng and 100ng from left to right in this order.
FIG. 3 is an electrophoretic detection map of DNA extracted by the method of the present invention. The numbers 1-6 in the figure and the sample loading amount are the same as those in figure 1. Lambda DNA as a quantitative standard was loaded with 10ng, 30ng, 50ng and 100ng from left to right in this order.
FIG. 4 is a diagram showing the full-length electrophoretic detection of 16S rRNA gene amplified directly using DNA extracted by the method of the present invention as a template. In the figure, the number 0 represents Tris-HCl (pH8.0) with a template of 10mmol/L, the numbers 1-6 sequentially represent templates from S-HF + UP, B-HF + UP, S-HF + TD, B-HF + TD, S-HF + CI and B-HF + CI, the sample loading amount of PCR is 2uL, and the repeated numbers represent three times of extraction of the same sample. M is a DNA molecular weight standard (TaKaRa, Dalian, China) which is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp (bp: base pair) from top to bottom in sequence.
FIG. 5 is a diagram showing the detection of the full-length electrophoresis of the amplified 16S rRNA gene using DNA extracted by the kit method directly as a template. The numbering, loading and DNA molecular weight standards in the figure are the same as in FIG. 4.
FIG. 6 shows the copy number of the 16S rRNA gene of bacteria detected by fluorescent quantitative PCR.
FIG. 7 is a DGGE detection diagram of an amplified 16S rRNAV3 region using DNA extracted by a kit method and the method of the present invention as a template, wherein the numbers 1-6 on the left sequentially indicate that a certain kit method is adopted, the templates are from S-HF + UP, B-HF + UP, S-HF + TD, B-HF + TD, S-HF + CI and B-HF + CI, and the loading amount of DGGE is 2 μ g; the numbers of the right side are 1-6, which sequentially show that the method is adopted, the templates are from S-HF + UP, B-HF + UP, S-HF + TD, B-HF + TD, S-HF + CI and B-HF + CI, and the sample loading amount of DGGE is 2 mu g.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and procedures, and unless otherwise indicated, reagents and apparatus used in the present invention are conventional in the art.
Example 1: extraction of microbial DNA from artificial wetland gravel surface
Collecting artificial wetland gravel samples:
in the embodiment, gravel samples are collected from a group of horizontal subsurface flow constructed wetlands for treating domestic sewage, and the wetland device has the length of 80cm, the width of 60cm and the height of 80 cm. The wetland substrate is gravel (the particle size is 1-2 cm), the filling height is 60cm, and a plant-free group, a canna group and a rebuttoning group are respectively arranged. The device hydraulic load is 2.5m/d, and the whole day moves in succession, gathers the matrix sample respectively in nearly water inlet end and nearly water outlet end, and 0 ~ 10cm department matrix in top layer mixes to the top layer sample, and 30 ~ 40cm department matrix in bottom mixes to the bottom sample, and 6 samples in total are put to three device, and 3 of them are the top layer sample, and 3 are the bottom sample, and the aseptic sealed bag of each about 200g of matrix of gathering is packed into for use, and each sample is named respectively: S-HF + UP (horizontal undercurrent does not have plant group surface layer matrix), B-HF + UP (horizontal undercurrent does not have plant group bottom layer matrix), S-HF + TD (horizontal undercurrent rebirth group surface layer sample), B-HF + TD (horizontal undercurrent rebirth group bottom layer sample), S-HF + CI (horizontal undercurrent canna group surface layer sample), B-HF + CI (horizontal undercurrent canna group bottom layer sample).
Based on the collected samples, the embodiment provides a method for quantitatively extracting the microbial DNA on the gravel surface of an artificial wetland (used for treating sewage discharged by urban residents every day), which comprises the following steps:
s1, sample pretreatment
S11, collecting artificial wetland gravels, filling the artificial wetland gravels into an aseptic sealed bag, slowly pouring the gravels in the sealed bag into a 500mL aseptic polyethylene bottle until the weight of the gravels is 100g, adding 100mL aseptic normal saline (0.85%) into the bottle, and uniformly shaking the bottle at the temperature of 15 ℃ of a shaking table at the rotating speed of 225rpm for 3 hours to sufficiently elute the biofilm on the surface of the gravels.
S12, sucking the suspension into a sterile centrifuge tube with a proper volume, centrifuging at 12000rpm at 4 ℃ for 10min, discarding the supernatant, continuously adding the suspension, centrifuging in the same way, and storing the centrifuge tube containing the precipitate in a refrigerator at-20 ℃ for later use after the precipitate in the tube reaches the detected set precipitate quality (0.5 g).
S13, taking out the centrifuge tube storing 0.5g of precipitate, standing at normal temperature for 3min for unfreezing, adding 0.4g of sterile quartz sand (with the particle size of about 1mm) into the precipitate, adding 1.5mL of de-corrosion buffer solution [100mmol/L of Tris-HCl (pH8.0), 100mmol/L of Na ] into the precipitate2P2O7，100mmol/L Na2EDTA(pH8.0)，1％PVP，100mmol/L NaCl，0.05％TritonX-100，pH 8.0]Vortex and mix evenly, centrifuge for 5min at 12000rpm room temperature, abandon the supernatant, get the precipitation and prepare for DNA extraction.
S2.DNA extraction
S21, adding 1mL of 0.5mol/L calcium chloride into the precipitate, uniformly mixing by vortex, centrifuging at 12000rpm for 5min at room temperature, and removing the supernatant.
S22, adding 1mL of 0.05mol/L sodium oxalate into the precipitate, uniformly mixing by vortex, centrifuging at 12000rpm for 5min at room temperature, and removing the supernatant.
S23, adding 800 mu L of DNA extracting solution (100mM Tris-HCl, 1.5mol/L NaCl, 1% CTAB, pH8.0) and 100 mu L of lysozyme (100mg/mL) into the precipitate, vortex and uniformly mixing, and placing the mixture in a shaking table at 37 ℃ and 225rpm for 30 min.
S24, performing short-time centrifugation, adding 100uL of 20% SDS, performing vortex mixing, performing water bath at 65 ℃ for 1h, and shaking up for several times at intervals.
S25.12000rpm room temperature centrifugation for 5min, collecting the middle liquid phase layer 900 microliter to a new 2mL centrifuge tube; adding 800 μ L of DNA extract and 90 μ L of 20% SDS into the precipitate, mixing uniformly by vortex, then bathing at 65 ℃ for 10min, shaking up for several times by reversing the interval, centrifuging the mixture in the same way, and collecting 800 μ L of the intermediate liquid layer to a new 2mL centrifuge tube.
S26, adding equal volume of chloroform isoamyl alcohol (24:1) into the collected supernatant fluid, extracting for 10min, then centrifuging at 12000rpm for 10min, and collecting an upper liquid phase layer.
S27, adding 0.1 volume time of 3mol/L sodium acetate (pH5.2) and 0.6 volume time of isopropanol into the collected liquid phase layer, and standing at-20 ℃ for 20 min.
S28.12000rpm centrifugation for 10min, and discarding the supernatant; the precipitate was washed by adding 1mL of 70% ethanol, centrifuged at 12000rpm for 5min, the supernatant was discarded, and the washing was repeated once.
S29.60 ℃ drying for 5min, adding 50 μ L10mmol/L Tris-HCl (pH8.0), dissolving the precipitate with finger flick, and combining the same samples of the two tubes into 100 μ L.
The 16S rDNA gene full-length PCR amplification effect of gDNA was examined using 16S rDNA primers 27F (5 'AGAGAGTTTGATCCTGGCTCAG 3') and 1492R (5 'GGTTACCTTGTTACGACTT 3'). The PCR amplification system specifically consists of: 5 μ L of 10 XBuffer, 5 μ L of dNTP (2mmol/L), 1.5 μ L of forward primer 27F (10 μmol/L), 1.5 μ L of reverse primer 1492R (10 μmol/L), 0.5 μ L of LTaq DNA pol-ymerase (TaKaRa, Dalian, China), 4 μ L of MgCl2(25mmol/L), 1. mu.L of DNA template, and sterile double distilled water to 50. mu.L. Thermal cycling was performed using a PCR thermal cycler (Bio-rad S1000, US) under the following specific thermal cycling conditions: performing touchdown PCR amplification, pre-denaturing at 94 ℃ for 5min, denaturing at 94 ℃ for 1min, annealing at 63.5 ℃ for 1min, cooling the annealing temperature per cycle by 1 ℃, extending at 72 ℃ for 3min for 10 cycles, denaturing at 94 ℃ for 1min, annealing at 53.5 ℃ for 1min, extending at 72 ℃ for 3min, extending at 72 ℃ for 20 cycles, and extending at 72 ℃ for 15 min. mu.L of the PCR reaction product was detected by electrophoresis on a 2% agarose gel in 1 XTAE buffer.
The electrophoresis method comprises the following steps: agarose gel electrophoresis of genomic DNA 2. mu.L of DNA lysate was mixed with 0.5. mu.L of 6 Xloading buffer, spotted onto 1% agarose gel (containing 1 XGelred), subjected to electrophoresis in TAE buffer (pH8.0) for 95V18min, and then analyzed by photography using a gel imaging system (Bio-rad, US).
TABLE 1 DNA purity of substrate microorganism extracted in this example
Example 2: extraction of microbial DNA from artificial wetland gravel surface
Collecting artificial wetland gravel samples:
in the embodiment, gravel samples are collected from a group of horizontal subsurface flow constructed wetlands for treating domestic sewage, the length, the width and the height of each wetland device are 80cm, 60cm and 80cm, the wetland substrate is gravel (the particle size is 1-2 cm), the filling height is 60cm, and a plant-free group, a canna group and a rebuttoning group are respectively arranged. The device hydraulic load is 2.5m/d, and the whole day moves in succession, gathers the matrix sample respectively in nearly water inlet end and nearly water outlet end, and 0 ~ 10cm department matrix in top layer mixes to the top layer sample, and 30 ~ 40cm department matrix in bottom mixes to the bottom sample, and 6 samples in total are put to three device, and 3 of them are the top layer sample, and 3 are the bottom sample, and the aseptic sealed bag of each about 200g of matrix of gathering is packed into for use, and each sample is named respectively: S-HF + UP (horizontal undercurrent does not have plant group surface layer matrix), B-HF + UP (horizontal undercurrent does not have plant group bottom layer matrix), S-HF + TD (horizontal undercurrent rebirth group surface layer sample), B-HF + TD (horizontal undercurrent rebirth group bottom layer sample), S-HF + CI (horizontal undercurrent canna group surface layer sample), B-HF + CI (horizontal undercurrent canna group bottom layer sample).
Based on the collected samples, the embodiment provides a method for quantitatively extracting the microbial DNA on the gravel surface of an artificial wetland (used for treating sewage discharged by urban residents every day), which comprises the following steps:
s1, sample pretreatment
S11, collecting artificial wetland gravels, filling the artificial wetland gravels into an aseptic sealed bag, slowly pouring the gravels in the sealed bag into a 500mL aseptic polyethylene bottle until the weight of the gravels is 100g, adding 100mL aseptic normal saline (0.85%) into the bottle, and uniformly shaking the bottle at the temperature of 15 ℃ of a shaking table at the rotating speed of 225rpm for 3 hours to sufficiently elute the biofilm on the surface of the gravels.
S12, sucking 2mL of suspension into a 2mL sterile centrifuge tube (weighing and recording m of an empty tube)0) Centrifugation at 12000rpm for 10min at 4 ℃ and discarding the clear supernatant, the tubes containing the pellet are weighed and recorded as m1The mass of the precipitate is m1-m0Continuously adding the suspension with the volume of 1/(m)1-m0) 2, after centrifugation as above, 0.5g of precipitate, equivalent to 1/(m)1-m0) The gravel is added, and the centrifuge tube containing the sediment is stored in a refrigerator at the temperature of 20 ℃ below zero for standby.
Weighing 30g of gravel, placing the gravel in an oven to dry for 48 hours at 70 ℃, weighing the dried gravel, and recording the weight as m2. Thus, m2/(30m1-30m0) This is the dry weight of the matrix in 0.5g of the pellet, and the total DNA extracted from this 0.5g pellet is designated mgDNA. Calculating the formula: the matrix DNA content (ng/g) is 30mgDNA(m1-m0)/m2。
S13, taking out the centrifuge tube storing 0.5g of precipitate, standing at normal temperature for 3min for unfreezing, adding 0.4g of sterile quartz sand (with the particle size of about 1mm) into the precipitate, adding 1.5mL of de-corrosion buffer solution [100mmol/L of Tris-HCl (pH8.0), 100mmol/L of Na ] into the precipitate2P2O7，100mmol/L Na2EDTA(pH8.0)，1％PVP，100mmol/L NaCl，0.05％TritonX-100，pH 8.0]Vortex and mix evenly, centrifuge for 5min at 12000rpm room temperature, abandon the supernatant, get the precipitation and prepare for DNA extraction.
S3.DNA extraction
S21, adding 1mL of 0.5mol/L calcium chloride into the precipitate, uniformly mixing by vortex, centrifuging at 12000rpm for 5min at room temperature, and removing the supernatant.
S22, adding 1mL of 0.05mol/L sodium oxalate into the precipitate, uniformly mixing by vortex, centrifuging at 12000rpm for 5min at room temperature, and removing the supernatant.
S23, adding 800 mu L of DNA extracting solution (100mM Tris-HCl, 1.5mol/L NaCl, 1% CTAB, pH8.0) and 100 mu L of lysozyme (100mg/mL) into the precipitate, vortex and uniformly mixing, and placing the mixture in a shaking table at 37 ℃ and 225rpm for 30 min.
S24, performing short-time centrifugation, adding 100uL of 20% SDS, performing vortex mixing, performing water bath at 65 ℃ for 1h, and shaking up for several times at intervals.
S25.12000rpm room temperature centrifugation for 5min, collecting the middle liquid phase layer 900 microliter to a new 2mL centrifuge tube; adding 800 μ L of DNA extract and 90 μ L of 20% SDS into the precipitate, mixing uniformly by vortex, then bathing at 65 ℃ for 10min, shaking up for several times by reversing the interval, centrifuging the mixture in the same way, and collecting 800 μ L of the intermediate liquid layer to a new 2mL centrifuge tube.
S26, adding equal volume of chloroform isoamyl alcohol (24:1) into the collected supernatant fluid, extracting for 10min, then centrifuging at 12000rpm for 10min, and collecting an upper liquid phase layer.
S27, adding 0.1 volume time of 3mol/L sodium acetate (pH5.2) and 0.6 volume time of isopropanol into the collected liquid phase layer, and standing at-20 ℃ for 20 min.
S28.12000rpm centrifugation for 10min, and discarding the supernatant; the precipitate was washed by adding 1mL of 70% ethanol, centrifuged at 12000rpm for 5min, the supernatant was discarded, and the washing was repeated once.
S29.60 ℃ drying for 5min, adding 50 μ L10mmol/L Tris-HCl (pH8.0), dissolving the precipitate with finger flick, and combining the same samples of the two tubes into 100 μ L.
The 16S rDNA gene full-length PCR amplification effect of gDNA was examined using 16S rDNA primers 27F (5 'AGAGAGTTTGATCCTGGCTCAG 3') and 1492R (5 'GGTTACCTTGTTACGACTT 3'). The PCR amplification system specifically consists of: 5 μ L of 10 XBuffer, 5 μ L of dNTP (2mmol/L), 1.5 μ L of forward primer 27F (10 μmol/L), 1.5 μ L of reverse primer 1492R (10 μmol/L), 0.5 μ L of LTaq DNA pol-ymerase (TaKaRa, Dalian, China), 4 μ L of MgCl2(25mmol/L), 1. mu.L of DNA template, and sterile double distilled water to 50. mu.L. Thermal cycling was performed using a PCR thermal cycler (Bio-rad S1000, US) under the following specific thermal cycling conditions: performing touchdown PCR amplification, pre-denaturing at 94 ℃ for 5min, denaturing at 94 ℃ for 1min, annealing at 63.5 ℃ for 1min, cooling the annealing temperature per cycle by 1 ℃, extending at 72 ℃ for 3min for 10 cycles, denaturing at 94 ℃ for 1min, annealing at 53.5 ℃ for 1min, extending at 72 ℃ for 3min, extending at 72 ℃ for 20 cycles, and extending at 72 ℃ for 15 min. mu.L of the PCR reaction product was detected by electrophoresis on a 2% agarose gel in 1 XTAE buffer.
The electrophoresis method comprises the following steps: agarose gel electrophoresis of genomic DNA 2. mu.L of DNA lysate was mixed with 0.5. mu.L of 6 Xloading buffer, spotted onto 1% agarose gel (containing 1 XGelred), subjected to electrophoresis in TAE buffer (pH8.0) for 95V18min, and then analyzed by photography using a gel imaging system (Bio-rad, US).
TABLE 2 the amount and purity of DNA obtained from the substrate microorganism extracted in this example
Example 3 comparative test
In order to compare the extraction effects of the present invention (examples 1 and 2, the following data and samples are referred to as the present invention) with those of the conventional non-putrefaction treatment method and kit method, the applicant performed experiments using the non-putrefaction treatment method and kit method at the same time, and specifically performed the following operations:
(1) the procedure was carried out without calcium chloride and sodium oxalate treatment, and the other steps were the same as in example 1 or example 2, and the formulation of the DNA extract was (100mM Tris-HCl, 100mM EDTA, 200mM NaCl, 2% PVPP, 3% CTAB, pH 8.0).
(2) Use of
DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, Calif., USA) extracts genomic DNA, and the liquid and beads in the Powerbead Tubes are all transferred to a 2mL centrifuge tube containing 0.5g of the pellet, and the remaining steps are performed according to the Kit procedures.
The matrix samples used in both methods were the same as in the present invention, i.e., the sample treatment was performed by eluting with a shaker and centrifuging to 0.5g of precipitate, and the extracted DNA was dissolved in 100. mu.L of 10mmol/L Tris-HCl (pH 8.0).
DNA concentration and purity measurement
(1) Agarose gel electrophoresis of genomic DNA 2. mu.L of DNA lysate was mixed with 0.5. mu.L of 6 Xloading buffer, spotted onto 1% agarose gel (containing 1 XGelred), subjected to electrophoresis in TAE buffer (pH8.0) for 95V18min, and then analyzed by photography using a gel imaging system (Bio-rad, US).
The DNA extracted according to the method (FIG. 3) of step S1 in example 1 or example 2 and the kit method (FIG. 2) has higher integrity and no obvious RNA band, and the result of electrophoretic quantification also shows that the amount of the extracted DNA is higher than that of the kit method; the DNA extracted without the de-rotting treatment (FIG. 1) is less in amount, has weaker bands, is obviously degraded, and has stronger RNA bands. This shows that the extraction method of the invention has higher DNA yield, higher integrity of DNA, and better removal effect on impurities by removing the corrosion.
(2) Determination of DNA yield and purity by ultramicro Spectrophotometer 1. mu.L of extracted gDNA stock solution was directly taken and placed on a Nanodrop ND-2000 ultramicro Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) to determine its concentration and OD260/280And OD230/260。
The DNA obtained by extraction according to the method of S1 in example 1, as shown in Table 1, had a total DNA content exceeding 1.5. mu.g per single extraction, OD260/280And OD260/230The total content is about 1.8, the result is stable, and the sample sending requirement of metagenome sequencing can be met no matter in the total amount or the quality; the total amount of DNA extracted by the kit method is only part of samples which can meet the sample sending requirement of metagenome sequencing, and compared with the method, the stability is poorer, and the total amount of DNA extracted by the kit method is extremely obviously less than that of the invention (p is less than 0.01); the quality and quality of the DNA of the microorganisms on the surface of the large-particle substrate of the constructed wetland extracted by the invention are better than those of the DNA of the microorganisms on the surface of the large-particle substrate of the constructed wetland
DNA Isolation Kit。
TABLE 3 comparison of the DNA yield and purity of the substrate microorganism extracted by the present invention and the kit method
And (3) PCR detection:
(1) the 16S rDNA gene full-length PCR amplification effect of gDNA was examined using 16S rDNA primers 27F (5 'AGAGAGTTTGATCCTGGCTCAG 3') and 1492R (5 'GGTTACCTTGTTACGACTT 3'). The PCR amplification system specifically consists of: 5 μ L of 10 XBuffer, 5 μ L of dNTP (2mmol/L), 1.5 μ L of forward primer 27F (10 μmol/L), 1.5 μ L of reverse primer 1492R (10 μmol/L), 0.5 μ L of LTaq DNA pol-ymerase (TaKaRa, Dalian, China), 4 μ L of MgCl2(25mmol/L), 1. mu.L of DNA template, and sterile double distilled water to 50. mu.L. Thermal cycling was performed using a PCR thermal cycler (Bio-rad S1000, US) under the following specific thermal cycling conditions: performing touchdown PCR amplification, pre-denaturing at 94 ℃ for 5min, denaturing at 94 ℃ for 1min, annealing at 63.5 ℃ for 1min, cooling the annealing temperature per cycle by 1 ℃, extending at 72 ℃ for 3min for 10 cycles, denaturing at 94 ℃ for 1min, annealing at 53.5 ℃ for 1min, extending at 72 ℃ for 3min, extending at 72 ℃ for 20 cycles, and extending at 72 ℃ for 15 min. mu.L of the PCR reaction product was detected by electrophoresis on a 2% agarose gel in 1 XTAE buffer.
The DNA obtained by the method and kit method of example 1 or example 2 was extracted and used as a template to amplify the full length of the 16SrRNA gene. As shown in FIGS. 4 and 5, neither of the control samples amplified a band of the target size in 10mmol/L Tris-HCl (pH 8.0); one of the samples marked as 6 in the samples extracted by the kit method fails to amplify a band with a target size; however, all samples extracted by the method of the invention can amplify target bands with the size of about 1500bp, and no obvious primer dimer exists. Therefore, the DNA with higher purity can be obtained by the method of the invention and can be directly used for PCR amplification and bacterial 16S rRNA gene information analysis.
(2) The bacterial 16S rRNA gene copy number of the substrate was determined by real-time quantitative PCR using primers 341F (5 'CCTACGGGAGGCAGCAG 3') and 534R (5 'ATTACCGCGGCTGCTGG 3'). The real-time quantitative PCR system specifically comprises the following components: mu.L of LSYBR Premix Ex TaqTM (Takara, Japan), 0.4. mu.L of each primer (10. mu.M), 2. mu.L of template DNA, and sterile deionized water were filled up to 20. mu.L. Measured using a real-time quantitative PCR instrument (Bio-rad CFX96, US) with a specific amplification procedure of 95 ℃ pre-denaturation for 30 s; 40 cycles of the following process: denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 20s, and extension at 72 ℃ for 30 s.
The DNA extracted according to the method of S1 and the kit method in example 1 or example 2 is directly used as a template for real-time quantitative PCR, and the result shows that the copy number of the bacterial 16S rRNA gene measured by the method of the invention is remarkably higher than that of the kit method (p is less than 0.01, figure 6), which shows that compared with the kit method, the extraction method of the invention has higher cell lysis degree, can reflect the whole bacterial biomass of the sample more and can describe the bacterial biomass in the artificial wetland system more accurately and comprehensively.
(3) PCR amplification of the 16S rRNAV3 region
The bacterial community structure composition of the matrix was detected by Denaturing Gradient Gel Electrophoresis (DGGE) using the same primers as real-time quantitative PCR but with the addition of a GC clamp 5 ' CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGC ACGGGGGG3 ' at the 5 ' end of 341F. The PCR amplification system specifically consists of: mu.L of 10 XBuffer, 5. mu.L of dNTP (2mmol/L), 1.5. mu.L of forward primer 27F (10. mu. mol/L), 1.5. mu.L of reverse primer 1492R (10. mu. mol/L), 0.5. mu.L of Taq DNA polymerase (TaKaRa, Dalian, China), 4. mu.L of LMgCl2(25mmol/L), 1. mu.L of DNA template, and sterile double distilled water to 50. mu.L. Thermal cycling was performed using a PCR thermal cycler (Bio-rad S1000, US) under the following specific thermal cycling conditions: adopting touchdown PCR amplification, pre-denaturing at 94 ℃ for 10min, denaturing at 94 ℃ for 30s, annealing at 65 ℃ for 30s, cooling the annealing temperature of each cycle by 0.5 ℃, extending at 72 ℃ for 1min for 19 cycles, denaturing at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extending at 72 ℃ for 1min, and extending at 72 ℃ for 10min for 14 cycles. mu.L of the PCR reaction product was detected by electrophoresis on a 2% agarose gel in 1 XTAE buffer, and the band of interest of each PCR product sample was quantified using quantitative DNA. 10 μ L of 10 × Loading Buffer (TaKaRa, Dalian, China) was added to each PCR product and mixed well, and the mixture was concentrated overnight in an oven at 60 ℃, and according to the quantitative result of electrophoresis, sterile deionized water was added to each product sample to adjust the band concentration to 100 ng/. mu.L, and the Loading amount of DGGE was 20 μ L. By using DcodeTMA gene mutation detection system (Bio-rad, US) separates PCR reaction products to prepare 8% polyacrylamide gel, and the concentration range of a denaturant is 40-60% (100% of the denaturant contains 7mol/L of urea and 40% of deionized formamide).
The electrophoresis conditions are as follows: electrophoresis is carried out for 12h at 60 ℃ and 110V. ElectrophoresisThe results were stained with Gel Red nucleic acid dye, visualized with a Gel imaging system (Bio-rad, US) and analyzed with Quantity One software. The richness (S) of the sample is expressed by the number of bands, and the fragrance-Vera index (H') is calculated by the formula: h ═ Σ (p)i)(lnpi). Wherein p isiThe value is the ratio of the intensity density of each DNA band to the sum of the intensities of the DNA bands in each lane; lnpiIs piThe natural logarithm of (c).
The DNA obtained by extraction according to the method and kit method in example 1 or example 2 was used as a template to amplify the bacterial 16S rRNA V3 region for DGGE analysis, and FIG. 7 shows that the sample bands are clearer and the bands are clearly separated from each other compared with the kit method. As can be seen from Table 2, the abundance and diversity of the bacteria obtained by the method are higher than those obtained by the kit method, which indicates that the DNA extracted by the method contains higher information amount of microbial diversity than that obtained by the kit method, and the diversity of the microbial community of the constructed wetland substrate can be more effectively reflected.
TABLE 4 bacterial abundance and diversity results of the invention and kit method
Claims (4)
1. A qualitative and/or quantitative extraction method of microbial total DNA on the surface of a large-particle substrate of an artificial wetland is characterized in that the large-particle substrate filled in the artificial wetland is taken as a material, a shaking table oscillation method is adopted to elute a biological membrane on the surface of the large-particle substrate, the biological membrane is centrifuged to obtain a precipitate, the precipitate is subjected to de-corrosion treatment by a de-corrosion buffer solution and a calcium chloride solution, cells are cracked by an SDS-lysozyme method, chloroform extraction is carried out, and isopropanol precipitation is carried out to obtain DNA; the elution of the large-particle substrate surface biological membrane is carried out according to the following steps: adding the large-particle matrix into physiological saline, placing in a shaking table, adjusting the temperature to 15 ℃, and shaking at the rotating speed of 225rpm for 3 h;
the method specifically comprises the following steps:
s1, eluting and removing corrosion of a matrix biological membrane;
s2, extracting DNA;
wherein, step S1 includes the following steps:
s11, collecting the large-particle matrix, adding sterile normal saline, and oscillating to elute the biological membrane on the surface of the large-particle matrix to obtain turbid liquid;
s12, sucking the suspension obtained in the step S11 into a sterile centrifuge tube with a proper volume, centrifuging, removing the supernatant, continuously adding the suspension, centrifuging again, and storing the centrifuge tube containing the precipitate in a refrigerator at-20 ℃ for later use after the precipitate in the tube reaches the preset precipitate quality;
s13, taking out the sterile centrifuge tube with the preserved precipitate, standing at normal temperature for thawing, adding sterile quartz sand into the precipitate, adding a decay-removing buffer solution into the precipitate, uniformly mixing by vortex, centrifuging, removing supernatant, and performing DNA extraction operation on the obtained precipitate in the next step; the composition of the de-corrosion buffer solution is 100mmol/L pH8.0Tris-HCl and 100mmol/L Na2P2O7、100mmol/LpH8.0Na2EDTA, 1% PVP, 100mmol/L NaCl, 0.05% Triton X-100; the pH value of the de-corrosion buffer solution is 8.0;
step S2 includes the following steps:
s21, adding 0.5mol/L calcium chloride into the precipitate obtained in the step S13, uniformly mixing in a vortex manner, centrifuging, and then removing supernatant to obtain precipitate;
s22, adding 0.05mol/L sodium oxalate into the precipitate obtained in the step S21, uniformly mixing in a vortex mode, carrying out centrifugal treatment, and then removing supernatant to obtain the precipitate;
s23, adding the DNA extracting solution and lysozyme into the precipitate obtained in the step S22, uniformly mixing by vortex, and carrying out shaking table treatment;
the DNA extracting solution consists of 100mM Tris-HCl, 1.5mol/L NaCl and 1% CTAB, and the pH value is 8.0; the concentration of the lysozyme is 100 mg/mL;
s24, adding 100uL of 20% SDS after short-time centrifugation, uniformly mixing by vortex, treating in a water bath, and shaking up for several times by reversing at intervals;
s25, after centrifugal treatment, collecting an intermediate liquid phase layer, and repeatedly washing and precipitating once;
s26, adding mixed liquor of chloroform and isoamylol with the same volume into the collected supernatant fluid for extraction, and collecting an upper liquid phase layer after centrifugal treatment; the mixing volume ratio of the chloroform to the isoamyl alcohol is 24: 1;
s27, adding 0.1 time volume of 3mol/L sodium acetate and 0.6 time volume of isopropanol into the collected liquid phase layer, and standing at-20 ℃ for 20 min;
s28, centrifuging and then discarding the supernatant; adding 1mL of 70% ethanol into the precipitate for washing, centrifuging again, discarding the supernatant, and repeatedly washing once to obtain the precipitate;
s29, drying and dissolving the precipitate obtained in the step S28 to obtain a DNA solution;
step S12, the centrifugation is performed for 10min at 4 ℃ and 12000 rp; step S13, the centrifugal treatment is carried out for 5min under the conditions of room temperature and 12000 rp; step S21, the centrifugation treatment is centrifugation for 5min at 12000rpm and room temperature; step S22, the centrifugation treatment is centrifugation for 5min at 12000rpm and room temperature; step S25, the centrifugation treatment is centrifugation for 5min at 12000rpm and room temperature; step S26, the centrifugation treatment is centrifugation for 10min at 12000rpm and room temperature; step S28, centrifuging at 12000rpm at room temperature for 10min, and step S28, centrifuging at 12000rpm at room temperature for 5 min;
step S13, the grain size of the sterile quartz sand is 1 mm; the shaking treatment condition in step S23 is to treat at 37 deg.C and 225rpm for 30 min; the water bath treatment temperature of the step S24 is 65 ℃, and the water bath treatment time is 1 h; the extraction time of the step S26 is 10 min; the pH value of the sodium acetate in the step S27 is 5.2;
the drying and DNA dissolution of step S29 is carried out according to the following steps: drying at 60 ℃ for 5min, adding 50 μ L of 10mmol/L Tris-HCl with pH8.0, flicking with finger to dissolve precipitate, and combining the same sample DNA of the two tubes to 100 μ L.
2. The method for qualitatively and/or quantitatively extracting the microbial total DNA on the large-particle substrate surface of the constructed wetland according to claim 1, wherein the large-particle substrate is made of materials including gravel, volcanic rock or zeolite.
3. According to the rightThe method for qualitatively and/or quantitatively extracting the microbial total DNA on the large-particle substrate surface of the constructed wetland according to claim 1, which is characterized by comprising the steps of claim 1 and further comprising the following operations: step S11 further includes the step of recording the mass M of the large granular matrix and the volume V of sterile saline; step S12 further includes recording the total volume V of the suspension added1A step of; calculating the mass M of the large-particle matrix corresponding to the precipitate1Is composed of
Weighing the large-particle matrix, drying, weighing, and recording the mass as m2(ii) a The total DNA extracted from the in-tube pellet that reached the quality set for the detection of the pellet is marked mgDNA; the calculation formula is as follows: the matrix DNA content (ng/g) is 30mgDNA(m1-m0)/m2(ii) a Empty pipe weighing m0Tubes containing the precipitate were weighed and recorded as m1The mass of the precipitate is m1-m0The constant 30 is based on 30g of large particle substrate.
4. The application of the method for qualitatively and/or quantitatively extracting the total DNA of the microorganisms on the large-particle substrate surface of the artificial wetland according to any one of claims 1 to 3, which is characterized in that the extraction method is combined with a PCR (polymerase chain reaction) method and an electrophoresis method and is applied to the qualitative and/or quantitative detection of the total DNA of the microorganisms on the large-particle substrate surface of the artificial wetland.
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