CN114350651A - Method for efficiently extracting microbial DNA on aerobic fermentation micro-interface - Google Patents
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Images
Abstract
The invention discloses a method for efficiently extracting microbial DNA on an aerobic fermentation micro-interface, which comprises the steps of wall breaking and purification. By limiting operation parameters such as vortex, oscillation time, centrifugal time and the like in the extraction process and matching with the use of a washing buffer solution, the interference of organic matters can be reduced, so that DNA in an aerobic fermentation micro-interface environment can be extracted, microorganisms on a 1mm particle micro-interface can be treated, the sample treatment capacity is increased, the purity and the yield of the finally extracted environmental DNA are high, the extraction cost is low, the mechanical damage to the DNA is small, and the integrity is better.
Description
Technical Field
The invention relates to the technical field of biological analysis, in particular to a method for efficiently extracting microbial DNA on an aerobic fermentation micro-interface.
Background
9000 ten thousand tons of kitchen waste are generated in China every year, and if the kitchen waste is not treated in time, the production and the life of residents are influenced. Along with the propelling of the household garbage classification work, the kitchen garbage collection amount can continuously rise. The kitchen waste is not suitable for landfill and incineration due to high water content and low heat value; moreover, land is occupied by landfill, underground water is polluted, and air is polluted by incineration. Aerobic fermentation is a better mode for treating the kitchen waste, and can enable the nutrient elements in the kitchen waste to return to soil.
The aerobic fermentation microorganisms have complex composition, various types, large quantity and various functions, and the research on community structure and genetic function diversity is generally regarded by scholars in the fields of microbiology, ecology and the like, so that the aerobic fermentation microorganisms become a hot spot of the current research, wherein DNA extraction is a key step in the research on the composting microorganisms. Because of the tight combination of microorganisms and compost particles and the interference of organic matters, the acquisition of high-quality compost microorganism DNA is difficult to achieve.
The existing literature shows that the specific surface area of organic matters is in direct proportion to the degradation rate, so that the particle size has important influence on the period length of the kitchen waste aerobic composting and the composting effect. It is also disclosed that in the aerobic fermentation stack, the outer layer microorganisms on the aerobic fermentation granules are subjected to aerobic reaction, and the inner layer microorganisms are subjected to anaerobic reaction. Different action types of microorganisms exist inside and outside the aerobic fermentation pile body granules. The existing extraction method is a macroscopic interface, the micro interface is smaller in scale, the dynamics of a plurality of micro interfaces are superposed, the dynamics of microorganisms are not reflected from the smaller scale, and the existing extraction method is complex in operation and long in required time, so that the extraction efficiency is low.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for efficiently extracting microbial DNA on an aerobic fermentation micro-interface, and by the method, the genomic DNA which has high purity, high concentration and high integrity and meets the requirement of genome sequencing can be obtained, and the DNA purity does not obviously change, and meanwhile, the column adsorption and elution are not needed, so that the variety of experimental materials and the usage amount of reagents are reduced, the experimental time is shortened, and the reagent usage amount and the experimental cost are saved.
The purpose of the invention is realized by the following technical scheme:
a method for efficiently extracting microbial DNA on an aerobic fermentation micro-interface comprises the following steps:
(1) randomly selecting aerobic fermentation sample particles about 1 mm;
(2) adding 5-10 mu L of Buffer solution Buffer 1 and 0.65-1.22 mu L of Buffer 2 into the sample obtained in the step (1), and oscillating to fully mix the sample, the Buffer 1 and the Buffer 2;
(3) adding 1.25-2.50 mu L of Buffer 3 into the mixed solution in the step (2), shaking up to fully mix the mixed solution, centrifuging, and sucking supernatant;
(4) sucking 5-10 mu L of fully and uniformly mixed Buffer 4, adding into the supernatant obtained in the step (3), and shaking to fully mix the solution;
(5) centrifuging the solution fully mixed in the step (4), sucking the supernatant and discarding;
(6) adding 25-50 mu L of Buffer 5 solution into the mixture left in the step (5), uniformly shaking, continuing centrifuging, sucking the supernatant again and discarding;
(7) repeating the step (6) for 2-3 times, and then placing the remaining mixture at room temperature for standing to fully volatilize the ethanol;
(8) adding 10-20 mu L of Buffer 6 solution into the mixture obtained in the step (7), shaking and uniformly mixing, rotating at a high speed for centrifugation, and sucking supernatant fluid, so that the microbial DNA on the aerobic fermentation micro-interface is positioned in the supernatant fluid;
the Buffer 1 reagent is as follows: sodium phosphate solution with the concentration of 0.10-0.30 mol/L;
the Buffer 2 reagent is obtained by mixing 2 wt% of hexadecyl trimethyl ammonium bromide, 500-700mM NaCl, 0.2% of mercaptoethanol, 20 wt% of SDS and 10mg/mL of protease K, TE Buffer solution, and adjusting the pH value to 8.0;
the Buffer 3 reagent is a DNA protein washing solution which is prepared by mixing DNA protein washing solution with a volume ratio of 24: 1, chloroform and isoamylol;
the Buffer 4 reagent is an alkoxide-based silica sol-gel mixture containing a silica matrix;
the Buffer 5 reagent is ethanol with the purity of 70 percent;
the Buffer 6 reagent is TE Buffer solution which is a known substance and is a mixed solution of Tris and EDTA. I.e., 10mmol/L Tris-HCl, 0.1mmol/L EDTA, and adjusting the pH to 7.0-8.0.
Further, the rotation speed of the centrifugation in the step (3) is 14000 Xg, and the centrifugation time is 5 min.
Further, the rotation speed of the centrifugation in the steps (5) and (6) is 1000 Xg, and the centrifugation time is 1 min.
Further, the rotation speed of the centrifugation in the step (8) is 14000 Xg, and the centrifugation time is 1 min.
The invention has the following beneficial effects:
the method for efficiently extracting the microbial DNA on the aerobic fermentation micro-interface is simple to operate, saves experimental time, has higher extraction efficiency, and higher purity and yield of the extracted DNA, and the purity of the DNA extracted by the method can reach the precision of the existing kit, and has low extraction cost, small mechanical damage to the DNA and better integrity.
Drawings
FIG. 1 is a diagram showing a comparison of DNA agarose gel electrophoresis detection extracted in different embodiments.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments, and the objects and effects of the present invention will become more apparent, it being understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.
The test was conducted at the university of agriculture, china, institute of organic recycling (suzhou). Firstly, carrying out aerobic composting on kitchen waste, wherein the method comprises the following steps:
(1) adding 15-20% of sawdust according to the fresh weight of the kitchen waste to adjust the C/N ratio to 20-40: 1; the materials are mixed evenly.
(2) The material in (1) was divided into 6 parts on average and charged into a 50kg composting reactor.
(3) 3 of the reactors were not inoculated; is denoted as CK.
(4) Setting the aeration rate of the reactor to 0.20m3·min-1·m-3(ii) a Aerating for 30min, stopping for 30min, and circulating all the time; turning over every 3 days.
(5) Aerobic composting was carried out for 17 days.
In the embodiment of the invention, when extracting microorganisms on an aerobic fermentation micro-interface, firstly, a product in a high-temperature period of the aerobic fermentation of the kitchen waste is collected, a sample with the depth being the high temperature position in an aerobic fermentation pile body is collected, 3 parallel samples are collected in total, and the samples are stored at the temperature of minus 20 ℃ for later use.
The buffers 1 to 6 used in the following two examples are:
the Buffer 1 reagent is: sodium phosphate solution with the concentration of 0.10-0.30 mol/L;
buffer 2 reagent, which is obtained by mixing 2 wt% of hexadecyl trimethyl ammonium bromide, 500-700mM NaCl, 0.2% of mercaptoethanol, 20 wt% of SDS and 10mg/mL of protease K, TE Buffer solution, and adjusting the pH value to 8.0;
the Buffer 3 reagent is a DNA protein washing solution which is prepared by mixing DNA protein and a Buffer solution in a volume ratio of 24: 1, chloroform and isoamylol;
the Buffer 4 reagent is an alkoxide-based silica sol-gel mixture containing a silica matrix;
the Buffer 5 reagent is ethanol with the purity of 70 percent;
buffer 6 reagent is TE Buffer.
Example 1 (three replicates)
(1) Randomly taking sample particles of about 1mm and adding the sample particles into a proper centrifugal tube;
(2) adding 10 mu L of Buffer solution Buffer 1 and 1.22 mu L of Buffer 2 into the centrifuge tube added with the sample particles, and oscillating for 30 s;
(3) adding 2.50 μ L Buffer 3 into the centrifuge tube, shaking, mixing, adjusting the rotation speed of the centrifuge to 14000 Xg, centrifuging for 5min, and transferring the supernatant to 1 new centrifuge tube;
(4) fully and uniformly mixing the Buffer 4, sucking 10 mu L of Buffer 4, adding into the centrifuge tube, and shaking for 1-2min to uniformly mix the Buffer 4 and the centrifuge tube;
(5) regulating the rotation speed of a centrifuge to 1000 Xg, centrifuging for 1min, and sucking and discarding supernatant;
(6) adding 50 μ L Buffer 5 solution, shaking uniformly, adjusting the rotation speed of the centrifuge to 1000 Xg, centrifuging for 1min, sucking and discarding the supernatant;
(7) and (4) after repeating the step (6) for two to three times, opening the tube cover of the centrifugal tube, and standing for 20-30min at room temperature so as to fully volatilize the ethanol.
(8) Adding 20 mu L of Buffer 6 solution, shaking and mixing uniformly, adjusting the rotating speed of a centrifuge to 14000 Xg, centrifuging for 1min, sucking 10 mu L of supernatant into a new proper centrifuge tube, and allowing the microbial DNA on the aerobic fermentation micro-interface to be located in the supernatant for later-stage experiments.
Example 2 (three replicates)
(1) Randomly taking sample particles of about 1mm, and adding the sample particles into a proper centrifugal tube;
(2) adding 5 mu L of Buffer solution Buffer 1 and 0.65 mu L of Buffer 2 into the centrifuge tube added with the sample particles, and oscillating for 30 s;
(3) adding 1.25 μ L Buffer 3 into the centrifuge tube, shaking, mixing, adjusting the rotation speed of the centrifuge to 14000 Xg, centrifuging for 5min, and transferring the supernatant to 1 new centrifuge tube;
(4) fully and uniformly mixing the Buffer 4, sucking 5 mu L of Buffer 4, adding the Buffer 4 into the centrifuge tube, and shaking for 1-2min to uniformly mix the Buffer 4 and the centrifuge tube;
(5) regulating the rotation speed of a centrifuge to 1000 Xg, centrifuging for 1min, and sucking and discarding supernatant;
(6) adding 25 μ L Buffer 5 solution, shaking uniformly, adjusting the rotation speed of the centrifuge to 1000 Xg, centrifuging for 1min, sucking and discarding the supernatant;
(7) repeating the step (6) for two to three times; opening the tube cover of the centrifugal tube, and standing at room temperature for 20-30min to fully volatilize ethanol.
(8) Adding 10 μ L of Buffer 6 solution, shaking, mixing, adjusting the rotation speed of the centrifuge to 14000 Xg, centrifuging for 1min, sucking 10 μ L of supernatant into a new proper centrifuge tube, and allowing the microorganism DNA on the aerobic fermentation micro-interface to be located in the supernatant for later experiments.
Comparative example
The extraction was carried out using FASTDNA extraction kit FAST DNA SPIN KIT from mobilo corporation, as follows:
0.5g of each sample was weighed into a lysine Matrix E tube. 978ul of Sodium Phosphate Buffer was added to the lysis Matrix E tube to which the sample had been added. Then adding 122ul MT Buffer, placing the mixture on a rapid nucleic acid extractor, setting the speed to be 6.0 and the time to be 40s, and fully mixing the samples; 14000 Xg for 5-10 minutes. Transferring the supernatant into clean 2.0ml microcentrifuge tubes respectively, adding 250ul PPS, and shaking up and down for 7 times to mix the materials fully and uniformly; 14000 Xg for 5 minutes, and respectively transferring the supernatant into a clean 15ml centrifuge tube; mixing the Binding Matrix evenly, sucking 1.0 and adding the mixture into a 15ml centrifuge tube; the vortex instrument or the manual work is shaken for 2 minutes to be bound with the DNA, and then the centrifugal tube is placed on a frame to be kept stand for 3 minutes; carefully remove 500ul of supernatant. The remaining solution was again mixed and approximately 600ul was pipetted into the SPIN Filter, centrifuged at 14000 × g for 1 minute, the lower grasping tube was emptied, the remaining solution was then added to the SPIN Filter, centrifuged again at 14000 × g for 1 minute, and the grasping tube was emptied. 500ul of prepared SEWS-M (100 ml of 100% ethanol added) was added to the SPIN Filter and flicked for several times; 14000 Xg for 1 minute; clearing the grabbing pipe, repeating the step 12, and clearing again; then 14000 Xg was centrifuged again for 2 minutes to remove the washing solution sufficiently, the grasping tube was discarded, and the SPIN Filter was allowed to stand at room temperature for 10 to 20 minutes in place of a new one in order to evaporate ethanol sufficiently to prevent the influence on the next test. Slowly adding 50ul DES, centrifuging at 14000 Xg for 5min, repeating once, dissolving DNA into DES, placing DNA into lower grasping tube, discarding SPIN Filter, and storing at-20 deg.C.
DNA purity of the microorganism DNA samples extracted from the micro-interfaces of the aerobic fermentation products extracted in examples 1 and 2 and comparative example was measured by a Dano-drop ultramicro-detection system, and the results are shown in Table 1.
TABLE 1 microbial DNA concentration and quality control parameters on the micro-interface of aerobic fermentation products
The DNA extracted in the above examples 1, 2 and comparative examples has the highest DNA concentrations of 20.7ng/uL, 22.7ng/uL and 130.8ng/uL respectively, and the A260/A280 ratios of 1.65, 1.73 and 1.81 respectively, as detected by a Dano-drop ultramicro detection system. From the experimental results, the data of each of examples 1 and 2 was substantially stable in three replicates, so as to prove that the experiment has no error. According to the experimental results, when the method is used for experimental operation, the operation is simple, the experimental time is saved, the DNA extraction efficiency is higher, the time and the labor are saved, and the method is more economical and more economical.
(2) The DNA extracted in the above example 1, example 2 and comparative example was subjected to PCR amplification with the following primer sequences: f' GTGCCAGCMGCCGCGGTAA; r' GGACTACVSGGGTATCTAAT, and carrying out gel electrophoresis detection on the amplification product, wherein the electrophoresis result is shown in figure 1. The samples corresponding to the right starting lane 1 are deoxyribonucleic acid molecular weight standards (1000 bp, 700bp, 500bp, 400bp, 300bp, 200bp and 100bp from top to bottom), the right starting lane 2 is a blank control, lanes 3-11 are results of the compost sample DNA extracted in the examples 1 and 2 and the comparative example, and the right electrified lanes 3-5 are the compost sample DNA extracted in the example 1; the right electrified lanes 6-8 are the compost sample DNA extracted in example 2; right electrification lanes 9-11 are DNA agarose electrophoresis images extracted in comparative examples. The results show that: the effect of extracting DNA from compost samples in examples 1 and 2 was as good as that of the kit of the comparative example.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and although the invention has been described in detail with reference to the foregoing examples, it will be apparent to those skilled in the art that various changes in the form and details of the embodiments may be made and equivalents may be substituted for elements thereof. All modifications, equivalents and the like which come within the spirit and principle of the invention are intended to be included within the scope of the invention.
Claims (4)
1. A method for efficiently extracting microbial DNA on an aerobic fermentation micro-interface is characterized by comprising the following steps:
(1) randomly selecting aerobic fermentation sample particles about 1 mm;
(2) adding 5-10 mu L of Buffer solution Buffer 1 and 0.65-1.22 mu L of Buffer 2 into the sample obtained in the step (1), and oscillating to fully mix the sample, the Buffer 1 and the Buffer 2;
(3) adding 1.25-2.50 mu L of Buffer 3 into the mixed solution in the step (2), shaking up to fully mix the mixed solution, centrifuging, and sucking supernatant;
(4) sucking 5-10 mu L of fully and uniformly mixed Buffer 4, adding into the supernatant obtained in the step (3), and shaking to fully mix the solution;
(5) centrifuging the solution fully mixed in the step (4), sucking the supernatant and discarding;
(6) adding 25-50 mu L of Buffer 5 solution into the mixture left in the step (5), uniformly shaking, continuing centrifuging, sucking the supernatant again and discarding;
(7) repeating the step (6) for 2-3 times, and then placing the remaining mixture at room temperature for standing to fully volatilize the ethanol;
(8) adding 10-20 mu L of Buffer 6 solution into the mixture obtained in the step (7), shaking and uniformly mixing, rotating at a high speed for centrifugation, and sucking supernatant fluid, so that the microbial DNA on the aerobic fermentation micro-interface is positioned in the supernatant fluid;
the Buffer 1 reagent is as follows: sodium phosphate solution with the concentration of 0.10-0.30 mol/L;
the Buffer 2 reagent is obtained by mixing 2 wt% of hexadecyl trimethyl ammonium bromide, 500-700mM NaCl, 0.2% of mercaptoethanol, 20 wt% of SDS and 10mg/mL of protease K, TE Buffer solution, and adjusting the pH value to 8.0;
the Buffer 3 reagent is a DNA protein washing solution which is prepared by mixing DNA protein washing solution with a volume ratio of 24: 1, chloroform and isoamylol;
the Buffer 4 reagent is an alkoxide-based silica sol-gel mixture containing a silica matrix;
the Buffer 5 reagent is ethanol with the purity of 70 percent;
the Buffer 6 reagent is TE Buffer solution.
2. The method for efficiently extracting the microbial DNA on the aerobic fermentation micro-interface according to claim 1, wherein the rotation speed of the centrifugation in the step (3) is 14000 Xg, and the centrifugation time is 5 min.
3. The method for efficiently extracting the microorganism DNA on the aerobic fermentation micro-interface according to claim 1, wherein the rotation speed of the centrifugation in the steps (5) and (6) is 1000 Xg, and the centrifugation time is 1 min.
4. The method for efficiently extracting the microbial DNA on the aerobic fermentation micro-interface according to claim 1, wherein the rotation speed of the centrifugation in the step (8) is 14000 Xg, and the centrifugation time is 1 min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050205005A1 (en) * | 2001-04-06 | 2005-09-22 | California Institute Of Technology | Microfluidic protein crystallography |
| US20090014388A1 (en) * | 2005-02-28 | 2009-01-15 | Jian Wen | Grafted Photo-Polymerized Monolithic Column |
| CN106011221A (en) * | 2016-06-21 | 2016-10-12 | 天津师范大学 | Method for regulating and controlling lawn compost matrix bacterium identification and analysis through carbon nanomaterials |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050205005A1 (en) * | 2001-04-06 | 2005-09-22 | California Institute Of Technology | Microfluidic protein crystallography |
| US20090014388A1 (en) * | 2005-02-28 | 2009-01-15 | Jian Wen | Grafted Photo-Polymerized Monolithic Column |
| CN106011221A (en) * | 2016-06-21 | 2016-10-12 | 天津师范大学 | Method for regulating and controlling lawn compost matrix bacterium identification and analysis through carbon nanomaterials |
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