CN101148664B - Method for extracting solid-state fermentation microorganism total DNA - Google Patents
Method for extracting solid-state fermentation microorganism total DNA Download PDFInfo
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- CN101148664B CN101148664B CN2007100356764A CN200710035676A CN101148664B CN 101148664 B CN101148664 B CN 101148664B CN 2007100356764 A CN2007100356764 A CN 2007100356764A CN 200710035676 A CN200710035676 A CN 200710035676A CN 101148664 B CN101148664 B CN 101148664B
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Abstract
The present invention provides process of extracting total DNA of solid fermented microbe. The process includes the following steps: washing the sample with phosphate buffer solution, ultrasonic crushing and CTAB solution extracting solid fermented microbe cell, precipitating the coarse DNA extracted liquid in pre-cooled isopropanol and washing in 70 % concentration pre-cooled alcohol solution, purifying with purifying kit and eliminating RNA with ribonuclease. The process of the present invention can obtain DNA in high yield and extract DNAs of bacteria, actinomycete and fungi, so as to understand the microbial diversity of the solid fermented sample quickly and comprehensively.
Description
Technical field
The present invention relates to the DNA extraction method, be specifically related to microorganism total DNA extracting method in the solid state fermentation.
Background technology
Solid state fermentation is meant and utilizes the nature substrate to do the carbon source and the energy, or utilizes inert substrate to do solid-state upholder, and its system is anhydrous or approach anhydrous any fermenting process, and microbial growth and formed product are all on the surface of matrix.Serious day by day along with energy dilemma and environmental problem, solid-state fermentation technology causes the great interest of people with its distinctive advantage (as there not being " three wastes " discharging); Fully this technology of development and use solid state fermentation is produced the required leavened prod of people simultaneously, has received countries in the world scientific worker's concern; And microorganism plays the part of crucial role in solid ferment process.Therefore, species diversity and the bacterium colony succession situation of microorganism in the research solid state fermentation will help us more in depth to understand the mechanism that solid state fermentation takes place, thereby realize control and optimization to its technology better.
At present, traditional microscopy, flat band method and biolog method are mainly used in the diversity and the bacterium colony succession of microbe colony in the research solid state fermentation sample, and these methods are all by means of culture of microorganism, and this will cause losing a lot of non-cultivation species information, thereby makes research not comprehensive.Yet be based upon on the Protocols in Molecular Biology diversity and the bacterium colony succession of research microbe colony, what overcome microorganism can not cultivation property, for the diversity and the bacterium colony succession of microbe colony in the research solid state fermentation sample provides new approach.
In the research of most Protocols in Molecular Biology, crucial extraction at DNA.The method that is used for DNA extraction at present is a lot, but mainly concentrates on the extraction of DNA of bacteria, and is relatively simple.But the method that can extract bacterium, actinomycetes, fungal DNA simultaneously is rarer.Therefore, seek a kind of method that can extract bacterium, actinomycetes, fungal DNA simultaneously, change being very important for the bacterium colony of these three kinds of bacterium of overall understanding in the solid state fermentation sample.
Summary of the invention
The objective of the invention is to solve above-mentioned the deficiencies in the prior art, a kind of method that can extract bacterium, actinomycetes, fungal DNA simultaneously is provided, with the diversity of microorganism in the overall understanding solid state fermentation sample, thereby better bring into play the application of Microbial resources in solid state fermentation.
Purpose of the present invention realizes by following scheme:
A kind of method for extracting solid-state fermentation microorganism total DNA may further comprise the steps:
A. the washing of sample: the solid state fermentation sample must wash three times with the 0.1M phosphate buffered saline buffer;
The extraction of b.DNA: add Virahol in the solid state fermentation sample after the above-mentioned washing, use the ultrasonic wave ice-bath ultrasonic, after the sample fragmentation, high speed centrifugation to sample precipitates fully, removes supernatant liquor; Add hexadecyl trimethyl ammonium bromide (CTAB) extract in precipitation, after 65 ℃ of water-bath complete reactions, add isopyknic chloroform-primary isoamyl alcohol, high speed centrifugation is complete to precipitation, and reclaims water; Reclaiming the CTAB-NaCl solution that aqueous phase adds 1/10 volume, after complete reaction is treated in 65 ℃ of water-baths, add isopyknic chloroform-primary isoamyl alcohol, high speed centrifugation is complete to precipitation again, and reclaims water; At the pre-cold isopropanol that reclaims 0.6 times of volume of aqueous phase adding, and after fully reaction was treated in-20 ℃ of placements, high speed centrifugation was complete to precipitation again, removes supernatant liquor; Precipitation precooled ethanol repeated washing and drying, dried precipitation is dissolved in the TE damping fluid, is slightly carried DNA;
The purifying of c.DNA: after the purified test kit purifying of the above-mentioned DNA that slightly carries, add rnase in its purified product, 37 ℃ of water-bath digestion are to remove RNA.
Add Virahol 4mL in described every 1g solid state fermentation sample, broken 5.2s in the described ultrasonic procedure, stop 2.4s, amplitude 45%, described chloroform-primary isoamyl alcohol volume ratio is 24: 1, add CTAB extract 1000 μ L in the described every 1g precipitation, also contain percent by volume in the CTAB extract of described adding and be 2% beta-mercaptoethanol, described CTAB-NaCl solution composition is: 10mg/mL CTAB; 0.7mol/L NaCl, described Virahol, chloroform, primary isoamyl alcohol massfraction are all＞99%, the rnase final concentration of described adding is 0.5 μ g/mL.
Advantage of the present invention is: at present, mainly concentrate on DNA extraction of bacterium and follow-up research for the research of the molecular ecology of environmental sample.And microbe species is profuse in the environmental sample, and it is not enough only wherein bacterial colony being carried out diversity analysis, also will analyze other a few class bacterium and interaction.Therefore be necessary to seek a kind of method that can obtain total DNA of the actual composition situation of microorganism in the former environmental sample of true reflection, and can be used in subsequently that enzyme is cut, connection, PCR and gradient gel electrophoresis and set up clone library and the dna sequence dna order-checking, thereby obtain comprehensive microorganism hereditary information.The present invention is directed to solid ferment process is the coefficient process of multiclass microorganism, provide a kind of can study simultaneously, comprehensively fungi, bacterium, actinomycetic dynamic change with and the multifarious approach of kind.This method can simultaneously successful extraction solid state fermentation sample in total DNA of bacterium, actinomycetes, fungi also can obtain higher, purer DNA output, and the purpose band that amplifies bacterium, actinomycetes, fungi that all can success and obtain the PCR product of suitable high yield, and can be used for analysis such as follow-up genetic diversity.It is the method that a kind of quick, both economical suitable solid state fermentation sample microorganism total DNA extracts.This law does not need special instrument in addition, and experimental cost is relatively low.Therefore present method provides a kind of quick, economic method for the molecular ecology research of research solid state fermentation sample.
Description of drawings
Fig. 1: after slightly carrying and the agarose gel electrophoresis figure of the later total DNA of solid state fermentation sample of purifying;
Wherein Marker is from the dna molecular amount mark (λ DNA/HindIII) of biotech firm's purchase, and 1,2,3 represent parallel sample respectively, and numeral is the size of DNA, and unit is bp.
Fig. 2: the agarose gel electrophoresis figure of the 16SrDNA product of pcr amplification purify DNA fungi 18SrDNA product and actinomycetes, bacterium.
Wherein Marker is from the dna molecular marker (100bp DNA ladder) of biotech firm's purchase, F1-F3 represents the parallel sample of fungi PCR product respectively, A1-A3 represents the parallel sample of actinomycetes PCR product respectively, B1-B3 represents the parallel sample of bacteria PCR product respectively, numeral is the size of DNA, and unit is bp.
Embodiment
Embodiment 1: use method of the present invention the solid state fermentation sample to be carried out the extraction of microorganism total DNA:
1, solid state fermentation sample
The solid state fermentation sample is taken from 5L and is adorned laboratory scale solid-state fermenter, main raw is to be cut into the long straw 180g of 10～20mm after cleaning, drying, take from the uncontamination soil 90g on Yue Lu mountain, and fluid matrix 650ml, make its former water ratio between 70%～80%.Solid-state fermenter is positioned in 30 ℃ of thermostat containers, and other condition natures are cultivated sampling after 30 days.Sampling optimization is got three samples altogether and is made as 1,2,3 respectively in sample below 3cm.
The moiety of described fluid matrix is: KH
2PO
42.0gL
-1, MgSO
47H
2O 0.5gL
-1, ammonium tartrate 0.2gL
-1, liquid microelement 70mLL
-1, the pH nature; Wherein, the composition of liquid microelement is NaCl 1.0gL
-1, CoCl
26H
2O 0.18gL
-1, Na
2MoO
42H
2O 0.01gL
-1, ZnSO
47H
2O 0.1gL
-1, CaCl
20.1gL
-1, CuSO
45H
2O 0.01gL
-1, MnSO
4H
2O 0.5gL
-1, FeSO
47H
2O 0.1gL
-1, AlK (SO
4)
212H
2O0.01gL
-1, MgSO
47H
2O 3.0gL
-1, HBO
30.01gL
-1, NTA 1.5gL
-1
2, the washing of sample
Before extracting DNA, sample is washed, to reduce extracellular dna and the especially pollution of humic acid material of dissolved organic matter.The 0.5g sample is added in the phosphate buffered saline buffer (pH8.0) of 3mL 100mmol/L, vibrator at the uniform velocity shakes 30min with 180r/min, then with the centrifugal 10min of 5500r/min; Precipitation is used for subsequent experimental after the washing once more.
Described 100mmol/L pH8.0 phosphate buffered saline buffer is: 5.3ml 0.2mol/L KH
2PO
4With 94.7mL 0.2mol/LK
2HPO
4Mix the back and be settled to the 200mL gained with ultrapure water.
3, the extraction of DNA
The Virahol that adds 2mL massfraction＞99% in washed sample is used Model450 ultrasonic disruption instrument (Branson, USA) the broken 10min (broken 5.2s stops 2.4s, amplitude 45%) of ice bath behind the mixing; The all product of solid state fermentation after the fragmentation are with 13000r/min, and centrifugal 15min removes supernatant liquor.(described CTAB extract consists of: 20mg/mL CTAB to add 500 μ L CTAB extracts in the precipitation; 100mmol/L Tris-Cl, pH8.0; 20mmol/L disodium ethylene diamine tetraacetate (EDTA), pH8.0; 1.4mol/L NaCl), described 500 μ LCTAB take out the beta-mercaptoethanol that has also added 10 μ L massfraction＞99% in the topic liquid, 65 ℃ of water-baths after 2 hours, add 500 μ L volume ratios again and be chloroform (massfraction＞99%)-primary isoamyl alcohol (massfraction＞98.5%) mixing solutions of 24: 1, with 10000r/min, centrifugal 10min also reclaims water again.(described CTAB-NaCl solution composition is: 10mg/mL CTAB to add the CTAB-NaCl solution of 1/10 volume at aqueous phase; 0.7mol/LNaCl), after 1 hour, add isopyknic 24: 1 (V/V) chloroform-primary isoamyl alcohol 65 ℃ of water-baths again, again with 10000r/min, centrifugal 10min also reclaims water.Aqueous phase adds the Virahol of massfraction＞99% of 0.6 times of volume-20 ℃ precooling, places precipitation 1.5h at-20 ℃, with the centrifugal 15min of 10000r/min, removes supernatant liquor again.The massfraction of-20 ℃ of precoolings of precipitation usefulness is 70% ethanol repeated washing and drying.Dried precipitation is dissolved in the TE damping fluid of 300 μ L, and (described TE damping fluid contains: 10mM Tris-Cl, pH8.0; 1mM EDTA), slightly carried DNA.After slightly carrying the purified test kit purifying of DNA (described purification kit is the common DNA product purification test kit of sky, Beijing with biochemical company limited), adding final concentration in its purified product is 0.5 μ g/mL RNaseA, at 37 ℃ of water-bath digestion 2h, to remove RNA.
4, slightly carry with the agarose gel electrophoresis of purifying DNA fragment size and detecting
Slightly propose agarose gel electrophoresis detection with the purifying DNA fragment size, with containing 0.5 μ g/mL bromination second shallow lake (EB), agarose concentration is the gel of 10mg/mL, used dna molecular amount mark (Marker) is the λ DNA/HindIII of sky, Beijing with biochemical company limited, used electrophoretic buffer is the 1*TAE electrophoretic buffer, with the voltage electrophoresis 25min of 100V.Electrophoresis finishes later gel in the detection of taking pictures of the Gel Doc2000 of U.S. BIORAD company gel imaging system, the results are shown in Figure 1, can see slightly carry with purifying after the length of dna fragmentation all be about 23kb.
1*TAE electrophoretic buffer wherein: 40mM Tris alkali, 20mM acetate, 2mM EDTA; Describedly contain the 10mg/mL sepharose that 0.5 μ g/mL bromination second is formed sediment: the 1g agarose, add 100ml 1*TAE electrophoretic buffer heating for dissolving, to be cooled after, the 10 μ g/mL bromination second that add 5 μ L are formed sediment.
5, amplification of 18SrDNA fungi and 16SrDNA actinomycetes and bacteria PCR and product detect
(1) the 18SrDNA amplification universal primer of selecting fungi is right as primer to NU-SSU-0817 (5 '-TTA GCA TGG AATAAT RRA ATA GGA-3 ') and NU-SSU-1196 (3 '-TCT GGA CCT GGT GAG TTT CC-5 '), and the length that amplifies the 18SrDNA sequence is about 422bp.
The PCR reaction system of 50 μ L is: 4 μ L are purify DNA, 4 μ L dNTP mixing solutionss (final concentration is 1pmol/ μ L), each 1.5 μ L (concentration is 10 μ M) of primer, 10 * Buffer, 6 μ L, Taq archaeal dna polymerase (Promege, USA) 1 μ L (enzyme activity unit 2.5U) adds aseptic deionized water and complements to 50 μ L.Amplification condition is 94 ℃ of pre-sex change 3min; Next be 94 ℃ of sex change 30s, 55 ℃ of annealing 20s, 72 ℃ are extended 40s, 35 circulations; 72 ℃ are extended 8min, stop at 4 ℃.
(2) select actinomycetic 16SrDNA amplification universal primer to F243 (5 '-GGA TGA GCC CGC GGCCTA-3 ') and R531 (5 '-CGG CCG CGG CTG CTG GCA CGT A-3 ') as the pcr amplification primer, this primer is to 226～243 bit bases and 513～528 bit bases of the 16S rDNA that corresponds respectively to E1 coli, and the amplified production size is about 308bp.
The PCR reaction system of 50 μ L is: 8 μ L are purify DNA, 4 μ LdNTP mixing solutionss (final concentration is 1pmol/ μ L), each 1.5 μ L (concentration is 10 μ M) of primer, 10 * Buffer, 6 μ L, Ex Taq DNA HS warm start polysaccharase (Promege, USA) 0.5 μ L (enzyme activity unit 2.5U), adding aseptic deionized water, to complement to 50 μ L. amplification conditions be 94 ℃ of sex change 30s, 63 ℃ of annealing 1min, 72 ℃ are extended 2min, 35 circulations; 72 ℃ are extended 10min, stop at 4 ℃.
(3) selecting bacteria 16SrDNA amplification universal primer is right as primer to 341F (5 '-CCT ACG GGA GGC AGC AG-3 ') and 907R (5 '-CCG TCAATT CCT TTG AGT TT-3 '), this primer is to corresponding respectively to 341 bit base to 927 bit bases of E1 coli, and amplification length is about 586bp.
The PCR reaction system of 50 μ L is: 4 μ L are purify DNA, 4 μ L dNTP mixing solutionss (final concentration is 1pmol/ μ L), each 1.5 μ L (concentration is 10 μ M) of primer, 10 * Buffer, 6 μ L, Taq archaeal dna polymerase (Promege, USA) 1 μ L (enzyme activity unit 2.5U) adds aseptic deionized water and complements to 50 μ L.Amplification condition is 94 ℃ of pre-sex change 5min; Next be 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 60s, 35 circulations; 72 ℃ are extended 8min, stop at 4 ℃.
Pcr amplification product all adopts the 10mg/mL agarose gel electrophoresis equally, and except that used dna molecular amount is labeled as sky, Beijing with the 100bp DNA ladder Marker of biochemical company limited, all the other are with method 4.The results are shown in Figure 2, as can be seen from the figure, fungi, the actinomycetes bacterium universal primer is to the equal purpose band that can PCR goes out separately, and specific amplification is strong, illustrates that this method can study Study on Diversity of three major types microorganism in the solid state fermentation sample and mutual succession situation simultaneously.
Claims (6)
1. method for extracting solid-state fermentation microorganism total DNA may further comprise the steps:
A. the washing of sample: the solid state fermentation sample must wash three times with the 0.1M phosphate buffered saline buffer;
The extraction of b.DNA: add Virahol in the solid state fermentation sample after the above-mentioned washing, use the ultrasonic wave ice-bath ultrasonic, after the sample fragmentation, high speed centrifugation to sample precipitates fully, removes supernatant liquor; Add the CTAB extract in precipitating, add CTAB extract 1000 μ L in every 1g precipitation, 65 ℃ of water-baths after the complete reaction, add isopyknic chloroform-primary isoamyl alcohol, and high speed centrifugation extremely precipitates fully, and reclaims water; Reclaiming the CTAB-NaCl solution that aqueous phase adds 1/10 volume, after complete reaction is treated in 65 ℃ of water-baths, add isopyknic chloroform-primary isoamyl alcohol, high speed centrifugation is complete to precipitation again, and reclaims water; At the pre-cold isopropanol that reclaims 0.6 times of volume of aqueous phase adding, and after fully reaction was treated in-20 ℃ of placements, high speed centrifugation was complete to precipitation again, removes supernatant liquor; Precipitation precooled ethanol repeated washing and drying, dried precipitation is dissolved in the TE damping fluid, is slightly carried DNA;
The purifying of c.DNA: after the purified test kit purifying of the above-mentioned DNA that slightly carries, add rnase in its purified product, 37 ℃ of water-bath digestion are to remove RNA;
Consisting of of above-mentioned CTAB extract: 20mg/mL CTAB; 100mmol/L Tris-Cl, pH8.0; 20mmol/L disodium ethylene diamine tetraacetate (EDTA), pH8.0; 1.4mol/L NaCl; Percent by volume is 2% beta-mercaptoethanol;
Consisting of of above-mentioned CTAB-NaCl solution: 10mg/mL CTAB; 0.7mol/L NaCl.
2. method for extracting solid-state fermentation microorganism total DNA according to claim 1 is characterized in that adding in every 1g solid state fermentation sample among the described step b Virahol 4mL.
3. method for extracting solid-state fermentation microorganism total DNA according to claim 1 and 2 is characterized in that among the described step b, broken 5.2s stops 2.4s in the ultrasonic procedure, amplitude 45%.
4. method for extracting solid-state fermentation microorganism total DNA according to claim 1 and 2 is characterized in that among the described step b that chloroform-primary isoamyl alcohol volume ratio is 24: 1.
5. method for extracting solid-state fermentation microorganism total DNA according to claim 1 and 2 is characterized in that among the described step b, and Virahol, chloroform, primary isoamyl alcohol massfraction are all＞99%.
6. method for extracting solid-state fermentation microorganism total DNA according to claim 1 and 2 is characterized in that among the described step c, and the rnase final concentration of adding is 0.5 μ g/mL.
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| CN102643795A (en) * | 2012-04-05 | 2012-08-22 | 湖北省农业科学院经济作物研究所 | Method for extracting total DNAs of soil microorganisms through PVP pretreatment |
| CN102643801B (en) * | 2012-05-10 | 2013-03-27 | 南京林业大学 | Extraction method for glycocalyx-generating bacterium genome DNA (Deoxyribonucleic Acid) suitable for whole-genome sequencing |
| CN104862303A (en) * | 2015-05-30 | 2015-08-26 | 福建出入境检验检疫局检验检疫技术中心 | Method for extracting DNA of puffer fish by utilizing ultrasonic treatment |
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Non-Patent Citations (7)
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| HowelerM ET-AL.A quantitative analysis ofDNA extraction and purification from compost.J Microbiol Methods54 1.2003,54(1),37-45. |
| HowelerM ET-AL.A quantitative analysis ofDNA extraction and purification from compost.J Microbiol Methods54 1.2003,54(1),37-45. * |
| Zh. H. Yang ET-AL.Comparison of methods for total community DNA extractionand purification from compost.Appl Microbiol Biotechnol74.2006,74918-925. * |
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