CN101261204B - Method for simultaneously extracting compost sample PLFA and DNA - Google Patents
Method for simultaneously extracting compost sample PLFA and DNA Download PDFInfo
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- CN101261204B CN101261204B CN200810031165XA CN200810031165A CN101261204B CN 101261204 B CN101261204 B CN 101261204B CN 200810031165X A CN200810031165X A CN 200810031165XA CN 200810031165 A CN200810031165 A CN 200810031165A CN 101261204 B CN101261204 B CN 101261204B
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Abstract
The invention provides a method for simultaneously extracting and purifying phospholipid fatty acid PLFA and deoxyribonucleic acid DNA from the same composting sample, and the extracting method is based on an extraction method of lipid, after phase separation, the lipid enters an organic phase while the DNA is left in a water phase and a solid phase to realize the simultaneous extraction of the PLFA and the DNA finally; the extracting method of the invention has the advantages of simplifying the procedures for extracting the lipid of the composting sample and the DNA, ensuring the identity of the information source of the sample, well reducing the error caused by composting sampling to the measuring method, and being more meaningful when the quantity of the samples is small.
Description
Technical field
The present invention relates to PLFA of microorganism in the compost sample and the mixing extracting method of DNA.
Background technology
Microorganism is the key factor of compost processing, make its more effective degradation of organic substances with the control composting process, improves composting efficiency, and various microbial numbers, composition and interaction thereof are understood and be necessary in the research composting process.Because most of environmental microorganism all is in survival can not cultivation conditions, make that to understand the actual structure of community of microorganism relatively more difficult.Foranalysis of nucleic acids and lipid analysis all try hard to overcome this difficult point in recent years, and these methods all have can directly extract the advantage of cellular component to be used to analyze of living from environmental sample.
PLFA (Phospholipid fatty acid) spectrum analysis method is the representative of biochemical method.Phosphatide is the important component part that constitutes the active somatic cell film, and different microorganisms has different PLFA kind and quantity.First functional group in the PLFA molecule and side chain are different and different because of the type of microorganism usually, therefore every kind of sample has unique PLFA spectrogram, the PLFA spectrogram difference of different strain has diversity on height selectivity basis, can be used as the label of different groups in the micropopulation.It more is that composition and the biomass that comes from the microorganism in the sample changes that the PLFA spectrogram changes, and the content of phospholipid in the biosome inner cell can be thought relatively significantly constant usually.Because PLFA spectrum analysis method can more completely provide the biomass information and the implicit microorganism type information of PLFA molecule of viable microbial, PLFA spectrum analysis method is generally used for estimating that the group of microbes biomass and definite microorganism forms.The extraction of DNA is the element task of molecular ecology research.Different microbial populations has different dna sequence dnas or length, and its sequence of dna fragmentation that molecular weight is identical may be different.From the variation of dna fingerprint figure, can seek the genome sequence column information of critical function flora, means such as binding molecule clone and probe hybridization can also realize separating of functional flora, be used to study the structure and the dynamic change of complicated microbial ecosystem, for optimizing population structure, regulating system function and finding that new important microbe function monoid provides feasible approach.
When studying so complicated biological community structure of compost sample and community dynamic, it is not enough adopting a kind of method.If the applied in any combination of distinct methods can be learnt from other's strong points to offset one's weaknesses, avoided because the inevitable deviation that method principle itself is brought, therefore, the whole bag of tricks applied in any combination has been become a kind of trend that the compost microbe structure of community changes of analyzing, but PLFA method and foranalysis of nucleic acids method all require to extract the material of representing biological information from compost sample, and extraction step is all more loaded down with trivial details, and the applied in any combination of the method that is combined into of extracting method provides may.
Summary of the invention
The objective of the invention is to overcome above-mentioned the deficiencies in the prior art, a kind of method that can extract PLFA and DNA in the compost sample simultaneously be provided, this method accurately, efficient, and required sample size is less.
For achieving the above object, the present invention is by the following technical solutions:
The extraction of a.PLFA
A-1. take by weighing compost sample in test tube, add methylene chloride, methyl alcohol, phosphate buffered solution respectively, the volume ratio of Jia Ruing is 1: 2: 1 successively;
A-2. low-temperature dark standing over night after the ultrasonic homogenize;
A-3. with the centrifugal layering of above-mentioned test tube, liquid phase is transferred in the new glass tube, adds methylene chloride and pure water, makes methylene chloride: methyl alcohol: the volume ratio of phosphate buffer is 1: 1: 0.9, be used for continue extracting PLFA, solid phase is preserved down to extract DNA in-20C;
A-4. after above-mentioned liquid phase being left standstill 24h, shift water and be used for deposit D NA in new centrifuge tube a, organic phase is filtered, remove possibility residual particles suspension, filtrate is used N
2Add n-hexane dissolution after the drying;
A-5. the lipid of previous step n-hexane dissolution is crossed silicagel column, use normal hexane, chloroform, acetone, methanol wash pillar successively, collect methanol wash liquid, N
2Dry;
A-6. dissolve dried lipid material with methyl alcohol and toluene mixed solution, methyl alcohol in the described mixed solution: the volume ratio of toluene is 1: 1, add KOH solution water-bath heating and make its esterification, be cooled to room temperature, add in the acetic acid and excessive KOH, add the chloroform extraction after shaking up, the phase of anhydrating, add deionized water wash again, make the inorganics of organic phase part be dissolved in water as much as possible, the bottom organic phase is the esterification PLFA that is dissolved in the chloroform;
B.DNA extracts
B-1. get the solid matter of being preserved among the step a-3, add the humic acid in the phosphate buffer washing removal sample;
B-2. sample washing back interpolation DNA extraction liquid and proteinase (Proteinase) K digest, and wherein, sample size: DNA extraction liquid: Proteinase K is 1g: 1ml: 0.005ml;
B-3. previous step gained material is transferred among the centrifuge tube b, and adds SDS, also add SDS among the centrifuge tube a in step a-4 in addition, the protein in two centrifuge tube water-bath simultaneously vitellophags will contain the centrifugal layering of centrifuge tube b of solid phase then;
B-4. collect the supernatant among the centrifuge tube b, transfer among the centrifuge tube a, the slight mixing;
B-5. add isopyknic chloroform and isoamylol mixed solution to centrifuge tube a, chloroform in the described mixed solution: the volume ratio of isoamylol is 24: 1, puts upside down slightly that to mix the back centrifugal, draws water and is transferred in the new centrifuge tube;
B-6. the previous step aqueous phase adds 0.6 times of volume isopropanol precipitating 1h, and is centrifugal then;
B-7. precipitate with air-dry behind the ice precooled ethanol repeated washing;
B-8. add TE damping fluid dissolving DNA and place-20 ℃ to preserve down;
Filtrate is used N among the described step a-4
2Can add a methylene chloride when adding n-hexane dissolution after the drying.
Advantage of the present invention:
The present invention provides a kind of thinking for PLFA and the DNA that extracts simultaneously in the compost sample, to the applied in any combination generation impetus of compost microbe colony assay method.The information that can provide more comprehensive objectively microflora to form and change from different angles environmental microorganism structure of community and community dynamic is is provided simultaneously.Nucleic acid and lipid analysis can provide complementary information and every kind of method can both confirm the result of other method.Two kinds of methods all depend on the direct extraction of compost sample, in order to obtain more information about microbial ecological aspect in the compost sample, have very big advantage in conjunction with these two kinds of methods.
The mixing extracting method of mentioning among the present invention makes the researcher put the quantity of information maximization that obtains the compost sample from the same space, simplified the program of compost sample lipid and DNA extraction, shortened the time that sample message extracts, guarantee the homogeneity in sample message source, reduced the error that the compost sampling causes detection method preferably.Especially when the fewer or sampling ratio of sample size than under the situation of difficult, seem particularly useful.
Description of drawings
Fig. 1 is PLFA and a DNA extraction process flow diagram in the compost sample.
Embodiment
1.PLFA extraction (as shown in Figure 1)
(1-1) take by weighing the 5.00g compost sample in the glass tube of band teflon lid, add 5mL methylene chloride, 10mL methyl alcohol respectively, (pH 7.4,0.05mol/L) for the 5mL phosphate buffered solution;
(1-2) behind the ultrasonic homogenize 10min, 4 ℃ of following lucifuge standing over night;
(1-3) centrifugal 20min, rotating speed 8000rmp.Liquid phase is transferred in the new glass tube, adds 5mL methylene chloride and 4mL pure water again, is used for continuing to extract PLFA; Solid phase is preserved down to extract DNA in-20 ℃;
(1-4) leave standstill 24h after, shift water and in new centrifuge tube a, be used for deposit D NA; Organic phase is filtered with the 4# fibrous filter membrane; Filtrate adds 2mL n-hexane dissolution (solute effect is bad can to add a methylene chloride) after drying up with Nitrogen evaporator (DC-12 type, scientific instrument company limited is composed in last Hai'an);
(1-5) lipid of n-hexane dissolution is crossed silicagel column (500mg, 100-200 order, water generation science and technology (Shanghai) Co., Ltd.), uses 5mL normal hexane, 5mL chloroform, 5mL acetone, 5mL methanol wash pillar successively; Collect methanol wash liquid, dry up with Nitrogen evaporator (DC-12 type, scientific instrument company limited is composed in last Hai'an);
(1-6) (described methyl alcohol: the volume ratio of toluene is 1: 1, contains internal standard compound C19: 0) dissolve the lipid material that dries up, add 0.5mL 2.0M KOH, 37 ℃ of water-baths heating 15min with 0.5mL methyl alcohol-toluene mixed solution; Be cooled to room temperature, add 5mL 0.2M acetic acid, add the 2mL chloroform after shaking up, the phase of anhydrating, add deionized water wash after, the bottom organic phase is the esterification PLFA that is dissolved in the chloroform.
2.DNA extract (as shown in Figure 1)
(2-1) get the solid matter of being preserved among the step 1-3, add the phosphate buffer (pH8.0) of 10mL 120mmol/L, 150r/min at the uniform velocity shakes 15min, with the centrifugal 10min of 4700r/min, removes supernatant then;
(2) the DNA extraction liquid of above-mentioned washed precipitation solid matter and 4ml (0.1mol/L Tris-HCl, 0.1mol/LEDTA, 0.1mol/L phosphate (Na
3PO
4), 1.5mol/L NaCl, 1%CTAB (mass percent), pH are 8.0) mix, add 20 μ l Proteinase Ks (10mg/ml, TIANGEN Biotech (Beijing) Co., Ltd.); 225rpm37 ℃ of shaking table shakes 30min;
(3) previous step gained material is transferred among the centrifuge tube b, and add 100 μ l 20%SDS (mass percent), in centrifuge tube a, add 400 μ l 20%SDS (mass percent) in addition, two centrifuge tubes are simultaneously at 65 ℃ of following water-bath 2h, put upside down gently every 10-15min, contain centrifuge tube b centrifugal 10min under 4700r/min of solid phase then;
(4) supernatant among the collection centrifuge tube b is transferred among the centrifuge tube a, the slight mixing;
(5) add isopyknic chloroform and isoamylol mixed solution (described chloroform: the volume ratio of isoamylol is 24: 1) and to centrifuge tube a, slightly put upside down mixing, the centrifugal 5min of room temperature 4700r/min draws water and is transferred in the new centrifuge tube;
(6) water adds 0.6 times of volume isopropanol precipitating 1h, the centrifugal 15min of 15000r/min then;
(7) precipitate with air-dry behind 70% (mass percent) the ice precooled ethanol repeated washing;
(8) add 600 μ L TE damping fluid dissolving DNAs and place-20 ℃ to store down.
Claims (2)
1. one kind is extracted PLFA in the compost sample and the method for DNA simultaneously, it is characterized in that may further comprise the steps:
The extraction of a.PLFA
A-1. take by weighing compost sample in test tube, add methylene chloride, methyl alcohol, phosphate buffered solution respectively, the volume ratio of Jia Ruing is 1: 2: 1 successively;
A-2. low-temperature dark standing over night after the ultrasonic homogenize;
A-3. with the centrifugal layering of above-mentioned test tube, liquid phase is transferred in the new glass tube, adds methylene chloride and pure water, makes methylene chloride: methyl alcohol: the volume ratio of phosphate buffer is 1: 1: 0.9, be used for continuing to extract PLFA, solid phase is preserved down to extract DNA in-20 ℃;
A-4. after the previous step liquid phase being left standstill 24h, shift water and be used for deposit D NA in new centrifuge tube a, organic phase is filtered, remove possibility residual particles suspension, filtrate is used N
2Add n-hexane dissolution after the drying;
A-5. the lipid of previous step n-hexane dissolution is crossed silicagel column, use normal hexane, chloroform, acetone, methanol wash pillar successively, collect methanol wash liquid, N
2Dry;
A-6. dissolve dried lipid material with methyl alcohol and toluene mixed solution, methyl alcohol in the described mixed solution: the volume ratio of toluene is 1: 1, add KOH solution water-bath heating and make its esterification, be cooled to room temperature, add in the acetic acid and excessive KOH, add the chloroform extraction after shaking up, the phase of anhydrating, add deionized water wash again, make the inorganics of organic phase part be dissolved in water as much as possible, the bottom organic phase is the esterification PLFA that is dissolved in the chloroform;
B.DNA extracts
B-1. get the solid matter of being preserved among the step a-3, add the humic acid in the phosphate buffer washing removal sample;
B-2. sample washing back interpolation DNA extraction liquid and proteinase (Proteinase) K digest, and wherein, sample size: DNA extraction liquid: Proteinase K is 1g: 1ml: 0.005ml;
B-3. previous step gained material is transferred among the centrifuge tube b, and adds SDS, also add SDS among the centrifuge tube a in step a-4 in addition, the protein in two centrifuge tube water-bath simultaneously vitellophags will contain the centrifugal layering of centrifuge tube b of solid phase then;
B-4. collect the supernatant among the centrifuge tube b, transfer among the centrifuge tube a, the slight mixing;
B-5. add isopyknic chloroform and isoamylol mixed solution to centrifuge tube a, chloroform in the described mixed solution: the volume ratio of isoamylol is 24: 1, puts upside down slightly that to mix the back centrifugal, draws water and is transferred in the new centrifuge tube;
B-6. the previous step aqueous phase adds 0.6 times of volume isopropanol precipitating 1h, and is centrifugal then;
B-7. precipitate with air-dry behind the ice precooled ethanol repeated washing;
B-8. add TE damping fluid dissolving DNA and place-20 ℃ to preserve down.
2. a kind of PLFA in the compost sample and method of DNA extracted simultaneously according to claim 1 is characterized in that filtrate is used N among the described step a-4
2Add a methylene chloride when adding n-hexane dissolution after the drying simultaneously.
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| CN102798566A (en) * | 2011-05-23 | 2012-11-28 | 中国科学院研究生院 | Method for efficiently extracting phospholipid fatty acid in soil sample |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1733791A (en) * | 2005-09-06 | 2006-02-15 | 湖南大学 | Extraction and purification method of general DNA of compost microbe |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1733791A (en) * | 2005-09-06 | 2006-02-15 | 湖南大学 | Extraction and purification method of general DNA of compost microbe |
Non-Patent Citations (4)
| Title |
|---|
| 喻曼等.PLFA法研究稻草固态发酵中的微生物群落结构变化.环境科学28 11.2007,28(11),2603-2608. |
| 喻曼等.PLFA法研究稻草固态发酵中的微生物群落结构变化.环境科学28 11.2007,28(11),2603-2608. * |
| 杨朝晖等.用于分子生态学研究的堆肥DNA提取方法.环境科学27 8.2006,27(8),1613-1617. |
| 杨朝晖等.用于分子生态学研究的堆肥DNA提取方法.环境科学27 8.2006,27(8),1613-1617. * |
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