CN102732504B - Method for extracting microorganism macrogenome from oil/gas pool environment - Google Patents
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Abstract
The invention provides a simple method for directly extracting microorganism macrogenome from an oil/gas pool environment sample, which comprises the following steps: an organic phase/water phase is extracted to obtain an microbe component in an oil sample/oil-water sample, vacuum filtering is carried out on the water phase enrichment microbes, cells are cracked by lysozyme, protease, sodium dodecyl sulfate (SDS) and the like, passes through the cetyl trimethyl ammonium bromide (CTAB) under high salt condition to remove organic macro-molecule components like protein and polysaccharide, extraction is carried out through nucleic acid and deposition is carried out, thereby the unbiased oil/gas pool microorganism macrogenome component can be obtained. The method of the invention has the advantages that the process is fast and simple, the cost is low, the whole extraction process can be completed in 10 hours; the obtained genome enables high purity, the ratio of OD260nm to OD280nm is between 1.7 and 2.0, the electrophoresis result detects that the integral genome possesses no degradation condition. The method of the invention aims at the oil/gas pool complex environments, and is suitable for extraction of other microbe genome in the soil and water body polluted by petroleum.
Description
Technical field
The present invention relates to Molecular Ecology Techniques field, specifically by directly extracting the grand genomic simple and easy method of microorganism in the complex environment of oil/gas Tibetan.
Background technology
One of emphasis of microbe oil production/gas technology and oil/gas Tibetan microbiological prospecting technology is to understand the Global Information of microorganism in the complex environment of oil/gas Tibetan.Be limited to the limitation of current technique means, only have the microorganism of about 1% left and right to can be artificial culture and understanding, last century end for this reason, researcher has been developed technique of metagenome, and metagenomics is by extracting all microbe genome DNAs, the structure genomic library in a certain environment and library being screened to a kind of method of finding and finding new functional gene and active metabolite.
Handelsman in 1998 etc. propose grand genomic concept first, and be defined as: all genomes of a kind of microorganism in environmental sample are as research object, take functional gene screening and sequencing analysis as research means, for microbial diversity, population structure, evolutionary relationship, functionally active, mutual cooperation relation, and and environment between pass be the new microbe research method of research direction, and used for the first time this noun of Meta-genomics.Its elementary tactics flow process is: the enrichment of sample and gene (group); Extract the genomic dna in specific environment; Build macro genome DNA library; Screening goal gene; And goal gene active result is expressed.Metagenomics has been avoided the process of microorganism separation and Culture, has greatly expanded the space that utilizes of Microbial resources, is new developing direction of modern genetic engineering and study hotspot.
The primary prerequisite of metagenomics research is to obtain nature truly without the microbial genome information of preference, but the complicacy of hiding environment due to oil/gas, its operation difficulty relatively.
It is a kind of extreme ecotope of uniqueness that oil/gas is hidden, and it is containing abundant microbe groups; These microorganisms have multiple metabolic type, breeding growth by itself in oil/gas is hidden and the effect of meta-bolites, can be at MEOR(Microbial Enhanced Oil Recovery, microbial enhanced oil recovery) and MPOG(Microbiological Prospecting of Oil and Gas, Microbial prospecting of oil and gas) in play a significant role.
MEOR refers to that microbial inoculum and nutritive medium that surface separation is cultivated inject oil reservoir, or inject merely nutritive medium and activate the microorganism in oil reservoir, make its growth and breeding in oil reservoir, generation is conducive to improve the meta-bolites of recovery ratio, to improve the oil production method of oil recovery rate, be commonly called as microbe oil production, its mechanism is:
1. change former oil composition, reduce its viscosity
Microorganism in growth metabolism process, the carbon source using the normal paraffin in crude oil as self growth, thus change the carbochain composition of crude oil; On the other hand, the biological enzyme discharging when microorganism growth, can degrading crude oil, makes the fracture of crude oil carbochain, and high carbon chain crude oil becomes low carbon chain crude oil, and heavy constituent reduces, and light constituent increases, zero pour and reduced viscosity, thus play the effect of viscosity reduction, wax control.
2. the displacement of reservoir oil environment that changes crude oil, makes it be easier to extraction ground
(1) produce tensio-active agent
The metabolism meeting of microorganism produces tensio-active agent, and these bio-surfactants can reduce oil water interfacial tension, reduce water displacing oil capillary force.In addition, the wettability that bio-surfactant can change oil/gas hides rock, makes it become from oleophylic hydrophilic, and the oil film that is adsorbed on like this rock surface splits away off, and oil reservoir remaining oil saturation reduces, thereby improves oil recovery factor.
(2) produce biogas
Most of microbe all can produce gas in metabolic process, and as carbonic acid gas, hydrogen, nitrogen etc., these gas dissolvings, in water, can reduce viscosity of crude, improves crude oil flow ability.In addition, the existence of these gases also can increase sand pressure, indirectly reduces remaining oil saturation.Meanwhile, the Jamin effect of bubble also can increase flow resistance, improves injected water sweep efficiency.
(3) produce acid and organic solvent
The metabolism of microorganism produces relatively low-molecular-weight organic acid, also has part mineral acid, and their energy dissolved carbon hydrochlorates, increase rock porosity on the one hand, have improved rate of permeation; On the other hand, release of carbon dioxide, has improved sand pressure, thereby reduces viscosity of crude, has improved the flow capacity of crude oil.The metabolic process of microorganism also can produce the organic solvent such as alcohol, ester, can change rock surface character and crude oil physical properties, and the crude oil that is adsorbed on rock surface is released, and is easy to extraction ground.
(4) form biological polymer
In the metabolic process of microorganism, can produce biological polymer.These biological polymers can, at optionally macropore plugging of oil reservoir high permeability zones, be adjusted the intake profile of Deplug, and it is long-pending that increase injected water is swept pasta.Biological polymer can also increase the viscosity of water, reduces water-oil ratio, improves the oily efficiency of sweeping of injected water, thereby improves recovery ratio.
3. the direct effect of microorganism
Microorganism grows at rock surface, can occupy pore space and drive away crude oil.In addition, microorganism can be adsorbed onto rock surface, under oil film, grows, and finally oil film is pushed open, and crude oil is discharged.
Microbial Prospecting of Oil and Gas (Microbial Prospection for Oil and Gas, MPOG) be a kind of method of carrying out oil-gas exploration according to earth's surface microbial profile situation, mainly study the correlationship of microbiological anomaly and underground deep hydrocarbon-bearing pool near surface pedosphere.The Application Areas of MPOG comprises: (1) unworked country oil and gas prediction, and it is according to being: the high value of specified microorganisms exceptions area is the reliability index of Hydrocarbon leakage in stratum normally; (2) mature exploration area evaluating reservoir: Microbiological Oil And Gas Exploration Techniques is predicted for hydrocarbon potential district early stage of its application.But over nearly 10 years, this technology is obtaining breakthrough aspect the explanation of Measurement results.Geomicrobiology technology company of the U.S. takes the lead in the method among the evaluation of underground oil and gas reservoir distribution situation, set up " microorganism reservoir characteristics evaluation technique (MRC-Microbial Reservoir Characterization) ", the range of application of this technology has been extended to development field by Exploration Domain.
Due to the restriction of traditional method, the indicator microoraganism of MPOG research at present mainly concentrates on a few bacterial strains such as methanogen, and in geology soil, probably there are a large amount of microbe species that can be used for indication, therefore, use quantitative PCR, the advantage that high-flux sequence detects, can hide situation in conjunction with actual oil/gas, carry out quantitative examinations extensive, many bacterial classifications, utilize the data conclusion of many indexs to increase the success ratio of MPOG prediction., can compare the different microorganisms of different areas, different soil property, DIFFERENT DEPOSITIONAL ENVIRONMENTS meanwhile, filter out the new indication flora that is applicable to MPOG application.
There is no now both at home and abroad any about the grand genome of oil at MEOR, the correlative study in MPOG and MRC and application.Therefore, provide workable and simple and practical efficient method by this inventive method for obtaining microorganism complete genome group, can farthest obtain complete sample gene group information.
Summary of the invention
For overcoming the problems referred to above, the invention provides a kind of extract and purifying oil/gas is hidden grand genomic method in complex environment, the method can be hidden sample for most oil/gas, simple to operate, economy, environmental friendliness, high-quality and high-efficiency.
The present invention extracts oil/gas and hides that environment macro is genomic obtains comprising step:
(1) extraction of organic phase/water obtains microorganism component in oil sample/profit sample.Specifically, in crude oil, add sherwood oil/normal hexane mixture (1-3:1 v/v) of isopyknic 10-50% ethanol and half volume, fully shake up; Staticly make the layering of profit phase, extract lower floor's water with sebific duct.Fall in water a small amount of crude oil and other impurity with filter paper filtering; (2) by water that step 1 obtains through 0.22 μ m millipore filtration vacuum filtration, enrichment thalline is on filter membrane; (3) step 2 gained microorganism cells is carried out to cracking, specifically, filter membrane is shredded and puts into 10mL centrifuge tube, add 100-500 μ L N,O-Diacetylmuramidase (10mg/mL is dissolved in 10mM Tris-HCl, pH8.0), 37 ℃ of water-bath 0.5-2 h; Add SDS and the 10-50 μ L Proteinase K (20mg/mL) of 50-300 μ L10%, 37 ℃ of water-bath 1-4 h; (4) under high salt condition, remove step 3 by CTAB and obtain protein and the organic macromolecule (as polysaccharide) in component, specifically, add the 5M NaCl of 100-500 μ L to put upside down and mix, add 100-500 μ L 5%CTAB, 50-80 ℃ of water-bath 10-60min; (5) component that step 4 obtains is carried out to nucleic acid extracting and precipitation, specifically, cooling rear sucking-off supernatant, broken film washs once with 100-500 μ L aseptic deionized water, sucking-off supernatant, by the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1, put upside down and mix 0.5-5min, leave standstill 1-5min; The centrifugal 10-30min of 12000rpm, sucking-off supernatant.Add the Virahol of 0.5-1 times of volume, place 3h or placement for-20 ℃ and spend the night; The centrifugal 10-30min of 12000rpm, carefully outwells supernatant, and precipitation adds the cold washing with alcohol of 1-5mL 75% once, coolly volatilize totally to ethanol, adds the dissolving of 10-100 μ L aseptic deionized water.
Extraction object samples of the present invention can be crude oil sample, also includes, but not limited to water injection well oil mixing with water sample, oil-polluted soils, petroleum pollution water body, the sample such as water, soil of natural air reservoir surrounding enviroment simultaneously.To different samples, the mode of microorganism enrichment is slightly different, as for profit sample (oil-contg is less than 5%), mainly collect water by filtration, then the former oil sample of collecting on filter paper is carried out to vortex concussion with PBS damping fluid resuspended, again filter and collect water, then merge water and obtain microorganism component.Obtain after the microorganism component after enrichment, working method reaches unanimity.
Through 0.22 μ m millipore filtration vacuum filtration, in theory can more than 99.5% environmental microorganism of enrichment, therefore can fully obtain oil/gas and hide all information of microorganism.
Add N,O-Diacetylmuramidase, mainly for the gram positive bacterium that is difficult to broken wall under normal condition; In conjunction with anionic detergent SDS and Proteinase K processing, can under relatively gentle condition, promote bacteria cell wall to break, thereby discharge genome simultaneously.SDS can also suppress the activity of intracellular DNase simultaneously, and DNA stable environment is provided.
Under hypersaline environment, CTAB can be in conjunction with the organic macromolecule such as protein and polysaccharide in cracking component, and nucleic acid component keeps dissolved state, separates thereby obtain.
Mixture with isopyknic chloroform: primary isoamyl alcohol=24:1 carries out extracting, can further remove residual protein; Then by isopropanol precipitating, obtain complete whole grand genome.
The grand genome that this aspect method obtains, can be used for follow-up PCR reaction, and library is set up, the various molecular biology operations such as Southern.Method is simple, and without complex apparatus, total overall reaction can complete in 10 hours; Environmental friendliness, to environment and experimenter's toxicological harmless effect.
The main advantage of present method is as follows:
1 the present invention hides the extraction of grand genome composition in complex environment mainly for oil/gas, compared with conventional microbe to screen technology, the present invention has got around complicated microbial cultivation process, take into account can culturing micro-organisms and uncultured microbe simultaneously, therefore can reflect to the full extent that oil/gas hides the truth of microbial profile in complex environment;
2 are started with by method provided by the present invention, based on the in-depth analysis to grand genome component, can greatly shorten the construction cycle of microbial strains and meta-bolites, improve the understanding to indigenous microorganism, in microorganism and functional molecular sieve screening process, accomplish to shoot the arrow at the target;
3 with regard to the present invention self, compared with conventional genome extraction process, exist with low cost, repeatable high, the sample that obtains without preference, for object microorganism extensive, wait many advantages;
4 oil/gas are hidden the crude oil component, wax, bituminous matter, the arene that in environment, exist, and the material such as soil ulmin, vegetable jelly polyose of introducing because of construction, other environmental factorss etc., can cause considerable influence to conventional molecular biology operation, the present invention is directed to above-mentioned knotty problem, in technique, carry out improving one by one, therefore improved to the full extent the purification efficiency of grand genome composition.
Accompanying drawing explanation
The grand genome of Fig. 1 extracts situation summary view; In Fig. 1, Line1 is embodiment 1 illustration, and Line2 is embodiment 2 illustrations, and Line3-4 is embodiment 3 illustrations.Line1: PetroChina Company Limited.'s block crude oil sample microbial genome, Line2: PetroChina Company Limited.'s block profit sample microbial genome, Line3: PetroChina Company Limited.'s block earth's surface greasy filth sample microbial genome, Line4: PetroChina Company Limited.'s block polluted-water sample microbial genome.
16S rDNA sequence PCR fragment after Fig. 2 purifying.Line1: come from PetroChina Company Limited.'s block crude oil sample, Line2: come from PetroChina Company Limited.'s block profit sample, Line3: come from PetroChina Company Limited.'s block earth's surface greasy filth sample, Line4: come from PetroChina Company Limited.'s block polluted-water sample.
Embodiment
By embodiment, application method of the present invention is described below.Scope shown in volumetric parameter is not limited to describe, as need strengthen extraction scale, can amplify in proportion.Should be understood that these examples are only not used in the scope of application of restriction the inventive method for the present invention is described.
The embodiment 1 grand genomic simple and easy method of microorganism that abstracts from crude oil
1. get about 100mL crude oil and be placed in glass beaker, 70 ℃ of water-baths 30 minutes.
2. add equal-volume 30%(V/V) sherwood oil of ethanol and crude oil half volume: normal hexane (V/V=1:1) mixture, fully shakes up.
3. staticly make the layering of profit phase, extract lower floor's water with sebific duct.Fall in water a small amount of crude oil and other impurity with filter paper filtering.
4. 0.22 μ m millipore filtration vacuum filtration for liquid after filtering, enrichment thalline is on filter membrane.
5. filter membrane is shredded and puts into 10mL centrifuge tube, add 360 μ L N,O-Diacetylmuramidases (10mg/mL is dissolved in 10mM Tris-HCl, pH8.0), 37 ℃ of water-bath 1h.
6. add SDS and the 25 μ L Proteinase Ks (20mg/mL) of 115 μ L 10%, 37 ℃ of water-bath 2h.
7. add the 5M NaCl of 400 μ L to put upside down and mix, add 280 μ L 5%CTAB, 65 ℃ of water-bath 20min.
8. cooling rear sucking-off supernatant, broken film washs once with 200 μ L aseptic deionized waters, and sucking-off supernatant, by the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1, puts upside down and mixes 1min, leaves standstill 2min.
9.12000rpm centrifugal 30min, sucking-off supernatant.Add the Virahol of 0.6 times of volume, place 3h or placement for-20 ℃ and spend the night.
10. the centrifugal 20min of 12000rpm, carefully outwells supernatant, and precipitation adds cold 75% washing with alcohol of 1mL once, coolly volatilize totally to ethanol, adds the 30 μ L aseptic deionized waters dissolving grand genome compositions that obtain.
The 11. genome compositions that obtain are shown in Fig. 1 Line1, and deposition condition is lower 30 minutes of 75V.
1. get about 100mL oil-water mixture and be placed in glass beaker, 70 ℃ of water-bath 30min.
2. use filter paper filtering oil-water mixture, collect water.
3. the crude oil of staying filter paper top mixes with the long-pending aseptic PBS of diploid, and fully stirring is collected water with filter paper filtering, in triplicate.
4. merge and filter rear water, with 0.22 μ m millipore filtration vacuum filtration, enrichment thalline is on filter membrane.
5. filter membrane is shredded and puts into 10mL centrifuge tube, add 360 μ L N,O-Diacetylmuramidases (10mg/mL is dissolved in 10mM Tris-HCl, pH8.0), 37 ℃ of water-bath 1h.
6. add SDS and the 25 μ L Proteinase Ks (20mg/mL) of 115 μ L10%, 37 ℃ of water-bath 2h.
7. add the 5M NaCl of 400 μ L to put upside down and mix, add 280 μ L 5%CTAB, 65 ℃ of water-bath 20min.
8. cooling rear sucking-off supernatant, broken film washs once with 200 μ L aseptic deionized waters, and sucking-off supernatant, by the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1, puts upside down and mixes 1min, leaves standstill 2min.
9.12000rpm centrifugal 30min, sucking-off supernatant.Add the Virahol of 0.6 times of volume, place 3h or placement for-20 ℃ and spend the night.
10. the centrifugal 20min of 12000rpm, carefully outwells supernatant, and precipitation adds cold 75% washing with alcohol of 1mL once, coolly volatilize totally to ethanol, adds the 30 μ L aseptic deionized waters dissolving grand genome compositions that obtain.
The 11. genome compositions that obtain are shown in Fig. 1 Line2, and deposition condition is lower 30 minutes of 75V.
1. get about 10g oil-containing soil and be placed in glass beaker, add 100mL PBS, repeat to stir 70 ℃ of water-bath 30min.
2. use filter paper filtering mixture, collect water.
3. the crude oil and the aseptic PBS of soil 100mL that stay filter paper top dissolve, and fully stir, and collect water with filter paper filtering, in triplicate.
4. merge and filter rear water, with 0.22 μ m millipore filtration vacuum filtration, enrichment thalline is on filter membrane.
5. filter membrane is shredded and puts into 10mL centrifuge tube, add 360 μ L N,O-Diacetylmuramidases (10mg/mL is dissolved in 10mM Tris-HCl, pH8.0), 37 ℃ of water-bath 1h.
6. add SDS and the 25 μ L Proteinase Ks (20mg/mL) of 115 μ L 10%, 37 ℃ of water-bath 2h.
7. add the 5M NaCl of 400 μ L to put upside down and mix, add 280 μ L 5%CTAB, 65 ℃ of water-bath 20min.
8. cooling rear sucking-off supernatant, broken film washs once with 200 μ L aseptic deionized waters, and sucking-off supernatant, by the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1, puts upside down and mixes 1min, leaves standstill 2min.
9.12000rpm centrifugal 30min, sucking-off supernatant.Add the Virahol of 0.6 times of volume, place 3h or placement for-20 ℃ and spend the night.
10. the centrifugal 20min of 12000rpm, carefully outwells supernatant, and precipitation adds cold 75% washing with alcohol of 1mL once, coolly volatilize totally to ethanol, adds the 30 μ L aseptic deionized waters dissolving grand genome compositions that obtain.
The 11. genome compositions that obtain are shown in Fig. 1 Line3, and 4, respectively by obtaining in mud sample and water sample; Deposition condition is lower 30 minutes of 75V.
1. using the grand genome component by method was obtained in embodiment 1-3 as template, template add-on can suitably be diluted according to actual abundance, reference value is in single PCR system, to add the grand genome component of 10-100ng, as too lowly in the sequence copy of required amplification can suitably improve.
2. use bacterial 16 S rDNA sequence universal primer: 8F(5 '-TTTGATCCTGGCTCAG-3 ') and 1492R(5 '-GGTTACCTTGTTACGACTT-3 ').
3. PCR reaction system is 25 μ L, and reaction conditions is 94 ℃ of sex change 4min; Next carry out 30 circulating reactions: 94 ℃ of sex change 45 s, 50 ℃ of annealing 45 s, 72 ℃ are extended 90 s; Then 72 ℃ are extended 10 min again, finally in 4 ℃ of preservations.
4. obtain 16S rDNA sequence PCR product and clone and be connected in commercialization or self-control T carrier by TA after purified, will connect product and transform DH5 α competent cell, coat corresponding resistant panel structure library, and screening positive clone.
5. select positive colony measure insert 16S rDNA partial sequence, and with GenBank in known array carry out homology comparison, determine the kind of corresponding microorganism.
6. obtaining grand genome component can be equally other diversity analysis technique means microflora's Back ground Information is provided, downstream correlation technique comprises, but be not limited to, rDNA amplified fragments restriction endonuclease analysis (ARDRA), deformation gradient gel electrophoresis (DGGE), temperature gradient gel elec-trophoresis (TGGE) (TGGE) chain conformation polymorphism analysis (SS-CP), Terminal restriction fragment length polymorphism are analyzed (T-PFLP), random amplification fragment length polymorphism is analyzed (RAPD), amplified fragment length polymorphism (AFLP), etc.
The method of the invention is quick and easy, with low cost, and whole leaching process can complete within 10 hours; Institute's genome purity that obtains is high, OD
260nm: OD
280nmratio is between 1.7-2.0, and electrophoresis result detects genome entirety without degraded situation.The inventive method is not only hidden complex environment for oil/gas, is also applicable to the extraction of microbial genome in other petroleum-polluted soil and water body.
Claims (5)
1. hide environment and extract the grand genomic method of microorganism from oil/gas, it is characterized in that: comprise step:
(1) extraction of organic phase/water obtains microorganism component in liquid phase sample: in liquid phase sample, add and the isopyknic 10-50%(V/V of sample) ethanol and sherwood oil/normal hexane mixture of sample half volume, described sherwood oil/normal hexane mixture PetroChina Company Limited.'s ether/normal hexane (V/V)=1-3:1, fully shakes up; Staticly make the layering of profit phase, extract lower floor's water with sebific duct; Fall water Crude Oil and other impurity with filter paper filtering;
(2) by water that step 1) obtains through 0.22 μ m millipore filtration vacuum filtration, enrichment thalline is on filter membrane;
(3) to step 2) gained microorganism cells carries out cracking: filter membrane shredded and puts into 10mL centrifuge tube, add 100-500 μ L lysozyme soln, N,O-Diacetylmuramidase is dissolved in 10mM Tris-HCl, the lysozyme soln that acquisition ultimate density is 5-20mg/mL, pH8.0,37 ℃ of water-bath 0.5-2h; Add 50-300 μ L 10%(W/V) SDS and 10-50 μ L concentration 20mg/mL Proteinase K, 37 ℃ of water-bath 1-4h;
(4) under high salt condition, remove step 3) by CTAB and obtain protein and the polysaccharide in component: put upside down and mix to the 5M NaCl that adds 100-500 μ L in the whole solution of step (3), add 100-500 μ L 5% CTAB(cetyl trimethylammonium bromide), 50-80 ℃ of water-bath 10-60min;
(5) component that step 4) obtains is carried out to nucleic acid extracting and precipitation: be cooled to sucking-off supernatant after room temperature, broken film washs once with 100-500 μ L aseptic deionized water, sucking-off supernatant, by the supernatant liquid mixing of twice sucking-off, add isopyknic mixture, mixture is chloroform: primary isoamyl alcohol=24:1, puts upside down and mixes 0.5-5min, leaves standstill 1-5min; The centrifugal 10-3min of 12000rpm, sucking-off supernatant; Add the Virahol of 0.5-1 times of volume, place 3h or placement for-20 ℃ and spend the night; The centrifugal 10-30min of 12000rpm, outwells supernatant, and precipitation adds cold 75% washing with alcohol of 1-5mL once, dries in the air to ethanol and volatilize totally, adds the 10-100 μ L aseptic deionized water dissolving grand genome composition that obtains.
2. the method for claim 1, is characterized in that: the described liquid phase sample of step (1) comprises crude oil sample or oil-water mixture.
3. the method for claim 1, is characterized in that: the microorganism comprising in the described liquid phase sample of step (1) comprises, one or two or more kinds in Gram-negative bacteria, gram-positive microorganism, archeobacteria, fungi.
4. the method for claim 1, is characterized in that: in step (3) lysis, N,O-Diacetylmuramidase, SDS, Proteinase K are combined use, and final concentration when wherein N,O-Diacetylmuramidase is processed is 5-20mg/mL; It is 1-3% that SDS uses final concentration, and it is 0.2-2 mg/mL that Proteinase K uses final concentration.
5. the method for claim 1, is characterized in that: step (4) NaCl used and CTAB final concentration are respectively 1-4 M and 0.5-2.0%.
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| CN103667256B (en) * | 2013-11-19 | 2016-01-20 | 克拉玛依市金山石油化工有限公司 | Freezing method extracts the method for environmental microorganism STb gene in oilfield sewage |
| CN104087573B (en) * | 2014-06-18 | 2016-06-08 | 福建农林大学 | The extracting method of microbe genome DNA in a kind of water planting liquid |
| CN105986025A (en) * | 2015-03-03 | 2016-10-05 | 核工业北京地质研究院 | Research method for relation between microorganisms and uranium mineralization in sandstone type uranium ore deposit |
| CN105821033B (en) * | 2016-05-11 | 2019-06-11 | 清华大学 | A kind of extracting method of the macro genome of cellulose degradation flora |
| WO2017209990A1 (en) | 2016-05-31 | 2017-12-07 | Exxonmobil Upstream Research Company | METHODS FOR lSOLATING NUCLEIC ACIDS FROM SAMPLES |
| US10570735B2 (en) | 2016-07-01 | 2020-02-25 | Exxonmobil Upstream Research Comapny | Methods to determine conditions of a hydrocarbon reservoir |
| CN106754895B (en) * | 2017-03-01 | 2019-08-13 | 中国石油大学(北京) | Extract the method and kit of crude oil total dna |
| CN110511262B (en) * | 2019-08-01 | 2022-11-15 | 中海石油环保服务（天津）有限公司 | Extracting solution for extracting macro-proteome in oil sludge sand and extracting method |
| CN110438119A (en) * | 2019-08-23 | 2019-11-12 | 北京润世能源技术有限公司 | A kind of extracting method of crude oil microbe genome DNA |
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