CN101864488B - PCR-DGG primer for extracting sample microorganism total DNA in microbial enhanced oil recovery - Google Patents
PCR-DGG primer for extracting sample microorganism total DNA in microbial enhanced oil recovery Download PDFInfo
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Abstract
The invention relates to a method for extracting the total DNA of a microorganism sample during the microbial oil recovery process, which comprises the steps of selecting a proper PCR-DGGE primer for extracting the total DNA of a microorganism sample by adopting the method for monitoring an advantageous flora in microbial enhanced oil recovery, wherein the sequence of the PCR-DGGE primer is DQF338: 5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAG-3' DQR534:5'-ATTACCGCGGCTGCTGG-3'. By adopting the above primer, the actual amount of microorganisms in an oil deposit can be accurately reflected. Meanwhile, the distribution, migration and change of the oil recovery microorganisms in the oil deposit can be accurately and quickly analyzed. Thus, the method provides an important theatrical instruction for improving oil recovery ratio.
Description
Technical field:
The present invention relates to a kind of PCR-DGGE primer that in the microbe oil production process, extracts the sample microorganism total DNA in the oil extraction in oil field field.
Background technology:
Along with the development of tertiary oil recovery technology, Daqing oil field enters the high water-cut development later stage, and for low permeability oil field, Microbial Enhanced Oil Recovery has preferably application prospect.For the stable high yield of keeping the oil field, and Sustainable development has great importance.
The parsing of microbial community in oil reservoir and cognition are the bases of development and application Microbial Enhanced Oil Recovery.Adopt traditionally the method for pure culture to study and be familiar with microbial community in oil reservoir, because the method that tradition is cultivated exists: (1) does not take into full account and the simulated environment condition, causes only having the microorganism (0.01%～1%) of only a few to survive under culture conditions and to be cultivated; (2) culture conditions has different selectivity and concentration effect to microorganism, thereby detected microorganism the relative situation on kind, quantity and the function be by select and enrichment after the result, the truth in can not entirely accurate ground reflection oil reservoir; (3) can't study exactly and understand the rule of microbial ecosystem in the oil reservoir; (4) can't instruct people scientifically to regulate and control microbial community in oil reservoir and reach problems such as improving the recovery ratio purpose.So utilize traditional cultural method that there are larger deviation in cognition and the practical situation of microbial community in oil reservoir, thereby cause and to regulate and control accurately and efficiently microbial community in oil reservoir, also can't improve efficiently oil recovery factor.
At present, the microorganism that is separated to by pure culture technigne only accounts for natural about 0.1%～15%, development along with molecular biology and phylogenetic methods, especially based on pcr amplification and the probe hybridization technology of 16SrRNA gene, can make our the more accurate microbial population diversity of being familiar with all sidedly in the particular ecosystem, also fewer to the research report of the oil pool microorganisms ecosystem by molecular biological method.Wherein Orphan etc. is by the composition of high temperature oil field biological community structure of sample total DNA having been set up 16S rDNA Clone library analysis; Watanabel etc. by fluorescence in situ hybridization (FISH), make up the library, microflora in the local water of crude oil storage hole be studied, and by competitive PCR (cPCR) dominant microflora is wherein carried out quantitative analysis.All results of study show that there is very high microbial diversity in the crude oil reservoir, and these results make people further understand crude oil reservoir microbial ecosystem.
Summary of the invention:
In order to overcome the deficiency that exists in the background technology, the invention provides a kind of PCR-DGGE primer that in the microbe oil production process, extracts the sample microorganism total DNA, utilize this primer to carry out the dominant microflora monitoring and can accurately reflect the true quantity of oil pool microorganisms, can be accurately and resolve rapidly oil extraction microbial and in oil reservoir, distribute, move and change, provide important theoretical direction for improving oil recovery.
Technical scheme of the present invention is: method of the present invention comprises the following steps:
(1), at first getting oil well produced liquid is sample, and the total DNA of microorganism in the sample is extracted;
(2), the suitable PCR-DGGE primer of screening, by to the known primer that is used for PCR-DGGE, screen;
(3), the optimization of the pcr amplification system of screening primer;
(4), denaturing gradient gel electrophoresis (DGGE) is optimized;
(5), the glue of advantage band reclaims order-checking;
(6), the comparison of the network of advantage band and analysis.
The extraction of total DNA in the such scheme: 1. the 1mL oil well is adopted liquid vibration 5min, then 3000r/min is centrifugal, stays supernatant liquor; 2. 10 * the PBS that adds 2 times of volumes washs, vibration 5min, and then the centrifugal 5min of 12000r/min removes supernatant liquor; 3. add 10 * PBS damping fluid, 100 μ L, then ultrasonic 40s; 4. use Hua Shun " DNA in a small amount bacterium extracts test kit ", carry out follow-up DNA extraction, then be kept in-20 ℃ of refrigerators;
Screening is fit to the PCR-DGGE primer of the DNA of step (1) extraction in the such scheme, and definite primer is DQF338:5 '-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGG CACGGGGGGACTCCTACGGGAGGCAGCAG-3 '; DQR534:5 '-ATTACCGCGGCTGCTGG-3 ';
The pcr amplification system of primer in the such scheme: (20 μ L) is as follows for the PCR reaction system: 10 * buffer, 2.0 μ L, 2.0mmol/L dNTP 2 μ L, each 1 μ L of 10pmol/L primer, Taq DNA enzyme 0.3 μ L; The pcr amplification condition is 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, and 52 ℃ of annealing 30s, 72 ℃ are extended 2min, 30 circulations, 72 ℃ are extended 20min.
Denaturing gradient gel electrophoresis in the such scheme (DGGE) condition: the 1. preparation of denatured gradient glue: use gradient mixing device, the polyacrylamide gel of preparation 6% (w/w) and 8% (w/w), denaturing agent concentration from 40% to 60%, 100% denaturing agent is the mixture of deionized formamide of the urea and 40% (w/w) of 7mol/L, and wherein the concentration of denaturing agent and acrylamide increases progressively downwards successively from the top of glue; 2. the application of sample of PCR sample: after the complete polymerization of denatured gradient glue, offset plate is put into the electrophoresis chamber that electrophoretic buffer is housed, add the loading hole after 10 * sample loading buffer of getting PCR sample 5 μ l and 5 μ l mixes; 3. electrophoresis and dyeing: the primer amplification segment, under the voltage of 130V, 60 ℃ of electrophoresis 7h; The primer electrophoresis carries out silver with gel and dyes after finishing; 4. glue figure scanning: the gel after will dyeing obtains glue figure after scanning with UMAX PowerLook 1000 transmission scan instrument.
The glue of advantage band reclaims order-checking in the such scheme: reclaim test kit with glue and cut the glue recovery, be connected with the pGEM-T carrier afterwards, be transformed into intestinal bacteria TOP10 competent cell; Add penbritin Amp (5 μ g/mL) and X-gal in the LB solid medium, blue hickie screening transformant; Extract plasmid, detect with carrier primer T7:5 '-TAATACGACTCACTATAGGG-3 ' and SP6:5 '-ATTTAG GTGACACTATAGAAT-3 '; Then go order-checking.
The comparison of the network of advantage band and analysis in the such scheme: the known array in the sequence of measuring and the GenBank database carries out the similarity comparative analysis, determines whether to be oil recovery bacterial classification and dominant bacteria.
The present invention has following beneficial effect: the present invention's " Molecular Ecology of Microbiology " non-pure culture technigne take microbial DNA, RNA as research object, be not subjected to the restriction of microorganism Culturability, also overcome selectivity and cultivated the shortcoming that can not accurately reflect the true quantity of oil pool microorganisms, can more accurate, directly and all sidedly reflect structure and the diversity of group.Can be accurately and resolve rapidly oil extraction microbial and in oil reservoir, distribute, move and change, this not only has important directive significance to research and the regulation and control of biological community structure in the microbial oil displacement process, but also be the various oil pool microorganisms resources of more effective acquisition, regulation and control microbial community in oil reservoir, improving oil recovery provides important theoretical direction.
Description of drawings:
Fig. 1 is the 16S rRNA V3～V6 district DGGE collection of illustrative plates of single port oil well before, during and after the microbe oil production;
Fig. 2 is the PCR-DGGE cloned sequence and the systematic evolution tree of their the most similar sequences in GenBank;
16S rRNA V3 before, during and after Fig. 3 microbe oil production～V6 district DGGE collection of illustrative plates;
The systematic evolution tree of Fig. 4 PCR-DGGE cloned sequence and their the most similar sequences in GenBank.
Embodiment:
The invention will be further described below in conjunction with embodiment:
(1), under anaerobic get oil well produced water, water sample is carried out total DNA extract: 1. with 1mL recovered water vibration 5min, then 3000r/min is centrifugal, stays supernatant liquor; 2. 10 * the PBS that adds 2 times of volumes washs, vibration 5min, and then the centrifugal 5min of 12000r/min removes supernatant liquor; 3. add 10 * PBS damping fluid, 100 μ L, then the ultrasonic 40s of ultrasonic cell disintegration instrument (S-450D); 4. be " DNA in a small amount bacterium extracts test kit " with Hua Shun, carry out follow-up DNA extraction, then be kept in-20 ℃ of refrigerators; Use the PBS damping fluid higher background value is eliminated in the washing of oil reservoir sample effectively, be conducive to the extraction of follow-up test kit; Another important prerequisite that the sufficient cracking of bacterium in the sample is DNA extraction.
(2), the suitable PCR-DGGE primer of screening, by to the known primer that is used for PCR-DGGE, definite primer is: DQF338:5 '-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCA GCAG-3 '; DQR534:5 '-ATTACCGCGGCTGCTGG-3 '.
(3), the optimization of pcr amplification system of screening primer, the pcr amplification system of primer: (20 μ L) is as follows for the PCR reaction system: 10 * buffer, 2.0 μ L, 2.0mmol/L dNTP 2 μ L, each 1 μ L of 10pmol/L primer, Taq DNA enzyme 0.3 μ L.The pcr amplification condition is 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, and 52 ℃ of annealing 30s, 72 ℃ are extended 2min, 30 circulations, 72 ℃ are extended 20min.
(4), denaturing gradient gel electrophoresis (DGGE) is optimized, denaturing gradient gel electrophoresis (DGGE) deposition condition: the 1. preparation of denatured gradient glue: use gradient mixing device, the polyacrylamide gel of preparation 6% and 8%, Molecular Weight for Polyacrylamide 300-900 ten thousand, denaturing agent concentration is (100% denaturing agent is the mixture of deionized formamide of the urea and 40% (w/w) of 7mol/L) from 40% (w/w) to 60% (w/w), and wherein the concentration of denaturing agent and acrylamide increases progressively downwards successively from the top of glue.2. the application of sample of PCR sample: after the complete polymerization of denatured gradient glue, offset plate is put into the electrophoresis chamber that electrophoretic buffer is housed, add the loading hole after 10 * sample loading buffer of getting PCR sample 5 μ l and 5 μ l mixes.3. electrophoresis and dyeing: the primer amplification segment, under the voltage of 130V, 60 ℃ of electrophoresis 7h.After the primer electrophoresis finishes, gel is carried out silver dye it and the results are shown in Figure 1.4. glue figure scanning: the gel after will dyeing obtains glue figure after scanning with UMAX PowerLook 1000 transmission scan instrument.
(5), the glue of advantage band reclaims order-checking: reclaim with glue and be connected with pGEM-T carrier (buying from Promega company) after test kit is cut glue and reclaimed, be transformed into intestinal bacteria TOP10 competent cell (be Time Inc.'s purchase by the sky).Add penbritin Amp (5 μ g/mL) and X-gal in the LB solid medium, blue hickie screening transformant.Extract plasmid, detect with carrier primer T7:5 '-TAATACGACTCACTATAGGG-3 ' and SP6:5 '-ATTTAGGTGACACTATAGAAT-3 '.Then go order-checking.
(6), the comparison of the network of advantage band and analysis: the known array in the sequence of measuring and the GenBank database carries out the similarity comparative analysis, result such as Fig. 2, main dominant population is Pseudomonas sp., Acinetobacter sp., Clostridia bacterium, Thermomicrobium sp., Agrobacterium sp., Firmicutes bacterium, candidate division, also has simultaneously the bacterial classification of many unknowns.The oil recovery bacterial classification is Clostridia bacterium, shows that in the microbe oil production process it no longer is dominant strain that this bacterial strain becomes, strong explanation the general problem of microbe oil production effect.Need oil production technology further to improve.
Experiment is carried out glue recovery and cloning and sequencing for different bands, sees Fig. 3 and Fig. 4.Main dominant population is Acinetobacter johnsonii, Pseudomonas fluorescens., Pseudomonas sp., Bosea sp., Syntrophothermus lipocalidus, Aeromonas media.
Claims (1)
1. PCR-DGGE primer that in the microbe oil production process, extracts the sample microorganism total DNA, its sequence is DQF338:5 '-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCA GCAG-3 '; DQR534:5 '-ATTACCGCGGCTGCTGG-3 '.
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| CN102071917A (en) * | 2010-12-30 | 2011-05-25 | 中国石油天然气股份有限公司 | Microbial multi-turn huff and puff oil production method |
| CN103088134B (en) * | 2013-01-22 | 2014-07-30 | 华东理工大学 | Method for detecting microorganism with hydrocarbon degradation function under anaerobic conditions |
| CN103667255B (en) * | 2013-11-19 | 2016-01-20 | 克拉玛依市金山石油化工有限公司 | The DNA extraction method of petroleum microorganism in raw petroleum environmental sample |
| CN104830662B (en) * | 2014-02-10 | 2016-09-14 | 中国石油化工股份有限公司 | A kind of quantitative assessment antibacterial apparatus and method chemotactic to crude oil |
| 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 |
| CN105201474A (en) * | 2015-10-23 | 2015-12-30 | 中国石油化工股份有限公司 | Method for improving recovery ratio of indigenous microbial enhanced oil recovery |
| WO2017209990A1 (en) | 2016-05-31 | 2017-12-07 | Exxonmobil Upstream Research Company | METHODS FOR lSOLATING NUCLEIC ACIDS FROM SAMPLES |
| CN108796096A (en) * | 2017-05-03 | 2018-11-13 | 奥为（天津）环保科技有限公司 | A method of monitoring oil degradation Bacterial community variation |
| CN109289498A (en) * | 2018-10-17 | 2019-02-01 | 浙江大学城市学院 | A kind of identification of municipal sewage plant's foul gas feature and deodorization technology |
| CN110863809B (en) * | 2019-10-22 | 2022-01-28 | 中国石油化工股份有限公司 | Method for compositely displacing oil by utilizing electric field and microorganisms |
| CN112646901A (en) * | 2020-12-24 | 2021-04-13 | 山西医科大学 | Kit and method for rapidly detecting food pathogenic bacteria vibrio parahaemolyticus |
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| CN1566327A (en) * | 2003-06-09 | 2005-01-19 | 大庆油田有限责任公司 | Viscosity reduction bacterium for improving petroleum recovery efficiency and its use |
| CN1236053C (en) * | 2004-05-17 | 2006-01-11 | 大庆油田有限责任公司 | Bacterium for degrding petroleum and its use |
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