CN102925563A - Method for quantifying microorganisms for producing lipopeptide surfactant in microbial flooding reservoir - Google Patents
Method for quantifying microorganisms for producing lipopeptide surfactant in microbial flooding reservoir Download PDFInfo
- Publication number
- CN102925563A CN102925563A CN2012103983477A CN201210398347A CN102925563A CN 102925563 A CN102925563 A CN 102925563A CN 2012103983477 A CN2012103983477 A CN 2012103983477A CN 201210398347 A CN201210398347 A CN 201210398347A CN 102925563 A CN102925563 A CN 102925563A
- Authority
- CN
- China
- Prior art keywords
- lipopeptid
- concentration
- plasmid
- microorganism
- pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for quantifying indigenous microorganisms for producing lipopeptide surfactant in microbial flooding reservoir. Primers are designed according to lipopeptide synthetase genes of microorganisms for producing lipopeptide surfactant, and fluorescent quantitative PCR (polymerase chain reaction) is performed. In optimized reaction procedures and reaction system, the lipopeptide synthetase genes obtained from a standard lipopeptide-producing strain is connected with a carrier to construct a recombinant plasmid used as a standard sample for real-time quantitative PCR; the standard sample is subjected to 10-time gradient dilution and used as a template for fluorescent quantitative PCR, and a standard curve is drawn; a microbial flooding reservoir sample is subjected to simple treatment and used as a template for fluorescent quantitative PCR; and whether data of replicate samples are identical and whether initial templates having different numbers of copies have the same amplification efficiency are judged according to linear relationship R2 and amplification efficiency E. When being used for detecting microorganisms for producing lipopeptide surfactant in reservoir under optimized reaction conditions, the lipopeptide synthetase specific primers have higher stability and sensitivity, thereby being widely used in the field of microbial enhanced oil recovery.
Description
Technical field
The present invention relates to the quantivative approach that a kind of microbial flooding oil reservoir produces lipopeptid class tensio-active agent microorganism, microorganism belonging to genus oil recovery field.
Background technology
The biological tensio-active agent of production by biological is one of dominant mechanism of microorganism raising oil recovery factor.The biological tensio-active agent of production by biological mainly contains two kinds of lipopeptid and glycolipids.Lipopeptid is one of best bio-surfactant of the effect of hitherto reported, because its special structure, can help microorganism cells to adhere to the hydrocarbons surface and separate hydrocarbon metabolism, reduce surface tension, under extreme conditions still can keep its activity, microorganism be improved the oil recovery factor technology have extremely important value.Biosurfactant production is that microorganism raising oil recovery factor has two kinds of methods at present, and a kind of ground method of crying is about to microorganism ground fermentation to produce biological tensio-active agent and injects oil reservoir; The another kind of ground laxative remedy of crying is about to the endogenous microbes of oil reservoir or the inoculating microbe of injection and produces bio-surfactant at the ground bottom fermentation.
But the microorganism that at present produces the lipopeptid tensio-active agent about how in the accurate quantitative analysis microbial flooding oil reservoir there is no methods involving, main by measure oil extraction circle size and surface tension size indirectly judge Surfactant Producing Microorganism how much.In the prior art, " oil spreading is to the qualitative and quantitative of bio-surfactant " literary composition that periodical " chemical engineer " 2005 the 01st is interim, introduced a kind of utilize between oil extraction loop diameter and the surfactant concentration all linear, the microorganism by measuring oil extraction loop diameter size qualitative analysis biosurfactant production what.Periodical " chemistry and biotechnology " the 27th volume in 2010, the 2nd interim " separation and the Methanogenesis of Lipopeptide Biosurfactants bacterial strain produced in a strain " literary composition has been introduced by capillary mensuration the microorganism of biosurfactant production has been carried out qualitative analysis.Above-mentioned two pieces of documents utilize respectively the size of oil extraction loop diameter and the amount that capillary size is come the characterising biological tensio-active agent, and then qualitative reflection produce tensio-active agent microorganism what, working method is simple, but its shortcoming is only can qualitatively judge having or not or what of product table agent microorganism alive, can't quantitatively judge the amount of product table agent microorganism alive, and have poor repeatability and the low deficiency of accuracy.
Summary of the invention
The object of the invention is to overcome the deficiency that above-mentioned prior art exists, provide a kind of microbial flooding oil reservoir to produce the quantivative approach of lipopeptid class tensio-active agent microorganism.The method can realize fast to producing the quantitative of lipopeptid class tensio-active agent microorganism in the microbial flooding oil reservoir.
The technical problem scheme that the present invention solves provides the quantivative approach that a kind of microbial flooding oil reservoir produces lipopeptid class tensio-active agent microorganism, and its concrete steps are as follows:
1, design and synthetic primer
The lipopeptid synthase gene of lipopeptid class tensio-active agent microorganism is produced in search in GeneBank, and its homology is compared, and the conserved regions sequence of Select gene is according to design of primers principle design pair of primers;
2, the optimization of quantitative fluorescent PCR response procedures and reaction system and foundation
By to Mg
2+The selection of concentration, primer concentration and annealing temperature, take minimum Ct value (cycle number that the fluorescent signal in the quantitative fluorescent PCR process in each reaction tubes is experienced when reaching the threshold value of setting) and the highest RFU(relative intensity of fluorescence value) as screening index, determine reaction system and response procedures;
3, the preparation of standard substance plasmid
Use round pcr and obtain lipopeptid synthase gene fragment from produce lipopeptid microorganism subtilis, be connected with the T carrier, construction recombination plasmid is measured recombinant plasmid A
260(absorbancy when wavelength is 260nm) calculates plasmid copy number, and this recombinant plasmid that calculates copy number is the standard substance plasmid of real-time quantitative PCR;
4, Criterion curve
Get step 3 standard substance plasmid, multiple proportions relation with 10 times is carried out gradient dilution, respectively as the qPCR(real-time fluorescence quantitative PCR) template of reaction increases, and corresponding threshold cycle number is figure, the production standard curve with the logarithmic value of difference dilution gradient plasmid copy number.
5, method sensitivity and stability assessment
(1) method sensitivity examination
Will be by 10 times of dilutions, copy number is 1 * 10
7Copies/ μ l～1 * 10
1The standard substance plasmid of copies/ μ l is as template, and reaction system and response procedures in the step 2 detect respectively, investigates and detects lower limit, and susceptibility is estimated;
(2) method stability assessment
Minutes three days carry out respectively three times independent experiment, each concentration do three parallel, come the stable or repeatable of evaluation method by mean value and the variation coefficient to experimental result Ct value.
Primer in the described step 1 to sequence is:
srfAF 5’-CAAAAKCGCAKCATACCACKTTGAG-3’
srfAR 5’-TCA TAR AGC GGC AYA TAT TGA TGC-3’
Reaction system in the described step 2 is made as 20 μ l, composing system: reaction buffer is 10 * Taq PCR buffer, Mg
2+Concentration is 3.5mM, and dNTPs concentration is 0.2mM, Taq enzyme 2.5U, and the concentration of primer is 0.1 μ M, 20 * Sybr Green I, 1 μ l, template 2 μ l, sterilized water 7 μ l.
PCR response procedures in the described step 2 is: 95 ℃ of 5min of (1) denaturation, (2) phosphor collection, 72 ℃ of 20s of 58 ℃ of 15s of 95 ℃ of 15s, carrying out fluorescent signal after each loop ends collects, 40 circulations of increasing, (3) solubility curve analysis, 65 ℃ progressively rise to 95 ℃, collect first order fluorescence every 0.2 ℃.
Annealing region is 55 ℃～65 ℃ in the described step 2, Mg
2+Concentration range is 1 μ M, 1.5 μ M, 2.5 μ M, 3.5 μ M, 4.5 μ M, 6 μ M, and the primer concentration scope is 0.l μ M, 0.2 μ M, 0.4 μ M, 0.6 μ M., 0.8 μ M, μ M l.0.
Described a kind of microbial flooding oil reservoir produces the quantivative approach of lipopeptid class tensio-active agent microorganism, the on-site sampling that also comprises microbial flooding oil reservoir sample, its sampling procedure is: (1) adopts the vial with plug and screw-cap, clean up with deionized water, with air in the high pure nitrogen displacement bottle, behind plug and sealing of lid, 121 ℃ of sterilizations of high pressure steam 20min, (2) open wellhead sampling mouth valve, access liquid with waste liquid barrel, make the 15L～20L of liquid Channel Group, (3) open sampling container, connect thief hole, treat that injected water is full of sampling container, screw capping sealing, (4) are labelled on sampling container, place to send the laboratory back under 4 ℃ of conditions in 4h and analyze.
Described a kind of microbial flooding oil reservoir produces the quantivative approach of lipopeptid class tensio-active agent microorganism, also comprise described microbial flooding oil reservoir sample preparation, the sample preparation step is: (1) gets the oil reservoir production fluid sample in the anaerobism pipe that contains high pure nitrogen, getting 1ml is injected in the centrifuge tube of 1.5 ml of sterilization, (2) abundant vibration 5 min on vibrator, centrifugal 1 min of 13 000 rpm, from pipe gently abandon supernatant liquor 900 μ l, (3) in remaining 100 μ l, add stroke-physiological saline solution 900 μ l, 5 min fully vibrate on vibrator, centrifugal 2 min of (4) 13 000 rpm, abandon supernatant liquor 980 μ l, abundant vibration 2 min, gained 20 μ l are template.
The preparation of described step 3 Plays product plasmid, concrete grammar is as follows:
(1) lipopeptid synthase gene fragment obtains
Produce the total DNA of extraction the lipopeptid microorganism subtilis overnight culture from 1 ml, producing lipopeptid microorganism bacillus subtilis bacterium culture medium is Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L, pH value 7.3, determine purity and the concentration of DNA by ultraviolet spectrophotometer and gel electrophoresis, with srfAF/ srfAR primer to carrying out the real-time quantitative PCR reaction.Reaction system and amplification program are as follows:
20 μ l systems: reaction buffer is 10 * Taq PCR buffer, Mg
2+Concentration is 3.5mM, and dNTPs concentration is 0.2mM, Taq enzyme 2.5U, and the concentration of primer is 0.1 μ M, template: 3 μ l, sterilized water 7 μ l.
Amplification program: 1. 95 ℃ of sex change 5min, 2. 72 ℃ of 45s of 58 ℃ of 30s of 95 ℃ of 30s, 30 circulations, 3. 72 ℃ of 10min.
By agarose gel electrophoresis the PCR product is identified, then under ultraviolet lamp, downcut the goal gene band, reclaim test kit with glue the PCR product is carried out the purifying recovery, namely obtain the lipopeptid synthase gene fragment of purifying.
(2) connection of recombinant plasmid, conversion, positive colony screening and evaluation
The T carrier is connected with mol ratio 1:4 ratio with lipopeptid synthase gene fragment, 16 ℃ of ligation 1h, connect product and transform DH5 α competent cell, the method that adopts the dull and stereotyped first screening of amicillin resistance LB long hollow piece of jade with rectangular sides lipolysaccharide and bacterium colony PCR again to screen is determined positive bacterium colony, the positive bacterium colony of amplification in amicillin resistance LB nutrient solution is sent to bacterium liquid at last order-checking and is again verified.
(3) the standard substance template is quantitative
Extract the recombinant plasmid that empirical tests contains goal gene, measure the A of plasmid by ultraviolet spectrophotometer
260Value, calculate the molecule copy number according to relative molecular mass and Avogadro constant, recombinant plasmid is diluted to the standard substance plasmid that different multiples is real-time quantitative PCR reaction.
The present invention compared with prior art has the following advantages:
1, sensitivity and accuracy are high, high specificity;
2, simple to operate, repeatable high;
3, detection time section, measure as a result required time less than 2h from the upper machine examination of extracting of sample DNA;
4, can realize high throughput testing to a plurality of samples simultaneously.
Description of drawings
Figure l: the microbial flooding oil reservoir produces the amplification of the quantitative susceptibility of lipopeptid microorganism.From left to right be followed successively by 1 * 10
7Copies/ μ l, 1 * 10
6Copies/ μ l, 1 * 10
5Copies/ μ l, 1 * 10
4Copies/ μ l, 1 * 10
3Copies/ μ l, 1 * 10
2Copies/ μ l and 1 * 10
1Copies/ μ l.
Fig. 2: the microbial flooding oil reservoir produces the typical curve of lipopeptid microorganism detection.X-coordinate represents the log value of original template amount, and ordinate zou represents the Ct value of each concentration sample.
Fig. 3: the microbial flooding oil reservoir produces the detected result of lipopeptid microorganism triplicate.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is done and to describe in further detail.Should be appreciated that these embodiment only are used for explanation the present invention, and be not used in restriction claimed scope of the present invention.
1, design, synthetic primer
The lipopeptid synthase gene of lipopeptid class tensio-active agent microorganism is produced in search in GeneBank, its homology is compared, and the conserved regions sequence of Select gene, according to design of primers principle design primer pair, synthetic by precious biotechnology (Dalian) company limited.
The right sequence of primer is
srfAF 5’-CAAAAKCGCAKCATACCACKTTGAG-3’
srfAR 5’-TCA TAR AGC GGC AYA TAT TGA TGC-3’
2, the optimization of quantitative fluorescent PCR response procedures and reaction system and foundation
The optimization of reaction conditions mainly comprises Mg
2+The selection of concentration, primer concentration and probe concentration ratio and annealing temperature.In the Real-TimePCR response procedures, the fixation reaction system, adjust different annealing temperature (55 ℃～65 ℃), detect take same concentrations product lipopeptid microorganism subtilis TH-2 genomic dna as template, screen as standard take minimum Ct value and RFU, determine optimum annealing temperature.Under optimum annealing temperature, select Mg
2+Concentration is respectively 1 μ M, 1.5 μ M, 2.5 μ M, 3.5 μ M, 4.5 μ M, 6 μ M, under best primer concentration and probe concentration proportioning, and preferred best Mg
2+Concentration.Test is produced lipopeptid microorganism subtilis TH-2 genomic dna as template, at best Mg take same concentrations
2+Under the concentration, primer concentration is respectively 0.l μ M, 0.2 μ M, 0.4 μ M, 0.6 μ M., 0.8 μ M, μ M l.0, take minimum Ct value and RFU as screening index, and preferred primer concentration.
Reaction system and response procedures after optimizing are:
Reaction system is made as 20 μ l, composing system: reaction buffer is 10 * Taq PCR buffer, Mg
2+Concentration is 3.5mM, and dNTPs concentration is 0.2mM, Taq enzyme 2.5U, and the concentration of primer is 0.1 μ M, 20 * Sybr Green I, 1 μ l, template 2 μ l, sterilized water 7 μ l.
The qPCR response procedures is: 95 ℃ of 5min of (1) denaturation, (2) phosphor collection, 72 ℃ of 20s of 58 ℃ of 15s of 95 ℃ of 15s, carry out fluorescent signal after each loop ends and collect 40 circulations of increasing, (3) solubility curve analysis, 65 ℃ progressively rise to 95 ℃, collect first order fluorescence every 0.2 ℃.
3, the preparation of standard substance plasmid
Use round pcr and from produce lipopeptid microorganism subtilis genomic dna, obtain the lipopeptid synthase gene, be connected with the T carrier, construction recombination plasmid, as the standard substance of real-time quantitative PCR, concrete grammar is as follows:
(1) acquisition of lipopeptid synthase gene fragment
Produce the total DNA of extraction the lipopeptid microorganism subtilis overnight culture from 1 ml, determine purity and the concentration of DNA by ultraviolet spectrophotometer and gel electrophoresis.React carrying out real-time quantitative PCR with srfAF/ srfAR primer.Reaction system and amplification program are as follows:
20 μ l systems: reaction buffer is 10 * Taq PCR buffer, Mg
2+Concentration is 3.5mM, and dNTPs concentration is 0.2mM, Taq enzyme 2.5U, and the concentration of primer is 0.1 μ M, template: 3 μ l, distilled water polishing.
Amplification program: 95 ℃ of sex change 5min; 30 circulations of 72 ℃ of 45s of 58 ℃ of 30s of 95 ℃ of 30s, by agarose gel electrophoresis the PCR product is identified, then under ultraviolet lamp, downcut the goal gene band, reclaim test kit with glue the PCR product is carried out the purifying recovery, namely obtain the lipopeptid synthase gene fragment of purifying.
(2) connection of recombinant plasmid, conversion, positive colony screening and evaluation:
The T carrier is connected with the mol ratio of srfA gene fragment with 1:4, and 16 ℃ of ligation times are 1h.Connect product and transform DH5 α competent cell, the method that adopts the first screening of amicillin resistance LB agar plate and bacterium colony PCR again to screen is determined positive bacterium colony, the positive bacterium colony of amplification in amicillin resistance LB nutrient solution is sent to bacterium liquid at last order-checking and is finally verified.
(3) the standard substance plasmid is quantitative
Extract the recombinant plasmid that empirical tests contains the lipopeptid synthase gene, measure the A of plasmid by ultraviolet spectrophotometer
260Value, calculate plasmid copy number according to relative molecular mass and Avogadro constant, recombinant plasmid is diluted to the standard substance plasmid that different concns is real-time quantitative PCR reaction with 10 times.
4, Criterion curve
Get step 3 standard substance plasmid, carry out gradient dilution with 10 times multiple proportions relation, the template as the qPCR reaction increases respectively, corresponding threshold cycle number is figure, the production standard curve with the logarithmic value of difference dilution gradient plasmid copy number.
The result shows that method of the present invention is 1 * 10
7Copies/ μ l～1 * 10
1In the copies/ μ l scope, typical curve is good linear relationship, R
2=0.998, amplification efficiency E=99.6%.
5, method stability and sensitivity examination
(1) method sensitivity examination
Will be by 10 times of dilutions, copy number is 1 * 10
7Copies/ μ l～1 * 10
1The standard substance plasmid of copies/ μ l detects respectively with fluorescence quantitative PCR detection system of the present invention as template, investigates and detects lower limit, and susceptibility is estimated.
(2) method stability assessment
Minutes three days carry out respectively three times independent experiment, each concentration do three parallel, come the stable or repeatable of evaluation method by mean value and the variation coefficient to experimental result Ct value.
Table 1 oil reservoir produces the quantivative approach Detection of Stability of lipopeptid class tensio-active agent microorganism
The quantivative approach of table 2 oil reservoir product lipopeptid class tensio-active agent microorganism is the variation coefficient in the daytime
The plasmid copy number that present method can detect is minimum to be 10copies/ μ l, has higher detection sensitivity.
To the bacterium liquid duplicate detection of 6 kinds of different concns 3 times, the Ct value that amplification obtains sees Table 1, the Ct value variation coefficient and (is 1.31% to the maximum, all less than 5%) in the reasonable scope.Minutes three days carry out respectively three independent experiments and the results are shown in Table 2, the variation lines number average is less than 5%, in concentration 10
4Copies/ μ l～10
5Repeatability is relatively good during copies/ μ l, and present method stability stability is best during this concentration range.
Embodiment 2: the comparison that the quantivative approach of microbial flooding oil reservoir product lipopeptid class tensio-active agent microorganism and traditional mensuration oil extraction circle and surface tension methods analyst oil reservoir produce the lipopeptid microorganism
The 6 mouthfuls of produced liquid in oil well of sharp oil field block of winning victory respectively after corresponding the processing, carry out respectively the oil spreading test, surface tension test and produce the lipopeptid quantitatively analysing microorganism.
The different produced liquid in oil well oil extraction of table 3 circle, surface tension and product lipopeptid microorganism detection by quantitative result
As can be seen from the results, oil spreading and measurement of surface tension really can reflect in the oil reservoir sample produce the Lipopeptide Biosurfactants microorganism existence whether, and utilize these data between each sample, to compare, judge what of biosurfactant production microorganism, but the result who lacks direct quantitative, so that between the different batches data, the comparability variation, and owing to be the mensuration of Surfactant, be subjected to easily the interference of other chemical surfactants of using in the oilfield development process etc.
Sequence table (SEQUENCE LISTING)
＜110〉Oil Mined Technology Research Inst., Shengli Oil Field
＜120〉a kind of oil reservoir produces the quantivative approach of lipopeptid class tensio-active agent microorganism
<130>
<160>1
<170>
<210>1
<211>274
<212>DNA
＜213〉subtilis
<220>
<223>
<400>1
CAAAATCGCA TCATACCACT TTGAGTACGG CGCTGCAGGC AGTCTGGAGC GTATTGATCA GCCGCTATCA GCAGTCTGGC GATTTGGCCT TCGGTACAGT TGTTTCAGGG CGTCCCGCGG AAATCAAAGG CGTTGAACAT ATGGTTGGGC TGTTTATCAA CTCGTCCCGA GACGTGTGAA GCTGTCTGAG GGTATCACAT TTAACGGCTT GCTCAAGCAG CTGCAGGAGC AATCGCTGCA GTCTGAGCCG CATCAATATA TGCCGCTTTA TGA
Claims (6)
1. a microbial flooding oil reservoir produces the quantivative approach of lipopeptid class tensio-active agent microorganism, comprises following implementation step:
(1), design and synthetic primer
The lipopeptid synthase gene of lipopeptid class tensio-active agent microorganism is produced in search in GeneBank, and its homology is compared, and the conserved regions sequence of Select gene is according to design of primers principle design pair of primers;
(2), the optimization of quantitative fluorescent PCR response procedures and reaction system and foundation
By to Mg
2+The selection of concentration, primer concentration and annealing temperature take minimum Ct value and the highest RFU as screening index, is determined reaction system and response procedures;
(3), the preparation of standard substance plasmid
Use round pcr and obtain lipopeptid synthase gene fragment from produce lipopeptid microorganism subtilis, be connected with the T carrier, construction recombination plasmid is measured recombinant plasmid A
260Calculate plasmid copy number, this recombinant plasmid that calculates copy number is the standard substance plasmid of real-time quantitative PCR;
(4), Criterion curve
Get step (3) standard substance plasmid, multiple proportions relation with 10 times is carried out gradient dilution, respectively as the qPCR(real-time fluorescence quantitative PCR) template of reaction increases, and corresponding threshold cycle number is figure, the production standard curve with the logarithmic value of difference dilution gradient plasmid copy number;
(5), method sensitivity and stability assessment
1. method sensitivity examination
Will be by 10 times of dilutions, copy number is 1 * 10
7Copies/ μ l～1 * 10
1The standard substance plasmid of copies/ μ l is as template, and reaction system and response procedures in the step (2) detect respectively, investigates and detects lower limit, and susceptibility is estimated;
2. method stability assessment
Minutes three days carry out respectively three times independent experiment, each concentration do three parallel, come the stable or repeatable of evaluation method by mean value and the variation coefficient to experimental result Ct value;
It is characterized in that the primer in the described step (1) to sequence is:
srfAF 5’-CAAAAKCGCAKCATACCACKTTGAG-3’
srfAR 5’-TCA TAR AGC GGC AYA TAT TGA TGC-3’
Reaction system in the described step (2) is made as 20 μ l, composing system: reaction buffer is 10 * Taq PCR buffer, Mg
2+Concentration is 3.5mM, and dNTPs concentration is 0.2mM, Taq enzyme 2.5U, and the concentration of primer is 0.1 μ M, 20 * Sybr Green I, 1 μ l, template 2 μ l, sterilized water 7 μ l;
PCR response procedures in the described step (2) is: 1. 95 ℃ of 5min of denaturation, 2. phosphor collection, 72 ℃ of 20s of 58 ℃ of 15s of 95 ℃ of 15s, carrying out fluorescent signal after each loop ends collects, 40 circulations of increasing, 3. solubility curve analysis, 65 ℃ progressively rise to 95 ℃, collect first order fluorescence every 0.2 ℃.
2. a kind of microbial flooding oil reservoir according to claim 1 produces the quantivative approach of lipopeptid class tensio-active agent microorganism, it is characterized in that described implementation step also comprises the on-site sampling of microbial flooding oil reservoir sample, and sampling procedure is:
(1) employing cleans up with deionized water with the vial of plug and screw-cap, with an air in the high pure nitrogen displacement bottle, and behind plug and sealing of lid, 121 ℃ of 20min that sterilize of high pressure steam;
(2) open wellhead sampling mouth valve, access liquid with waste liquid barrel, make the 15L～20L of liquid Channel Group;
(3) open sampling container, connect thief hole, treat that injected water is full of sampling container, the screw capping sealing;
(4) labelled on sampling container, place and in 4h, send the laboratory back under 4 ℃ of conditions and analyze.
3. a kind of microbial flooding oil reservoir according to claim 1 and 2 produces the quantivative approach of lipopeptid class tensio-active agent microorganism, it is characterized in that described implementation step also comprises microbial flooding oil reservoir sample preparation, and the sample preparation step is:
(1) gets oil reservoir production fluid sample in the anaerobism pipe that contains high pure nitrogen, get 1ml and be injected in the centrifuge tube of 1.5 ml of sterilization;
(2) abundant vibration 5 min on vibrator, centrifugal 1 min of 13 000 rpm, from pipe gently abandon supernatant liquor 900 μ l;
(3) add stroke-physiological saline solution 900 μ l in remaining 100 μ l, 5 min fully vibrate on vibrator; Centrifugal 2 min of (4) 13 000 rpm abandon supernatant liquor 980 μ l, 2 min that fully vibrate, and gained 20 μ l are
Template.
4. a kind of microbial flooding oil reservoir according to claim 1 produces the quantivative approach of lipopeptid class tensio-active agent microorganism, it is characterized in that, annealing region is 55 ℃～65 ℃ in the described step (2), Mg
2+Concentration range is 1 μ M, 1.5 μ M, 2.5 μ M, 3.5 μ M, 4.5 μ M, 6 μ M, and the primer concentration scope is 0.l μ M, 0.2 μ M, 0.4 μ M, 0.6 μ M., 0.8 μ M, μ M l.0.
5. a kind of microbial flooding oil reservoir according to claim 1 produces the quantivative approach of lipopeptid class tensio-active agent microorganism, it is characterized in that, and the preparation of described step (3) Plays product plasmid, concrete preparation method is as follows:
(1) lipopeptid synthase gene fragment obtains
Produce the lipopeptid microorganism subtilis overnight culture from 1 ml and to extract total DNA, determine purity and the concentration of DNA by ultraviolet spectrophotometer and gel electrophoresis, with srfAF/ srfAR primer to carrying out the real-time quantitative PCR reaction;
20 μ l systems: reaction buffer is 10 * Taq PCR buffer, Mg
2+Concentration is 3.5mM, and dNTPs concentration is 0.2mM, Taq enzyme 2.5U, and the concentration of primer is 0.1 μ M, template: 3 μ l, sterilized water 7 μ l;
Amplification program: 1. 95 ℃ of sex change 5min, 2. 72 ℃ of 45s of 58 ℃ of 30s of 95 ℃ of 30s, 30 circulations, 3. 72 ℃ of 10min;
By agarose gel electrophoresis the PCR product is identified, then under ultraviolet lamp, downcut the goal gene band, reclaim test kit with glue the PCR product is carried out the purifying recovery, namely obtain the lipopeptid synthase gene fragment of purifying;
(2) connection of recombinant plasmid, conversion, positive colony screening and evaluation
The T carrier is connected with mol ratio 1:4 ratio with lipopeptid synthase gene fragment, 16 ℃ of ligation 1h, connect product and transform DH5 α competent cell, the method that adopts the dull and stereotyped first screening of amicillin resistance LB long hollow piece of jade with rectangular sides lipolysaccharide and bacterium colony PCR again to screen is determined positive bacterium colony, the positive bacterium colony of amplification in amicillin resistance LB nutrient solution is sent to bacterium liquid at last order-checking and is again verified;
(3) the standard substance template is quantitative
Extract the recombinant plasmid that empirical tests contains goal gene, measure the A of plasmid by ultraviolet spectrophotometer
260Value, calculate the molecule copy number according to relative molecular mass and Avogadro constant, recombinant plasmid is diluted to the standard substance plasmid that different multiples is real-time quantitative PCR reaction.
6. a kind of microbial flooding oil reservoir according to claim 5 produces the quantivative approach of lipopeptid class tensio-active agent microorganism, it is characterized in that described product lipopeptid microorganism bacillus subtilis bacterium culture medium is Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L, pH value 7.3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210398347.7A CN102925563B (en) | 2012-10-19 | 2012-10-19 | Method for quantifying microorganisms for producing lipopeptide surfactant in oil reservoir |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210398347.7A CN102925563B (en) | 2012-10-19 | 2012-10-19 | Method for quantifying microorganisms for producing lipopeptide surfactant in oil reservoir |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102925563A true CN102925563A (en) | 2013-02-13 |
| CN102925563B CN102925563B (en) | 2015-03-04 |
Family
ID=47640486
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210398347.7A Active CN102925563B (en) | 2012-10-19 | 2012-10-19 | Method for quantifying microorganisms for producing lipopeptide surfactant in oil reservoir |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102925563B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103760268A (en) * | 2014-01-16 | 2014-04-30 | 华东理工大学 | Method for quantitatively detecting lipopeptide |
| CN104313178A (en) * | 2014-11-27 | 2015-01-28 | 中国石油化工股份有限公司 | Fluorogenic quantitative PCR detecting method of surfactant-producing viable bacteria |
| CN104450891A (en) * | 2014-11-17 | 2015-03-25 | 中国科学院微生物研究所 | Method and system for digital quantitative analysis of nucleic acid amplification based on micro-droplet |
| 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 |
| CN108070462A (en) * | 2018-01-16 | 2018-05-25 | 江苏龙蟠科技股份有限公司 | Environment protection type biodegradable glass cleaning solution |
| CN108373912A (en) * | 2018-02-02 | 2018-08-07 | 中国石油化工股份有限公司华北油气分公司石油工程技术研究院 | A kind of salt-resisting high-temperature-resisting foam discharging agent and preparation method thereof |
| US10435737B2 (en) | 2014-11-17 | 2019-10-08 | Institute Of Microbiology, Chinese Academy Of Sciences | Droplet generating apparatus, system, and method |
| CN110863809A (en) * | 2019-10-22 | 2020-03-06 | 中国石油化工股份有限公司 | Method for compositely displacing oil by utilizing electric field and microorganisms |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321746A (en) * | 2011-08-08 | 2012-01-18 | 中国海洋石油总公司 | Method for identifying Bacillus subtilis for producing lipopeptide surfactants |
-
2012
- 2012-10-19 CN CN201210398347.7A patent/CN102925563B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321746A (en) * | 2011-08-08 | 2012-01-18 | 中国海洋石油总公司 | Method for identifying Bacillus subtilis for producing lipopeptide surfactants |
Non-Patent Citations (2)
| Title |
|---|
| 吕应年等: "脂肽类生物表面活性剂的研究进展", 《生物技术通报》 * |
| 王大威等: "一株产生脂肽的枯草芽孢杆菌的分离鉴定及脂肽对原油的作用", 《微生物学报》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103760268B (en) * | 2014-01-16 | 2015-11-18 | 华东理工大学 | A kind of method that lipopeptid quantitatively detects |
| CN103760268A (en) * | 2014-01-16 | 2014-04-30 | 华东理工大学 | Method for quantitatively detecting lipopeptide |
| US11066695B2 (en) | 2014-11-17 | 2021-07-20 | Beijing Dawei Bio Ltd. | Droplet generating apparatus, system, and method |
| CN104450891A (en) * | 2014-11-17 | 2015-03-25 | 中国科学院微生物研究所 | Method and system for digital quantitative analysis of nucleic acid amplification based on micro-droplet |
| CN104450891B (en) * | 2014-11-17 | 2017-06-06 | 中国科学院微生物研究所 | Digital nucleic acid amplification quantitative analysis method and system based on microlayer model |
| US11674170B2 (en) | 2014-11-17 | 2023-06-13 | Beijing Dawei Bio Ltd. | Droplet generating method |
| US10435737B2 (en) | 2014-11-17 | 2019-10-08 | Institute Of Microbiology, Chinese Academy Of Sciences | Droplet generating apparatus, system, and method |
| CN104313178A (en) * | 2014-11-27 | 2015-01-28 | 中国石油化工股份有限公司 | Fluorogenic quantitative PCR detecting method of surfactant-producing viable bacteria |
| 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 |
| CN108070462A (en) * | 2018-01-16 | 2018-05-25 | 江苏龙蟠科技股份有限公司 | Environment protection type biodegradable glass cleaning solution |
| CN108070462B (en) * | 2018-01-16 | 2020-10-09 | 江苏龙蟠科技股份有限公司 | Biodegradable environment-friendly glass cleaning fluid |
| CN108373912A (en) * | 2018-02-02 | 2018-08-07 | 中国石油化工股份有限公司华北油气分公司石油工程技术研究院 | A kind of salt-resisting high-temperature-resisting foam discharging agent and preparation method thereof |
| CN110863809A (en) * | 2019-10-22 | 2020-03-06 | 中国石油化工股份有限公司 | Method for compositely displacing oil by utilizing electric field and microorganisms |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102925563B (en) | 2015-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102925563B (en) | Method for quantifying microorganisms for producing lipopeptide surfactant in oil reservoir | |
| CN107513561A (en) | Detect primer, kit and the method for P. aeruginosa and C.perfringens | |
| CN102174653A (en) | Haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method | |
| CN103966318A (en) | Method for revealing and distinguishing paddy field formic acid utilization type methanogenic archaea in situ by adopting DNA-based stable isotope probing technology | |
| CN104232783B (en) | Quick detection method for cow brucella attenuated vaccine strain A19 | |
| CN104059975A (en) | Providencia O3, O4, O8, O12, O13 and O20 specific nucleotides and application thereof | |
| CN101676405A (en) | Mycobacterium tuberculosis fluorescence quantitative PCR detection method and kit thereof | |
| CN104726594A (en) | Quintuple fluorescent PCR detection kit for foodborne pathogenic bacteria | |
| CN103555842A (en) | Animal chlamydia TaqMan-MGB probe multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) detection primers, kit and method | |
| CN103088133A (en) | Detection method for sulfate reducing bacteria | |
| CN107513562A (en) | Detect pseudomonas aeruginosa and ExoU primer, kit and method in water | |
| CN103993090A (en) | Specific nucleotides for providencia O31, O41, O42, O43 and O50 and application of specific nucleotides | |
| CN103397108A (en) | HCLV (hog cholera lapinized virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection kit | |
| CN109234432A (en) | A kind of primer, probe and kit based on recombinase polymeric enzymatic amplification method detection soybean samping off | |
| Ma et al. | Research of the impact of elevated CO2 on soil microbial diversity | |
| CN107287328A (en) | Detect primer, kit and the method for pseudomonas aeruginosa and toxA in water | |
| CN104561312A (en) | Kit and detection method for detecting endogenus component from bear by means of fluorescence PCR | |
| CN104450930A (en) | Molecular detection method of vibrio parahaemolyticus and application thereof | |
| CN103290107A (en) | Construction method of SSR fingerprint suitable for identification of rice hybrid authenticity | |
| CN103740839B (en) | The botulinal universal test kit of fluorescence quantitative PCR detection and nondiagnostic detection method | |
| CN101440406A (en) | Method for detecting stability of functional flora in water treatment system | |
| CN104531861A (en) | Molecular detection method of enterobacter sakazakii and application of molecular detection method | |
| CN107447001A (en) | Detect pseudomonas aeruginosa and ExoS primer, kit and method in water | |
| CN104152576A (en) | Fast quantitative detection method for methanogenic archaea in oil-water well output liquid | |
| CN107653331A (en) | Detect pseudomonas aeruginosa and ExoY primer, kit and method in water |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhang Shaodong Inventor after: Cao Yanbin Inventor after: Song Yongting Inventor after: Guo Liaoyuan Inventor after: Li Caifeng Inventor after: Hao Bin Inventor after: Hu Jing Inventor after: Cao Gongze Inventor after: Pan Yongqiang Inventor after: Wang Jing Inventor after: Xu Peng Inventor after: Du Chunan Inventor before: Zhang Shaodong Inventor before: Wu Xiaoling Inventor before: Hao Bin Inventor before: Song Yongting Inventor before: Guo Liaoyuan Inventor before: Gao Guangjun Inventor before: Song Zhiyong Inventor before: Liu Tao Inventor before: Li Caifeng Inventor before: Cao Gongze |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: ZHANG SHAODONG HAO BIN SONG YONGTING GUO LIAOYUAN GAO GUANGJUN SONG ZHIYONG LIU TAO LI CAIFENG CAO GONGZE WU XIAOLING TO: ZHANG SHAODONG LI CAIFENG HAO BIN HU JING CAO GONGZE PAN YONGQIANG WANG JING XU PENG DU CHUNAN CAO YANBIN SONG YONGTING GUO LIAOYUAN |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |