CN108796096A - A method of monitoring oil degradation Bacterial community variation - Google Patents
A method of monitoring oil degradation Bacterial community variation Download PDFInfo
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- CN108796096A CN108796096A CN201710303202.7A CN201710303202A CN108796096A CN 108796096 A CN108796096 A CN 108796096A CN 201710303202 A CN201710303202 A CN 201710303202A CN 108796096 A CN108796096 A CN 108796096A
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- oil degradation
- degradation bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The present invention relates to a kind of methods of monitoring oil degradation bacteria structure of community variation, belong to technical field of microbial ecology.This method extracts oil degradation bacteria total DNA using New Probe bacteria total DNAs extracts kits；Annealing temperature, recurring number in DNA profiling amount, primer amount and program in PCR amplification system are optimized；To denaturing gradient gel electrophoresis（DGGE）Middle gel strength, electrophoretic voltage, electrophoresis time, APS amounts, TEMED amounts are also optimized.The present invention had both overcome the shortcomings that pure culture technigne, had the characteristics that easy, quick, efficient, stable.This method is that dynamic change of the monitoring oil degradation flora in practical degradation process provides a kind of method with exploitation different phase oil degradation function bacterium.
Description
Technical field
The present invention relates to a kind of methods of monitoring oil degradation bacteria structure of community variation, belong to microbial ecological technology neck
Domain.
Background technology
The PCR-DGGE technological break-throughs constraint of traditional synthetic medium is probed into bacterial community composition from gene level and is become
Change, can directly analyze the mixed bacterial structure in complex sample, more truly reflect the more of microorganism under natural conditions
Sample.The technology includes mainly the extraction of DNA, PCR amplification, three big key step of DGGE gel electrophoresises.DNA is PCR-DGGE skills
The core starting materials of art, impurity is more in general petroleum environments sample, and the extraction of DNA can also become complicated.DNA mass is research
The guarantee of biological community structure in petroleum environments, generally use traditional method for extracting DNA of bacteria in research, but this process pilot scale
Agent preparation is cumbersome, dosage is few, when be easy to causeing reagent waste and operating cost；Import reagent box extraction DNA is mostly used at present, by
In expensive, it is difficult to universal to use.In a PCR step, the randomness of PCR is stronger and influenced by factors, usually
Many non-target fragment products, such as heteroduplex, dimer etc. are will produce, it is various that these by-products can influence bacterial community
The authenticity of property.DNA profiling amount, primer amount, annealing temperature, recurring number are to influence the principal element of PCR specific amplifications.DNA
Extraction quality and PCR stablize the basis that amplification is denaturing gradient gel electrophoresis, the essential condition for influencing its effect includes solidifying
Gum concentration, electrophoretic voltage, electrophoresis time, the improper effect to collection of illustrative plates of deposition condition have a direct impact.
In view of PCR-DGGE technologies the research of oil degradation bacteria structure of community the problem of, it is necessary to be directed to each skill
Shortcoming in art is improved, and to mature technology condition, ensures the DNA mass of extraction, is stablized amplification target fragment, is obtained
Take clear DGGE collection of illustrative plates.
Invention content
In order to overcome the shortcomings of in background technology, the present invention establishes a kind of improved PCR-DGGE methods for monitoring sea
The variation of oil degradation bacteria structure of community, this method can really reflect oil degradation mistake during foreign oil pollution is biological prosthetic
The dynamic change of flora in journey is conducive to develop different phase oil degradation function bacterium.
Technical scheme of the present invention includes the following steps：
a）It is extracted using kit to containing oil culture solution petrochina degradation bacteria total DNA；
b）Using extracted DNA as template, PCR amplification goes out target fragment；
c）Pcr amplified fragment is detached by denaturing gradient gel electrophoresis, obtains finger-print；
d）The recycling sequencing of gesture band glue；
e）Sequence result is at U.S. biology information technology center（NCBI）Sequence analysis and the analysis of sequence are carried out in database.
Said program step a）In：Oil degradation bacteria total DNA is extracted using New Probe bacteria total DNA extracts kits.
Said program step b）In：DNA profiling amount, primer amount, annealing temperature, recurring number are optimized, after optimization
PCR system is 25 μ L, 10 X Master, 3 μ L DNA profilings, 1 μ L forward primers, 1 μ L reverse primers, 20 μ L
ddH2O；After optimization amplification program be 94 DEG C of 4 min of pre-degeneration, 94 DEG C denaturation 1 min, 55 DEG C annealing 1 min, 72 DEG C prolong
Stretch 1 min, 25 cycles, 72 DEG C of 6 min of final extension.
Said program step b）In：To gel strength in denaturing gradient gel electrophoresis condition, electrophoretic voltage, electrophoresis time into
Gone optimization, deposition condition includes acrylamide gel a concentration of 8%, and gel is denaturalized ranging from 30%-60%, in gel solution plus
Enter 100 μ L APS and 18 μ L TEMED；PCR product is with sample loading buffer with 4：1 mixes, totally 20 μ L；Electrophoresis temperature is 60
DEG C, 60 v of electrophoretic voltage, 16 h of electrophoresis time.
The beneficial effects of the invention are as follows：a）New Probe bacteria total DNAs extracts kits that the present invention uses with into oral examination
Agent box is compared, relative low price, is suitable for daily monitoring, while can get the bacteria total DNA of high quality；B) present invention is right
DNA profiling amount, primer amount, annealing temperature, recurring number are improved in PCR conditions so that expanding effect is good, stability higher；
c）Gel strength, electrophoretic voltage, electrophoresis time in DGGE conditions is optimized in the present invention, to improve separating effect,
Available clearly finger-print, is conducive to atlas analysis and gel extraction；d）The present invention can be simultaneously to the oil of different time
Degradation flora is monitored, and accurately reflects the dynamic change of oil degradation bacteria structure of community；e）Be conducive to exploitation not same order
Section oil degradation function bacterium.
Description of the drawings
Fig. 1 is embodiment petrochina degradation bacteria total DNA agarose electrophoresis test map.
Fig. 2 is PCR primer agarose electrophoresis test map in embodiment.
Fig. 3 is DGGE finger-prints in embodiment.
Specific implementation mode
In order to better understand the present invention, with reference to the embodiment content that the present invention is furture elucidated.
Using oil culture solution as research object, and oil is as sole carbon source in culture solution.Experimental period is 2 months, 7 d
Sampling is primary.Take the oil-containing culture solution of 4 mL in centrifuge tube, 11500 r/min centrifuge 4 min and obtain bacterium precipitation, in strict accordance with
New Probe bacteria total DNA extracts kit specifications operate, and extract oil degradation bacteria total DNA.Oil degradation bacteria total DNA at
As result such as Fig. 1.
It selects primers F 357 and R518 as primer pair, is carried out as template using the oil degradation bacteria total DNA of kit extraction
Amplification.Wherein amplification system be 25 μ L, 10 X Master, 3 μ L DNA profilings, 1 μ L forward primers, 1 μ L reverse primers,
20 μL ddH2O；Amplification program be 94 DEG C of 4 min of pre-degeneration, 94 DEG C denaturation 1 min, 55 DEG C annealing 1 min, 72 DEG C prolong
Stretch 1 min, 25 cycles, 72 DEG C of 6 min of extension.Oil degradation bacteria PCR product imaging results such as Fig. 2.
PCR target fragments are detached by DGGE gel electrophoresises, deposition condition is gel strength 8%, denaturation range
30%-60%, 60 DEG C of electrophoresis temperature, voltage 60v, electrophoresis time 16h.After electrophoresis, by the uniform shakedown of SYBR Green solution
In gel surface, it is protected from light dyeing 30min.After dyeing, ddH is used2O slowly rinses gel, carefully opens gel bottom simultaneously
Use ddH2O is rinsed, and gel is finally transferred to 2000 gel imaging systems of Gel Doc and is observed.Oil degradation bacteria DGGE refers to
The imaging results of line collection of illustrative plates such as Fig. 3.
Claims (6)
1. a kind of method of monitoring oil degradation bacteria structure of community variation, characterized in that step is：
a）Oil degradation bacteria total DNA is extracted using New Probe bacteria total DNA extracts kits；
b）In PCR amplification system in DNA profiling amount, primer amount and PCR amplification program annealing temperature, recurring number optimization；
c）Denaturing gradient gel electrophoresis（DGGE）The optimization of middle gel strength, electrophoretic voltage, electrophoresis time, APS amounts, TEMED amounts；
d）The recycling of oil degradation bacteria dominant band is sequenced；
e）The analysis of dominant band sequencing result.
2. a kind of method of monitoring oil degradation bacteria structure of community variation according to claim 1, it is characterized in that：Total DNA
In extraction, takes the culture solution containing oil of 4 mL, 11500 r/min to centrifuge 4 min and obtain bacterium precipitation, use New Probe bacteriums
Genome DNA extraction kit extracts total DNA in being precipitated from bacterium, final DNA profiling liquor capacity is 100 μ L.
3. a kind of method of monitoring oil degradation bacteria structure of community variation according to claim 1, step b）Middle PCR amplification
After condition optimizing, specific steps include selecting primer pair F357/R518 as primer, and the ends forward primer 5' are pressed from both sides plus GC, positive
Primer is F357-GC-clamp（5'-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCCCCG CCC CCC
TAC GGG AGG CAG CAG-3'）, reverse primer F357-GC-clamp（5'-CGC CCG CCG CGC CCC GCG
CCC GGC CCG CCG CCCCCG CCC CCC TAC GGG AGG CAG CAG-3'）, using oil degradation bacteria total DNA as mould
Plate carries out PCR amplification, and PCR system is reversed including 25 μ L, 10 X Master, 3 μ L DNA profilings, 1 μ L forward primers, 1 μ L
Primer, 20 μ L ddH2O, amplification program include 94 DEG C of 4 min of pre-degeneration, 94 DEG C of 1 min of denaturation, 55 DEG C of 1 min of annealing, 72
DEG C extend 1 min, 25 cycles, 72 DEG C final extend 6 min.
4. a kind of method of monitoring oil degradation bacteria structure of community variation according to claim 1, step c）Middle denaturation ladder
After spending Gel electrophoresis conditions optimization, specific steps include acrylamide gel a concentration of 8%, and gel is denaturalized ranging from 30%-60%,
100 μ L APS and 18 μ L TEMED are added in gel solution；PCR product is with sample-loading buffer with 4：1 mixes, totally 20 μ L；Electricity
Swim temperature be 60 DEG C, 60 v of voltage, 16 h of electrophoresis time.
5. a kind of method of monitoring oil degradation bacteria structure of community variation according to claim 1, characterized in that step d）
In：The gel extraction dominant band from gel uses 30 μ L ddH2O impregnates, and 6-12 h is retained at 4 DEG C, to solution PCR amplification
After be sequenced.
6. a kind of method of monitoring oil degradation bacteria structure of community variation according to claim 1, characterized in that step e）
Middle sequencing result and U.S. biology information technology center（NCBI）Database known array carries out tetraploid rice, determines that oil drops
Solve bacterium structure of community.
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Citations (6)
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|---|---|---|---|---|
| CN1456684A (en) * | 2003-03-26 | 2003-11-19 | 中国科学院生态环境研究中心 | Induction design plan for PCR-DGGE research on environment microorgan population |
| KR20070013870A (en) * | 2005-07-27 | 2007-01-31 | 한국생명공학연구원 | Primer sets specific to the oil-degrading bacterium, nocardia sp. h17-1 and a monitoring method of the bacterium by using the said primer sets |
| CN101042049A (en) * | 2007-04-30 | 2007-09-26 | 南开大学 | Method for rapid tracking monitoring variation of oil production inpouring bacterium |
| CN101864488A (en) * | 2010-05-30 | 2010-10-20 | 大庆石油管理局 | Method for monitoring advantageous flora in microbial enhanced oil recovery |
| CN102653791A (en) * | 2012-04-25 | 2012-09-05 | 中国水产科学研究院珠江水产研究所 | Method for confirming geographic fish sources on basis of PCR (Polymerase Chain Reaction)-DGGE (Denaturing Gradient Gel Electrophoresis) technology |
| CN104152394A (en) * | 2014-06-29 | 2014-11-19 | 北京大学工学院包头研究院 | Method for directionally activating microorganisms with oil recovery functions in crude oil |
-
2017
- 2017-05-03 CN CN201710303202.7A patent/CN108796096A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456684A (en) * | 2003-03-26 | 2003-11-19 | 中国科学院生态环境研究中心 | Induction design plan for PCR-DGGE research on environment microorgan population |
| KR20070013870A (en) * | 2005-07-27 | 2007-01-31 | 한국생명공학연구원 | Primer sets specific to the oil-degrading bacterium, nocardia sp. h17-1 and a monitoring method of the bacterium by using the said primer sets |
| CN101042049A (en) * | 2007-04-30 | 2007-09-26 | 南开大学 | Method for rapid tracking monitoring variation of oil production inpouring bacterium |
| CN101864488A (en) * | 2010-05-30 | 2010-10-20 | 大庆石油管理局 | Method for monitoring advantageous flora in microbial enhanced oil recovery |
| CN102653791A (en) * | 2012-04-25 | 2012-09-05 | 中国水产科学研究院珠江水产研究所 | Method for confirming geographic fish sources on basis of PCR (Polymerase Chain Reaction)-DGGE (Denaturing Gradient Gel Electrophoresis) technology |
| CN104152394A (en) * | 2014-06-29 | 2014-11-19 | 北京大学工学院包头研究院 | Method for directionally activating microorganisms with oil recovery functions in crude oil |
Non-Patent Citations (1)
| Title |
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| 汤瑶: "基于 PCR-DGGE 技术的原油降解菌群落结构分析", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
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