CN109423455A - A kind of Rhodococcus equi and its identification method and application - Google Patents
A kind of Rhodococcus equi and its identification method and application Download PDFInfo
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Abstract
The present invention relates to a kind of Rhodococcus equi of microorganisms technical field and its identification method and applications.For the gene order of Rhodococcus equi 16S rDNA as shown in SEQ ID NO.1, it in the deposit number of China typical culture collection center is CCTCC NO:M2016109 that bacterial strain, which is SINOPEC05,.Abundance of the bacterial strain above the oil-gas reservoir in soil is positively correlated with oil-gas reservoir floating gaseous hydrocarbon concentration, can be used as oil gas microorganism, Indication of Oil-Gas hiding top Hydrocarbon leakage high level region.Simultaneously, abundance of the bacterial strain above the oil-gas reservoir in soil is higher, without amplification cultivation, by improved primer can the 16S rDNA conserved sequence to the Rhodococcus equi SINOPEC05 in soil expand, it can accurately and efficiently judge the concentration of oil-gas reservoir floating gaseous hydrocarbon, solve that traditional Physiology and biochemistry detection cycle is long, the deficiency high to sample culturing object purity requirement.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of Rhodococcus equi and its identification method and application.
Background technique
The core of microbial prospecting technology is the identification of oil and gas indication microorganism.It is ground in exploration process under normal conditions
The relative abundance of the objective microbe studied carefully in the environment is very low, therefore need to stimulate oil-gas reservoir under traditional special condition of culture
The quick breeding of hydrocarbon oxidation bacterium in environment and non-oil-gas reservoir environment.Although the above method can obtain the quantity of microorganism
Information, but the quantity information is in fact higher than the micro organism quantity in situ environment at geometric multiple, inevitably covers
The difference in oil-gas reservoir and non-oil-gas reservoir environment on micro organism quantity has been covered, while can not also obtain uncultured microorganisms
Information.In addition, the growth of oil gas microorganism except being influenced by hydro carbons, also suffers from the influence of ambient enviroment, as soil humidity,
PH value, salinity or in which crucial ion, nutriment and disturbance etc..If the power relatively of development of microorganisms be by environment because
Caused by element, it will cause rich accumulation of oil and gas or poor illusions.
Rhodococcus equi (Rhodococcus equi) is former to be known as corynebacterium equi, is once attributed to corynebacterium, but in recent years
After foreign scholar analyzes its cell wall, it is found that some corynebacterias do not meet the characteristic of corynebacterium, then arranged
Enter Rhod.Rhod can survive in the environment of pollution under field conditions (factors), therefore can be used as connecing for bio-decontaminated
Kind mediator, and good effect has been obtained using Rhodococcus sp degraded oil dirt.Above spring scenery oilfield is hidden in soil
It was found that Rhodococcus equi accounts for biggish abundance, in order to further determine the relationship between the bacterium and Microbial Prospecting of Oil and Gas, to its allusion quotation
The acquisition of type bacterial strain and detection technique carry out developmental research.
Currently, there are two types of the common Testing and appraisal methods of Rhodococcus equi: the identification of 1. Physiology and biochemistries, it is pure by what is isolated
Bacterium carries out qualitative research to microbial physiology biochemistry, and this method period is long and proposes higher requirement to culture purity;2.
Gene carries out the sequencing of 16S rDNA after extracting, this method still needs to obtain pure culture progress full genome extraction, then by general
16S rDNA primer obtains conserved sequence and is sequenced.
Therefore, in order to quick, the accurate characteristic information for obtaining Rhodococcus equi above oil gas field, it is badly in need of to its typical case
The acquisition of bacterial strain and the detection technique in mixed system carry out developmental research.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of Rhodococcus equi and its mirror in view of the deficiencies of the prior art
Determine method, abundance of the bacterial strain above the oil-gas reservoir in soil is higher, and the Rhodococcus equi is above the oil-gas reservoir in soil
Abundance is positively correlated with oil-gas reservoir floating gaseous hydrocarbon concentration, therefore the bacterial strain can be used as oil gas microorganism, Indication of Oil-Gas hiding top
Hydrocarbon leakage high level region.
The present invention also provides a kind of methods using Rhodococcus equi Indication of Oil-Gas hiding floating gaseous hydrocarbon concentration, should
Method is expanded using the 16S rDNA conserved sequence of Rhodococcus equi described in improved primer pair, according to mesh in amplified production
The brightness for marking band, can accurately and efficiently judge the concentration of oil-gas reservoir floating gaseous hydrocarbon.
For this purpose, one aspect of the present invention provides a kind of Rhodococcus equi, the gene order of 16S rDNA such as SEQ ID NO.1
It is shown.
According to the present invention, the bacterial strain of the Rhodococcus equi is SINOPEC05, in China typical culture collection center
Deposit number is CCTCC NO:M2016109.
According to the present invention, the biological property of the Rhodococcus equi are as follows: colonial morphology of the bacterial strain on solid medium
Are as follows: the colonial morphology on solid medium is rounded, white, flat, smooth wet, edge blurry, under the microscope
Cell is positive in variform, round and smooth, Gram's staining reaction.
In certain embodiments of the present invention, the solid medium is methanol solid medium or butanol solid
Culture medium;Specifically, the composition of the methanol solid medium is following (in 1L deionized water):
And/or the composition of the butanol solid medium is following (in 1L deionized water):
In some preferred embodiments of the invention, the pH of the methanol solid medium and ethyl alcohol solid medium
Value is 7-8;Preferably, the pH value of the methanol solid medium and ethyl alcohol solid medium is 7.
According to the present invention, abundance and oil-gas reservoir floating gaseous hydrocarbon of the Rhodococcus equi above the oil-gas reservoir in soil are dense
Degree is positively correlated.
Second aspect of the present invention provides a kind of identification method of Rhodococcus equi as described in the first aspect of the invention, packet
It includes and 16S rDNA sequence or the partial sequence sequencing of 16S rDNA, and the 16S of the strain to be tested is carried out to strain to be tested
The consistency of rDNA sequence or the partial sequence of 16S rDNA and the sequence of such as SEQ ID NO.1 is preferably described 95% or more
The consistency of the partial sequence and the sequence such as SEQ ID NO.1 of the 16S rDNA sequence or 16S rDNA of strain to be tested exists
97% or more, more preferably the 16S rDNA sequence of the strain to be tested or the partial sequence of 16S rDNA and such as SEQ ID NO.1
Sequence consistency 99% or more, the 16S rDNA sequence of the most preferably described strain to be tested or the partial order of 16S rDNA
The consistency of the sequence of column and such as SEQ ID NO.1 then identifies that the strain to be tested is the Ma Hongqiu 99.5% or more
Bacterium.
In the present invention, using primer pair 1 (SEQ ID NO:2 and SEQ ID NO:3), primer pair 2 (SEQ ID NO:4 and
SEQ ID NO:5), primer pair 3 (SEQ ID NO:6 and SEQ ID NO:7) and (SEQ ID NO:8 and the SEQ ID of primer pair 4
One of) NO:9 it is used as primer pair, carries out PCR amplification with the partial sequence of the 16S rDNA obtained, and will be described
The partial sequence of 16S rDNA is sequenced to identify the strain to be tested compared with the corresponding sequence of such as SEQ ID NO:1.
In other embodiments of the invention, the method also includes combining the biological characteristics of strain to be tested
To identify the strain to be tested compared with the biological characteristics of the Rhodococcus equi described in the first aspect present invention.Specifically
Ground, the biological characteristics are as follows: the colonial morphology on solid medium is rounded, white, flat, smooth wet, side
Edge is fuzzy, and in variform, round and smooth, Gram's staining reaction is positive for cell under the microscope.The solid medium
For methanol solid medium or butanol solid medium;The pH value of the solid medium is 7-8.
In general, it is reflected using the partial sequence of the 16S rDNA sequence of microorganism or 16S rDNA to microorganism
Determine to have the advantages that for example as follows: quick kind analysis is carried out to unknown sample;Tutorial message is provided for biochemical identification;For difficulty
It the use of 16SrDNA identification is only available identification of means to obtain the bacterium of pure culture, such as bacterial parasite.
But some microorganisms, since interspecific difference is small, kind cannot be identified by relying solely on 16S rDNA identification.Need it
Its identification method supplement, such as the biological characteristics of microorganism.
Third aspect present invention provides the Rhodococcus equi of one kind as described in relation to the first aspect and hides floating gaseous state in Indication of Oil-Gas
Application in hydrocarbon concentration.
According to the present invention, the described application specifically includes the following steps:
A extracts the full-length genome of Soil Microorganism above oil-gas reservoir, obtains the template for being used for PCR amplification;
B, design primer carry out PCR amplification to template, obtain pcr amplification product;
C carries out agarose gel electrophoresis to pcr amplification product, according to the brightness of target stripe, judges that oil-gas reservoir is floated
The concentration of gaseous hydrocarbon.
In certain embodiments of the present invention, the primer is selected from a pair of following primer centering:
Upstream primer 1:5 '-CGAAAGCGTGGGTAGCGAACAGGATTAG-3 ',
Downstream primer 1:5 '-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3 ';
Upstream primer 2:5 '-CTCAACTGCGGGCTTGCAGGCGATACGG-3 ',
Downstream primer 2:5 '-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3 ';
Upstream primer 3:5 '-CGGGTTGTAAACCTCTTTCAGCAGGGAC-3 ',
Downstream primer 3:5 '-GATCTGCGATTACTAGCGACTCCGACTTCA-3 ';
With,
Upstream primer 4:5 '-GGGACTGAGACACGGCCCAGACTCCTAC-3 ',
Downstream primer 4:5 '-GATCTGCGATTACTAGCGACTCCGACTTCA-3 '.
In the present invention, the meaning of term " oil-gas reservoir above soil " are as follows: in oil-gas reservoir oil gas high abundance range it is vertical on
Soil in 1 meter of square near surface.
The invention has the benefit that the present invention is from screening one plant of Rhodococcus equi in soil above typical oil and gas reservoirs
SINOPEC05, abundance of the bacterial strain above the oil-gas reservoir in soil are positively correlated with oil-gas reservoir floating gaseous hydrocarbon concentration, can use
Make oil gas microorganism, Indication of Oil-Gas hiding top Hydrocarbon leakage high level region.Meanwhile the bacterial strain is above the oil-gas reservoir in soil
Abundance is higher, is not necessarily to amplification cultivation, can be to the 16S of the Rhodococcus equi SINOPEC05 in soil by improved primer
RDNA conserved sequence is expanded, and according to the brightness of target stripe in amplified production, can accurately and efficiently be judged in oil-gas reservoir
The concentration of floating gaseous hydrocarbon, solves that traditional Physiology and biochemistry detection cycle is long, the deficiency high to sample culturing object purity requirement.Separately
Outside, which can survive in the environment of pollution, the inoculation mediator as bio-decontaminated.
Detailed description of the invention
Illustrate the present invention below in conjunction with attached drawing.
Fig. 1 shows phylogenetic tree of the Rhodococcus equi SINOPEC05 based on 16S rDNA of the invention.
Fig. 2 is the Gram's staining figure of Rhodococcus equi SINOPEC05 of the present invention.
Fig. 3 is the electron microscope of Rhodococcus equi SINOPEC05 of the present invention.
Culture presevation
Classification naming: Rhodococcus equi (Rhodococcus equi);Strain number: SINOPEC05
Preservation mechanism: China typical culture collection center
Preservation mechanism abbreviation: CCTCC
Address: Wuhan University's Life Science College
Preservation date: on March 14th, 2016
Collection is registered on the books number: CCTCC NO:M2016109.
Specific embodiment
To be readily understood by the present invention, below in conjunction with attached drawing, the present invention will be described in detail.
To seek a kind of indicator microoraganism for accurately and efficiently judging oil-gas reservoir floating gaseous hydrocarbon concentration, the present invention passes through
Unremitting research and probe is in from one plant of bacterial strain abundance is screened above oil-gas reservoir in soil with oil-gas reservoir floating gaseous hydrocarbon concentration
Positively related microorganism fungus kind is accredited as Rhodococcus equi SINOPEC05 (Rhodococcus equi through separation
SINOPEC05);Abundance of the bacterial strain above the oil-gas reservoir in soil is higher, is not necessarily to amplification cultivation, is by improved primer
Can the 16S rDNA conserved sequence to the Rhodococcus equi SINOPEC05 in soil expand, according to target patch in amplified production
The brightness of section, can accurately and efficiently judge the concentration of oil-gas reservoir floating gaseous hydrocarbon, solve traditional Physiology and biochemistry detection cycle
Long, high to sample culturing object purity requirement deficiency.The present invention is based on what above-mentioned discovery was made.
Therefore, Rhodococcus equi according to the present invention can accurately and efficiently judge oil-gas reservoir floating gaseous hydrocarbon concentration,
Acquisition is screened and cultivated using following screening and culturing medium (methanol medium or butanol culture medium);Wherein, the methanol
The composition of culture medium is following (in 1L deionized water):
And/or the composition of the butanol culture medium is following (in 1L deionized water):
Specifically, the composition of the methanol medium is as follows: KH2PO41.0g/L, Na2HPO4·12H2O 2.9g/L,
MgSO4·7H2O 0.32g/L, (NH4)2SO43.0g/L, CaCl20.2g/L, KNO31.0g/L, methanol 2.0g/L;
The composition of the butanol culture medium is as follows: KH2PO41.0g/L, Na2HPO4·12H2O 2.9g/L, MgSO4·
7H2O 0.32g/L, (NH4)2SO43.0g/L, CaCl20.2g/L, KNO31.0g/L, butanol 2.0g/L.
The methanol medium and the pH value of butanol culture are equal are as follows: 7.0.
The screening technique of Rhodococcus equi of the present invention the following steps are included:
(1) it is formed according to above-mentioned culture medium and prepares culture medium, through sterilizing under high temperature (121 DEG C) high pressure (0.15MPa)
Then 20min is used after ultraviolet radiation sterilization 20min in clean bench.2% (weight is added in liquid medium
Amount/volume) agar, be poured into after autoclave sterilization dissolves in culture dish cooling, prepare corresponding solid culture
Base plate.
(2) 10g spring scenery oilfield hiding top pedotheque is weighed, is added in the sterilized physiological saline of 100ml,
It is stood after fulling shake.100 μ L of supernatant is taken to be inoculated into screening and culturing medium in superclean bench, in 30 DEG C of temperature, shaking table
Shaking table culture 3d under revolving speed 200r/min.Culture is diluted to 10 respectively using the normal saline solution after sterilizing-5、10-6With
10-7Afterwards, 100 μ L dilutions uniform coated plate on corresponding solid screening and culturing medium is taken respectively, is subsequently placed at 30 DEG C and is cultivated.
The monoclonal colonies grown can be observed in solid culture primary surface after 3d, adopted with transfer needle picking typical case's monoclonal colonies
It is reinoculated in solid screening and culturing medium with streak plating and carries out purifying culture, purifying obtains the pure of the bacterial strain afterwards three times
Culture.
(3) morphology and molecular biology identification are carried out to the pure culture of the bacterial strain
Find that the colonial morphology on solid medium of the bacterial strain is rounded, white, flat, smooth wet through observation
Profit, edge blurry.Cell under the microscope is in variform, round and smooth, and micro- electron microscope is as shown in Fig. 3.
Gram stain analysis is carried out to the bacterial strain, it is found that the bacterial strain is gram-positive bacteria, Gram's staining figure is such as
Shown in Fig. 2.
DNA extraction is carried out to the pure culture of above-mentioned bacterial strains, simultaneously identification is sequenced in PCR amplification, then passes through 16S rDNA gram
Grand gene analysis is sequenced, and will have been stepped in measured sequence and GenBank/EMBL/DDBJ database using BLAST
The sequence of record carries out tetraploid rice analysis, utilizes soil above the methods of bioinformatics analysis exploration area typical oil and gas reservoirs
Middle species abundance and opposite composition characteristic, obtain one plant of higher novel strain of bacterial abundance, the bacterial strain is identified and names
For Rhodococcus equi SINOPEC05,16S rDNA sequence length is 1427kp, and full length sequence is as shown in SEQ ID NO.1.Fig. 1
Show phylogenetic tree of the Rhodococcus equi SINOPEC05 based on 16S rDNA of the invention.The bacterial strain is in March, 2016
14, preservation is carried out in China typical culture collection center (referred to as: CCTCC), deposit number: CCTCC NO:
M2016109。
Identified, abundance of the Rhodococcus equi above the oil-gas reservoir in soil is in oil-gas reservoir floating gaseous hydrocarbon concentration
It is positively correlated.Therefore, the answering in Indication of Oil-Gas hiding floating gaseous hydrocarbon concentration the present invention provides a kind of above-mentioned Rhodococcus equi
With.
According to the present invention, the described application specifically includes the following steps:
A extracts the full-length genome of Soil Microorganism above oil-gas reservoir, obtains the template for being used for PCR amplification;
B carries out PCR expansion to template according to the 16S rDNA sequence design specific primer of Rhodococcus equi SINOPEC05
Increase, obtains pcr amplification product;
The primer is selected from a pair of following primer centering:
Upstream primer 1:5 '-CGAAAGCGTGGGTAGCGAACAGGATTAG-3 ',
Downstream primer 1:5 '-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3 ';
Upstream primer 2:5 '-CTCAACTGCGGGCTTGCAGGCGATACGG-3 ',
Downstream primer 2:5 '-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3 ';
Upstream primer 3:5 '-CGGGTTGTAAACCTCTTTCAGCAGGGAC-3 ',
Downstream primer 3:5 '-GATCTGCGATTACTAGCGACTCCGACTTCA-3 ';
With,
Upstream primer 4:5 '-GGGACTGAGACACGGCCCAGACTCCTAC-3 ',
Downstream primer 4:5 '-GATCTGCGATTACTAGCGACTCCGACTTCA-3 '.
Above-mentioned primer pair can carry out PCR amplification to ambient soil sample DNA, be quickly, in precise Identification pedotheque
It is no that there are the Rhodococcus equis;
C carries out agarose gel electrophoresis to pcr amplification product, according to the brightness of target stripe, judges that oil-gas reservoir is floated
The concentration of gaseous hydrocarbon;Specifically, the brightness of target stripe is stronger, and the concentration of oil-gas reservoir floating gaseous hydrocarbon is higher.
Embodiment
To keep the present invention easier to understand, below in conjunction with embodiment, present invention be described in more detail, these realities
Apply example only serve it is illustrative, it is not limited to application range of the invention.If raw material used in the present invention or component nothing
Specified otherwise can be made by commercial sources or conventional method.
Embodiment 1: pedotheque above oil-gas reservoir is identified using Rhodococcus equi SINOPEC05.
(1) four pairs of specific primers are designed according to the 16S rDNA conserved sequence of Rhodococcus equi SINOPEC05, be respectively as follows:
Upstream primer 1:5 '-CGAAAGCGTGGGTAGCGAACAGGATTAG-3 ',
Downstream primer 1:5 '-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3 ';
Upstream primer 2:5 '-CTCAACTGCGGGCTTGCAGGCGATACGG-3 ',
Downstream primer 2:5 '-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3 ';
Upstream primer 3:5 '-CGGGTTGTAAACCTCTTTCAGCAGGGAC-3 ',
Downstream primer 3:5 '-GATCTGCGATTACTAGCGACTCCGACTTCA-3 ';
Upstream primer 4:5 '-GGGACTGAGACACGGCCCAGACTCCTAC-3 ',
Downstream primer 4:5 '-GATCTGCGATTACTAGCGACTCCGACTTCA-3 '.
Wherein, the size for the target stripe that primer 1 expands is 355bp, and the size of the target stripe of primer 2 amplification is
501bp, the size for the target stripe that primer 3 expands are 919bp, and the size for the target stripe that primer 4 expands is 1023bp.
(2) full-length genome of Soil Microorganism is extracted.
Soil and each 0.5g of Soil Background above oil-gas reservoir are weighed, is added in Lysing Matrix E Tube respectively, is used
Cell crushing instrument carry out clasmatosis (speed 4.5m/s, 30s, 4 time), broken cell according toSPIN Kit
The specification of for Soil (MP Biomedicals Biomedicines, Inc., U.S. product) carries out subsequent operation, obtains soil
The full-length genome of microorganism in sample, the template as PCR amplification.
(3) PCR amplification is carried out respectively using 4 pairs of primers.
PCR amplification system is equal are as follows:
Following reagent is sequentially added into 0.2mL PCR pipe:
Adding distilled water to final volume is 50 μ l.
PCR amplification program is equal are as follows:
94 DEG C of initial denaturation 5min;
94 DEG C of denaturation 30s, 57 DEG C of annealing 90s, 72 DEG C of extension 1min, 30 recycle;
72 DEG C of extension 10min.
(4) agarose gel electrophoresis.
Above-mentioned pcr amplification product is carried out to 1% agarose gel electrophoresis, is mentioned in soil above oil-gas reservoir as the result is shown
In the full-length genome taken amplification obtain with design equal length genetic fragment (primer pair 1:355bp, primer pair 2:501bp,
Primer pair 3:919bp, primer pair 4:1023bp), and corresponding piece is not amplified in the full-length genome extracted in Soil Background
Section.Glue recycling is carried out to target stripe and is sequenced, sequencing result shows that target when expanded band is design of primers is protected
Keep sequence.Illustrate that the bacterial strain can be used as oil gas microorganism, it being capable of specific Indication of Oil-Gas hiding top Hydrocarbon leakage high level region.
It should be noted that embodiment described above for explaining only the invention, is not constituted to of the invention any
Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word used in it is descriptive
With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it relates to
And specific method, material and embodiment, it is not intended that the present invention is limited to particular case disclosed in it, on the contrary, this hair
It is bright to can be extended to other all methods and applications with the same function.
SEQUENCE LISTING
<110>Sinopec Group;China Petroleum & Chemical Co., Ltd., Research Institute of Petroleum Exploration and Development
<120>a kind of Rhodococcus equi and its identification method and application
<130> 2017
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 1427
<212> DNA
<213>Rhodococcus equi (the 16S rDNA of SINOPEC05)
<400> 1
ccagtggcgc gtgcttacac atgcagtcga gcggtaaggc cctttcgggg gtacacgagc 60
ggcgaacggg tgagtaacac gtgggtgatc tgccctgcac tctgggataa gcctgggaaa 120
ctgggtctaa taccggatat gagctcctgt cgcatggcgg gggttggaaa ggtttactgg 180
tgcaggatgg gcccgcggcc tatcagcttg ttggtggggt aatggcctac caaggcgacg 240
acgggtagcc ggcctgagag ggcgaccggc cacactggga ctgagacacg gcccagactc 300
ctacgggagg cagcagtggg gaatattgca caatgggcga aagcctgatg cagcgacgcc 360
gcgtgaggga tgacggcctt cgggttgtaa acctctttca gcagggacga agcgcaagtg 420
acggtacctg cagaagaagc accggccaac tacgtgccag cagccgcggt aatacgtagg 480
gtgcgagcgt tgtccggaat tactgggcgt aaagagctcg taggcggttt gtcgcgtcgt 540
ctgtgaaaac ccgcagctca actgcgggct tgcaggcgat acgggcagac ttgagtactg 600
caggggagac tggaattcct ggtgtagcgg tgaaatgcgc agatatcagg aggaacaccg 660
gtggcgaagg cgggtctctg ggcagtaact gacgctgagg agcgaaagcg tgggtagcga 720
acaggattag ataccctggt agtccacgcc gtaaacggtg ggcgctaggt gtgggtttcc 780
ttccacggga tccgtgccgt agctaacgca ttaagcgccc cgcctgggga gtacggccgc 840
aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag cggcggagca tgtggattaa 900
ttcgatgcaa cgcgaagaac cttacctggg tttgacatat accggaaacg cctagagata 960
ggtgccccct tgtggtcggt atacaggtgg tgcatggctg tcgtcagctc gtgtcgtgag 1020
atgttgggtt aagtcccgca acgagcgcaa cccttgtcct gtgttgccag cgcgtaatgg 1080
cggggactcg caggagactg ccggggtcaa ctcggaggaa ggtggggatg acgtcaagtc 1140
atcatgcccc ttatgtccag ggcttcacac atgctacaat ggccggtaca gagggctgcg 1200
ataccgtgag gtggagcgaa tcccttaaag ccggtctcag ttcggatcgg ggtctgcaac 1260
tcgaccccgt gaagtcggag tcgctagtaa tcgcagatca gcaacgctgc ggtgaatacg 1320
ttcccgggcc ttgtacacac cgcccgtcac gtcatgaaag tcggtaacac ccgaagccgg 1380
tggcctaacc cctcgtggag ggagccgtcg aaggtggatc ggcgggg 1427
<210> 2
<211> 28
<212> DNA
<213>upstream primer 1(artificial sequence)
<400> 2
cgaaagcgtg ggtagcgaac aggattag 28
<210> 3
<211> 28
<212> DNA
<213>downstream primer 1(artificial sequence)
<400> 3
acaagggttg cgctcgttgc gggactta 28
<210> 4
<211> 28
<212> DNA
<213>upstream primer 2(artificial sequence)
<400> 4
ctcaactgcg ggcttgcagg cgatacgg 28
<210> 5
<211> 28
<212> DNA
<213>downstream primer 2(artificial sequence)
<400> 5
acaagggttg cgctcgttgc gggactta 28
<210> 6
<211> 28
<212> DNA
<213>upstream primer 3(artificial sequence)
<400> 6
cgggttgtaa acctctttca gcagggac 28
<210> 7
<211> 30
<212> DNA
<213>downstream primer 3(artificial sequence)
<400> 7
gatctgcgat tactagcgac tccgacttca 30
<210> 8
<211> 28
<212> DNA
<213>upstream primer 4(artificial sequence)
<400> 8
gggactgaga cacggcccag actcctac 28
<210> 9
<211> 30
<212> DNA
<213>downstream primer 4(artificial sequence)
<400> 9
gatctgcgat tactagcgac tccgacttca 30
Claims (10)
1. a kind of Rhodococcus equi, the gene order of 16S rDNA is as shown in SEQ ID NO.1.
2. Rhodococcus equi according to claim 1, which is characterized in that the bacterial strain of the Rhodococcus equi is SINOPEC05,
The deposit number of China typical culture collection center is CCTCC NO:M2016109.
3. Rhodococcus equi according to claim 1 or 2, which is characterized in that the biological property of the Rhodococcus equi are as follows:
Colonial morphology on solid medium is rounded, white, flat, smooth wet, edge blurry, and cell under the microscope is in
Variform, round and smooth, Gram's staining reaction is positive.
4. Rhodococcus equi according to claim 3, which is characterized in that the solid medium is methanol solid medium
Or butanol solid medium;The pH value of the solid medium is 7-8.
5. Rhodococcus equi described in any one of -4 according to claim 1, which is characterized in that the Rhodococcus equi is in oil-gas reservoir
Abundance in necromancer earth is positively correlated with oil-gas reservoir floating gaseous hydrocarbon concentration.
6. a kind of method of identification Rhodococcus equi as described in any one of claim 1-5 comprising carry out 16S to strain to be tested
RDNA sequence or the sequencing of the partial sequence of 16S rDNA, and the portion of the 16S rDNA sequence of the strain to be tested or 16S rDNA
The consistency of sub-sequence and the sequence such as SEQ ID NO.1 is 95% or more, the 16S rDNA sequence of the preferably described strain to be tested
Or the consistency of the partial sequence of 16S rDNA and the sequence such as SEQ ID NO.1 is 97% or more, the more preferable bacterium to be measured
The 16S rDNA sequence or the partial sequence of 16S rDNA and the consistency of the sequence such as SEQ ID NO.1 of strain 99% or more,
The 16S rDNA sequence of the most preferably described strain to be tested or the partial sequence and one of the sequence such as SEQ ID NO.1 of 16S rDNA
Cause property then identifies that the strain to be tested is the Rhodococcus equi 99.5% or more.
7. according to the method described in claim 6, it is characterized in that, the method also includes combining the biology of strain to be tested
To reflect to the strain to be tested compared with the biological characteristics of characteristic Rhodococcus equi described in any one of claim 1-5
It is fixed;
Preferably, the biological characteristics are as follows: the colonial morphology on solid medium is rounded, white, flat, smooth
Wet, edge blurry, in variform, round and smooth, Gram's staining reaction is positive for cell under the microscope;
It is further preferred that the solid medium is methanol solid medium or butanol solid medium;The solid training
The pH value for supporting base is 7-8.
8. a kind of answering in Indication of Oil-Gas hiding floating gaseous hydrocarbon concentration such as Rhodococcus equi of any of claims 1-7
With.
9. application according to claim 8, which comprises the following steps:
A extracts the full-length genome of Soil Microorganism above oil-gas reservoir, obtains the template for being used for PCR amplification;
B, design primer carry out PCR amplification to template, obtain pcr amplification product;
C carries out agarose gel electrophoresis to pcr amplification product and judges oil-gas reservoir floating gaseous hydrocarbon according to the brightness of target stripe
Concentration.
10. application according to claim 9, which is characterized in that the primer is selected from a pair of following primer centering:
Upstream primer 1:5 '-CGAAAGCGTGGGTAGCGAACAGGATTAG-3 ',
Downstream primer 1:5 '-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3 ';
Upstream primer 2:5 '-CTCAACTGCGGGCTTGCAGGCGATACGG-3 ',
Downstream primer 2:5 '-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3 ';
Upstream primer 3:5 '-CGGGTTGTAAACCTCTTTCAGCAGGGAC-3 ',
Downstream primer 3:5 '-GATCTGCGATTACTAGCGACTCCGACTTCA-3 ';
With,
Upstream primer 4:5 '-GGGACTGAGACACGGCCCAGACTCCTAC-3 ',
Downstream primer 4:5 '-GATCTGCGATTACTAGCGACTCCGACTTCA-3 '.
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CN109423458A (en) * | 2017-08-30 | 2019-03-05 | 中国石油化工股份有限公司 | A kind of Rhodococcus sp and its identification method and application |
CN109423458B (en) * | 2017-08-30 | 2022-08-19 | 中国石油化工股份有限公司 | Rhodococcus and identification method and application thereof |
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