CN109423455B - Rhodococcus equi, and identification method and application thereof - Google Patents
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Abstract
The invention relates to Rhodococcus equi, an identification method and application thereof, belonging to the technical field of microorganisms. The gene sequence of the rhodococcus equi 16S rDNA is shown in SEQ ID NO.1, and the strain is SINOPEC05 with the preservation number of CCTCC NO: m2016109. The abundance of the strain in the soil above the oil and gas reservoir is in positive correlation with the concentration of the floating gaseous hydrocarbon in the oil and gas reservoir, and the strain can be used as an oil and gas microorganism and indicates a high-value hydrocarbon leakage area above the oil and gas reservoir. Meanwhile, the strain has high abundance in soil above an oil and gas reservoir, amplification culture is not needed, the 16S rDNA conserved sequence of Rhodococcus equi SINOPEC05 in the soil can be amplified through improved primers, the concentration of floating gaseous hydrocarbons in the oil and gas reservoir can be accurately and efficiently judged, and the defects of long period and high requirement on the purity of a sample culture in the traditional physiological and biochemical detection process are overcome.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to rhodococcus equi, and an identification method and application thereof.
Background
The core of microbial exploration technology is the identification of hydrocarbon-indicating microorganisms. The relative abundance of target microorganisms studied in exploration procedures is typically low in the environment, and thus rapid propagation of hydrocarbon oxidizing bacteria in hydrocarbon reservoir environments and non-hydrocarbon reservoir environments is stimulated under conventional specific culture conditions. Although the method can obtain the quantity information of the microorganisms, the quantity information is actually geometrically multiplied higher than the quantity of the microorganisms in an in-situ environment, so that the difference of the quantity of the microorganisms in an oil-gas reservoir and a non-oil-gas reservoir environment is inevitably covered, and meanwhile, the information of the non-culturable microorganisms cannot be obtained. In addition, the growth of oil and gas microorganisms is affected by hydrocarbons and the surrounding environment, such as the humidity, pH, salinity of the soil or key ions, nutrients and disturbances therein. If the relative strength of microbial development is caused by environmental factors, it can lead to the illusion of oil and gas enrichment or depletion.
Rhodococcus equi (Rhodococcus equi) originally called Corynebacterium, was assigned to Corynebacterium, but in recent years, foreign scholars analyzed the cell wall and found that some Corynebacterium did not meet the characteristics of Corynebacterium, and was subsequently classified into Rhodococcus. Rhodococcus can survive in a polluted environment under natural conditions and thus can be used as an inoculation medium for biological decontamination, and has obtained good effects on degrading oil dirt by using Rhodococcus. The rhodococcus equi is found to be in large abundance in the soil above the oil and gas reservoir of the vernal light oil field, and in order to further determine the relationship between the rhodococcus equi and the oil and gas microbial exploration, the acquisition and detection technology of typical strains of the rhodococcus equi is developed and researched.
At present, two common detection and identification methods for rhodococcus equi exist: 1. performing physiological and biochemical identification, and performing qualitative research on the physiological and biochemical characteristics of microorganisms through separated pure bacteria, wherein the method has long period and puts higher requirements on the purity of a culture; 2. after gene extraction, 16S rDNA sequencing is carried out, and the method still needs to obtain pure culture for whole gene extraction and then obtain a conserved sequence through a universal 16S rDNA primer for sequencing.
Therefore, in order to rapidly and accurately obtain the characteristic information of rhodococcus equi above the oil and gas field, the development and research of the acquisition of typical strains and detection technology in a mixed system are urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing the Rhodococcus equi and the identification method thereof aiming at the defects of the prior art, the abundance of the Rhodococcus equi in the soil above the oil-gas reservoir is higher, and the abundance of the Rhodococcus equi in the soil above the oil-gas reservoir is positively correlated with the concentration of floating gaseous hydrocarbons in the oil-gas reservoir, so that the Rhodococcus equi can be used as oil-gas microorganisms to indicate a high-value hydrocarbon leakage area above the oil-gas reservoir.
The invention also provides a method for indicating the concentration of the floating gaseous hydrocarbon in the oil and gas reservoir by using the rhodococcus equi, the method adopts an improved primer to amplify the 16S rDNA conserved sequence of the rhodococcus equi, and the concentration of the floating gaseous hydrocarbon in the oil and gas reservoir can be accurately and efficiently judged according to the brightness of a target strip in an amplification product.
Therefore, the invention provides Rhodococcus equi, the 16S rDNA gene sequence of which is shown in SEQ ID NO. 1.
According to the invention, the Rhodococcus equi strain is SINOPEC05, and the preservation number in China center for type culture Collection is CCTCC NO: m2016109.
According to the invention, the Rhodococcus equi is characterized biologically by: the colony morphology of the strain on the solid culture medium is as follows: the colony on the solid culture medium is round, white, flat, smooth and moist, has blurred edges, the cells under a microscope are in various shapes and smooth, and the gram staining reaction is positive.
In some embodiments of the invention, the solid medium is methanol solid medium or butanol solid medium; specifically, the composition of the methanol solid medium is as follows (in 1L of deionized water):
and/or the composition of the butanol solid medium is as follows (in 1L deionized water):
in some preferred embodiments of the present invention, the methanol solid medium and the ethanol solid medium each have a pH of 7 to 8; preferably, the pH value of the methanol solid culture medium and the pH value of the ethanol solid culture medium are both 7.
According to the invention, the abundance of rhodococcus equi in the soil above the oil and gas reservoir is positively correlated with the concentration of floating gaseous hydrocarbons in the oil and gas reservoir.
In a second aspect, the present invention provides a method of identifying Rhodococcus equi according to the first aspect of the invention, which comprises the steps of carrying out 16S rDNA sequence or 16S rDNA partial sequence sequencing on a strain to be tested, and the consistency of the 16S rDNA sequence or the 16S rDNA partial sequence of the strain to be detected and the sequence of SEQ ID No.1 is more than 95%, preferably, the consistency of the 16S rDNA sequence or the 16S rDNA partial sequence of the strain to be detected and the sequence of SEQ ID No.1 is more than 97%, more preferably, the consistency of the 16S rDNA sequence or the 16S rDNA partial sequence of the strain to be detected and the sequence of SEQ ID No.1 is more than 99%, most preferably, the consistency of the 16S rDNA sequence or the 16S rDNA partial sequence of the strain to be detected and the sequence of SEQ ID No.1 is more than 99.5%, and then identifying the strain to be detected as the rhodococcus equi.
In the invention, one of a primer pair 1(SEQ ID NO:2 and SEQ ID NO:3), a primer pair 2(SEQ ID NO:4 and SEQ ID NO:5), a primer pair 3(SEQ ID NO:6 and SEQ ID NO:7) and a primer pair 4(SEQ ID NO:8 and SEQ ID NO:9) is used as a primer pair, PCR amplification is carried out to obtain a partial sequence of the 16S rDNA, and the partial sequence of the 16S rDNA is sequenced and compared with a corresponding sequence of the SEQ ID NO:1 to identify the strain to be detected.
In other embodiments of the present invention, the method further comprises identifying the test strain in conjunction with comparing the biological properties of the test strain to the biological properties of Rhodococcus equi in the first aspect of the invention. Specifically, the biological characteristics are as follows: the colony on the solid culture medium is round, white, flat, smooth and moist, and has blurred edges, the cells under the microscope are in various forms and smooth, and the gram staining reaction is positive. The solid culture medium is a methanol solid culture medium or a butanol solid culture medium; the pH value of the solid culture medium is 7-8.
In general, the identification of microorganisms using their 16S rDNA sequence or partial 16S rDNA sequence has advantages such as: performing rapid species analysis on an unknown sample; providing guidance information for biochemical identification; for bacteria that are difficult to obtain pure cultures, such as parasites, etc., identification using 16SrDNA is the only available means of identification.
However, some microorganisms cannot identify species by the 16S rDNA identification alone because of small interspecies differences. Other methods of identification are needed to complement, for example, the biological properties of the microorganism.
In a third aspect the present invention provides the use of rhodococcus equi as described in the first aspect for indicating the concentration of an buoyant gaseous hydrocarbon in a hydrocarbon reservoir.
According to the invention, the application specifically comprises the following steps:
a, extracting a whole genome of a microorganism in soil above an oil and gas reservoir to obtain a template for PCR amplification;
b, designing a primer to perform PCR amplification on the template to obtain a PCR amplification product;
and C, performing agarose gel electrophoresis on the PCR amplification product, and judging the concentration of the floating gaseous hydrocarbon in the oil and gas reservoir according to the brightness of the target band.
In some embodiments of the invention, the primer is selected from one of the following primer pairs:
an upstream primer 1: 5'-CGAAAGCGTGGGTAGCGAACAGGATTAG-3', and the adhesive tape is used for adhering the film to a substrate,
a downstream primer 1: 5'-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3', respectively;
an upstream primer 2: 5'-CTCAACTGCGGGCTTGCAGGCGATACGG-3', and the adhesive tape is used for adhering the film to a substrate,
a downstream primer 2: 5'-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3', respectively;
an upstream primer 3: 5'-CGGGTTGTAAACCTCTTTCAGCAGGGAC-3' the flow of the air in the air conditioner,
a downstream primer 3: 5'-GATCTGCGATTACTAGCGACTCCGACTTCA-3', respectively;
and the combination of (a) and (b),
an upstream primer 4: 5'-GGGACTGAGACACGGCCCAGACTCCTAC-3', and the adhesive tape is used for adhering the film to a substrate,
a downstream primer 4: 5'-GATCTGCGATTACTAGCGACTCCGACTTCA-3' is added.
In the present invention, the term "soil above the reservoir" means: and the soil within 1 meter above the vertical direction of the high abundance range of the oil and gas in the oil and gas reservoir and near the earth surface.
The beneficial effects of the invention are as follows: the Rhodococcus equi Sinopec05 is screened from soil above a typical oil and gas reservoir, the abundance of the Rhodococcus equi Sinopec05 in the soil above the oil and gas reservoir is in positive correlation with the concentration of floating gaseous hydrocarbons in the oil and gas reservoir, and the Rhodococcus equi Sinopec05 can be used as oil and gas microorganisms to indicate a high-value hydrocarbon leakage area above the oil and gas reservoir. Meanwhile, the abundance of the strain in the soil above the oil and gas reservoir is high, amplification culture is not needed, the 16S rDNA conserved sequence of Rhodococcus equi SINOPEC05 in the soil can be amplified through an improved primer, the concentration of the floating gaseous hydrocarbon in the oil and gas reservoir can be accurately and efficiently judged according to the brightness of a target strip in an amplification product, and the defects that the traditional physiological and biochemical detection period is long and the requirement on the purity of a sample culture is high are overcome. In addition, the strain can survive in a contaminated environment and serve as an inoculation medium for biological decontamination.
Drawings
The invention will be explained below with reference to the drawings.
FIG. 1 shows a 16S rDNA-based phylogenetic tree of Rhodococcus equi Sinopec05 of the present invention.
FIG. 2 is a gram stain of Rhodococcus equi Sinopec05 of the present invention.
FIG. 3 is an electron micrograph of Rhodococcus equi Sinopec05 of the present invention.
Strain preservation
And (3) classification and naming: rhodococcus equi (Rhodococcus equi); the strain number is as follows: SINOPEC05
The preservation organization: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (China center for cell communication)
Address: wuhan university Life sciences college
The preservation date is as follows: 2016 (3 months) and 14 days
Registration number of the preservation center: CCTCC NO: m2016109.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention briefly described above will be rendered by reference to the appended drawings.
In order to find an indicating microorganism for accurately and efficiently judging the concentration of the floating gaseous hydrocarbon in the oil-gas reservoir, through continuous research and exploration, a microorganism strain with the strain abundance being in positive correlation with the concentration of the floating gaseous hydrocarbon in the oil-gas reservoir is screened from the soil above the oil-gas reservoir, and is separated and identified as Rhodococcus equi SINOPEC05 (Rhodococcus equi SINOPEC 05); the strain has high abundance in soil above an oil and gas reservoir, amplification culture is not needed, the 16S rDNA conserved sequence of Rhodococcus equi SinopeC05 in the soil can be amplified through an improved primer, the concentration of floating gaseous hydrocarbons in the oil and gas reservoir can be accurately and efficiently judged according to the brightness of a target fragment in an amplification product, and the defects that the traditional physiological and biochemical detection period is long and the requirement on the purity of a sample culture is high are overcome. The present invention has been made based on the above findings.
Therefore, the rhodococcus equi can accurately and efficiently judge the concentration of the floating gaseous hydrocarbon in the oil-gas reservoir, and is obtained by screening and culturing the following screening culture medium (methanol culture medium or butanol culture medium); wherein, the composition of the methanol culture medium is as follows (in 1L deionized water):
and/or the composition of the butanol culture medium is as follows (in 1L of deionized water):
specifically, the composition of the methanol medium is as follows: KH (Perkin Elmer) 2 PO 4 1.0g/L，Na 2 HPO 4 ·12H 2 O 2.9g/L，MgSO 4 ·7H 2 O 0.32g/L，(NH 4 ) 2 SO 4 3.0g/L，CaCl 2 0.2g/L，KNO 3 1.0g/L and methanol 2.0 g/L;
the butanol medium had the following composition: KH (Perkin Elmer) 2 PO 4 1.0g/L，Na 2 HPO 4 ·12H 2 O 2.9g/L， MgSO 4 ·7H 2 O 0.32g/L，(NH 4 ) 2 SO 4 3.0g/L，CaCl 2 0.2g/L，KNO 3 1.0g/L and butanol 2.0 g/L.
The pH values of the methanol culture medium and the butanol culture medium are as follows: 7.0.
the screening method of rhodococcus equi comprises the following steps:
(1) the culture medium is prepared according to the above culture medium composition, sterilized at high temperature (121 deg.C) under high pressure (0.15MPa) for 20min, and then sterilized in a clean bench by ultraviolet irradiation for 20 min. Adding agar of 2% (weight/volume) into the liquid culture medium, sterilizing at high temperature and high pressure, dissolving, pouring into a culture dish, cooling, and making into corresponding solid culture medium plate.
(2) Weighing 10g of a soil sample above the oil-gas reservoir of the vernal light oilfield, adding the soil sample into 100ml of sterilized normal saline, fully shaking and standing. Taking 100 mu L of supernatant in a clean bench, inoculating the supernatant into a screening culture medium, and performing shake culture at 30 ℃ and 200r/min of shaking table rotation speed for 3 d. The culture was diluted to 10 with sterilized normal saline solution -5 、10 -6 And 10 -7 Then, 100. mu.L of each dilution was uniformly plated on the corresponding solid screening medium, followed by incubation at 30 ℃. After 3d, growth of monoclonals on the surface of the solid medium was observedAnd (3) picking a typical monoclonal colony by using an inoculating needle, re-inoculating the typical monoclonal colony into a solid screening culture medium by adopting a streaking culture method, and performing purification culture for three times to obtain a pure culture of the strain.
(3) Morphological and molecular biological identification of pure culture of the strain
The colony morphology of the strain on a solid culture medium is observed to be circular, white, flat, smooth and moist and blurred in edges. The microscopic image of the microscopically round cells with various shapes is shown in FIG. 3.
Gram stain analysis is carried out on the strain, the strain is found to be gram-positive bacteria, and the gram stain graph is shown in figure 2.
DNA extraction, PCR amplification and sequencing identification are carried out on a pure culture of the strain, then sequencing is carried out by a 16S rDNA clone gene analysis method, the BLAST is used for carrying out homology comparison analysis on the measured sequence and the registered sequence in a GenBank/EMBL/DDBJ database, the methods such as bioinformatics and the like are used for analyzing the abundance and relative composition characteristics of species in soil above a typical oil and gas reservoir in a exploration area, a novel strain with higher microorganism abundance is obtained, the strain is identified and named as Rhodococcus equi SINOPEC05, the length of the 16S rDNA sequence is 1427kp, and the full-length sequence is shown as SEQ ID NO. 1. FIG. 1 shows a 16S rDNA-based phylogenetic tree of Rhodococcus equi Sinopec05 of the present invention. The strain is preserved in China center for type culture Collection (CCTCC for short) in 2016, 3 months and 14 days, and the preservation number is as follows: CCTCC NO: m2016109.
Through identification, the abundance of the rhodococcus equi in the soil above the oil and gas reservoir is positively correlated with the concentration of the floating gaseous hydrocarbon in the oil and gas reservoir. Accordingly, the present invention provides a use of rhodococcus equi as described above for indicating the concentration of an elevated gaseous hydrocarbon in a hydrocarbon reservoir.
According to the invention, the application specifically comprises the following steps:
extracting a whole genome of a microorganism in soil above an oil and gas reservoir to obtain a template for PCR amplification;
b, designing a specific primer according to a 16S rDNA sequence of Rhodococcus equi Sinopec05, and carrying out PCR amplification on the template to obtain a PCR amplification product;
the primer is selected from one of the following primer pairs:
an upstream primer 1: 5'-CGAAAGCGTGGGTAGCGAACAGGATTAG-3' the flow of the air in the air conditioner,
a downstream primer 1: 5'-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3';
an upstream primer 2: 5'-CTCAACTGCGGGCTTGCAGGCGATACGG-3' the flow of the air in the air conditioner,
a downstream primer 2: 5'-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3';
an upstream primer 3: 5'-CGGGTTGTAAACCTCTTTCAGCAGGGAC-3' the flow of the air in the air conditioner,
a downstream primer 3: 5'-GATCTGCGATTACTAGCGACTCCGACTTCA-3', respectively;
and the combination of (a) and (b),
an upstream primer 4: 5'-GGGACTGAGACACGGCCCAGACTCCTAC-3', and the adhesive tape is used for adhering the film to a substrate,
a downstream primer 4: 5'-GATCTGCGATTACTAGCGACTCCGACTTCA-3' are provided.
The primer pair can carry out PCR amplification on the DNA of the environmental soil sample, and quickly and accurately identify whether the Rhodococcus equi exists in the soil sample;
c, performing agarose gel electrophoresis on the PCR amplification product, and judging the concentration of the floating gaseous hydrocarbon in the oil and gas reservoir according to the brightness of a target band; specifically, the stronger the brightness of the target band, the higher the concentration of buoyant gaseous hydrocarbons in the reservoir.
Examples
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
Example 1: and (4) identifying a soil sample above the oil and gas reservoir by using rhodococcus equi Sinopec 05.
(1) Four pairs of specific primers were designed according to the 16S rDNA conserved sequence of rhodococcus equi SINOPEC05, and were:
an upstream primer 1: 5'-CGAAAGCGTGGGTAGCGAACAGGATTAG-3', and the adhesive tape is used for adhering the film to a substrate,
a downstream primer 1: 5'-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3', respectively;
an upstream primer 2: 5'-CTCAACTGCGGGCTTGCAGGCGATACGG-3' the flow of the air in the air conditioner,
a downstream primer 2: 5'-ACAAGGGTTGCGCTCGTTGCGGGACTTA-3';
an upstream primer 3: 5'-CGGGTTGTAAACCTCTTTCAGCAGGGAC-3' the flow of the air in the air conditioner,
a downstream primer 3: 5'-GATCTGCGATTACTAGCGACTCCGACTTCA-3', respectively;
an upstream primer 4: 5'-GGGACTGAGACACGGCCCAGACTCCTAC-3' the flow of the air in the air conditioner,
a downstream primer 4: 5'-GATCTGCGATTACTAGCGACTCCGACTTCA-3' is added.
Wherein, the size of the target band amplified by the primer 1 is 355bp, the size of the target band amplified by the primer 2 is 501bp, the size of the target band amplified by the primer 3 is 919bp, and the size of the target band amplified by the primer 4 is 1023 bp.
(2) Extracting the whole genome of the microorganism in the soil.
Weighing 0.5g of soil above the oil-gas reservoir and 0.5g of background soil respectively, adding into the lysine Matrix E Tube, performing cell disruption with cell disruptor (speed 4.5m/s, 30s, 4 times), and processing the disrupted cells according to the formula
The instructions of the SPIN Kit for Soil (MP Biomedicals biomedical corporation, USA) are followed to obtain the whole genome of the microorganism in the Soil sample as a template for PCR amplification.
(3) 4 pairs of primers were used for PCR amplification.
The PCR amplification systems are all as follows:
the following reagents were added to a 0.2mL PCR tube in order:
double distilled water was added to a final volume of 50. mu.l.
The PCR amplification procedures were all as follows:
pre-denaturation at 94 ℃ for 5 min;
denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 90s, extension at 72 ℃ for 1min, and 30 cycles;
extension for 10min at 72 ℃.
(4) Agarose gel electrophoresis.
The PCR amplification product is subjected to 1% agarose gel electrophoresis, and the result shows that the gene segments with the same length as the designed gene segments (primer pair 1: 355bp, primer pair 2: 501bp, primer pair 3: 919bp and primer pair 4: 1023bp) are amplified in the whole genome extracted from the soil above the oil-gas reservoir, and the corresponding segments are not amplified in the whole genome extracted from the background soil. And (3) carrying out glue recovery and sequencing on the target band, wherein the sequencing result shows that the amplified band is a target conserved sequence when the primer is designed. The strain can be used as an oil and gas microorganism and can specifically indicate a high-value hydrocarbon leakage area above an oil and gas reservoir.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined within the scope of the claims and modifications may be made without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all alternative means and applications having the same functionality.
SEQUENCE LISTING
<110> petrochemical industry, Inc. of China; petroleum exploration and development research institute of China petrochemical industry Limited company
<120> Rhodococcus equi, and identification method and application thereof
<130> 2017
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 1427
<212> DNA
<213> Rhodococcus equi (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 (1)
1. A Rhodococcus equi (B)Rhodococcus equi) The gene sequence of the 16S rDNA is shown in SEQ ID NO. 1; the Rhodococcus equi strain is SinOPEC05, and the preservation number in China center for type culture Collection is CCTCC NO: m2016109;
the abundance of the rhodococcus equi in the soil above the oil and gas reservoir is positively correlated with the concentration of the floating gaseous hydrocarbon in the oil and gas reservoir.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01229000A (en) * | 1988-03-09 | 1989-09-12 | Snow Brand Milk Prod Co Ltd | 2(3-hydroxy-1,3,5(10)-estriene-17-yl)propionic acid |
| WO2013082309A1 (en) * | 2011-11-29 | 2013-06-06 | Kiverdi, Inc. | Process for growing natural or engineered high lipid accumulating strain on crude glycerol and/or other sources of waste carbon for the production of oils, fuels, oleochemicals, and other valuable organic compounds |
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| KR20080007861A (en) * | 2006-07-18 | 2008-01-23 | (주)네오팜 | Novel microorganisms having biodegradation ability for oil and bioremediation method for oil-contaminated sites |
| RU2322400C1 (en) * | 2006-07-28 | 2008-04-20 | Институт биологии Коми научного центра Уральского отделения Российской академии наук | Method for cleaning oil out of bodies of water, swamp territories, polluted waters of barns and slurry accumulators |
| CN101935630B (en) * | 2010-07-02 | 2013-04-17 | 大连百奥泰科技有限公司 | Rhodococcus equi strain and use thereof in petroleum microorganism yield increase |
| CN102732424A (en) * | 2011-04-15 | 2012-10-17 | 大连百奥泰科技有限公司 | Compounded oil extraction microbe and its application in thickened oil and extra thickened oil exploitation |
| CN104164225B (en) * | 2013-05-16 | 2017-11-28 | 中国石油化工股份有限公司 | A kind of method of viscosity reduction composition and reducing thick oil viscosity |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01229000A (en) * | 1988-03-09 | 1989-09-12 | Snow Brand Milk Prod Co Ltd | 2(3-hydroxy-1,3,5(10)-estriene-17-yl)propionic acid |
| WO2013082309A1 (en) * | 2011-11-29 | 2013-06-06 | Kiverdi, Inc. | Process for growing natural or engineered high lipid accumulating strain on crude glycerol and/or other sources of waste carbon for the production of oils, fuels, oleochemicals, and other valuable organic compounds |
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| 马红球菌的高温烃降解特性及其驱油效果;谢英等;《新疆石油地质》;20121201;第33卷(第6期);715-719 * |
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