CN103421899A - Specific primer for quickly detecting nitrate reducing bacteria in oil filed and using method of specific primer - Google Patents
Specific primer for quickly detecting nitrate reducing bacteria in oil filed and using method of specific primer Download PDFInfo
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- CN103421899A CN103421899A CN2013103447953A CN201310344795A CN103421899A CN 103421899 A CN103421899 A CN 103421899A CN 2013103447953 A CN2013103447953 A CN 2013103447953A CN 201310344795 A CN201310344795 A CN 201310344795A CN 103421899 A CN103421899 A CN 103421899A
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Abstract
The invention discloses a specific primer for quickly detecting nitrate reducing bacteria in an oil field and a using method of the specific primer, and relates to not only the microorganism field, but also the technical field of well logging in oil fields. The using method comprises the following steps: designing a reliable PCR (Polymerase Chain Reaction) primer according to the nucleotide sequence of a nitrite reductase Nir coding gene nir, performing the separation and cultivation of the nitrate reducing bacteria on the samples in the oil fields, extracting the genome DNA as a template, and analyzing the amplification results through AGE (agarose gel electrophoresis) after the primer is amplified, so as to build the PCR method for screening and identifying the nitrate reducing bacteria. The primer and the using method have the advantages that the detection method is simple, convenient and fast, the result is accurate and sensitive, the real condition of the nitrate reducing bacteria in to-be-detected samples can be responded in time, and an effective method is provided for detecting the nitrate reducing bacteria.
Description
Technical field
The present invention relates to the microbiology field, also relate to the oil reservoir logging technical field simultaneously.
Background technology
Nitrate reduction bacterium (DNB) is present in reservoir media with sulphate reducing bacteria (SRB) jointly, produce hydrogen sulfide in the Growth of Sulfate Reducing Bacteria process, make the acidifying of oil reservoir water sample, caused serious harm and loss (burn into perforation, run-down, fouling, obstruction, the even spontaneous combustion blast of breakout tank).Import the nitrate of lower concentration/nitrite composition to oil field system, alternative vitriol becomes electron acceptor(EA), thereby impel the natural rapid hyperplasia of DNB and the diffusion be present in oil field system, and with SRB competition living space and matrix the time, matrix in the preferential choice for use oil field system of DNB, can stop SRB to obtain needed nutrition, thereby control the metabolic activity of SRB.DNB is significant in the biological control strategy of SRB.
But correlative study at present mainly concentrates on some culturing micro-organisms.Yet bacterium also exists and a kind ofly under normal culture condition, can not grow and produce the state of bacterium colony, be called alive but can not cultivate (viable but nonculturable, VBNC) state.Research shows, in various habitats, only have the small portion microorganism can use the laboratory method separation and Culture, and the kind of not cultivated has represented huge diversity.Have too the bacterium that can not cultivate in a large number in oil field system DNB monoid, these DNB quantity are stable and metabolic activity is arranged, thereby can bring into play very important effect equally in oil field system SRB control.But existing disappearance dilution method can only detect the DNB that can cultivate, thereby detected result is on the low side.The dilution method that simultaneously disappeares detects and exists the cycle long, the shortcomings such as non-specific height.For a long time to the inadequate system of classification identification research of DNB and deeply, not yet set up the method for press traditional taxonomy method evaluation DNB, the denitrogenation phenotype can not be inferred from phylogeny.Therefore, also there is very big difficulty in the conventional method research DNB based on 16S rRNA order-checking.
Summary of the invention
The present invention seeks to propose a kind of Auele Specific Primer for rapid detection oil field nitrate reduction bacterium that sense cycle is short, verification and measurement ratio is high.
Primer of the present invention is two pairs, and concrete sequence is respectively:
1F:?5’-gtcaacgtcaaggaaaccgg-3’
2F:?5’-gtgaacgtgaaggagacggg-3’
1R:?5’-gacttcggatgcgtcttga-3’
2R:?5’-gagttcgggtgggtcttga-3’。
At first the present invention finds distinctive specific function gene in nitrate reduction bacterium (DNB), and this gene does not exist in other microorganisms.Nitrate reduction bacterium (DNB) thoroughly is reduced to nitrogen by nitrate needs four steps, and wherein second step (be about to nitrite and be reduced to nitrogen protoxide) is the committed step in whole reaction path.This step is by nitrite reductase Nir realization, and Nir does not exist in other microorganisms.Therefore, the present invention, according to the PCR primer of the nucleotide sequence reliable design of nitrite reductase Nir encoding gene nir, identifies the PCR method of DNB to set up examination.
Another purpose of the present invention is to propose to utilize above primer to carry out rapid detection oil field nitrate reduction bacterium method.
The present invention carries out nitrate reduction bacterium separation and Culture by the Oil Field sample, and the extraction genomic dna is template, after being increased with above primer, and the analysing amplified result of recycling agarose gel electrophoresis, thus set up the PCR method that the nitrate reduction bacterium is identified in examination.
The present invention utilizes round pcr to identify the nitrate reduction bacterium (DNB) that the identical but relationship of ecological functions may be far away, but adopt in above two pairs of primer rapid detection samples and whether contain nitrate reduction bacterium (DNB), especially comprise the nitrate reduction bacterium (DNB) that can not cultivate, high specificity.This detection method is easy fast, result is accurately sensitive, can react in time the time of day of nitrate reduction bacterium (DNB) in sample, for the detection of nitrate reduction bacterium (DNB) provides effective ways, solve can not effectively detect at present can not cultivate DNB and conventional DNB detect in long, the problem such as specificity is weak and positive rate is on the low side of cycle.
The accompanying drawing explanation
Fig. 1 is the pcr amplification effect electrophoretic analysis pictures of two pairs of primers of the present invention for the identification of DNB.
Fig. 2 is two pairs of primers of the present invention for the identification of the part pcr amplification of DNB in Oil Field sample electrophoretic analysis picture as a result.
Embodiment
1, build the concrete sequence of primer sets:
Analyze the nucleotide sequence (the part representative series sees appendix) of nitrite reductase Nir encoding gene nir with bioinformatics softwares such as DNAstar, and carry out choosing the conserved regions fragment in these gene nucleotide series after sequence analysis, then select respectively each 20 left and right of representative base in its upstream and downstream conserved regions, the specific PCR primer of identifying as nitrate reduction bacterium (DNB) respectively, designed two pairs of primers.Primer can synthesize voluntarily, also can be synthetic by biotech company.
The concrete sequence of primer is respectively:
1F:?5’-gtcaacgtcaaggaaaccgg-3’
2F:?5’-gtgaacgtgaaggagacggg-3’
1R:?5’-gacttcggatgcgtcttga-3’
2R:?5’-gagttcgggtgggtcttga?-3’
2, pcr amplification system:
Extract sample gene group DNA and carry out nitrate reduction bacterium (DNB) examination as template.
50 μ l pcr amplification systems comprise:
3, pcr amplification:
At first 95 ℃ of denaturation 5 mim; Then be the amplification of 35 circulations, each circulation comprises three steps: 95 ℃ of 35 S, 52 ℃ of 1 min, 72 ℃ of 1 min; Last 72 ℃ are extended 10 min.
Amplified production carries out electrophoretic analysis with 1% sepharose.
4, expanding effect:
Extract respectively the genomic dna of Pseudomonas stutzeri, secondary coccus, 6 plasmid DNA that can not cultivate the gene clone of nitrate reduction bacterium, with intestinal bacteria, micrococcus luteus, Corynebacterium diphtheriae and the negative contrast of staphylococcic genome DAN, with primer of the present invention, increased.
As shown in Figure 1,1-4 is respectively the negative controls such as intestinal bacteria, micrococcus luteus, Corynebacterium diphtheriae, staphylococcus to result; 5-12 is respectively Pseudomonas stutzeri, secondary coccus, and 6 can not be cultivated the gene clone of nitrate reduction bacterium.
Visible: the positive findings that all can increase in can cultivating and can not cultivating the nitrate reduction bacterium, and the band that can not increase in the non-nitrate reduction bacterium such as intestinal bacteria, micrococcus luteus, Corynebacterium diphtheriae and staphylococcus.
5, primer of the present invention is applied in the detection to the Oil Field sample:
10 parts of Oil Field samples are carried out to nitrate reduction bacterium separation and Culture, wherein cultivate the positive for 3 parts, cultivate negative for 7 parts.
Get this 10 duplicate samples, extracting respectively genomic dna is template, with this primer, by above method, is increased.
Result is as shown in Figure 2: 1,2 be respectively intestinal bacteria and common water sample; 3～5 are respectively the Oil Field sample of 3 parts of nitrate reduction bacterium separation and Culture positives; 6～12 are respectively 7 parts cultivates negative Oil Field sample.
Visible: as all can to amplify positive band in educable 3 duplicate samples, in 7 duplicate samples that can not cultivate, have 5 parts to amplify positive band.Show that the positive rate that primer of the present invention detects is much higher than the disappearance dilution method.
gtcaacgtcaaggaaaccgg
gtgaacgtgaaggagacggg
gacttcggatgcgtcttga
gagttcgggtgggtcttga
Claims (2)
1. for the Auele Specific Primer of rapid detection oil field nitrate reduction bacterium, described primer is two pairs, and concrete sequence is respectively:
1F:?5’-gtcaacgtcaaggaaaccgg-3’
2F:?5’-gtgaacgtgaaggagacggg-3’
1R:?5’-gacttcggatgcgtcttga-3’
2R:?5’-gagttcgggtgggtcttga-3’?。
2. as claimed in claim 1 for the using method of the Auele Specific Primer of rapid detection oil field nitrate reduction bacterium, it is characterized in that the Oil Field sample is carried out to nitrate reduction bacterium separation and Culture, the extraction genomic dna is template, after being increased with above primer, the analysing amplified result of recycling agarose gel electrophoresis, thus the PCR method that the nitrate reduction bacterium is identified in examination set up.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948905A (en) * | 2010-09-10 | 2011-01-19 | 天津亿利科石油技术发展有限公司 | Method for detecting nitrate reducing bacteria on site in oil field |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948905A (en) * | 2010-09-10 | 2011-01-19 | 天津亿利科石油技术发展有限公司 | Method for detecting nitrate reducing bacteria on site in oil field |
Non-Patent Citations (3)
| Title |
|---|
| GESCHE B等: "Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes （nirK and nirS）To Detect Denitrifying Bacteria in Environmental Samples", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 庄文等: "油田采出液中硝酸盐还原菌的分离培养及对硫酸盐还原菌的抑制研究", 《科学技术与工程》 * |
| 谭燕等: "油井采出液中硝酸盐还原菌的分离培养", 《应用与环境生物学报》 * |
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