Specific primer for quickly detecting nitrate reducing bacteria in oil filed and using method of specific primer
A nitrate and specific technology, applied in the fields of microbiology and oilfield logging, can solve the problems of high non-specificity, difficulty in studying DNB, and low detection results, and achieve strong specificity, simple and fast detection methods, and accurate and sensitive results Effect
Active Publication Date: 2015-01-21
CHINA PETROLEUM & CHEM CORP +1
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Problems solved by technology
However, the existing extinction dilution method can only detect DNB that can be cultured, so the detection result is low
At the same time, the extinction dilution method has the disadvantages of long cycle time and high non-specificity.
For a long time, the research on the classification and identification of DNB has not been systematic and in-depth, and the method of identifying DNB according to the traditional taxonomic method has not been established, and the denitrification phenotype cannot be inferred from the phylogeny
Therefore, the conventional method based on 16S rRNA sequencing is also very difficult to study DNB
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[0016] 1. Construct the specific sequence of the primer set:
[0017] Use bioinformatics software such as DNAstar to analyze the nucleotide sequence of the gene nir encoding nitrite reductase Nir (see the attachment for some representative sequences), and select the conserved region fragments in the nucleotide sequences of these genes after performing homology comparisons , and then select about 20 representative bases in its upstream and downstream conserved regions, respectively, as specific PCR primers for the identification of nitrate-reducing bacteria (DNB), and design two pairs of primers. Primers can be synthesized by themselves or by biotechnology companies.
[0018] The specific sequences of the primers are:
[0019] 1F: 5’-gtcaacgtcaaggaaaccgg-3’
[0020] 2F: 5’-gtgaacgtgaaggagacggg-3’
[0021] 1R: 5'-gacttcggatgcgtcttga-3'
[0022] 2R: 5’-gagttcgggtgggtcttga-3’
[0023] 2. PCR amplification system:
[0024] Genomic DNA was extracted from the sample as a templa...
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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 [0001] The invention relates to the field of microbiology, and also relates to the field of oil field logging technology. Background technique [0002] Nitrate-reducing bacteria (DNB) and sulfate-reducing bacteria (SRB) co-exist in the oil reservoir environment. During the growth of sulfate-reducing bacteria, hydrogen sulfide is produced, which acidifies the water samples of the reservoir, causing serious damage and loss (corrosion , perforation, leakage, scaling, blockage, and even spontaneous combustion explosion of pipeline storage tanks). Introducing low-concentration nitrate / nitrite components into the oilfield system can replace sulfate as an electron acceptor, thereby promoting the rapid proliferation and diffusion of DNB that naturally exists in the oilfield system, and when competing with SRB for living space and substrates, DNB preferentially uses substrates in oilfield systems that prevent SRB from obtaining the nutrients they need, thereby contro...
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IPC IPC(8): C12Q1/68C12N15/11C12Q1/04
Inventor 姚峰吴伟林杨海滨孟章进吴波何永强
Owner CHINA PETROLEUM & CHEM CORP