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Extraction method of oil reservoir microbial genome DNA

An extraction method and microbial technology, applied in recombinant DNA technology, DNA preparation and other directions, can solve the problems of reducing DNA yield and purity, a large number of membranes, and the impact of extraction operations, and achieve improved sample processing capacity, increased possibility, and extraction cost. low effect

Inactive Publication Date: 2015-05-20
CHINA PETROLEUM & CHEM CORP +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limitations of current technical means, only about 1% of microorganisms can be cultivated and recognized artificially. For this reason, researchers have developed various molecular biology analysis techniques that do not depend on cultivation. Microbial genome information, but due to the particularity and complexity of the oil reservoir environment, there are some problems in its operation, the most important problem is that the concentration of microorganisms in the oil reservoir sample is too low, and to collect a sufficient amount of bacteria, it must be processed A large number of samples, collect bacteria; oil reservoir samples contain crude oil pollution, adsorbed bacteria, affect the subsequent extraction operation, reduce the yield and purity of DNA
[0004] The existing bacterial collection methods in oil reservoirs mainly include two types of bacterial collection by filtration and centrifugal collection. The filtration adopts flat membrane filtration, which has the problems of low filtration flux, a large number of membrane sheets, and a large workload for subsequent membrane treatment.

Method used

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  • Extraction method of oil reservoir microbial genome DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] (1) Organic solvent extraction removes crude oil and releases microorganisms at the oil-water interface

[0025] Take 3L of each oil well output fluid and 3L of one injected water sample. Add 500ml of petroleum ether to the liquid phase of the sample, shake and mix well, and then let it stand for 1 hour to separate the organic phase and the aqueous phase, and remove the lower aqueous phase.

[0026] (2) Collect bacteria

[0027] The obtained aqueous phase was filtered through a 0.22 μm hollow cellulose membrane filter by using a peristaltic pump to control the pressure to 0.5 MPa, and bacteria were collected.

[0028] (3) Lysis of microbial cells

[0029] First, cut the hollow cellulose membrane of the filter, mix it with quartz sand, put it into a centrifuge tube containing 800 μl extraction buffer, add 450 μl 1xTE, 50 μl 10 mg / mL lysozyme and 10 μl 100 mg / mL helicase, and mix well by blowing and aspiration , incubate at 37°C for 1 hour, invert and mix every 15 minut...

Embodiment 2

[0034] (1) Organic solvent extraction removes crude oil and releases microorganisms at the oil-water interface

[0035] Take 3L of each oil well output fluid and 3L of one injected water sample. Add 600ml of petroleum ether to the liquid phase of the sample, shake and mix well, and then let it stand for 2 hours to separate the organic phase and the aqueous phase, and remove the lower aqueous phase.

[0036] (2) Collect bacteria

[0037] The obtained aqueous phase was filtered through a 0.22 μm hollow cellulose membrane filter by using a peristaltic pump to control the pressure to 0.5 MPa, and bacteria were collected.

[0038] (3) Lysis of microbial cells

[0039] First, cut the hollow cellulose membrane of the filter, mix it with quartz sand, put it into a centrifuge tube containing 1000 μl extraction buffer, add 450 μl 1xTE, 50 μl 10 mg / mL lysozyme and 10 μl 100 mg / mL helicase, and mix by pipetting , incubate at 37°C for 1 hour, invert and mix every 15 minutes, then add 600...

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Abstract

The invention relates to the technical field of microbial enhanced oil recovery, and in particular relates to an extraction method of oil reservoir microbial genome DNA. The method comprises the following steps: removing interference of crude oil in samples with extraction by means of organic solvent extraction; releasing microbes on an oil-water interface; filtering water phase by using a hollow fiber membrane to enrich microbes; removing organic macromolecular components such as proteins by phenol and chloroform in cooperation with lysis cells such as lysozyme, helicase, protease and lauryl sodium sulfate according to a physical wall-breaking method under a high-salinity condition; subsequently, performing nucleic acid extraction and precipitation to obtain the oil reservoir microbial genome DNA. The method is quick and simple, and the whole extraction process can be completed in 10 hours; the extraction efficiency is high, and the purity of the obtained genome is high.

Description

1. Technical field [0001] The invention relates to the technical field of microbial oil recovery, in particular to a method for extracting oil reservoir microbial genome DNA. 2. Background technology [0002] Microbial oil recovery improves oil recovery through the comprehensive action of microbial activities and metabolites in the reservoir. Compared with other oil displacement methods, it has many outstanding advantages. First, it has a wide range of applications. Shengli Oilfield has 1 billion tons of reserves suitable for microbial oil recovery. Second, the cost is low and the construction is simple. Third, it does not harm the reservoir, and there is no problem of post-treatment of produced fluid, which meets the requirements of sustainable development. [0003] The growth and metabolism of microorganisms in oil reservoirs is the material basis and main research object of microbial oil recovery technology. Therefore, in-depth and systematic investigation and analysis ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 郝滨李彩风曹功泽王静宋欣袁长忠徐闯徐鹏王刚曹嫣镔宋永亭
Owner CHINA PETROLEUM & CHEM CORP
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