CN107216998B

CN107216998B - Automatic device for rapidly measuring bacterial content in oil field sewage and finished oil

Info

Publication number: CN107216998B
Application number: CN201610163852.1A
Authority: CN
Inventors: 孙冰; 徐伟; 文松
Original assignee: China Petroleum and Chemical Corp; Sinopec Qingdao Safety Engineering Institute
Current assignee: China Petroleum and Chemical Corp; Sinopec Qingdao Safety Engineering Institute
Priority date: 2016-03-22
Filing date: 2016-03-22
Publication date: 2020-05-19
Anticipated expiration: 2036-03-22
Also published as: CN107216998A

Abstract

The invention relates to an automatic device for rapidly measuring the content of bacteria in oil field sewage and finished oil, which mainly solves the problems of low measurement speed, inconvenience and inaccurate result in the prior art. The invention solves the problems well by adopting an automatic device for rapidly measuring the bacterial content in the oil field sewage and the finished oil, which comprises a box body, a bacterial enrichment and nucleic acid molecule extraction device, wherein the bacterial enrichment and nucleic acid molecule extraction device comprises a reagent tube placing position (1), a reagent tube placing position (4), a filter membrane (2), an adsorption layer (3), a reaction tank (5), a light source (6), an optical filter (7), a photoreceptor (8) and at least four valves.

Description

Automatic device for rapidly measuring bacterial content in oil field sewage and finished oil

Technical Field

The invention relates to an automatic device for rapidly measuring the content of bacteria in oil field sewage and finished oil.

Background

Corrosive destruction of materials by the vital activity of microorganisms such as bacteria is referred to as microbial corrosion (MIC). MIC is a ubiquitous phenomenon. A large number of bacteria are present in oil and gas fields, oil tanks, gas stations, oil lines and even in automobile fuel tanks. The economic losses due to corrosion are more than one hundred billion dollars per year. The most predominant of these are Sulfate Reducing Bacteria (SRB), which cause corrosion losses of over 77% of the U.S. production wells. SRB can reduce sulfate into hydrogen sulfide by using organic matters in the environment as a carbon source and utilizing hydrogen generated in a bacterial biofilm in an anaerobic environment, energy is obtained from oxidation-reduction reaction, and metal is oxidized and corroded in the process. Currently, there are three main detection methods for SRB, which are: culture detection technology, microscope detection technology and biochemical direct detection technology. The culture detection technology is a method which is widely applied at present, and the principle is that a sample is subjected to gradient dilution, culture is carried out on a proper culture medium, and finally quantification is carried out according to the dilution multiple and the positive reaction of bacteria. The method is mainly based on an extinct dilution method recommended by API of the American Petroleum institute, and is also specified in detail in SY/T0532-2012 'extinct dilution method for bacteria analysis method of oilfield injection water' in the national Petroleum industry standard. Related patents (such as CN102329851A, CN103983636A, CN1769472A, CN200510010459, etc.) have been reported, and currently, SRB dedicated test bottles are available in the market. The culture detection technology has the advantages of simple operation, low cost and higher result accuracy. The main disadvantages are that the time is long (2-14 days), and the culture of the strains is incomplete, which results in low quantitative results. Once fungus growth appears in the negative control group, the experiment needs to be redone, and the re-sampling result is difficult to be ensured to be consistent with the original sample.

The microscopic detection technique combines a specific staining method with a fluorescence counting method, selectively labels fluorescent groups on the surface of the SRB, and then counts under a fluorescence microscope for quantification. However, the method still needs short-term culture to improve the fungus concentration, and the culture period is about two days. Although relatively simple to operate and low in quantitative cost, high specificity of the dye molecule is required and no information about bacterial activity can be provided.

The biochemical direct detection technique comprises various specific modes (Guanshuxiaye Haichun fangying oil field sewage sulfate reducing bacteria detection technique research progress chemistry and biological engineering 20122917), can detect certain protein inside or on the SRB (such as adenosine-5' -phosphate sulfate reductase APS (such as GB2354072B, CN103983636A, EP272916A 1) determined by enzyme linked immunosorbent assay ELISA method), can detect SRB specific nucleic acid molecule sequence (such as polymerase chain reaction PCR method [ CN105112543A, CN1769472A, CN200510010459 ], fluorescence in situ hybridization FISH method [ Wangming Yi Liang Xiao Bing Zhang from Weizhongqing sulfate reducing bacteria identification and detection method research progress microbiology 20052581 ], and can detect SRB metabolite (such as potential method or indicator method determination H₂S) [ Li Wan Yi Zhao Zi Guang sulfate reducing bacteria amount detection method research-sulfur ion selective electrode method chemical sensor 19911164 ]. These assaysThe method has high accuracy, high specificity and short detection period (about a few hours), but has complex operation, and can be generally only used for experiments by trained professionals in special biochemical laboratories. The APS detection method is relatively simple, has already commercialized kit sale abroad, can realize quick on-the-spot measurement, but the shortcoming of this method has: 1) all bacterial species with APS were detected simultaneously, with possible high results; 2) the result takes the color change as an index, and the quantification is inaccurate; 3) bacterial lysis may not be complete resulting in low detection results.

In summary, a rapid, convenient and accurate on-site measurement method and equipment are lacking at present. This patent describes a specific, automated, portable, quantitative detection device for bacteria in a sample based on nucleic acid molecular amplification technology.

Disclosure of Invention

The invention aims to solve the technical problems of low measuring speed, inconvenience and inaccurate result in the prior art, and provides a novel automatic device for rapidly measuring the content of bacteria in oil field sewage and finished oil. The device has the advantages of rapidness, convenience and accuracy.

In order to solve the problems, the technical scheme adopted by the invention is as follows: an automatic device for rapidly measuring the content of bacteria in oil field sewage and finished oil comprises a box body, bacteria enrichment and nucleic acid molecule extraction equipment, the bacteria enrichment and nucleic acid molecule extraction equipment comprises a reagent tube placing position 1, a reagent tube placing position 4, a filter membrane 2, an adsorption layer 3, a reaction tank 5, a light source 6, an optical filter 7, a photoreceptor 8 and at least four valves, the bottom of the reagent tube placement position 1 is communicated with the space above the filter membrane 2, the space below the filter membrane 2 is divided into two paths, one path is connected with the inlet end of a valve a, the other path is connected with the inlet end of a valve b, the outlet end of the valve a is connected with a waste liquid pool, the outlet end of the valve b is connected with the inlet side of an adsorption layer 3 through a pipeline, the pipeline at the outlet side of the adsorption layer 3 is divided into two paths, one path is connected with the inlet end of a valve c, the outlet end of the valve c is connected with the waste liquid pool, and the other path is; the bottom of the reagent tube placing position 4 is connected with a pipeline between the valve b and the inlet side of the adsorption layer through the pipeline; the light source 6 provides dye molecule excitation energy for the reaction tank 5, and the generated fluorescence is received by the photoreceptor 8 after passing through the optical filter 7; the top in space above the filter membrane 2, the bottom of the reagent tube placing position 4 all are equipped with the metal spine, can puncture the aluminium foil of reagent bottom of the tube when the reagent tube is placed on the reagent tube placing position 1 or the reagent tube placing position 4.

In the above-described embodiment, preferably, when the reagent vessel is placed at the reagent vessel placement position 1, a piston is placed at the upper portion of the reagent vessel, and the reagent in the reagent vessel is pressed downward into the space above the filter membrane 2.

In the above technical solution, preferably, the bacteria enrichment and nucleic acid molecule extraction device is integrated in the box body.

In the above technical solution, preferably, the valves a to d are program-controlled solenoid valves, or micro-fluidic micro-valve technology is adopted, and the valves are controlled by an air pump to open and close the pipelines.

In the above technical solution, preferably, an opening or a light-transmitting material is disposed above the reaction cell 5, a reaction mother solution or a freeze-dried reagent ball is disposed inside the reaction cell, and the nucleic acid eluent flows into the reaction cell 5 and is mixed with the predetermined solution or the reagent ball.

In the above technical solution, preferably, the signal output by the photoreceptor 8 is sent to the signal processing unit for analysis.

In the above technical solution, preferably, the reagent vessel placement position 1 or 4 is disposed at a higher position, so that the liquid flows down to a lower position.

In the above technical solution, preferably, when the PCR method is adopted, a rapid heating-cooling module is required to be disposed at the periphery of the reaction cell 5 to realize temperature cycling of denaturation-annealing-extension; when the isothermal amplification method is adopted, only a heating module needs to be arranged around the reaction tank.

In the above technical solution, preferably, the replacement of the reagent tube is completed by an automatic control system.

In the present invention, bacterial enrichment is achieved by passing the sample through a filter. After the sample is added, the aluminum foil package at the bottom of the sample tube is punctured by the metal tip at the lower part, the liquid flows into the upper part of the filter membrane 2, at the moment, pressure is applied to the upper part to push the liquid to pass through the filter membrane, solid matters larger than the aperture of the filter membrane are remained on the filter membrane, and the liquid flows out. At the moment, the valve a is opened, the valve b is closed, and the waste liquid flows out to the waste liquid pool. When the sample is water phase material such as oil field waste water, a hydrophilic filter membrane is adopted, and the size of the filter membrane is below 0.5 micron so as to ensure that the target microorganism can be retained on the filter membrane. When the sample is oil phase substance such as product oil, lipophilic filter membrane is adopted. After the sample completely flows through the filter membrane, the program automatically controls the sample tube to replace the ethanol solution preset in the box body, and the pressurizing process is repeated to wash the filter membrane. The rinsing is continued with deionized water or a buffer of a suitable pH. And after the washing is finished, adding the bacterial lysate, starting pressurization after a certain time, closing the valve a, opening the valve b, and allowing the bacterial lysate to enter the nucleic acid extraction unit. The nucleic acid extraction unit is internally provided with a DNA adsorption column (in the adsorption layer 3), when the bacterial lysate flowing into the upstream flows through the adsorption layer 3, the DNA in the bacterial lysate is adsorbed on the adsorption layer 3, and the liquid phase flows into the waste liquid pool. At this time, valve c is opened and valve d is closed. The washing liquid is pre-arranged in the test tube at the reagent tube placing position 4 and flows through the adsorption layer 3 by pressure to take away impurities. The test tube at the reagent tube placing position 4 is replaced to repeat the washing process. When the washing is finished, the solution in the test tube at the reagent tube placement position 4 is replaced with a small amount of buffer solution with proper pH. At this time, valve c is closed and valve d is opened. The buffer solution elutes the DNA as it passes through the adsorption layer 3, and is washed into the reaction cell 5. The reaction tank 5 is preset with various components required by nucleic acid amplification reaction, and can be prepared mother liquor, or can be used for long-term storage, and the freeze-dried reagent can be placed in the reaction tank and dissolved by the buffer solution. The amplifying and detecting unit consists of a reaction cell 5, a light source (halogen lamp or LED)6, a light filter 7 and a photoreceptor (CCD or PMT) 8. When a nucleic acid amplification reaction occurs in the reaction cell 5, the dye molecules in the reaction cell 5 are excited by light emitted from the light source 6, and the generated fluorescence is received by the photoreceptor 8. The concentration of the dye molecule is positively correlated with the concentration of the nucleic acid molecule in the solution, from which the initial concentration of the nucleic acid molecule can be judged.

The device comprises three units of bacteria enrichment, nucleic acid molecule extraction and amplification reading, is an integrated portable automatic instrument, and can realize field measurement. The bacteria enrichment unit realizes the separation and enrichment of bacteria by adopting a mode that a sample flows through a filtering membrane, and the required time is greatly shortened compared with a culture method. Nucleic acid molecule extraction and amplification reading are both realized on a microfluidic platform. The extraction part adopts the silicon dioxide adsorption column technology to adsorb DNA molecules, and the reagents are respectively pushed to flow through the adsorption columns under the pressure controlled by a program, so that pure nucleic acid molecules are finally obtained. In the amplification readout unit, a PCR method or a loop-mediated isothermal amplification (LAMP) method may be selectively employed. Compared with the conventional PCR method, the LAMP method has higher specificity, higher tolerance to impurities in a reaction medium, higher reaction efficiency, shorter detection time (which can be shortened to 20 minutes), no denaturation-annealing-extension temperature cycle and reduced requirement on a temperature control module. All operations are controlled by programs, manual operation is not needed except for sample adding, and the parallelism and convenience of testing are improved.

Drawings

FIG. 1 is a schematic external view of the apparatus of the present invention;

FIG. 2 is a schematic structural diagram of modules in the apparatus according to the present invention;

in fig. 1 and 2, 1 is a reagent tube placement position; 2 is a filter membrane; 3 is an adsorption layer; 4 is a reagent tube placing position; 5 is a reaction tank; 6 is a light source; 7 is an optical filter; 8 is a receptor; a to d are valves.

The present invention will be further illustrated by the following examples, but is not limited to these examples.

Detailed Description

[ example 1 ]

An automatic device for rapidly measuring the content of bacteria in oil field sewage and finished oil is shown in figures 1 and 2. The device comprises a box body, bacteria enrichment and nucleic acid molecule extraction equipment, wherein the bacteria enrichment and nucleic acid molecule extraction equipment comprises a reagent tube placement position 1, a reagent tube placement position 4, a filter membrane 2, an adsorption layer 3, a reaction tank 5, a light source 6, a light filter 7, a photoreceptor 8 and at least four valves, the bottom of the reagent tube placement position 1 is communicated with the space above the filter membrane 2, the space below the filter membrane 2 is divided into two paths, one path is connected with the inlet end of a valve a, the other path is connected with the inlet end of a valve b, the outlet end of the valve a is connected with a waste liquid tank, the outlet end of the valve b is connected with the inlet end of the adsorption layer 3 through a pipeline, the pipeline at the outlet side of the adsorption layer 3 is divided into two paths, one path is connected with the inlet end of a valve c, the outlet end; the bottom of the reagent tube placing position 4 is connected with a pipeline between the valve b and the inlet side of the adsorption layer through the pipeline; the light source 6 provides dye molecule excitation energy for the reaction tank 5, and the generated fluorescence is received by the photoreceptor 8 after passing through the optical filter 7; the top in space above the filter membrane 2, the bottom of the reagent tube placing position 4 all are equipped with the metal spine, can puncture the aluminium foil of reagent bottom of the tube when the reagent tube is placed on the reagent tube placing position 1 or the reagent tube placing position 4.

The device of the invention is integrated in a metal casing of 0.5m 0.4m, and a user only needs to open the panel as required to load about 1mL of liquid sample.

In order to make the advantages and technical solutions of the present invention clearer and clearer, the steps of the present invention are described in detail below with reference to specific embodiments.

The liquid flow in the device is realized by pressure driving, wherein the reagent tube placing position 1 and the reagent tube placing position 4 are arranged at higher positions, so that the liquid flows to lower positions conveniently, and the upper rubber piston generates thrust in the downward movement process, so that the liquid flows through the filter membrane and the adsorption column. The bottom positions of the reagent tube placing position 1 and the reagent tube placing position 4 are made of aluminum foil materials, and when entering the designated positions, the aluminum foil materials can be punctured by a metal needle point fixed below the aluminum foil materials, so that the liquid inside the aluminum foil materials flows out. The position 1 of the reagent tube placing position is preset with ethanol flushing fluid, buffer flushing fluid and cell lysis solution (containing lysozyme, CTAB or SDS); the 4-position preset solution is washing solution (twice) and buffer eluent.

And (3) selecting a filter membrane 2: can select for use the filter membrane of different properties according to the difference of sample, the part at filter membrane 2 place is detachable, is located the front end of device, can be dismantled by the user. The filter membrane can be selected from nylon, polyvinylidene fluoride, cellulose, polypropylene, PTFE, ceramic and the like, and different filter membranes are selected according to the hydrophilicity and hydrophobicity of a sample and possibly contained impurities. The nucleic acid adsorption column in the adsorption layer 3 is made of silicon dioxide material, can specifically adsorb DNA and RNA molecules, and controls the adsorption and desorption of nucleic acid molecules by adjusting the pH value of the solution.

The valves a to d can be program controlled electromagnetic valves, and can also adopt micro-fluidic micro-valve technology, and the air pump controls the opening and closing of the pipeline.

An opening or a light-transmitting material is arranged above the position of the reaction tank 5, reaction mother liquor or a freeze-dried reagent ball is preset inside the reaction tank, and the nucleic acid eluent flows into the reaction tank 5 and then is mixed with the preset solution or the reagent ball. At this time, the heating module starts to operate, and the nucleic acid amplification reaction starts. As the reaction proceeds, the dye in the solution binds to the formed nucleic acid molecule (SYBR Green detection) or the dye molecule capable of emitting fluorescence is generated in the solution (Qaqman detection) or the dye in the solution binds to the reaction by-product to emit light (LAMP calcein detection), the fluorescence is excited by the light source, the receptor is detected, and the detected signal is sent to the signal processing unit for analysis (this part is not shown in the schematic diagram).

Quantitative detection of SRB in oil field wastewater

Since the sample is an aqueous solution, a cellulose membrane with a pore size of 0.2 μm is used. The user need change the filter membrane part at first, collects oil field waste water 1mL with specific sample cell, opens the cover and puts into the device with it, sets for relevant parameter on the panel, starts the detection procedure. The whole process lasts for 30-120 minutes.

For SRB species, SRB specific nucleic acid amplification kits are used, wherein PCR primers and probe design can be referred to relevant literature or designed by oneself. For LAMP reaction, no related literature reports exist at present, no finished kit exists in the market, and primer design and development are needed to be further carried out on a 16S rDNA region of SRB.

After the detection is finished, the real-time change trend of the fluorescence can be obtained, the concentration of the SRB in the sample can be calculated according to the kinetic data of the reaction, and the calculation method is preset in a program. The results can be displayed on a panel, exported to a computer, sent by mail or short message, or directly uploaded to the cloud for statistics.

It is noted that those skilled in the art, having the benefit of the teachings of this specification, may effect these and other changes as may be made without departing from the scope of the invention as defined by the appended claims.

Claims

1. The utility model provides a measure automation equipment of bacterial content in oil field sewage and finished product oil rapidly, includes box, bacterium enrichment and nucleic acid molecule extraction equipment include that reagent pipe places position (1) and reagent pipe place position (4), filter membrane (2), adsorbed layer (3), reaction tank (5), light source (6), light filter (7), photoreceptor (8) and four at least valves, reagent pipe places position (1) bottom and filter membrane (2) top space intercommunication, and filter membrane (2) below space divide into two the tunnel, links to each other with valve a entry end all the way, and another way links to each other with valve b entry end, and valve a exit end links to each other with the waste liquid pond, and valve b exit end passes through the pipeline and links to each other with adsorbed layer (3) entrance side, and the pipeline of adsorbed layer (3) outlet side divide into two the tunnel, links to each other with valve c entry end all the way, the outlet end of the valve c is connected with the waste liquid tank, and the other path of the valve c is connected with the reaction tank (5) after passing through a valve d; the bottom of the reagent tube placing position (4) is connected with a pipeline between the valve b and the inlet side of the adsorption layer (3) through a pipeline; the light source (6) provides dye molecule excitation energy for the reaction tank (5), and generated fluorescence penetrates through the optical filter (7) and is received by the photoreceptor (8); metal sharp thorns are arranged at the top of the space above the filter membrane (2) and at the bottom of the reagent tube placing position (4), and when the reagent tube is placed on the reagent tube placing position (1) or the reagent tube placing position (4), the aluminum foil at the bottom of the reagent tube can be punctured; when the reagent tube is placed on the reagent tube placement position (1), a piston is placed on the upper portion of the reagent tube, and the reagent in the reagent tube is pressed downward into the space above the filter membrane (2).

2. The automated apparatus for rapid measurement of bacterial content in oilfield wastewater and product oil of claim 1, wherein the bacteria enrichment and nucleic acid molecule extraction device is integrated into the housing.

3. The automatic device for rapidly measuring the content of bacteria in the oil field sewage and the finished oil according to claim 1, wherein the valves a to d are program-controlled solenoid valves or micro-valve technology with micro-flow control, and the air pump controls the opening and closing of the pipeline.

4. The automatic device for rapidly measuring the content of bacteria in the oilfield wastewater and the finished oil according to claim 1, wherein an opening or a light-transmitting material is arranged above the reaction tank (5), a reaction mother solution or a freeze-dried reagent ball is arranged in the reaction tank, and the nucleic acid eluent flows into the reaction tank (5) and then is mixed with a preset solution or the reagent ball.

5. The automated apparatus for rapid measurement of bacterial content in oilfield effluents and finished oils according to claim 1, wherein the signal output by the photoreceptor (8) is sent to a signal processing unit for analysis.

6. The automated apparatus for rapid measurement of bacterial content in oilfield wastewater and product oil according to claim 1, wherein the reagent tube placement site (1) or the reagent tube placement site (4) is disposed at a higher position to facilitate liquid flow down to a lower position.

7. The automatic device for rapidly measuring the bacterial content in the oilfield sewage and the finished oil according to claim 1 is characterized in that when the PCR method is adopted, a rapid heating-cooling module is required to be arranged at the periphery of the reaction tank (5) to realize the temperature cycle of denaturation-annealing-extension; when the isothermal amplification method is adopted, only a heating module needs to be arranged around the reaction tank.

8. The automated apparatus for rapidly measuring the bacterial content in oilfield wastewater and product oil of claim 1, wherein the replacement of the reagent tube is accomplished by an automatic control system.