CN113817716A - Method for extracting nucleic acid from living body and water treatment system - Google Patents
Method for extracting nucleic acid from living body and water treatment system Download PDFInfo
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- CN113817716A CN113817716A CN202110639506.7A CN202110639506A CN113817716A CN 113817716 A CN113817716 A CN 113817716A CN 202110639506 A CN202110639506 A CN 202110639506A CN 113817716 A CN113817716 A CN 113817716A
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- nucleic acid
- water
- sodium hypochlorite
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- water treatment
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- C12N15/09—Recombinant DNA-technology
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C—CHEMISTRY; METALLURGY
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C—CHEMISTRY; METALLURGY
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Abstract
The technical problem is solved. The invention provides a biological nucleic acid extraction method capable of extracting biological nucleic acid such as microorganisms with high efficiency and high extraction (recovery) rate, and a water treatment system using the method. The solution is provided. The method for extracting nucleic acid from a living body comprises: a sodium hypochlorite addition step of adding sodium hypochlorite to a sample solution for extracting nucleic acid from a living body so that the concentration of the sample solution is within a concentration range determined in advance by an experiment; and a nucleic acid extraction step of extracting nucleic acid of a living body to be extracted from a sample solution to which sodium hypochlorite is added.
Description
Technical Field
The present invention relates to a method for extracting nucleic acid from a living body and a water treatment system.
Background
For example, in all technical fields centered on the technical fields of food, medical and pharmaceutical products, agriculture, and the like, research and development have been actively conducted on methods for extracting and recovering nucleic acids from various organisms such as viruses, prokaryotes (eubacteria, archaea), eukaryotes (algae, protists, fungi, slime mold), microorganisms such as small animals (rotifers and the like), animals and plants, human tissues, and the like, for the purpose of genome analysis, qualitative (detection, identification)/quantitative, mass replication (amplification, proliferation), and the like.
The research and development efforts relating to such a nucleic acid extraction method are effective for speeding up, increasing the accuracy, and increasing the efficiency of an examination using a gene amplification method such as a PCR (Polymerase Chain Reaction) method.
In addition, the quantitative pcr (qpcr) method is sometimes used for quantitative examination of microorganisms and the like contained in treated water, and further for management of discharged water such as compliance (compliance) of drainage standards, environmental standards and the like, in order to prevent adverse effects on the environment, fishery, human beings and the like when treated water obtained by treating various kinds of wastewater and the like is discharged to public waters such as rivers, lakes, seas, underground water and the like.
Here, in order to ensure the accuracy of the examination of microorganisms by the quantitative PCR method, that is, the accuracy of quantification, it is important to improve the efficiency of extraction and recovery of nucleic acids from microorganisms contained in sample water (sample liquid) obtained by sampling or the like as much as possible.
In contrast, in patent document 1, nucleic acids derived from microorganisms can be efficiently recovered by subjecting the extraction solution before and after the treatment of the microbial lytic enzyme reaction to pressure cycle treatment or ultrasonic treatment.
In order to increase the recovery rate of nucleic acids derived from microorganisms, a method of adding tRNA that is coprecipitated with DNA, polysaccharides such as glycogen, and carriers such as polyacrylamide to ethanol precipitation is often used.
Documents of the prior art
Patent document
Patent document 1: japanese patent laid-open publication No. 2013-042670
Disclosure of Invention
Problems to be solved by the invention
However, conventional methods for extracting and purifying nucleic acids, such as pressure cycle treatment, ultrasonic treatment, and the use of carriers, have problems of very complicated operation, a large amount of time required, and high cost.
Therefore, a method for extracting nucleic acids from a living body such as a microorganism more easily and efficiently with an excellent nucleic acid extraction rate than in the past has been strongly desired.
In view of the above circumstances, an object of the present invention is to provide a method for extracting nucleic acid from a living organism, such as a microorganism, which can extract nucleic acid from the living organism more efficiently and with a remarkably excellent extraction (recovery) rate, and a water treatment system using the method.
Technical scheme
One embodiment of the method for extracting nucleic acid from an organism of the present invention comprises: a sodium hypochlorite addition step of adding sodium hypochlorite to a sample solution for extracting nucleic acid from a living body so that the concentration of the sample solution is within a concentration range determined in advance by an experiment; and a nucleic acid extraction step of extracting nucleic acid of the microorganism to be extracted from the sample solution to which the sodium hypochlorite is added.
One aspect of the water treatment system of the present invention includes: a water treatment unit for improving the quality of water to be treated; a biological quantification unit for quantifying the amount of biological substances in the treated water after the water has been treated by the water treatment unit, using the method for extracting nucleic acid from biological substances; and a water discharge unit configured to discharge the treated water when the biological quantity determined by the biological quantity determining unit is equal to or less than a preset set value.
The term "organism" in the present invention includes not only microorganisms (organisms whose size cannot be visually distinguished without using a microscope or the like, including viruses), but also tissues (including blood and the like) constituting a part of multicellular organisms such as humans and animals and plants.
The "sample solution" in the present invention refers to a "liquid sample" for extracting nucleic acids from a living body.
Effects of the invention
According to the method for extracting nucleic acid from a living body of the present invention, nucleic acid from a living body such as a microorganism can be extracted more efficiently and with a remarkably excellent extraction (recovery) rate.
According to the water treatment system of the present invention, by performing the examination of the treated water using the above-described method for extracting nucleic acid from a living body, highly reliable water treatment can be realized.
Drawings
FIG. 1 is a diagram showing an example of a water treatment system according to one embodiment, and shows an example of a flow of a method for extracting nucleic acid from microorganisms.
FIG. 2 is a diagram showing an example of an amplicon amplification curve for determining a concentration range of sodium hypochlorite in a method for extracting nucleic acid from a living body (microorganism) according to one embodiment.
FIG. 3 is a diagram showing an example of the relationship between the sodium hypochlorite concentration and the initial concentration of nucleic acid in a nucleic acid isolation method for a living body (microorganism) according to one embodiment.
FIG. 4 is a graph showing the relationship between pH and the dissolution pattern of hypochlorous acid.
Detailed Description
Hereinafter, a method for extracting nucleic acid from a living body and a water treatment system according to one embodiment will be described with reference to fig. 1 to 4.
In the present embodiment, the water treatment system of the present invention will be described as a water treatment system for treating wastewater such as industrial wastewater, discharging the wastewater to public water areas, or reusing the treated water. In this embodiment, the method for extracting nucleic acid from a biological body according to the present invention will be described as a water treatment system for extracting nucleic acid from microorganisms (including viruses and biological bodies) in water to be treated by the water treatment system according to the present embodiment, and checking and managing the treatment state of the treated water before discharge and reuse.
The water treatment system and the method for extracting nucleic acid from a living body according to the present embodiment are not limited to the application, use, and method thereof, and can be applied to all cases requiring water treatment and nucleic acid extraction from a living body.
In the present embodiment, the "method for extracting nucleic acid from a living body" will be hereinafter referred to as "method for extracting nucleic acid from a microorganism".
(Water treatment System)
As shown in fig. 1, a water treatment system 1 according to the present embodiment includes: a water storage unit 2 for temporarily storing wastewater (water to be treated) W1 such as industrial wastewater that may contain microorganisms to be detected; a water treatment section 3 for purifying the treated wastewater W1, that is, for improving the water quality; a microorganism quantifying unit (organism quantifying unit) 4 for measuring (quantifying) the amount of microorganisms (organism amount) in the treated water W2 using a sample solution (sample, sample water) W2' sampled from the treated water W2 purified by the water treatment unit 3; and a drainage unit 5 configured to drain the treated water W2 when the amount of microorganisms in the microorganisms quantifying unit 4 is equal to or less than a preset set value.
The water treatment section 3 performs, for example, biological treatment such as an activated sludge process, physicochemical treatment such as advanced treatment such as coagulation and precipitation, filtration, and UV irradiation, and performs appropriate necessary treatment such as removal, separation, concentration adjustment, and pH adjustment of harmful substances such as N (nitrogen), P (phosphorus), C (carbon such as organic carbon), and heavy metals in the wastewater, and performs purification treatment of the wastewater W1 to be treated.
The microorganism quantifying unit 4 is provided for quantifying microorganisms contained in the treated water W2 with respect to the treated water W2 treated by the water treatment unit 3 and determining whether or not to discharge the water.
In the microorganism quantifying unit 4, for example, when the treated water W2 is discharged from the drainage unit 5 to public waters such as rivers, lakes and marshes, seas, and the like, or when the treated water W2 is reused as reclaimed water, microorganisms that may have adverse effects or adverse phenomena on the natural environment, industries such as fishery and agriculture, and humans are detected, and the amount of the microorganisms is quantified. In the treated water W2, not only may adverse effects or adverse phenomena occur due to the presence of microorganisms, but also microorganisms preferably present may be detected and quantified.
Examples of the microorganism (organism) in the present embodiment include: staphylococci, escherichia coli, salmonella, pseudomonas aeruginosa, vibrio cholerae, shigella, anthrax, tubercle bacillus, clostridium botulinum, clostridium tetani, streptococcus, bacteria, candida, aspergillus, norovirus, rotavirus, influenza virus, adenovirus, coronavirus, measles virus, rubella virus, hepatitis virus, herpes virus, HIV, etc., all viruses, prokaryotes (eubacteria, archaea), eukaryotes (algae, protists, fungi, slime), small animals (rotifers, etc.), and the like. That is, the microorganism in the present embodiment is required to be detected and quantified by all microorganisms (including viruses). In the method for extracting nucleic acid from a living body of the present invention, the living body may include a tissue or the like constituting a part of a multicellular organism, and the living body is not necessarily limited to a microorganism.
Incidentally, in the case where the treated water W2 is discharged to public waters such as rivers, seas, and the like, it is required to sufficiently consider the influence on fishery. For example, in farms for puffer fish, small fish (ハマチ), oysters, salmon, and the like, much labor and cost are required to prevent diseases and the like caused by microorganisms. Therefore, it is important to consider not only the influence on the vicinity of the discharge treated water W2 but also the influence on a wide area. The same applies to agriculture and other industries, and it is needless to say that the influence on animals, plants, humans, and the like is also required to be considered.
The microorganism quantifying unit 4 of the present embodiment destroys (dissolves) cell walls, cell membranes, cytoplasm, other tissues surrounding nucleic acids, and the like of the microorganism to be detected/quantified with respect to the sample liquid W2' sampled from the treated water W2, and extracts nucleic acids (DNA and/or RNA). In the case of viruses, nucleic acids are extracted by disrupting the capsid, envelope (envelope), and the like.
Then, the extracted nucleic acid is amplified and quantified by a PCR (polymerase chain reaction) method such as a quantitative PCR method.
As is well known, the quantitative PCR (qPCR) method is a method of measuring a fluorescent substance generated by a series of PCR up to a determined cycle number, and is capable of measuring nucleic acid synthesis in qPCR examination and determining the amount (nucleic acid amount) of a mold DNA (mainly cDNA) from fluorescence intensity. Therefore, the present invention is used not only in the fields of food and medical care, but also in quantitative inspection of microorganisms and the like contained in treated water W2 when treated water W2 obtained by treating water such as various waste waters W1 is discharged to public waters such as rivers, lakes and marshes, the sea, and groundwater, and further in management of discharged water such as compliance of drainage standards, environmental standards, and the like. The main quantitative PCR method includes i) MPN-PCR (Most basic Number-Polymerase Chain Reaction: maximum likelihood polymerase chain reaction), ii) competitive PCR, iii) real-time quantitative PCR, and the like.
The amount of nucleic acid (gene) can be calculated by measuring the Ct value (threshold Cycle). The Ct value is the number of PCR cycles at which a constant amount of an amplification product is obtained, and is inversely proportional to the initial amount of a target nucleic acid, and therefore the amount of the nucleic acid can be calculated by measuring the Ct value.
Specifically, for example, first, an amplification curve arranged at equal intervals is obtained from the initial amount of DNA using a standard solution column obtained by diluting a sample containing a known amount of DNA in a stepwise manner. A threshold value is set at a point where the amplification curve rises, and a point intersecting the amplification curve is obtained as a Ct value. Next, a linear calibration curve is created between the Ct value and the initial DNA amount. Then, the Ct value is measured similarly for a sample containing an unknown amount of DNA, and the initial amount of DNA of the sample containing the unknown amount of DNA is determined by checking with a calibration curve.
As a simpler quantitative method, a straight line portion may be selected from an amplification curve obtained as a result of PCR, and the fluorescence intensity in a section calculated by extrapolating the straight line portion may be used as the initial nucleic acid amount.
Here, the method of quantifying the amount of nucleic acid by the quantitative microorganism determining section 4 of the present embodiment may be any method as long as it can quantify the amount of nucleic acid of the microorganism, and for example, a culture method, a FRET (fluorescence resonance energy transfer) method, a CPRINS-FISH (Cycling in situ amplification-fluorescence in situ hybridization) method, or the like may be used, and the method may not be limited to a PCR method (quantitative PCR method).
In the water treatment system 1 of the present embodiment, when it is determined that the amount of microorganisms in the treated water W2 is appropriate based on the quantitative examination of microorganisms in the microorganism quantifying unit 4, the treated water W2 is sent to the water drain unit 5, and is appropriately discharged from the water drain unit 5 to an industrial water area or reused as reclaimed water. When it is determined that the amount of microorganisms in the treated water W2 is not appropriate, the treated water W2 is returned to the water reservoir 2 through the return line 6 before being sent to the drain 5 or from the drain 5, for example.
Here, conventionally, as a method for extracting nucleic acid from a microorganism to be subjected to nucleic acid amplification pretreatment, for example, there has been used: a method of heat-treating microorganisms, a method of adding a surfactant, a phenol extraction method, an osmotic shock method, a freeze-thaw method, an enzymatic digestion method, use of a kit for DNA extraction, an ultrasonic treatment method, a French press method, use of a homogenizer, and the like. In addition, there are cases where two or more of these methods are combined.
However, as a result of repeated studies by the inventors of the present application, it has been confirmed that the conventional method for extracting nucleic acid from a living body such as a microorganism requires much time, labor, and cost, and thus the efficiency of nucleic acid extraction is low. Therefore, it is required to develop a method capable of extracting nucleic acids from organisms such as microorganisms more easily, efficiently and efficiently with excellent nucleic acid extraction efficiency, and the inventors of the present application have made studies on this point and completed the method for extracting nucleic acids from organisms of the present invention.
(method of extracting nucleic acid from organism such as microorganism)
The method for extracting nucleic acid from a microorganism of the present embodiment includes: an oxidizing agent addition step of adding an oxidizing agent to the sample liquid W2' for nucleic acid extraction of a microorganism so as to have a concentration within a concentration range determined in advance by an experiment, as a pre-step (pretreatment step) for nucleic acid amplification of the microorganism; and a nucleic acid extraction step of extracting nucleic acid of the microorganism to be extracted from the sample solution W2' to which the oxidizing agent is added.
The oxidizing agent addition step of the present embodiment is a sodium hypochlorite addition step, and hypochlorous acid (sodium hypochlorite solution) is added as the oxidizing agent.
The method for extracting nucleic acid from a microorganism according to the present embodiment includes: and a sodium hypochlorite concentration determining step (oxidizing agent concentration determining step) for determining a sodium hypochlorite concentration range of the oxidizing agent.
In the above-described test in the sodium hypochlorite concentration determining step, a plurality of sample solutions W2 'having different sodium hypochlorite concentrations were prepared, and an amplicon amplification curve (a curve showing the relationship between PRC cycle and fluorescence intensity) as shown in fig. 2 was prepared for each sample solution W2' by the quantitative PCR method. Then, the concentration range of sodium hypochlorite was determined based on the obtained amplicon amplification curve.
Then, the sample solution W2' obtained by neutralizing sodium hypochlorite added as needed was subjected to conventional operations of nucleic acid extraction, purification, and quantification. For example, the sample solution W2' was subjected to extraction and purification of a nucleic acid of a microorganism according to the protocol using an examination Kit such as Extrap Soil DNA Kit Plus ver.2 (manufactured by Nippon environmental Co., Ltd.).
Therefore, in the method for extracting nucleic acid from microorganisms of the present embodiment and the water treatment system 1 using the method, based on the results of repeated studies by the present inventors, as the previous steps for extracting nucleic acid by a PCR method or the like, a sodium hypochlorite concentration determining step and a sodium hypochlorite adding step are included, and sodium hypochlorite (sodium hypochlorite solution) is added to the sample solution W2' so as to have a concentration corresponding to the microorganism from which nucleic acid is extracted. Thus, nucleic acids can be extracted with an extraordinarily excellent nucleic acid extraction rate (nucleic acid recovery rate) as compared with conventional nucleic acids. As shown in FIGS. 2 and 3, as a result of repeated studies by the inventors of the present application, it was confirmed that the effect of improving the nucleic acid extraction rate was 10 to 100 times as high as that of the conventional method (0 ppm by weight (hereinafter referred to as ppm)). In FIG. 3, the ordinate represents the relative value of the initial concentration of DNA when the initial concentration of untreated DNA is 1, and the abscissa represents the concentration of sodium hypochlorite.
That is, based on the particularly remarkable findings and results of research by the inventors of the present application, it is possible to achieve nucleic acid extraction that is remarkably superior to conventional nucleic acid extraction only by adding an appropriate amount of sodium hypochlorite to a sample at a concentration corresponding to the microorganism to be subjected to nucleic acid extraction (difficulty and easiness in disrupting cell walls, cell membranes, and the like).
In other words, with respect to various microorganisms to be extracted nucleic acid, which have different difficulties and ease of lysis (destruction) of cell walls, cell membranes, cytoplasm, capsids, envelopes and the like, nucleic acids can be extracted at a very excellent nucleic acid extraction rate by appropriately destroying cell walls, cell membranes, cytoplasm, capsids, envelopes and the like without damaging the nucleic acids by merely adjusting the concentration of sodium hypochlorite.
Thus, an epoch-making method for extracting nucleic acid from a microorganism can be realized and provided, which can easily, efficiently and effectively extract nucleic acid from a microorganism without requiring complicated operations and the like as in the conventional methods.
Further, by performing an experiment using the quantitative PCR method, amplicon amplification curves were prepared for a plurality of sample solutions W2' having different concentrations of sodium hypochlorite, and the optimal concentration range of sodium hypochlorite in nucleic acid extraction of the microorganism to be subjected to nucleic acid extraction was determined.
Thus, in the sodium hypochlorite addition step, by adding sodium hypochlorite to the sample solution so as to fall within the concentration range of sodium hypochlorite determined by the test, it is possible to extract nucleic acids with a high nucleic acid extraction rate and high reliability. That is, the extraction of nucleic acid from a microorganism can be more reliably, easily, efficiently, and effectively achieved.
Here, as shown in FIGS. 2 and 3 as an example, in the sodium hypochlorite addition step of the method for extracting nucleic acid from a microorganism according to the present embodiment, the concentration of sodium hypochlorite is in the range of 0.5 to 8ppm, preferably 0.5 to 2 ppm. This concentration range is obtained based on the findings of experiments for nucleic acid extraction from various microorganisms and organisms performed by the inventors of the present application.
The water treatment system 1 of the present embodiment further includes: a water storage unit 2 for temporarily storing wastewater W1 before the microbial biomass is quantified by the microbial quantifying unit 4; and a return line 6 for returning the treated water W2 to the water storage unit 2 when the amount of microorganisms in the microorganism quantifying unit 4 exceeds a set value.
The water treatment system 1 of the present embodiment further includes: a water feeding device 7 having a pump or the like for feeding the treated water W2 to the water discharge unit 5 or the water storage unit 2 based on the result of the microbial biomass determination in the microbial biomass determination unit 4; and a control unit 8 for controlling the driving of the water supply device 7.
Further, by providing the water storage part 2 and the return line 6, the amount of microorganisms in the treated water W2 can be efficiently and effectively detected by the method for extracting nucleic acid using microorganisms, and when it is determined that the treated water W2 cannot be discharged to the outside through the water discharge part 5, the treated water W2 can be returned to the water storage part 2 through the return line 6. This enables the treated water W2 to be reprocessed, enables higher-level treatment to be performed, and enables efficient and reliable water treatment.
Further, the nucleic acid isolation method using microorganisms comprising the water supply device 7 and the controller 8 enables the amount of microorganisms in the treated water W2 to be detected efficiently and effectively, and the controller 8 controls the driving of the water supply device 7 based on the result of the judgment as to whether or not the treated water W2 can be discharged to the outside through the water discharge unit 5, thereby automatically returning the treated water W2 to the water storage unit 2 or sending the treated water to the water discharge unit 5 for discharge. This enables further efficient and reliable water treatment.
Here, in the water treatment system 1 of the present embodiment, the water treatment unit 3 includes, for example, a filter unit 3a that performs a filtration treatment on water or a sterilization unit 3b that performs a sterilization treatment on water.
For example, when the filtration unit 3a is constituted by a single filtration unit such as sand filtration, MF membrane, UF membrane, or RO membrane or by combining a plurality of filtration units, the microbial nucleic acid isolation method of the present embodiment can efficiently and effectively detect the microbial biomass of the treated water W2, and thus can easily sort a desired combination of filtration units or filtration units. This enables more efficient and reliable water treatment.
Further, for example, when the sterilization unit is constituted by sterilizing by UV irradiation, sterilizing by ozone, adding a bactericide or the like, singly or in combination of a plurality of units, the microbial nucleic acid extraction method of the present embodiment can efficiently and effectively detect the microbial biomass of the treated water W2, and thus can easily sort out a combination of a necessary sterilization unit and a sterilization unit. This enables more efficient and reliable water treatment.
Accordingly, when the treated water W2 is returned to the water storage unit 2 and further to the water treatment unit 3 through the return line 6, the treated water W2 can be reprocessed, and further advanced treatment can be performed by appropriately selecting and combining the filter unit, the sterilization unit, and the like, and thus, highly efficient and reliable water treatment can be realized.
Here, when a microorganism is sterilized by adding a bactericide (bactericide) to the wastewater W1 or the return treated water W2, for example, a bactericide capable of destroying a biofilm formed by a cell wall or an aggregate of microorganisms and sterilizing is preferable as the bactericide. Examples thereof include halogen-based bactericides, ozone, and hydrogen peroxide. Examples of the halogen-based bactericide include hypochlorous acid (HClO) and salts thereof (e.g., sodium hypochlorite), monochloramine (NH)2Cl), bromochlorohydantoin (Br, Cl-DMH), bromosulfamic acid (BrNHSO)3H) Bromochloramine (NH)4Br + HClO), and the like. Among them, from the viewpoint of the intensity of bactericidal activity, a halogen-based bactericide or ozone is preferable, and hypochlorous acid and salts thereof are more preferable.
In addition, in the case where a bactericide is added, it is desirable to perform neutralization treatment as needed. For example, when sodium hypochlorite is used as the bactericide, sodium thiosulfate and the like are used as the neutralizer.
(examples)
Next, the method for extracting nucleic acid from microorganisms of the present embodiment will be described in more detail below, with reference to an example in which a waste liquid (for example, waste water W1 containing an absorbent (slurry) used in the limescale process) generated when sulfur oxides are removed from waste gas discharged from thermal power plants or the like by a wet flue gas desulfurization apparatus is purified, and the method for extracting nucleic acid from microorganisms of the present embodiment is applied to the examination of the amount of microorganisms in treated water W2 after treatment.
Here, there is a case where the absorption liquid which has been brought into contact with the exhaust gas and absorbed the sulfur oxides has a high Chemical Oxygen Demand (COD). As a result of repeated studies by the inventors of the present application, it is found that this is mainly caused by the proliferation of sulfur oxidizing bacteria (for example, bacteria belonging to the genus thermothiobacillus) as autotrophic bacteria in the waste liquid of the absorbent solution in which sulfur oxides are absorbed. When sulfur-oxidizing bacteria grow, heterotrophic bacteria (for example, bacteria of the genus Pseudomonas (Pseudomonas)) that grow using the products (organic substances) of the sulfur-oxidizing bacteria grow, and organic substances such as sugars are also produced from the grown heterotrophic bacteria, so that the COD of the waste liquid may increase.
When the COD is higher than the drainage standard, the wastewater cannot be discharged to public waters such as rivers, and therefore, the water treatment system 1 performs a purification treatment.
On the other hand, it is known that when treated water W2 containing sulfur-oxidizing bacteria, which is not specified in the drainage standards, is discharged and drained through a sewer pipe or the like, concrete corrosion of a sewage treatment facility such as a sewer pipe or the like occurs.
Specifically, sulfate ions in anaerobic sewage are reduced by sulfate-reducing bacteria contained in the sewage to generate hydrogen sulfide, and the hydrogen sulfide is released from the sewage into the air, and is absorbed and dissolved by dew condensation or the like on the concrete surface. In this state, when sulfur-oxidizing bacteria are present on the surface of the concrete in an aerobic state, hydrogen sulfide absorbed by dew condensation or the like is oxidized by the sulfur-oxidizing bacteria to generate sulfuric acid, and the concrete is dissolved by the sulfuric acid, so-called concrete corrosion occurs. If such microorganisms cause concrete corrosion of underground sewers or the like, the concrete is changed to clay and loses the endurance of concrete structures, and unexpected sudden road collapse, foundation subsidence, and the like may occur as a function of sewer facilities.
Therefore, it is preferable that the treated water W2 obtained by purifying the wastewater W1 containing the absorbent having absorbed the sulfur oxides is quantitatively determined and confirmed as bacterial load of sulfur-oxidizing bacteria and sulfate-reducing bacteria as microorganisms, and then discharged to public waters or the like.
In the present example, in order to examine the number of sulfur-oxidizing bacteria in the treated water, the nucleic acid extraction method of microorganisms and the water treatment system 1 according to the present embodiment were applied, and the number of sulfur-oxidizing bacteria in the wastewater was detected by a PCR method using a primer set for amplification of a known gene of sulfur-oxidizing bacteria. The amount of the oxidizing bactericide is adjusted based on the amount of the detected sulfur-oxidizing bacteria, and the treated water W2 is sterilized (sterilized) and discharged.
Incidentally, the absorbing liquid to be used may be any absorbing sulfur oxides, and for example, a slurry obtained by dissolving (dispersing) limestone (lime gypsum method), an aqueous sodium hydroxide solution (caustic soda method), or a slurry obtained by dissolving (dispersing) magnesium hydroxide (magnesium method) may be used. In the present embodiment, a description will be given of an example in which a slurry obtained by dissolving (dispersing) limestone is used as an absorbing liquid.
Furthermore, sulfur-oxidizing bacteria of microorganisms that are targets for nucleic acid extraction are sulfur compounds (S)2-) Reduced inorganic sulfur compounds such as Sulfur Oxide (SO), thiosulfuric acid, polythionic acid, sulfurous acid, etc. can be used as bacteria for energy for propagation. Examples of the sulfur oxidizing bacteria include: bacteria that temporarily precipitate sulfur in their cells, such as sulfobacillus beijerinckii (Beggiatoa), sulfobacillus (Thiothrix), and sulfobacillus (thiobacillus); mesophilic bacteria that do not accumulate sulfur in the body, such as Thiobacillus (Thiobacillus), Thermithiobacillus (Thermithiobacillus), Thiomonas (Thiomonas), and Thiospirillum (Thiomicrospira); thermophilic bacteria such as high-temperature trichogenous bacteria (Thermothrix); acidovorax (Acidianus) genus, Staphylococcus aureus (Metallosp)And hyperthermophilic bacteria such as Haera) genus and Sulfolobus (Sulfolobus) genus. In the present embodiment, as an example, it is considered that the sulfur oxidizing bacteria present in the desulfurization apparatus are bacteria belonging to the genus Thermithiobacillus (Thermithiobacillus).
In the present example, the amount of the sulfur-oxidizing bacteria in the treated water W2 was detected and quantified by a PCR method using a primer set for amplification of a known gene of the sulfur-oxidizing bacteria.
The cell amount of the sulfur-oxidizing bacteria in the treated water W2 was detected as the amount of known genes of the sulfur-oxidizing bacteria. The known gene may be any gene specific to sulfur-oxidizing bacteria as long as it is a gene possessed by the sulfur-oxidizing bacteria, and for example, a gene related to sulfur metabolism may be used. Paracoccus sulfuration pathway (PSO pathway, also called sox pathway) and S4 intermediateprathway (S4 pathway) exist in sulfur metabolism. In the PSO pathway, the enzyme encoded by the sox gene cluster (soxXAYZBCD) plays a major role. Specific examples of the enzyme include soxyyz, soxXA, soxB, and soxCD. Among them, the known gene is preferably the soxB gene. The soxB gene of a sulfur-oxidizing bacterium is composed of, for example, the base sequence shown in SEQ ID No. 1.
On the other hand, examples of the gene related to the S4 pathway include a thiosulfate dehydrogenase gene, a sulfite dehydrogenase gene, and a Tetrathionate hydrolase (4 THase) gene.
The primer set for amplifying a known gene can be designed by a known method in consideration of the nucleotide sequence, Tm value, GC content, and the like of the target gene.
For example, the soxB gene, particularly the amplification primer set for the region consisting of the base sequence represented by sequence No. 1, includes, for example, one or more primers selected from the group consisting of table 1 and 1) to 8) below, and the like.
1) A forward primer (forward primer) comprising the base sequence represented by SEQ ID No. 2 and a reverse primer (reverse primer) comprising the base sequence represented by SEQ ID No. 3;
2) a forward primer consisting of the base sequence represented by SEQ ID No. 4 and a reverse primer consisting of the base sequence represented by SEQ ID No. 5;
3) a forward primer consisting of the base sequence represented by SEQ ID No. 6 and a reverse primer consisting of the base sequence represented by SEQ ID No. 7;
4) a forward primer consisting of the base sequence represented by SEQ ID No. 8 and a reverse primer consisting of the base sequence represented by SEQ ID No. 9;
5) a forward primer consisting of the base sequence represented by SEQ ID No. 10 and a reverse primer consisting of the base sequence represented by SEQ ID No. 11;
6) a forward primer consisting of the base sequence represented by SEQ ID No. 12 and a reverse primer consisting of the base sequence represented by SEQ ID No. 13;
7) a forward primer consisting of the base sequence represented by SEQ ID No. 14 and a reverse primer consisting of the base sequence represented by SEQ ID No. 15;
8) a forward primer consisting of the base sequence represented by SEQ ID No. 16 and a reverse primer consisting of the base sequence represented by SEQ ID No. 17;
[ Table 1]
The above-mentioned primer set is an example of a primer set that can be used. Since the primer can be designed to have a length of about 15 bases or more and 30 bases or less, a primer pair in which the base length is increased or decreased by, for example, about 1 base, 2 bases, 3 bases, 5 bases, or 10 bases can be similarly used in the above-mentioned primer pair in view of the GC content and Tm value.
In the PCR method targeting the known genes described above, the amount of the sulfur-oxidizing bacteria contained in the wastewater W1 can be quantified as the amount of the known genes of the sulfur-oxidizing bacteria. In this case, by performing two-stage operation of extracting nucleic acid from sulfur-oxidizing bacteria contained in the treated water W2 and then performing PCR on the obtained extract as a casting sample, the measurement results can be obtained in a short time of 3 hours or more and 1 day or less.
(method of extracting DNA from sulfur-oxidizing bacteria)
In the pretreatment for quantifying the amount of bacteria by the PCR method, for example, extraction and purification of DNA of sulfur-oxidizing bacteria were performed based on a gypsum sample collected from a desulfurization apparatus according to the experimental procedure using the extract Soil DNA Kit Plus ver.2 (manufactured by nippon iron environmental co., ltd.).
In the method for extracting DNA from sulfur-oxidizing bacteria of the present embodiment (method for extracting nucleic acid from microorganisms of the present embodiment), first, 1cc of sodium hypochlorite solutions having different sodium hypochlorite concentrations were added to 1cc of a sample solution of a target biological suspension so that the sodium hypochlorite concentration (final concentration) was 0.5ppm (case 1), 1ppm (case 2), 2ppm (case 3), 4ppm (case 4), 8ppm (case 5), and 16ppm (case 6), and 6 kinds of sample solutions (mixed solutions) W2' of cases 1 to 6 were generated and vibrated for 30 minutes using a vortex mixer or the like. The temperature of the sample liquid W2' was set to 25 ℃.
After the vibration treatment, it is preferable to add a solution of a neutralizing agent such as sodium thiosulfate to each of the 6 sample solutions W2 'which are mixed solutions of the target biological suspension and sodium hypochlorite to neutralize residual chlorine remaining in each sample solution W2'. In this example, a sodium thiosulfate solution was used, and an equivalent amount of the sodium thiosulfate solution corresponding to the amount of sodium hypochlorite added was added to perform neutralization treatment.
Next, each sample solution W2', which is a mixture of the target biological suspension, the sodium hypochlorite solution, and the sodium thiosulfate solution, was centrifuged at 13500rpm for 10 minutes using a centrifuge.
Then, in the present example, the nucleic acid (DNA) of the thiooxidated bacteria was extracted and purified according to the experimental procedure using, for example, the extract Soil DNA Kit Plus ver.2 (manufactured by nippon environmental co., ltd.) from the pellet obtained by the centrifugal separation treatment and the solid-liquid separation (the pellet subjected to the pretreatment for the quantification of the amount of the bacteria by the PCR method).
The experimental procedures for the extraction and purification of nucleic acids from sulfur-oxidizing bacteria are as follows.
1) To a rectification tube (Bead Tubes) were added: particles of the sample: 0.5g (incidentally, 500. mu.L in the case of a liquid sample); extraction Buffer (Extraction Buffer, mixture of major ingredient/disodium phosphate): 950 μ L; lysis Solution (Lysis Solution, mixture of main component/sodium lauryl sulfate): 50 μ L.
When concentration is required, it is desirable to filter/capture microorganisms in a sample using a membrane filter and extract nucleic acids from the filter.
2) Stir with vortex mixer for 5 seconds.
3) Bead milling (Beads milling) (4-6 m/s or 30-45 seconds at 4200-6800 rpm).
4) Centrifugation was carried out (14000 Xg, 5 min, 4 ℃ C.).
5) Transfer 600 μ L of supernatant to a 1.5mL tube and add PP Solution: 300 μ L.
6) The tube of 5) was inverted and mixed about 10 times, and stirred.
7) Centrifugation was carried out (14000 Xg, 5 min, 4 ℃ C.).
8) Transfer 800. mu.L of supernatant to a 2mL tube.
9) To the tube of 8) was added: MBs Solution (mixture of main ingredient/guanidine hydrochloride, ferric oxide): 50 μ L, Binding Solution (Binding Solution, mixture of principal component/guanidine thiocyanate): 890. mu.L.
10) The tube of 9) was mixed by inversion for about 2 minutes, and sufficiently stirred.
11) Spin-down (spin-down) the tube of 10) was placed on a magnetic stage.
12) After magnetic collection was performed for 1 minute or more, the supernatant was removed using a micropipette.
13) Add wash Solution (wash Solution, mixture of principal component/guanidine thiocyanate) to tube of 12): 800 μ L, stirred well by a vortex mixer (low speed).
14) The tube of 13) was decelerated, placed on a magnetic stage, magnetism was collected for 1 minute or more, and then the supernatant was removed using a micropipette.
15) Adding 70% ethanol solution: 1mL, and stirred well with a vortex mixer (low speed).
16) The tube of 15) was decelerated, magnetic collection was performed for 1 minute or more by a magnetic stage, and then ethanol was removed by a micropipette.
17) The steps from 15) to 16) are repeated again.
18) The magnetic particles were air-dried at room temperature for about 10 minutes with the lid of the tube open.
19) Addition of lysis solution (TE buffer, sterile water, etc.): after 100. mu.L, the mixture was sufficiently stirred by a vortex mixer (low speed).
20) While stirring several times in the middle, heating the mixture for 5 to 10 minutes by using a heating block (or water bath) at 65 ℃.
21) After the tube is placed on a magnetic table for magnetism collection, the solution is moved to a new tube.
Thus, nucleic acid is eluted, and an eluate (sample solution) containing nucleic acid is obtained.
Next, the amount of the soxB gene was quantified by real-time PCR using the purified sample as a casting mold and using 5 to 8 primer pairs among the 8 primer pairs shown in Table 1. The reaction conditions for PCR were: after the reaction was carried out at 98 ℃ for 2 minutes as the initial denaturation, 45 cycles of "DNA denaturation reaction at 98 ℃ for 10 seconds, annealing at 55 ℃ for 10 seconds, and extension reaction at 68 ℃ for 30 seconds" were carried out.
Thus, as a result of the real-time PCR method using the primer pair 8 of the primer pair and having the cycle number of 45 cycles, the amplification curve of each amplicon (curve of the relationship between PRC cycle and fluorescence intensity) when the sodium hypochlorite concentration was changed as shown in fig. 2 was obtained.
Thus, it was confirmed that nucleic acids can be extracted with a very excellent nucleic acid extraction rate by appropriately destroying cell walls, cell membranes, cytoplasm, capsids, envelopes and the like only by pretreatment with sodium hypochlorite and adjusting the concentration of sodium hypochlorite. Furthermore, it was also confirmed from the amplification curves of the respective amplicons that if sodium hypochlorite is added to the sample solution W2' so that the concentration of sodium hypochlorite becomes 0.5 to 8ppm, nucleic acids of various microorganisms can be appropriately extracted at a higher extraction rate than in the conventional nucleic acid extraction methods.
As described above, based on the results of the quantification of microorganisms in the treated water W2 of the wastewater W1, a bactericide or the like is added to the treated water W2 as needed. In this case, the amount of the oxidizing bactericide used for sterilization can be appropriately adjusted according to the amount of the sulfur-oxidizing bacteria. Further, the amount of the oxidizing bactericide to be used may be appropriately adjusted according to the type of the oxidizing bactericide to be used. For example, when sodium hypochlorite is used as the oxidizing bactericide, the amount of sodium hypochlorite to be added may be set so that the final concentration in the treated water W2 becomes 1ppm or more and 70ppm or less.
The sterilization time of the oxidizing bactericide may be, for example, about 1 minute to 24 hours, preferably about 1 minute to 12 hours, and more preferably about 1 minute to 6 hours. When the sterilization time is equal to or more than the lower limit value, a more sufficient sterilization effect can be obtained, and when the sterilization time is equal to or less than the upper limit value, damage such as corrosion to the desulfurization apparatus can be reduced.
While the embodiments of the method for extracting a biological nucleic acid and the water treatment system 1 of the present invention have been described above, the method for extracting a biological nucleic acid and the water treatment system 1 of the present invention are not limited to the above-described embodiments, and may be modified as appropriate without departing from the scope of the invention.
For example, in the present embodiment,: in the sodium hypochlorite addition step, sodium hypochlorite is added to the sample solution W2' for extracting nucleic acid from a microorganism (organism) so that the concentration thereof is within a concentration range determined in advance by the test.
In the present embodiment, the following are provided: in the test in the sodium hypochlorite concentration determining step, a plurality of sample solutions W2' having different sodium hypochlorite concentrations were prepared, an amplicon amplification curve was prepared for each sample solution by the quantitative PCR method, and the sodium hypochlorite concentration range was determined based on the amplicon amplification curve.
On the other hand, as shown in FIG. 4, hypochlorous acid changes into hypochlorous acid (HClO) depending on the pH) Hypochlorous acid ion (ClO)-) Dissolved chlorine gas (Cl)2) Three dissolution forms, the proportions of which vary depending on the pH.
Therefore, even when the concentration of sodium hypochlorite is constant, if the pH of the sample liquid W2' is adjusted, the proportion (concentration) of hypochlorous acid (HClO) having a strong oxidizing power can be adjusted, and as a result, the concentration of hypochlorous acid effective for nucleic acid extraction can be adjusted by adjusting the pH only according to the difficulty of breaking the cell wall, cell membrane, cytoplasm, capsid, envelope, or the like of the microorganism to be extracted, or by adjusting and setting the concentration of sodium hypochlorite according to the pH of water W1 or W2 to be examined, and as a result, nucleic acid can be extracted with a very excellent nucleic acid extraction rate, as in the present embodiment.
In the present embodiment, the following are provided: in order to appropriately destroy the cell wall, cell membrane, cytoplasm, capsid, envelope, etc. of a living body such as a microorganism to be subjected to nucleic acid extraction, sodium hypochlorite is added to the sample solution W2' as a pretreatment
On the other hand, the same effect can be obtained even with an oxidizing agent other than sodium hypochlorite. In addition, as described above, the form of the nucleic acid-extracting microorganism, which is dissolved in other oxidizing agents, and is suitable for disrupting the cell wall, cell membrane, cytoplasm, capsid, envelope, and the like of the organism such as the microorganism to be nucleic acid-extracted, also varies depending on pH.
Therefore, it can be said that the same action and effect as in the present embodiment can be obtained by selecting an oxidizing agent, adjusting the concentration and pH, and the like, depending on the difficulty of disrupting cell walls, cell membranes, cytoplasm, capsid, envelope, and the like.
As such an oxidizing agent, all known oxidizing agents can be used. For example, a halogen-based monochloramine (NH)2Cl), bromochlorohydantoin (Br, Cl-DMH), bromosulfamic acid (BrNHSO)3H) Bromochloramine (NH)4Br + HClO), and the like. Further, ozone, hydrogen peroxide, potassium permanganate, ferrous iron, ferric iron, 0-valent iron, and the like can be cited.
It was confirmed by repeated studies of the inventors that: for example, in view of the purpose and connection of improving the nucleic acid extraction rate by disrupting cell walls, cell membranes, cytoplasm, capsids, envelopes, and the like of a living body, it is more preferable to use an oxidizing agent having a higher oxidizing power in a neutral region such as sodium hypochlorite and a lower oxidizing power in a solution having a higher acidity and alkalinity (an oxidizing agent having a tendency as shown in fig. 4) than an oxidizing agent having a higher oxidizing power in an acidic solution such as iodic acid and a lower oxidizing power in a basic solution.
In the present embodiment, the following are provided: the method for extracting nucleic acid using the microorganism of the present invention will be described in order to quantify the amount of the microorganism in the treated water treated in the water treatment system. In the examples, the microorganisms to be extracted from nucleic acids are described as sulfur oxidizing bacteria.
On the other hand, the method for extracting nucleic acid from a living body and the water treatment system using the same of the present invention can be applied to the extraction of nucleic acid from a living body such as microorganisms contained in water in rivers, lakes and marshes, ponds, sea areas, and any other water. That is, the use and the like thereof are not necessarily limited.
In addition, in the present embodiment and example, it is assumed that: sodium hypochlorite was added to the sample solution to conduct pretreatment, and the sample solution subjected to the pretreatment was subjected to nucleic acid extraction in this step using a conventionally known nucleic acid extraction kit, and the amount of microorganisms was quantified by the PCR method.
The nucleic acid extraction kit may be constituted so as to include sodium hypochlorite added to the sample solution in advance. In this case, a nucleic acid extraction kit with high reliability can be realized and provided, which can further improve the nucleic acid extraction efficiency more efficiently than before.
Finally, the contents described in the embodiments are grasped as follows, for example.
(1) The method for extracting nucleic acid from an organism according to an embodiment of the present invention comprises: a sodium hypochlorite addition step of adding sodium hypochlorite to a sample solution (sample water, sample solution W2') for extracting nucleic acid from a living body so that the concentration of the sample solution falls within a concentration range determined in advance by a test; and a nucleic acid extraction step of extracting nucleic acid of a living body to be extracted from a sample solution to which sodium hypochlorite is added.
The method for extracting nucleic acid from a living body according to (1) above is based on the finding and research results that have been found by the inventors of the present application that nucleic acid can be extracted at a particularly superior nucleic acid extraction rate (nucleic acid recovery rate) to the conventional one by adding sodium hypochlorite to a living body such as a microorganism that extracts nucleic acid from a sample solution at a concentration that corresponds to the concentration of the living body, and that can realize nucleic acid extraction at a particularly superior level to the conventional one by adding sodium hypochlorite to the sample at a suitable amount to a concentration that corresponds to the living body to be extracted from nucleic acid.
That is, for example, in the case of cell walls, cell membranes, cytoplasm, and viruses, it is possible to appropriately destroy cell walls, cell membranes, cytoplasm, capsids, envelopes, and the like by adjusting the concentration of sodium hypochlorite alone for various organisms extracted with nucleic acids, which have different difficulties in dissolving (destroying) capsids, envelopes, and the like, and to extract nucleic acids with a very excellent nucleic acid extraction rate.
Therefore, an epoch-making method for extracting nucleic acid from a microorganism, which can easily, efficiently and effectively extract nucleic acid from a living body such as a microorganism, can be realized and provided without requiring complicated operations and the like as in the conventional methods.
(2) A method for extracting a nucleic acid from a living organism according to another aspect of the present invention is the method for extracting a nucleic acid from a living organism according to (1) above, comprising: determining a sodium hypochlorite concentration range in the experiment in the sodium hypochlorite concentration determination step, a plurality of sample solutions having different sodium hypochlorite concentrations are prepared, an amplicon amplification curve is prepared for each sample solution by a quantitative PCR method, and the sodium hypochlorite concentration range is determined based on the amplicon amplification curve.
In the method for extracting nucleic acid from a living body according to item (2), an amplicon amplification curve is prepared for a plurality of sample solutions having different concentrations of sodium hypochlorite by performing a test using a quantitative PCR method, and the optimal concentration range of sodium hypochlorite for extracting nucleic acid from a living body to be extracted can be determined.
Thus, in the sodium hypochlorite addition step, by adding sodium hypochlorite to the sample solution so as to fall within the concentration range of sodium hypochlorite determined by the test, it is possible to extract nucleic acids with a high nucleic acid extraction rate and high reliability. That is, nucleic acid extraction from a living body can be achieved more reliably, easily, efficiently, and effectively.
(3) A method for extracting nucleic acid from a living organism according to another aspect of the present invention is the method for extracting nucleic acid from a living organism according to the above (1) or (2), wherein the living organism is a microorganism.
In the method for extracting nucleic acid from a living body according to the above (3), based on the findings of the experiments on nucleic acid extraction from various microorganisms conducted by the inventors of the present application, if sodium hypochlorite is added to a sample solution, nucleic acid of various microorganisms can be appropriately extracted at a higher nucleic acid extraction rate than in the conventional method.
(4) A water treatment system according to one aspect of the present invention includes: a water treatment unit (water treatment unit 3) for improving the quality of water to be treated (wastewater W1); a biological quantitative determination unit (microbial quantitative determination unit, microbial quantitative determination unit 4) for performing nucleic acid extraction of a sample solution obtained from the treated water (treated water W2) treated by the water treatment unit using a biological nucleic acid extraction method to determine the amount of biological substances in the treated water; and a drainage unit (drainage unit 5) for draining the treated water when the amount of the living organism quantified by the living organism quantifying unit is equal to or less than a preset set value, the method for extracting nucleic acid from a living organism comprising: a sodium hypochlorite addition step of adding sodium hypochlorite to a sample solution for extracting nucleic acid from a living body so that the concentration of the sample solution is within a concentration range determined in advance by an experiment; and a nucleic acid extraction step of extracting nucleic acids of the microorganisms to be extracted from the sample solution to which sodium hypochlorite is added.
The water treatment system according to (4) above, which is provided with the biological quantitative section for quantifying the amount of the biological substance in the water treated by the water treatment section by using the method for extracting nucleic acid from a biological substance comprising the sodium hypochlorite addition step and the nucleic acid extraction step, can exhibit the effect of the method for extracting nucleic acid from a biological substance according to (1) above. This makes it possible to realize highly reliable water treatment and to appropriately ensure the reliability of water discharged from the water discharge unit.
(5) A water treatment system according to another aspect of the present invention is the water treatment system according to (4) above, wherein the water treatment unit includes: and a filtering unit (filtering unit 3a) for filtering the water.
In the water treatment system of the above (5), when the filter unit is constituted by a single filter unit such as sand filtration, an MF membrane, a UF membrane, or an RO membrane or by combining a plurality of filter units, the amount of the organism in the treated water can be detected efficiently and effectively by using the method for nucleic acid isolation of an organism of any of the above (1) to (3), and thus a desired combination of the filter unit and the filter unit can be easily selected. This enables more efficient and reliable water treatment.
(6) A water treatment system according to another aspect of the present invention is the water treatment system according to (4) above, wherein the water treatment unit includes: and a sterilization unit (sterilization unit 3b) for sterilizing the water.
In the water treatment system of the above (6), when the sterilizing unit such as UV irradiation sterilization, ozone sterilization, hypochlorous acid sterilization, or the like is used singly or in combination in a plurality to constitute the sterilizing unit, the amount of the living body in the treated water can be detected efficiently and effectively by using the method for extracting nucleic acid from a living body of any of the above (1) to (3), and thus a combination of the sterilizing unit and the sterilizing unit required for the separation can be easily selected. This enables more efficient and reliable water treatment.
(7) A water treatment system according to another aspect of the present invention is the water treatment system according to any one of the above (4) to (6), further including: a water storage unit (water storage unit 2) for temporarily storing water before the biomass is quantified by the biomass quantifying unit; and a return line (return line 6) for returning the treated water to the water storage unit when the biological quantity determined by the biological quantity determining unit exceeds a set value.
In the water treatment system described in the above (7), the method for extracting nucleic acid from a living body according to any one of the above (1) to (3) is used, and the amount of the living body in the treated water can be efficiently and effectively detected, and the treated water can be returned to the water storage unit through the return line when it is determined that the treated water cannot be discharged to the outside through the water discharge unit. This enables the reuse of the treated water, and further enables the implementation of higher-level treatment, thereby realizing efficient and highly reliable water treatment.
(8) A water treatment system according to another aspect of the present invention is the water treatment system according to (7) above, further including: a water supply device (water supply device 7) for supplying water to the water discharge unit or the water storage unit based on the result of the quantitative determination of the amount of living organisms in the living organism quantitative determination unit; and a control unit (control unit 8) for controlling the driving of the water supply device.
In the water treatment system according to the above (8), the method for extracting nucleic acid from a living body according to any one of the above (1) to (3) is used, whereby the amount of the living body in the treated water can be efficiently and effectively detected, and the control unit controls the driving of the water feeding device based on the result of the judgment as to whether the treated water can be discharged to the outside through the water discharge unit, whereby the treated water can be automatically returned to the water storage unit or discharged to the water discharge unit. This enables more efficient and reliable water treatment.
Description of the reference numerals
1 Water treatment System
2 water storage part
3 Water treatment section
3a filter part
3b bacteria removing part
4 microorganism quantifying unit and microorganism amount determining unit (organism quantifying unit, organism amount determining unit)
5 drainage part
6 Return line
7 water supply device
8 control part
W1 waste water (water)
W2 treated water
W2' sample liquid (sample water)
Claims (8)
1. A method for nucleic acid extraction from an organism, comprising:
a sodium hypochlorite addition step of adding sodium hypochlorite to a sample solution for extracting nucleic acid from a living body so that the concentration of the sample solution is within a concentration range determined in advance by an experiment; and
a nucleic acid extraction step of extracting nucleic acid of the living body to be extracted from the sample solution to which the sodium hypochlorite is added.
2. The method for extracting nucleic acid from an organism according to claim 1,
the method comprises the following steps: a sodium hypochlorite concentration determining step of determining the concentration range of the sodium hypochlorite,
in the test in the sodium hypochlorite concentration determining step, a plurality of sample solutions having different concentrations of the sodium hypochlorite are prepared, an amplicon amplification curve is created for each sample solution by a quantitative PCR method, and the concentration range of the sodium hypochlorite is determined based on the amplicon amplification curve.
3. The method for extracting nucleic acid from an organism according to claim 1 or 2,
the organism is a microorganism.
4. A water treatment system is provided with:
a water treatment unit for improving the quality of water to be treated;
a biological quantitative determination unit for performing nucleic acid extraction of the sample liquid obtained from the treated water obtained by treating the water in the water treatment unit by using a biological nucleic acid extraction method to determine an amount of biological substances in the treated water; and
a water discharge unit configured to discharge the treated water when the biological quantity determined by the biological quantity determining unit is equal to or less than a preset set value,
wherein the nucleic acid extraction method of the organism comprises the following steps: a sodium hypochlorite addition step of adding sodium hypochlorite to a sample solution for extracting nucleic acid from a living body so that the concentration of the sample solution is within a concentration range predetermined by an experiment; and a nucleic acid extraction step of extracting nucleic acid of the living body to be extracted from the sample solution to which the sodium hypochlorite is added.
5. The water treatment system of claim 4,
the water treatment section includes: and a filtering part for filtering the water.
6. The water treatment system of claim 4,
the water treatment section includes: and a sterilization unit for sterilizing the water.
7. The water treatment system according to any one of claims 4 to 6, further comprising:
a water storage unit that temporarily stores the water before the biomass amount is determined by the biomass determining unit; and
and a return line for returning the treated water to the water storage unit when the biological quantity determined by the biological quantity determining unit exceeds the set value.
8. The water treatment system according to claim 7, further comprising:
a water supply device for supplying the treated water to the water discharge unit or the water storage unit based on a result of quantifying the biological quantity in the biological quantity quantifying unit; and
and a control unit for controlling the driving of the water supply device.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000310641A (en) * | 1999-04-28 | 2000-11-07 | Nippon Steel Corp | Method for inspecting microorganism |
| CN1531601A (en) * | 2001-04-23 | 2004-09-22 | ������ɽ���� | PCR-based monitoring in wastewater biotreatment systems |
| CN1842495A (en) * | 2004-02-13 | 2006-10-04 | 三菱重工业株式会社 | Method of liquid detoxification and apparatus therefor |
| WO2010068802A1 (en) * | 2008-12-10 | 2010-06-17 | University Of Washington | RATIOMETRIC PRE-rRNA ANALYSIS |
| CN108473346A (en) * | 2016-03-08 | 2018-08-31 | 富士电机株式会社 | Drainage processing method and disintegrating system in waste pipe |
-
2020
- 2020-06-19 JP JP2020105918A patent/JP2022000003A/en active Pending
-
2021
- 2021-06-08 CN CN202110639506.7A patent/CN113817716A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000310641A (en) * | 1999-04-28 | 2000-11-07 | Nippon Steel Corp | Method for inspecting microorganism |
| CN1531601A (en) * | 2001-04-23 | 2004-09-22 | ������ɽ���� | PCR-based monitoring in wastewater biotreatment systems |
| CN1842495A (en) * | 2004-02-13 | 2006-10-04 | 三菱重工业株式会社 | Method of liquid detoxification and apparatus therefor |
| WO2010068802A1 (en) * | 2008-12-10 | 2010-06-17 | University Of Washington | RATIOMETRIC PRE-rRNA ANALYSIS |
| JP2012511331A (en) * | 2008-12-10 | 2012-05-24 | ユニヴァーシティ オブ ワシントン | Ratiometric analysis of rRNA precursors |
| CN108473346A (en) * | 2016-03-08 | 2018-08-31 | 富士电机株式会社 | Drainage processing method and disintegrating system in waste pipe |
Non-Patent Citations (2)
| Title |
|---|
| MARELIZE BOTES ET AL.: ""Application of quantitative PCR for the detection of microorganisms in water"", 《ANAL BIOANAL CHEM》, vol. 405, 25 September 2012 (2012-09-25), pages 91 - 108, XP037066296, DOI: 10.1007/s00216-012-6399-3 * |
| 余昆英等: ""PCR技术在水体微生物检测 中的应用效果"", 《深圳中西医结合杂志》, vol. 26, no. 9, 31 May 2016 (2016-05-31), pages 73 - 75 * |
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