JP2022000003A - Nucleic acid extraction method and water treatment system for organism - Google Patents

Abstract

To provide a method for extracting nucleic acid from an organism and a water treatment system using the same, which enable more efficient extraction of nucleic acid from an organism, such as a microorganism, with a remarkably excellent high extraction (recovery) rate.SOLUTION: The method comprises the steps of: adding sodium hypochlorite to a sample solution for extracting nucleic acid from an organism so that the concentration thereof is within the concentration range predetermined by the test; and extracting the nucleic acid from the organism, which is a target for nucleic acid extraction from the sample solution to which the sodium hypochlorite is added.SELECTED DRAWING: Figure 1

Description

The present disclosure relates to a nucleic acid extraction method and a water treatment system for a living body.

For example, for genome analysis, qualitative (detection, identification) / quantification, mass replication (amplification, proliferation), etc., in all technical fields centered on food, medical / pharmaceutical, and agricultural technical fields, viruses and eukaryotes ( Techniques for extracting / recovering nucleic acids from microorganisms such as eubacteria (eubacteria), eukaryotes (algae, protozoa, fungi, slime), small animals (worms, etc.), and various living organisms such as animals, plants, and human tissues. Research and development is being actively carried out.

The results of research and development on such a nucleic acid extraction method have been utilized for speeding up, improving accuracy, and improving efficiency of tests using a gene amplification method such as the PCR (polymerase chain reaction) method.

In addition, the quantitative PCR (qPCR) method has an adverse effect on the environment, fisheries, people, etc. when the treated water after water treatment such as various wastewaters is discharged to public water areas such as rivers, lakes, seas, and groundwater. In order to prevent this, it may be used for the management of discharged water, such as quantitative inspection of microorganisms contained in treated water, and compliance with wastewater standards, environmental standards, etc.

Here, in order to ensure the inspection accuracy of microorganisms by the quantitative PCR method, that is, the quantitative accuracy, for example, the extraction efficiency of the nucleic acid of the microorganism contained therein from the sample water (sample solution) obtained by sampling or the like / It is important to increase the recovery rate as much as possible.

On the other hand, in Patent Document 1, the extraction solution before and after the microbial lysing enzyme reaction treatment is subjected to pressure cycle treatment or ultrasonic treatment so that the microorganism-derived nucleic acid can be efficiently recovered. ..

Further, in order to increase the recovery rate of nucleic acid derived from microorganisms, a method of adding a carrier such as tRNA co-precipitating with DNA, a polysaccharide such as glycogen, or polyacrylamide is often used when performing ethanol precipitation.

Japanese Unexamined Patent Publication No. 2013-042670

However, conventional nucleic acid extraction and purification methods such as pressure cycle treatment, ultrasonic treatment, and the use of carriers have problems such as extremely complicated operation, a large amount of time, and high cost.

Therefore, there has been a strong demand for a method capable of extracting nucleic acid of a living body such as a microorganism more easily, efficiently, and effectively with an excellent nucleic acid extraction rate as compared with the conventional method.

In view of the above circumstances, the present disclosure discloses a nucleic acid extraction method for a living body that enables more efficient extraction of nucleic acid of a living body such as a microorganism with a remarkably excellent high extraction (recovery) rate, and a water treatment using the same. The purpose is to provide a system.

One aspect of the method for extracting nucleic acid in a living body of the present disclosure is to add sodium hypochlorite to a sample solution for extracting nucleic acid in a living body so that the concentration is within a concentration range predetermined in advance by a test. The present invention includes a step of adding sodium hypochlorite and a nucleic acid extraction step of extracting the 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 disclosure is a water treatment unit that improves the water quality of the water to be treated, and the treated water after the water is treated by the water treatment unit using the above-mentioned biological nucleic acid extraction method. It is provided with a biometric unit for quantifying the biometric amount in the water, and a drainage unit for discharging the treated water when the biometric amount quantified by the biometric unit is equal to or less than a preset set value.

The term "living organism" as used in the present disclosure refers to not only microorganisms (including living organisms and viruses whose existence cannot be discerned with the naked eye without using a microscope), but also multicellular organisms such as humans, animals and plants. Includes tissues that make up a part (including blood).
Further, the "sample solution" in the present disclosure means a "liquid sample" for performing nucleic acid extraction of a living body.

According to the method for extracting nucleic acid in a living body of the present disclosure, it becomes possible to extract nucleic acid in a living body such as a microorganism more efficiently and with a remarkably excellent high extraction (recovery) rate.

According to the water treatment system of the present disclosure, it is possible to realize highly reliable water treatment by inspecting the treated water using the above-mentioned nucleic acid extraction method for living organisms.

It is a figure which shows an example of the water treatment system of one aspect, and is the figure which shows an example of the flow of the nucleic acid extraction method of a microorganism. It is a figure which shows an example of the amplicon amplification curve used for determining the concentration range of sodium hypochlorite in the nucleic acid extraction method of the living body (microorganism) of one aspect. It is a figure which shows an example of the relationship between the sodium hypochlorite concentration and the nucleic acid initial concentration in one aspect of the nucleic acid extraction method of a living body (microorganism). It is a figure which shows the relationship between pH and the dissolved form of hypochlorous acid.

Hereinafter, a method for extracting nucleic acid of a living body and a water treatment system according to an embodiment will be described with reference to FIGS. 1 to 4.

Here, in the present embodiment, it is assumed that the water treatment system of the present disclosure is a water treatment system for treating wastewater such as industrial wastewater and discharging it to a public water area, or for reusing the treated water. .. Further, in the present embodiment, the nucleic acid extraction method of the living body of the present disclosure is used to extract the nucleic acid of a microorganism (including a virus: living body) in the treated water treated by the water treatment system of the present embodiment, and before the release and reuse. The explanation will be given as it is used to confirm and manage the treatment status of treated water.
However, the water treatment system of the present embodiment and the nucleic acid extraction method of a living body do not need to be limited in its application, use and method as in the present embodiment, and are applied to cases where any water treatment and nucleic acid extraction of a living body are required. It is possible.
In the present embodiment, the "nucleic acid extraction method for living organisms" is hereinafter referred to as "nucleic acid extraction method for microorganisms".

(Water treatment system)
As shown in FIG. 1, the water treatment system 1 of the present embodiment has a water storage unit 2 that temporarily stores wastewater (water to be treated) W1 such as industrial wastewater that may contain microorganisms to be detected, and wastewater W1. Is purified, that is, a sample solution sampled from the treated water W2 after being purified by the water treatment unit 3 using the water treatment unit 3 for improving the water quality and the nucleic acid extraction method of the microorganism of the present embodiment. Using the sample, sample water) W2', the amount of microorganisms in the treated water W2 is measured (quantified) by the microorganism quantification unit (bioquantification unit) 4 and the microorganism quantification unit 4. It is configured to include a drainage unit 5 for discharging the treated water W2 when the value is equal to or less than a preset value.

The water treatment unit 3 is subjected to biological treatment such as activated sludge method and physicochemical treatment such as coagulation sedimentation, filtration and advanced treatment such as UV irradiation, and N (nitrogen), P (phosphorus), P (phosphorus) in wastewater. C (carbon such as organic carbon), removal of harmful substances such as heavy metals, separation, concentration adjustment, and other necessary treatments such as pH adjustment are performed, and the wastewater W1 to be treated is purified.

The microorganism quantification unit 4 is provided for quantifying the microorganisms contained in the treated water W2 with respect to the treated water W2 after being treated by the water treatment unit 3 and determining the necessity of discharge.

In the microorganism quantification unit 4, for example, when the treated water W2 is discharged from the drainage unit 5 to a public water area such as a river, a lake, or the sea, or when the treated water W2 is reused as regenerated water, the natural environment. Detects microorganisms that can cause adverse effects on industries such as fisheries and agriculture, people, etc., and inconvenient events, and quantifies the amount of microorganisms. It should be noted that, of course, it is possible not only to cause an adverse effect or an inconvenient event due to the presence of microorganisms in the treated water W2, but also to detect and quantify microorganisms that are preferably present.

Examples of the microorganism (living body) in the present embodiment include staphylococcus, Escherichia coli, Salmonella, pyogenic, cholera, diarrhea, charcoal, tuberculosis, botulinum, tetanus, rentha, and tinea. Candida, Aspergillus, Norovirus, Rotavirus, Influenza virus, Adenovirus, Coronavirus, Meadow virus, Ruin virus, Hepatitis virus, Herpesvirus, HIV, etc. Examples include algae, protozoa, fungi, slime molds), small animals (worms, etc.). That is, the microorganisms in the present embodiment are all microorganisms (including viruses) that need to be detected and quantified. In addition, the living body in the nucleic acid extraction method of the living body of the present disclosure includes tissues constituting a part of multicellular organisms, and the living body does not necessarily have to be limited to microorganisms.

Incidentally, when the treated water W2 is discharged into a public water area such as a river or the sea, it is required to fully consider the influence on the fishing industry. For example, in aquaculture farms such as blowfish, yellowtail, oysters, and salmon, a great deal of labor and cost are required to prevent the onset of diseases derived from microorganisms. Therefore, it is important to consider not only the influence on the vicinity of the discharge point where the treated water W2 is discharged but also the influence on a wide area. The same applies to other industries such as agriculture, and it is naturally required to consider the impact on animals, plants and people.

The microorganism quantification unit 4 of the present embodiment destroys the cell wall, cell membrane, cytoplasm, and other tissues existing so as to surround nucleic acid with respect to the sample liquid W2'sampled from the treated water W2. (Dissolve) and extract nucleic acid (DNA and / or RNA). In the case of a virus, the capsid, envelope, etc. are destroyed to extract nucleic acid.

Then, the extracted nucleic acid is amplified and quantified by using a PCR (polymerase chain reaction) method such as a quantitative PCR method.
As is well known, the quantitative PCR (qPCR) method is a method for measuring a fluorescent substance generated by a series of PCRs up to a predetermined number of cycles. Nucleic acid synthesis in a qPCR test is measured, and template DNA is measured from the fluorescence intensity. The amount of (mainly cDNA) (nucleic acid amount) can be determined. Therefore, not only in the food and medical fields, but also in the treated water W2 when the treated water W2 after water treatment such as various wastewaters W1 is discharged to public water areas such as rivers, lakes, marshes, seas, and groundwater. It is also used for quantitative inspection of microorganisms, and for management of discharged water such as compliance with wastewater standards and environmental standards. Examples of the main quantitative PCR method include i) MPN-PCR method, ii) competitive PCR method, and iii) quantitative real-time PCR method.

The amount of nucleic acid (gene) can be calculated by measuring the Ct value (Thrshhold Cycle). The Ct value means the number of PCR cycles that results in a constant amount of amplification product, and since the Ct value is inversely proportional to the initial amount of the target nucleic acid, the amount of nucleic acid is calculated by measuring the Ct value. Can be done.

Specifically, for example, first, an amplification curve arranged at equal intervals according to the initial amount of DNA is obtained by using a standard liquid train in which a sample containing a known amount of DNA is serially diluted. A threshold value is set at the point where the amplification curve rises, and the point where the amplification curve intersects is obtained as the Ct value. Next, a straight calibration curve is created between the Ct value and the initial DNA amount. Then, the Ct value is measured in the same manner for the sample containing the unknown DNA amount, and the initial DNA amount of the sample containing the unknown DNA amount is obtained by comparing with the calibration curve.

As a simpler quantification method, a linear portion is selected from the amplification curves obtained as a result of PCR, and the fluorescence intensity in the section calculated by extrapolating the portion is used as the initial nucleic acid amount. May be good.

Here, the method for quantifying the amount of nucleic acid in the microorganism quantification unit 4 of the present embodiment is, for example, a culture method, a FRET (Fluorescent resonance energy transfer) method, or CPRINS, if it is possible to quantify the amount of nucleic acid in the microorganism. -The FISH (Cycling primed in situ hybridization) method or the like may be used, and the method is not necessarily limited to the 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 inspection of microorganisms in the microorganism quantification unit 4, the treated water W2 is supplied to the drainage unit 5. Is discharged from the drainage section 5 to an industrial water area or reused as reclaimed water as appropriate. When it is determined that the amount of microorganisms in the treated water W2 is appropriate, for example, the treated water W2 is returned from the drainage unit 5 to the water storage unit 2 through the return line 6 before being sent to the drainage unit 5. ..

Here, conventionally, as a method for extracting a nucleic acid of a microorganism as a pretreatment for amplifying a nucleic acid, for example, a method of heat-treating a microorganism, a method of adding a surfactant, a phenol extraction method, an osmotic shock method, a freeze-thaw method, and the like. Enzyme digestion methods, use of DNA extraction kits, sonication methods, French press methods, use of homogenizers, etc. are used. In addition, there are cases where two or more of these methods are combined.

However, through diligent research by the inventor of the present application, it has been confirmed that the conventional nucleic acid extraction method for living organisms such as microorganisms requires a great deal of time, labor and cost, and the nucleic acid extraction efficiency may be low. Therefore, it is required to develop a method capable of extracting living nucleic acids such as microorganisms more easily, efficiently and effectively with excellent nucleic acid extraction efficiency, and the inventor of the present application has focused on research on this point and disclosed the present invention. We have invented a method for extracting nucleic acids from living organisms.

(Nucleic acid extraction method for living organisms such as microorganisms)
The method for extracting nucleic acid of a microorganism of the present embodiment is within a concentration range predetermined by a test with respect to a sample solution W2'for extracting nucleic acid of a microorganism as a pre-step (pretreatment step) for amplifying the nucleic acid of the microorganism. It is provided with an oxidant addition step of adding an oxidant so as to have a concentration of the above, and a nucleic acid extraction step of extracting the nucleic acid of the nucleic acid to be extracted from the sample solution W2'to which the oxidant is added.
Further, the oxidant addition step of the present embodiment is a sodium hypochlorite addition step, and hypochlorous acid (sodium hypochlorite solution) is added as an oxidant.

Further, the method for extracting a microorganism nucleic acid of the present embodiment includes a sodium hypochlorite concentration determining step (oxidizing agent concentration determining step) for determining a concentration range of sodium hypochlorite as an oxidizing agent.
In the test in the step of determining the sodium hypochlorite concentration, a plurality of sample solutions W2'with different sodium hypochlorite concentrations were prepared, and a quantitative PCR method was used for each sample solution W2'. An amplicon amplification curve (curve of the relationship between the PRC cycle and the fluorescence intensity) as shown in FIG. 2 is created. Then, the concentration range of sodium hypochlorite is determined based on the obtained amplicon amplification curve.

Then, the conventional nucleic acid extraction, purification, and quantification operations are performed on the sample liquid W2'after the neutralization treatment of the added sodium hypochlorite as needed. For example, Extrap Soil DNA Kit Plus ver. Using a test kit such as 2 (manufactured by Nippon Steel Environment Co., Ltd.), the nucleic acid of the microorganism is extracted and purified from the sample solution W2'according to the protocol.

Therefore, in the nucleic acid extraction method for microorganisms of the present embodiment and the water treatment system 1 using the method, the following is based on the results of the diligent research of the inventor of the present application as a pre-step before performing nucleic acid extraction such as the PCR method. Sodium hypochlorite concentration determination step and sodium hypochlorite addition step are provided, and sodium hypochlorite (sodium hypochlorite solution) is provided so that the concentration of the sample solution W2'is suitable for the microorganism that extracts nucleic acid. Is added. This makes it possible to extract nucleic acid with a significantly superior nucleic acid extraction rate (nucleic acid recovery rate) as compared with the conventional method. As a result of the diligent research of the inventor of the present application, as shown in FIGS. 2 and 3, the effect of improving the nucleic acid extraction rate by 10 to 100 times as compared with the conventional method (0 wt ppm (hereinafter referred to as ppm)). Has been confirmed. The vertical axis of FIG. 3 is the relative value of the initial DNA concentration when the initial concentration of untreated DNA is 1, and the horizontal axis is the sodium hypochlorite concentration.

That is, based on the exceptionally remarkable findings and research results by the inventor of the present application, an appropriate amount of hypochlorite is used so as to have a concentration corresponding to the microorganism (difficulty / ease of destruction of cell wall, cell membrane, etc.) to be extracted with nucleic acid. By simply adding sodium acid acid to the sample, it becomes possible to realize much better nucleic acid extraction than before.

In other words, nucleic acids can be extracted by simply adjusting the concentration of sodium hypochlorite for various nucleic acid target microorganisms that have different difficulty / ease of lysis (destruction) of cell walls, cell membranes, cytoplasms, capsids, envelopes, etc. Nucleic acid can be extracted with a very excellent nucleic acid extraction rate by suitably destroying the cell wall, cell membrane, cytoplasm, capsid, envelope, etc. without damaging the nucleic acid.

Therefore, it is possible to realize and provide an epoch-making microbial nucleic acid extraction method capable of easily, efficiently and effectively extracting microbial nucleic acid without the need for complicated work as in the past. ..

In addition, by conducting a test using a quantitative PCR method and creating an amplicon amplification curve for a plurality of sample solutions W2'with different concentrations of sodium hypochlorite, nucleic acid extraction of the nucleic acid to be extracted. The optimum concentration range of sodium hypochlorite can be determined.
As a result, in the sodium hypochlorite addition step, by adding sodium hypochlorite to the sample solution so as to be within the concentration range of sodium hypochlorite determined in the test, the nucleic acid extraction rate and thus the reliability are high. It becomes possible to realize nucleic acid extraction. That is, it becomes possible to more reliably, easily, efficiently and effectively extract nucleic acid of microorganisms.

Here, as shown in FIGS. 2 and 3 as an example, in the sodium hypochlorite addition step of the method for extracting the nucleic acid of the microorganism of the present embodiment, the concentration is 0.5 to 8 ppm, preferably 0.5 to 2 ppm. Is the concentration range of sodium hypochlorite. This concentration range is based on the findings of the nucleic acid extraction tests of various microorganisms and living organisms conducted by the inventor of the present application.

Further, in the water treatment system 1 of the present embodiment, the amount of microorganisms exceeds the set value in the water storage unit 2 that temporarily stores the wastewater W1 before the amount of microorganisms is quantified by the microorganism quantification unit 4 and the microorganism quantification unit 4. In some cases, it is provided with a return line 6 for returning the treated water W2 to the water storage unit 2.
Further, the water treatment system 1 of the present embodiment includes a water supply device 7 having a pump or the like for sending the treated water W2 to the drainage unit 5 or the water storage unit 2 based on the quantification result of the amount of microorganisms in the microorganism quantification unit 4. It further includes a control unit 8 that controls the drive of the water supply device 7.

Then, by providing the water storage unit 2 and the return line 6, the amount of microorganisms in the treated water W2 is efficiently and effectively detected by using the microbial nucleic acid extraction method, and the treated water W2 is sent to the outside through the drainage unit 5. When it is determined that the water cannot be discharged, the treated water W2 can be returned to the water storage unit 2 by the return line 6. As a result, the treated water W2 can be reprocessed, more advanced treatment can be performed, and more efficient and highly reliable water treatment can be realized.

Further, by providing the water supply device 7 and the control unit 8, the amount of microorganisms in the treated water W2 can be efficiently and effectively detected by using the method for extracting microorganisms, and the treated water W2 can be discharged to the outside through the drainage unit 5. The control unit 8 drives and controls the water supply device 7 according to the result of determining whether or not to discharge the water, and the treated water W2 can be automatically returned to the water storage unit 2 or sent to the drainage unit 5 for discharge. This makes it possible to realize even more efficient and highly reliable water treatment.

Here, in the water treatment system 1 of the present embodiment, the water treatment unit 3 is configured to include, for example, a filtration unit 3a for filtering water or a sterilization unit 3b for sterilizing water. ..

For example, when the filtration unit 3a is composed of one or a plurality of filtration means such as sand filtration, MF membrane, UF membrane, RO membrane, etc., the treated water is treated by using the nucleic acid extraction method of the microorganism of the present embodiment. Since the amount of microorganisms in W2 can be detected efficiently and effectively, a necessary combination of filtration means and filtration means can be easily selected. This makes it possible to realize more efficient and reliable water treatment.

Further, for example, when the sterilizing unit is composed of one or a plurality of sterilizing means such as UV irradiation sterilization, ozone sterilization, and addition of a sterilizing agent, the treatment is performed using the microorganism nucleic acid extraction method of the present embodiment. Since the amount of microorganisms in water W2 can be detected efficiently and effectively, a necessary combination of sterilizing means and sterilizing means can be easily selected. This makes it possible to realize more efficient and reliable water treatment.

Therefore, when the treated water W2 is returned to the water storage unit 2 and thus the water treatment unit 3 through the return line 6, the treated water W2 can be retreated, and the filtration means and the sterilization means can be appropriately selected and combined. In this way, more advanced treatment can be applied, and more efficient and reliable water treatment can be realized.

Here, when a sterilizing agent (bactericidal agent) is added to the wastewater W1 or the return treated water W2 to sterilize the microorganisms, for example, the sterilizing agent is a biofilm composed of a cell wall or an aggregate of microorganisms. It is preferable that the film can be destroyed and sterilized. For example, halogen-based disinfectants, ozone, hydrogen peroxide and the like can be mentioned. Examples of the halogen-based bactericide include hypochlorous acid (HClO) and salts thereof (for example, sodium hypochlorite, etc.), ammonia chloramine (NH ₂ Cl), bromchlordimethylhydantoin (Br, Cl-DMH), and the like. Examples thereof include bromsulfamic acid (BrNHSO ₃ H) and _{bromchloramine (NH 4} Br + HClO). Of these, halogen-based bactericides or ozone are preferable, and hypochlorous acid and salts thereof are more preferable, because of their strong bactericidal activity.

In addition, when a disinfectant is added, it is desirable to neutralize it as necessary. For example, when sodium hypochlorite is used as the disinfectant, sodium thiosulfate or the like can be mentioned as the neutralizing agent.

(Example)
Next, hereinafter, wastewater containing an absorption liquid (slurry) used in the limestone method (for example, wastewater) generated when sulfur oxides are removed from exhaust gas discharged from a thermal power plant or the like by a wet exhaust gas desulfurization apparatus. The present embodiment cites an example in which the microorganism nucleic acid extraction method of the present embodiment is applied to the inspection of the amount of microorganisms in the treated water W2 after the treatment by purifying W1 and using the microorganism nucleic acid extraction method of the present embodiment. The method for extracting the nucleic acid of the microorganism and the water treatment system 1 will be described more specifically.

Here, there are cases where the absorption liquid that has come into contact with the exhaust gas and has absorbed the sulfur oxide has a high chemical oxygen demand (COD). According to the diligent research by the inventor of the present application, this is partly due to the proliferation of sulfur-oxidizing bacteria (for example, the bacterium belonging to the genus Thermithiobacillus), which are autotrophic bacteria, in the drainage of the absorption liquid that has absorbed sulfur oxides. Obtained as a finding. In addition, when sulfur-oxidizing bacteria grow, heterotrophic bacteria (for example, Pseudomonas spp.) That grow using the products (organic substances) of sulfur-oxidizing bacteria grow, and the grown heterotrophic bacteria also produce organic substances such as sugars. Therefore, there is a risk that the COD of the drainage will increase.

If the COD is higher than the wastewater standard, the wastewater cannot be discharged to a public water area such as a river, so that the water treatment system 1 performs purification treatment.

On the other hand, it is known that when treated water W2 containing sulfur-oxidizing bacteria, which is not specified in the wastewater standard, is discharged and drained through a sewage pipe or the like, concrete corrosion of a sewage treatment facility such as a sewage pipe is generated.
Specifically, sulfate-reducing bacteria contained in sewage reduce sulfate ions in anaerobic sewage to generate hydrogen sulfide, hydrogen sulfide is released from the sewage into the air, and dew condensation on the concrete surface. Absorbed and dissolved in such as. In this state, if sulfur-oxidizing bacteria are present on the aerobic concrete surface, hydrogen sulfide absorbed by dew condensation is oxidized by sulfur-oxidizing bacteria to generate sulfuric acid, and this sulfuric acid melts the concrete, causing so-called concrete corrosion. appear. When concrete corrosion such as sewerage in the ground occurs due to such microorganisms, the concrete becomes like clay and the bearing capacity of the concrete structure is lost, and the function as a sewerage facility is of course unexpectedly sudden. It may cause the collapse of the road and the subsidence of the ground.

For this reason, the amount of sulfur-oxidizing bacteria and sulfate-reducing bacteria, which are microorganisms, is quantified and confirmed with respect to the treated water W2 obtained by purifying the wastewater W1 containing the absorbing liquid that has absorbed sulfur oxides, and then discharged into public water areas. It is desirable to do.

In this embodiment, in order to inspect the amount of sulfur-oxidizing bacteria in such treated water, the microorganism nucleic acid extraction method and the water treatment system 1 of the present embodiment are applied, and the sulfur-oxidizing bacteria cells in the wastewater are applied. The amount was detected by the PCR method using a primer pair for amplification of a known gene of sulfur-oxidizing bacteria. The amount of the oxidizing fungicide is adjusted according to the amount of the detected sulfur-oxidizing bacteria, and the treated water W2 is sterilized (sterilized) and then discharged.

By the way, the absorption liquid used is arbitrary as long as it can absorb sulfur oxides, for example, a slurry in which limestone is dissolved (dispersed) (lime gypsum method), an aqueous solution of sodium hydroxide (caustic soda method), and the like. A slurry (magnesium method) in which magnesium hydroxide is dissolved (dispersed) can be applied. In this embodiment, as an example, a case where a slurry in which limestone is dissolved (dispersed) is used as an absorption liquid will be described.

Further, sulfur-oxidizing bacteria are microorganisms of the nucleic acid extraction target is utilized sulfide (S ^2-), elemental sulfur (SO), thiosulfate, polythionic acid, the oxidation energy of the reduced inorganic sulfur compounds sulfite as energy growth It is a bacterium that does. Sulfur-oxidizing bacteria include, for example, bacteria that temporarily deposit sulfur in the body such as Beggiatoa, Thiothrix, Thiobacillus; Sexual bacteria; thermophilic bacteria such as Thermothrix; ultrahyperthermic bacteria such as Acidianus, Metallosphaera, Sulflobus and the like. In this embodiment, as an example, the sulfur-oxidizing bacterium existing in the desulfurization apparatus is described as being a bacterium belonging to the genus Thermithiobacillus.

Then, in this example, the amount of sulfur-oxidizing bacteria in the treated water W2 is detected and quantified by a PCR method using a primer pair for amplifying a known gene of sulfur-oxidizing bacteria.

The amount of sulfur-oxidizing bacteria in the treated water W2 is detected as the amount of known genes of sulfur-oxidizing bacteria. The known gene may be a gene possessed by a sulfur-oxidizing bacterium and is unique to the sulfur-oxidizing bacterium, and for example, a gene involved in sulfur metabolism can be used. Paracoccus sulfur oxidation passage (also referred to as PSO pathway or sox pathway) and S4 intermediatepathway (S4 pathway) exist in sulfur metabolism. The enzyme encoded by the sox gene cluster (soxXAYZBCD) plays a major role in the PSO pathway. Specific examples of the enzyme include soxYZ, soxXA, soxB, soxCD and the like. Among them, the soxB gene is preferable as a known gene. The soxB gene of a sulfur-oxidizing bacterium consists of, for example, the base sequence represented by SEQ ID NO: 1.

On the other hand, examples of genes involved in the S4 pathway include thiosulfate dehydrogenase gene, sulfite dehydrogenase gene, tetrathionate hydralolase (4THase) gene and the like.

A known gene amplification primer pair can be designed by a known method in consideration of the base sequence, Tm value, GC content, etc. of the target gene.
For example, as the primer pair for amplification of the soxB gene, particularly the region consisting of the base sequence represented by SEQ ID NO: 1, one or more primers selected from the group consisting of Table 1 and 1) to 8) below, for example. Equality can be mentioned.

1) A forward primer consisting of the base sequence represented by SEQ ID NO: 2 and a reverse primer consisting of 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;

The above primer pair is an example of a usable primer pair. Normally, the primer can be designed to have a length of about 15 bases or more and 30 bases or less. Therefore, in the above primer pair, the base length is set to, for example, 1 base, 2 bases, 3 bases, or 5 bases in consideration of the GC content and the Tm value. Similarly, those having an increase or decrease of about 10 bases can also be used.

In the above-mentioned PCR method targeting known genes, the amount of sulfur-oxidizing bacteria contained in the wastewater W1 can be quantified as the amount of known genes of sulfur-oxidizing bacteria. At this time, by performing a two-step operation of extracting nucleic acid from the sulfur-oxidizing bacteria contained in the treated water W2 and then performing PCR using the obtained extract as a template sample, a short period of 3 hours or more and 1 day or less is performed. Measurement results can be obtained in time.

(DNA extraction method for sulfur-oxidizing bacteria)
Here, in the pretreatment for quantifying the amount of cells by the PCR method, for example, Extrap Soil DNA Kit Plus ver. Using 2 (manufactured by Nippon Steel Environment Co., Ltd.), DNA of sulfur-oxidizing bacteria is extracted and purified from the gypsum sample collected from the desulfurization apparatus according to the protocol.

In the DNA extraction method of sulfur-oxidizing bacteria of this example (nucleic acid extraction method of microorganisms of this example), first, the sodium hypochlorite concentration (final concentration) is 0.5 ppm (Case1), 1 ppm (Case2), and 2 ppm. (Case3), 4 ppm (Case4), 8 ppm (Case5), 16 ppm (Case6) Sodium hypochlorite with different sodium hypochlorite concentration with respect to 1 cc of sample solution of the target biological suspension. Add 1 cc of the solution to generate 6 kinds of sample liquids (mixed liquids) W2'of Case1 to Case6, and shake each for 30 minutes using a vortex mixer or the like. The temperature of the sample liquid W2'was set to 25 ° C.

After the shaking treatment in this way, a neutralizing agent solution such as sodium thiosulfate was added to each of the six sample solutions W2', which is a mixed solution of the target biological suspension and sodium hypochlorite, and each was added. It is desirable to neutralize the residual chlorine remaining in the sample solution W2'. In this example, a sodium thiosulfate solution is used, and an equivalent amount of the sodium thiosulfate solution corresponding to the amount of added sodium hypochlorite is added for 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 is centrifuged at 13500 rpm for 10 minutes using a centrifuge.

Then, in this embodiment, for example, from the pellets obtained by centrifugation and solid-liquid separation in this way (pellets after pretreatment for quantifying the amount of cells by the PCR method), for example. Exec Soil DNA Kit Plus ver. 2 (manufactured by Nippon Steel Environment Co., Ltd.) is used to extract and purify the nucleic acid (DNA) of sulfur-oxidizing bacteria according to the protocol.

The protocol for extracting and purifying nucleic acid of sulfur-oxidizing bacteria is as follows.

1) In Bed Tubes, sample pellets: 0.5 g (by the way, 500 μL in the case of liquid sample), Extension Buffer (main component / mixture of disodium hydrogen phosphate): 950 μL, Lysis Solution (main component / sodium dodecyl sulfate). Mixture): 50 μL is added.
When concentration is required, it is desirable to filter and capture microorganisms in the sample using a membrane filter and extract nucleic acid from this filter.

2) Stir with a vortex mixer for 5 seconds.

3) Perform bead beating (4 to 6 m / sec or 4200 to 6800 rpm for 30 to 45 seconds).

4) Centrifuge (14000 xg, 5 minutes, 4 ° C).

5) Transfer 600 μL of the supernatant to a 1.5 mL tube and add PP Solution (sodium acetate solution): 300 μL.

6) Mix the tube of 5) by inversion about 10 times and stir.

7) Centrifuge (14000 xg, 5 minutes, 4 ° C).

8) Transfer 800 μL of the supernatant to a 2 mL tube.

9) Add 50 μL of MBs Solution (mixture of main component / guanidine hydrochloride and iron sesquioxide) and 890 μL of Binding Solution (mixture of main component / guanidine thiocyanate) to the tube of 8).

10) Mix the tube of 9) by inversion for about 2 minutes and stir well.

11) Spin down the tube of 10) and set it on the magnetic stand.

12) After magnetizing for 1 minute or more, remove the supernatant using a micropipette.

13) Add 800 μL of Washing Solution (mixture of main component / guanidin thiocyanate) to the tube of 12), and stir well with a vortex mixer (low speed).

14) Spin down the tube of 13), set it on a magnetic stand, collect magnetism for 1 minute or more, and then remove the supernatant using a micropipette.

15) Add 1 mL of 70% ethanol solution and stir well with a vortex mixer (low speed).

16) Spin down the tube of 15), collect magnetism on a magnetic stand for 1 minute or longer, and then remove ethanol using a micropipette.

17) The steps 15) to 16) are repeated again.

18) With the tube lid open, air dry the magnetic particles at room temperature for about 10 minutes.

19) Eluate (TE buffer, sterile water, etc.): After adding 100 μL, stir well with a vortex mixer (low speed).

20) Heat with a heat block (or water bath) at 65 ° C. for 5 to 10 minutes while stirring several times in the middle.

21) After setting the tube on the magnetic stand and collecting magnetism, transfer the eluate to a new tube.

As a result, an eluate (sample solution) containing the eluate of nucleic acid can be obtained.

Next, using the purified sample as a template, the amount of the soxB gene was quantified by a real-time PCR method using primer pairs 5 to 8 among the eight types of primer pairs shown in Table 1 above. The reaction conditions for PCR are that after performing a reaction at 98 ° C for 2 minutes as initial denaturation, "DNA denaturation reaction at 98 ° C for 10 seconds, annealing at 55 ° C for 10 seconds, and extension reaction at 68 ° C for 30 seconds" are performed for 45 cycles. did.

As a result, each amplicon amplification curve when the sodium hypochlorite concentration is changed as shown in FIG. 2, which is the result of the real-time PCR method in which the primer pair 8 is used among the primer pairs and the number of cycles is 45 cycles. (Curve of relationship between PRC cycle and fluorescence intensity) can be obtained.

Therefore, simply by performing a pretreatment to add sodium hypochlorite and adjusting the concentration of sodium hypochlorite, the cell wall, cell membrane, cytoplasm, capsid, envelope, etc. are suitably destroyed, and excellent nucleic acid extraction is performed. It was demonstrated that nucleic acid can be extracted at a rate. Further, from each amplicon amplification curve, if sodium hypochlorite is added to the sample solution W2'so that the sodium hypochlorite concentration is 0.5 to 8 ppm, it is suitable for a nucleic acid extraction rate superior to the conventional one. It was also demonstrated that nucleic acids of various microorganisms can be extracted.

As described above, a bactericide is added to the treated water W2 as necessary based on the quantification result of microorganisms in the treated water W2 of the waste water W1. At this time, the amount of the oxidizing fungicide used for sterilization can be appropriately adjusted according to the amount of cells of sulfur-oxidizing bacteria. Further, it can be appropriately adjusted according to the type of the oxidizing fungicide to be used. For example, when sodium hypochlorite is used as the oxidizing bactericidal agent, the amount of sodium hypochlorite added can be such that the final concentration in the treated water W2 is 1 ppm or more and 70 ppm or less.

The sterilization time with the oxidizing fungicide can be, for example, about 1 minute or more and 24 hours or less, preferably 1 minute or more and 12 hours or less, and more preferably 1 minute or more and 6 hours or less. When the sterilization time is not less than the above lower limit value, a more sufficient sterilization effect can be obtained, while when it is not more than the above upper limit value, damage such as corrosion to the desulfurization apparatus can be reduced.

Although the living body nucleic acid extraction method and the embodiment of the water treatment system 1 of the present disclosure have been described above, the living body nucleic acid extraction method and the water treatment system 1 according to the present disclosure are not limited to the above-described embodiment. , Including examples of changes, etc., can be changed as appropriate within the range that does not deviate from the purpose.

For example, in the present embodiment, in the sodium hypochlorite addition step, the concentration of the sample liquid W2'for extracting nucleic acid of a microorganism (living body) is set to be within the concentration range predetermined by the test. Sodium chlorite was added.
Further, in the present embodiment, in the test in the step of determining the sodium hypochlorite concentration, a plurality of sample solutions W2'with different sodium hypochlorite concentrations are prepared, and a quantitative PCR method is used to prepare each sample solution. On the other hand, an amplicon amplification curve was created, and the concentration range of sodium hypochlorite was determined based on the amplicon amplification curve.

On the other hand, as shown in FIG. 4, hypochlorous acid has three types ^{, hypochlorous acid (HClO), hypochlorite ion (ClO −} ), and dissolved chlorine gas (Cl _{2), depending on the pH.} It changes to a dissolved form, and the dissolved ratio of these three types varies depending on the pH.
Therefore, even when the concentration of sodium hypochlorite is constant, the proportion (concentration) of hypochlorite (HClO) having strong oxidizing power can be adjusted by adjusting the pH of the sample solution W2'. It is possible to adjust the pH according to the difficulty of destroying the cell wall, cell membrane, cytoplasm, capsid, envelope, etc. of the nucleic acid to be extracted, or to adjust the pH according to the pH of the water W1 and W2 to be inspected. By simply adjusting and setting the concentration of sodium, the concentration of hypochlorite effective for nucleic acid extraction can be adjusted as a result, and nucleic acid is extracted with a very excellent nucleic acid extraction rate as in the present embodiment. It will be possible to do.

In this embodiment, sodium hypochlorite is added to the sample solution W2'as a pretreatment in order to suitably destroy the cell wall, cell membrane, cytoplasm, capsid, envelope, etc. of a living body such as a microorganism to be extracted with nucleic acid. I made it.
On the other hand, even an oxidizing agent other than sodium hypochlorite can exert the same effect. Further, as described above, the dissolved morphology of other oxidants also changes depending on the pH, and by extension, the morphology suitable for destroying the cell wall, cell membrane, cytoplasm, capsid, envelope, etc. of the living body such as the microorganism to be extracted with nucleic acid.
Therefore, by selecting an oxidizing agent and adjusting the concentration and pH according to the difficulty of destroying the cell wall, cell membrane, cytoplasm, capsid, envelope, etc., the same action and effect as in this embodiment can be obtained. It can be said that it can be done.
As such an oxidizing agent, any known oxidizing agent can be applied. For example, halogen-based ammonia chloramine _(NH 2 Cl), bromine chloro dimethyl hydantoin (Br, Cl-DMH), bromine sulfamate (BrNHSO ₃ H), such as bromine chloramine _(NH 4 Br + HClO) can be exemplified. Further, ozone, hydrogen peroxide, potassium permanganate, ferrous iron, ferric iron, zero-valent iron and the like can also be mentioned.
For example, for the purpose of destroying the cell wall, cell membrane, cytoplasm, capsid, envelope, etc. of a living body to improve the nucleic acid extraction rate, the oxidizing power is strong in an acidic solution such as iodic acid, and in a basic solution. Compared to oxidants that tend to have weak oxidative power, the oxidative power is stronger in neutral solution such as sodium hypochlorite, and the stronger the acidity and basicity, the lower the oxidative power in solution. It has been confirmed by the intent of the inventor that the application of the agent (oxidizing agent showing the tendency as shown in FIG. 4) is more preferable.

Further, in the present embodiment, it has been described that the nucleic acid extraction method of the microorganisms of the present disclosure is used to quantify the amount of microorganisms in the treated water after being treated by the water treatment system. Further, in the examples, the microorganism to be extracted with nucleic acid was described as a sulfur-oxidizing bacterium.
On the other hand, the method for extracting nucleic acid of a living body according to the present disclosure, and a water treatment system using the method, can use nucleic acid of a living body such as microorganisms contained in water of rivers, lakes, ponds, sea areas, and any other water. Applicable for extraction. That is, it is not necessary to limit its use and the like.

Further, in the present embodiment and the examples, sodium hypochlorite is added to the sample solution to perform pretreatment, and the sample solution subjected to this pretreatment is subjected to the present step using a conventionally known nucleic acid extraction kit. Nucleic acid extraction was performed and the amount of microorganisms was quantified using the PCR method.
Here, the nucleic acid extraction kit may be configured with sodium hypochlorite to be added to the sample solution in advance. In this case, it becomes possible to realize and provide a highly reliable nucleic acid extraction kit, which is more efficient than the conventional one and can improve the nucleic acid extraction rate.

Finally, the content described in the embodiment is grasped as follows, for example.

(1) The method for extracting nucleic acid in a living body according to one aspect of the present disclosure is a concentration within a concentration range predetermined by a test with respect to a sample solution (sample water, sample solution W2') for extracting nucleic acid in a living body. It is provided with a sodium hypochlorite addition step of adding sodium hypochlorite and a nucleic acid extraction step of extracting a nucleic acid of a living body to be nucleic acid extracted from a sample solution to which sodium hypochlorite is added.

In the method for extracting nucleic acid of a living body according to (1) above, by using the result of the diligent research of the inventor of the present application, that is, the concentration is adjusted according to the living body such as a microorganism that extracts nucleic acid from a sample solution. Nucleic acid extraction target based on the exceptionally remarkable findings and research results by the inventor of the present application that nucleic acid can be extracted with a much better nucleic acid extraction rate (nucleic acid recovery rate) than before by adding sodium chlorite. By simply adding an appropriate amount of sodium hypochlorite to the sample so that the concentration corresponds to that of the living body, it becomes possible to realize much better nucleic acid extraction than before.

That is, for example, in the case of cell walls, cell membranes, cytoplasms, and viruses, it is only necessary to adjust the concentration of sodium hypochlorite for living organisms that extract various nucleic acids with different difficulty in lysis (destruction) such as capsids and envelopes. It is possible to extract nucleic acid with a very excellent nucleic acid extraction rate by preferably destroying the cell wall, cell membrane, cytoplasm, capsid, envelope and the like.

Therefore, it is possible to realize and provide an epoch-making method for extracting nucleic acid of a microorganism, which can easily, efficiently and effectively extract nucleic acid of a living body such as a microorganism without the need for complicated work as in the past. be able to.

(2) The method for extracting nucleic acid of a living body according to another aspect of the present disclosure is the method for extracting nucleic acid of a living body according to the above (1), and determines the concentration range of sodium hypochlorite. In the test in the step of determining the sodium hypochlorite concentration, multiple sample solutions with different sodium hypochlorite concentrations were prepared, and quantitative PCR method was used to amplify the amplicon for each sample solution. Create a curve and determine the sodium hypochlorite concentration range based on the amplicon amplification curve.

In the above-mentioned (2) method for extracting nucleic acid of a living body, a test using a quantitative PCR method is carried out, and an amplicon amplification curve is created for a plurality of sample solutions having different concentrations of sodium hypochlorite. , The optimum concentration range of sodium hypochlorite for nucleic acid extraction of a living body to be extracted with nucleic acid can be determined.
As a result, in the sodium hypochlorite addition step, by adding sodium hypochlorite to the sample solution so as to be within the concentration range of sodium hypochlorite determined in the test, the nucleic acid extraction rate and thus the reliability are high. It becomes possible to realize nucleic acid extraction. That is, it becomes possible to more reliably, easily, efficiently and effectively extract nucleic acid of a living body.

(3) The method for extracting nucleic acid of a living body according to another aspect of the present disclosure is the method for extracting nucleic acid of a living body according to the above (1) or (2), wherein the living body is a microorganism.

The method for extracting nucleic acid in a living body according to (3) above is superior to the conventional method if sodium hypochlorite is added to the sample solution based on the findings of the nucleic acid extraction test of various microorganisms conducted by the inventor of the present application. It is possible to preferably extract nucleic acids of various microorganisms with the nucleic acid extraction rate.

(4) The water treatment system according to one aspect of the present disclosure includes a water treatment unit (water treatment unit 3) for improving the water quality of the water to be treated (waste W1) and a sample liquid for extracting nucleic acid of a living body. In contrast, the sodium hypochlorite addition step in which sodium hypochlorite is added so that the concentration is within the concentration range determined in advance by the test, and the microorganism to be extracted from the sample solution to which sodium hypochlorite is added. Using a biological nucleic acid extraction method equipped with a nucleic acid extraction step for extracting the nucleic acid of the above, nucleic acid extraction is performed on the sample solution obtained from the treated water (treated water W2) after being treated in the water treatment unit, and the treated water is treated. A biometric unit (microbiological amount quantification unit, microbial amount determination unit 4) for quantifying the amount of biometric content in the water, and drainage for discharging treated water when the biometric amount quantified by the biometric amount is less than a preset set value. A unit (drainage unit 5) is provided.

In the water treatment system of (4) above, a nucleic acid extraction method of a living body including a sodium hypochlorite addition step and a nucleic acid extraction step is used to quantify the amount of living body in water after treatment in a water treatment unit. By providing the biometric unit, the action and effect of the nucleic acid extraction method for the living body according to (1) above can be achieved. As a result, highly reliable water treatment can be realized, and by extension, the reliability of the water discharged from the drainage portion can be suitably ensured.

(5) The water treatment system according to another aspect of the present disclosure is the water treatment system of the above (4), and the water treatment unit includes a filtration unit (filtration unit 3a) for filtering water.

In the water treatment system of (5) above, for example, when the filtration unit is configured by using one or a plurality of filtration means such as sand filtration, MF membrane, UF membrane, RO membrane, etc., the above (1) to (1) to ( By efficiently and effectively detecting the biological amount of the treated water by using any of the biological nucleic acid extraction methods of 3), it is possible to easily select a necessary combination of filtration means and filtration means. This makes it possible to realize more efficient and reliable water treatment.

(6) The water treatment system according to another aspect of the present disclosure is the water treatment system of (4) above, and the water treatment unit includes a sterilization unit (sterilization unit 3b) for sterilizing water. ..

In the water treatment system of (6) above, for example, when the sterilization unit is composed of one or a plurality of sterilization means such as UV irradiation sterilization, ozone sterilization, and hypochlorite sterilization, the above (1) to (1) to By efficiently and effectively detecting the biological amount of the treated water by using any of the biological nucleic acid extraction methods of (3), it is possible to easily select a necessary combination of sterilizing means and sterilizing means. This makes it possible to realize more efficient and reliable water treatment.

(7) The water treatment system according to another aspect of the present disclosure is the water treatment system according to any one of (4) to (6) above, and the water before the biometric amount is quantified by the biometric unit is temporarily used. It is further provided with a water storage unit (water storage unit 2) for storing in the water storage unit and a return line (return line 6) for returning the treated water to the water storage unit when the biological amount quantified by the biometric unit exceeds the set value.

In the water treatment system of the above (7), the biological amount of the treated water is efficiently and effectively detected by using the nucleic acid extraction method of the living body according to any one of the above (1) to (3), and the treated water is used. If it is determined that the water cannot be discharged to the outside through the drainage section, the treated water can be returned to the water storage section by the return line. As a result, the treated water can be reprocessed, more advanced treatment can be performed, and more efficient and reliable water treatment can be realized.

(8) The water treatment system according to another aspect of the present disclosure is the water treatment system of (7) above, and sends water to the drainage section or the water storage section based on the quantification result of the biological amount in the biometric section. A water supply device (water supply device 7) for this purpose and a control unit (control unit 8) for controlling the drive of the water supply device are further provided.

In the water treatment system of the above (8), the biological amount of the treated water is efficiently and effectively detected by using the nucleic acid extraction method of the living body according to any one of the above (1) to (3), and the treated water is used. The control unit drives and controls the water supply device according to the result of determining whether or not the water can be discharged to the outside through the drainage unit, and the treated water can be automatically returned to the water storage unit or discharged to the drainage unit. This makes it possible to realize even more efficient and highly reliable water treatment.

1 Water treatment system 2 Water storage unit 3 Water treatment unit 3a Filtration unit 3b Sterilization unit 4 Microbial quantification unit, microbial amount determination unit (bioquantification unit, biometric amount determination unit)
5 Drainage unit 6 Return line 7 Water supply device 8 Control unit W1 Drainage (water)
W2 treated water W2'sample liquid (sample water)

Claims (8)

Sodium hypochlorite addition step of adding sodium hypochlorite so that the concentration is within the concentration range predetermined by the test to the sample solution for nucleic acid extraction of the living body, and the above-mentioned hypochlorite. The present invention comprises a nucleic acid extraction step of extracting the nucleic acid of the microorganism to be extracted from the sample solution to which sodium is added.
Nucleic acid extraction method for living organisms. The sodium hypochlorite concentration determination step for determining the concentration range of the sodium hypochlorite is provided.
In the test in the step of determining the sodium hypochlorite concentration, a plurality of sample solutions having different concentrations of the sodium hypochlorite were prepared, and a quantitative PCR method was used to amplify the amplicon for each of the sample solutions. A curve is created and the concentration range of the sodium hypochlorite is determined based on the amplicon amplification curve.
The method for extracting nucleic acid of a living body according to claim 1. The living body is a microorganism,
The method for extracting nucleic acid of a living body according to claim 1 or 2. A water treatment unit that improves the quality of the water to be treated,
A step of adding sodium hypochlorite to a sample solution for nucleic acid extraction of a living body so that the concentration is within a concentration range predetermined by a test, and the hypochlorite addition step. Using a living body nucleic acid extraction method comprising a nucleic acid extraction step of extracting the nucleic acid of the living body to be nucleic acid extracted from the sample solution to which sodium was added, the water was obtained from the treated water after being treated by the water treatment unit. A biometric unit that extracts nucleic acid from the sample solution and quantifies the amount of biometrics in the treated water.
A drainage unit for discharging the treated water when the biological amount quantified by the biometric unit is equal to or less than a preset set value is provided.
Water treatment system. The water treatment unit includes a filtration unit that filters the water.
The water treatment system according to claim 4. The water treatment unit includes a sterilization unit for sterilizing the water.
The water treatment system according to claim 4. A water storage unit that temporarily stores the water before the biometric amount is quantified by the biometric unit, and a water storage unit.
Further provided with a return line for returning the treated water to the water storage unit when the biological amount quantified by the biometric unit exceeds the set value.
The water treatment system according to any one of claims 4 to 6. A water supply device for sending the treated water to the drainage section or the water storage section based on the quantification result of the biological amount in the biometric section.
A control unit that controls the drive of the water supply device is further provided.
The water treatment system according to claim 7.

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