Method for extracting and concentrating nucleic acid and apparatus for extracting and concentrating nucleic acid

Abstract

According to an embodiment of the present invention, a method for extracting and concentrating a nucleic acid is provided. The method for extracting and concentrating a nucleic acid comprises: a first step for removing at least a portion of a solid component from raw water containing the solid component and at least one type of nucleic acid-containing body selected from the group consisting of viruses and microorganisms; a second step for extracting, from a treated liquid obtained via the first step, the nucleic acid contained in the nucleic acid-containing body, thereby obtaining a nucleic acid extract liquid; and a third step for concentrating the nucleic acid extraction liquid. Vacuum filtration is used in the first step and / or the third step.

Description

Nucleic acid extraction and concentration method and nucleic acid extraction and concentration apparatus

[0001] Embodiments of the present invention relate to a nucleic acid extraction and concentration method and a nucleic acid extraction and concentration apparatus.

[0002] The global pandemic of coronavirus disease 2019 (COVID-19) has caused long-term and enormous damage to human life and health, as well as far-reaching impacts on social and economic activities. The main transmission routes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are droplet infection and contact infection between humans. However, SARS-CoV-2 viral genes have also been detected in human feces from pre-symptomatic individuals. For this reason, wastewater epidemiological surveys that detect genes of microorganisms and viruses causing infectious diseases from wastewater have been attempted around the world including the United States and European countries, and the utilization of information obtained therefrom is under discussion.

[0003] Wastewater epidemiological surveys are expected to serve as a new tool to support infection control measures in the living with COVID era. While clinical tests performed on infected individuals can identify confirmed cases, infected persons who do not undergo testing, and those who cannot be tested due to insufficient testing capacity, remain undetected. Therefore, the infection status of an entire region may not be fully reflected in some cases. On the other hand, wastewater epidemiological surveys, by testing genes of microorganisms and viruses contained in wastewater, make it possible to grasp the spread of infectious diseases in a region, including infections from asymptomatic individuals who have not received testing. Furthermore, methods for predicting the epidemic trend of viral infection by detecting viruses contained in wastewater are also under investigation.

[0004] Quantification of the concentration of microorganisms and viruses in wastewater is carried out by real-time PCR (Polymerase chain reaction) method, similarly to methods used for testing infected individuals. A technical challenge in detecting microorganisms and viruses from wastewater lies in the pretreatment step before loading a sample into real-time PCR. When detecting genes of viruses, microorganisms and the like excreted in feces from wastewater, the detection target is diluted by a large amount of wastewater. Therefore, the nucleic acid to be detected is at a very low concentration.

[0005] When detecting low concentrations of nucleic acids, PCR testing is difficult. Therefore, it is necessary to perform concentration procedures for microorganisms or viruses as a pretreatment before PCR testing. However, the series of procedures in this pretreatment require specialized skills and are time-consuming.

[0006] International Publication No. 2003 / 040687

[0007] The object of the present invention is to provide a simple nucleic acid extraction and concentration method, and a nucleic acid extraction and concentration apparatus that can easily extract and concentrate nucleic acids.

[0008] According to the embodiment, a nucleic acid extraction and concentration method is provided. The nucleic acid extraction and concentration method includes a first step of removing at least a portion of the solid components from raw water containing at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms and solid components; a second step of extracting nucleic acids contained in the nucleic acid-containing substance from the processed liquid obtained in the first step to obtain a nucleic acid extract; and a third step of concentrating the nucleic acid extract. Vacuum filtration is used in at least one of the first or third step.

[0009] Figure 1 is a flowchart showing a nucleic acid extraction and concentration method according to one embodiment. Figure 2 is a diagram showing an overview of the nucleic acid extraction and concentration method according to one embodiment. Figure 3 is a block diagram showing an example of a nucleic acid extraction and concentration apparatus according to another embodiment. Figure 4 is a graph showing the measurement results for Example 1 and Comparative Example 1. Figure 5 is a graph showing the measurement results for Example 2 and Comparative Example 2. Figure 6 is a graph showing the measurement results for Example 3 and Comparative Example 3. Figure 7 is a diagram showing an overview of the nucleic acid extraction and concentration method according to a comparative example. Embodiment

[0010] When detecting microorganisms or viruses using PCR (polymerase chain reaction) testing, it is necessary to remove suspended solids (SS) from the sample as a pretreatment, and then extract and concentrate the nucleic acids, which are the genetic information of the microorganisms or viruses, while preserving their gene sequence information.

[0011] As a pretreatment, solid components in the sample are removed by centrifugation, followed by the extraction of nucleic acids, and the resulting nucleic acid extract is concentrated. Concentration is performed by passing the nucleic acid extract through a nucleic acid concentration column to adsorb the nucleic acids onto the column, washing the column, and then eluting the adsorbed nucleic acids. Each of the nucleic acid adsorption, washing, and elution processes involves centrifugation.

[0012] The centrifugation performed in each of the above steps—solid component removal, nucleic acid adsorption to the column, washing, and elution—tends to process only small amounts of liquid at a time. Therefore, multiple centrifugations may be required for each step. Consequently, the more steps involving centrifugation there are, the longer the sample preparation time becomes and the more complicated the operation becomes. This problem tends to be more pronounced when the amount of sample used for pretreatment is large, or when the concentration ratio of nucleic acids in the concentration process is high.

[0013] Furthermore, when nucleic acid extraction and concentration are performed manually, there is a problem in that the number of samples that can be processed at one time is limited to about 10. Therefore, if the entire process can be performed by an instrument, it is thought that the number of samples that can be processed at one time can be increased. Moreover, if nucleic acid extraction and concentration can be automated by an instrument, the differences due to the analyst's technique will be less likely to occur compared to when it is performed manually, and the data obtained by tests performed after the sample preparation can be stabilized, which is considered useful.

[0014] However, if the pre-processing steps are complex, the equipment used for pre-processing can become complex in structure. Therefore, the more steps involved, including centrifugation, the more complex the equipment tends to become. As a result, realizing equipment for pre-processing is difficult.

[0015] Based on the above circumstances, the inventors have found that nucleic acid extraction and concentration can be simplified by reducing the number of steps, including centrifugation, in the pretreatment process.

[0016] (First Embodiment) According to the first embodiment, a nucleic acid extraction and concentration method is provided. The nucleic acid extraction and concentration method includes a first step of removing at least a portion of the solid components from raw water containing at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms and solid components; a second step of extracting nucleic acids contained in the nucleic acid-containing substance from the processed liquid obtained in the first step, i.e., raw water from which at least a portion of the solid components has been removed, to obtain a nucleic acid extract; and a third step of concentrating the nucleic acid extract. Vacuum filtration is used in at least one of the first or third step.

[0017] In this specification, nucleic acid extraction and concentration method means a method that includes the extraction and concentration of nucleic acids.

[0018] The first step is to remove at least a portion of the solid components from the raw water. The raw water contains at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms, and solid components.

[0019] The solid component may be, for example, a suspended solid (SS) in the raw water. The solid component may be an impurity in the extraction and concentration of nucleic acids from the raw water. Therefore, by removing at least a portion of the solid component in the first step, the purity of the nucleic acid obtained by the nucleic acid extraction and concentration method according to the embodiment can be improved.

[0020] Furthermore, as will be described later, the second step, performed after the first step, can be carried out using a nucleic acid extraction column. The third step, performed after the second step, can be carried out using a nucleic acid concentration column. If a large amount of solid components remain in the processing solution used in the second step and / or the nucleic acid extract used in the third step, it can cause the column to clog. Therefore, by removing at least a portion of the solid components in the first step, the processing efficiency of the second and / or third steps can be improved. Consequently, the efficiency of nucleic acid extraction and concentration can be improved.

[0021] In the nucleic acid extraction and concentration method according to this embodiment, vacuum filtration is used in at least one of the first or third step.

[0022] In this specification, vacuum filtration refers to the use of reduced pressure as a means to facilitate separation when separating a portion containing a target component from a mixture of multiple components. In contrast, centrifugation refers to the use of centrifugal force to facilitate separation when separating a portion containing a target component from a mixture of multiple components.

[0023] Vacuum filtration is preferable because it allows for the continuous separation of the target portion from a mixture, making it a simple and efficient method. Therefore, it is particularly effective when separating and removing solid components from a large sample, or when separating nucleic acid-containing portions from a sample with low nucleic acid concentrations to concentrate the nucleic acids.

[0024] When vacuum filtration is used in the first step, at least a portion of the solid components can be removed from the raw water without centrifugation. When vacuum filtration is used in the third step, centrifugation in concentration can be reduced. In either of the above cases, the number of steps involving centrifugation in nucleic acid extraction and concentration can be reduced. Therefore, nucleic acid extraction and concentration can be performed simply according to the nucleic acid extraction and concentration method according to the embodiment.

[0025] A nucleic acid extraction and concentration method according to an embodiment will be further described with reference to the drawings.

[0026] Figure 1 is a flowchart showing a nucleic acid extraction and concentration method according to an example of the first embodiment. Figure 2 is a diagram showing an overview of the nucleic acid extraction and concentration method according to an example of the embodiment. Figures 1 and 2 show, as an example, a nucleic acid extraction and concentration method in which vacuum filtration is used in all three steps: the first, second, and third steps.

[0027] For example, wastewater or sewage can be used as the sample for this nucleic acid extraction and concentration method. Raw water is obtained by performing a raw water preparation step S0 on the sample. The raw water is subjected to a first step S1 to remove at least a portion of the solid components in the raw water to obtain a treated solution. Next, a second step S2 is performed to extract nucleic acids contained in nucleic acid-containing materials from the treated solution to obtain a nucleic acid extract. A third step S3 is performed to concentrate the nucleic acid extract. The concentrated solution obtained in the third step S3 is subjected to an analysis step S4 as needed.

[0028] The following explains each step.

[0029] <Raw Water Preparation Process> The raw water preparation process is the process of preparing raw water from a sample. The sample may be prepared as raw water as is. The raw water preparation process S0 may include, for example, protease treatment. Protease treatment can be performed, for example, by adding protease to sewage 2, which is a sample containing nucleic acid-containing material 3 and solid components not shown, and holding it, as shown in Figure 2. The liquid obtained in this way can be obtained as raw water 4. The proteins contained in the nucleic acid-containing material can be broken down by protease treatment. In addition, the action of nucleolytic enzymes can be inhibited. Therefore, nucleic acids can be easily extracted, and the purity of the obtained nucleic acids can be improved, which is preferable.

[0030] <First Step> The first step S1 is a step in which at least a portion of the solid components in the raw water is removed to obtain a treated liquid.

[0031] An example of performing the first step S1 by vacuum filtration will be explained with reference to Figure 2.

[0032] The first step can be carried out, for example, using a vacuum filter equipped with a filtration filter. The vacuum filter used in the first step will be referred to as the first vacuum filter.

[0033] As shown in Figure 2, the first vacuum filter can be configured, for example, by connecting a filter 5 and a suction bell 6, and further connecting a pump (not shown) to the suction port 7 of the suction bell 6. A filtrate collection container 8 is installed inside the suction bell 6.

[0034] An example of the first step using the first vacuum filter described above will be explained with reference to Figures 1 and 2.

[0035] As shown by arrow A1, raw water 4 is poured into the filtration filter 5. At this time, the inside of the suction bell 6 is depressurized by the pump connected to the suction port 7, allowing the raw water 4 to be filtered under reduced pressure. Specifically, solid components that do not pass through the filtration filter 5 remain on the filtration filter 5, and the filtrate that has passed through the filtration filter 5 can be collected in the filtrate collection container 8. This filtrate is referred to as the processed liquid 9.

[0036] The filtration filter preferably includes a first filter with a pore size / retained particle size of 1 μm or less. Pore size / retained particle size refers to either the pore size or the retained particle size. For example, a pore size / retained particle size of 1 μm or less means that the pore size is 1 μm or less, or the retained particle size is 1 μm or less. When a first filter with a pore size / retained particle size of 1 μm or less is included, the amount of residual solid components in the processing solution can be reduced. In addition, the particle size of the solid components remaining in the processing solution may be reduced. Therefore, it is possible to suppress the occurrence of problems in processes after the first step. For example, when a nucleic acid extraction column is used in the second step, and when a nucleic acid concentration column is used in the third step, it is possible to suppress clogging of the column. The pore size / retained particle size of the first filter can be, for example, 0.22 μm or more and 1 μm or less.

[0037] The filtration filter preferably includes one or more second filters with a pore size / retained particle size of 1 μm or more before the first filter, and may include two or more. When one or more second filters with a pore size / retained particle size of 1 μm or more are included, clogging of the first filter can be suppressed. When two or more second filters are included, clogging can be suppressed even further. The pore size / retained particle size of the second filter can be, for example, 1 μm or more and 25 μm or less. The pore size / retained particle size of the second filter can be larger than 1 μm.

[0038] A filtration filter comprising a first filter and a second filter may have a configuration in which the second filter and the first filter are provided in this order along the direction in which raw water travels (liquid contact direction). That is, raw water poured onto the filtration filter may first come into contact with one or more second filters, pass through the second filters, then come into contact with the first filter provided therebelow and pass through the first filter. The filtration filter may have a configuration in which the second filter and the first filter are stacked in this order along the liquid contact direction. With such a stacked configuration, raw water can be passed through the first filter and the second filter in a single suction filtration step. Therefore, the number of man-hours required for suction filtration can be reduced, so that suction filtration can be carried out more simply.

[0039] <Second Step> The treatment liquid obtained in the first step S1 is provided in the second step S2. An additive may be added to the treatment liquid. Examples of the additive include ethanol, isopropanol (IPA: isopropanol), polyethylene glycol (PEG: polyethylene glycol), and buffer solutions. One or more types of additives may be added to the treatment liquid. When two or more types of additives are used, for example, at least one selected from the group consisting of ethanol, isopropanol and polyethylene glycol can be used in combination with a buffer solution.

[0040] The second step can be performed using, for example, a suction filtration device. The suction filtration device used in the second step is referred to as a second suction filtration device. The second suction filtration device may comprise a nucleic acid extraction column.

[0041] The second suction filtration device includes, for example, a nucleic acid extraction column 10 and a pump (not shown) connected downstream of the nucleic acid extraction column 10. A method of suction filtration using the second suction filtration device is described below.

[0042] When the second step S2 is performed using the second suction filtration device comprising a nucleic acid extraction column, the second step S2 may include adsorbing nucleic acid onto the nucleic acid extraction column S21, washing the nucleic acid extraction column S22, and eluting nucleic acid from the nucleic acid extraction column S23.

[0043] First, perform nucleic acid adsorption S21 on a nucleic acid extraction column. As indicated by arrow A2, the treatment liquid 9 is poured into the nucleic acid extraction column 10. At this time, pressure is reduced by a pump (not shown) connected downstream of the nucleic acid extraction column 10, whereby the treatment liquid 9 passes through the inside of the nucleic acid extraction column 10. At this time, nucleic acids contained in the treatment liquid 9 can be adsorbed onto the nucleic acid extraction column 10.

[0044] Next, washing S22 of the nucleic acid extraction column is performed. The washing can be performed, for example, by pouring a washing liquid (not shown) into the nucleic acid extraction column 10 and reducing pressure with a pump connected downstream of the nucleic acid extraction column 10. When the washing liquid passes through the inside of the nucleic acid extraction column 10, the nucleic acid extraction column 10 having nucleic acids adsorbed thereon is washed, and impurities can be removed from the inside of the nucleic acid extraction column.

[0045] Next, nucleic acid extraction S23 from the nucleic acid extraction column is performed. Nucleic acid extraction can be performed by pouring an eluate (not shown) into the nucleic acid extraction column 10 and reducing pressure with a pump connected downstream of the nucleic acid extraction column 10. In this way, nucleic acids adsorbed on the nucleic acid extraction column 10 can be eluted into the eluate. The eluate that has passed through the nucleic acid extraction column 10 is obtained as a nucleic acid extract 11.

[0046] As described above, nucleic acid extraction using reduced pressure filtration can be performed. The obtained nucleic acid extract 11 contains nucleic acids 12.

[0047] When performing the second step S2 using a nucleic acid extraction column, it is preferable that one or more steps among S21 to S23 are performed by reduced pressure filtration, and it is more preferable that all of S21 to S23 are performed by reduced pressure filtration. In any of the above cases, it can be said that reduced pressure filtration is used in the second step S2.

[0048] The washing S22 of the nucleic acid extraction column can be performed one or more times. The type of the washing liquid may be one type or two or more types. When the washing S22 of the nucleic acid extraction column is performed two or more times, one type of washing liquid may be used for two or more washes, or the washing may be performed by changing the type of the washing liquid used in each time.

[0049] <Third Step> In the third step S3, the nucleic acid extract obtained in the second step S2 is provided. Additives may be added to the nucleic acid extract. As additives, for example, those similar to those described in the second step can be used.

[0050] The third step can be carried out, for example, using a vacuum filter. The vacuum filter used in the third step will be referred to as the third vacuum filter. The third vacuum filter may be equipped with a nucleic acid concentration column.

[0051] The third vacuum filter includes, for example, a nucleic acid concentration column 13 and a pump (not shown) connected downstream of the nucleic acid concentration column 13. A method of vacuum filtration using the third vacuum filter is described below.

[0052] When the third step S3 is performed using a third vacuum filter equipped with a nucleic acid concentration column, the third step S3 may include nucleic acid adsorption S31 onto the nucleic acid concentration column, washing of the nucleic acid concentration column S32, and nucleic acid extraction S33 from the nucleic acid concentration column.

[0053] First, nucleic acid adsorption S31 onto the nucleic acid concentration column is performed. As shown by arrow A3, the nucleic acid extract 11 is poured into the nucleic acid concentration column 13. At this time, the nucleic acid extract 11 passes through the nucleic acid concentration column 13 by reducing the pressure with a pump (not shown) connected downstream of the nucleic acid concentration column 13. At this time, the nucleic acids contained in the nucleic acid extract 11 can be adsorbed onto the nucleic acid concentration column 13.

[0054] Next, the nucleic acid concentration column 13 is washed. Washing can be performed, for example, by pouring a washing solution (not shown) into the nucleic acid concentration column 13 and reducing the pressure using a pump connected downstream of the nucleic acid concentration column 13. As the washing solution passes through the nucleic acid concentration column 13, the nucleic acid adsorbed on the column is washed, and impurities can be removed from the nucleic acid concentration column.

[0055] Next, nucleic acid extraction is performed from the nucleic acid concentration column 13. Nucleic acid extraction can be performed by pouring an eluate (not shown) into the nucleic acid concentration column 13 and reducing the pressure using a pump connected downstream of the nucleic acid concentration column 13. In this way, nucleic acids adsorbed on the nucleic acid concentration column 13 can be eluted into the eluate. The eluate that has passed through the nucleic acid concentration column is obtained as the concentrate 14.

[0056] As described above, nucleic acid concentration can be performed using reduced-pressure filtration. The obtained concentrate 14 contains nucleic acid 12. The nucleic acid 12 retains its genetic sequence.

[0057] When performing the third step S3 using a nucleic acid concentration column, it is preferable to perform one or more of the steps S31 to S33 by vacuum filtration, and it is more preferable to perform all of S31 to S33 by vacuum filtration. In either of the above cases, vacuum filtration can be used in the third step S3.

[0058] The nucleic acid concentration column washing S32 can be performed one or more times. The washing solution can be one or more types. If the nucleic acid concentration column washing S32 is performed two or more times, the type of washing solution used may be changed each time.

[0059] The nucleic acids contained in the concentrate obtained in the third step may retain the gene sequence. Retaining the gene sequence means that the nucleic acids can be used to identify microbial species or virus species through genetic analysis.

[0060] From the viewpoint of simplifying the nucleic acid extraction and concentration method by reducing steps involving centrifugation, it is preferable to perform the first and third steps by vacuum filtration. It is particularly preferable that the process from raw water to obtaining the concentrate does not involve centrifugation. Centrifugal separators tend to be expensive. Also, equipment including centrifugal separators tends to be large. Large centrifugal separators tend to take time to obtain the desired centrifugal force. If centrifugation is not included, there is no need to use a centrifuge, so nucleic acid extraction and concentration can be performed at low cost, simply and in a short time. For this reason, it is particularly preferable to perform all of the first, second, and third steps by vacuum filtration.

[0061] <Analysis Step> The concentrated solution obtained in step 3 may be subjected to analysis step S4 as needed. Analysis step S4 is a step in which the genes of the concentrated solution are analyzed.

[0062] Analysis step S4 is a step in which the concentrated solution 14 is mixed with, for example, a measuring reagent to form a measurement sample 15, which is then subjected to a measuring instrument 16 to obtain a measurement result 17. As the measuring instrument 16, for example, known nucleic acid analysis devices such as real-time PCR, next-generation sequencers (NGS), and mass spectrometers can be used. Such nucleic acid analysis devices can identify the base sequence of nucleic acids. By comparing the obtained base sequence with a database, the species of organism or virus from which the nucleic acid originates can be identified.

[0063] As explained earlier, the nucleic acids contained in the concentrate may retain their gene sequence. Therefore, by subjecting the concentrate to genetic analysis, the microbial or viral species of nucleic acid-containing organisms in the raw water can be identified. In other words, the microbial or viral species of nucleic acid-containing organisms in the sample can be identified.

[0064] <Comparative Example Method> A method for extracting and concentrating nucleic acids from sewage, as described in the comparative example below, will be explained with reference to Figure 7.

[0065] The sample used in the comparative example method is the same as the sample used in the nucleic acid extraction and concentration method according to the embodiment described above. The sample is subjected to a raw water preparation step S0 to obtain raw water. The raw water is subjected to an SS removal step S11 to remove solid components from the raw water by centrifugation to obtain a supernatant. Next, a primary purification step S12 is performed to extract nucleic acids contained in the nucleic acid-containing material from the supernatant to obtain an extract. A secondary purification step S13 is performed to concentrate the extract by centrifugation. The concentrated solution obtained in the secondary purification step S13 is subjected to an analysis step S14.

[0066] The following explains each step.

[0067] <Raw Water Preparation Process> The raw water preparation process S0 is the same as described in the nucleic acid extraction and concentration method according to the embodiment, so the explanation is omitted.

[0068] <SS Removal> SS removal is performed as follows: Raw water 4 is poured into a centrifugal tube 106 and placed in a centrifuge 105. The centrifuge 105 rotates the centrifugal tube 106 in the direction indicated by arrow A101 to centrifuge the raw water 4. Centrifugation separates solid components (not shown) from the supernatant 109. The supernatant 109 obtained by the above SS removal process is subjected to primary purification.

[0069] <Primary Purification> For primary purification, a nucleic acid extraction column 110 and a pump (not shown) connected downstream of the nucleic acid extraction column 110 are used.

[0070] As shown by arrow A102, the supernatant 109 is poured into the nucleic acid extraction column 110. At this time, the supernatant 109 passes through the nucleic acid extraction column 110 due to the reduced pressure exerted by a pump connected downstream of the nucleic acid extraction column 110. At this time, the nucleic acids contained in the supernatant 109 are adsorbed onto the nucleic acid extraction column 110. In this way, nucleic acid adsorption onto the nucleic acid extraction column is performed.

[0071] Next, the nucleic acid extraction column 110 is washed. Washing can be performed, for example, by pouring a washing solution (not shown) into the nucleic acid extraction column 110 and reducing the pressure using a pump connected downstream of the nucleic acid extraction column 110. As the washing solution passes through the nucleic acid extraction column 110, any remaining impurities in the nucleic acid extraction column 110 are washed away.

[0072] Next, nucleic acid extraction is performed from the nucleic acid extraction column 110. Nucleic acid extraction can be performed by pouring an eluent (not shown) into the nucleic acid extraction column 110 and reducing the pressure using a pump connected downstream of the nucleic acid extraction column 110. The nucleic acids adsorbed on the nucleic acid extraction column 110 are eluted by the eluent to obtain the extract 111.

[0073] Primary purification can be performed as described above. The resulting extract 111 contains nucleic acid 112.

[0074] <Secondary Purification> Next, the extract 111 is poured into the nucleic acid concentration column 113 and placed in the centrifuge 105. The centrifuge 105 rotates the nucleic acid concentration column 113 in the direction indicated by arrow A103 and performs centrifugation. Due to centrifugation, the extract 111 passes through the nucleic acid concentration column 113. At this time, the nucleic acids contained in the extract 111 are adsorbed onto the nucleic acid concentration column 113. The portion that is not adsorbed onto the nucleic acid concentration column 113 (flow-through) is separated to the outside of the nucleic acid concentration column 113 by centrifugal force.

[0075] Next, the nucleic acid concentration column 113 is washed as follows. A washing solution (not shown) is poured into the nucleic acid concentration column 113 and placed in the centrifuge 105. The column is centrifuged in the centrifuge 105 in the same manner as described above. The washing solution passes through the nucleic acid concentration column 113 due to centrifugal force, thereby washing the inside of the nucleic acid concentration column 113.

[0076] Next, nucleic acid extraction from the nucleic acid concentration column 113 is performed as follows. An eluate (not shown) is poured into the nucleic acid concentration column 113 and placed in the centrifuge 105. The centrifuge 105 is used to perform centrifugation in the same manner as described above. As the eluate passes through the nucleic acid concentration column 113 due to centrifugal force, the nucleic acids adsorbed on the nucleic acid concentration column 113 are eluted into the eluate. The liquid that has passed through the nucleic acid concentration column 113 is obtained as the concentrate 114. Secondary purification can be performed in this manner.

[0077] <Analysis Process> The obtained concentrate 114 contains nucleic acid 112. The concentrate 114 is mixed with, for example, a measuring reagent to form a measurement sample 115, which is then subjected to a measuring instrument 116 to obtain a measurement result 117. As the measuring instrument 116, a known nucleic acid analysis device such as a real-time PCR device can be used.

[0078] As described above, the nucleic acid extraction and concentration method of the comparative example includes centrifugation in the SS removal step and the secondary purification step. Therefore, multiple centrifugations are required in each of the SS removal step and the secondary purification step. Consequently, compared to the nucleic acid extraction and concentration method of the example, there are problems such as the time required being longer or the operation being more complicated.

[0079] According to the embodiment, a nucleic acid extraction and concentration method is provided. The nucleic acid extraction and concentration method includes a first step of removing at least a portion of the solid components from raw water containing at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms, and solid components; a second step of extracting nucleic acids contained in the nucleic acid-containing substance from the processed liquid obtained in the first step to obtain a nucleic acid extract; and a third step of concentrating the nucleic acid extract. Vacuum filtration is used in at least one of the first or third step. Therefore, nucleic acid extraction and concentration can be performed easily.

[0080] (Second Embodiment) According to the second embodiment, a nucleic acid extraction and concentration apparatus is provided. The nucleic acid extraction and concentration apparatus includes a first unit that removes at least a portion of the solid components from raw water containing at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms and solid components to obtain a processed liquid; a second unit that extracts nucleic acids contained in the nucleic acid-containing substance from the processed liquid to obtain a nucleic acid extract; and a third unit that concentrates the nucleic acid extract. At least one of the first unit or the third unit includes a vacuum filter.

[0081] If the first unit includes a vacuum filter, the first unit can obtain a treated liquid by removing at least a portion of the solid components from the raw water through vacuum filtration. Therefore, at least a portion of the solid components can be removed from the raw water without centrifugal separation.

[0082] If the third unit includes a vacuum filter, the third unit can concentrate the nucleic acid extract by vacuum filtration. In either of the above cases, the number of steps including centrifugation in the nucleic acid extraction and concentration process can be reduced. Therefore, the nucleic acid extraction and concentration apparatus according to the embodiment can be performed simply.

[0083] Referring to Figure 3, an example of a nucleic acid extraction and concentration apparatus according to this embodiment will be described.

[0084] The nucleic acid extraction and concentration apparatus 100 comprises a first unit 31, a second unit 32, a third unit 33, a collection container 34, a waste liquid tank 35, a filtration filter 41, reagent input units 42-43, valves 52-53, piping L1-L3, piping L10, L20-L21, L30-L32, L40-L41, and a pump P.

[0085] The first unit 31 is a vacuum filter comprising a filtration filter 41 and a chamber (not shown). The filtration filter 41 comprises a contact side 41a that comes into contact with the raw water introduced and a discharge side 41b that discharges the filtrate as a treated liquid. The same filtration filter as described in the first embodiment can be used.

[0086] The second unit 32 is a vacuum filter equipped with a nucleic acid extraction column (not shown). The nucleic acid extraction column has an inlet for supplying the processing solution and an outlet for discharging the nucleic acid extract.

[0087] The third unit 33 is a vacuum filter equipped with a nucleic acid concentration column (not shown). The nucleic acid concentration column has an inlet for supplying nucleic acid extract and an outlet for discharging the concentrate.

[0088] The first to third units are arranged in the order of first unit 31, second unit 32, and third unit 33, along the direction in which the liquid to be processed flows. Along the above direction, the second unit 32 is located downstream of the first unit 31. The third unit 33 is located downstream of the second unit 32.

[0089] Here, the liquids to be processed refer to, for example, the raw water to be processed by the first unit, the processing solution to be processed by the second unit, and the nucleic acid extract to be processed by the third unit. The direction in which the liquids to be processed flow can be said to be the direction in which the raw water flows from the point where the raw water is introduced (for example, the first unit 31) as the starting point to the point where the concentrated solution is collected (for example, the collection container 34) as the ending point, through the processing solution and the nucleic acid extract, until it is collected as the concentrated solution.

[0090] Such a nucleic acid extraction and concentration apparatus may have a channel for exhaust and / or waste liquid in addition to the channel through which the liquid to be processed flows.

[0091] First, let's explain the configuration of the flow path through which the liquid to be processed flows.

[0092] One end of pipe L1 is connected to the discharge side 41b of the filtration filter 41 of the first unit 31. The other end of pipe L1 is connected to the inlet of the nucleic acid extraction column of the second unit 32. In addition to the other end of pipe L1, a reagent supply unit 42 is connected to the inlet of the nucleic acid extraction column. One end of pipe L20 is connected to the outlet of the nucleic acid extraction column. The other end of pipe L20 is connected to a valve 52. One end of pipe L2 and one end of pipe L21 are connected to the valve 52. The other end of pipe L2 is connected to the inlet of the nucleic acid concentration column of the third unit 33. In addition to the other end of pipe L2, a reagent supply unit 43 is connected to the inlet of the nucleic acid concentration column. One end of pipe L30 is connected to the outlet of the nucleic acid concentration column. The other end of pipe L30 is connected to a valve 53. One end of pipe L3 and one end of pipe L31 are connected to the valve 53. The other end of pipe L3 is connected to a collection container 34.

[0093] Next, we will describe the flow path through which exhaust and / or waste liquids pass.

[0094] One end of pipe L10 is connected to a chamber (not shown) provided in the first unit. The other end of pipe L10 is connected to one end of pipe L40.

[0095] The piping L21 connected to valve 52 is in communication with piping L40.

[0096] The piping L31 connected to valve 53 is in communication with piping L40.

[0097] Pipe L32 is installed downstream of valve 53 and pipe L3. A collection container 34 is connected to one end of pipe L32. The other end of pipe L32 is in communication with pipe L31. In other words, pipe L32 is installed so that, along the direction of exhaust flow, the order is valve 53, pipe L3, collection container 34, and pipe L32.

[0098] The other end of pipe L40 is connected to pump P. One end of pipe L41 is further connected to pump P. The other end of pipe L41 is connected to waste liquid tank 35.

[0099] The process of nucleic acid extraction and concentration using the nucleic acid extraction and concentration apparatus according to this embodiment will be explained.

[0100] The raw water supplied to the first unit 31 comes into contact with the contact side 41a of the filtration filter 41. When the pump P starts to reduce pressure, air in the chamber is drawn through the piping L10 in the direction of arrow A11. This air is sent as exhaust to piping L40, which is connected to the end of piping L10. The exhaust is sent by the pump P to piping L41 and then to the waste liquid tank 35, which is connected to the end of piping L41. As a result of this pressure reduction in the chamber, the raw water is filtered under reduced pressure by the filtration filter 41. Due to the reduced pressure filtration, solid components that do not pass through the filtration filter 41 remain on the filtration filter 41, and the filtrate that has passed through the filtration filter 41 is discharged from the discharge side 41b. The filtrate is obtained as the treated liquid. The treated liquid is sent to the second unit 32 through piping L1.

[0101] A filtrate collection container (not shown) may be provided on the discharge side 41b of the filtration filter. The filtrate collection container can be connected to the piping L1. In this case, the filtrate obtained by vacuum filtration can be collected in the filtrate collection container and sent to the piping L1.

[0102] As described above, the first unit can remove at least a portion of the solid component from raw water containing at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms, and the solid component. That is, the first step described in the first embodiment can be carried out. The first unit 31 may be the first vacuum filter described in the first embodiment.

[0103] The processing solution supplied to the nucleic acid extraction column of the second unit 32 is first mixed with an additive supplied from the reagent input unit 42, and then supplied to the nucleic acid extraction column. The same additive as described in the first embodiment can be used. When pump P starts to reduce pressure, the liquid supplied to the nucleic acid extraction column passes through the nucleic acid extraction column. As a result, the nucleic acids contained in the processing solution are adsorbed onto the nucleic acid extraction column. The unadsorbed portion (non-adsorbed portion) is discharged from the outlet of the nucleic acid extraction column and sent to piping L20.

[0104] Valve 52, connected to the end of pipe L20, controls whether the liquid discharged from the nucleic acid extraction column is supplied to pipe L2 or pipe L21. For example, it controls the supply of the liquid to be processed to pipe L2 and the waste liquid to pipe L21. The non-adsorbed portion, which is the waste liquid, is sent to pipe L21 under the control of valve 52. The non-adsorbed portion is sent through pipe L21 in the direction of arrow A21 and merges into pipe L40, which is connected to pipe L21. Pump P sends the liquid in pipe L40 to waste liquid tank 35, which is connected to the end of pipe L41. In this way, the non-adsorbed portion is collected in the waste liquid tank 35 as waste liquid. Thus, nucleic acid adsorption S21 onto the nucleic acid extraction column, as described above, can be performed.

[0105] Next, the reagent supply unit 42 supplies the washing solution to the nucleic acid extraction column. When the pump P starts to reduce pressure, the washing solution passes through the nucleic acid extraction column. This cleans the inside of the nucleic acid extraction column. The washing solution that has passed through the nucleic acid extraction column is discharged from the outlet of the nucleic acid extraction column and sent to the piping L20. After that, it is collected in the waste liquid tank 35 as waste liquid, similar to the non-adsorbed portion described earlier. Thus, the washing of the nucleic acid extraction column S22 described earlier can be performed.

[0106] Next, the eluate is supplied from the reagent supply unit 42 to the nucleic acid extraction column of the second unit 32. When the pump P connected to the nucleic acid extraction column starts to reduce pressure, the eluate passes through the nucleic acid extraction column. As a result, the nucleic acids adsorbed on the nucleic acid extraction column are eluted into the eluate. The eluate that has passed through the nucleic acid extraction column is the nucleic acid extract. The nucleic acid extract is discharged from the outlet of the nucleic acid extraction column and sent to piping L20. The nucleic acid extract is sent to piping L2 under the control of valve 52. The nucleic acid extract is sent through piping L2 in the direction of arrow A20 and supplied to the third unit 33 connected to the end of piping L2. Thus, nucleic acid extraction S23 from the nucleic acid extraction column described above can be performed.

[0107] As described above, the second unit can extract nucleic acids contained in the nucleic acid-containing material from the processing solution obtained in the first step to obtain a nucleic acid extract. Therefore, the second step described in the first embodiment can be carried out. The second unit 32 may be the second vacuum filter described in the first embodiment.

[0108] The nucleic acid extract supplied to the third unit 33 is first mixed with an additive supplied from the reagent input unit 43, and then supplied to the nucleic acid concentration column. The same additive as described in the first embodiment can be used. When pump P starts to reduce pressure, the liquid supplied to the nucleic acid concentration column passes through the nucleic acid concentration column. As a result, the nucleic acids contained in the nucleic acid extract are adsorbed onto the nucleic acid concentration column. The unadsorbed portion (non-adsorbed portion) is discharged from the outlet of the nucleic acid concentration column and sent to piping L30.

[0109] Valve 53, connected to the end of pipe L30, controls whether the liquid and / or gas in pipe L30 is supplied to pipe L3 or pipe L31. For example, it controls the supply of the liquid to be treated to pipe L3, and the waste liquid and / or exhaust to pipe L31. The non-adsorbed portion, which is the waste liquid, is sent to pipe L31 under the control of valve 53. The non-adsorbed portion is sent through pipe L31 in the direction of arrow A31 and merges into pipe L40, which is connected to pipe L31. Subsequently, as described above, the liquid in pipe L40 is collected as waste liquid in the waste liquid tank 35.

[0110] Next, the reagent supply unit 43 is supplied with washing solution to the nucleic acid concentration column. When the pump P starts to reduce pressure, the washing solution passes through the nucleic acid concentration column. This cleans the inside of the nucleic acid concentration column. The washing solution that has passed through the nucleic acid concentration column is discharged from the outlet of the nucleic acid concentration column and sent to the piping L30. After that, it is collected as waste liquid in the waste liquid tank 35, similar to the non-adsorbent portion described earlier. Thus, the washing of the nucleic acid concentration column S32 described earlier can be performed.

[0111] Next, the eluate is supplied from the reagent input unit 43 to the nucleic acid concentration column of the third unit 33.

[0112] At this time, valve 53 controls the liquid and / or gas in piping L30 to be sent to piping L3. Therefore, when pump P starts to depressurize, the air in piping L3, L30 and the collection container 34 is sent to piping L32 as exhaust, and further sent in the direction of A31 through piping L31 which is connected to piping L32. The exhaust merges into piping L40 which is connected to L31. The exhaust is sent to piping L41 by pump P and then to the waste liquid tank 35 connected to the end of piping L41.

[0113] The eluate supplied from the reagent input unit 43 to the nucleic acid concentration column of the third unit 33 is drawn towards the collection container 34 by the reduced pressure described above. As a result, the eluate passes through the nucleic acid concentration column. This causes the nucleic acids adsorbed on the nucleic acid concentration column to eluate into the eluate. The eluate that has passed through the nucleic acid concentration column is the concentrate. The concentrate is discharged from the outlet of the nucleic acid concentration column and sent to piping L30. The concentrate is sent to piping L3 under the control of valve 53 and collected in the collection container 34 connected to the end of piping L3. In this way, the concentrate is obtained by reduced pressure filtration. Thus, nucleic acid extraction S33 from the nucleic acid concentration column can be performed.

[0114] The piping L3 may include a pump (not shown). This pump can send the concentrated liquid, controlled by the valve 53, to a collection container 34 connected to the end of the piping L3.

[0115] As described above, the third unit can concentrate the nucleic acid extract. Therefore, the third step of concentrating the nucleic acid extract, as described in the first embodiment, can be carried out. The third unit 33 may be the third vacuum filter described in the first embodiment.

[0116] According to the nucleic acid extraction and concentration apparatus described above, the first to third steps described in the first embodiment can be carried out, and vacuum filtration can be used in at least one of the first or third steps. Therefore, the nucleic acid extraction and concentration method according to the first embodiment can be carried out.

[0117] A vacuum filter is a unit that has a configuration capable of performing vacuum filtration. As explained earlier, in the example shown in Figure 3, the first unit 31, the second unit 32, and the third unit 33 are each connected to the same pump located outside each unit. The first unit 31, the second unit 32, or the third unit 33 may each be connected to different pumps, or each may have a pump inside. In any of the above cases, the first unit 31, the second unit 32, and the third unit 33 can each perform vacuum filtration. Therefore, in any of the above cases, it can be said that the first unit 31, the second unit 32, and the third unit 33 are each vacuum filters.

[0118] If the first unit 31, the second unit 32, or the third unit 33 each has a pump inside, the filtrate as the processing liquid may be collected in a filtrate collection container installed inside the first unit, and the processing liquid in the filtrate collection container may be supplied to the second unit. In this case, the piping and valves connecting the first unit and the second unit can be omitted. Alternatively, a collection container may be installed inside the second unit to collect the nucleic acid extract, and the nucleic acid extract in the collection container may be supplied to the third unit. In this case, the piping and valves connecting the second unit and the third unit can be omitted. Details of the above configurations can be made in the same way as described in the embodiments described later, for example.

[0119] Each of the valves 52 and 53 can be a dispenser capable of automatic dispensing. When valve 52 is a dispenser, it can automatically control whether the liquid discharged from the second unit 32 is sent to piping L2 or L21. When valve 53 is a dispenser, it can automatically control whether the liquid discharged from the third unit 33 is sent to piping L3 or L31.

[0120] Each of the reagent supply units 42 and 43 may be a dispenser or a liquid delivery pump. In this case, each of the reagent supply units 42 and 43 can automatically control the type, amount, and timing of reagents such as additives, washing solutions, and eluents, and supply them to the second unit 32 or the third unit 33.

[0121] The nucleic acid extraction and concentration apparatus described herein preferably does not include a centrifuge. For example, it is preferable to have a configuration illustrated in Figure 3, in which a vacuum filter and a dispensing machine or liquid delivery pump are connected by piping. With the above configuration, since a centrifuge is not included, the nucleic acid extraction and concentration apparatus can be manufactured at low cost. Furthermore, if a dispensing machine or liquid delivery pump is included, it is possible to provide an apparatus that can perform nucleic acid extraction and concentration automatically at low cost. When nucleic acid extraction and concentration can be performed automatically, it is possible to reduce the differences caused by the analyst's technique compared to when nucleic acid extraction and concentration is performed manually. Therefore, for example, it is preferable because it is possible to stabilize the data obtained by tests performed after processing with the nucleic acid extraction and concentration apparatus according to the second embodiment. The nucleic acid extraction and concentration apparatus described herein may consist only of a vacuum filter and a dispensing machine or liquid delivery pump.

[0122] The nucleic acid extraction and concentration apparatus according to the second embodiment includes a first unit that removes at least a portion of the solid components from raw water containing at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms, and solid components, to obtain a processed liquid; a second unit that extracts the nucleic acid contained in the nucleic acid-containing substance from the processed liquid to obtain a nucleic acid extract; and a third unit that concentrates the nucleic acid extract. At least one of the first unit or the third unit includes a vacuum filter. Therefore, according to this embodiment, a nucleic acid extraction and concentration apparatus that can easily extract and concentrate nucleic acids can be provided.

[0123] (Example 1) Sewage to which inactivated coronavirus was added was used as a sample.

[0124] The raw water preparation process, the first process, the second process, and the third process were carried out as follows.

[0125] <Raw Water Preparation Process> The sample was treated with protease. 40 mL of the sample was mixed with 500 μm protease solution. The mixture was incubated at room temperature for 30 minutes to obtain the raw water.

[0126] <First Step> First, the first vacuum filter to be used in the first step was assembled as follows. A suction bell for Kiriyama funnels (model number: VKB-50, manufacturer: Kiriyama Seisakusho) was prepared as the suction bell. A PP Büchner funnel (φ55 mm, model number: 0043800, manufacturer: Kartell) was prepared as the Büchner funnel. A quantitative filter paper No. 5C (model number: 01531055, manufacturer: Advantec Toyo) was prepared as the first filter, and a quantitative filter paper No. 5A (model number: 01511055, manufacturer: Advantec Toyo) was prepared as the second filter. The pore size / retained particle size of the first filter was 1 μm, and the pore size / retained particle size of the second filter was 7 μm.

[0127] An empty 100 mL iBoy® container was placed inside the suction bell to serve as a filtrate collection container. A Buchner funnel was connected to this suction bell. A second filter was placed on top of a first filter as the filtration filter. This filtration filter was placed on the Buchner funnel with the second filter on top. A pump was connected to the suction port of the suction bell.

[0128] The entire volume of raw water was filtered under reduced pressure using the first vacuum filter described above. The filtrate was collected in a 100 mL iBoy container. This was used as the treated solution.

[0129] <Second Step> The second vacuum filter used in the second step was assembled by installing a PureYield Midi Binding Column, which serves as the nucleic acid extraction column, into a VacMan Vacuum Manifold and connecting the VacMan Vacuum Manifold to a pump.

[0130] As additives, Binding Buffer 1 (BBD), Binding Buffer 2 (BBE), and isopropanol were prepared. 12 mL of Binding Buffer 1 (BBD) and 1 mL of Binding Buffer 2 (BBE) were added to a 100 mL iBoy containing the processing solution. The mixture was thoroughly mixed by shaking the iBoy more than 10 times. 48 mL of isopropanol was then added and mixed thoroughly again. This mixture was poured into the nucleic acid extraction column, and the pressure was reduced with a pump to ensure that the entire mixture passed through the nucleic acid extraction column.

[0131] Two washing solutions, Column Wash 1 (CWE) and Column Wash 2 (RWA), were prepared. 5 ml of Column Wash 1 (CWE) was poured into the nucleic acid extraction column and passed through under reduced pressure. Next, 20 ml of Column Wash 2 (RWA) was poured into the nucleic acid extraction column and passed through under reduced pressure. After the solution had completely passed through the nucleic acid extraction column, the valve was repeatedly opened and closed to completely remove any liquid adhering to the bottom of the nucleic acid extraction column. Then, reduced pressure was continued for 3 minutes to completely dry the nucleic acid extraction column.

[0132] After drying was complete, an eluator was placed directly below the column. The eluator used had a connection port at the top and a suction port at the bottom. A 1.5 ml tube was set inside the eluator as a collection container, and then the nucleic acid extraction column, which had been completely dried using the procedure described above, was placed in the connection port of the eluator. A pump was connected to the suction port of the eluator.

[0133] Nuclease-free water heated to 60°C was prepared as the eluate. 500 μL of the eluate was poured into the nucleic acid extraction column, and the inside of the eluator was depressurized by pumping, allowing it to pass through the nucleic acid extraction column. Another 500 μL of the eluate was poured into the nucleic acid extraction column and allowed to pass through the nucleic acid extraction column by pumping. Thus, the entire volume of 1000 μL of the eluate was collected in a collection container by vacuum filtration. This was used as the nucleic acid extract.

[0134] <Step 3> The vacuum filter used in Step 3 was assembled as follows: A PureYield Mini Column, used as a nucleic acid concentration column, was placed in a VacMan Vacuum Manifold. The tip of an autoclaved (A / C) 10 mL tip was connected to the nucleic acid concentration column. The VacMan Vacuum Manifold was connected to the pump.

[0135] As additives, Binding Buffer 1 (BBD), Binding Buffer 2 (BBE), and isopropanol were prepared. 400 μL of Binding Buffer 1 (BBD) and 100 μL of Binding Buffer 2 (BBE) were added to 1000 μL of nucleic acid extract and mixed. This mixture was dispensed into two 1.5 ml tubes, 740 μL each. 740 μL of isopropanol was added to each of the two 1.5 ml tubes and mixed thoroughly.

[0136] The mixtures prepared in two 1.5 ml tubes were poured into 10 ml tips, and the mixtures were then injected into a nucleic acid concentration column connected to the 10 ml tips. The mixture was then pumped under reduced pressure to ensure that the entire volume of the mixture passed through the nucleic acid concentration column. After the mixture had completely passed through, the 10 ml tips were removed from the nucleic acid concentration column.

[0137] Two washing solutions, Column Wash 1 (CWE) and Column Wash 2 (RWA), were prepared. 300 μL of Column Wash 1 (CWE) was poured into the nucleic acid concentration column and passed through under reduced pressure. Next, 500 μL of Column Wash 2 (RWA) was poured into the nucleic acid concentration column and passed through under reduced pressure. Another 500 μL of Column Wash 2 (RWA) was poured into the nucleic acid concentration column and passed through under reduced pressure. After the solution had completely passed through the nucleic acid concentration column, the valve was repeatedly opened and closed to completely remove any liquid adhering to the bottom of the nucleic acid concentration column. Then, reduced pressure was continued for 3 minutes to completely dry the nucleic acid concentration column.

[0138] After drying was complete, an eluator was placed directly below the column. The eluator used had a connection port at the top and a suction port at the bottom. A 1.5 ml tube was set inside the eluator as a collection container, and then the nucleic acid concentration column, which had been completely dried using the procedure described above, was placed in the connection port of the eluator. A pump was connected to the suction port of the eluator.

[0139] Nuclease-free water heated to 60°C was prepared as the eluate. 40 μL of the eluate was poured into the nucleic acid concentration column and allowed to stand for 1 min. Then, the inside of the eluator was depressurized by pumping, and the eluate was passed through the nucleic acid concentration column. Another 40 μL of the eluate was poured into the nucleic acid concentration column and passed through the column by pumping. Thus, approximately 60-70 μL of the eluate was collected in a collection container by vacuum filtration. This was obtained as the concentrate.

[0140] (Example 2) A different type of sewage was used as the sample compared to Example 1. No inactivated coronavirus was added. Except for the above, the concentrate was obtained in the same manner as in Example 1.

[0141] (Example 3) A different type of sewage sample was used as the sample, compared to that used in Examples 1 and 2. Inactivated coronavirus was not added. Except for the above, the concentrated solution was obtained in the same manner as in Example 1.

[0142] (Comparative Example 1) A sample prepared in the same manner as in Example 1 was subjected to the same raw water preparation process as in Example 1 to obtain raw water. Nucleic acid extraction and concentration were performed using the Direct capture method as follows.

[0143] <SS Removal> The raw water was centrifuged at 3000xg for 10 minutes at room temperature. 20 ml of the supernatant after centrifugation was transferred to two 50 mL tubes, taking care not to include any precipitate. The resulting supernatant was used as the treatment solution.

[0144] <Primary Purification> The vacuum filter was assembled in the same manner as described in the second step of Example 1.

[0145] 6 mL of Binding Buffer 1 (BBD) and 0.5 mL of Binding Buffer 2 (BBE) were added to each of the two 50 mL tubes containing the processing solution, and thoroughly mixed. Then, 24 mL of isopropanol was added and thoroughly mixed.

[0146] This mixture (total volume 100 mL) was poured into one nucleic acid extraction column in several portions, and the pressure was reduced with a pump to ensure that the entire volume of the mixture passed through the nucleic acid extraction column. Subsequent washing and elution of the nucleic acid extraction column were carried out in the same manner as described in the second step of Example 1, and a total volume of 1000 μL of eluate was collected. This was used as the extract. <Secondary purification> 400 μL of Binding Buffer 1 and 100 μL of Binding Buffer 2 were added to 1000 μL of the extract and mixed. This mixture was dispensed into two 1.5 ml tubes, 750 μL each. 750 μL of isopropanol was added to each of the two 1.5 ml tubes and mixed well to obtain the sample solution for centrifugation.

[0147] The sample solution for centrifugation was adsorbed onto the nucleic acid concentration column as follows: First, a PureYield Mini Column, used as the nucleic acid concentration column, was set on a PureYield Collection Column. 750 μL of the sample solution for centrifugation was poured onto the nucleic acid concentration column and centrifuged at 10,000 xg for 1 min to allow it to pass through the nucleic acid concentration column. After centrifugation, the flow-through that had passed through the nucleic acid concentration column was discarded. The above centrifugation process was repeated until the entire volume of the sample solution for centrifugation had passed through the nucleic acid concentration column.

[0148] Two washing solutions, Column Wash 1 (CWE) and Column Wash 2 (RWA), were prepared. 300 μL of Column Wash 1 was poured into the nucleic acid concentration column and centrifuged at 10,000 xg for 1 minute to allow it to pass through. Next, 500 μL of Column Wash 2 (RWA) was poured into the nucleic acid concentration column and centrifuged at 10,000 xg for 1 minute, repeating this process twice. The flow-through was discarded, and the nucleic acid concentration column was centrifuged at 10,000 xg for 30 seconds.

[0149] The nucleic acid concentration column was placed in a new 1.5 ml tube.

[0150] Nuclease-free water warmed to 60°C was prepared as the eluate. 20 μL of the eluate was poured into a nucleic acid concentration column and allowed to stand for 1 min. After centrifugation at 10,000 xg for 1 min, the eluate was passed through the nucleic acid concentration column. This procedure was repeated twice, and the total volume of 40 μL of eluate was collected in a 1.5 ml tube.

[0151] (Comparative Example 2) The same sewage used in Example 2 was used as the sample. No inactivated coronavirus was added. Except as described above, the concentrated solution was obtained in the same manner as in Comparative Example 1.

[0152] (Comparative Example 3) The same sewage used in Example 3 was used as the sample. No inactivated coronavirus was added. Except as described above, the concentrated solution was obtained in the same manner as in Comparative Example 1.

[0153] <Real-time PCR> (Example 1) The concentrated solution obtained by the method of Example 1 described above was measured by real-time PCR as described below.

[0154] For detecting the coronavirus, we used Takara Bio's real-time PCR device (Thermal Cycler Dice® Real Time System III).

[0155] As a positive control, we used synthetic nucleic acids containing the gene region targeted by real-time PCR.

[0156] The positive control solution used to create the calibration curve was prepared as follows. First, the concentration was adjusted to 10 pmol / μL, and then the copy number of nucleic acids contained in the positive control solution was calculated using Avogadro's number. From the calculated results, 10 ７ A positive control stock solution containing copies of nucleic acid was prepared. Serial dilutions were performed from this stock solution, and the nucleic acid content in one PCR reaction solution was 10 ５ ,10 ４ ,10 ３ ,10 ２ ,10 １ Positive control solutions, which serve as copies, were prepared separately.

[0157] Two primer sets, P2 and N1, were prepared for use in PCR. Table 1 shows the sequences of the primers constituting the P2 primer set, and Table 2 shows the sequences of the primers constituting the N1 primer set. In both P2 and N1, FAM (fluorescein) was used as the probe labeling dye.

[0158]

[0159]

[0160] The above primer sets were mixed to the compositions shown in Tables 3 and 4 below and used as a primer mix. Tables 3 and 4 show the concentration of each primer or probe in the PCR reaction solution as x1 concentration.

[0161]

[0162]

[0163] The PCR reaction mixture was prepared by mixing 10 μL of real-time PCR enzyme containing reverse transcriptase (x2), 1 μL of TaqMan primer mix (x20), 5 μL of RT-PCR Grade Water, and 4 μL of PCR sample, so that the total volume per reaction of the PCR reaction mixture was 20 μL.

[0164] The concentrated solution obtained in Example 1 was used as the PCR sample.

[0165] Furthermore, a positive control reaction solution was prepared in the same manner as described above, except that the positive control solution described earlier was used as the PCR sample instead of the concentrated solution. A positive control reaction solution was prepared for each copy number of the positive control solution prepared by the serial dilution described above.

[0166] Furthermore, for both the PCR reaction solution and the positive control reaction solution, two types of reaction solutions were prepared: a P2 reaction solution using P2 primer mix as the TaqMan primer mix, and an N1 reaction solution using N1 primer mix as the TaqMan primer mix. A total of four PCR reaction solutions were prepared for each sample, such that N=2 for both the P2 and N1 reaction solutions.

[0167] The PCR reaction solution and positive control reaction solution prepared as described above were simultaneously subjected to real-time PCR as follows. First, the reverse transcription reaction was performed at 52°C for 5 minutes. Then, the real-time PCR reaction was performed. PCR was performed by repeating a cycle of 95°C for 10 seconds, followed by 45 cycles of 95°C for 10 seconds and 60°C for 60 seconds.

[0168] The validity of the measurement was evaluated by measuring the PCR reaction solution and the positive control reaction solution in parallel.

[0169] Furthermore, a calibration curve was created from the measurement results of a positive control whose nucleic acid copy number was known. From this calibration curve, the nucleic acid copy number in the PCR reaction solution using the concentrated solution obtained in Example 1 as the sample was quantified for each of the four reaction solutions. The average value of the four quantified copy numbers was calculated. Furthermore, the copy number per liter of sewage (copies / L) was calculated from the average value and the concentration ratio. The concentration ratio is the value obtained by dividing the volume of the concentrated solution by the volume of the sample used for nucleic acid extraction and concentration. The copy number (copies / L) is a numerical value that indicates how many molecules of nucleic acid identified as coronavirus are present per liter of sewage. The copy number (copies / L) was used as the measurement result for Example 1.

[0170] (Examples 2, 3, Comparative Examples 1-3) Measurements were performed by real-time PCR in the same manner as described for Example 1, except that the PCR sample was changed to the concentrated solution obtained in Examples 2, 3, and Comparative Examples 1-3, respectively.

[0171] <Evaluation> Figures 4 to 6 show bar graphs of the number of copies per liter of sewage (copies / liter) calculated for each example and comparative example. By comparing Example 1 with Comparative Example 1, Example 2 with Comparative Example 2, and Example 3 with Comparative Example 3, all of which were measured using the same sample, the detection sensitivity of the nucleic acid extraction and concentration method used in the examples was confirmed compared to the direct capture method.

[0172] From the results obtained in the above evaluation, the copy number per liter (copies / L) obtained by the nucleic acid extraction and concentration method performed in Examples 1 to 3 was superior in two of the examples compared to the copy number per liter obtained by the Direct capture method performed in Comparative Examples 1 to 3. In other words, the copy number per liter in the examples was at least equivalent to that of the comparative examples and was not inferior to that of the comparative examples. That is, it became clear that the nucleic acid extraction and concentration method according to the examples was able to extract nucleic acids while retaining gene sequence information to the same extent as the Direct capture method.

[0173] Furthermore, upon examining the nucleic acid extraction column used in the second step, the degree of contamination on the surface of the nucleic acid extraction column was comparable to that of the nucleic acid extraction column used in the primary purification in the Direct Capture method. Also, similar to the Direct Capture method, no clogging of the column occurred. From this, it became clear that the nucleic acid extraction and concentration method performed in Examples 1 to 3 differed from the Direct Capture method in that solid substances were separated by vacuum filtration instead of centrifugation, but it was still possible to sufficiently remove solid components.

[0174] Furthermore, the time required for nucleic acid extraction and concentration using the Direct capture method, as performed in Examples 1-3, was approximately the same as that required for nucleic acid extraction and concentration using the Direct capture method, as performed in Comparative Examples 1-3.

[0175] Comparative Examples 1 to 3 involved centrifugation, requiring repeated centrifugation operations. The methods used in Examples 1 to 3 employed vacuum filtration in the first and third steps, allowing for continuous separation of the target portion in both steps. This reduced the number of steps and enabled nucleic acid extraction and concentration with simple operations.

[0176] Since coronavirus was detected in Example 1, it is considered that the inactivated coronavirus added to the sewage in Example 1 was detected. Furthermore, coronavirus was also detected in Examples 2 and 3, in which no inactivated coronavirus was added. From this, it is considered that the sewage used as samples in Examples 2 and 3 contained coronavirus, and that this coronavirus was detected.

[0177] From the above results, it became clear that the nucleic acid extraction and concentration methods performed in Examples 1 to 3 are simpler than the Direct Capture method because they reduce the number of steps, including centrifugation, and that they can extract and concentrate nucleic acids while preserving gene sequence information to the same extent as the Direct Capture method.

[0178] While several embodiments of the present invention have been described, these embodiments are presented as examples only and are not intended to limit the scope of the invention. These novel embodiments can be carried out in a variety of other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims and their equivalents.

[0179] 1...Nucleic acid extraction and concentration method, 2...Wastewater, 3...Nucleic acid-containing material, 4...Raw water, 5...Filtration filter, 6...Suction bell, 7...Suction port, 8...Filtrate collection container, 9...Processed solution, 10...Nucleic acid extraction column, 11...Nucleic acid extract, 12...Nucleic acid, 13...Nucleic acid concentration column, 14...Concentrated solution, 15...Measurement sample, 16...Measuring equipment, 17...Measurement results, 101...Nucleic acid extraction and concentration method, 105...Centrifuge, 106...Centrifuge tube, 109...Supernatant, 111...Extract, 112... Nucleic acid, 113... Nucleic acid concentration column, 114... Concentrated solution, 115... Measurement sample, 116... Measuring instrument, 117... Measurement result, 100... Nucleic acid extraction and concentration device, 31... Unit 1, 32... Unit 2, 33... Unit 3, 34... Collection container, 35... Waste liquid tank, 41... Filtration filter, 42-43... Reagent input unit, 52-53... Valve, L1-L3, L10, L20-L21, L30-L32, L40-L41... Piping, P... Pump.

Claims

1. A nucleic acid extraction and concentration method comprising: a first step of removing at least a portion of the solid components from raw water containing at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms and solid components; a second step of extracting nucleic acids contained in the nucleic acid-containing substance from the processed liquid obtained in the first step to obtain a nucleic acid extract; and a third step of concentrating the nucleic acid extract, wherein vacuum filtration is used in at least one of the first or third step.

2. The nucleic acid extraction and concentration method according to claim 1, wherein vacuum filtration is used in the first, second, and third steps.

3. The nucleic acid extraction and concentration method according to claim 1, wherein the first step includes obtaining a filtrate as the processing solution by removing at least a portion of the solid components from the raw water using a vacuum filter equipped with a filtration filter.

4. The nucleic acid extraction and concentration method according to claim 3, wherein the filtration filter includes a first filter having a pore size of 1 μm or less, or a retained particle size of 1 μm or less.

5. The nucleic acid extraction and concentration method according to claim 4, wherein the filtration filter comprises one or more second filters having a pore size of 1 μm or less, or a retained particle size of 1 μm or more, prior to the first filter.

6. The nucleic acid extraction and concentration method according to claim 5, wherein the filtration filter comprises two or more of the second filters.

7. A nucleic acid extraction and concentration apparatus comprising: a first unit that obtains a processed liquid by removing at least a portion of the solid components from raw water containing at least one nucleic acid-containing substance selected from the group consisting of viruses and microorganisms and a solid component; a second unit that obtains a nucleic acid extract by extracting the nucleic acid contained in the nucleic acid-containing substance from the processed liquid; and a third unit that concentrates the nucleic acid extract, wherein at least one of the first unit or the third unit includes a vacuum filter.

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