WO2017013731A1 - Procédé de production de solution de virus purifié, procédé de détection de virus, et élément de purification de virus - Google Patents

Procédé de production de solution de virus purifié, procédé de détection de virus, et élément de purification de virus Download PDF

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WO2017013731A1
WO2017013731A1 PCT/JP2015/070663 JP2015070663W WO2017013731A1 WO 2017013731 A1 WO2017013731 A1 WO 2017013731A1 JP 2015070663 W JP2015070663 W JP 2015070663W WO 2017013731 A1 WO2017013731 A1 WO 2017013731A1
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virus
functional group
solution
humic acid
acid
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PCT/JP2015/070663
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English (en)
Japanese (ja)
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平橋　智裕
大英 中熊
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Dic株式会社
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Priority to PCT/JP2015/070663 priority Critical patent/WO2017013731A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a method for producing a purified virus solution, a virus detection method, and a virus purification member.
  • a method of concentrating a sample used for virus detection by ultracentrifugation or polyethylene glycol method is known.
  • Patent Document 1 reports a method of capturing a virus through a solution containing the virus in a surface-treated hollow fiber and evaluating the concentration of the virus. In that case, ultrafiltration is performed as necessary. In some cases, secondary concentration is performed on a membrane.
  • the virus concentration method as described above has a problem that humic acid, which is a substance that inhibits the NAT method present in the environment, is also recovered at the same time.
  • Humic acid is a kind of humic substance that exists in the environment, and it is a high-molecular organic material that is chemically and biologically synthesized from the degradation products of plant residues, microorganisms, and plankton deceased by microorganisms. It is a mixture of acids. Humic substances, also called humic substances, are soluble in alkaline solutions, but there are humic acids that form precipitates in acidic solutions, fulvic acids that are soluble at any pH, and humic substances (or humic substances) that are insoluble in alkalis. Since humic substances are derived from animals and plants, they exist in every place in the environment, such as soil, seawater, river lakes, drainage, and waste.
  • humic substances are amorphous polymer substances because they are derived from animals and plants, but many have a three-dimensional network structure having many aromatic rings.
  • humic acid is often a polyphenol type carboxylic acid having at least one acidic group such as a hydroxyl group or a carboxyl group, and has a chelating property of metals due to its structure, and is used industrially as a chelating agent. It is done.
  • Patent Document 2 reports a method for improving the virus detection rate by adsorbing impurities such as humic acid on silica gel having specific pores.
  • impurities such as humic acid
  • Silica gel having specific pores since it is nonspecific adsorption depending on the size of the pores, there is a problem that it is insufficient for quantitative measurement.
  • An object of the present invention is to provide a method for producing a purified virus solution capable of specifically recovering and purifying only virus while trapping humic acid as a contaminant, and a virus purification member capable of purifying the virus. Is an issue.
  • Another object of the present invention is to provide a virus detection method using the purified virus solution obtained by the method for producing the purified virus solution.
  • Production of a purified virus solution comprising a step of bringing a sample solution containing virus and humic acid into contact with a substrate having a functional group A having a pKa of a conjugate acid of 8.0 to 9.9 on the surface A method for producing, wherein the functional group A is a single functional group; (2) The production method according to (1), further comprising a step of bringing the base material brought into contact with the sample solution into contact with a recovered solution having a pH of 10 to 12; (3) The production method according to (1) or (2), wherein the substrate is a hollow fiber membrane; (4) The production method according to any one of (1) to (3), wherein the functional group A is an amino group; (5) The production method according to any one of (1) to (4), wherein the pH of the sample solution is 3.0 to 9.0; (6) A step of bringing a sample solution containing virus and humic acid into contact with a substrate having a functional group A having a conjugate acid pKa of 8.0 to 9.9 on the surface, and a recovery
  • the method for producing a purified virus solution of the present invention is capable of specifically recovering and concentrating only virus without concentrating humic acid, which is a contaminant, so that it contains a highly sensitive and quantitative virus containing virus. It is possible to provide a test solution.
  • the base material having the functional group A of the present invention can adsorb virus and humic acid in the sample liquid.
  • the pKa of the conjugate acid of the functional group A is 8.0 to 9.9 and it is a single functional group
  • the recovered solution is brought into contact with the substrate, only the virus remains adsorbed with humic acid. Therefore, it is possible to obtain a purified virus solution from which humic acid has been removed (that is, a virus solution in which only the virus is specifically concentrated with little or no concentration of humic acid). Since the humic acid is selectively removed from the purified virus solution obtained, the subsequent virus detection step can be carried out accurately and quantitatively.
  • a sample solution containing virus and humic acid is obtained by turbidity of a sample containing virus in water.
  • a sample containing virus in water For example, if it is a liquid sample such as environmental water or water for daily use, it may be diluted with sterilized water. If so, a supernatant turbid with sterilized water, a washing solution obtained by washing the solid sample surface with sterilized water, or the like can be used as the sample solution.
  • the virus applicable to this embodiment is not particularly limited, and is a double-stranded DNA virus, a single-stranded DNA virus, a double-stranded RNA virus, a single-stranded RNA virus, a single-stranded RNA reverse transcription virus, or a double-stranded virus.
  • Examples include DNA reverse transcription viruses.
  • Examples of the double-stranded DNA virus include adenovirus and podovirus.
  • Examples of the single-stranded DNA virus include parvovirus.
  • RNA virus examples include rotavirus and reovirus.
  • RNA virus examples include single-stranded RNA viruses acting as mRNA of enterovirus, hepatitis A virus (HAV), hepatitis C virus (HCV) norovirus, feline calicivirus, etc .; measles virus, rabies virus, Marburg Examples thereof include single-stranded RNA viruses that act as complementary strands of mRNA such as viruses, Ebola viruses, and influenza viruses.
  • HAV hepatitis A virus
  • HCV hepatitis C virus
  • feline calicivirus etc .
  • measles virus rabies virus
  • Marburg examples thereof include single-stranded RNA viruses that act as complementary strands of mRNA such as viruses, Ebola viruses, and influenza viruses.
  • RNA reverse transcription virus examples include human immunodeficiency virus and foamy virus.
  • Examples of the double-stranded DNA reverse transcription virus include hepatitis B virus.
  • viruses that can be applied to the present form are adenoviruses, polyomaviruses, hepatitis A viruses (HAV), noroviruses, enteroviruses, polioviruses, hepatitis E viruses that exist in water and can infect humans ( HEV), rotavirus, Aichi virus, parecovirus and reovirus, preferably norovirus, HAV, HEV, enterovirus and rotavirus.
  • the above viruses may be applied alone or in combination of two or more.
  • the humic acid that can be contained in the sample liquid of the present embodiment is not particularly limited, and is known, for example, humic acid that may exist in nature, humic acid that differs from humic acid in nature at least in part in chemical structure. Derivatives and the like. These humic acids may be contained alone in the sample solution, or two or more kinds may be mixed and contained.
  • Humic acid has a complicated structure as described above, but humic acid can be detected and evaluated by measuring absorbance.
  • the pH of the sample solution is preferably 3.0 to 9.0, and more preferably 8.0 to 9.9. It is preferable that the pH of the sample solution is in the above range because the charge balance between the virus and humic acid becomes suitable.
  • the pH may be adjusted by a known and commonly used method, and may be adjusted with various acid bases or with an inorganic salt buffer. When the sample has a desired pH, it can be used as it is without adjustment.
  • the method for producing a purified virus solution comprises a step of bringing a sample solution containing a virus and humic acid into contact with a substrate having a functional group A having a pKa of a conjugate acid of 8.0 to 9.9 on the surface.
  • the functional group A is a single functional group.
  • the substrate having the functional group A having a pKa of the conjugate acid of the present invention of 8.0 to 9.9 on the surface may be any substrate having the functional group A on the surface. Having the functional group A on the surface means that the substrate itself may have a functional group, a compound having the functional group A may be immobilized on the surface of the substrate, or a compound having the functional group A. It can also be obtained by applying to the substrate surface.
  • the material of the base material is not particularly limited, but is preferably a hydrophobic material, specifically, an olefin resin, a styrene resin, an acrylic resin, a sulfone resin, a polyimide resin, a urethane resin, an ester resin.
  • a hydrophobic material specifically, an olefin resin, a styrene resin, an acrylic resin, a sulfone resin, a polyimide resin, a urethane resin, an ester resin.
  • examples thereof include resins, ether resins, and cellulose blended esters.
  • the olefin resin examples include polyethylene (for example, low density polyethylene, linear low density polyethylene, ultra-low density polyethylene, medium density polyethylene, high density polyethylene, etc.), polypropylene, polybutene, polybutylene, polybutadiene, ethylene- Examples include ethylene such as a propylene copolymer (random copolymer) and / or a copolymer of propylene and another ⁇ -olefin (particularly a random copolymer).
  • polyethylene for example, low density polyethylene, linear low density polyethylene, ultra-low density polyethylene, medium density polyethylene, high density polyethylene, etc.
  • polypropylene polybutene
  • polybutylene polybutadiene
  • ethylene- Examples include ethylene such as a propylene copolymer (random copolymer) and / or a copolymer of propylene and another ⁇ -olefin (particularly a random copolymer).
  • styrenic resin examples include high impact polystyrene (HIPS), methyl methacrylate / butadiene / styrene copolymer, styrene / maleic anhydride copolymer, styrene / acrylonitrile copolymer, in addition to a single polymer of styrene. .
  • HIPS high impact polystyrene
  • methyl methacrylate / butadiene / styrene copolymer examples include methyl methacrylate / butadiene / styrene copolymer, styrene / maleic anhydride copolymer, styrene / acrylonitrile copolymer, in addition to a single polymer of styrene. .
  • acrylic resins include (meth) acrylic monomers ((meth) acrylic acid, (meth) acrylic acid C1-18 alkyl ester, hydroxyalkyl (meth) acrylate, glycidyl (meth) acrylate, (meth) Acrylonitrile, etc.) or copolymers, for example, poly (meth) acrylic acid esters such as poly (meth) methyl acrylate, methyl methacrylate- (meth) acrylic acid copolymer, methyl methacrylate-acrylic acid ester- (Meth) acrylic acid copolymer, methyl methacrylate- (meth) acrylic acid ester copolymer, (meth) acrylic acid ester-styrene copolymer (MS resin, etc.) and the like.
  • poly (meth) acrylic acid esters such as poly (meth) methyl acrylate, methyl methacrylate- (meth) acrylic acid copolymer, methyl methacrylate-acrylic acid ester- (Meth
  • sulfone resins examples include polysulfone resins, polyether sulfone resins, and polyaryl sulfone resins.
  • polyimide resin examples include polyetherimide resin, polyamideimide resin, and polybenzimidazole resin.
  • urethane resins examples include diisocyanates (aliphatic diisocyanates such as hexamethylene diisocyanate, alicyclic diisocyanates such as 1,4-cyclohexane diisocyanate and isophorone diisocyanate, tolylene diisocyanate, diphenylmethane-4,4'-diisocyanate.
  • diisocyanates aliphatic diisocyanates such as hexamethylene diisocyanate, alicyclic diisocyanates such as 1,4-cyclohexane diisocyanate and isophorone diisocyanate, tolylene diisocyanate, diphenylmethane-4,4'-diisocyanate.
  • Aromatic diisocyanates such as 1,5-naphthalene diisocyanate or hydrogenated diisocyanates thereof, araliphatic diisocyanates such as xylylene diisocyanate or hydrogenated diisocyanates thereof, and polyols (for example, polyester polyol, polytetra Polyether polyol such as methylene ether glycol, polycarbonate polyol, etc.) and, if necessary, obtained by reaction with a chain extender Such as that polyurethane-based resin and the like.
  • polyols for example, polyester polyol, polytetra Polyether polyol such as methylene ether glycol, polycarbonate polyol, etc.
  • ester resins include aromatic polyesters such as polyethylene terephthalate, polybutylene terephthalate, and polyethylene naphthalate, and polyesters obtained by condensation of diols and dicarboxylic acids used in coating resins and the like.
  • ether resins examples include poly (thio) ether resins, such as polyoxyalkylene resins (stabilized polyoxymethylene glycol or homo- or copolyacetal resins, polyoxypropylene glycol, polyoxytetramethylene glycol, etc. Polyoxy C1-4 alkylene diols), polyphenylene ether resins, polyphenylene ether ketone resins, polysulfide resins (polythioether resins such as polyphenylene sulfide or copolymers thereof), polyether ketone resins (polyether ether ketone resins) Resin).
  • polyoxyalkylene resins stabilized polyoxymethylene glycol or homo- or copolyacetal resins, polyoxypropylene glycol, polyoxytetramethylene glycol, etc. Polyoxy C1-4 alkylene diols
  • polyphenylene ether resins polyphenylene ether ketone resins
  • polysulfide resins polythioether resins such as polyphen
  • the functional group A in the present invention has a pKa of a conjugate acid of 8.0 to 9.9, preferably 8.5 to 9.9, more preferably 9.0 to 9.5, and is on the surface of the substrate.
  • functional group A is a single functional group.
  • the functional group A is a single functional group
  • the functional group A (functional group having a pKa of 8.0 to 9.9) existing on the surface of the substrate is a single one. Means that it consists of At this time, the functional group A may be present directly on the substrate or may be present via another element (for example, an intervening group).
  • the functional group A is a single functional group for polyethyleneimine and the like in which a plurality of types of functional groups A exist.
  • Whether or not the functional group A is single can be determined by measuring the number of inflection points of the titration curve obtained when the substrate containing the functional group A is titrated with an acid.
  • the value of “pKa of the conjugate acid of the functional group A” is a calculated value calculated from the titration value when the base material containing the functional group A is titrated with an acid, according to the Henderson Hasselbalch equation. Shall be adopted. That is, since the “pKa of the conjugate acid of the functional group A” is calculated in a form introduced into the base material, for example, when the functional group A is “—CH 2 NH 2 ”, It may be different from the pKa value of the conjugate acid of the corresponding amine “CH 3 NH 2 ”.
  • the “functional group A” means a minimum group having a pKa of 8.0 to 9.9 and satisfying the pKa.
  • the functional group A includes “—CH 2 NHCH 2 CH 2 OH”, “—NHCH 2 CH 2 OH”, “ Five groups of —CH 2 CH 2 OH ”,“ —CH 2 OH ”and“ —OH ”are conceivable.
  • the pKa of the conjugate acid of “—CH 2 CH 2 OH”, “—CH 2 OH”, and “—OH” is less than 8.0, these groups are not the functional group A.
  • amino groups include primary amino groups such as 1-hydroxy-2-aminoethyl group; secondary amino groups such as 1-hydroxy-2- (N-methylamino) ethyl group; 1-hydroxy-2- (N , N-dimethylamino) ethyl group, N, N-dimethylaminoethoxycarbonyl group, tertiary amino group such as N, N-diethylaminoethoxycarbonyl group and the like.
  • conjugated acid refers to AH + in the following formula.
  • the virus and humic acid are adsorbed on the functional group A. This is probably because the functional group A having a pKa of the conjugate acid of 8.0 to 9.9 is positive, and the virus and humic acid are negative, so that electrical coupling occurs. .
  • a compound having an amino group may be applied to the substrate surface.
  • examples of the compound having an amino group that can be used include amino group-containing silane coupling agents such as N- ⁇ (aminoethyl) ⁇ -aminopropyltrimethoxysilane, and various known resins having an amino group.
  • a compound having a functional group A may be bonded to the surface of the substrate. For example, there is a method of reacting ammonia or diethylamine via an epoxy group included on the surface of the substrate.
  • the substrate that has been brought into contact with the sample solution can obtain a purified virus solution, for example, by bringing a recovered solution having a pH of 10 to 12 into contact therewith. That is, in one embodiment, the method for producing a purified virus solution according to the present invention further comprises a step of bringing the substrate that has been brought into contact with the sample solution into contact with a recovered solution having a pH of 10 to 12.
  • a recovery solution having a pH of 10 to 12 When contacted with a recovery solution having a pH of 10 to 12, the field becomes alkaline, so that the charge of the functional group A becomes neutral, the electrical bond between the virus and the functional group A is released, and the virus is detached. Conceivable.
  • humic acid is not detached from the base material, a purified virus solution can be obtained. This is probably because the hydrophobic structure of humic acid is adsorbed by interacting with the substrate.
  • a recovery solution When the virus is released by contacting with humic acid, even humic acid can be easily eluted, and humic acid can be mixed into the purified virus solution. The reason for this is not clear, but due to the binding of multiple functional groups to the virus, it is difficult for the virus to be detached even if it is brought into contact with the recovery solution, and if it is not contacted with a higher alkaline recovery solution, the virus is detached. This is probably because humic acid is also desorbed and eluted at the same time.
  • the shape of the substrate of the present invention is not particularly limited, and examples thereof include a porous membrane shape, a hollow fiber shape, a flat membrane shape, and a bead shape, but a hollow fiber shape is preferable from the viewpoint of contact efficiency with the sample liquid, A hollow fiber shape made of a porous membrane is particularly preferred.
  • the base material of the present invention When bringing the base material of the present invention into contact with the sample liquid, various methods can be used. In the case of a batch type, the base material and the sample solution can be brought into contact with each other by placing them in a beaker, a test tube, a microtube, a chip, or the like and stirring. When stirring, a stirring blade or a stirring bar may be used, or stirring may be performed with a vibration mixer or the like.
  • the base material may be packed in a column or tube, and the sample solution may be passed through, or the base material may be fixed on a carrier and then passed through.
  • a purified virus liquid can be obtained by recovering the recovered liquid from which the virus has been eluted.
  • the substrate and the recovered solution may be separated.
  • a known and commonly used method may be used as the separation method, and methods such as filtration, decantation, and centrifugation may be used, or only the liquid may be collected using a syringe or the like.
  • the obtained purified virus solution can be subjected to a virus detection step.
  • the virus detection step is a step of detecting and identifying a virus.
  • a virus detection method a morphological identification method from an isolated culture has been used in the past, but a nucleic acid amplification test (NAT) method represented by a polymerase chain reaction method (PCR) or the like is now used.
  • NAT nucleic acid amplification test
  • PCR polymerase chain reaction method
  • the NAT method is advantageous in that it can detect a virus for which a culture method has not been established and can be detected in a short period of time because the number of days of culture is not required.
  • the virus When the virus concentration is low in the purified virus solution obtained in the recovery step, the virus may be concentrated by a known and conventional method. Specifically, positively charged membrane method or negatively charged membrane method, polyethylene glycol precipitation method, cellulose Examples include an agglomeration method and an ultrafiltration method.
  • the purified virus solution obtained in the present invention can be detected with high sensitivity because humic acid that inhibits the NAT method is removed or almost separated.
  • the purified virus solution is subjected to the NAT method, it is preferable to perform a pretreatment for extracting nucleic acids from the virus.
  • Extraction of viral nucleic acid is not particularly limited, and a phenol / chloroform extraction method, an extraction method using a surfactant or a protease in combination, or the like is used.
  • viral nucleic acid purification can also be performed by a liquid phase extraction method, an ethanol precipitation method, a spin column method, or the like.
  • the extracted nucleic acid is examined by the NAT method.
  • the NAT method is a general term for a method of artificially amplifying a minute amount of a gene such as a virus and detecting it with high sensitivity.
  • transcription-mediated amplification ( TMA) method strand displacement reaction (LAMP) method
  • isothermal gene amplification (ICAN) method strand displacement reaction (ICAN) method
  • nucleic acid sequence amplification (NASBA) method ligase chain reaction (LCR) method and the like.
  • nucleic acid amplification is performed using primers specific to the virus, and if amplification is observed, it is found that the virus is present.
  • Reference Example 1 Base Material Preparation Example A high-density polyethylene (HIZEX 2200J, manufactured by Mitsui Petrochemical Co., Ltd.) having a density of 0.968 g / cm 3 and a melt index of 5.5 is used. Using a spinneret for hollow fiber shaping having a diameter of 5 mm and a discharge cross-sectional area of 1.06 cm 2 , spinning was performed at a spinning temperature of 160 ° C. and wound around a bobbin with a spinning draft 1890. The obtained unstretched hollow fiber had an inner diameter of 324 ⁇ m and a film thickness of 48 ⁇ m.
  • HIZEX 2200J manufactured by Mitsui Petrochemical Co., Ltd.
  • the unstretched hollow fiber was heat-treated at 115 ° C. for 24 hours at a constant length. Subsequently, the film was stretched 1.8 times at a deformation rate of 7500% / min at room temperature, and then heated in a heating furnace at 100 ° C. until the total stretching ratio was 3.8 times so that the deformation rate was 220% / min. The stretched yarn was further subjected to thermal shrinkage in a heating furnace at 125 ° C. until the total draw ratio became 2.3 times to obtain a drawn yarn.
  • the resulting porous hollow fiber membrane had an inner diameter of 294 ⁇ m and a film thickness of 40 ⁇ m.
  • Example 1 1) The hollow fiber obtained in Reference Example 1 was added to a 2.5 wt% ethanol / water mixed solution (50 ° C.) in an ethylene-vinyl alcohol copolymer (Nippon Synthetic Chemical Products, ethylene content 44 mol%, weight average molecular weight 90000). The film was immersed for 100 seconds and kept at 80 ° C. for 80 seconds under ethanol saturated steam at 50 ° C., and then the solvent was further dried for 80 seconds for hydrophilic treatment. The hollow fibers thus obtained (about 13 cm, about 150 fibers: 19.5 mg of immobilized resin) were immersed in a test tube containing 20 mL of acetone, 16 mL of epichlorohydrin, and 4 mL of 40% NaOH aqueous solution. . The reaction was carried out at 30 to 40 ° C. for 5 hours while applying ultrasonic waves, and after completion of the reaction, the reaction product was washed with acetone and water and vacuum dried to obtain a hollow fiber having an epoxy group introduced therein.
  • the hollow fiber introduced with the epoxy group prepared above was immersed in a 20% ethanolamine aqueous solution and reacted at 40 ° C. for 15 hours. After completion of the reaction, the reaction product was washed with water to obtain a hollow fiber having an amino group (conjugated acid pKa8.9) introduced therein.
  • the amount of the functional group was measured by titrating the hollow fiber, the amount of the functional group introduced was 0.040 ⁇ mol / cm 2 (inner surface area).
  • humic acid was quantified by measuring absorbance at 260 nm.
  • phage RNA was extracted from each fraction using QIAGEN MinElute Virus Spin kit (Qiagen Co., Ltd.), and the amount of RNA was quantified by a real-time RT-PCR method. Primer and probe sequences used for the measurement were prepared according to the literature (Journal of Virological Methods 149 (2008), p123-128), and the measurement was performed using a StepOnePlus real-time PCR system (Life Technologies Japan, Inc.).
  • the amount of phage and humic acid contained in the non-adsorbed fraction with respect to the mixed solution of phage and humic acid before the substrate contact treatment (hereinafter referred to as untreated) Were 8% and 4%, respectively. Therefore, it was found that the adsorption rate to the base material in the module was 92% for phage and 96% for humic acid, respectively.
  • the recovery rate of the phage and humic acid in the eluted fraction with respect to the untreated phage / humic acid mixed solution was 64 respectively. %, 46%.
  • the phage recovery rate (humic acid recovery rate / phage recovery rate) relative to the humic acid recovery rate obtained above was calculated as a recovery rate ratio, and was 0.72.
  • the recovery ratio means that the smaller the value, the more humic acid could be separated.
  • Example 2 A hollow fiber introduced with an epoxy group produced in the same manner as in Example 1 was immersed in 28% aqueous ammonia and reacted at 20 ° C. for 15 hours. After completion of the reaction, the reaction product was washed with water to obtain a hollow fiber having an amino group (conjugated acid pKa 9.5) introduced therein. When the amount of the functional group was measured by titrating the hollow fiber, the amount of the functional group introduced was 0.022 ⁇ mol / cm 2 (inner surface area).
  • a module for a small amount of sample was prepared in the same manner as in Example 1, and 1 mL of phage / humic acid mixed solution was adsorbed, and recovery evaluation was performed with 1 mL of 1 mmol / L NaOH (pH: 11).
  • the adsorption rates of phage and humic acid in the non-adsorbed fraction with respect to the untreated phage / humic acid mixed solution were 95% and 100%, respectively.
  • the recovery rates of phage and humic acid in the eluted fraction were 69% and 32%, respectively. Therefore, the recovery rate ratio is 0.46.
  • Example 3 A hollow fiber introduced with an epoxy group prepared in the same manner as in Example 1 was immersed in a 20% diethylamine aqueous solution and reacted at 40 ° C. for 15 hours. After completion of the reaction, the reaction product was washed with water to obtain a hollow fiber having an amino group (conjugated acid pKa 9.9) introduced therein. When the amount of the functional group was measured by titrating the hollow fiber, the amount of the functional group introduced was 0.025 ⁇ mol / cm 2 (inner surface area).
  • a module for a small amount of sample was prepared in the same manner as in Example 1, and the collection and evaluation by adsorption of 1 mL of the phage / humic acid mixed solution and 1 mL of 10 mmol / L NaOH (pH: 12) were performed.
  • the adsorption rates of phage and humic acid in the non-adsorbed fraction with respect to the untreated phage / humic acid mixture were 96% and 100%, respectively.
  • the recovery rates of phage and humic acid in the eluted fraction were 63% and 46%, respectively. Therefore, the recovery rate ratio is 0.73.
  • Example 1 A hollow fiber introduced with an epoxy group prepared in the same manner as in Example 1 was immersed in a 5% polyallylamine aqueous solution and reacted at 40 ° C. for 15 hours. After completion of the reaction, the reaction product was washed with water to obtain a hollow fiber having an amino group (conjugated acid pKa10.6) introduced therein. When the amount of the functional group was measured by titrating the hollow fiber, the amount of the functional group introduced was 0.015 ⁇ mol / cm 2 (inner surface area).
  • a module for a small amount of sample was prepared in the same manner as in Example 1, and 1 mL of phage / humic acid mixed solution was adsorbed, and recovery evaluation was performed with 1 mL of 1 mmol / L NaOH (pH: 11). Since the amount of phage and humic acid in the non-adsorbed fraction with respect to the untreated phage / humic acid mixture was 0%, the adsorption rates of phage and humic acid were calculated to be 100% and 100%, respectively. In addition, after passing through the phage / humic acid mixture, elution was performed with the recovery solution. With respect to the untreated phage / humic acid mixture, the phage and humic acid in the eluted fraction were hardly recovered. The recovery rate was 0%. For this reason, the recovery ratio could not be calculated.
  • Example 2 A hollow fiber introduced with an epoxy group prepared in the same manner as in Example 1 was immersed in a 5% poly- ⁇ -lysine aqueous solution and reacted at 40 ° C. for 15 hours. After completion of the reaction, the reaction product was washed with water to obtain a hollow fiber into which an amino group (conjugated acid pKa 7.6) was introduced. When the amount of the functional group was measured by titrating the hollow fiber, the amount of the functional group introduced was 0.042 ⁇ mol / cm 2 (inner surface area).
  • a module for a small amount of sample was prepared in the same manner as in Example 1, and 1 mL of phage / humic acid mixed solution was adsorbed, and recovery evaluation was performed with 1 mL of 1 mmol / L NaOH (pH: 11).
  • the adsorption rates of phage and humic acid in the non-adsorbed fraction with respect to the untreated phage / humic acid mixture were 100% and 87%, respectively.
  • the recovery rates of phage and humic acid in the eluted fraction were 71% and 65%, respectively. Therefore, the recovery ratio is 0.92.
  • Example 3 A hollow fiber introduced with an epoxy group produced in the same manner as in Example 1 was immersed in an aqueous solution of 5% polyethyleneimine (Wako Pure Chemical Industries, Ltd., average molecular weight 600) and reacted at 40 ° C. for 15 hours. After completion of the reaction, the reaction product was washed with water to obtain a hollow fiber into which an amino group (conjugated acid pKa 8.8; average value) was introduced. When the amount of the functional group was measured by titrating the hollow fiber, the amount of the functional group introduced was 0.021 ⁇ mol / cm 2 (inner surface area).
  • a module for a small amount of sample was prepared in the same manner as in Example 1, and the collection and evaluation with 1 mL of phage / humic acid mixed solution and 1 mL of 10 mmol / L NaOH (pH: 11) were performed.
  • the adsorption rates of phage and humic acid in the non-adsorbed fraction with respect to the untreated phage / humic acid mixture were 100% and 97%, respectively.
  • the recovery rates of phage and humic acid in the eluted fraction were 70% and 68%, respectively. Therefore, the recovery ratio is 0.97.
  • the recovery rate ratio is particularly small, and phages can be selectively separated.
  • the resulting solution is a purified virus in which only the virus is suitably concentrated. It can be said that it is a liquid.
  • the method for producing a purified virus solution of the present invention can be suitably used for a virus inspection method.

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Abstract

La présente invention aborde le problème consistant à fournir : un procédé de production d'une solution de virus purifié, grâce à quoi il devient possible de collecter et de purifier seulement un virus de manière spécifique, tout en piégeant l'acide humique qui est un contaminant ; et un élément de purification de virus qui permet la mise en œuvre de la purification de virus susmentionnée. L'invention porte sur un procédé de production d'une solution de virus purifié, comprenant une étape consistant à mettre en contact une solution échantillon contenant un virus et de l'acide humique avec un élément de base qui comprend, sur sa surface, un groupe fonctionnel A dont un acide conjugué a une valeur de pKa de 8,0 à 9,9, ledit procédé étant caractérisé en ce que le groupe fonctionnel A comprend un groupe fonctionnel unique.
PCT/JP2015/070663 2015-07-21 2015-07-21 Procédé de production de solution de virus purifié, procédé de détection de virus, et élément de purification de virus WO2017013731A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010193720A (ja) * 2009-02-23 2010-09-09 Asahi Kasei Chemicals Corp アニオン交換基が固定された多孔膜を用いたウイルスの分離方法
WO2015111606A1 (fr) * 2014-01-23 2015-07-30 Dic株式会社 Procédé pour produire une solution de virus et procédé pour détecter un virus
JP2015167536A (ja) * 2014-03-10 2015-09-28 Dic株式会社 精製ウイルス液の製造方法、ウイルス検出方法及びウイルス精製部材

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010193720A (ja) * 2009-02-23 2010-09-09 Asahi Kasei Chemicals Corp アニオン交換基が固定された多孔膜を用いたウイルスの分離方法
WO2015111606A1 (fr) * 2014-01-23 2015-07-30 Dic株式会社 Procédé pour produire une solution de virus et procédé pour détecter un virus
JP2015167536A (ja) * 2014-03-10 2015-09-28 Dic株式会社 精製ウイルス液の製造方法、ウイルス検出方法及びウイルス精製部材

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AKIHIKO HATA ET AL.: "Concentration of Organic Matter During a Virus Concentration and Inhibition on Virus Detection", JAPAN SOCIETY ON WATER ENVIRONMENT SYMPOSIUM KOENSHU, vol. 15, 2012, pages 275 - 280 *
HATA A. ET AL.: "Organic substances interfere with reverse transcription-quantitative PCR- based virus detection in water samples.", APPL. ENVIRON. MICROBIOL., vol. 81, no. 5, March 2015 (2015-03-01), pages 1585 - 1593, XP055348033 *
LEWIS G.D. ET AL.: "Influence of environmental factors on virus detection by RT-PCR and cell culture.", J. APPL. MICROBIOL., vol. 88, no. 4, 2000, pages 633 - 640, XP055348027 *
STRAUB T.M. ET AL.: "Removal of PCR inhibiting substances in sewage sludge amended soil.", WAT. SCI. TECH., vol. 31, no. 5-6, 1995, pages 311 - 315, XP055348026 *
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