WO2015111606A1 - Procédé pour produire une solution de virus et procédé pour détecter un virus - Google Patents

Procédé pour produire une solution de virus et procédé pour détecter un virus Download PDF

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Publication number
WO2015111606A1
WO2015111606A1 PCT/JP2015/051489 JP2015051489W WO2015111606A1 WO 2015111606 A1 WO2015111606 A1 WO 2015111606A1 JP 2015051489 W JP2015051489 W JP 2015051489W WO 2015111606 A1 WO2015111606 A1 WO 2015111606A1
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virus
solution
beads
humic acid
hydrophobic
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PCT/JP2015/051489
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English (en)
Japanese (ja)
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大英 中熊
久由 新井
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Dic株式会社
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Priority to JP2015559086A priority Critical patent/JPWO2015111606A1/ja
Publication of WO2015111606A1 publication Critical patent/WO2015111606A1/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
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18151—Methods of production or purification of viral material

Definitions

  • the present invention relates to a virus solution production method and a virus detection method.
  • 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 is concentrated and recovered by the method as described above, and the obtained virus solution is detected and identified by various methods.
  • a nucleic acid amplification test (NAT) method typified by the polymerase chain reaction (PCR) or the like is often performed.
  • the NAT method is a method of artificially amplifying nucleic acid contained in a virus and detecting it with high sensitivity. When detecting with a virus-specific primer or after amplification of a gene, identification is performed by hybridization or the like. .
  • 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 high molecular organic acids synthesized chemically and biologically from the decomposition products of plant residues, microorganisms, and plankton remains. It is a mixture of 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.
  • the sample solution containing the virus is Step 1 in which the inorganic salt concentration is adjusted to 0.5 to 200 mM and the pH 4.0 to 9.0, and the hydrophobic beads. Then, the step 2 of bringing the test solution into contact with the hydrophobic beads having a critical surface tension of 30 to 45 mN on the bead surface, and the step 3 of obtaining the purified virus solution by separating the hydrophobic beads and the test solution. It has been found that the above-mentioned problems can be solved by providing a method for producing a purified virus solution characterized by having the above.
  • the present inventors have found a method for producing a purified virus solution, in which the sample solution further contains humic acid.
  • the present inventors have found a method for producing the purified virus solution, wherein the hydrophobic beads have a surface containing high-density polyethylene, polystyrene, polymethyl methacrylate, or 2-phenoxyethyl acrylate.
  • the present inventors have found a method for producing a purified virus solution in which the hydrophobic beads have a BET specific surface area of 100 to 1200 m 2 / g or more.
  • the present inventors have found a method for producing a purified virus solution in which the hydrophobic beads have a cumulative 10% diameter of volume distribution of 1 ⁇ m or more and a cumulative 90% particle size distribution of 1000 ⁇ m or less.
  • the present inventors have found a virus detection method characterized by subjecting the purified virus solution obtained by any of the virus solution production methods described above to a virus detection step.
  • the method for producing a virus solution of the present invention is capable of specifically recovering and concentrating only the virus without concentrating humic acid, which is a contaminant. Can be provided.
  • the present invention includes a step 1 of adjusting a virus-containing sample solution to a test solution having an inorganic salt concentration of 0.5 to 200 mM and a pH of 4.0 to 9.0; Characterized in that it comprises a step 2 of bringing the test solution into contact with hydrophobic beads having a surface tension of 30 to 45 mN and a step 3 of obtaining the purified virus solution by separating the hydrophobic beads and the test solution.
  • the present invention provides a method for producing a purified virus solution, and a virus detection method comprising subjecting the purified virus solution to a virus detection step.
  • the hydrophobic beads of the present invention have a property of selectively adsorbing relatively strong hydrophobic humic acids over viruses.
  • the virus-containing sample liquid in the present invention is an aqueous medium liquid containing a virus.
  • the sample solution containing the virus may be obtained by turbidizing the sample that seems to contain the virus in the water.
  • the sample containing the virus includes environmental water such as river water, lake water, seawater, and rainwater, well water, tap water, and bottles. Living water such as drinking water such as drinking water, industrial water such as sewage, drainage, pool water, agricultural water, industrial water and refrigerant water; food, soil, animals and plants, body fluids such as blood, etc. should be used as samples Can do.
  • the virus-containing sample solution is obtained by turbidity of the virus-containing sample in water.
  • it is a liquid sample such as environmental water or domestic water, it may be diluted with sterilized water, and if it is a solid sample A supernatant turbid with sterilized water, a washing solution obtained by washing the solid sample surface with sterilized water, or the like can also be used as the sample solution.
  • the sample solution containing virus may be appropriately diluted depending on the amount of sample and the amount of virus. However, when it is used in step 2, the inorganic salt concentration is 0.5 to 200 mM and the pH is 4.0 to 9. It is necessary to adjust to 0 to obtain a test solution.
  • Inorganic salts include phosphates, citrates, hydrochlorides, acetates, sulfates, nitrates, carbonates, formates, and salts include potassium, sodium, lithium, calcium, magnesium, and the like. .
  • PH may be adjusted by a known and commonly used method, may be adjusted with various acid bases, and is preferably adjusted with the above-mentioned inorganic salt buffer.
  • sample solution containing virus already has an inorganic salt concentration of 0.5 to 200 mM and a pH of 4.0 to 9.0, it can be used as it is without adjustment.
  • Step 2 the test solution obtained in Step 1 is contacted with hydrophobic beads having a bead surface critical surface tension of 30 to 45 mN.
  • the hydrophobic bead of the present invention has a property of selectively adsorbing a relatively strong hydrophobic humic acid over a virus.
  • the hydrophobic beads of the present invention are hydrophobic beads having a critical surface tension of 30 to 45 mN on the bead surface, and the material is not particularly limited, but the surface is preferably high-density polyethylene, polystyrene, polymethyl methacrylate, It preferably contains 2-phenoxyethyl acrylate. Hydrophobic beads may be composed of a single material or may be obtained by combining multiple materials, and the surface of the beads is modified so that the critical surface tension is 30 to 45 mN. May be good.
  • the hydrophobic beads of the present invention may use several percent of a cross-linking agent to increase strength.
  • cross-linking agents include divinyl benzene, divinyl benzene / ethyl vinyl benzene mixture, ethylene glycol dimethacrylate, trimethylol.
  • a radical polymerizable polyfunctional monomer such as propane trimethacrylate can be used.
  • Polymethyl methacrylate and poly (2-phenoxyethyl acrylate) can be cross-linked by these cross-linking agents, and the obtained cross-linked polymethyl methacrylate and cross-linked poly (2-phenoxyethyl acrylate) are preferable because of their excellent strength.
  • the value of the critical surface tension can be measured according to JIS K-6768: 1999, using, for example, a wetting index standard solution manufactured by Wako Pure Chemical Industries, Ltd. Specifically, a plurality of standard solutions having different surface tensions are prepared step by step, and one of the standard solutions is dropped onto the smooth sheet surface of the target material, and the droplets are applied to the film surface. It can be determined by setting the surface tension value of the upper limit standard solution that can be wetted for 2 seconds or more as the critical surface tension without causing the liquid film of the spread and dropped standard solution to break.
  • the size of the hydrophobic beads of the present invention is preferably such that the 10% diameter cumulative volume distribution is 1 ⁇ m or more and the 90% cumulative size distribution is 1000 ⁇ m or less. This is because when the cumulative 10% diameter of the bead volume distribution is 1 ⁇ m or more and the cumulative size is 90% and the particle size distribution is 1000 ⁇ m or less, the beads can be contacted efficiently. More preferably, the cumulative 10% diameter of the volume distribution is 30 ⁇ m or more and the cumulative 90% particle size distribution is 500 ⁇ m or less.
  • the volume distribution and particle size can be measured by microscopy.
  • the hydrophobic beads preferably have a BET surface area of 100 to 1200 m 2 / g. This is preferably 100 m 2 / g or more because the contact efficiency between the test solution and the beads is improved, and if it is 1200 m 2 / g or less, the pore diameter is relatively large, so humic acid is captured in the pores on the bead surface. It is because it is easy to do.
  • the BET specific surface area is a value measured by a nitrogen substitution method using a BET specific surface area measuring instrument (manufactured by Yuasa Ionics Co., Ltd .: Autosorb 1-C).
  • the hydrophobic beads and the test solution can be brought into contact with each other by placing them in a beaker, a test tube, a microtube or a chip and stirring them.
  • a stirring blade or a stirring bar may be used, or stirring may be performed with a vibration mixer or the like.
  • hydrophobic beads may be packed in a column or tube and then the test solution may be passed, or the hydrophobic beads may be fixed on a carrier and then passed.
  • Step 3 of the present invention a purified virus solution from which humic acid has been removed can be obtained by separating the hydrophobic beads and the test solution brought into contact with each other in Step 2.
  • 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.
  • Step 4 The purified virus liquid obtained in step 3 is preferably subjected to a virus detection step in step 4.
  • 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 may be concentrated by a known and commonly used method. Specifically, the positively charged membrane method, the negatively charged membrane method, the polyethylene glycol precipitation method, There are external filtration methods.
  • the purified virus solution obtained in the present invention can be detected with high sensitivity since the humic acid that inhibits the NAT method is removed.
  • 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.
  • Humic acid Nacalai Tesque Co., Ltd.
  • Q ⁇ phage NBRC20012
  • the prepared humic acid aqueous solution were added to purified water to a final concentration of 5 ⁇ 10 10 PFU / mL and 500 ppm, respectively, to prepare phage / humic acid solution 1.
  • Example 1 In a polypropylene microtube, 100 ⁇ l of Superite DAX-8 (manufactured by Supelco), which has been washed as hydrophobic beads, is taken as a volume after spin-down, and 0.2 mL of the pH 1 solution of Preparation 1 and pH 7. After adding 0.8 ml of 0 sodium phosphate buffer (buffer concentration 100 mM), the mixture was mixed by inversion for 10 minutes. The supernatant was collected by spinning down the beads to obtain a purified virus solution.
  • Superite DAX-8 manufactured by Supelco
  • the amount of humic acid was measured by measuring the 260 nm light absorbency of the obtained purified virus liquid.
  • the humic acid recovery rate was calculated by the following formula.
  • Phage RNA was extracted from the obtained purified virus solution according to the QIAamp Virtual RNA Mini protocol (Qiagen Co., Ltd.), diluted 100 times, and then the RNA amount 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 Q ⁇ recovery rate (phage recovery rate) was calculated by the following formula.
  • Example 2 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 1 except that the buffer used was a sodium citrate buffer having a pH of 5.0 (buffer concentration 100 mM).
  • Example 3 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 2 except that the beads used were changed from Superlite DAX-8 to Sepa beads SP2MGS (manufactured by Mitsubishi Chemical Corporation).
  • Example 4 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 3 except that the addition amount of beads was changed from 100 ⁇ l to 30 ⁇ l.
  • Example 5 The recovery rates of phage and humic acid were evaluated in the same manner as in Example 1 except that the inversion mixing time was changed from 10 minutes to 60 minutes.
  • Example 6 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 3 except that the buffer used was a sodium citrate buffer having a pH of 5.0 (buffer concentration 50 mM).
  • Example 7 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 6 except that the beads used were changed from Superlite DAX-8 to Diaion HP2MG (Mitsubishi Chemical Corporation) and the addition amount was changed to 300 ⁇ l.
  • Example 8 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 6 except that the beads used were changed from Superlite DAX-8 to Diaion HP20 (manufactured by Mitsubishi Chemical Corporation) and the addition amount was 900 ⁇ l.
  • a phage / humic acid solution was prepared by adding 0.2 ml of the phage / humic acid solution 1 of Preparation Example 1 and 0.8 ml of pH 7.0 sodium phosphate buffer (buffer concentration 100 mM) to a polypropylene microtube. Pass the above-mentioned phage-humic acid solution through a column made by packing 300 ⁇ l of Superlite DAX-8 in a ⁇ 2 ⁇ 4 silicon tube, and further 4 ml of pH 7.0 sodium phosphate buffer (buffer concentration 0.1 M). did. The recovered liquid was evaluated in the same manner as in Example 1 for the recovery rate of phage and humic acid.
  • Example 10 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 9 except that the amount of beads used in the column was changed to 1200 ⁇ l and the buffer used was changed to sodium phosphate buffer pH 7.0 (buffer concentration 10 mM).
  • Example 11 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 9 except that the amount of beads used in the column was changed to 1800 ⁇ l and the buffer used was changed to sodium phosphate buffer pH 7.0 (buffer concentration 1.0 mM). .
  • Example 12 The recovery rate of phage and humic acid was evaluated in the same manner as in Example 7 except that the buffer used was changed to a pH 4.0 sodium citrate buffer (buffer concentration 50 mM) and the inversion mixing time was changed from 10 minutes to 60 minutes. .
  • ⁇ Comparative example 1> Implemented except that the beads used were changed to silica gel beads (silica gel 60N (63-210 um) manufactured by Kanto Chemical Co., Inc.), the amount of beads was changed to 1000 ⁇ l, and the buffer used was changed to sodium phosphate buffer pH 7.0 (buffer concentration 10 mM). In the same manner as in Example 1, the recovery rates of phage and humic acid were evaluated.
  • ⁇ Comparative example 2> The recovery rates of phage and humic acid were evaluated in the same manner as in Example 7 except that the beads used were changed from Superlite DAX-8 to Diaion SA-10A (Mitsubishi Chemical Corporation).
  • ⁇ Comparative example 4> The recovery rate of phage and humic acid was evaluated in the same manner as in Example 1 except that the buffer used was changed to a pH 2.0 hydrochloric acid-potassium chloride buffer (buffer concentration 200 mM) and beads were not used.
  • PMMA Crosslinked polymethyl methacrylate (Crosslinking agent: Ethylene glycol dimethacrylate)
  • PS Cross-linked polystyrene (Crosslinking agent: Divinylbenzene)
  • Anion exchange resin Compound in which trimethylammonium group is introduced into crosslinked polystyrene
  • the particle size of the beads in the table is as follows.
  • the virus purification method and virus detection method of the present invention can be suitably used for virus inspection methods.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

　Le but de la présente invention concerne un procédé pour produire une solution purifiée de virus pouvant récupérer spécifiquement un virus seul et le purifier sans concentrer l'acide humique, qui est un contaminant ; et un procédé pour détecter un virus à l'aide d'une solution de virus obtenue par ce procédé de purification de virus. Le problème ci-dessus est résolu par un procédé de production d'une solution purifiée de virus, caractérisé en ce qu'il présente une étape (1) d'ajustement d'une solution d'échantillon contenant un virus à une solution de test présentant une concentration en sel inorganique de 0,5-200 mM et un pH de 4,0-9,0, une étape (2) de mise en contact de la solution de test avec des billes hydrophobes, les billes hydrophobes présentant une tension superficielle critique à leur surface de 30-45 mN et une étape (3) de séparation des billes hydrophobes et de la solution de test et d'obtention d'une solution purifiée de virus.
PCT/JP2015/051489 2014-01-23 2015-01-21 Procédé pour produire une solution de virus et procédé pour détecter un virus WO2015111606A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017013731A1 (fr) * 2015-07-21 2017-01-26 Dic株式会社 Procédé de production de solution de virus purifié, procédé de détection de virus, et élément de purification de virus
WO2017013732A1 (fr) * 2015-07-21 2017-01-26 Dic株式会社 Procédé de production de solution de virus purifié, et méthode de détection de virus
CN107436332A (zh) * 2017-08-01 2017-12-05 清华大学 一种分离并测定腐殖酸不同极性及分子量组分的方法
WO2021002257A1 (fr) 2019-07-04 2021-01-07 株式会社カネカ Procédé de purification d'un virus ou d'une particule de type virus
WO2022044727A1 (fr) 2020-08-28 2022-03-03 株式会社カネカ Procédé de purification d'une substance utile

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JP2011155919A (ja) * 2010-02-02 2011-08-18 Fisheries Research Agency ノロウイルス検出用材料および該材料を用いるノロウイルスの検出方法

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017013731A1 (fr) * 2015-07-21 2017-01-26 Dic株式会社 Procédé de production de solution de virus purifié, procédé de détection de virus, et élément de purification de virus
WO2017013732A1 (fr) * 2015-07-21 2017-01-26 Dic株式会社 Procédé de production de solution de virus purifié, et méthode de détection de virus
CN107436332A (zh) * 2017-08-01 2017-12-05 清华大学 一种分离并测定腐殖酸不同极性及分子量组分的方法
CN107436332B (zh) * 2017-08-01 2020-05-22 清华大学 一种分离并测定腐殖酸不同极性及分子量组分的方法
WO2021002257A1 (fr) 2019-07-04 2021-01-07 株式会社カネカ Procédé de purification d'un virus ou d'une particule de type virus
WO2022044727A1 (fr) 2020-08-28 2022-03-03 株式会社カネカ Procédé de purification d'une substance utile
KR20230056661A (ko) 2020-08-28 2023-04-27 가부시키가이샤 가네카 유용 물질의 정제 방법

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