WO2015111606A1 - Method for producing virus solution and method for detecting virus - Google Patents

Method for producing virus solution and method for detecting 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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French (fr)
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/en
Publication of WO2015111606A1 publication Critical patent/WO2015111606A1/en

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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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Abstract

　The purpose of the present invention is: to provide a method for producing purified virus solution capable of specifically recovering a virus alone and purifying same without concentrating humic acid, which is a contaminant; and to provide a method for detecting a virus by using a virus solution obtained by this virus purification method. The above problem is solved by providing a method for producing purified virus solution characterized in having a step (1) for adjusting a sample solution containing a virus to a test solution having an inorganic salt concentration of 0.5-200 mM and a pH of 4.0-9.0, a step (2) for bringing the test solution into contact with hydrophobic beads, wherein the hydrophobic beads have a critical surface tension at the bead surface of 30-45 mN, and a step (3) for separating the hydrophobic beads and test solution and obtaining a purified virus solution.

Description

ウイルス液の製造方法及びウイルス検出方法Virus liquid production method and virus detection method
　本発明は、ウイルス液の製造方法、及びウイルス検出方法に関する。 The present invention relates to a virus solution production method and a virus detection method.
　安全志向が高まる近年、河川水や海水といった環境水、水道水や井戸水等の生活用水、土壌や食品等、我々を取り巻く環境に対する安全性の担保が非常に重要視されている。特に環境水や生活水が各種ウイルスによって汚染されると、近隣住民へと爆発的に感染し、甚大な健康被害を生じる恐れがあり、とくに開発途上地域における生活用水のウイルス汚染は大きな問題となっている。 In recent years, safety-consciousness has been increasing, and safety of the environment surrounding us, such as environmental water such as river water and seawater, domestic water such as tap water and well water, soil and food, etc. has been regarded as very important. Especially when environmental water and domestic water are polluted by various viruses, it may explodely infect neighboring residents and cause serious health damage. Especially, virus contamination of domestic water in developing areas becomes a big problem. ing.
　これらを鑑み、環境中のウイルス汚染を恒常的に監視することは、人々の安全安心を維持していく上で非常に重要であり、そのためには簡便で正確にウイルス量を評価する方法を確立することが急務である。 In view of these, it is very important to constantly monitor the virus contamination in the environment in order to maintain people's safety and security. To that end, a simple and accurate method for evaluating the viral load has been established. There is an urgent need to do.
　しかし、様々な環境、特に環境水中のウイルス量はごく少量であるため、検出することが非常に難しいということが課題となっている。そこで、ウイルスを濃縮回収したうえで、ウイルスを検出する方法が試みられてきた。 However, the problem is that it is very difficult to detect because the amount of virus in various environments, especially environmental water, is very small. Therefore, methods for detecting viruses after concentrating and recovering viruses have been attempted.
　例えば、ウイルス検出に用いる試料を、超遠心やポリエチレングリコール法などによって濃縮する方法などが知られている。 For example, a method of concentrating a sample used for virus detection by ultracentrifugation or polyethylene glycol method is known.
　また、特許文献１においては、表面処理された中空糸にウイルスを含む液を通じてウイルスを捕捉し、ウイルスの濃度を評価する方法が報告されており、その際には、必要に応じて限外ろ過膜で２次濃縮を行う場合もある。 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.
　上記のような方法でウイルスを濃縮回収し、得られたウイルス液を、各種方法で検出、同定を行う。一般的によく行われるのが、ポリメラーゼ連鎖反応法（ＰＣＲ）等に代表される核酸増幅検査（ＮＡＴ）法である。ＮＡＴ法は、ウイルスに含まれる核酸を人工的に増幅して高感度に検出する方法であり、ウイルスに特異なプライマーによって検出を行う場合や、遺伝子を増幅させた後にハイブリダイゼーション等で同定を行う。 The virus is concentrated and recovered by the method as described above, and the obtained virus solution is detected and identified by various methods. In general, 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. .
　しかし、上記のようなウイルス濃縮方法では、環境中に存在するＮＡＴ法を阻害する物質であるフミン酸も同時に回収してしまうという問題があった。 However, 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.
　フミン酸とは、環境中に存在する腐植物質の一種であり、植物残渣や微生物、プランクトンの遺骸が微生物による分解を受け、その分解生成物から化学的、生物的に合成された高分子有機酸の混合物である。腐植物質はフミン質とも呼ばれ、アルカリ溶液に可溶だが、酸性溶液では沈殿を形成するフミン酸、どのｐＨでも可溶なフルボ酸、及びアルカリに不溶なヒューミン（またはフムス質）とが存在する。フミン質は、動植物由来物質であることから、土壌・海水・河川湖沼水や排水・廃棄物等、環境中のあらゆる場所に存在する。フミン質は、動植物由来であることから不定形の高分子物質であるが、芳香環族を多数有する三次元網目状構造をもつものが多い。その中でもフミン酸は、水酸基やカルボキシル基と言った酸性基を一つ以上有するポリフェノール型カルボン酸であることが多く、その構造から金属類のキレート性を有し、工業的にはキレート剤として用いられる。 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. The 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. Of these, 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.
　上記問題を解決する為に、特許文献２においては、特定の細孔を持つシリカゲルにフミン酸等の夾雑物を吸着させることによる、ウイルス検出率の向上方法が報告されている。しかし、細孔の大きさに依存した非特異的な吸着であるため、定量的測定には不十分であるといった問題があった。 In order to solve the above problems, 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. However, since it is nonspecific adsorption depending on the size of the pores, there is a problem that it is insufficient for quantitative measurement.
国際公開第２０１２／１４４５５４号International Publication No. 2012/144554 特開２０１１－１５５９１９号公報JP2011-155919A
　本発明の課題は、夾雑物であるフミン酸を濃縮することなく、ウイルスのみを特異的に回収し精製することができるウイルス液の製造方法を提供することを課題とする。また、該ウイルスの精製方法によって得られるウイルス液を用いたウイルス検出方法を提供することを課題とする。 An object of the present invention is to provide a method for producing a virus solution that can specifically recover and purify only virus without concentrating humic acid, which is a contaminant. Another object of the present invention is to provide a virus detection method using a virus solution obtained by the virus purification method.
　本発明者らは鋭意検討の結果、ウイルスを含有するサンプル液を、無機塩濃度が０．５～２００ｍＭ及びｐＨ４．０～９．０の試験液に調整する工程１と、疎水性ビーズであって、ビーズ表面の臨界表面張力が３０～４５ｍＮである疎水性ビーズに、上記試験液を接触させる工程２と、上記疎水性ビーズと試験液とを分離して精製ウイルス液を得る工程３とを有することを特徴とする、精製ウイルス液の製造方法を提供することで、上記課題を解決できることを見出した。 As a result of intensive studies, the present inventors have determined that 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.
　また、上記サンプル液が、さらにフミン酸を含有するものである、精製ウイルス液の製造方法を見出した。 Further, the present inventors have found a method for producing a purified virus solution, in which the sample solution further contains humic acid.
　また、上記疎水性ビーズが、表面が高密度ポリエチレン、ポリスチレン、ポリメチルメタクリレート、２－フェノキシエチルアクリレートを含有するものである、上記精製ウイルス液の製造方法を見出した。 Further, 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.
　また、上記疎水性ビーズが、ＢＥＴ比表面積が１００～１２００ｍ２／ｇ以上である、精製ウイルス液の製造方法を見出した。 Further, 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.
　また、記疎水性ビーズが、体積分布の累積１０％径が１μｍ以上かつ、累積９０％粒度分布が１０００μｍ以下である、精製ウイルス液の製造方法を見出した。 Further, 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 μm or more and a cumulative 90% particle size distribution of 1000 μm or less.
　また、上記何れかに記載のウイルス液の製造方法で得られた精製ウイルス液を、ウイルス検出工程にかけることを特徴とするウイルス検出方法を見出した。 In addition, 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.
　本発明のウイルス液の製造方法は、夾雑物であるフミン酸を濃縮することなく、ウイルスのみを特異的に回収し濃縮することができることから、ウイルス検出方法に対し感度よく定量的な精製ウイルス液を提供することが可能である。 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.
　本発明は、ウイルスを含有するサンプル液を、無機塩濃度が０．５～２００ｍＭ及びｐＨ４．０～９．０の試験液に調整する工程１と、疎水性ビーズであって、ビーズ表面の臨界表面張力が３０～４５ｍＮである疎水性ビーズに、上記試験液を接触させる工程２と、上記疎水性ビーズと試験液とを分離して精製ウイルス液を得る工程３とを有することを特徴とする、精製ウイルス液の製造方法、及び上記精製ウイルス液をウイルス検出工程に供することを特徴とするウイルス検出方法を提供するものである。 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.
　本発明の疎水性ビーズは、疎水性の比較的強いフミン酸をウイルスよりも選択的に吸着する性質を有する。試験液のｐＨを弱酸性から中性領域に設定することで、ウイルス粒子及び核酸の破壊を防ぎつつ、無機塩濃度を調整することでフミン酸の電離を解消し、ビーズに吸着しやすくしたものである。そのため、得られた精製ウイルス液は、フミン酸が選択的に除去されているため、その後のウイルス検出工程を精度よく定量的に実施することができる。 The hydrophobic beads of the present invention have a property of selectively adsorbing relatively strong hydrophobic humic acids over viruses. By setting the pH of the test solution from weakly acidic to neutral, preventing the destruction of virus particles and nucleic acids, and adjusting the inorganic salt concentration to eliminate humic acid ionization and make it easier to adsorb to beads It is. Therefore, 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.
〔工程１〕
　本発明におけるウイルスを含有するサンプル液とは、ウイルスが含まれる水媒体の液体である。ウイルスを含有するサンプル液は、ウイルスを含有すると思われるサンプルを水に混濁すればよく、ウイルスを含有するサンプルとは、河川水・湖沼水・海水・雨水といった環境水、井戸水・水道水・ボトルドウォーターといった飲料水や下水・排水・プール水・農業用水・工業用水・冷媒水といった産業用水のような生活用水；食品、土壌、動植物、血液等の体液など、様々なものをサンプルとして用いることができる。 [Step 1]
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.
　ウイルスを含有するサンプル液は、ウイルスを含有するサンプルを水に混濁することによって得られ、例えば環境水や生活用水のような液体サンプルであれば滅菌水で希釈すればよく、固形サンプルであれば滅菌水で混濁した上清や、固形サンプル表面を滅菌水で洗浄した洗浄液等もサンプル液として用いることができる。 The virus-containing sample solution is obtained by turbidity of the virus-containing sample in water. For example, if 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.
　ウイルスを含有するサンプル液はサンプル量やウイルス量によって適時希釈濃度を変更すればよいが、工程２に供する際には、無機塩濃度を０．５～２００ｍＭに、ｐＨを４．０～９．０に調整し、試験液とする必要がある。 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.
　無機塩としては、リン酸塩、クエン酸塩、塩酸塩、酢酸塩、硫酸塩、硝酸塩、炭酸塩、蟻酸塩、が挙げられ、塩としてはカリウム、ナトリウム、リチウム、カルシウム、マグネシウム等が挙げられる。 Inorganic salts include phosphates, citrates, hydrochlorides, acetates, sulfates, nitrates, carbonates, formates, and salts include potassium, sodium, lithium, calcium, magnesium, and the like. .
　ｐＨは公知慣用の方法で調整すればよく、各種酸塩基で調整してもよいし、上記無機塩のバッファーで調整するのが好ましい。 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.
　ウイルスを含有するサンプル液が、すでに無機塩濃度０．５～２００ｍＭかつｐＨを４．０～９．０である場合は、無調整でそのまま試験液とすることができる。 If the 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.
〔工程２〕
　上記工程１で得られた試験液を、ビーズ表面の臨界表面張力が３０～４５ｍＮである疎水性ビーズと接触させるのが工程２である。 [Step 2]
In 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.
　ウイルスを含有する試験液がフミン酸を含有する場合、そのままウイルス検出工程に供しても、フミン酸により核酸増幅検査法が阻害されてしまう為、ウイルス検出を精度よく実施することができない。本発明の疎水性ビーズは、疎水性の比較的強いフミン酸をウイルスよりも選択的に吸着する性質を有する。試験液のｐＨを弱酸性から中性領域に設定することで、ウイルス粒子及び核酸の破壊を防ぎつつ、無機塩濃度を調整することでフミン酸の電離を解消し、ビーズに吸着しやすくしたものである。そのため、得られた精製ウイルス液は、フミン酸が選択的に除去されているため、その後のウイルス検出工程を精度よく定量的に実施することができる。 When the test solution containing virus contains humic acid, even if it is subjected to the virus detection process as it is, the nucleic acid amplification test method is inhibited by humic acid, so that virus detection cannot be performed with high accuracy. The hydrophobic bead of the present invention has a property of selectively adsorbing a relatively strong hydrophobic humic acid over a virus. By setting the pH of the test solution from weakly acidic to neutral, preventing the destruction of virus particles and nucleic acids, and adjusting the inorganic salt concentration to eliminate humic acid ionization and make it easier to adsorb to beads It is. Therefore, 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.
　本発明の疎水性ビーズとは、ビーズ表面の臨界表面張力が３０～４５ｍＮである疎水性ビーズであって、材質は特に限定は無いが、好ましくは表面が高密度ポリエチレン、ポリスチレン、ポリメチルメタクリレート、２－フェノキシエチルアクリレートを含有するものであることが好ましい。疎水性ビーズは、単一の素材からなるものでもよいし、複数の素材を複合して得られるビーズであってもよく、ビーズ表面が臨界表面張力が３０～４５ｍＮであるように表面修飾を行ったものでもよい。 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.
　本発明の疎水性のビーズは、強度を高めるために数％の架橋剤を用いてもよく、そういった架橋剤としては、ジビニルベンゼンや、ジビニルベンゼン・エチルビニルベンゼン混合物、エチレングリコールジメタクリレート、トリメチロールプロパントリメタクリレート等のラジカル重合性の多官能モノマーを用いることができる。 The hydrophobic beads of the present invention may use several percent of a cross-linking agent to increase strength. Examples of such 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.
　これら架橋剤によってポリメチルメタクリレート、ポリ（２－フェノキシエチルアクリレート）を架橋することができ、得られる架橋ポリメチルメタクリレート、架橋ポリ（２－フェノキシエチルアクリレート）は、強度に優れるため好ましい。 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.
　臨界表面張力の値は、例えば、和光純薬工業株式会社製ぬれ指数標準液を用い、ＪＩＳ Ｋ ６７６８：１９９９に準じて測定できる。具体的には、段階的に表面張力が異なる複数の標準液を準備し、その中の一つの標準液を用いて対象となる素材の平滑なシート表面上に滴下し、液滴を膜面に広げ、滴下した標準液の液膜が破れを生じることなく、２秒以上濡らすことのできる上限の標準液の表面張力値を臨界表面張力とすることで決定できる。 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.
　本発明の疎水性ビーズの大きさは、体積分布の累積１０％径が１μｍ以上かつ、大きさが累積９０％粒度分布が１０００μｍ以下であるものが好ましい。ビーズの体積分布の累積１０％径が１μｍ以上かつ、大きさが累積９０％粒度分布が１０００μｍ以下であると、効率的に試験液と接触できる為である。より好ましくは、体積分布の累積１０％径が３０μｍ以上かつ、累積９０％粒度分布が５００μｍ以下であるものである。 The size of the hydrophobic beads of the present invention is preferably such that the 10% diameter cumulative volume distribution is 1 μm or more and the 90% cumulative size distribution is 1000 μm or less. This is because when the cumulative 10% diameter of the bead volume distribution is 1 μm or more and the cumulative size is 90% and the particle size distribution is 1000 μm or less, the beads can be contacted efficiently. More preferably, the cumulative 10% diameter of the volume distribution is 30 μm or more and the cumulative 90% particle size distribution is 500 μm or less.
　体積分布及び粒径の測定方法は、顕微鏡法によって測定することができる。 The volume distribution and particle size can be measured by microscopy.
　上記疎水性ビーズは、ＢＥＴ表面積が１００～１２００ｍ２／ｇであることが好ましい。これは、１００ｍ２／ｇ以上であると試験液とビーズとの接触効率が向上するため好ましく、１２００ｍ２／ｇ以下であれば孔径が比較的大きいため、フミン酸をビーズ表面の孔内で捕捉しやすいため好ましいからである。 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.
　本発明におけるＢＥＴ比表面積とは、ＢＥＴ式比表面積測定器（ユアサアイオニクス株式会社製：オートソーブ１－Ｃ）を用い窒素置換法にて測定した値である。 In the present invention, 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).
　本発明の疎水性ビーズと試験液を接触させる場合、様々な方法を用いることができる。　 When contacting the hydrophobic beads of the present invention with the test solution, various methods can be used. *
　バッチ式の場合、疎水性ビーズと試験液をビーカーや試験管、マイクロチューブやチップ等に入れ、撹拌することで接触させることができる。撹拌する場合、撹拌翼や撹拌子を使用してもよいし、振動式ミキサー等で撹拌してもよい。 In the case of the batch type, 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. When stirring, a stirring blade or a stirring bar may be used, or stirring may be performed with a vibration mixer or the like.
　フロー式の場合、疎水性ビーズをカラムやチューブに詰めた上、試験液を通液してもよいし、担体に疎水性ビーズを固定したうえで通液してもよい。 In the case of the flow type, 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.
〔工程３〕
　本発明の工程３では、工程２において接触させた疎水性ビーズと試験液とを分離することで、フミン酸を除去した精製ウイルス液を得ることができる。分離の方法は公知慣用の方法を用いればよく、濾過、デカンテーション、遠心分離等の方法を用いることができるし、注射器等を用いて液のみを採取してもよい。 [Step 3]
In 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.
〔工程４〕
　工程３で得られた精製ウイルス液は、工程４にてウイルス検出工程に供することが好ましい。 [Step 4]
The purified virus liquid obtained in step 3 is preferably subjected to a virus detection step in step 4.
　ウイルス検出工程とは、ウイルスを検出、及び同定する工程である。ウイルス検出方法としては、古くは単離培養からの形態的同定法が用いられていたが、現在はポリメラーゼ連鎖反応法（ＰＣＲ）等に代表される核酸増幅検査（ＮＡＴ）法が用いられる。ＮＡＴ法は、培養方法が確立していないウイルスを検出できるうえ、培養日数が不要なため短期間での検出が可能と言う利点がある。 The virus detection step is a step of detecting and identifying a virus. As 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. 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.
　工程３で得られる精製ウイルス液中で、ウイルス濃度が低い場合、公知慣用の方法でウイルスを濃縮してもよく、具体的には陽電荷膜法や陰電荷膜法、ポリエチレングリコール沈殿法、限外ろ過法などがある。本発明で得られる精製ウイルス液は、ＮＡＴ法を阻害するフミン酸が除去されているため、感度よく検出することが可能である。 If the virus concentration in the purified virus solution obtained in step 3 is low, 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.
　精製ウイルス液をＮＡＴ法に供する場合、ウイルスから核酸を抽出する前処理を行うことが好ましい。ウイルス核酸（ＤＮＡ，ＲＮＡ）の抽出は、特に限定されるものではなく、フェノール・クロロホルム抽出法、界面活性剤やプロテアーゼを併用した抽出法等が用いられる。また、ウイルス核酸精製に関しても、液相抽出法、エタノール沈殿法やスピンカラム法等により行うことができる。 When 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 (DNA, RNA) 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. Moreover, 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.
　抽出された核酸は、ＮＡＴ法により検査を行うが、ＮＡＴ法はウイルス等の微量の遺伝子を人工的に増幅して高感度に検出する方法の総称であり、ＰＣＲ法の他、転写媒介増幅（ＴＭＡ）法、鎖置換反応（ＬＡＭＰ）法、等温遺伝子増幅（ＩＣＡＮ）法、核酸配列増幅（ＮＡＳＢＡ）法、リガーゼ連鎖反応（ＬＣＲ）法等が挙げられる。 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. In addition to the PCR method, transcription-mediated amplification ( TMA) method, strand displacement reaction (LAMP) method, isothermal gene amplification (ICAN) method, nucleic acid sequence amplification (NASBA) method, ligase chain reaction (LCR) method and the like.
　例えばＰＣＲ法の場合、ウイルスに特異的なプライマーを用いて核酸増幅を行い、増幅が認められればウイルスが存在することが判明する。 For example, in the case of the PCR method, nucleic acid amplification is performed using primers specific to the virus, and if amplification is observed, it is found that the virus is present.
　以下、実施例を用いて本発明を説明するが、本発明は実施例の記載に制限されるものではない。また、実施例において特段の記載がない場合、部及び％は質量基準である。 Hereinafter, the present invention will be described using examples, but the present invention is not limited to the description of the examples. Moreover, when there is no special description in an Example, a part and% are mass references | standards.
〈調製例１〉　ファージ・フミン酸溶液１の調製
　精製水にフミン酸（ナカライテスク株式会社）を１０００ｐｐｍとなるように溶解し、水酸化ナトリウム溶液を用いてｐＨ７．３に調整した。さらに、精製水にＱβファージ（ＮＢＲＣ２００１２）及び上記調整したフミン酸水溶液をそれぞれ終濃度５×１０１０ＰＦＵ／ｍＬ、５００ｐｐｍとなるように添加し、ファージ・フミン酸溶液１を調製した。 <Preparation Example 1> Preparation of Phage / Humic Acid Solution 1 Humic acid (Nacalai Tesque Co., Ltd.) was dissolved in purified water to 1000 ppm and adjusted to pH 7.3 using a sodium hydroxide solution. Furthermore, Qβ phage (NBRC20012) and 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.
〈実施例１〉
ポリプロピレンマイクロチューブに、疎水性ビーズとして洗浄処理を行ったＳｕｐｅｌｉｔｅ　ＤＡＸ－８（Ｓｕｐｅｌｃｏ社製）をスピンダウン後の容積として１００μｌ取り、調製例１のファージ・フミン酸溶液１を０．２ｍＬ、ｐＨ７．０のリン酸ナトリウムバッファ（バッファ濃度１００ｍＭ）を０．８ｍｌ加えた後、１０分間転倒混和した。ビーズをスピンダウンして上澄みを回収し、精製ウイルス液を得た。 <Example 1>
In a polypropylene microtube, 100 μl 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.
・フミン酸量の測定
　得られた精製ウイルス液の２６０ｎｍ吸光度を測定することで、フミン酸量を測定した。 -Measurement of the amount of humic acid The amount of humic acid was measured by measuring the 260 nm light absorbency of the obtained purified virus liquid.
　測定したフミン酸量から、フミン酸回収率を下記式により算出した。 From the measured amount of humic acid, the humic acid recovery rate was calculated by the following formula.
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000001
　フミン酸回収率の値が小さいほど、ファージ・フミン酸溶液１からフミン酸を好適に除去することができたといえる。 It can be said that the smaller the value of the humic acid recovery rate, the better the humic acid could be removed from the phage-humic acid solution 1.
・ＲＮＡ量の測定
　得られた精製ウイルス液を、ＱＩＡａｍｐ　Ｖｉｒａｌ　ＲＮＡ　Ｍｉｎｉ　プロトコール（株式会社キアゲン）に従ってファージＲＮＡを抽出し、１００倍に希釈した後、リアルタイムＲＴ－ＰＣＲ法にてＲＮＡ量を定量した。測定に用いたプライマー及びプローブの配列は文献（Ｊｏｕｒｎａｌ　ｏｆ　Ｖｉｒｏｌｏｇｉｃａｌ　Ｍｅｔｈｏｄｓ　１４９（２００８），ｐ１２３－１２８）に従って作製し、測定はＳｔｅｐＯｎｅＰｌｕｓリアルタイムＰＣＲシステム（ライフテクノロジースジャパン株式会社）を使用した。 -Measurement of RNA amount 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.).
　測定したＲＮＡ量から、Ｑβ回収率（ファージ回収率）を下記式により算出した。 From the measured RNA amount, the Qβ recovery rate (phage recovery rate) was calculated by the following formula.
Figure JPOXMLDOC01-appb-M000002
Figure JPOXMLDOC01-appb-M000002
　Ｑβ回収率の値が大きいほど、精製工程においてウイルスは除去されておらず、不純物が選択的に除去されたといえる。 It can be said that the larger the value of Qβ recovery rate, the more virus was not removed in the purification process and the impurities were selectively removed.
〈実施例２〉
　使用バッファをｐＨ５．０のクエン酸ナトリウムバッファ（バッファ濃度１００ｍＭ）にした以外は実施例１と同様にして、ファージ，フミン酸の回収率を評価した。 <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).
〈実施例３〉
　使用ビーズをＳｕｐｅｌｉｔｅ　ＤＡＸ－８からセパビーズＳＰ２ＭＧＳ（三菱化学株式会社製）に変更した以外は実施例２と同様にして、ファージ，フミン酸の回収率を評価した。 <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).
〈実施例４〉
　ビーズの添加量を１００μｌから３０μｌに変更した以外は実施例３と同様にして、ファージ，フミン酸の回収率を評価した。 <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 μl to 30 μl.
〈実施例５〉
　転倒混和時間を１０分から６０分に変えた以外は実施例１と同様にして、ファージ，フミン酸の回収率を評価した。 <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.
〈実施例６〉
　使用バッファをｐＨ５．０のクエン酸ナトリウムバッファ（バッファ濃度５０ｍＭ）にした以外は実施例３と同様にして、ファージ，フミン酸の回収率を評価した。 <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).
〈実施例７〉
　使用ビーズをＳｕｐｅｌｉｔｅ　ＤＡＸ－８からダイアイオンＨＰ２ＭＧ（三菱化学株式会社製）に変更し、添加量を３００μｌにした以外は実施例６と同様にして、ファージ，フミン酸の回収率を評価した。 <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 μl.
〈実施例８〉
　使用ビーズをＳｕｐｅｌｉｔｅ　ＤＡＸ－８からダイアイオンＨＰ２０（三菱化学株式会社製）に変更し、添加量を９００μｌにした以外は実施例６同様にして、ファージ，フミン酸の回収率を評価した。 <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 μl.
〈実施例９〉
　ポリプロピレンマイクロチューブに調製例１のファージ・フミン酸溶液１を０．２ｍＬ、ｐＨ７．０のリン酸ナトリウムバッファ（バッファ濃度１００ｍＭ）を０．８ｍｌ加えてファージ・フミン酸液を調製した。３００μｌのＳｕｐｅｌｉｔｅ　ＤＡＸ－８をφ２×４のシリコンチューブに詰めて作製したカラムに、上記ファージ・フミン酸液を通じ、更に４ｍｌのｐＨ７．０のリン酸ナトリウムバッファ（バッファ濃度０．１Ｍ）を通液した。回収した液を実施例１と同様にしてファージ，フミン酸の回収率を評価した。 <Example 9>
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 μl of Superlite DAX-8 in a φ2 × 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.
〈実施例１０〉
　カラムに使用したビーズ量を１２００μｌにし、使用バッファをｐＨ７．０のリン酸ナトリウムバッファ（バッファ濃度１０ｍＭ）に変更した以外は実施例９と同様にして、ファージ，フミン酸の回収率を評価した。 <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 μl and the buffer used was changed to sodium phosphate buffer pH 7.0 (buffer concentration 10 mM).
〈実施例１１〉
　カラムに使用したビーズ量を１８００μｌにし、使用バッファをｐＨ７．０のリン酸ナトリウムバッファ（バッファ濃度１．０ｍＭ）に変更した以外は実施例９と同様にしてファージ，フミン酸の回収率を評価した。 <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 μl and the buffer used was changed to sodium phosphate buffer pH 7.0 (buffer concentration 1.0 mM). .
〈実施例１２〉
　使用バッファをｐＨ４．０のクエン酸ナトリウムバッファ（バッファ濃度５０ｍＭ）に変え、転倒混和時間を１０分から６０分に変えた以外は実施例７と同様にして、ファージ，フミン酸の回収率を評価した。 <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. .
〈比較例１〉
　使用ビーズをシリカゲルビーズ（シリカゲル６０Ｎ（６３－２１０ｕｍ）関東化学株式会社製）に変え、ビーズ量を１０００μｌにし、使用バッファをｐＨ７．０のリン酸ナトリウムバッファ（バッファ濃度１０ｍＭ）に変更した以外は実施例１と同様にして、ファージ，フミン酸の回収率を評価した。 <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 μl, 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.
〈比較例２〉
　使用ビーズをＳｕｐｅｌｉｔｅ　ＤＡＸ－８からダイアイオンＳＡ－１０Ａ（三菱化学株式会社製）に変更した以外は実施例７と同様にして、ファージ，フミン酸の回収率を評価した。 <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).
〈比較例３〉
　使用バッファをｐＨ７．０のリン酸ナトリウムバッファ（バッファ濃度３００ｍＭ）に変更した以外は実施例１と同様にしてファージ、フミン酸の回収率を評価した。 <Comparative Example 3>
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 sodium phosphate buffer having a pH of 7.0 (buffer concentration: 300 mM).
〈比較例４〉
　使用バッファをｐＨ２．０の塩酸－塩化カリウムバッファ（バッファ濃度２００ｍＭ）に変え、ビーズを使用しない以外は実施例１と同様にして、ファージ，フミン酸の回収率を評価した。 <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.
〈比較例５〉
　使用バッファをｐＨ１２．０の水酸化ナトリウム水溶液に変え、ビーズを使用しない以外は実施例１と同様にして、ファージ，フミン酸の回収率を評価した。 <Comparative Example 5>
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 sodium hydroxide aqueous solution of pH 12.0 and no beads were used.
上記実施例及び比較例の結果を、表１～３に示した。 The results of the above examples and comparative examples are shown in Tables 1 to 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表の内容の説明
ＰＭＭＡ：架橋ポリメチルメタクリレート（架橋剤：エチレングリコールジメタクリレート）
ＰＳ：架橋ポリスチレン（架橋剤：ジビニルベンゼン）
陰イオン交換樹脂：架橋ポリスチレンにトリメチルアンモニウム基を導入した化合物 Description of Table Contents 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
　表中におけるビーズの粒径は以下の通りである。 The particle size of the beads in the table is as follows.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　本発明のウイルス精製方法及びウイルス検出方法は、ウイルス検査方法に対し好適に使用可能である。 The virus purification method and virus detection method of the present invention can be suitably used for virus inspection methods.

Claims (7)

  1. 　ウイルスを含有するサンプル液を、無機塩濃度が０．５～２００ｍＭ及びｐＨ４．０～９．０の試験液に調整する工程１と、
    　疎水性ビーズであって、ビーズ表面の臨界表面張力が３０～４５ｍＮである疎水性ビーズに、上記試験液を接触させる工程２と、
    　上記疎水性ビーズと試験液とを分離して精製ウイルス液を得る工程３とを有することを特徴とする、精製ウイルス液の製造方法。     Adjusting the sample solution containing the virus to a test solution having an inorganic salt concentration of 0.5 to 200 mM and a pH of 4.0 to 9.0;
    A step 2 of contacting the test solution with a hydrophobic bead, wherein the bead surface has a critical surface tension of 30 to 45 mN;
    A method for producing a purified virus solution, comprising the step 3 of separating the hydrophobic beads and the test solution to obtain a purified virus solution.
  2. 　上記サンプル液が、さらにフミン酸を含有するものである、請求項１に記載の精製ウイルス液の製造方法。 The method for producing a purified virus solution according to claim 1, wherein the sample solution further contains humic acid.
  3. 　上記疎水性ビーズが、表面が高密度ポリエチレン、ポリスチレン、ポリメチルメタクリレート、ポリ（２－フェノキシエチルアクリレート）を含有するものである、請求項１または２に記載の精製ウイルス液の製造方法。 The method for producing a purified virus solution according to claim 1 or 2, wherein the hydrophobic beads have a surface containing high-density polyethylene, polystyrene, polymethyl methacrylate, or poly (2-phenoxyethyl acrylate).
  4. 　上記疎水性ビーズが、ＢＥＴ表面積が１００～１２００ｍ２／ｇである、請求項１～３の何れかに記載の精製ウイルス液の製造方法。 The method for producing a purified virus solution according to any one of claims 1 to 3, wherein the hydrophobic beads have a BET surface area of 100 to 1200 m 2 / g.
  5. 　上記疎水性ビーズが、体積分布の累積１０％径が１μｍ以上かつ、累積９０％粒度分布が１０００μｍ以下である、請求項１～４の何れかに記載の精製ウイルス液の製造方法。 The method for producing a purified virus solution according to any one of claims 1 to 4, wherein the hydrophobic beads have a cumulative 10% diameter of volume distribution of 1 µm or more and a cumulative 90% particle size distribution of 1000 µm or less.
  6. 　ウイルスを含有するサンプル液を、無機塩濃度が０．５～２００ｍＭ及びｐＨ４．０～９．０の試験液に調整する工程１と、
    　疎水性ビーズであって、ビーズ表面の臨界表面張力が３０～４５ｍＮである疎水性ビーズに、上記試験液を接触させる工程２と、
    　上記疎水性ビーズと試験液とを分離して精製ウイルス液を得る工程３と、
    　上記精製ウイルス液をウイルス検出工程に供する工程４とを有することを特徴とする、ウイルス検出方法。     Adjusting the sample solution containing the virus to a test solution having an inorganic salt concentration of 0.5 to 200 mM and a pH of 4.0 to 9.0;
    A step 2 of contacting the test solution with a hydrophobic bead, wherein the bead surface has a critical surface tension of 30 to 45 mN;
    Step 3 of separating the hydrophobic beads and the test solution to obtain a purified virus solution,
    And a step 4 of subjecting the purified virus solution to a virus detection step.
  7. 　上記ウイルス検出工程が、核酸増幅検査法である、請求項６に記載のウイルス検出方法。 The virus detection method according to claim 6, wherein the virus detection step is a nucleic acid amplification test method.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017013731A1 (en) * 2015-07-21 2017-01-26 Dic株式会社 Method for producing purified virus solution, method for detecting virus, and virus purification member
WO2017013732A1 (en) * 2015-07-21 2017-01-26 Dic株式会社 Method for producing purified virus solution, and method for detecting virus
CN107436332A (en) * 2017-08-01 2017-12-05 清华大学 A kind of method for separating and determining humic acid opposed polarity and molecular weight component
WO2021002257A1 (en) 2019-07-04 2021-01-07 株式会社カネカ Method for purifying virus or virus-like particle
WO2022044727A1 (en) 2020-08-28 2022-03-03 株式会社カネカ Method for purifying useful substance

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5987089A (en) * 1982-11-11 1984-05-19 Mitsubishi Chem Ind Ltd Removal of organic substance in water
JP2011155919A (en) * 2010-02-02 2011-08-18 Fisheries Research Agency Material for detecting norovirus and method for detecting norovirus using the material

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5987089A (en) * 1982-11-11 1984-05-19 Mitsubishi Chem Ind Ltd Removal of organic substance in water
JP2011155919A (en) * 2010-02-02 2011-08-18 Fisheries Research Agency Material for detecting norovirus and method for detecting norovirus using the material

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ABBASZADEGAN M. ET AL.: "Detection of Enteroviruses in Groundwater with the Polymerase Chain Reaction", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 59, no. 5, 1993, pages 1318 - 1324, XP055076740 *
BLONDEAU R. ET AL.: "Fractionation of humic substances by hydrophobic interaction chromatography", JOURNAL OF CHROMATOGRAPHY, vol. 351, 1986, pages 585 - 589, XP026474577 *
KAZUYUKI SUGITA: "Surface Modification of Plastics. 1. Surface Characteristics of Polymeric Materials and Their Modification", JOURNAL OF PRINTING SCIENCE AND TECHNOLOGY, vol. 35, no. 4, 1998, pages 202 - 210, XP055214787 *
PEURAVUORI J. ET AL.: "Comparative study for separation of aquatic humic-type organic constituents by DAX-8, PVP and DEAE sorbing solids and tangential ultrafiltration: elemental composition, size-exclusion chromatography, UV-vis and FT-IR", TALANTA, vol. 65, 2005, pages 408 - 422, XP027859668 *
SATOSHI KADOWAKI ET AL.: "Hydrophobic- Hydrophilic Fractionation of Dissolved Organic Matter in Natural Waters Using Styrene Divinylbenzene Copolymer Resins", JOURNAL OF JAPAN SOCIETY ON WATER ENVIRONMENT, vol. 32, no. 4, 2009, pages 205 - 211, XP055214803 *
SCHRIEWER A. ET AL.: "Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8", J OURNAL OF MICROBIOLOGICAL METHODS, vol. 85, 2011, pages 16 - 21, XP028173556 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017013731A1 (en) * 2015-07-21 2017-01-26 Dic株式会社 Method for producing purified virus solution, method for detecting virus, and virus purification member
WO2017013732A1 (en) * 2015-07-21 2017-01-26 Dic株式会社 Method for producing purified virus solution, and method for detecting virus
CN107436332A (en) * 2017-08-01 2017-12-05 清华大学 A kind of method for separating and determining humic acid opposed polarity and molecular weight component
CN107436332B (en) * 2017-08-01 2020-05-22 清华大学 Method for separating and measuring components with different polarities and molecular weights of humic acid
WO2021002257A1 (en) 2019-07-04 2021-01-07 株式会社カネカ Method for purifying virus or virus-like particle
WO2022044727A1 (en) 2020-08-28 2022-03-03 株式会社カネカ Method for purifying useful substance
KR20230056661A (en) 2020-08-28 2023-04-27 가부시키가이샤 가네카 Methods for Purifying Useful Substances

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