JP2001258551A - Particle for concentration of virus, virus concentration reagent using the particle, method for concentration of virus and method for detecting virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a virus concentration particle capable of easily treating a number of specimens at a time by a simple means without producing adverse effect on a nucleic acid amplification test, a method for concentrating virus using the particle and a method for detecting virus. SOLUTION: The particle for the concentration of virus contains a magnetic material. The invention also relates to a virus concentration reagent, a virus concentrating method and a virus detection method using the particle.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to virus-concentrating particles for concentrating a virus in a sample, a virus-concentrating kit containing the virus-concentrating particles, a virus-concentrating method, and a virus-testing method.

[0002]

2. Description of the Related Art Viruses are one of the causes of various diseases of humans, animals and plants, and the identification of the causative virus is very important for the inspection and diagnosis thereof. As a conventional virus inspection / diagnosis method, immunological measurement of a virus antigen or an antiviral antibody is generally performed. However, several weeks to several months after virus infection, the amount of the virus or the amount of the anti-virus antibody is small, and thus it may not be detected by these immunological assays. This undetectable period is called a window period (blank period), and when such a patient makes a blood donation, the blood donation is often sufficiently infectious and an unspecified number of transfusions May be dangerous to patients and patients using blood products. Therefore, in order to shorten the window period as much as possible, there is an urgent need to develop a technique capable of detecting a virus under the limit of immunological measurement with high sensitivity.

[0003] In recent years, the possibility of detecting even a trace amount of virus has been opened up by a nucleic acid amplification technique represented by the polymerase chain reaction method (hereinafter, PCR method). However, detection of a trace amount of virus by PCR or the like also requires special facilities and advanced techniques, and it is extremely difficult to simply carry out detection in a general facility. In order to solve these problems, a technique of concentrating a virus in a specimen has been used.

[0004] A typical example of the conventional virus concentration method is an ultracentrifugation method, which requires expensive equipment and a long time, and it is difficult to process a large number of samples at the same time. hard. In addition, since a hepatitis B virus surface antigen (HBs antigen) binds to heparin, a chromatography method using a heparin sepharose carrier has been reported, but it is also difficult to treat a large number of samples at the same time. In addition, there is a method of precipitating the virus using ammonium sulfate, polyethylene glycol, a combination of a polyanion and a divalent ion, and the like. However, it is necessary to purify the sample after precipitation and separation of the virus due to problems such as PCR inhibition of the reagents to be mixed. There were difficulties.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems of the virus concentration method, and to easily process a large number of samples at the same time by simple means, and to cope with automation. An object of the present invention is to provide a virus concentration particle which is easy and does not adversely affect a nucleic acid amplification test, a virus concentration reagent, a virus concentration method and a virus detection method using the particle.

[0006]

As a result of the research, the present inventors have developed the following virus concentrating particles and a concentrating method as means for solving the above-mentioned problems. That is, the present invention first provides virus concentration particles containing a magnetic substance. The virus concentration particles preferably have an anionic group (including a salt state) on the particle surface.
The present invention secondly provides a virus concentrating reagent comprising the above-mentioned virus concentrating particles and a polyvalent metal compound. Further, the present invention provides, thirdly, a step of adding the above-mentioned virus concentrating particles to a sample which may contain a virus and binding the virus to the particles, and then, And a method for concentrating the virus, comprising the steps of separating and collecting the virus from the virus. In a preferred embodiment, a polyvalent metal compound is added to the sample along with the virus-enriched particles. Further, the present invention provides, fourthly, a step of adding the above-mentioned virus concentrating particles to a sample which may contain a virus, and binding the virus to the particles. And a method for detecting a virus, comprising the steps of: concentrating a virus by separating and collecting the virus from a target; and subjecting the virus thus concentrated to a nucleic acid amplification test. In a preferred embodiment, a polyvalent metal compound is added to the sample along with the virus-enriched particles.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. <Virus concentration particles> In the present invention, virus concentration particles are particles that are separated and concentrated after adsorbing viruses in a sample such as blood or body fluid, and the viruses separated and concentrated by the particles are nucleic acids. It is used for extraction / test / diagnosis, especially for test / diagnosis involving nucleic acid amplification. The virus concentration particles of the present invention contain a magnetic substance and preferably have an anionic group (including a salt state) on the particle surface.

[0008] The virus concentration particles of the present invention comprise a base material containing a magnetic substance. It is preferable that this magnetic material is contained only in the inside of the particle and is not exposed on the surface. In the present invention, the inclusion of a magnetic substance in the virus concentration particles makes it possible to collect the particles by applying the magnetism of the particles, and it is not necessary to perform an operation such as centrifugation, thereby shortening the inspection time. In addition, it is easy to respond to automation of inspection and diagnosis. Such magnetic materials include, for example, triiron tetroxide (Fe ₃ O ₄ ), γ-iron trioxide (γ-F
Various ferrites such as e ₂ O ₃ ), metals such as iron, manganese, cobalt, and chromium, and alloys of these metals can be used.

The content of the magnetic substance is preferably at least 10% by weight, particularly preferably 20 to 100% by weight, based on the whole particles for virus concentration. If the amount is too small, the virus concentrating particles will not have good magnetic separability. As a result, in the virus separation / concentration method described below, virus concentrating particles are separated from a sample such as blood or body fluid. Since it takes a considerably long time to perform the above, high temporal efficiency may not be obtained, which is not preferable.

Examples of the anionic group in the present invention include a carboxyl group and a sulfone group (the sulfone group may be present as a part of a sulfate group). These anionic groups may be present in the form of a salt. In the present invention, an anionic group may inhibit a nucleic acid amplification method such as a PCR method when eluted in water, a buffer solution, blood, or a body fluid, and thus needs to be chemically bonded to base particles. . Its abundance is usually 1 × 10 ⁻¹² mol or more per 1 g of virus concentration particles on average,
Typically, it is 1 × 10 ⁻¹² to 1 × 10 ⁻² mol, preferably 1 × 10 ⁻¹¹ to 1 × 10 ⁻³ mol, and more preferably 1 × 10 ⁻¹¹ to 1 × 10 ⁻⁴ mol. Is a mole. If the amount of the anionic group is too small, the virus concentrating ability may be insufficient. Without limitation, usually
It is often difficult to introduce more than 1 × 10 ^-2 mol of anionic groups per gram of virus concentrating particles.

The base particles of the virus concentration particles of the present invention include, for example, particles having a sulfone group on the surface (referred to as “surface sulfonated particles”) and particles having a carboxyl group on the surface (“surface carboxylic acid particles”). Particles of a synthetic polymer particle obtained by seed polymerization or graft polymerization of a sulfone group-containing monomer and / or a carboxyl group-containing monomer, hydrogel particles comprising a sulfone group-containing monomer and a crosslinkable monomer, Polyanion compound-immobilized particles,
Examples include sulfated polysaccharide gel particles, but are not limited thereto, and any particles having an anionic group on at least a part of the surface can be used.

The surface sulfonated particles include, for example, functional groups capable of being sulfonated on at least the surface thereof, for example, unsaturated double bonds, aromatic groups, primary or secondary amino groups on the main chain or side chains, It consists of a (co) polymer of a high molecular compound having a primary alkyl halide group, an aliphatic aldehyde, an aliphatic ketone, an aliphatic carboxylic acid, an aliphatic carboxylic anhydride, a hydroxyl group, etc., and at least a part of the surface thereof. Can be used to obtain particles having a sulfone group. Examples of the high molecular compound constituting the particles capable of surface sulfonation include (co) polymers of sulfonable monomers such as styrene, α-methylstyrene, vinylnaphthalene, divinylbenzene, butadiene, isoprene, and vinyl alcohol. And addition-polymerized polymer compounds such as (co) polymers of these monomers and other polymerizable monomers; polycarbonate, polyester, polyester ether, polyaryl ether, polyalkylene oxide, polysulfone, polyether sulfone , Polyamide, polyimide, polyetherimide, polyetherketone, polyurethane, acetaldehyde condensate of aromatic compound, polycondensation polymer compound such as polyether and the like.

[0013] The sulfonation of the particles composed of these polymer compounds is carried out by (1) converting unsulfonated polymer particles into concentrated sulfuric acid, fuming sulfuric acid, sulfuric anhydride, sulfuric anhydride-dioxane complex, sulfuric anhydride-pyridine complex, This can be achieved by a method of treating particles with chlorosulfonic acid or the like, or (2) a method of copolymerizing a sulfonic acid monomer with another monomer.

Examples of the surface carboxylic acid particles include (co) polymer particles of a carboxylic acid-containing monomer.
Here, the carboxylic acid-containing monomer (hereinafter referred to as “carboxylic acid monomer”) is a polymerizable monomer having an addition-polymerizable unsaturated bond and a carboxyl group in the molecule. Specific examples include acrylic acid, methacrylic acid, crotonic acid, itaconic acid, fumaric acid, and maleic acid. These (co) polymer particles can be synthesized by a usual emulsion polymerization or suspension polymerization technique.

The particles obtained by seed-polymerizing or graft-polymerizing a sulfonic group-containing monomer (hereinafter referred to as a “sulfonic acid monomer”) and / or a carboxylic acid monomer on a synthetic polymer particle are made of a synthetic polymer. Seed particles may be seed (co) polymerized or graft (co) polymerized with a sulfonic acid monomer and / or a carboxylic acid monomer, and optionally other copolymerizable monomers. be able to. Such particles can be prepared by adding a monomer and a radical generator to seed particles dispersed in water or a water / polar solvent containing a surfactant, and adding 50 to 1 particles.
It can be synthesized by reacting at 00 ° C. Here, components forming the synthetic polymer of the seed particles include aromatic compounds containing a polymerizable double bond such as styrene, α-methylstyrene, vinyltoluene, and vinylnaphthalene; acrylonitrile, methacrylonitrile, vinylidene cyanide, and the like. A polymerizable crosslinkable compound such as divinylbenzene and ethylene glycol dimethacrylate; vinyl chloride, vinylidene chloride, vinyl methyl ethyl ketone, vinyl methyl ether, vinyl acetate,
Vinyl formate, allyl acetate, methallyl acetate, acrylamide, methacrylamide, N-methylol methacrylamide, N-isopropylacrylamide, glycidyl acrylate, glycidyl methacrylate, acrolein, methacrolein, allyl alcohol, 2-hydroxyethyl acrylate, 2-hydroxy Ethyl methacrylate, 2-methoxyethyl acrylate, n-butyl acrylate, sec-butyl acrylate, isobutyl acrylate, t-butyl acrylate, n-butyl methacrylate, sec-butyl methacrylate, isobutyl methacrylate, t-butyl methacrylate, methyl acrylate, ethyl acrylate , N-propyl acrylate, isopropyl acrylate, methyl methacrylate, ethyl meth Polymerizable double bond-containing compounds such as acrylate, n-propyl methacrylate, and isopropyl methacrylate; and (co) polymerizable cyclic compounds such as ethylene oxide, propylene oxide, 2-methyltetrahydroxyfuran, styrene oxide, butylene oxide, and glycidyl ether. Polymers and the like can be mentioned.

The sulfonic acid monomers include isoprenesulfonic acid, ethylenesulfonic acid, vinylsulfonic acid, styrenesulfonic acid, α-methylstyrenesulfonic acid,
Acrylamide-2-methylpropanesulfonic acid, sulfoethyl acrylate, sulfonated dicyclopentadiene and the like can be mentioned.

Other copolymerizable monomers include aliphatic diene compounds such as 1,3-butadiene and isoprene; and aromatic compounds containing polymerizable double bonds such as styrene, α-methylstyrene and vinyltoluene. Alkyl (meth) acrylates such as methyl acrylate, ethyl acrylate, 2-ethylhexyl acrylate, methyl methacrylate, ethyl methacrylate, 2-hydroxyethyl acrylate, and 2-hydroxyethyl methacrylate;
Polymerizable cyanide compounds such as acrylonitrile and methacrylonitrile; vinyl chloride, vinylidene chloride, vinyl methyl ethyl ketone, vinyl methyl ether, vinyl acetate, vinyl formate, allyl acetate, methallyl acetate, acrylamide, methacrylamide, N-methylol methacrylamide, N- Polymerizable double bond-containing compounds such as isopropylacrylamide, glycidyl acrylate, glycidyl methacrylate, acrolein, methacrolein, allyl alcohol, ethylene oxide, propylene oxide, 2-
Examples include polymerizable cyclic compounds such as methyltetrahydroxyfuran, styrene oxide, butylene oxide, and glycidyl ether.

The hydrogel particles comprising a sulfonic acid monomer and a water-soluble cross-linkable monomer include, for example, the above-mentioned sulfonic acid monomer and a water-soluble cross-linkable monomer such as N, N'-methylenebisacrylamide. Hydrogel particles composed of a solid polymer can be used. Such particles are mixed vigorously with an aqueous solution of a sulfonic acid monomer, a water-soluble crosslinkable monomer, and a radical reaction initiator while adding a surfactant to a non-polar solvent to form a water-in-oil reverse micelle. After forming
It is obtained by reacting at 00 ° C.

The polyanion compound-immobilized particles include:
A polyanion having a plurality of sulfone groups and / or carboxyl groups in the molecule can be directly applied to a particle having a functional group such as an epoxy group, an amino group, an aldehyde group, a carboxyl group, a hydroxyl group, an acid chloride group or a coupling agent or a spacer. Particles supported on the surface of the material through the intermediary. Here, as the polyanion, for example, a polymer compound obtained by (co) polymerizing a sulfonic acid monomer and / or a carboxylic acid monomer and, if necessary, other monomers; a sulfated polysaccharide Phosphotungstic acid; polyphosphoric acid, and the like.

Examples of the solid acid particles include particles obtained by coating the surface of a granular material such as glass or silica with a polymer having a sulfone group or a carboxyl group. In this case, the carboxyl group can be introduced by introducing an amino group to the surface of the granular material with γ-aminopropyltriethoxysilane and reacting the amino group with succinic anhydride or succinic acid in the presence of carbodiimide. Further, the sulfone group can be introduced by introducing an aldehyde group by glutaraldehyde treatment after subjecting the granular material to aminosilane treatment, and then reacting the aldehyde group with 2-aminoethanesulfonic acid.

A sulfonic acid monomer, a carboxylic acid monomer, a polymer compound obtained by (co) polymerizing a sulfonic acid monomer and another monomer, and a carboxylic acid monomer and another monomer Specific examples of the (co) polymerized polymer compound are as described above.

Examples of the sulfated polysaccharide include heparin, dextran sulfate, cellulose sulfate, guardlan sulfate, chondroitin sulfate, heparan sulfate, dextran sulfate, chitin sulfate, chitosan sulfate, dermatan sulfate, amylopectin sulfate, ketalan sulfate, xylan sulfate, and karonin sulfate. ,
Examples include pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, starch sulfate, polyglucose sulfate, laminarin sulfate, galactan sulfate, levan sulfate, mepesulfate, fucoidan, and the like.

Examples of the sulfated polysaccharide gel particles include gel particles made of sulfonated polysaccharides obtained by sulfated crosslinked or uncrosslinked particles of cellulose, agarose, dextran, chitin, chitosan or the like. .

The virus concentrating particles of the present invention can be produced as follows. 1) base particles are synthetic polymers (for example, surface sulfonic acid particles, surface carboxylic acid particles, particles obtained by seed-polymerizing or graft-polymerizing a sulfonic acid-containing monomer and / or a carboxylic acid monomer onto synthetic polymer particles; In the case of polyanion compound-immobilized particles using a synthetic polymer compound) (a) Polymerization is carried out by dispersing a magnetic material in a monomer. Examples of the polymerization method include ordinary methods such as emulsion polymerization, suspension polymerization, and dispersion polymerization. (B) After polymer particles are synthesized by the method (a), a magnetic layer is formed on the surface of the particles. (C) A layer of a polymer or a natural polymer is further formed on the surface of the particles obtained in the above (a) or (b). 2) When the base particles are a natural polymer (for example, a polyanion compound-immobilized particle using a natural polymer compound, a sulfated polysaccharide gel particle, etc.) (a) After dispersing the magnetic material in an aqueous solution of the natural polymer ,
Crosslink natural polymers into water-insoluble particles. (B) A polymer or natural polymer layer is further formed on the surface of the particles obtained in (a).

The magnetic properties of the virus concentrating particles obtained as described above have a magnetic coercive force of 4000 to 160,000 A / m, particularly 800 when subjected to a magnetic field of 1,200,000 A / m (ampere per meter). 8,000 A / m is preferable, and the residual magnetic flux density is 1 × 10 ⁻⁴ wb · m
/ Kg or less, particularly preferably 2.0 × 10 ^{−6 to} 20 × 10 ⁻⁶ wb · m / kg. If the coercive force is too small or the residual magnetic flux density is too small, the natural magnet (magnetic flux density is, for example, 0.1
(0.4 T), magnetic force is not sufficiently large between the virus concentrating particles, and it may be difficult to separate and collect the particles with high efficiency by the natural magnet. Not preferred. On the other hand, if the coercive force is too large or the residual magnetic flux density is too large, the magnetic force acting between the particles will be excessive, and the particles will be separated and separated.
After recovery, it may be difficult to redisperse.

In order to concentrate the virus in a sample such as plasma or serum using the virus-concentrated particles of the present invention, the virus is usually used not in a column chromatography but in a batch method. Therefore, the particle size of the particles is usually from 0.08 μm to 300 μm, preferably
It is 0.1 μm to 100 μm. If the particle size is within this range, it can be used for the purpose of the present invention even if the particle size is not uniform. Further, the particle shape of the virus concentration particles of the present invention need not be spherical, and may be irregularly shaped particles. The particle diameter of the non-spherical particles is an average of the longest diameter and the shortest diameter of each particle.
If the particle size is too small, good magnetic separability may not be obtained, which is not preferable. Also, if the particle size is too large,
It is not preferable because the efficiency of capturing the virus is reduced and the virus cannot be concentrated sufficiently.

The base particles of the virus concentrating particles of the present invention may be porous or non-porous. However, if the virus particles are porous, when the virus particles enter the inside of the particles, it may be difficult to separate the virus from the particles. It is not preferable that the porous body exists continuously from the inside to the inside of the particle. Examples of the virus concentration particles of the present invention include commercially available magnetic particles such as Magacell (CORTEX BIOCHEM), Dynabeads (Dynal).
), SeraMag (Seradyne) and the like can be used as they are, or if necessary, sulfonated.

<Virus Concentration Method> Next, the virus concentration method using the virus concentration particles of the present invention will be specifically described. The virus concentration particles of the present invention have a concentration ability for various viruses, for example, hepadnavirus (hepatitis B virus, etc.), adenovirus, flavivirus (Japanese encephalitis virus, etc.), herpes virus,
(Herpes simplex virus, varicella-zoster virus, cytomegalovirus, EB virus, etc.), poxvirus, parvovirus (adeno-associated virus, etc.), orthomyxovirus (influenza virus, etc.), rhabdovirus (rabies virus, etc.), retro Viruses (such as acquired immunodeficiency syndrome virus) and hepatitis C virus can be concentrated.

Samples to be subjected to virus concentration by the virus concentration particles of the present invention include various body fluids such as plasma, serum, cell lysate, urine, saliva, and cell culture. Such a sample may be used as a sample as it is, or may be used as a sample after being diluted for any purpose.

The amount of the virus concentration particles of the present invention added to a sample depends on the concentration of the virus contained in the sample.
Usually 0.05-50% by weight of sample, preferably 0.1-10% by weight
It is. If the addition amount is too small, the number of viruses that can bind to the virus concentration particles is limited, so that the concentration efficiency is deteriorated. On the other hand, if the addition amount is too large, a large amount of elimination liquid is required to desorb the bound virus at a later stage, and the concentration efficiency is reduced.

In the present invention, by adding a polyvalent metal compound to the sample together with the virus concentration particles, the binding ratio of the virus to the virus concentration particles can be further increased. Here, as the polyvalent metal, for example, Be,
Mg, Sr, Ba, Ti, Zr, Cr, Mo, W, Mn, Fe, Ru, Os, Co,
Rh, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd, Hg, Al, Ga, S
i, Ge, Sn, Pb, P, As, Sb, Bi and the like.Polyvalent metal compounds include chlorides, hydroxides, carbonate compounds, sulfate compounds, nitrate compounds, and acetates of these polyvalent metals. Among compounds, chloric acid compounds, etc., they are compounds that dissociate in water to generate divalent or higher cations. As these polyvalent metal compounds, manganese chloride, copper sulfate and the like are preferable. The amount of the polyvalent metal compound used is usually 0.1 to 100 mm in the concentration of the reaction solution when the particles for virus concentration and the sample are mixed.
ol / l.

The virus-concentrating particles adsorbing the virus in the sample are separated from the sample by magnetic separation, centrifugation or spontaneous sedimentation. Since the virus concentration particles of the present invention usually have a particle size range of 0.01 to 100 μm, centrifugation can be sufficiently performed with a centrifuge in addition to magnetic separation. The magnetically or centrifugally separated virus concentrating particles are washed with a low-concentration buffer, if necessary, and then are subjected to virus particle separation and nucleic acid extraction. Nucleic acid extraction can be performed while the virus remains attached to the particles. That is, for example, a small amount of buffer solution is added to the separated virus concentration particles, and the nucleic acid of the virus can be directly extracted by heating, or a commercially available nucleic acid extraction reagent can be directly added. It is also possible to extract viral nucleic acids.

As a method for separating the virus from the particles, there is a method in which the virus is dissociated from the virus concentration particles by the action of a salt solution. High concentration potassium bromide, sodium bromide, 1.5M sodium chloride, 1m as salt solution
M phosphotungstic acid or the like can be used. Accordingly, the present invention provides, as one aspect thereof, the above-described virus detection method.

<Virus Detection Method> This virus detection method comprises the steps of: concentrating a virus by separating and collecting from a sample particles according to the present invention to which the virus is bound as described above; The method has a step of subjecting to a nucleic acid amplification test.

Virus nucleic acid amplification test (NAT; nuclei
The method of c acid amplification test is not particularly limited. For example, PCR (Polymerization chain) of Roche
reaction) method, TMA (Transcript
ion Mediated amplification-hybridization protectio
n assay) method, Abbott's LCR (Ligase chain reacti
on) method or the like can be used. In the virus detection method using this virus concentration method, since the virus in the sample subjected to the nucleic acid amplification test has already been concentrated, even if the amount of the virus contained in the original sample or sample is extremely small, Viruses can be detected efficiently.

[0036]

Hereinafter, embodiments of the present invention will be described.
The present invention is not limited to these examples. The measurement of the particle diameter of the particles and the abundance of the anionic group present on the particle surface are values measured as follows. Measurement of particle diameter: The particle diameter is measured by taking a photograph with an optical microscope, a scanning electron microscope or a transmission electron microscope, and taking a photograph 200
The particle size of each particle was measured, and the average value was determined.・ Abundance of anionic groups: 90 g of ion-exchanged water in about 10 g of particles
g and 30 g of a cation-anion exchange resin mixture (Amberlite MB3 manufactured by Organo) were added, followed by gentle stirring for 1 hour. The ion-exchange resin was removed by filtration using a nylon mesh (48 mesh; pore size: 295 μm). After quantifying the amount of particles, conductivity titration was performed using a 0.01 mol / l sodium hydroxide solution to determine the amount of anionic groups. I asked.

[Example 1] Preparation of magnetic surface sulfonated styrene / divinylbenzene particles Magnetic styrene / divinylbenzene particles (manufactured by DYNO) 10
100 g of concentrated sulfuric acid (98% by weight) at 50 ° C.
After stirring for minutes, the particles were washed with pure water and subsequently with phosphate buffered saline (hereinafter referred to as “PBS”) to obtain magnetic sulfonated styrene / divinylbenzene particles. Table 1 shows the particle size and the amount of the anionic group.

Example 2 Preparation of Magnetic Carboxylic Acid Particles Acetone was added to oil-based magnetic fluid “Fericolloid HC50” (manufactured by Taiho Kogyo Co., Ltd.) to precipitate and precipitate the particles. Ferrite superparamagnetic material having a lipophilic surface (particle diameter: 0.01
μm). To 40 parts of the superparamagnetic material, 95 parts of cyclohexyl methacrylate, 5 parts of methacrylic acid and 3 parts of benzoyl peroxide (polymerization initiator) are added, and the superparamagnetic material is uniformly dispersed by mixing and stirring this system to obtain a monomer composition. Was prepared. On the other hand, 10 parts of polyvinyl alcohol, 0.05 part of sodium laurate and 0.1 part of polyethylene oxide nonyl phenyl ether were added to water 1
000 parts to prepare an aqueous medium. The above-mentioned monomer composition is added to the obtained aqueous medium (aqueous phase), the system is preliminarily stirred with a homomixer, and then subjected to a dispersion treatment with an ultrasonic disperser to obtain an oil having an average particle diameter of 2 μm. A suspension (oil droplet dispersion) in which droplets (oil phase) were dispersed in an aqueous medium was prepared.

Next, the resulting suspension was charged into a two-liter three-necked flask equipped with a stirrer.
C. and polymerizing the monomer in the oil droplets (suspension polymerization) for 5 hours while stirring under a nitrogen atmosphere, thereby dispersing the magnetic polymer particles of the present invention in an aqueous medium. (Magnetic polymer latex) was prepared. The obtained particles were dispersed in a 5 mM aqueous sodium hydroxide solution and treated at 80 ° C. for 12 hours to obtain surface carboxylic acid particles. Table 1 shows the particle size and the amount of anionic group present.
Shown in

Example 3 Preparation of Sulfonic Acid Monomer Seed Polymer Particles 8 g of styrene / divinylbenzene particles (manufactured by DYNO) were
100 g of water containing 0.03 g of sodium dodecyl sulfate
Dispersed. After adding 1 g of styrene and 1 g of sodium styrene sulfonate and heating to 80 ° C., 0.01 g of potassium persulfate was added and reacted for 10 hours. After the reaction,
The particles were washed with pure water and subsequently with PBS to obtain magnetic styrene sulfonic acid / styrene seed polymer particles. Table 1 shows the particle size and the amount of the anionic group.

Example 4 Preparation of Magnetic Polystyrene Sulfonic Acid-Immobilized Particles A pressure-resistant reaction vessel was charged with 35 g of styrene, 0.44 g of n-butyllithium and 200 g of cyclohexane.
After polymerization for 4 hours while maintaining the temperature at 0 to 90 ° C., the reaction was terminated by blowing carbon dioxide into the reaction vessel. Then, after distilling off the solvent and unreacted monomers under reduced pressure,
100 g of ether was added for dilution to obtain a polymer solution. To this polymer solution, 41 g of concentrated sulfuric acid was added little by little, and after stirring was continued at 50 ° C. for 5 hours, the solvent was distilled off under reduced pressure. The obtained product was neutralized with sodium hydroxide and purified by dialysis. Thus, terminal carboxylic acid-modified sodium polystyrene sulfonate (molecular weight: 26,000) was obtained.

Next, 10 g of the particles of Example 2 were dispersed in 50 g of water, 0.1 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (WSC) was added, and the mixture was reacted at 4 ° C. for 1 hour. After washing with cold water, 0.5 g of hexamethylenediamine was added, and the mixture was reacted at room temperature for 2 hours, followed by washing with pure water to obtain hexamethylenediamine-immobilized particles. To this, 0.1 g of WSC and 0.5 g of polystyrene sulfonic acid modified with a terminal carboxylic acid were added and reacted at room temperature for 2 hours. Then, the particles were washed with pure water and then with PBS to obtain sodium polystyrene sulfonate-immobilized particles. Was. Table 1 shows the particle size and the amount of the anionic group.

Example 5 Preparation of Particles Having Water-Soluble Polysaccharide Heparin was dissolved in an aqueous 0.04 mol / ml HCl solution at 100 ° C. for 1.5 hours.
The reaction was allowed to proceed for a period of time to prepare partially de-N-sulfated heparin, which was purified by reprecipitation in ethanol. Using the water-soluble carbodiimide reagent EDC · HCl (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide / hydrochloride), the amino residue of the de-N-sulfated heparin and the magnetic particles having a carboxyl group are dissolved in 10 mmol. / MlMES buffer solution (pH6.0)
The reaction was carried out at 2 ° C. for 2 hours to obtain heparin-containing magnetic particles.

Example 6 Preparation of Particles Containing Water-Soluble Polysaccharide Dextran sulfate was oxidized by stirring for 30 minutes in a 0.1 mol / l sodium periodate solution to give a 0.01 mol / l sodium carbonate buffer (pH 9. It was dialyzed against 5) and purified. This dextran sulfate, aminodiamine-containing magnetic particles obtained by reacting ethylenediamine and carboxyl group-containing magnetic particles with EDC / HCl were mixed, reacted at 4 ° C. for 12 hours, and coupled by Schiff base formation. . Further, sodium borohydride was added to reduce the Schiff base to obtain dextran sulfate-containing magnetic particles.

Example 7 4 g of acidic gelatin having an isoelectric point of pH 9 was dissolved in warm water at 40 ° C. to make 100 ml, and 10%
Was adjusted to pH 9 using a sodium hydroxide solution. Dissolve 4 g of dextran sulfate in water to 100 ml, and add
Was heated. 25 ml of the gelatin solution thus obtained
And a dextran sulfate solution (25 ml) were mixed, and the mixture was poured into a 30% by volume ethyl alcohol solution (150 ml) preliminarily heated to 40 ° C., followed by thorough stirring. 0.8 ml of a 10% sodium hexametaphosphate solution, 1 ml of a 10% alkylsulfomaleic acid solution and 300 μl of ferricolloid W-35 (manufactured by Taiho Kogyo Co., Ltd.) having an average particle size of 150 ° were added thereto, and the mixture was stirred well.

Next, while maintaining the temperature at 40 ° C., a 10% by volume acetic acid solution was added dropwise to adjust the pH to 7.2, thereby forming particles.
After the obtained particle dispersion was ice-cooled, glutaraldehyde (0.7 g) was added, and the mixture was stirred well and allowed to stand on ice overnight. The particle dispersion was centrifuged at 2000 rpm for 10 minutes, washed three times with 0.005% alkylsulfone maleic acid, dispersed in a 4% by volume formalin solution, and left at 5 ° C. for 1 week. Then, it was washed three times with distilled water and suspended in physiological saline.

Test Example 1 Virus Concentration—Nucleic Acid Quantification Virus concentration was performed using the particles obtained in Examples 1 to 4. Human plasma 1 for which the presence of HCV has been confirmed
100 mL of particle suspension (10% by weight) and 1 M Mn in mL
75 μL of a Cl ₂ solution was added, and the mixture was rotated and stirred at room temperature for 10 minutes. After completion of the reaction, the mixture was separated on a magnetic tube stand, and the supernatant was discarded to obtain particles. 50 μL of a saturated potassium bromide aqueous solution was added to the particles obtained in the above operation, and the mixture was stirred for 5 minutes and then separated by a magnetic tube stand to obtain a supernatant (separation and concentration fractionation). Final volume was approximately 50 μL.

2 μL was taken from the separated and concentrated fraction, and the nucleic acid was extracted by an ordinary method. The plasma used for separation and concentration is
A dilution series was prepared using human plasma confirmed to be nucleic acid negative for each virus, and this was used as a sample for calibration. Nucleic acids were extracted from these samples using a conventional method. This extracted nucleic acid is generally used for the 5′UTR region of the HCV gene.
The amount of HCV-RNA was quantified by the T-PCR method.

[Comparative Example] In parallel with the assay of Test Example 1, a heparin solution (160 / USPunits) was added to 1 mL of human plasma in which the presence of the same HCV as that used in Test Example 1 was confirmed.
/ mg was dissolved in 5 mL of a 0.15 M sodium chloride solution), 10 μL was added, and 1 M MnCl ₂ 75 μL was further added.
L was added and reacted at room temperature for 20 minutes. 150 after completion of reaction
The mixture was separated by a low-speed microcentrifuge at 00 rpm for 10 minutes, the supernatant was discarded, and a precipitate was obtained.
L was added to solubilize the precipitate (separation and concentration fractionation). Final volume was approximately 50 μL. Then, the same evaluation as the above-mentioned test example was performed. Table 1 shows the recovery amount, the concentration ratio, and the time required for separation and concentration. In the method of the comparative example, PCR using heparin was performed.
Nucleic acid could not be detected due to inhibition. On the other hand, in each example, the virus recovery rate was almost 100%, and the concentration rate was as high as 25 times.

[0050]

[Table 1]

[0051]

As described above, by using the virus concentrating particles of the present invention, a large number of specimens can be easily and simultaneously concentrated by a simple means of applying magnetism. Does not adversely affect nucleic acid amplification processing. The virus concentration method using these particles can efficiently concentrate the virus from a sample containing a very small amount of virus in a short time, and the virus detection method can detect the virus with higher accuracy.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/553 (C12N 7/02 // (C12N 7/02 C12R 1:93) C12R 1:93) C12N 15/00 A (72) Inventor Shozo Nishida 2--11-24 Tsukiji, Chuo-ku, Tokyo Inside JSR Co., Ltd. (72) Inventor Mikio Hikami 2-11-24 Tsukiji, Chuo-ku, Tokyo JSR Co., Ltd. (72) Inventor Sumi Kasai 2-11-24 Tsukiji, Chuo-ku, Tokyo Inside JSR Corporation

Claims (7)

[Claims] 1. Virus concentrating particles containing a magnetic substance. 2. The method according to claim 1, wherein the particle surface contains an anionic group.
The virus-concentrated particles according to 1. 3. A virus concentration reagent comprising the particles according to claim 1 and a polyvalent metal compound. 4. A step of adding the virus concentrating particles according to claim 1 or 2 to a sample which may contain a virus, allowing the virus to bind to said particles, and then said virus bound to said particles. A method for concentrating virus, comprising separating and collecting particles from a sample. 5. The virus concentration method according to claim 4, wherein a polyvalent metal compound is added to the sample together with the virus concentration particles. 6. The step of adding the virus concentrating particles according to claim 1 or 2 to a sample which may contain a virus, allowing the virus to bind to the particles, and then the step of binding the virus to the particles. Concentrating the virus by separating and collecting the particles from the sample, and subjecting the virus thus concentrated to a nucleic acid amplification test,
Virus detection method. 7. The method according to claim 6, wherein a polyvalent metal compound is added to the sample together with the virus concentration particles.