JP2002045176A - Method for concentrating virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for concentrating virus in a specimen. SOLUTION: This method for concentrating virus is characterized by comprising a step wherein particles are added to a specimen which may contain virus, the virus, if any, is bound to the particles, and the resulting particles are collected using a filter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a virus concentration method for concentrating a virus in a specimen.

[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, etc. 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. It is an object of the present invention to provide a virus concentration method which is easy and does not adversely affect a nucleic acid amplification test.

[0006]

As a result of research, the present inventors have developed the following virus concentration method as a means for solving the above-mentioned problems. That is, the present invention provides a method for concentrating virus, which comprises a step of adding particles to a sample that may contain a virus, binding the virus to the particles, and then collecting the particles by a filter. To provide.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, the particle is a virus concentration particle that separates and concentrates after adsorbing a virus in a sample such as blood or body fluid, and the virus separated and concentrated by the particle is used for nucleic acid extraction, examination, diagnosis, In particular, it is used for testing and diagnosis involving nucleic acid amplification. Samples to be subjected to virus concentration by the method of the present invention include various body fluids such as plasma, serum, cell lysate, urine, saliva, and cell lysate.
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. In addition, as a virus that can be concentrated,
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., pox virus, parvovirus (adeno-associated virus, etc.), orthomyxovirus (influenza virus, etc.), rhabdovirus (rabies virus, etc.) ),
Retrovirus (acquired immunodeficiency syndrome virus, etc.),
Hepatitis C virus and the like.

The particles that can be used in the present invention include polymers, inorganic compounds, natural polysaccharides and composites thereof, and all of them need to be water-insoluble. The surface of the particles has an anionic group, a cationic group, and the like, if necessary, and may further contain a magnetic substance. Preferred particles in the present invention include polymer particles containing a magnetic material, a primary amino group, a secondary amino group,
It is a particle on which an amine compound containing a group selected from the group consisting of a quaternary amino group, a quaternary ammonium group, an imino group, an amidino group, an imidino group, a hydrazino group, and a nitrogen-containing cyclic group is immobilized. The average particle diameter of the particles used in the present invention is from 0.08 to 300 μm, and the method of the present invention is particularly suitable when particles having an average particle diameter of 5 μm or less are used. Further, the shape of the particles is not limited at all, and may be a fixed shape such as a spherical shape, or a collection of irregular shaped particles. The particle diameter of the non-spherical particles is an average of the longest diameter and the shortest diameter of each particle.

The use ratio of the particles of the present invention depends on the concentration of the virus contained in the sample and the shape of the particles, but is usually 0.001 to 50% by weight, preferably 0.005 to 10% by weight of the sample. If the amount of the particles used is too small, the number of viruses that can be adsorbed on the particles is limited, so that the concentration efficiency deteriorates. On the other hand, if the addition amount is too large, a large amount of elimination liquid is required to desorb the adsorbed virus at a later stage, and the concentration efficiency is reduced.

The particles adsorbing the virus in the sample are separated from the sample, and the present invention is characterized in that a filter is used in this step. The filter that can be used in the present invention has a maximum pore size smaller than the particle size of the particles used, and preferably has a maximum pore size of 0.1 to 5 μm.
A filter in the range of 0μ, a flat membrane, a hollow fiber membrane,
Non-woven fabrics and woven fabrics can be exemplified. When the specimen is a plasma or a culture solution, the maximum pore size is preferably 0.1 μm to 5 μm,
When cells and the like are included, the maximum pore size is preferably 5 to 50 μm. If the maximum pore diameter is 0.1 μm or less, a sufficient filtration amount cannot be obtained because the pore diameter becomes small. In addition, some viruses are trapped depending on the size if the pore size is 0.1 μ or less. The water permeability of the filter of the present invention is 10 ml.
/ min / m ² / mmHg or more, preferably 100 ml / min / m ² / m
The pressure is preferably at least mHg because the filtration pressure can be reduced.

In the case of a non-woven base material, a filter
The filament forming the material may be a monofilament or a multifilament, but if the average diameter is 100 μm or less, preferably 50 μm or less,
This is preferable because the surface area of the film increases and the number of adsorption sites increases. Further, the ratio of the porosity of the filter (porosity) is 20% or more, preferably 50% or more.
The material of the base material is not particularly limited, and a natural polymer such as cellulose or a derivative thereof, or a polymer material such as polyolefin, polyamide, polyimide, polyurethane, polyester, polysulfone, or polyacrylonitrile can be exemplified, but it is preferable. Preferably, the base film is made of a material having excellent dimensional stability and low swelling with respect to water, for example, polypropylene or polyvinylidene fluoride, and is preferably modified into a hydrophilic filter by surface treatment. .

In the present invention, by bringing the sample into contact with the above-mentioned filter, the virus-adsorbed particles can be collected by the filter.

Next, as a method for separating the virus from the particles separated by the filter, the particles and the virus are dissociated by a method such as the action of a salt solution. As the salt solution, a high concentration of potassium bromide, sodium bromide, 1.5 M sodium chloride, 1 mM phosphotungstic acid or the like can be used.

The virus concentrated by the method of the present invention can be subjected to other immunological tests such as a nucleic acid amplification test. For example, in the case of viral nucleic acid amplification test,
First, nucleic acids are extracted from the virus. The nucleic acid can be extracted by adding a small amount of buffer to the separated virus and heating it, or by directly adding a commercially available nucleic acid extraction reagent to extract the nucleic acid of the virus. This nucleic acid extraction can be performed while the virus remains bound to the particles. Virus nucleic acid amplification test (NA
The method of nucleic acid amplification test (T; nucleic acid amplification test) is not particularly limited. For example, PCR (Polymerizatio) of Roche
n chain reaction) method, TMA (Tr
anscription Mediated amplification-hybridization p
rotection assay), Abbott's LCR (Ligase chai
n reaction) 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 original sample or the virus contained in the sample is extremely small, Viruses can be detected efficiently.

[0015]

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 size: The particle size was determined by taking a photograph with an optical microscope, a scanning electron microscope or a transmission electron microscope, measuring the particle size of 200 particles, and calculating the average value.

Preparation of Magnetic Carboxylic Acid Particles Acetone is added to an oil-based magnetic fluid “Fericolloid HC50” (manufactured by Taiho Kogyo Co., Ltd.) to precipitate and precipitate the particles. Ferrite superparamagnetic material (particle diameter: 0.01
μm). To 40 parts of the superparamagnetic substance, 95 parts of cyclohexyl methacrylate, 5 parts of methacrylic acid and 3 parts of benzoyl peroxide (polymerization initiator) are added, and the superparamagnetic substance is uniformly dispersed by mixing and stirring the 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 obtained suspension was charged into a two-liter flask with a stirrer having a capacity of 2 liters, and the temperature of the system was raised to 75 ° C. while stirring under a nitrogen atmosphere.
Polymerize monomer in oil droplets over time (suspension polymerization)
Thereby, a magnetic particle dispersion (magnetic polymer latex) having an average particle diameter of 2.0 μm, in which the magnetic polymer particles of the present invention were dispersed in an aqueous medium, was prepared. The resulting particles were dispersed in a 5 mM aqueous sodium hydroxide solution,
C. for 12 hours to obtain surface carboxylic acid particles. Next, 1 g of the obtained surface carboxylic acid particles were dispersed in 50 g of water, 10 mg of an anti-HBV antibody was added, the mixture was reacted at 37 ° C. for 2 hours, and washed with cold water to obtain virus concentration particles.

Example 1 (1) The aqueous dispersion of the virus concentration particles obtained above was purified and then adjusted to a solid concentration of 5% by weight with physiological saline. (2) Human plasma containing 1 × 10 ⁵ copies / ml of HBV was serially diluted using viral DNA negative plasma, and HBV was reduced to 10 ⁴ to 1
Samples were prepared containing ^one copy / ml. 100 μL of the particle suspension (5% by weight) prepared in the above (1) and 100 μL of a 1M aqueous solution of zinc acetate were added to 5 ml of the sample, and the mixture was stirred at room temperature for 10 minutes to perform a virus adsorption treatment. After the virus adsorption treatment, the sample was set on a magnetic separation stand, the particles and the supernatant were separated, and the supernatant was removed. (3) 100 μL of a 1 mol / l aqueous solution of sodium thiocyanate was added to the virus-adsorbed particles to dissociate the virus from the particles. The suspension of the particles and the virus was filtered through a 0.45 μm pore size centrifugal filter (Ultrafree C3HV: trade name, manufactured by Millipore) to separate the particles. (4) From the filtrate obtained in (3) above, use the Smitest EX
Nucleic acids were extracted using R & D (trade name, Genome Science Institute) according to the attached protocol. Example 2 (1) The virus concentrate obtained by adsorbing the virus obtained in Example 1 (2) was added to the protein diluent 40 from Sumytest EX R & D.
0 μL was added, and the mixture was incubated at 55 ° C. for 30 minutes to lyse the virus, and the virus nucleic acid was brought into solution. (2) The mixture of the virus nucleic acid and the virus concentration particles obtained in (1) above is filtered through a 0.45 μm pore size centrifugal filter (Ultrafree C3 HV: trade name, manufactured by Millipore) to filter the particles. Different. (3) Stytest EX R is added to the filtrate obtained in (2) above.
250 μL of the protein solution was added from & D, incubated at 55 ° C. for 30 minutes, and the nucleic acid was extracted according to the attached procedure.

Example 3 (1) The virus concentrate obtained by adsorbing the virus obtained in Example 1 (2) was added to the protein diluent 40 from Sumitest EX R & D.
0 μL was added, and the mixture was incubated at 55 ° C. for 30 minutes to lyse the virus, and the virus nucleic acid was brought into solution. (2) To a mixture of the virus nucleic acid and the virus concentration particles obtained in (1) above, 300 μL of ethanol was added,
Apply to the silica matrix filter included in the DN easy Tissue Kit (trade name) manufactured by Qiagen.
After centrifugation for minutes, the nucleic acid was adsorbed on the filter. afterwards,
Wash the filter twice with the washing solution provided with Kit, and add 1 part of distilled water.
Eluting the nucleic acid adsorbed on the filter with 00 μL, applying the eluate to the filter again to elute the nucleic acid,
The nucleic acid was obtained by freeze-drying.

Comparative Example 1 A nucleic acid was extracted from 100 μL of a plasma sample containing HBV at 10 ⁴ to 10 ¹ copies / ml by using Smitest EX R & D. DNA was amplified from the extracts obtained in Examples 1 to 3 and Comparative Example 1 by the PCR method in the amplification process of 35 times of PCR. PCR
Primer sequence is Hiroaki Okamoto, Igakuno ayum
i, (1992), 162 (9), PP544-549. PCR obtained
The amplified product was subjected to electrophoresis using 3% agarose, DNA was visualized by ethidium bromide staining, and photographed on a Polaroid (registered trademark) photograph. The amount of the obtained target DNA was determined in four steps of-, +, ++, +++ from the fluorescence intensity of the band. Table 1 (Example 1), Table 2 (Example 2), Table 3
The results are shown in (Example 3) and Table 4 (Comparative Example 1).

[0020]

[Table 1]

[0021]

[Table 2]

[0022]

[Table 3]

[0023]

[Table 4] In the comparative example, 1 × 1
0 It was necessary that the sample had a virus concentration of ³ copies / ml or more, but by using the method of the present invention, Examples 1 to
As shown in 3, the virus nucleic acid was able to be detected from a sample having a virus concentration of 1 × 10 ¹ copy / ml or more.

[0024]

The virus-concentrating material of the present invention can be used for recognition, separation (removal), and concentration of viruses, so that it can be widely used in industries such as the food industry, the fermentation industry, and the pharmaceutical industry. Material. In addition, since the virus can be recognized even in a solution containing a protein component such as plasma, it is effective for prevention, treatment, diagnosis, and the like of virus contamination and virus infection in medical care and daily life. When the virus-concentrating material of the present invention is in the form of particles, a large number of specimens can be easily and simultaneously concentrated by a simple means of centrifugation or the action of magnetism. Does not adversely affect nucleic acid amplification processing.
In the virus concentration method using these particles, the virus can be efficiently concentrated in a short time from a specimen containing a very small amount of virus, and as a result, the virus can be detected with higher accuracy. In addition, the particles of the present invention can be subjected to immunochemical measurement after adsorbing a virus from a specimen. Further, when used as a porous membrane, it is easy to handle, has a selective interaction with a virus, is simple, and has a high processing speed.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Mikio Hijikata 2-11-24 Tsukiji, Chuo-ku, Tokyo JAE S.R. 2965-37 (72) Inventor: Kenji Tanaka 3-15-14-107 Yoga, Setagaya-ku, Tokyo F-term (reference) 4B065 AA95X BD14 BD21 CA46

Claims (2)

[Claims] 1. A method for concentrating virus, comprising the steps of adding particles to a sample that may contain a virus, binding the virus to the particles, and collecting the particles by a filter. 2. The method according to claim 1, wherein the particle size is 0.08 to 300 μm.