JP2001190273A - Virus concentration particles, virus concentration method using the particles, and virus detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a virus concentration particle which can easily process a large number of specimens simultaneously by simple means and does not adversely affect a nucleic acid amplification test, a virus concentration method using the particle, and Provide a virus detection method. SOLUTION: The virus concentration particles having a water-insoluble sulfated polysaccharide on the particle surface and having a particle diameter of 0.1 to 1,000 μm, a virus concentration method using the particles and a virus detection method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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.

In recent years, the possibility of detecting even a trace amount of virus has been opened 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.

[0003] A typical example of the conventional virus concentration method is an ultracentrifugation method. However, it is difficult to process a large number of samples at the same time because it requires expensive equipment and a long 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.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems of the virus concentration method, and it is possible to easily process a large number of samples at the same time by simple means, which has an adverse effect on nucleic acid amplification tests. It is an object of the present invention to provide a virus concentration particle which does not cause the virus, a virus concentration method and a virus detection method using the particle.

[0005]

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 concentrating particles having a particle diameter of 0.1 to 1,000 μm and having a water-insoluble sulfated polysaccharide on the particle surface. In addition, the present invention provides, secondly, a step of adding the above-mentioned virus concentrating particles to a sample that may contain a virus and allowing the virus to bind to the particles, and then removing the virus-bound particles to the sample. And a method for concentrating the virus, comprising the steps of separating and collecting the virus from the virus. Furthermore, the present invention provides, thirdly, a step of adding the above-mentioned virus concentrating particles to a sample that may contain a virus, and binding the virus to the particles, and then, removing the virus-bound particles into the sample. 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.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. <Virus Concentrating Particles> In the present invention, virus concentrating particles are particles that separate and concentrate after adsorbing a virus in a sample such as blood or body fluid, and are separated and concentrated by the particles.
The concentrated virus is used for nucleic acid extraction / test / diagnosis, particularly for test / diagnosis involving nucleic acid amplification. The virus concentration particles of the present invention are particles having a water-insoluble sulfated polysaccharide on the particle surface and having a particle size of 0.1 to 1,000 μm. The virus concentration particles of the present invention have a sulfone group on at least a part of the particles, preferably on the surface of the particles. The sulfone group may be present in a salt form.

In the present invention, the sulfone group, when eluted in water, a buffer solution, blood, or body fluid, may interfere with a nucleic acid amplification method such as a PCR method, and must be chemically bound to particles. . Its abundance is usually 1 × 10 ⁻¹² equivalent or more per 1 g of particles on average, typically 1 × 10 ⁻¹² equivalent.
^{-10 to} 2 × 10 ^-6 equivalents, preferably 1 × 10 ^-9 to
2 × 10 ⁻⁶ equivalent, more preferably 1 × 10 ⁻⁸ to 2
× 10 ^-6 equivalents. If the amount of the sulfone group is too small, the virus concentrating ability is often insufficient. At least a part of the surface of the virus concentration particles of the present invention is composed of a water-insoluble sulfated polysaccharide. Examples of such particles include the following sulfated polysaccharide particles, sulfated polysaccharide-immobilized particles, and the like.

Sulfated polysaccharides: Sulfated polysaccharide particles include, for example, a sulfone group obtained by treating crosslinked or non-crosslinked particles of a polysaccharide such as cellulose, agarose, dextran, chitin and chitosan with sulfuric acid. And polysaccharide particles obtained by cross-linking a sulfated polysaccharide.

[0009] Sulfated polysaccharide-immobilized particles: Sulfated polysaccharides are directly applied to particles 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. And particles carried via the carrier.

[0010] Sulfated polysaccharides 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 kaolinin sulfate. ,
Anti-viral properties such as pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, starch sulfate, polyglucose sulfate, laminarin sulfate, galactan sulfate, levan sulfate, mepesulfate, fucoidan, sulfated glycyrrhizin, etc. Polysaccharides.

When the virus concentration particles of the present invention are added to a sample solution containing a virus, the virus binds to the particles by a sulfone group present on the surface of the particle. This effect is considered to be due to the virus being positively charged. As a result, in order to concentrate virus in a specimen such as plasma or serum with high efficiency, it is usually used in a batch method instead of a column chromatography method. Therefore, the particle size is 0.1 μm
It is 1,000 μm, preferably 0.5 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 does not need to be spherical,
Irregular particles may be used. The particle diameter of the non-spherical particles is an average of the longest diameter and the shortest diameter of each particle. When the particle size is less than 0.1 μm, the number of rotations and the rotation time of centrifugation when separating the particles for virus concentration from blood or body fluid are increased, and the apparatus becomes large, or high time efficiency cannot be obtained, which is preferable. Absent. Also,
If the particle size exceeds 1,000 μm, the efficiency of capturing the virus decreases, and the virus may not be sufficiently concentrated, which is not preferable.

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.Therefore, pores having a pore size approximately equal to or larger than the virus size, for example, pores having a pore size of 10 nm or more are required. A porous body that continuously exists from the particle surface to the inside of the particle is not preferable.

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

Samples to be concentrated by the virus-concentrating particles of the present invention include plasma, serum, cell lysates, various body fluids such as urine and saliva, and cell lysates. 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-concentrating particles of the present invention added to a sample depends on the concentration of the virus contained in the sample.
Usually 0.01 to 30% by weight of sample, preferably 0.05 to 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 amount of addition is too large, a large amount of elimination liquid is required to remove the bound virus at a later stage, and the concentration efficiency is reduced.

The virus-concentrating particles adsorbing the virus in the sample are separated from the sample by centrifugation or spontaneous sedimentation. The virus concentration particles of the present invention are 0.1 to 1,000 μm.
Since it has a particle size range of m, centrifugation is possible with a small centrifuge. The centrifuged particles for virus concentration are optionally washed with a low-concentration buffer, and then subjected to the steps of separating from virus particles and extracting nucleic acids. Nucleic acid extraction
This can be done while the virus remains attached to the particles.
That is, for example, a small amount of a buffer solution is added to the separated virus concentration particles, and the virus nucleic acid can be directly extracted by heating.

As a method for separating the virus from the particles, there is a method in which a salt solution is allowed to act to dissociate the virus from the polyanion. 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. 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 separating and collecting particles according to the present invention to which the virus has been bound as described above from a sample and collecting the virus, and then concentrating the virus thus concentrated. 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.

[0020]

Hereinafter, embodiments of the present invention will be described.
The present invention is not limited to these examples. In addition, the particle diameter of a particle is a value 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.

Example 1 (1) 96 parts of styrene and 4 parts of methacrylic acid were emulsion-polymerized in water using potassium persulfate as a polymerization initiator to give a particle size of 0.1 part.
6 μm carboxy-modified polystyrene particles were synthesized. (2) Heparin was dissolved in 0.04 mol / ml HCl aqueous solution, 100 ° C,
Reaction for 1.5 hours to prepare partially de-N-sulfated heparin,
The precipitate was purified by reprecipitation in ethanol. 5 mg of the des-N-sulfated heparin and 100 mg of the carboxy-modified polystyrene particles obtained in the above (1) were combined with a carbodiimide reagent EDC.HCl (1-ethyl-
3 (3-dimethylaminopropyl) carbodiimide / sulfuric acid)
0.1 mg was reacted in a 10 mmol / ml MES buffer solution (pH 6.0) at 0 ° C. for 2 hours to obtain particles having heparin on the surface.

Example 2 (1) Dextran sulfate was oxidized by stirring in a 0.1 mol / l sodium periodate solution for 30 minutes, and dialyzed against a 0.01 mol / l sodium carbonate buffer (pH 9.5). And purified. (2) In the presence of EDC.HCl, 100 mg of the carboxy-modified polystyrene particles obtained in Example 1 (1) were reacted with 10 times equivalent of ethylenediamine of the carboxyl group of the particles,
Amino group-containing particles were obtained. (3) 5 mg of the dextran sulfate obtained in the above (1), and the above (2)
Was mixed with the amino group-containing particles obtained in the above, reacted at 4 ° C. for 12 hours, and coupled by Schiff base formation. Furthermore, sodium borohydride was added to reduce the Schiff base to obtain particles having dextran sulfate on the surface.

Test Example 1 Virus Concentration—Nucleic Acid Quantification Virus concentration was performed using the particles obtained in Examples 1 to 5 described above. Human plasma 1 for which the presence of HCV has been confirmed
100 μL of the particle suspension (10% by weight) was added to the mL, and the mixture was rotated and stirred at room temperature for 10 minutes. After completion of the reaction, the mixture was separated by a cooling microcentrifuge at 15,000 rpm for 5 minutes, and the supernatant was discarded to obtain particles. Add 50% saturated potassium bromide to the particles obtained in the above operation.
After adding μL and stirring for 5 minutes, the mixture was separated by a cooling microcentrifuge at 15000 rpm for 5 minutes to obtain a supernatant (separation and concentration fractionation). Final volume was approximately 50 μL. From the plasma used for separation and recovery, a dilution series was prepared in advance using human plasma that had been confirmed to be nucleic acid negative for each virus, and this was used as a measurement sample. Nucleic acids were extracted from this sample 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 determined by the CR method. Next, 2 μL was taken from the separated and concentrated fraction, a diluted sample was prepared in the same manner as above, and the amount of HCV-RNA was measured. Table 1 shows the recovery amount, the concentration ratio, and the time required for the separation and concentration fractionation.

[0024]

[Table 1]

[0025]

EFFECT OF THE INVENTION By using the virus concentration particles of the present invention, a large number of specimens can be easily and simultaneously concentrated by simple means, and the obtained sample containing the concentrated virus can be subjected to nucleic acid amplification treatment. Has no adverse effect on
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.

Continued on the front page (72) Inventor Shozo Nishida 2-11-24 Tsukiji, Chuo-ku, Tokyo Inside JSR Co., Ltd. (72) Inventor Sumi Kasai 2--11-11 Tsukiji, Chuo-ku, Tokyo FSR in JSR Corporation (reference) 4B063 QA01 QQ10 QR43 QR54 QS13 4B065 AA95X BC46 BD05 BD14 BD38 CA46

Claims (3)

[Claims] 1. Virus concentrating particles having a particle size of 0.1 to 1,000 μm having a water-insoluble sulfated polysaccharide on the surface of the particles. 2. The step of adding the virus concentrating particles according to claim 1 to a sample which may contain a virus, allowing the virus to bind to the particles, and then removing the particles thus bound to the sample from the sample. A method for concentrating virus, comprising the steps of separating and collecting. 3. adding the virus concentrating particles according to claim 1 to a sample which may contain a virus, allowing the virus to bind to the particles, and then removing the virus-bound particles from the sample. A virus detection method comprising the steps of: concentrating a virus by separating and collecting; and subjecting the virus thus concentrated to a nucleic acid amplification test.