Viral particle measurement method and viral particle measurement device

Abstract

The present invention efficiently quantifies the particle size distribution of viral particles, and has a staining step for adding a membrane-permeable fluorescent dye to a sample that includes viral particles to fluorescently stain contents of the viral particles, and, subsequent to the staining step, a measurement step for detecting luminescence of the membrane-permeable fluorescent dye to measure the particle size distribution of the viral particles.

Description

Technical Field

[0001] The present invention relates to a method for measuring viral particles and an apparatus for measuring viral particles.

Background Art

[0002] For example, as a method for measuring viruses in the air, as shown in Patent Document 1, a method is conceivable in which viruses floating in the air are collected, and the concentration of viruses contained in the air is measured by surface-enhanced Raman scattering spectroscopy.

[0003] Here, when performing quantification by surface-enhanced Raman scattering spectroscopy, it is necessary to establish a correlation between the signal amount of the detected surface-enhanced Raman scattered light and the amount of viruses to be analyzed. Since the signal amount of the surface-enhanced Raman scattered light greatly depends on the three-dimensional positional relationship between the generated electric field enhancement region and the analyte, in order to ensure quantification, the three-dimensional positional relationship between the noble metal fine particles on the substrate that generates surface-enhanced Raman scattering and the viruses that are the analyte needs to be controlled.

[0004] However, in practice, it is difficult to control this three-dimensional positional relationship, and a technique for performing quantification by surface-enhanced Raman scattering spectroscopy has not been established. Although Patent Document 1 also describes that "virus concentration can be measured" and "a signal corresponding to the amount of virus is output", there is no disclosure at all regarding the technique for controlling the above-described positional relationship. Also, no data indicating quantification has been disclosed at all.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, the present invention has been made to solve the above problems, and its main object is to efficiently quantify viral particles.

Means for Solving the Problems

[0007] That is, the method for measuring viral particles according to the present invention includes a staining step of adding a membrane-permeable fluorescent dye to a sample containing viral particles to fluorescently stain the contents of the viral particles, and after the staining step, a measurement step of detecting the emission of the membrane-permeable fluorescent dye to measure the particle size distribution of the viral particles.

[0008] With this method for measuring viral particles, the contents of the viral particles are stained with a membrane-permeable fluorescent dye, and the emission of the membrane-permeable fluorescent dye is detected to measure the particle size distribution. Therefore, viral particles can be identified from the information on the contents and particle size, and viral particles can be efficiently quantified.

[0009] In the staining step, the contents of the viral particles are fluorescently stained with the membrane-permeable fluorescent dye, and the viral particles are fluorescently stained with another fluorescent dye different from the membrane-permeable fluorescent dye. In the measurement step, light having an excitation wavelength corresponding to each of the membrane-permeable fluorescent dye and the other fluorescent dye is irradiated, and the emission of each of the membrane-permeable fluorescent dye and the other fluorescent dye is detected to measure the particle size distribution of the viral particles. With this configuration, not only the emission of the membrane-permeable fluorescent dye that stains the contents of the viral particles but also the emission of another fluorescent dye is detected, so that the measurement accuracy of the particle size distribution of the viral particles can be improved.

[0010] In the staining step, the contents of the viral particles are fluorescently stained with the membrane-permeable fluorescent dye, and the viral particles are fluorescently stained with another fluorescent dye different from the membrane-permeable fluorescent dye. In the measuring step, it is desirable to irradiate light having an excitation wavelength of one of the membrane-permeable fluorescent dye or the other fluorescent dye, and detect the emission of the other of the membrane-permeable fluorescent dye or the other fluorescent dye by fluorescence resonance energy transfer (FRET) to measure the particle size distribution of the viral particles. With this configuration, not only the emission of the membrane-permeable fluorescent dye that stains the contents of the viral particles but also the emission of another fluorescent dye by FRET can be detected, so that the measurement accuracy of the particle size distribution of the viral particles can be improved.

[0011] It is desirable that the other fluorescent dye fluorescently stains the surface of the viral particles.

[0012] The method for measuring viral particles of the present invention further includes a collection step of collecting viral particles floating in the air, and it is desirable that the collection step collects the viral particles in the liquid by passing the air through the liquid.

[0013] Further, the viral particle measuring apparatus of the present invention includes a light irradiation unit that irradiates light having an excitation wavelength of a membrane-permeable fluorescent dye to a sample in which the contents of viral particles are fluorescently stained with the membrane-permeable fluorescent dye, a light detection unit that detects the emission of the membrane-permeable fluorescent dye, and a particle size distribution calculation unit that calculates the particle size distribution of the viral particles using a detection signal obtained by the light detection unit.

Effects of the Invention

[0014] According to the present invention described above, viral particles can be efficiently quantified.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Explanation of Signs

[0016] 100 ··· Viral particle measuring device 2 ··· Light irradiation part 3 ··· Light detection part 4 ··· Particle size distribution calculation part

Mode for Carrying Out the Invention

[0017] Hereinafter, a viral particle measuring device and a viral particle measuring method according to an embodiment of the present invention will be described with reference to the drawings.

[0018] <Viral Particle Measuring Device> The viral particle measuring device 100 of the present embodiment measures, for example, the particle size distribution and the number concentration of viral particles floating in the air. Specifically, light is irradiated onto a sample in which nucleic acid (DNA or RNA), which is the content of viral particles, is fluorescently stained with a membrane-permeable fluorescent dye, and the emission of the membrane-permeable fluorescent dye is detected to measure the particle size distribution of viral particles. Note that as the air sampling locations, indoor, toilet, laboratory, hospital, factory, restaurant, store, live house, movie theater, public facilities, etc. can be considered.

[0019] In this specification, the "viral particle" holds biomolecules as its content, and examples thereof include viruses, vaccines, and virus-like substances (substances in which nucleic acid is artificially retained in biomolecules).

[0020] The particle size distribution is the number concentration, frequency, or probability of existence according to the particle size. This is a concept that includes the number concentration at a single particle size (generally simply expressed as "number concentration") or the total number concentration in a narrow range of particle size distributions.

[0021] Here, the membrane-permeable fluorescent dye is a membrane-permeable nucleic acid dye substance such as a DNA intercalator. Also, a hydrophobic monomer is desirable for the membrane-permeable fluorescent dye. Since the virus membrane is composed of lipids, a hydrophobic membrane-permeable fluorescent dye has more affinity and is more likely to penetrate the membrane. Specific examples of DNA intercalators include cyanine dyes such as DAPI, PI, ethidium bromide, SYBR-green I, SYBR-green II, pico green, and Hoechst dyes such as gelstar, acridine orange, Hoechst33258, and Hoechst33342. In addition, cell staining dyes such as Calcein-AM, BCECF-AM, CFSE, CytoRed, FDA, and Fura-2 can also be considered as membrane-permeable fluorescent dyes.

[0022] Specifically, as shown in FIG. 1, the viral particle measuring device 100 includes a light irradiation unit 2 that irradiates light with an excitation wavelength λ1 corresponding to the membrane-permeable fluorescent dye, a light detection unit 3 that detects the emission (fluorescence wavelength λ1') of the membrane-permeable fluorescent dye, and a particle size distribution calculation unit 4 that measures the particle size distribution of the viral particles modified with the membrane-permeable fluorescent dye using the detection signal obtained by the light detection unit 3.

[0023] The viral particle measuring device 100 is also provided with a cell installation part (not shown) where a cuvette cell 5 for batch measurement containing the sample is detachably installed. In FIG. 1, the light irradiation direction of the light irradiation unit 2 and the light detection direction of the light detection unit 3 are provided perpendicular to each other, but it is not limited to this.

[0024] The light irradiation unit 2 has a light source 21 that irradiates light with an excitation wavelength λ1 corresponding to the membrane-permeable fluorescent dye. The light source 21 is, for example, a laser light source. The light emitted from the light source 21 is guided to the cuvette cell 5 through an irradiation optical system 24 such as reflection mirrors 241 and 242 and a condenser lens 243.

[0025] The light detection unit 3 detects the emission of the membrane-permeable fluorescent dye that exits from the cuvette cell 5. In this embodiment, it is an imaging camera 31 such as a CCD camera, and outputs moving image data as a detection signal. The light detection unit 3 also has a filter 32 that blocks light with the excitation wavelength λ1 and transmits light with the fluorescence wavelength λ1'. This filter 32 is configured to selectively detect the emission of the membrane-permeable fluorescent dye.

[0026] The particle size distribution calculation unit 4 uses the moving image data to track the Brownian motion of the viral particles modified with the membrane-permeable fluorescent dye, and calculates the particle size and number of the viral particles based on the Stokes-Einstein equation from the diffusion rate. Then, the particle size distribution calculation unit 4 calculates the particle size distribution of the viral particles modified with the membrane-permeable fluorescent dye from the calculation result (see Figure 2). The particle size distribution calculation unit 4, for example, uses the moving image data obtained by imaging the emission of the membrane-permeable fluorescent dye to obtain the diffusion rate of the viral particles modified with the membrane-permeable fluorescent dye, and calculates the particle size distribution of the viral particles from the diffusion rate.

[0027] <Method for Measuring Viral Particles> Next, the method for measuring viral particles according to this embodiment will be described with reference to Figure 3.

[0028] (S1) Collection step First, viral particles floating in the air are collected. Here, as a collection method, for example, a method of collecting viral particles in a liquid by passing air through a liquid (collection liquid) such as pure water using an air sampler of a liquid cyclone method can be considered. In addition, by passing air through a filter member, viral particles are collected on the filter member, and the filter member may be immersed in a liquid to take in the viral particles into the liquid. Note that the air before passing through the liquid may be collected in a bag in advance, or may be passed through the liquid on-site without collecting it in a bag.

[0029] (S2) Staining step A membrane-permeable fluorescent dye is added to the sample prepared by the above collection step. Thereby, the nucleic acid (DNA or RNA) of the viral particles contained in the sample is fluorescently stained.

[0030] (S3) Measurement step The sample obtained by the staining step is placed in a cuvette cell 5, and the cuvette cell 5 is placed in the cell installation part of the viral particle measuring device 100. Then, in the viral particle measuring device 100, the cuvette cell 5 is irradiated with light having an excitation wavelength λ1 corresponding to the membrane-permeable fluorescent dye, and the emission (fluorescence wavelength λ1') of the membrane-permeable fluorescent dye is detected. Thereby, the particle size distribution calculation unit 4 calculates the particle size distribution of the viral particles modified with the membrane-permeable fluorescent dye based on the detection signal (moving image data) obtained by the light detection unit 3. The calculated particle size distribution of the viral particles (see Figure 2) is displayed on a display or the like of the viral particle measuring device 100.

[0031] <Effects of this embodiment> According to the method for measuring viral particles of this embodiment configured as described above, since the nucleic acid of the viral particles is stained with a membrane-permeable fluorescent dye and the particle size distribution is measured by irradiating light having the excitation wavelength of the membrane-permeable fluorescent dye, viral particles can be identified from the information on the content and the particle size, and the viral particles can be efficiently quantified. When a sample is measured using a particle size distribution measuring device, the SN deteriorates due to contaminants such as dust. However, in this embodiment, only viral particles can be selectively measured by staining specific contents.

[0032] <Other modified embodiments> Note that the present invention is not limited to the above-described embodiment.

[0033] For example, in the staining step of the above-described embodiment, while the contents of viral particles are fluorescently stained with a membrane-permeable fluorescent dye, the viral particles may be fluorescently stained with another fluorescent dye different from the membrane-permeable fluorescent dye. Here, another fluorescent dye different from the membrane-permeable fluorescent dye is a membrane-impermeable fluorescent dye that fluorescently stains specific proteins on the surface of viral particles. Then, in the measurement step, light having an excitation wavelength corresponding to each of the membrane-permeable fluorescent dye and the other fluorescent dye is irradiated, the emission of each of the membrane-permeable fluorescent dye and the other fluorescent dye is detected, and the particle size distribution of viral particles is measured.

[0034] The configuration of the viral particle measuring device 100 used in this measuring method can be as shown in FIG. 4. Specifically, the light irradiation unit 2 includes a plurality of light sources 21 and 22 that irradiate light having an excitation wavelength corresponding to each of the plurality of fluorescent dyes. The light sources 21 and 22 are, for example, laser light sources. Note that one light source may irradiate light including two excitation wavelengths.

[0035] The first light source 21 irradiates light having an excitation wavelength λ1 of the membrane-permeable fluorescent dye, and the second light source 22 irradiates light having an excitation wavelength λ2 of the membrane-impermeable fluorescent dye. Here, the excitation wavelengths λ1 and λ2 are different from the fluorescence wavelengths λ1' and λ2' generated by the emission of the two fluorescent dyes. Further, the light emitted from each of these light sources 21 and 22 is guided to the cuvette cell 5 through an irradiation optical system 24 such as reflection mirrors 241 and 242, a half mirror 244, and a condenser lens 243.

[0036] These light sources 21 and 22 are controlled by a control unit (not shown) to sequentially irradiate light of excitation wavelengths λ1 and λ2 corresponding to each fluorescent dye. Note that these light sources 21 and 22 can also be controlled to irradiate light simultaneously.

[0037] The light detection unit 3 detects the emission of each of the two fluorescent dyes emerging from the cuvette cell 5. In this embodiment, it is an imaging camera 31 such as a CCD camera, for example, and outputs moving image data as a detection signal. Further, the light detection unit 3 has a filter 32 that blocks light of excitation wavelengths λ1 and λ2 and transmits light of fluorescence wavelengths λ1' and λ2'. This filter 33 is configured to selectively detect the emission of each fluorescent marker.

[0038] Also, two fluorescent dyes (a membrane-permeable fluorescent dye and a membrane-impermeable fluorescent dye) may be configured to be excited by one type of excitation wavelength to detect two types of fluorescence. Further, the fluorescence of each of the two fluorescent dyes may be configured to be detected by different photodetectors (for example, a CCD camera).

[0039] The particle size distribution calculation unit 4 uses the moving image data to track the Brownian motion of the viral particles modified with two fluorescent dyes, and calculates the particle size and number of the viral particles based on the Stokes-Einstein equation from the diffusion rate. Then, the particle size distribution calculation unit 4 calculates the particle size distribution of the viral particles modified with two fluorescent dyes from the calculation result (see FIG. 2).

[0040] By using a membrane-permeable fluorescent dye that modifies the nucleic acid of the viral particle and a membrane-impermeable fluorescent dye that modifies a specific protein on the surface of the viral particle, it is possible to determine four cases for the sample: "not having both nucleic acid and specific protein", "having only nucleic acid", "having only specific protein", and "having both nucleic acid and specific protein".

[0041] Furthermore, in the staining step of the above-described embodiment, the content of the viral particles may be fluorescently stained with a membrane-permeable fluorescent dye, and the viral particles may be fluorescently stained with another fluorescent dye different from the membrane-permeable fluorescent dye. Here, another fluorescent dye different from the membrane-permeable fluorescent dye is a membrane-impermeable fluorescent dye that fluorescently stains a specific protein on the surface of viral particles, and is a fluorescent dye that causes fluorescence resonance energy transfer by the emission of the membrane-permeable fluorescent dye. Then, in the measurement step, light having the excitation wavelength of the membrane-permeable fluorescent dye is irradiated, and the emission of another fluorescent dye due to fluorescence resonance energy transfer is detected to measure the particle size distribution of the viral particles. Also, in the measurement step, light having the excitation wavelength of another fluorescent dye is irradiated, and the emission of the membrane-permeable fluorescent dye due to fluorescence resonance energy transfer is detected to measure the particle size distribution of the viral particles. In this measurement method, the viral particle measuring apparatus 100 may be configured to detect only the emission of another fluorescent dye, or to detect the emission of another fluorescent dye together with the emission of the membrane-permeable fluorescent dye.

[0042] As a reagent containing a membrane-permeable fluorescent dye added to the sample prepared by the collection step, the concentration of the fluorescent dye may be adjusted according to the particle concentration optimal for detection.

[0043] Also, the collection liquid for collecting the viral particles floating in the air may be adjusted to a pH at which it is easy to trap the viral particles in the air. To adjust the pH, a buffer solution such as an acetic acid buffer solution, a phosphate buffer solution, or a citric acid buffer solution may be added to the collection liquid.

[0044] Measurement may also be performed using a kit in which a fluorescent dye is added to the collection liquid and the collection and staining of viral particles are performed simultaneously. With such a kit, measurement can be performed immediately after the particles are collected.

[0045] After collection, the collection liquid may be stirred or shaken so that the collected viral particles are uniformly stained.

[0046] In addition, although the viral particle measuring apparatus 100 of the above embodiment measures the particle size distribution using the particle tracking method (PTA), it may use a dynamic light scattering method that calculates the particle size distribution based on the fluctuation of the emission intensity associated with the Brownian motion of the viral particles. Other optical analysis methods can also be applied.

[0047] In addition to the configuration of the viral particle measuring apparatus 100 of the above embodiment, as shown in FIG. 5, in the cell installation section 6, as a cuvette cell 5 for batch measurement, not only a standard cell 5A (see FIG. 5(a)), but also a small cell 5B (see FIG. 5(b)) may be configured to be installable.

[0048] The small cell 5B has a shape that equally divides the standard cell 5A in a plan view. Here, it has a shape that is quartered by bisecting the standard cell 5A vertically and horizontally. Furthermore, the four divided small cells 5B may be joined together to form an integral structure or a separate structure. Here, it is desirable to configure the plurality of small cells 5B using quartz glass with low thermal conductivity. Thereby, the temperature unevenness inside the cell can be reduced, and the influence on the Brownian motion of the viral particles can be reduced.

[0049] Furthermore, since the optical arrangements of the light sources 21 and 22 and the light detection unit 3 with respect to the cell installation section 6 are the same, when installing the small cell 5B, it is desirable to accommodate a jig 7 at the bottom of the cell installation section 6 and raise the bottom of the cell installation section 6. Since the cell installation section 6 is temperature-controlled at, for example, 25 degrees, it is desirable to use a member with good thermal conductivity such as copper for the jig 7.

[0050] With the above configuration, for example, when using the standard cell 5A, a 450 μl sample is required, whereas when using the small cell 5B, measurement can be performed with a 90 μl sample.

[0051] In the configuration where the small cell 5B is installed in the cell installation part 6, it may be configured to switch the small cell 5B to be measured within the cell installation part 6. For example, it is conceivable to switch the small cell 5B to be measured by rotating a plurality of small cells 5B within the cell installation part 6.

[0052] The light detection unit 3 of the above embodiment was an imaging camera such as a CCD camera, but it may also be one using a photomultiplier tube (PMT). When using a PMT, the sensitivity can be improved compared to the case of using a CCD camera, and even a small amount of light emission can be detected.

[0053] In the above staining step, electroporation (electroporation method) may be used. Thereby, staining of viral particles can be ensured. Also, by electroporation, even if the membrane-permeable fluorescent dye is hydrophilic or a dimer, it can be easily stained to viral particles.

[0054] Further, the viral particle measuring device 100 of the above embodiment may be configured to measure the zeta potential in addition to the particle diameter and the number (concentration) of viral particles. In this case, the average zeta potential of the entire viral particles modified with the membrane-permeable fluorescent dye is measured. Also, it is desirable to display the particle diameter, the number (concentration), and the zeta potential of the viral particles on a display device such as the display of the viral particle measuring device 100, for example, on a single screen such as on a three-dimensional graph.

[0055] Here, as a configuration for measuring the zeta potential, a configuration in which an electrode for measuring the zeta potential is provided in the cuvette cell 5 is conceivable. Also, in addition to the cuvette cell 5, a flow cell for continuous measurement may be used. And a configuration is adopted in which an electrode for measuring the zeta potential is provided in the flow cell. When using a flow cell, when measuring the zeta potential, the inflow and outflow of the sample into the cell are stopped for measurement, and the sample is allowed to flow after the measurement of the zeta potential is completed.

[0056] In the above embodiment, the particle size distribution of viral particles floating in the air was measured, but it may also measure the particle size distribution of viral particles in the human body. As a method for collecting viral particles in this case, methods such as collecting from exhaled breath, collecting from saliva or mucous membranes, etc. can be considered. Also, viral particles can be collected from livestock and animals in the same way. Furthermore, when measuring the particle size distribution of viral particles attached to the surface of an object, it is conceivable to wipe off the viral particles attached to the surface of the object with a collection sheet. Note that it is also possible to measure the particle size distribution of viral particles collected on a collection filter such as an air purifier. Moreover, it may be to collect a sample from waste liquid, purified water, sewage, a river, etc. and measure the particle size distribution of viral particles contained in the sample.

[0057] The content of the viral particle may be a biomolecule other than nucleic acid (for example, protein, lipid, sugar chain, etc.).

[0058] By using the viral particle measuring device of the above embodiment as a virus amount monitor, it is possible to grasp the infection state, perform an infection determination, or confirm the aseptic state.

[0059] In addition, various modifications and combinations of embodiments may be made as long as they do not contravene the spirit of the present invention.

Industrial Applicability

[0060] According to the present invention, the particle size distribution of viral particles can be efficiently quantified.

Claims

1. A staining step of adding a membrane-permeable fluorescent dye to a sample containing viral particles to fluorescently stain the contents of the viral particles; A measurement step of measuring the particle size distribution by detecting the emission of the membrane-permeable fluorescent dye and tracking the Brownian motion of the viral particles after the staining step. A method for measuring viral particles.

2. A staining step of adding a membrane-permeable fluorescent dye to a sample containing viral particles to fluorescently stain the contents of the viral particles and fluorescently staining the viral particles with another fluorescent dye different from the membrane-permeable fluorescent dye; A measurement step of irradiating light with an excitation wavelength corresponding to each of the membrane-permeable fluorescent dye and the other fluorescent dye after the staining step, detecting the emission of each of the membrane-permeable fluorescent dye and the other fluorescent dye, and measuring the particle size distribution of the viral particles. A method for measuring viral particles.

3. A staining step of adding a membrane-permeable fluorescent dye to a sample containing viral particles to fluorescently stain the contents of the viral particles and fluorescently staining the viral particles with another fluorescent dye different from the membrane-permeable fluorescent dye; A measurement step of irradiating light with an excitation wavelength of one of the membrane-permeable fluorescent dye or the other fluorescent dye after the staining step and detecting the emission of the other of the membrane-permeable fluorescent dye or the other fluorescent dye by fluorescence resonance energy transfer, and measuring the particle size distribution of the viral particles. A method for measuring viral particles.

4. The method for measuring viral particles according to claim 2 or 3, wherein the other fluorescent dye fluorescently stains the surface of the viral particles.

5. Further comprising a collection step of collecting viral particles floating in the air, The method for measuring viral particles according to any one of claims 1 to 4, wherein the collection step collects the viral particles in the liquid by passing the air through the liquid.

6. A light irradiation unit that irradiates light with an excitation wavelength of the membrane-permeable fluorescent dye on a sample in which the contents of the viral particles are fluorescently stained with the membrane-permeable fluorescent dye; A light detection unit that detects the emission of the membrane-permeable fluorescent dye; A viral particle measurement device comprising a particle size distribution calculation unit that calculates the particle size distribution by tracking the Brownian motion of the viral particles using a detection signal obtained by the light detection unit.

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