JP2007020565A - Method and system for detecting viruses in a sample - Google Patents

Abstract

A detection method and a detection apparatus for detecting a virus with high sensitivity are provided.
A step of preparing a sample solution by mixing a fluorescently labeled or fluorescent virus-binding substance with a sample, and a step of measuring a time course of a fluorescence signal of the sample solution using a confocal optical system A method for detecting a virus or / and a virus-related substance derived from a virus-infected cell in a sample, comprising: The virus-binding substance is selected from the group consisting of sugars, antibodies, proteins, peptides, nucleic acids, lipids, and low molecular compounds.
[Selection] Figure 2

Description

  The present invention relates to a rapid and sensitive test method for detecting a virus in a sample, and a reagent and an instrument for detecting a virus in a sample.

  Recently, viral infections such as new pneumonia and avian influenza have become a major threat worldwide. Viruses are one of the causes of various diseases in humans, animals and plants, and virus confirmation is very important for testing and diagnosis. In particular, in order to overcome viral infections for which antibiotics do not work, it is essential to establish rapid diagnostic methods together with the development of therapeutic drugs and to take immediate measures for public health as well as to help with treatment. . In addition, since various antiviral agents have begun to be applied clinically, a technique for detecting extremely low concentrations of viruses is also required in order to confirm their therapeutic effects. Furthermore, in order to ensure the safety of biopharmaceuticals produced by cell culture or the like, it has become important to detect viruses with high sensitivity.

  As a conventional virus inspection / diagnosis method, immunological measurement of a virus antigen or an antiviral antibody is generally used. However, during weeks to months after virus infection, the amount of virus or antiviral antibody is small, and in many cases it cannot be detected by these immunoassays. Therefore, there is an urgent need to develop a technology that can detect a virus under the limit of immunological measurement with high sensitivity in order to shorten the period during which it cannot be detected as much as possible. In addition, current inspection methods require engineers and equipment with specialized knowledge, and can only be performed at large hospitals, clinical laboratory laboratories, or at universities or public laboratory level or higher.

  In recent years, the possibility of detecting even a trace amount of virus has been opened by nucleic acid amplification techniques represented by the polymerase chain reaction (PCR) method. However, detection of a trace amount of virus by PCR or the like requires special facilities and advanced techniques, and it is extremely difficult to carry out easily in general facilities.

  On the other hand, in the case of human influenza, excellent therapeutic drugs have been put on the market in recent years, but the administration conditions are within 48 hours after infection, and thereafter, the therapeutic effect is extremely lowered. This suggests that there is a high risk that detection cannot be performed because the detection sensitivity is too low with the current simple test method, even though it is important to diagnose at a low viral load immediately after virus infection. . In addition, the accuracy is not sufficient, and in recent avian influenza, the final definitive diagnosis had to wait for the result of the virus isolation test. Influenza is currently concerned about the emergence of new viruses all over the world, and the establishment of a highly sensitive and accurate rapid test method is an urgent issue.

  Therefore, the present invention produces a virus detection method, a detection apparatus and a reagent therefor that can immediately respond to comprehensive public health measures including clinical sites and even animals, and is a rapid, highly sensitive and reliable diagnostic system. The purpose is to provide.

  The inventors have conducted extensive research to develop a test method for detecting a virus in a sample quickly and with a simple system, by combining a confocal optical system with an appropriate fluorescent labeling reagent. The inventors have found that virus detection can be performed quickly, with high sensitivity and with high reliability, and the present invention has been completed.

That is, the present invention is as follows.
1. In a sample, including a step of preparing a sample solution by mixing a fluorescently labeled or fluorescent virus-binding substance with a sample, and measuring a time course of a fluorescence signal of the sample solution using a confocal optical system And / or a virus-related substance derived from a virus-infected cell.
2. Item 2. The method according to Item 1, wherein the virus-binding substance is selected from the group consisting of sugars, antibodies, proteins, peptides, nucleic acids, lipids, and low-molecular chemical substances.
3. Item 2. The method according to Item 1, wherein the virus is in a particle state and binds to a fluorescent labeling substance on the surface of the virus.
4). Item 2. The method according to Item 1, wherein part or all of the virus particles are physically or chemically disrupted in advance.
5. Item 5. The method according to any one of Items 1 to 4, further comprising a pretreatment step for extraction and purification of a virus in the sample.
6). Item 6. The method according to any one of Items 1 to 5, wherein the virus has an average particle diameter of 1 to 1000 nm in the minor axis and 5 to 10,000 nm in the major axis.
7). Item 7. The method according to any one of Items 1 to 6, wherein the virus is an influenza virus.
8). A detection system for a virus or / and a virus-related substance derived from a virus-infected cell, comprising a fluorescently labeled or fluorescent virus-binding substance and a confocal optical system.
9. Item 9. The system according to Item 8, wherein the virus-binding substance is selected from the group consisting of sugars, antibodies, proteins, peptides, nucleic acids, lipids, and low-molecular chemical substances.
10. The fluorescence wavelength of the fluorescently labeled or fluorescent virus-binding substance is 350 to 800 nm, the molecular weight is 120 or more, and the confocal region of the confocal optical system is 10 ⁻¹⁶ to 10 ⁻¹⁰ liter. 10. The system according to item 8 or 9.
11. Item 11. The system according to any one of Items 8 to 10, wherein the virus has an average particle diameter of 1 to 1000 nm in the minor axis and 5 to 10,000 nm in the major axis.
12 Item 12. The system according to any one of Items 8 to 11, wherein the virus to be measured is an influenza virus.

Examples of viruses to be measured in the present invention include adenoviruses (human and monkeys, animal adenoviruses such as cows, pigs, dogs, mice, and birds), herpes viruses (herpes simplex virus, cytomegalovirus, varicella-zoster virus, EB virus, human herpesvirus type 6,7,8, animal herpesviruses such as bovine, horse, sheep, goat, carp, etc., poxvirus (decubitus virus, cowpox virus, vaccinia virus, monkeypox virus, infectious genus ), Polyomavirus (BK virus, JC virus, etc.), papillomavirus (human and animal papillomaviruses such as horse, sheep), parvovirus (human and animal parvovirus, adeno-associated virus, etc.), hepatitis Virus (A, B, C, D, E, F and G liver Viruses, TT viruses, etc.), picornaviruses (polioviruses, coxsackie viruses, echoviruses, enteroviruses, rhinoviruses, etc.), caliciviruses (noro (small spherical) viruses, etc.), astroviruses (human and animal astroviruses, etc.), Togavirus (rubella virus, chikungunya virus, western equine encephalitis virus, eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc.), flavivirus (Japanese encephalitis virus, West Nile fever virus, dengue virus, yellow fever virus, etc.), influenza virus (human) A, B and C influenza viruses, animal influenza viruses such as swine, horses and birds), paramyxovirus (parainfluenza virus, measles virus, mumps virus, RS virus) , Rinderpest virus, canine distemper virus, Newcastle disease virus, Hendra virus, Nipah virus, etc., rhabdovirus (rabies virus, vesicular stomatitis virus, etc.), filovirus (Ebola virus, Marburg virus etc.), coronavirus (SARS (SARS) Severe acute respiratory syndrome) virus, human and bovine, porcine, equine, chicken and other coronaviruses), arterivirus, bunyavirus (such as hantavirus), arenavirus (such as Lassa virus, lymphocytic choriomeningitis virus), Bornavirus, reovirus (rotavirus, orthoreovirus, etc.), retrovirus (HTLV (human T lymphocyte tropic virus, HIV (human immunodeficiency virus), SIV (monkey immunodeficiency virus), rous sarcoma virus, animal virus) Etc. b virus), insect virus (poxvirus, iridoviruses, parvoviruses, baculoviruses), plant viruses, such as bacteriophage and the like.

Samples that may contain viruses or / and virus-related substances derived from virus-infected cells include mammals such as blood, serum, plasma, saliva, cerebrospinal fluid, urine, feces, feces, lymph, tears, and various organs (Humans, cows, horses, pigs, wild boars, sheep, rabbits, especially humans), birds (chicken, ducks, quail, turkeys, ducks, pheasants, etc.), invertebrates (insects; silkworms, bees, ants, stag beetles, beetles) , Crustaceans; shrimp, crabs, etc.), plants (mulberry, red beans, broad beans, tomatoes, eggplant, cucumbers, melons, tobacco, chrysanthemums, lilies, roses, etc.) biological samples, foods (eggs, milk, soybeans, Examples include samples derived from the environment such as cereals such as wheat and rice, seafood, processed foods, rivers, and soils.
The virus-binding substance is not particularly limited as long as it specifically binds to the virus. Sugar, antibody, protein (including glycoprotein), peptide, nucleic acid, lipid (including glycolipid), low molecular chemical substance Etc. are widely exemplified. For example, fetuin can be preferably used as a virus-binding substance for detection of influenza virus. Preferred virus-binding substances include antibodies specific for viruses, proteins such as fetuin, and glycolipids such as ganglioside LysoGM3.

Specific combinations of viruses and virus-binding substances are exemplified below:
The most preferable virus-binding substance is a specific antibody of each virus and a receptor-like substance of each virus. When the receptor is a protein as shown below, it may be a full-length protein, a partial-length protein or a peptide containing a binding site with a virus. CD155 for poliovirus, CD46 and CD150 for measles virus, CD155 for rinderpest virus and canine distemper virus, laminin receptor for Sindbis virus, a-dystroglycan for Lassa fever virus and lymphocytic choriomeningitis virus, CAR for coxsackie virus, CD55 and avb3 integrin, CD55 and a2 b1 integrin for echovirus, aminopeptidase N for human coronavirus, Bgp1 for mouse hepatitis virus, CD4 for HIV and SIV, chemokine receptors (such as CXCR4 and CCR3) and galactosylceramide, for avian leukemia virus TVA and TVB, MCAT-1 and PiT-2 for murine leukemia virus, PiT-1 for feline leukemia virus and simian sarcoma virus, herpes simplex virus HveA, HveB, HveC, Prr1 and Prr2, against CD46 against human herpesvirus 6, CD4 against human herpesvirus 7, CR2 against EB virus, ICAM-1 and LDLR against rhinovirus, CAR against adenovirus, avb3 integrin and avb5 Examples include integrins. In addition, in a virus having a sugar chain as a receptor, a glycoprotein, glycolipid or free sugar chain containing the corresponding sugar chain is bound to the virus. For example, sialic acid for influenza viruses A and B, parainfluenza virus, reovirus 3, mouse polyoma virus, canine parvovirus and adenovirus, 9-O-acetylsialic acid for influenza virus C, human and N-acetyl-9-O-acetylsialic acid functions as a receptor in bovine coronavirus, galactosylceramide in HIV, and heparin sulfate functions as a receptor in herpes simplex virus and human cytomegalovirus.

  Moreover, an antiviral agent is mentioned as a virus binding substance. Neuramidase inhibitors such as zanamivir and oseltamivir phosphate for influenza viruses and viral ion channel inhibitors such as amantadine hydrochloride and rimantadine hydrochloride, virus-specific DNA or RNA polymerase inhibitors for various viruses and their in vivo metabolites such as simple Reverse transcriptase inhibitors for retroviruses such as acyclovir and vidarabine for herpes virus, ganciclovir for cytomegalovirus, zidovudine, didanosine, zalcitabine, sanylvudine and lamivudine for HIV, lamivudine for hepatitis B virus, etc. Efavirenz, nevirapine and delavirdine mesylate for HIV, or protease inhibitors for HIV, such as mesyl Examples include, but are not limited to, saquinavir, ritonavir, indinavir, nelfinavir mesylate, and amprenavir, and derivatives that have been chemically modified to enhance binding to viruses or to perform fluorescent labeling. Also included are antiviral drugs that are included and will be developed in the future. Further, lectins that bind to the sugar chain structure on the surface of the virus, such as HIV and concanavalin A, can be mentioned. In addition, nucleic acid sequence binding substances such as DNA, RNA, and peptide nucleic acid having a complementary sequence that binds to viral DNA or RNA can be mentioned.

  The virus-binding substance may be used alone and fluorescently labeled. The virus-binding substance may be a polymer (for example, a hydrophilic polymer such as chitosan, polyvinyl alcohol, polyacrylic acid, or polymethacrylic acid) or fine particles (for example, quantum dots). In this case, the polymer / virus binding substance may be further bonded to the polymer or the polymer / microparticle. By adopting such a configuration, a plurality of virus-derived substances such as proteins constituting the virus bind to a plurality of adjacent virus-binding substances, and the sensitivity can be further increased. The size of the fine particles is not particularly limited as long as they can be suspended for a certain period of time, and the material of the fine particles may be either organic fine particles such as polymer fine particles or inorganic fine particles such as quantum dots. The virus-binding substance may be adsorbed on a polymer or fine particles, and may be linked by a covalent bond via a spacer as necessary.

  It is also possible to use a combination of two or more virus-binding substances to determine similar virus types. For example, a virus-binding substance (a sugar chain having a terminal sialic acid in 2,6- or 2,3-linked mode) that is selective for human influenza virus or avian influenza virus is bound to a polymer or carrier as required. By using these to inspect a virus-containing specimen, the type / type of virus can be detected more reliably.

  As the fluorescent label, one having strong and stable fluorescence intensity is preferable. For example, Alexa, various types of rhodamine (rhodamine 6G, rhodamine green, TMR, TAMRA), Bodipy, Cy5, R6G, FAM, JOE, ROX, EDANS, and the like can be preferably used, but are not limited thereto. The fluorescent label is bound to a virus-binding substance according to a conventional method.

  The fluorescence wavelength of the fluorescent labeling reagent is exemplified by about 350 to 800 nm. The molecular weight of the fluorescent labeling reagent is not particularly defined, but is preferably 20000 or less, more preferably 120 to 80,000.

The confocal optical system used in the present invention is a technique conventionally used for a microscope (confocal microscope), and is applied to a fluorescence correlation spectroscopy (FCS) method and the like. The FCS has four parts: a laser unit that excites fluorescent molecules, a confocal optical system, a fluorescence detection unit, and a digital correlator that performs calculation and analysis. Although FCS has been applied to the detection of low molecular weight substances such as antigen-antibody reactions, SNP typing, DNA-protein interactions, and small molecule-protein interactions, it can detect viruses that are much larger than proteins and nucleic acids. It was not known at all that it could be applied to.
As the confocal optical system, a confocal (laser) microscope can be used, but the function of scanning the laser is not necessarily required, and it is only necessary to measure the fluorescence intensity at one point (for example, the sub-femtoliter region) in the solution.

The measurement by the confocal optical system (FCS) can be performed by the following procedures (i) to (v).
(i) The virus-binding substance in the sample is fluorescently labeled in advance. However, this operation can be omitted when the virus-binding substance has fluorescence.
(ii) Focus the laser beam to an area below femtoliter with an objective lens.
(iii) Hundreds to thousands of photons are generated within a millisecond or less during which the molecules pass through the focal region of the laser.
(iv) The fluorescence-labeled virus-binding substance in the solution increases the molecular size due to binding with the virus, so that the moving speed in the solution becomes slow.
(v) Analyze the change in movement speed and fluorescence intensity by autocorrelation method and observe the intermolecular interaction.

The confocal region of the confocal optical system is preferably about 10 ⁻¹⁶ to 10 ⁻¹⁰ liter, preferably about 10 ⁻¹⁶ to 10 ⁻¹³ liter.

A sample containing virus may be subjected to virus measurement after crushing by ultrasonic treatment, surfactant treatment, or the like. By doing in this way, it is possible to improve the measurement sensitivity of a virus.
The average particle diameter of the virus or its crushed material is about 1 to 1000 nm in the minor axis and about 5 to 10,000 nm in the major axis, preferably about 10 to 200 nm in the minor axis and about 10 to 1000 nm in the major axis. If the average particle diameter of the virus or its crushed material is too small, the detection sensitivity decreases, and if it is too large, detection becomes difficult.

The amount of virus in the sample solution is not particularly limited, and for example, about 1 × 10 ⁵ to 1 × 10 ¹⁵ pfu / ml is exemplified.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

The virus solution preparation method, virus infectivity titer measurement method, virus partial purification method, and virus particle crushing method used in this example are as follows.
<Preparation method of virus solution>
For A / New Caledonia / 20/99 (H1N1) and A / Hyogo / 73/2002 (H1N1) strains, virus solutions were prepared using tissue culture cells. MDCK (Madin-Darby canine kidney) cell monolayer culture is inoculated with virus so that the Multiplicity of Infection (MOI) is 0.01 (0.1% of cells are infected by calculation), and 5μg / ml trypsin (manufactured by Sigma) is added. The cells were cultured in serum-free Minimum Essential Medium (MEM, manufactured by Sigma) at 34 ° C. for 3 to 5 days. The culture supernatant was centrifuged at 2,500 g for 10 minutes, and the supernatant was stored as a virus solution at −80 ° C.
About A / Puerto Rico / 8/34 (H1N1), the virus liquid was prepared using the growing chicken egg. Seed virus is diluted 1x10 ³ to 10 ⁴ times (infectivity titer is 1x10 ³ to 10 ⁴ CIU / ml) and 200 μl is inoculated into the chorioallantoic cavity of 10-day-old eggs, and 2 to Cultured for 3 days. The collected chorioallantoic fluid was centrifuged at 2,500 g for 10 minutes, and the supernatant was stored as a viral fluid at -80 ° C.

CIU (Cell Infecting Unit) is a unit of the number of infectious virus particles, and 1 CIU is theoretically equal to one infectious particle.
<Measurement method of infection titer>
MDCK cells were inoculated with 4-fold serially diluted virus solution, cultured at 34 ° C. for 14 hours, and infected cells were fixed with ethanol. Indirect using anti-influenza virus rabbit polyclonal antibody as primary antibody (distributed by Dr. Yoshinobu Okuno, Osaka Prefectural Public Health Research Institute), followed by FITC-conjugated anti-rabbit IgG goat serum (manufactured by Medical Biology Institute) as secondary antibody Infected cells were labeled by the fluorescent antibody method, observed with a fluorescence microscope (manufactured by Zeiss), counted by image analysis, and the infectious titer was calculated.
result:
A / New Caledonia / 20/99 (H1N1): 3.9x10 ⁶ CIU / ml
A / Hyogo / 73/2002 (H1N1): 5.6x10 ⁷ CIU / ml
A / Puerto Rico / 8/34 (H1N1): 1.1x10 ⁸ CIU / ml
<Partial virus purification method>
A partially purified virus was produced for A / New Caledonia / 20/99 (H1N1). The virus solution prepared by the above method was layered on 60% glycerol phosphate buffered saline (PBS) stacked with 20% glycerol PBS, and centrifuged at 113,000g for 1 hour at 4 ° C (using Hitachi ultracentrifuge). . Collect a viral layer between 60% glycerol PBS and 20% glycerol PBS, layer on a 100% glycerol cushion, centrifuge at 113,000g, 4 ° C for 1 hour (using Hitachi ultracentrifuge), and concentrate. Purified virus. By this operation, the virus solution was purified and concentrated to a concentration of about 100 times.
<Virus particle crushing method>
Treat A / New Caledonia / 20/99 (H1N1) virus solution with ultrasound (Nippon Seiki Seisakusho) for 5 seconds, or 1% polyoxyethylene (10) octylphenol ether (Triton X-100, Wako Pure Chemicals) And the virus particles were crushed.
[Example 1] Detection of influenza virus with fluorescently labeled glycoprotein probe It is generally known that fetuin, a kind of glycoprotein, has a plurality of sugar chain structures on the surface to which influenza virus can bind. When influenza virus (New Caledonia strain) and fetuin are mixed and influenza virus particles are precipitated by centrifugation, the virus particles and fetuin form a complex in solution because fetuin bound to the virus particles co-precipitates. This is clear from the fact (FIG. 1).

  Therefore, a fluorescent dye rhodamine was introduced into fetuin to form an influenza virus detection probe using fluorescence correlation spectroscopy. A purified fetuin is commercially available (for example, Wako Pure Chemicals). A rhodamine-N-hydroxysuccinimide ester derivative was used for introducing the fluorescent group of fetuin. Rhodamine-N-hydroxysuccinimide ester derivatives can be prepared by known methods and are also commercially available (for example, Molecular Probe). Rhodamine-N-hydroxysuccinimide ester derivative was dissolved in DMSO at a concentration of 5 mg / ml, 80 μl was mixed with 400 μl of 10 mg / ml fetuin, then 40 μl of 1M sodium bicarbonate solution was added. After stirring at room temperature for 1 hour, excess fluorescent labeling reagent was removed from the fetuin after the reaction by gel filtration. The introduction amount of the fluorescent group was confirmed by measuring absorption at a wavelength of 570 nm with an ultraviolet-visible light spectrophotometer.

Rhodamine-labeled fetuin was mixed with influenza virus (New Caledonia strain) in phosphate buffer saline (PBS), and after 1 hour incubation, FCS-101 (FCS measurement apparatus; manufactured by Toyobo) was used with 30 μl of the mixture. Was used to measure the micro-space fluorescence intensity. As a result, a fluorescence intensity fluctuation pattern similar to that of fluorescent polystyrene beads was observed (FIG. 2). When the relationship between the virus titer and the frequency of the fluorescence intensity fluctuation signal was examined, a correlation was observed, and the detection limit was 1 × 10 ⁴ pfu / ml (FIG. 3A).

Similarly, it was shown that proliferating virus in the culture supernatant of influenza virus-infected cultured cells can also be detected by observing fluctuations in fluorescence intensity. This culture supernatant contained influenza virus of about 1 × 10 ⁷ pfu / ml, and the detection limit was shown to be about 2 × 10 ⁶ pfu / ml (4-fold dilution). Therefore, it is possible to detect with FCS-101 in both the purified virus solution and the infected cell culture supernatant (FIG. 3B).
(Example 2) Detection of influenza virus using fluorescently labeled sugar chains In order to distinguish between human and avian type, fluorescently labeled sugar chains having 2-6 linked sialic acid and 2-3 linked sialic acid Can be used as a probe. Rhodamine was introduced into ganglioside LysoGM3, which is a glycolipid, for the production of fluorescently labeled sugar chains having 2-3 linked sialic acid (FIG. 4).

  In 40 μl of dimethylformamide solvent, 130 nmol of rhodamine-N-hydroxysuccinimide ester derivative, 65 nmol of ganglioside LysoGM3, and a final concentration of 0.1% triethylamine were mixed. After reacting at room temperature overnight, purification was performed by reversed-phase high performance liquid chromatography to obtain fluorescently labeled ganglioside LysoGM3. The structure was confirmed by a time-of-flight mass spectrometer and NMR measurement.

Detection sensitivity by FCS-101 using 2-3 linked sialic acid fluorescent sugar chain probes for influenza PR8 strain that binds to 2-3 linked sialic acid and influenza Hygo strain that binds to 2-6 linked sialic acid Was examined. A fixed concentration of rhodamine labeled 2-3 conjugated sialic acid probe was mixed with a diluted solution of influenza virus (PR8 and Hygo strains) cultured in chicken eggs in phosphate buffered saline (PBS) and immediately mixed. Microspace fluorescence intensity was measured by FCS-101 using 30 μl of the solution. The detection limit of influenza PR8 strain with a 2-3-linked sialic acid fluorescent sugar chain probe is 1.5 × 10 ⁵ pfu / ml, whereas the detection limit of influenza Hyogo strain is 2.5 × 10 ⁶ pfu / ml, The result was an order of magnitude different. This is consistent with the known fact that the PR8 strain has a high affinity for 2-3 linked sialic acid (FIG. 5). From the above results, it was shown that species specificity can be identified using a sialic acid fluorescent sugar chain probe and influenza virus can be detected with high sensitivity.

Comparative example

Sensitivity test of influenza virus rapid diagnosis kit
1. Sample Influenza virus used ・ A / New Caledpnia / 20/99 (H1N1)
2. Test method Evaluation target ・ Espline influenza A, BN (Fujirebio)
・ Capilia FluA, B (Towns)
It was performed according to the operation method (manufacturer's protocol) of each influenza virus rapid diagnosis kit.
3. Test Results The detection sensitivities of Capilia FluA, B and Esprine influenza A, BN were both between 1.0 × 10 ⁶ pfu / ml and 1.0 × 10 ⁷ pfu / ml.

(Example 3) In order to further improve detection sensitivity and signal intensity of influenza virus with a sugar chain polymer type fluorescently labeled probe , the literature (Y. Makimura et al., Chemoenzymatic synthesis and application of a sialoglycopolymer with a chitosan backbone as a potent In accordance with inhibitor of human influenza virus hemagglutination., Carbohydrate Research, 2006, May 20) A virus detection probe was used. By using the fluorescent probe, it was expected that a plurality of proteins constituting the surface of the virus particle were bound to a plurality of adjacent sugar chain structures, resulting in high affinity. The structure of the fluorescent probe is shown in FIG. Chitosan was used as the hydrophilic polymer.

First, 2-6 type terminal sialic acid is recognized using a fluorescently labeled polymer (Rho-SGP α2-6 type, SGP: sialoglycopeptide) in which a sugar chain having terminal sialic acid of type 2-6 is fixed in tandem The influenza virus strain Hyogo and the influenza virus PR8 strain that recognizes type 2-3 terminal sialic acid were detected by the method of the present invention in the same manner as in Examples 1 and 2. The result is shown in FIG. The detection sensitivity of the fluorescent probe was a detection limit of 1 × 10 ⁵ pfu / ml in both strains, and the number of peaks as a signal increased. Compared with the low-molecular-weight sugar chain fluorescent probe, the improvement in detection sensitivity and signal intensity expected by polymerization was confirmed. On the other hand, since the Hyogo strain showed a signal intensity five times that of the PR8 strain, it reflected that the Hyogo strain showed higher affinity for the 2-6 type terminal sialic acid.

  Next, an influenza virus that recognizes type 2-6 terminal sialic acid using a fluorescently labeled polymer (Rho-SGP α2-3 type) in which a sugar chain having terminal type sialic acid of type 2-3 binding is fixed in tandem Detection of the Hyogo strain and the influenza virus PR8 strain that recognizes type 2-3 terminal sialic acid was carried out by the method of the present invention in the same manner as in Examples 1 and 2. The result is shown in FIG. The detection sensitivity of the fluorescent probe was equivalent to that of Rho-SGP α2-6 type, and the signal intensity was further improved. On the other hand, the signal intensity of the Hyogo strain was 1.5 times that of the PR8 strain, and there was no reversal between 2-6 type recognition and 2-3 type recognition, but the difference was greatly reduced. This was considered to reflect that the PR8 strain showed higher affinity for the 2-3 type terminal sialic acid. Therefore, by using the fluorescent probes of Rho-SGP α2-6 type and Rho-SGP α2-3 type together and comparing the signal intensity, high-sensitivity detection of influenza virus and discrimination between human and avian types are possible. It is considered possible.

Results of mixing fetuin with influenza virus (New Caledonia strain), precipitating virus particles by centrifugation, and detecting fetuin contained in the precipitate with anti-fetuin antibody. Variation in fluorescence intensity over time when fluorescently labeled glycoprotein (fetuin) and influenza virus are mixed. Detection of influenza virus FCS by fluorescently labeled glycoprotein (fetuin). Structure of 2-3-linked sialic acid fluorescent sugar chain probe Results of FCS detection of influenza PR8 strain (bird-type recognition) and Hygo strain (human-type recognition) using a 2-3-linked sialic acid fluorescent sugar chain probe (bird-type sugar chain). Structure of sugar chain polymer type fluorescent labeled probe The result of detecting influenza PR8 strain and Hygo strain using a sugar chain polymer type fluorescent labeled probe (Rho-SGP α2-6 type). The result of detecting influenza PR8 strain and Hygo strain using a sugar chain polymer type fluorescent labeled probe (Rho-SGP α2-3 type).

Claims (12)

A sample comprising a step of mixing a fluorescently labeled or fluorescent virus-binding substance and a sample to prepare a sample solution, and measuring a time course of a fluorescence signal of the sample solution using a confocal optical system A method for detecting a virus-related substance derived from a virus or / and a virus-infected cell. The method according to claim 1, wherein the virus-binding substance is selected from the group consisting of sugars, antibodies, proteins, peptides, nucleic acids, lipids, and low-molecular chemical substances. The method according to claim 1, wherein the virus is in a particle state and binds to a fluorescent substance on the surface of the virus. The method according to claim 1, wherein a part or all of the virus particles are physically or chemically disrupted in advance. The method according to claim 1, further comprising a pretreatment step for extraction and purification of the virus in the sample. The method according to any one of claims 1 to 5, wherein the average particle diameter of the virus is 1 to 1000 nm in the minor axis and 5 to 10,000 nm in the major axis. The method according to claim 1, wherein the virus is an influenza virus. A detection system for a virus or a virus-related substance derived from a virus-infected cell, comprising a fluorescently labeled or fluorescent virus-binding substance and a confocal optical system. The system according to claim 8, wherein the virus-binding substance is selected from the group consisting of sugars, antibodies, proteins, peptides, nucleic acids, lipids, and low-molecular chemical substances. The fluorescence wavelength of a fluorescently-labeled or fluorescent virus-binding substance is 350 to 800 nm, the molecular weight is 120 or more, and the confocal region of the confocal optical system is 10 ⁻¹⁶ to 10 ⁻¹⁰ liter. The system according to claim 8 or 9. The system according to any one of claims 8 to 10, wherein the average particle diameter of the virus is 1 to 1000 nm in the minor axis and 5 to 10,000 nm in the major axis. The system according to any one of claims 8 to 11, wherein the virus to be measured is an influenza virus.

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