Method and kit for detecting human norovirus

Abstract

To provide a method for accurately and quantitatively detecting human norovirus having activity.SOLUTION: A method for detecting a human norovirus according to the present invention is a method for detecting a human norovirus having an activity present in a test object, the method including the steps of preparing a magnetic bead-bound body in which an anti-human norovirus IgY antibody is bound to a surface of a magnetic bead, mixing the test object containing the human norovirus with the magnetic bead-bound body, collecting the magnetic bead-bound body, separating an RNA virus genome of the human norovirus bound to the magnetic bead-bound body, synthesizing cDNA from the separated RNA virus genome and amplifying the cDNA, and determining that the human norovirus having the activity is present when an amount of an amplification product obtained in the amplifying step is higher than a reference value.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method and kit for detecting human norovirus. [Background technology]

[0002] Human noroviruses are known to be major pathogens that infect the human intestine and cause viral infectious gastroenteritis.

[0003] The structure of human norovirus is shown in Figure 4. As shown in Figure 4(a), human norovirus 11a consists of an RNA viral genome 4 and a capsid 13 that encases the RNA viral genome. For human norovirus 11a to infect the human intestine, the capsid 13 must be adsorbed onto target cells in the human intestine. Therefore, as shown in Figures 4(b) and 4(c), human noroviruses 11b and 11c, in which the capsid 13 of human norovirus 11a has been completely inactivated using, for example, a disinfectant, are deemed to have no infectious function in the human intestine and to have no viral activity. For convenience of illustration, in Figures 4(b) and 4(c), the inactivated capsid 13 and the destroyed RNA viral genome 4 are represented by dashed lines. Therefore, when testing for human norovirus in the presence of active or inactive human norovirus, a method for accurately and quantitatively detecting whether the human norovirus in the test sample is active is important.

[0004] In this specification, "active human norovirus 2" refers to human norovirus 11a in which the RNA viral genome 4 is not destroyed and the capsid 13 is not inactivated (see FIG. 4(a)). On the other hand, in this specification, "inactive human norovirus 3" refers to human norovirus 11b in which the capsid 13 is inactivated but the RNA viral genome 4 is not destroyed (see FIG. 4(b)), and human norovirus 11c in which the RNA viral genome 4 is destroyed and the capsid 13 is inactivated (see FIG. 4(c)).

[0005] Conventionally, quantitative detection of human norovirus has been mainly performed by real-time PCR, as described in Non-Patent Document 1. Specifically, quantitative detection using real-time PCR involves first extracting the RNA viral genome from active or inactive human norovirus in a test sample, synthesizing cDNA from the RNA viral genome, and then amplifying the cDNA by real-time PCR to measure the amount of the amplified product. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Food Safety Inspection Notification No. 1105001, Attachment "Detection Methods for Norovirus" (November 5, 2003) [Non-patent document 2] Araud E, DiCaprio E, Ma Y, Lou F, Gao Y, Kingsley D, Hughes JH, Li J. 2016. Thermal inactivation of enteric viruses and bioaccumulation of enteric foodborne viruses in live oysters (Crassostrea virginica). Appl Environ Microbiol 82:2086-2099. Summary of the Invention [Problem to be solved by the invention]

[0007] However, in conventional quantitative detection methods using real-time PCR, the RNA viral genome extracted from human norovirus also includes the RNA viral genome 4 of human norovirus 11b, which does not have viral activity (infectivity) due to inactivated capsid 13, as shown in Figure 4(b). Therefore, cDNA is also synthesized from the RNA viral genome 4 of human norovirus 11b, and is amplified by real-time PCR. Therefore, the amount of cDNA amplification product measured in conventional methods includes noise from the amount of cDNA amplification product synthesized from the inactive human norovirus 11b viral genome 4, resulting in a lack of accuracy.

[0008] The present invention has been made in consideration of the above-mentioned problems, and its object is to provide a method for accurately and quantitatively detecting active human norovirus and a kit that can easily carry out the method. [Means for solving the problem]

[0009] To achieve the above-mentioned object, the present inventors conducted extensive research and discovered that by preparing magnetic bead conjugates in which anti-human norovirus IgY antibodies are bound to the surface of magnetic beads and mixing these magnetic bead conjugates with a test object containing active human norovirus, it is possible to recover the magnetic bead conjugates in a state in which the active human norovirus is bound. Furthermore, the present inventors discovered that by isolating the RNA viral genome of the active human norovirus bound to the recovered magnetic bead conjugates without destroying it, synthesizing cDNA from the isolated RNA viral genome and amplifying it by real-time PCR, measuring the amount of the amplified product, and comparing it with a reference value, it is possible to accurately and quantitatively detect active human norovirus present in a test object, thereby completing the present invention.

[0010] Specifically, the human norovirus detection method of the present invention is a method for detecting active human norovirus present in a test object, and is characterized by comprising the steps of: preparing a magnetic bead conjugate in which anti-human norovirus IgY antibodies are bound to the surface of magnetic beads; mixing the test object containing the human norovirus with the magnetic bead conjugate; recovering the magnetic bead conjugate; isolating the RNA viral genome of the human norovirus bound to the magnetic bead conjugate; synthesizing cDNA from the isolated RNA viral genome and amplifying the cDNA; and determining that the active human norovirus is present if the amount of the amplification product obtained by the amplification is higher than a reference value.

[0011] In the method for detecting human norovirus according to the present invention, magnetic bead conjugates are first prepared, in which anti-human norovirus IgY antibodies are bound to the surface of magnetic beads. The IgY antibodies constituting these magnetic bead conjugates exhibit affinity for the capsid of active human norovirus. Next, the prepared magnetic bead conjugates are mixed with a test object containing active human norovirus, and the magnetic bead conjugates are recovered. This procedure allows the magnetic bead conjugates to be recovered in a state in which active human norovirus present in the test object is bound. These procedures simultaneously extract active human norovirus present in the test object while simultaneously removing inactive human norovirus present in the test object. Next, the RNA viral genome of the human norovirus bound to the recovered magnetic bead conjugates is separated without disrupting it, and cDNA is synthesized from the RNA viral genome and amplified by real-time PCR or other methods. Finally, if the amount of the amplified product is higher than the reference value, the test object is determined to contain active human norovirus. This eliminates noise caused by cDNA amplification products synthesized from the RNA viral genome of human norovirus whose capsid has been inactivated but whose RNA viral genome has not been destroyed, which has been a problem in conventional quantitative detection methods for human norovirus that do not use IgY antibodies. Therefore, the detection method of the present invention can accurately and quantitatively detect active human norovirus present in a test sample.

[0012] In the method for detecting human norovirus according to the present invention, the test object may include a sample collected from a bivalve mollusks, or may include a sample collected from an oyster.

[0013] The human norovirus detection method of the present invention allows for accurate and quantitative detection of active human norovirus contained in test samples, making it suitable for detecting active human norovirus contained in bivalve mollusks, particularly oysters. Regarding oysters in particular, a technique for detecting active human norovirus using a glycan antigen, such as porcine stomach mucin, that has affinity for the capsid of active human norovirus has been proposed in Non-Patent Document 2. Specifically, Non-Patent Document 2 discloses a method for detecting human norovirus by recovering the glycan antigen in a state in which active human norovirus is bound, and isolating the RNA viral genome of the human norovirus bound to the recovered glycan antigen. However, oysters are known to contain glycans that have affinity for active human norovirus. Therefore, active human norovirus binds to both the glycan antigen and the oyster glycans. As a result, even if all the glycan antigens are recovered, not all active human norovirus is recovered, and some human norovirus remains bound to the oyster glycans. For this reason, conventional quantitative detection methods for human norovirus using glycan antigens lack accuracy, particularly due to the risk of active human norovirus remaining in oysters, making them unsuitable as detection methods for determining the safety of oysters for consumption. In contrast, the detection method of the present invention has been found to eliminate the above-mentioned problems because the affinity between the anti-human norovirus IgY antibody conjugated to magnetic beads and active human norovirus is significantly higher than the affinity between the glycans present in oysters and active human norovirus. Therefore, the detection method of the present invention is suitable for accurately and quantitatively detecting active human norovirus present in test subjects, including samples collected from oysters.

[0014] The kit of the present invention is a kit for carrying out the above-mentioned method for detecting human norovirus, and is characterized in that the kit includes a magnetic bead conjugate in which an anti-human norovirus IgY antibody is bound to the surface of magnetic beads.

[0015] The kit according to the present invention allows the above-described method for detecting human norovirus according to the present invention to be carried out easily. [Effects of the Invention]

[0016] The method for detecting human norovirus according to the present invention can provide a method for accurately and quantitatively detecting active human norovirus and a kit that can easily carry out the method. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram illustrating a method for detecting human norovirus according to one embodiment of the present invention. FIG. 1(a) is a schematic diagram illustrating a test object. FIG. 1(b) is a schematic diagram illustrating a step of mixing a test object with a magnetic bead conjugate. FIG. 1(c) is a schematic diagram illustrating a step of recovering the magnetic bead conjugate. FIG. 1(d) is a schematic diagram illustrating the recovered magnetic bead conjugate. FIG. 1(e), (f), and (g) are schematic diagrams illustrating a step of isolating an RNA virus genome. [Figure 2] FIG. 2 is a bar graph showing the amounts of amplification products of cDNA synthesized from the RNA viral genome of human norovirus, obtained by the methods of Examples 1 to 3 and Comparative Examples 1 to 3. [Figure 3] FIG. 3 is a bar graph showing the amounts of amplification products of cDNA synthesized from the RNA viral genome of human norovirus, obtained by the methods of Examples 4 to 6 and Comparative Examples 4 to 6. [Figure 4] Figure 4(a) is a schematic diagram showing the structure of an active human norovirus, while Figures 4(b) and 4(c) are schematic diagrams showing the structure of an inactive human norovirus. DETAILED DESCRIPTION OF THE INVENTION

[0018] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following description of preferred embodiments is merely exemplary in nature and is not intended to limit the present invention, its application, or its uses.

[0019] (Method for detecting human norovirus) As shown in FIG. 1, a method for detecting human norovirus according to one embodiment of the present invention uses magnetic bead conjugates 7 (see FIG. 1(b)) in which anti-human norovirus IgY antibodies 5 are bound to the surface of magnetic beads 6 to accurately and quantitatively detect active human norovirus 2 present in a test object 1. Each step of the method for detecting human norovirus according to this embodiment will be described below with reference to FIG. 1. In this embodiment, active human norovirus 2 and inactive human norovirus 3 follow the definitions described above.

[0020] Although not shown, the detection method according to this embodiment includes a step of preparing a magnetic bead conjugate 7 (see FIG. 1(b)) in which an IgY antibody 5 is bound to the surface of magnetic beads 6. For example, the step of preparing the magnetic bead conjugate 7 can be performed by adding an IgY antibody 5, magnetic beads 6, and a crosslinker agent (not shown) to a container, stirring the mixture at room temperature for a predetermined time, and purifying the resulting reaction product. The magnetic bead conjugate 7 may be one in which two or more IgY antibodies 5 are bound to the surface of a single magnetic bead 6. In this specification, the term "IgY antibody" refers to chicken egg immunoglobulin (IgY) contained in the yolk of a chicken egg. The IgY antibody 5 consists of two heavy chains and two light chains, forming a Y-shape, and has two variable regions at the ends of the chains that bind to the capsid of active human norovirus 2. Commercially available IgY antibodies 5 can be used, or alternatively, prepared IgY antibodies can be used. The production method is not particularly limited, but for example, human norovirus or human norovirus VLPs (virus-like particles) can be injected into chickens to immunize them, and the magnetic beads can be extracted by any method from the yolk of eggs laid by the chickens. The magnetic beads 6 are not particularly limited, but carboxyl-derivatized beads can be used, for example. The crosslinker agent is not particularly limited as long as it can bind the IgY antibody 5 and the magnetic beads 6, but for example, a zero-length crosslinker can be used. Examples of zero-length crosslinkers include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter also referred to as "EDC"), etc.

[0021] As shown in FIG. 1(b), the detection method according to this embodiment includes a step of mixing a test object 1 containing an active human norovirus 2 with a magnetic bead conjugate 7. For example, the step of mixing the test object 1 containing the active human norovirus 2 with the magnetic bead conjugate 7 can be performed by adding the magnetic bead conjugate 7 prepared as described above to a container 10a containing the test object 1 containing the active human norovirus 2 as shown in FIG. 1(a), and stirring the mixture at room temperature for a predetermined period of time. The purpose of this mixing step is to allow the IgY antibody 5 constituting the magnetic bead conjugate 7 to bind to the active human norovirus 2. The IgY antibody 5 binds to the active human norovirus 2 because the IgY antibody 5 contains a variable region at its end that binds to the capsid 13 of the active human norovirus 2. On the other hand, inactive human norovirus 3 does not bind to the IgY antibody 5 of the magnetic bead conjugate 7 because its capsid 13 is inactivated.

[0022] As shown in FIGS. 1(c) and 1(d), the detection method according to this embodiment includes a step of recovering the magnetic bead conjugates 7. For example, the step of recovering the magnetic bead conjugates 7 can be performed by drawing a magnet 9 close to the bottom and side surfaces of a container 10a to attract the magnetic bead conjugates 7 in the test subject 1, and then removing the supernatant liquid from the test subject 1 with a pipette. This operation does not need to be performed once, but may be performed multiple times by adding solvent as appropriate. The method is not limited to this. The purpose of this step of recovering the magnetic bead conjugates 7 is to remove inactive human norovirus 3 and other substances present in the test subject 1.

[0023] As shown in Figures 1(d), 1(e), 1(f), and 1(g), the detection method according to this embodiment includes a step of isolating the RNA viral genome 4 of active human norovirus 2 bound to magnetic bead conjugates 7. For example, the step of isolating the RNA viral genome 4 can be performed by heating a solution containing the magnetic bead conjugates 7 bound to active human norovirus 2 at 95°C for 5 minutes (see Figures 1(d) and 1(e)), then attracting the magnetic bead conjugates 7 to a container 10a by a magnet 9 from the outside of the bottom and side of the container (see Figure 1(f)), and then transferring the supernatant containing the RNA viral genome 4 to another container 10b with a pipette (see Figure 1(g)). In this way, heating the magnetic bead conjugates 7 bound to active human norovirus 2 inactivates the capsid without destroying the RNA viral genome 4 of the human norovirus. The heat-denatured capsid and heat-denatured IgY are then precipitated together with the magnetic beads using a magnet, allowing the RNA viral genome 4 to be isolated. The temperature conditions and heating time during the heat treatment can be changed as appropriate as long as the RNA viral genome 4 of the active human norovirus 2 is not destroyed.

[0024] Although not shown, the detection method according to this embodiment includes a step of synthesizing cDNA from the isolated RNA virus genome 4 and amplifying the cDNA. The step of amplifying cDNA can be performed by synthesizing cDNA from the isolated RNA virus genome 4, for example, according to the instructions in a cDNA synthesis kit (Toyobo), and amplifying the synthesized cDNA by real-time PCR. The stirring temperature and stirring time are not particularly limited. Real-time PCR can be performed using methods and conditions commonly used in the art, and are not particularly limited.

[0025] Although not shown, the detection method according to this embodiment includes a step of determining the presence of active human norovirus 2 when the amount of the amplified product from the cDNA is higher than a reference value. The reference value is not particularly limited, but because the amount of cDNA varies depending on the dilution factor of the test object 1, it is preferable to set the reference value according to the dilution factor. Alternatively, the amount of the amplified product from cDNA in a sample that has been subjected to a heat treatment or disinfection treatment that inactivates human norovirus may be used as a control and used as the reference value.

[0026] As shown in FIG. 1(a), the test object 1 is not particularly limited as long as it contains active human norovirus 2; it may also contain inactive human norovirus 3. As described above, inactive human norovirus 3 does not bind to the IgY antibody 5 of the magnetic bead conjugate 7 (see FIG. 1(b)), and is therefore removed from the test object 1 in the subsequent step of recovering the magnetic bead conjugate 7. Therefore, in this embodiment, even if the test object 1 contains inactive human norovirus 3, it does not affect the quantitative detection results of active human norovirus 2. Furthermore, the test object 1 may be diluted in advance at any ratio, for example, 10-fold or 100-fold. Furthermore, the test object 1 preferably includes a sample collected from bivalve mollusks, more preferably oysters. In particular, with regard to oysters, a technique for detecting active human norovirus 2 using a glycan antigen with affinity for active human norovirus 2, such as mucin derived from porcine stomach, has been proposed (Non-Patent Document 2). This technology involves recovering active human norovirus 2 bound to the above-mentioned glycan antigen, and isolating the RNA viral genome 4 of the human norovirus bound to the recovered glycan antigen to detect human norovirus. However, it is known that glycans with affinity for active human norovirus 2 are also present in oysters. Therefore, active human norovirus 2 binds to both the above-mentioned glycan antigen and the glycans present in oysters. Even if all of the glycan antigens are recovered, not all of the active human norovirus 2 is recovered, and some of the virus remains bound to the glycans present in the oysters. Therefore, when conventional quantitative detection methods for human norovirus are applied to oysters, they suffer from a lack of accuracy, particularly due to the risk of active human norovirus remaining in the oysters, making them unsuitable as detection methods for determining the safety of oysters for consumption. On the other hand, it has been found that the affinity between the IgY antibody 5 used in the human norovirus detection method of the present invention and active human norovirus 2 is significantly higher than the affinity between the glycans found in oysters and active human norovirus 2.Therefore, the detection method of the present invention does not have the above problems and is suitable as a method for accurately and quantitatively detecting active human norovirus 2 even in test subjects 1 that include samples collected from oysters.

[0027] Human noroviruses include any human norovirus known to infect humans, including, but not limited to, GI type human norovirus, GII type human norovirus, GIV type human norovirus, and combinations thereof.

[0028] (kit) A kit according to one embodiment of the present invention is a kit for easily carrying out the method for detecting human norovirus according to one embodiment of the present invention described above. In particular, the kit according to this embodiment includes at least a magnetic bead conjugate 7 in which an IgY antibody 5 is bound to the surface of magnetic beads 6. The kit also preferably includes a reverse transcriptase for synthesizing cDNA from the RNA virus genome 4. The kit also preferably includes reagents for performing real-time PCR. The magnetic bead conjugate 7 can be prepared as described above. [Example]

[0029] Examples are provided below to explain in detail the method for detecting human norovirus according to the present invention. These examples are intended to illustrate the present invention and are not intended to limit the scope of the invention.

[0030] (material) Sodium hypochlorite aqueous solution: Sodium hypochlorite was diluted with purified water to 200 ppm and used. Magnetic bead conjugate: 1 mL of MagnaBind carboxyl-derivatized beads (Thermo Fisher Scientific) was placed in a 1.5 mL microtube (Watson) and washed three times with phosphate-buffered saline (PBS). Anti-norovirus IgY antibody (EW Nutrition) was diluted with conjugation buffer (0.2 M 2-(N-morpholino)ethanesulfonic acid, 1.8% sodium chloride, pH 4.7) to a concentration of 5 mg / mL. 1 mL of the IgY antibody solution was added to the washed magnetic beads and suspended. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (Tokyo Chemical Industry Co., Ltd.) was used as a cross-linker and diluted to 10 mg / mL with conjugation buffer. 0.1 mL of the diluted EDC solution was added to the magnetic bead-IgY antibody mixture and stirred at room temperature for 30 minutes to prepare the magnetic bead conjugate.

[0031] Example 1 A 10-fold dilution of 2 μL of a human norovirus solution (obtained from the Hiroshima Prefectural Technology Research Institute, Health and Environment Center) was added to a microtube (Watson) containing 18 μL of PBS. Next, 10 μL of the 10-fold diluted norovirus solution was added to a new microtube containing 990 μL of PBS. The microtube was then closed and gently shaken to obtain a 100-fold dilution. 50 μL of magnetic bead conjugate was then added to the microtube, the microtube was closed, and the tube was placed in an R2200 RotoFlex plus (Argos Technologies) and stirred at 37°C for 1 hour. After stirring, the microtube was placed in a centrifuge (Funakoshi) and centrifuged for approximately 5 seconds. The microtube was then placed in a magnetic rack (Dynal) and left to stand for 1 minute, after which the supernatant was removed, leaving the magnetic bead conjugate intact. 60 μL of PBS was then added to the microtube, which was then placed in a block incubator (AS ONE) and heated at 95°C for 5 minutes. The microtube was placed on a magnetic rack and allowed to stand for 1 minute. The supernatant containing the human norovirus RNA viral genome was then collected and transferred to a new microtube. cDNA was synthesized from the extracted RNA according to the instructions of a cDNA synthesis kit (Toyobo). Using the synthesized cDNA as a template, real-time PCR was performed to amplify the cDNA using norovirus GII-specific primers (COG2F, COG2R) and a probe (RING2ALTPN4). The amount of the amplified product was then measured. Finally, the amount of the amplified product was compared with a known amount of norovirus genomic DNA (plasmid) to quantitatively analyze the amount of active human norovirus genome (copy number) in the test sample.

[0032] Example 2 Human norovirus was detected using the same method as in Example 1, except that 2 μL of a solution containing human norovirus was added as the test subject to a microtube containing 18 μL of PBS, diluted 10-fold, and then heated at 95°C for 5 minutes.

[0033] Example 3 Human norovirus was detected in the same manner as in Example 1, except that 18 μL of aqueous sodium hypochlorite solution was used instead of 18 μL of PBS and allowed to react at room temperature for 2 minutes to inactivate the norovirus.

[0034] Example 4 Human norovirus was detected in the same manner as in Example 1, except that a norovirus solution that was initially diluted 100 times instead of 10 times was used.

[0035] Example 5 Human norovirus was detected in the same manner as in Example 2, except that a norovirus solution that was initially diluted 100 times instead of 10 times was used.

[0036] Example 6 Human norovirus was detected in the same manner as in Example 3, except that a norovirus solution that was initially diluted 100 times instead of 10 times was used.

[0037] Comparative Example 1 As in Example 1 above, 10 μL of the 10-fold diluted norovirus solution was added to a new microtube containing 990 μL of PBS, the tube was closed, and the tube was gently shaken to perform a 100-fold dilution. Next, RNA viral genomes were extracted from this diluted solution using a QIAamp Viral RNA Mini Kit (QIAGEN) according to the kit's protocol. cDNA was synthesized from the resulting viral RNA according to the instructions of a cDNA synthesis kit (Toyobo). The synthesized cDNA was amplified by real-time PCR as in Example 1, and the amount of the amplified product was measured. Finally, the amount of the measured amplified product was compared with a known amount of norovirus genomic DNA (plasmid) to quantitatively analyze the amount of human norovirus genome (copy number) in the test sample.

[0038] Comparative Example 2 Human norovirus was detected using the same method as in Comparative Example 1, except that 2 μL of a solution containing human norovirus was added as the test subject to a microtube containing 18 μL of PBS, diluted 10-fold, and then heated at 95°C for 5 minutes.

[0039] Comparative Example 3 Human norovirus was detected using the same method as in Comparative Example 1, except that 18 μL of sodium hypochlorite aqueous solution was used instead of 18 μL of PBS and allowed to react at room temperature for 2 minutes to inactivate the norovirus.

[0040] Comparative Example 4 Human norovirus was detected in the same manner as in Comparative Example 1, except that a norovirus solution that had been initially diluted 100-fold instead of 10-fold was used.

[0041] Comparative Example 5 Human norovirus was detected in the same manner as in Comparative Example 2, except that a norovirus solution that had been initially diluted 100-fold instead of 10-fold was used.

[0042] Comparative Example 6 Human norovirus was detected in the same manner as in Comparative Example 3, except that a norovirus solution that had been initially diluted 100-fold instead of 10-fold was used.

[0043] The quantities of amplification products measured by the methods of Examples 1 to 3 and Comparative Examples 1 to 3 are shown in a bar graph in Figure 2. In Figure 2, the horizontal axis indicates the type of Example or Comparative Example, and the vertical axis indicates the quantity of amplification products. In Figure 3, the quantities of amplification products measured by the methods of Examples 4 to 6 and Comparative Examples 4 to 6 are shown in a bar graph. In Figure 3, the horizontal axis indicates the type of Example or Comparative Example, and the vertical axis indicates the quantity of amplification products.

[0044] As can be seen from Figure 2, the presence of active human norovirus was determined in both the test samples of Example 1 and Comparative Example 1. Because these test samples were not subjected to heat treatment or disinfection, it can be seen that the human norovirus remained active. Furthermore, it was confirmed that the amount of amplification product measured by the method of Example 1 was significantly lower than the amount of amplification product measured by the method of Comparative Example 1. This is thought to be due to the detection of noise due to amplification products caused by human norovirus whose capsid was inactivated but whose RNA viral genome was not destroyed in Comparative Example 1. On the other hand, the results of Example 2 and Comparative Example 2, which were subjected to heat treatment, indicated that the test sample of Example 2 was determined to be free of active human norovirus, while the test sample of Comparative Example 2 was determined to be free of active human norovirus. The heat treatments performed in Example 2 and Comparative Example 2 were performed under conditions that completely inactivated the human norovirus. These results confirmed the problem of conventional human norovirus detection methods that do not use IgY antibodies failing to provide accurate detection results, even though human norovirus is actually present in an inactive state. This is also thought to be due to the noise detected in Comparative Example 2. Finally, the results of Example 3 and Comparative Example 3, which were disinfected with a strong disinfectant, sodium hypochlorite aqueous solution, indicated that no active human norovirus was present in either test object. This is thought to be because the disinfection treatment completely destroyed the RNA viral genome of human norovirus in the test objects of Example 3 and Comparative Example 3, and no difference was observed in the amount of amplified product between the two. These results, particularly those shown in Examples 1 and 2 and Comparative Examples 1 and 2, demonstrate that the human norovirus detection method using an IgY antibody according to this example can detect active human norovirus more accurately and quantitatively than conventional human norovirus detection methods that do not use IgY antibodies.

[0045] Furthermore, as can be seen from Figure 3, the methods of Examples 4 to 6 demonstrated that active human norovirus present in the test sample could be accurately and quantitatively detected even when the solution containing human norovirus was initially diluted 100-fold rather than 10-fold. Results similar to those of Comparative Examples 1 to 3 were obtained for Comparative Examples 4 to 6. Although the amount of amplified product was confirmed to be reduced overall when the solution was diluted 100-fold, these dilution conditions did not hinder the determination of whether or not active human norovirus was present in the test sample. Furthermore, it was found that the reference value should be appropriately set depending on the dilution conditions of the solution containing human norovirus.

[0046] From the above results, it was confirmed that the method for detecting human norovirus according to this example is a method for accurately and quantitatively detecting active human norovirus. [Explanation of symbols]

[0047] 1. Inspection object 2. Active human norovirus 3. Inactive human norovirus 4. RNA virus genome 5. IgY antibodies 6. Magnetic beads 7. Magnetic bead conjugate 9. Magnet 10a, 10b container 11a, 11b, 11c Human noroviruses 13 Capsid

Claims

1. A method for detecting active human norovirus present in a test object, comprising: preparing a magnetic bead conjugate in which an anti-human norovirus IgY antibody is bound to the surface of the magnetic bead; mixing the test object containing the human norovirus with the magnetic bead conjugate; recovering the magnetic bead conjugate; isolating the RNA viral genome of the human norovirus bound to the magnetic bead conjugate; synthesizing cDNA from the isolated RNA virus genome and amplifying the cDNA; and determining that a human norovirus having the activity is present if the amount of the amplification product obtained by the amplification step is higher than a reference value.

2. The method for detecting human norovirus according to claim 1 , wherein the test object includes a sample collected from a bivalve molluscan shellfish.

3. The method for detecting human norovirus according to claim 2 , wherein the test object includes a sample collected from an oyster.

4. A kit for carrying out the method according to any one of claims 1 to 3, comprising: The kit comprises a magnetic bead conjugate in which an anti-human norovirus IgY antibody is bound to the surface of the magnetic bead.

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