Method for concentrating virus samples

Abstract

To provide a virus sample concentrating method that can rapidly concentrate a virus sample at a high concentration percentage.SOLUTION: This method involves pouring a virus-containing liquid 30 to a solid phase 10 formed of a polymer having a hydrophobic group to cause the virus 40 to be retained on the solid phase 10, and then eluting the virus 40 from the solid phase 10 using an organic solvent 70.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for concentrating a sample containing a virus. [Background technology]

[0002] There is an academic field called sewage epidemiology, which aims to understand the trends of infectious disease outbreaks by measuring the concentration of viruses in sewage, including human urine and feces. Many results from sewage epidemiology have been reported in the field of infection control for norovirus and poliovirus, and it is also thought that they can be applied to the novel coronavirus disease (COVID-19), the spread of which has become increasingly serious in recent years.

[0003] Viruses in samples are detected by nucleic acid amplification methods such as polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) using primers specific to the virus. However, since the virus concentration in sewage is generally low and often below the detection limit of nucleic acid amplification methods, it is necessary to concentrate the sample containing the virus in advance.

[0004] Conventionally, a method for concentrating a virus sample is known as a concentration and precipitation method using polyethylene glycol (PEG) (so-called PEG precipitation method). For example, Non-Patent Document 1 describes increasing the virus concentration in a sewage sample by the PEG precipitation method.

[0005] In recent years, a method for concentrating a virus sample using solid-phase extraction has also been proposed. Solid-phase extraction is a technique for selectively isolating or concentrating a target substance in a liquid by interacting with a solid phase. For example, Patent Document 1 describes that a virus sample can be concentrated by passing water containing the virus through a column packed with a solid phase having an anion exchange capacity, thereby adsorbing the virus to the carrier, and then passing an alkaline solution (a buffer solution with a pH of 9 or higher) through the column to elute the virus. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2004-129548 A [Non-patent literature]

[0007] [Non-Patent Document 1] Lewis and Metcalf, 1988, "Polyethylene Glycol Precipitation for Recovery of Pathogenic Viruses, Including Hepatitis A Virus and Human Rotavirus, from Oyster, Water, and Sediment Samples", Appl. Environ. Microbiol. vol 54(8), p. 1983-1988 Summary of the Invention [Problem to be solved by the invention]

[0008] However, in the early or convergence stages of an infectious disease epidemic, the number of infected people is low, so the amount of virus discharged into sewage is inevitably low. Therefore, in order to grasp the infection status at such times, a method capable of concentrating virus samples with a higher concentration rate is required. In addition, in order to utilize sewage epidemiology in infection spread prevention, it is necessary to obtain results showing the infection status quickly, so a method capable of concentrating virus samples in a shorter time has been required.

[0009] The present invention has been made in consideration of the above points, and an object of the present invention is to provide a method for concentrating a virus sample, which can rapidly concentrate a virus sample at a high concentration rate. [Means for solving the problem]

[0010] The method for concentrating a virus sample according to the present invention, which has been achieved to solve the above problems, comprises: The method includes contacting a liquid containing the virus with a solid phase made of a polymer having a hydrophobic group, thereby causing the virus to be retained on the solid phase, and then contacting the solid phase retaining the virus with an organic solvent, thereby eluting the virus from the solid phase. Effect of the Invention

[0011] According to the method for concentrating a virus sample according to the present invention, it is possible to rapidly concentrate a virus sample at a high concentration rate. [Brief description of the drawings]

[0012] [Figure 1] FIG. 2 is a schematic diagram showing a process of adsorbing a virus onto a solid phase in one embodiment of the present invention. [Diagram 2] FIG. 4 is a schematic diagram showing a virus elution step in this embodiment. [Diagram 3] FIG. 4 is a schematic diagram showing a nitrogen blowing step in the embodiment. [Figure 4] Graph showing the quantification results of the N1 gene in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The method for concentrating a virus sample according to the present embodiment (hereinafter referred to as the "method of the present embodiment") uses a solid phase extraction method to increase the virus concentration in a liquid sample. In the method of the present embodiment, a solid phase made of a polymer having a hydrophobic group is used as a solid phase for adsorbing viruses. As such a solid phase, for example, a hydrophilic lipophilic balanced solid phase (HLB solid phase) can be suitably used, but is not limited thereto. In addition, in the method of the present embodiment, an organic solvent is used as a solvent for eluting the viruses adsorbed on the solid phase (hereinafter referred to as the "elution solvent"). As the organic solvent, for example, an alcohol solvent such as methanol or ethanol can be suitably used, but the organic solvent in the present invention is not limited thereto. In addition, the virus targeted by the method of the present embodiment includes, for example, enveloped viruses such as coronaviruses such as the new coronavirus (SARS-CoV-2) or influenza viruses, but is not limited thereto, and the method of the present embodiment can also be applied to the concentration of a sample containing a non-enveloped virus such as norovirus or poliovirus. Furthermore, the "liquid containing viruses" in the method of this embodiment is typically sewage, but is not limited to this, and may be, for example, river water, seawater, or a liquid containing body fluids collected from humans.

[0014] The procedure of the standard virus concentration treatment in the method of this embodiment will be described below with reference to FIGS.

[0015] (1) Virus adsorption to solid phase First, a conditioning liquid (e.g., methanol and pure water) is passed through the cartridge 20 filled with the solid phase 10 made of a polymer having a hydrophobic group, thereby activating the solid phase 10. The method of passing the liquid through the solid phase 10 includes a pressurization method using a syringe or the like, a suction method using a suction manifold and a vacuum pump, a centrifugal separation method, and a natural drop method. In the method of this embodiment, any method may be used, but it is preferable to adopt a suction method that can pass water in a short time and has little risk of sample contamination. Thereafter, a sewage sample 30 collected from a predetermined location is passed through the cartridge 20 (FIG. 1). As a result, the viruses 40 in the sewage sample 30 are adsorbed to the solid phase 10 made of a polymer having a hydrophobic group. Meanwhile, a part of the impurities 50 in the sewage sample 30 flows out of the cartridge 20 without being adsorbed to the solid phase 10. The impurities 50 are powders or fine particles, and such impurities 50 may inhibit virus detection by PCR or the like, which will be described later. In the method of the present embodiment, the impurities 50 can be separated from the viruses 40 in the process of adsorbing the viruses 40 to the solid phase 10 (and in the process of eluting the viruses 40 from the solid phase 10, which will be described later), so that the final virus detection accuracy can be expected to be improved. Note that, before passing the sewage sample 30 through the cartridge 20, it is desirable to perform centrifugation or filtration in advance to remove impurities with a relatively large specific gravity or size.

[0016] (2) Virus elution from the solid phase Next, a solvent (e.g., pure water) that elutes the impurities but does not elute the viruses 40 is passed through the cartridge 20 to wash away the impurities in the solid phase, and then an organic solvent 70 is passed through the cartridge 20 to elute the viruses 40 adsorbed on the solid phase 10 and collect them in a predetermined collection container 80 (FIG. 2). The amount of the organic solvent 70 used in this step is smaller than that of the sewage sample 30 passed through the cartridge 20 in the above step (1) (e.g., about 1 / 100 to 1 / 20 of the amount of the sewage sample 30). As a result, the eluate 90 from the cartridge 20 contains the viruses 40 at a higher concentration than the sewage sample 30. Note that the impurities 50 that are not eluted by the organic solvent in this step remain in the cartridge 20 while being adsorbed on the solid phase 10. Note that it is known that viruses are destroyed when they are reacted with an organic solvent such as alcohol. However, the present inventors have found, as a result of intensive research, that even when the viruses 40 adsorbed to the solid phase 10 are eluted by the organic solvent 70 as in the method of this embodiment, it is possible to elute the virus nucleic acid (DNA or RNA) necessary for virus detection by PCR or the like described below without destroying it. Furthermore, in the series of processes for eluting the viruses from the solid phase 10 described above, it is expected that a part of the viruses 40 during the process will be detoxified by the organic solvent 70, thereby reducing the risk of virus infection for the practitioner.

[0017] (3) Nitrogen spraying Nitrogen gas is sprayed onto the eluate 90 obtained above to evaporate a part of the organic solvent in the eluate 90. This can further increase the concentration of the viruses 40 in the eluate 90. Since most of the eluate 90 is a highly volatile organic solvent, a higher volatilization effect can be obtained compared to the case of extraction using water. In this step, the eluate 90 may be heated while the nitrogen gas is sprayed onto it. In this case, it is possible to evaporate the organic solvent in a shorter time. The temperature at which the eluate 90 is heated is preferably about 35°C to 41°C so that the nucleic acid (DNA or RNA) contained in the viruses 40 is not destroyed. Since most of the eluate 90 is an organic solvent typified by alcohol, it can be sufficiently volatilized within the above temperature range.

[0018] 1 to 3 show an example in which the solid phase 10 is packed in a syringe-type cartridge 20, but the shape of the cartridge 20 is not limited to this, and may be any shape such as a top type or a disk type. Also, instead of the cartridge 20, the solid phase 10 may be packed in a predetermined column.

[0019] Furthermore, in the method of this embodiment, it is not necessary to reconcentrate the eluate 90 by blowing nitrogen (i.e., the above step (3)). In that case, the concentration rate will be lower than when nitrogen blowing is performed, but the operation time can be shortened. When nitrogen blowing is omitted, it is desirable to use a smaller amount of organic solvent in the above step (2) than when nitrogen blowing is performed (for example, about 1 / 400 to 1 / 100 of the amount of the sewage sample 30) in order to compensate for the decrease in the concentration rate.

[0020] The virus concentrate obtained by the method of this embodiment (i.e., the eluate 90 recovered in (2) above, or the eluate re-concentrated by blowing nitrogen in (3) above) can be used for virus detection by nucleic acid amplification. For example, when the virus to be detected is a DNA virus, the virus concentrate is subjected to a DNA extraction process, and then DNA is amplified and detected by PCR using primers specific to the virus to be detected. When the virus to be detected is an RNA virus, the virus concentrate is subjected to an RNA extraction process, and then DNA complementary to the RNA (complementary DNA: cDNA) is synthesized, and the DNA is amplified and detected by RT-PCR using primers specific to the virus to be detected. For the DNA extraction process, RNA extraction process, PCR, and RT-PCR, existing reagent kits can be used. Furthermore, detection of the amplified product by PCR or RT-PCR may be performed by the traditional method of electrophoresing the amplified product to confirm the presence or absence and density of a band, but it is preferable to perform it by real-time PCR, which has been widely used in recent years. Real-time PCR is a method for measuring DNA concentration by detecting in real time a fluorescent signal generated by the interaction between an amplified DNA fragment and a reagent, and can rapidly quantify the PCR or RT-PCR amplified product. Since the method of this embodiment can remove at least a portion of the impurities 50 contained in the sewage sample 30, the virus detection accuracy can be improved by subjecting the virus concentrate obtained by the method of this embodiment to virus detection by PCR or RT-PCR.

[0021] As described above, the method of the present embodiment utilizes solid-phase extraction, and therefore, compared to the above-mentioned conventional method for concentrating a virus sample using PEG precipitation, the operation is simpler, the time required for virus concentration can be significantly shortened, and a high concentration rate can be achieved. Specifically, while the conventional method for concentrating a virus using PEG precipitation requires about 12 to 24 hours to complete the concentration, the method of the present embodiment can complete the concentration of a virus sample in about 2 hours. In addition, the method of the present embodiment achieves a concentration rate that is about 2.5 to 5 times higher than that of the PEG precipitation method. Furthermore, the method of the present embodiment uses an organic solvent as an elution solvent, and the organic solvent can be easily evaporated by blowing nitrogen, so that the eluate can be easily reconcentrated. Therefore, a high concentration rate can be achieved in a shorter time than the above-mentioned conventional method for concentrating a virus sample using solid-phase extraction (see, for example, Patent Document 1). EXAMPLES

[0022] The virus sample was concentrated and virus-derived RNA was detected by the following procedure. The number of tests was n = 3. First, 100 μL (9.3 × 10 4copy) was added, and the supernatant was collected by centrifugation at 3,000 rpm for 5 minutes. The supernatant was then subjected to concentration of a virus sample by the method according to the present invention. Concentration of the virus sample was performed using an HLB solid phase as follows. That is, first, the solid phase was conditioned by passing 10 mL of methanol and 10 mL of ultrapure water through the solid phase in this order, and then the supernatant was passed through the solid phase at a flow rate of 5 to 10 mL / min. After that, the solid phase was washed with 10 mL of ultrapure water, and the virus was eluted from the solid phase using 2 mL of methanol. 50 μL was taken from the obtained eluate, and 5 μL of RNase-free water was added thereto, and nitrogen was blown while heating at 40 ° C., and the eluate was re-concentrated to 5 μL. Next, Ampdirect 2019-nCoV Detection Kit (Shimadzu Corporation), which is a detection reagent for the new coronavirus, was added to the re-concentrated sample, and RT-PCR was performed. RT-PCR was performed using a Light Cycler 96 (Roche), and the fluorescence from the amplified products was measured in real time.

[0023] As a result of the above, as shown in FIG. 4, amplification of the N1 gene specific to the novel coronavirus was observed in two of the three tests. This indicates that the eluate obtained by the solid-phase extraction in this example contains the novel coronavirus or its RNA. This example experimentally confirmed that a polymer solid phase having a hydrophobic group such as the HLB solid phase can adsorb enveloped viruses such as the novel coronavirus, but it is presumed that a similar solid phase can also adsorb viruses other than enveloped viruses. The HLB solid phase used in this example is a solid phase having both hydrophobic and hydrophilic groups, but a solid phase having only hydrophobic groups is also considered to be usable as a solid phase in the virus concentration method according to the present invention in the same manner as described above.

[0024] [Various aspects] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0025] (Item 1) A method for concentrating a virus sample according to one aspect of the present invention comprises: The method includes contacting a liquid containing the virus with a solid phase made of a polymer having a hydrophobic group, thereby causing the virus to be retained on the solid phase, and then contacting the solid phase retaining the virus with an organic solvent, thereby eluting the virus from the solid phase.

[0026] (Item 2) In the method for concentrating a virus sample according to item 1, the organic solvent may be an alcohol solvent.

[0027] (Item 3) The method for concentrating a virus sample according to item 1 or 2, The method may further include blowing nitrogen onto the eluate from the solid phase to evaporate at least a portion of the organic solvent contained in the eluate.

[0028] (Item 4) The method for concentrating a virus sample according to any one of items 1 to 3, The virus may be an enveloped virus.

[0029] The method for concentrating a virus sample described in paragraph 1 utilizes a solid-phase extraction method, and therefore, compared with the above-mentioned conventional method for concentrating a virus sample using PEG precipitation, the operation is simpler, the time required for virus concentration can be significantly shortened, and a high concentration rate can be achieved. In addition, in the method for concentrating a virus sample described in paragraph 1, an organic solvent is used to elute the virus from the solid phase, and it is expected that at least a part of the virus will be detoxified by the organic solvent, and the effect of reducing the risk of virus infection for the operator can be obtained.

[0030] According to the method for concentrating a virus sample described in item 3, the virus concentration in the eluate can be further increased in a short period of time with simple operations. [Explanation of symbols]

[0031] 10...Solid phase 20…Cartridge 30…Sewage sample 40…Virus 50…impurities 70...Organic solvent 80…Collection container 90...Eluate

Claims

1. A method for concentrating a virus sample, comprising: contacting an HLB solid phase with a liquid containing the enveloped virus, thereby retaining the enveloped virus on the HLB solid phase, and then contacting the HLB solid phase retaining the enveloped virus with an alcohol solvent, thereby eluting the enveloped virus from the HLB solid phase.

2. 2. The method for concentrating a virus sample according to claim 1, further comprising blowing nitrogen onto the eluate from the HLB solid phase to evaporate at least a portion of the alcohol solvent contained in the eluate.

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