JP2019083789A - Virus concentration methods - Google Patents

Abstract

To provide techniques for concentrating viruses to enable virus detection even in samples of thin viral concentrations.SOLUTION: The invention relates to a method for concentrating virus comprising the steps of: (a) adding a co-precipitant to a virus-containing sample; (b) adding a water-soluble monohydric alcohol of C3 or more to the sample to which the co-precipitant was added; and (c) recovering a virus-containing precipitation from the mixture obtained in the step (b). Then invention also relates to a nucleic acid detection method using the concentrate and can be used for food hygiene inspections and environmental inspections.SELECTED DRAWING: None

Description

  The present invention relates to a method for virus concentration and a method for nucleic acid detection using the concentrate. The present invention can be used for food hygiene inspection, environmental inspection and the like.

  Nucleic acid amplification is a technology that amplifies several copies of a target nucleic acid to detectable levels, that is, hundreds of millions of copies, and is not only in the field of life science research, but also in the medical field such as gene diagnosis and clinical examination, or in food and the environment. Are also widely used in the field of microbial testing and the like.

  The samples to be tested using the nucleic acid amplification method vary depending on the purpose. In the case of detection of infectious disease-causing bacteria, samples in which the causative bacteria are present, that is, urine, sputum, feces, blood, nasal fluid, vaginal fluid and the like. In the case of food hygiene inspection, it is food or feces and urine collected from a food handler, and in environmental hygiene inspection it is soil, river water, rain water, environmental water such as seawater, wipes from manufacturing facilities, etc. With regard to food poisoning caused by microbes, which have a latent period before the onset of characteristic symptoms, a rapid test method is required to prevent damage from spreading. It becomes a thing etc.

  Even in the nucleic acid amplification method, there is a detection limit, and it may be necessary to concentrate the nucleic acid or nucleic acid-containing substance (cell, virus, etc.) in the sample so that the amount of target nucleic acid becomes more than the detection limit of the nucleic acid amplification method. In the case of the above-described food sanitation inspection, wipes from manufacturing facilities and the like correspond.

  Various methods are known as methods for concentrating nucleic acids and nucleic acid-containing substances. There are various methods for virus concentration methods, for example, adsorption of virus on a carrier (Patent Document 1), and precipitation concentration technology using an organic solvent. As an organic solvent, there is a method of using polyethylene glycol (Patent Document 1), a method of using ethyl alcohol (Non-Patent Document 1), and the like.

  In the case of a method using a carrier, under the conditions for detection by nucleic acid amplification, the nucleic acid may be adsorbed to the carrier, and it is basically necessary to separate the carrier from the nucleic acid. In particular, since the anion exchange resin is used in the method of Patent Document 1, there is a possibility that nucleic acid may be adsorbed under nucleic acid amplification reaction conditions under which the salt concentration is not so high. In the method which does not use a carrier, such a problem does not particularly occur, but in the case where a substance which inhibits the nucleic acid amplification reaction is simultaneously precipitated together with the virus, an operation for removing the inhibitory substance is required. Incidentally, in the method of Patent Document 1, after the virus-containing solution eluted from the carrier is further subjected to precipitation concentration with polyethylene glycol, nucleic acid purification with a commercially available product for RNA preparation is performed on the precipitate. Also, in the method of Non-Patent Document 1, nucleic acid purification with a commercially available product for preparation of RNA is performed on a precipitate obtained by adding ethyl alcohol.

Japanese Patent Application Publication No. 2004-129548

Kuni Funakoshi, The Japanese Society of Food and Microbiology, 30 (3), 156-159, 2013

  An object of the present invention is to provide a technique for concentrating a virus which enables detection even in a sample having a low virus concentration.

  As a result of intensive investigations, the present inventors have found a stable virus concentration method that is not affected by the type of sample, and have completed the present invention.

The representative present invention is as follows.
[1] A method for virus concentration,
(A) adding a coprecipitant to a sample containing virus,
(B) adding a water-soluble monohydric alcohol having 3 or more carbon atoms to a sample to which a coprecipitant has been added, and (c) recovering a precipitate containing a virus from the mixture obtained in step (b),
Methods of virus concentration including:
[2] The method according to [1], wherein the water-soluble monohydric alcohol having 3 or more carbon atoms is normal propyl alcohol or isopropyl alcohol.
[3] The water-soluble monohydric alcohol having 3 or more carbon atoms is isopropyl alcohol, and added to the sample such that the final concentration of isopropyl alcohol is 40 to 60% (v / v), [ 1] The method described.
[4] The method according to [1], wherein the coprecipitant is a polysaccharide having glucose as a constituent sugar.
[5] A polysaccharide comprising glucose as a constituent sugar is glycogen, glycogen azure, starch, soluble starch, starch syrup, amylopectin, amylopectin, amylopectin anthranilate, amylopectin azul, insoluble amylopectin, soluble amylose, and amylose azure The method of [4], which is at least one polysaccharide selected.
[6] The method according to [5], wherein the polysaccharide having glucose as its constituent sugar is glycogen or a modified product thereof.
[7] The method according to [6], wherein glycogen or its modified product is added to the sample to a final concentration of 40 to 500 ug / mL in step (a).
[8] The method according to [1], wherein the virus-containing sample of step (a) is a suspension of the collected matter by wiping.
[9] The method according to [8], wherein the solvent of the suspension of the collected matter by wiping is a solvent selected from the group consisting of sterile water, phosphate buffered saline or saline.
[10] A method of detecting a virus, comprising the steps of: (a) adding a coprecipitant to a sample possibly containing a virus,
(B) adding a water-soluble monohydric alcohol having 3 or more carbon atoms to a sample to which a coprecipitant has been added,
(C) recovering the precipitate from the liquid mixture obtained in step (b),
(D) treating the precipitate obtained in step (c) by a nucleic acid recovery method, and (e) subjecting the treated product obtained in step (d) to nucleic acid synthesis,
Virus detection methods, including:
[11] The method according to [10], wherein the alcohol having 3 or more carbon atoms is normal propyl alcohol or isopropyl alcohol.
[12] The method according to [10], wherein the nucleic acid recovery method in step (d) is a step including the addition of a strong alkali to the precipitate.
[13] The method of [10], wherein the nucleic acid synthesis of step (e) comprises a reverse transcription reaction.
[14] The method of [10], wherein the nucleic acid synthesis in step (e) is by PCR.

  According to the present invention, it is possible to confirm the presence of virus by a conventional detection means even for a sample with a low virus concentration. Furthermore, depending on the method of nucleic acid extraction from the concentrate, the risk that may occur when separating and purifying the nucleic acid, that is, the risk of sample loss and carryover can be reduced. The effect becomes remarkable especially in the inspection for wiping off objects such as manufacturing equipment for preventing food poisoning in advance.

The examination 1 (ethyl alcohol precipitation and PEG precipitation) of the concentration method of the reference example 1 is shown. The examination 2 (ethyl alcohol precipitation and PEG precipitation) of the concentration method of the reference example 2 is shown. The influence of the wiping off sample of the reference example 3 is shown. The examination 1 (ethyl alcohol precipitation and isopropyl alcohol precipitation) of the concentration method of Example 1 is shown. Figure 2 shows a comparison of the method of preparation of the viral sample of Example 2. The effect of the wiped sample of Example 3 is shown. The comparison of stationary time * centrifugation time after alcohol addition of Example 4 is shown.

  Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.

  One aspect of the present invention is a method of concentrating a virus characterized by precipitation of a virus combining a coprecipitant and a water-soluble monohydric alcohol having 3 or more carbon atoms. As the alcohol, normal propyl alcohol or isopropyl alcohol can be suitably used.

  The sample to which the concentration method of the present invention is applied is not particularly limited as long as it is a sample that may contain a virus, but it is particularly effective for a sample of a liquid having a low virus concentration. Examples of such samples include environmental water (seawater, river water, lake water, sewage, household drainage, industrial drainage, etc.), and a suspension of collected material obtained by wiping operation with a swab or the like. The method of preparing the suspension from the swab is generally a method of using a commercially available swab with diluted solution and suspending the collected material by putting the swab after wiping operation in the diluted solution, As another method, the tip of the swab is cut out and placed in a cup of NucleoSpin (registered trademark) Forensic Filters (manufactured by Mach Liner Gel Co., Ltd.), and then a suitable solvent such as physiological saline, sterile water or phosphate buffered saline ( After suspending directly in PBS etc., it can also be implemented by recovery of the filtrate by centrifugation. The objects of wiping include food manufacturing equipment such as a kitchen, laboratory equipment, floors such as medical equipment and toilets, walls, work benches, sinks, various appliances and equipment, and the like. There is no particular limitation on the swab, and a swab or a swab commercially available for wiping test can be used. When solid matter such as sewage is contained, it is preferable to remove suspended solids by centrifugal treatment or filter treatment before carrying out the concentration method. In the method of the present invention, virus concentration is usually performed for each sample, but a plurality of samples may be mixed and then subjected to a concentration operation. When dealing with a large number of samples, it may be advantageous to pre-mix and concentrate multiple samples.

  The virus targeted by the concentration method of the present invention is not particularly limited, but includes viruses belonging to the calicivirus family (Norovirus (NoV), Sapovirus (SV), feline calicivirus (FCV), etc.). In addition, the feline calicivirus (FCV) mentioned above is a substitute virus widely used for evaluation of a disinfectant, a cleaning agent, etc. instead of the human norovirus (HuNoV) which can not be culture | cultivated easily.

A coprecipitant is used in combination in the virus concentration method of the present invention. As the coprecipitant, known substances such as water-soluble polymers, polyacrylamides, polyethylene glycols and the like are used, but polysaccharides are preferred for the present invention. Polysaccharides include those modified from sugars alone. For example, such modifications include dye modification. For example, examples of polysaccharides having glucose as a constituent sugar include glycogen, starch, soluble starch, amylopectin, insoluble amylopectin, amylose, glycogen azul, stachyazule, amylopectin azul, amylose azur, amylopectin anthranilate and the like. Glycogens (glycogen and its modified products) are preferably used in the present invention. Glycogens marketed as reagents can be used in the present invention. The final concentration after addition of glycogens added to the sample as a coprecipitant is, for example, 20 to 2,000 ug / mL. Alternatively, it is 30 to 1,000 ug / mL, preferably 40 to 500 ug / mL, more preferably 50 to 250 ug / mL.
In addition, since the coprecipitant may be present in the sample at a certain amount or more, if the liquid volume of the sample exceeds 800 uL, 200 ug of glycogen may be uniformly added to the sample.

  In the virus concentration and precipitation method using an organic solvent, alcohol, acetone, acetonitrile, etc. which are used in a conventional protein concentration precipitation method using a water-soluble organic solvent are used, but in the present invention, the number of carbon atoms is 3 or more. Water soluble monohydric alcohols are preferred. The water-soluble monohydric alcohol having 3 or more carbon atoms is not particularly limited, but normal propyl alcohol, isopropyl alcohol, normal butyl alcohol and the like, or structural isomers thereof are preferable. In addition, a plurality of water-soluble monohydric alcohols having 3 or more carbon atoms may be mixed and used. More preferably, normal propyl alcohol or isopropyl alcohol is used in the present invention. In addition, unless otherwise noted in the present invention, a commercially available reagent or a higher purity alcohol can be used.

  In the present invention, a water-soluble monohydric alcohol having 3 or more carbon atoms is added to the sample to which the coprecipitant has been added. The amount of the alcohol added is not particularly limited as long as the virus precipitates, but it is preferable not to increase the volume as much as possible for ease of operation, for example, in the case of isopropyl alcohol, relative to 1 volume of coprecipitant-added sample It is effective to add about 1 volume of isopropyl alcohol, that is, the final concentration of isopropyl alcohol is 40 to 60% (v / v), preferably 45 to 55% (v / v). In addition, in Non-Patent Document 1, there was a description that "ethanol was added and allowed to stand at room temperature for 30 minutes after being mixed by inversion several times." In the method of the present invention using isopropyl alcohol, isopropyl alcohol was used. There is no particular limitation on the standing time of the mixed solution obtained by the addition, and the virus can be recovered even by immediately centrifuging.

  In the method of the present invention, recovery of a precipitate containing a virus is carried out from a mixed solution obtained by adding a water-soluble monohydric alcohol having a carbon number of 3 or more to a sample. There is no particular limitation on the method for separating the precipitate and the liquid phase, and solid-liquid separation with a filter, precipitation using centrifugal force, and the like can be mentioned. In the present invention, in the case of the precipitation particularly utilizing centrifugal force, the centrifugal force is not particularly limited. For example, 1,000 × g to 20,000 × g or 2,000 × g to 19,000 × g , Preferably 3,000 x g to 18,000 x g, more preferably 4,000 x g to 17,000 x g, more preferably 5,000 x g to 16,000 x g, particularly preferably Is 6,000 × g to 15,000 × g. Also, the time taken for centrifugation is not particularly limited, and an appropriate time for virus precipitation may be selected. For example, the time of centrifugation is more than 0 minutes, less than 15 minutes, or more than 0 minutes, less than 10 minutes, preferably more than 0 minutes, less than 5 minutes. The removal of the supernatant after the formation of the precipitate is not particularly limited, and may be performed by decantation, aspiration and removal with a pipette, an aspirator or the like. The resulting precipitate may be further air-dried to remove the remaining water-soluble monohydric alcohol. The precipitate thus obtained corresponds to the concentration of virus in the sample.

  Another aspect of the present invention is a method of detecting a virus using the above-described enrichment method. When a method targeting nucleic acid is used for detection, it is necessary to recover the nucleic acid from the concentrate recovered as a precipitate in the above operation. Here, recovery of nucleic acid refers to an operation for making a nucleic acid derived from a virus in a concentrate in a detectable state, and is not limited to an operation for separating or purifying a nucleic acid derived from a virus. As a nucleic acid recovery method, a commercially available product for nucleic acid extraction can be used, and by adding an appropriate solution to the concentrate and heat treating it, it may be possible to make the virus-derived nucleic acid detectable.

In the present invention, a nucleic acid recovery method using a strongly alkaline hydroxide solution can be suitably used. The method comprises treating a nucleic acid in a virus contained in the concentrate to be usable as a template for nucleic acid synthesis by adding a strong alkali hydroxide solution to the concentrate obtained by the virus concentration method of the present invention How to Also, the concentrate treated in this way can be added directly to the nucleic acid synthesis reaction in the subsequent step to carry out the synthesis. The strongly alkaline hydroxide used in the method is not particularly limited, but is preferably a hydroxide of a Group 1 element or a hydroxide of a Group 2 element, or a mixture thereof. Alternatively, it may be a hydroxide of tetraalkylammonium. Although not particularly limited, hydroxides of lithium, sodium, potassium, rubidium or cesium, beryllium, magnesium, calcium, strontium, barium or radium hydroxide, europium, thallium hydroxide and the like are exemplified. Preferably, it comprises KOH, NaOH, LiOH or Ca (OH) ₂ or a combination thereof. More preferably, it is NaOH.

  The concentration of the strongly alkaline hydroxide solution may be appropriately adjusted according to the mixing ratio with the concentrate, but the final concentration after addition and mixing is, for example, 10 mM to 100 mM, alternatively 20 mM to 70 mM, preferably 30 mM to 60 mM. More preferably, it is 30-40 mM. The mixing ratio is not particularly limited, but the mixing ratio of the concentrate and the strongly alkaline hydroxide solution may be appropriately selected in consideration of the final concentration of the strongly alkaline hydroxide after mixing.

  For mixing the concentrate and the strongly alkaline hydroxide solution, stirring, spin down, end-over mixing, pipetting, etc. may be used, but the method and container used are not limited.

The alkaline mixed solution obtained by mixing the concentrate and the strongly alkaline hydroxide solution can be directly subjected to a virus nucleic acid detection operation after being left without heating. It is effective to improve the reproducibility of the test because the temperature condition of the standing step can be made the same, so that the standing step is performed at a low temperature using a cooling block, crush ice or the like. Moreover, you may store in a refrigerator or a refrigerator compartment.
The temperature of the mixture in this case is about 0 ° C to 30 ° C. The temperature of standing is, for example, 0 ° C to 25 ° C, preferably 0 ° C to 20 ° C, more preferably 0 ° C to 15 ° C, still more preferably 0 ° C to 10 ° C, and particularly preferably 0 ° C to 5 ° C.
It is also possible to carry out this step at room temperature. Room temperature refers to the ambient temperature at which workers can easily operate. Preferably it is 30 degrees C or less, More preferably, it is 25 degrees C or less.
There is no particular limitation on the alkali treatment time, that is, the time for which the above mixture is left to stand, but a range of 0 to 60 minutes is exemplified. In the method of the present invention, centrifugation may be performed immediately after mixing with a strongly alkaline hydroxide solution. It is natural that the processing time is appropriately adjusted according to the number of samples. Within the range of the above processing time, there is no big difference to the nucleic acid synthesis reaction.

  When the detection target is an RNA virus belonging to the calicivirus family, the alkaline mixed solution treated as described above is subjected to reverse transcription reaction to synthesize DNA (cDNA) complementary to RNA contained in the reaction solution. can do.

  A small amount of cDNA can be detected by using a nucleic acid amplification method. Although there is no limitation in particular in the nucleic acid amplification method, For example, LAMP method and PCR method can be used. There is also a Two-Step method in which the reverse transcription reaction and the DNA amplification reaction are carried out in independent reaction solutions, but the one-step method in which the reverse transcription reaction and the DNA amplification reaction are possible in one reaction solution, For example, the One-Step RT-PCR method is preferred.

An example of the reaction solution composition of the reverse transcription reaction includes one containing a reverse transcription primer having a sequence complementary to the RNA to be detected, a salt, deoxyribonucleotides, a reverse transcriptase, and a buffer. As the above salts, MgCl ₂ , KCl, etc. are used, but they may be changed to other salts as appropriate. As an example of the reaction solution composition of PCR, a pair of PCR primer pairs in which one primer is complementary to the DNA extension product of the other primer, salts, deoxyribonucleotides, thermostable DNA polymerase and buffer There is a solution containing a solution. As the above salts, MgCl ₂ , KCl, etc. are used, but they may be changed to other salts as appropriate. In the RT-PCR method, one of the primer for cDNA synthesis and one of the primer pair for PCR can also be used. There may be a reaction called Multiplex RT-PCR, in which a plurality of primers for cDNA synthesis and a primer pair for PCR are included so that a plurality of amplification products can be obtained in one reaction. In addition, a known substance to be added to the reverse transcription reaction solution or the nucleic acid amplification reaction solution may be included for the purpose of improving the reactivity and the like. Examples of the substance to be added to the reaction solution include, but are not limited to, proteins such as bovine serum albumin (BSA), surfactants such as Tween-20, betaines, and acidic polymers. Also, although dNTP is normally used in four types of dATP, dGTP, dCTP and dTTP, dUTP may be further added to the reaction solution in order to prevent contamination of the PCR amplification product as a template for other nucleic acid amplification reactions. . The nucleic acid amplification product obtained in this reaction will be incorporated with dUTP, and the aforementioned nucleic acid amplification can be carried out by causing Uracil-N-glycosyrase (UNG) having the activity of degrading uracil-containing DNA to act before the nucleic acid amplification reaction. It can disassemble things. In this way, it is possible to prevent contamination resulting from the amplification product obtained in the previously performed reaction from being mixed into the later reaction.

  When an alkaline mixed solution containing a concentrate is added to an enzyme reaction solution (for example, a reverse transcription reaction solution), if the buffer capacity of the enzyme reaction solution is an effective range, the mixed solution is directly added to the enzyme reaction solution. It can be added. When there is a concern that the pH of the enzyme reaction solution shifts to the alkaline side to reduce the enzyme activity, a step of neutralizing the strongly alkaline hydroxide may be included in the mixture solution. In addition to this, a method of pre-adding a substance capable of neutralizing the mixture to the enzyme reaction solution is exemplified, but the method is more preferable in view of simplification of the procedure.

  Using cDNA derived from RNA contained in the sample as a template, amplified and generated DNA fragments should be detected and identified by electrophoresis, melting curve method, various probe methods (Q probe, scorpion probe, hybrid probe etc.) Can. In addition, if it is a device equipped with a temperature raising / lowering function and a fluorescence measurement function, using TaqMan (registered trademark) probes, intercalating dyes, etc., DNA fragments generated during the reaction can be detected and identified in real time. it can.

  As one embodiment of the present invention, a kit used for the virus concentration method of the present invention is exemplified. Although the said kit does not specifically limit this invention, the kit for concentrating the virus contained in the collection by wiping method is illustrated. For example, a swab used for the wiping method, a solvent (saline, sterile water, PBS, etc.) for preparing a suspension of the collected matter collected by the swab, a kit containing a coprecipitant can be mentioned. The kit may be a kit containing a water-soluble monohydric alcohol having 3 or more carbon atoms, which may be prepared by the user.

  Furthermore, the above-described kit for virus concentration can be combined with a kit for virus detection. Although the present invention is not particularly limited, examples of the above-mentioned kit for virus detection include a virus detection kit belonging to the caliciviridae family. The component for virus detection contained in such a combination kit includes a reagent for making the nucleic acid derived from concentrated virus detectable. For example, in the case of detecting an RNA virus, a reagent for reverse transcription reaction which synthesizes cDNA using RNA from the target RNA virus as a template is exemplified. In addition, a reagent for a nucleic acid amplification reaction that amplifies the cDNA or a part thereof, and a reagent for detecting an amplified nucleic acid fragment are exemplified. As these reagents, in addition to the reagents described for the virus detection method of the present invention, those used for known RNA detection techniques can be used. In addition, primers and probes for specifically detecting a target RNA virus can be synthesized based on the sequence of genomic RNA of the RNA virus.

  EXAMPLES The present invention will be specifically described below by way of Examples, but the scope of the present invention is not limited to these Examples.

Reference Example 1 Examination of concentration method 1 (ethyl alcohol precipitation and PEG precipitation)
1/10 dilution of a stock solution of VR-782, Feline calicivirus, F-9 (Caliciviridae), feline calicivirus (FCV) purchased from American Type Culture Collection (ATCC) in saline (NaCl, 153 mM), and 1 A / 100 dilution was prepared. The virus stock solution, 1/10 dilution solution and 1 μL dilution solution were respectively added to 399 μL of physiological saline to prepare a virus sample. Two groups of virus samples of each concentration were prepared, and 5 μL of a 20 mg / mL glycogen solution was added as a coprecipitant to each group. Add 800 μL of ethyl alcohol to one group of virus samples (ethyl alcohol precipitated), 200 μL of 32% PEG 10000 and 200 μL of a 1.6M NaCl solution to the other group (PEG precipitated), mix and let stand at room temperature for 10 minutes did. Each solution is centrifuged at 15,000 × g for 15 minutes at 25 ° C., the supernatant is removed, and the resulting precipitate is treated with Product Code No. RR296A TaKaRa Norovirus GI / GII Detection Kit (for high-speed detection) (Takara Thirty μL of Lysis Buffer (NV) (manufactured by BIO CO., LTD.) Was added and dissolved by pipetting, and heated at 90 ° C. for 3 minutes to prepare an RNA sample. Using this as a sample, it was subjected to RT-PCR under the following reaction conditions using the kit, the following primer pair and probe, and the amount of viral RNA was measured by the Ct value. The N number of the virus suspension was 2, and the product Code No. TP-990 ThermalCycler Dice (registered trademark) Real Time System III (manufactured by Takara Bio Inc.) was used as the nucleic acid amplification apparatus.

(FCV Primer / Probe Mix)
10 μM FCV detection primer F (SEQ ID NO: 1)
10 μM FCV detection primer R (SEQ ID NO: 2)
5 μM FCV detection probe (SEQ ID NO: 3)

(Reaction solution for RT-PCR)
10 μL RNA sample 12 μL PCR Buffer (NV) 4
1 μL FCV Primer / Probe Mix
1.5 μL Enzyme Mix (NV) 4
0.5 μL sterile water

(Reaction conditions)
The reaction liquid (25 μL) was reacted under the following temperature cycle conditions.
42 ° C 5min,
95 ° C 30 sec,
45 cycles with 95 ° C 5sec-54 ° C 30sec as one cycle (FAM, ROX, Cy5 fluorescence detection)

  The PCR results are shown in FIG. In the figure, the vertical axis represents the Ct value, and the horizontal axis represents the virus precipitation method and the dilution ratio of the virus dilution. Hereinafter, the graph shows the result of N number 2 unless otherwise described. From FIG. 1, it was shown that when viral samples were concentrated by ethyl alcohol precipitation, more viral RNA was obtained as compared to PEG precipitation, and it was possible to concentrate the virus with a high recovery rate. This means that ethyl alcohol precipitation can detect a virus with one order of sensitivity as high as PEG precipitation.

Reference Example 2 Examination of concentration method 2 (ethyl alcohol precipitation and PEG precipitation)
The saline used for preparation of the virus sample in Reference Example 1 was distilled water or phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ 12 H ₂ O, 1.8 mM KH ₂ PO ₄ ) was substituted for the test. That is, 399 μL of each of physiological saline, distilled water, and PBS was prepared, and 1 μL of a stock solution of virus was added to prepare a virus sample, and then the procedure of Example 1 was repeated to measure the amount of viral RNA. The results are shown in FIG. In the figure, the vertical axis indicates the Ct value, the water on the horizontal axis indicates distilled water, and the saline indicates saline. From FIG. 2, when ethyl alcohol precipitation is performed, a large amount of viral RNA is recovered as in Reference Example 1 even if distilled water is used for preparation of a virus sample, but viral RNA obtained using PBS is It was shown to decrease.

Reference Example 3 Influence of Wipe Sample The tap of a water tap, a sink and a refrigerator was wiped off with a swab moistened with physiological saline in advance, Askru model number, 102257 sterile cotton swab No. 103 (manufactured by Osaki Medical Co., Ltd.). A swab without wiping was prepared as a negative control. The tip of these swabs was cut out, placed in the cup of the product Code No. U0988A NucleoSpin® Forensic Filters (Machliner Gel), and 600 μL of physiological saline was added to the cup. After vigorously mixing them with a vortex, they were centrifuged at 20,000 × g for 1 minute at 25 ° C. The obtained filtrate was divided into 300 μL portions, and 1 μL of a solution obtained by diluting 199 L of a physiological saline solution and 1/10 of a stock solution of FCV was added to each, and the amount of viral RNA was determined in the same manner as in Reference Example 1 as a 400 μL virus sample. It was measured. The measurement results are shown in FIG. From FIG. 3, it was shown that when virus samples were concentrated by ethyl alcohol precipitation, the amount of viral RNA obtained was largely different depending on the wiping environment.

Example 1 Examination of concentration method 1 (ethyl alcohol precipitation and isopropyl alcohol precipitation)
Ethyl alcohol precipitation and isopropyl alcohol precipitation were compared as a method of concentration of the virus sample. A precipitate was prepared in two operations of ethyl alcohol precipitation of Reference Example 1 and isopropyl alcohol precipitation in which ethyl alcohol in the method was replaced with 400 μL of isopropyl alcohol. In addition, when preparing the RNA sample by dissolving the precipitate, it was replaced with Lysis Buffer (NV), the precipitate was dissolved in 30 μL of 50 mM NaOH solution, and the RNA sample was allowed to stand at room temperature for 5 minutes simultaneously. . The results are shown in FIG. In the figure, RR296 (white) shows the result of dissolving the precipitate in Lysis Buffer (NV), and NaOH (black) shows the result of dissolving the precipitate in NaOH solution. From FIG. 4, when the virus sample was concentrated by isopropyl alcohol precipitation, although half amount of isopropyl alcohol was added, it was shown that the recovery amount of viral RNA was equivalent to ethyl alcohol precipitation. As a result, the liquid volume at the time of concentration can be reduced to two thirds, and the operation becomes simpler. The virus RNA recovery was shown to be equivalent to ethyl alcohol precipitation. Also, it was shown that when RNA samples were prepared with NaOH solution, a large amount of viral RNA could be recovered.

Example 2 Comparison of Methods for Preparing Virus Samples Saline and Phosphate Buffered Saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ · 12 H ₂ O, 1. H) used to prepare virus samples. The test was performed with 8 mM KH ₂ PO ₄ ). That is, 399 μL of each of physiological saline and PBS was prepared, and 1 μL of a stock solution of virus was added to prepare a virus sample, which was concentrated by isopropyl alcohol precipitation.
To 400 μL of the virus sample, 400 μL of isopropyl alcohol was added, and allowed to stand at room temperature for 10 minutes. Each solution is centrifuged at 15,000 × g for 15 minutes at 25 ° C., the supernatant is removed, the obtained precipitate is dissolved in 30 μL of 50 mM NaOH solution, and an RNA sample allowed to stand at room temperature for 2 minutes is prepared. did. This was used as a sample and subjected to RT-PCR in the kit and conditions used in Reference Example 1.
The results are shown in FIG. According to FIG. 5, when concentrated by isopropyl alcohol precipitation, viral RNA can be recovered similarly to saline even if the viral sample is prepared particularly with PBS, and the influence of the solution for preparing the viral sample is reduced. It has been shown.

Example 3 Effect of wiping sample The ethyl alcohol precipitation carried out in Reference Example 3 was carried out by replacing it with isopropyl alcohol precipitation. That is, 400 μL of isopropyl alcohol was added to each virus sample to prepare a precipitate. These precipitates were dissolved in 30 μL of 30 mM NaOH solution or 30 μL of 40 mM NaOH solution, and allowed to stand at room temperature for 5 minutes to prepare an RNA sample. The other operations were performed in the same manner as in Reference Example 3. The results are shown in FIG. From FIG. 6, it was shown that the method of concentrating the virus by isopropyl alcohol precipitation and dissolving it in the NaOH solution can stably measure the viral RNA with little change in the measured value of the viral RNA due to the wiping environment.

Example 4 Examination of stationary time and centrifugation time after addition of isopropyl alcohol Inactivated Norovirus purchased from ZeptoMetrix Corporation, Catalog Number: NATNOVI-6 MC (NATtrol (TM) Norovirus GI) and Catalog Number: NATNO VII-6 MC (NATtrol (TM) 2.) Prepare 2 μL each of the stock solution of Norovirus GII) and add it to 16 μL of physiological saline (NaCl, 153 mM) to prepare a 10-fold dilution, and then dilute stepwise and dilute 1000-fold and 10000-fold. The solution was prepared. To 399 μL of physiological saline, 1 μL each of each of the 1000-fold diluted solution and the 10000-fold diluted solution was added to prepare two groups of virus samples having different concentrations. After adding 5 μL of 20 mg / mL glycogen solution as coprecipitant to 400 μL of virus samples of 2 groups, add 400 μL of isopropyl alcohol and mix several times by inverting, let stand at room temperature 25 ° C for 10 minutes, 15, The virus sample group centrifuged at 000 × g for 15 minutes was designated as A-treated group. On the other hand, 400 μL of isopropyl alcohol was added, and after several times of inversion and mixing, the virus sample group centrifuged at 15,000 × g for 5 minutes was prepared as a B-treated group. Remove the supernatant of the A-treated group and B-treated group, dissolve the obtained precipitate in 6 μL of 42 mM NaOH solution, prepare an RNA sample that is allowed to stand at room temperature for 2 minutes, and prepare the total amount of TaKaRa norovirus GI / GII Using a detection kit (for high-speed detection) (Takara Bio Inc. RR296A), it was subjected to RT-PCR under the following reaction conditions. In addition, each virus sample group set N number to 3, and calculated | required the average Ct value. The product Code No. TP-990 ThermalCycler Dice (registered trademark) Real Time System III (manufactured by Takara Bio Inc.) was used as the nucleic acid amplification apparatus.

(Reaction solution for RT-PCR)
6.0 μL RNA sample 12.0 μL PCR Buffer (NV) 4
1.5 μL NVPrimer / Probe Mix 4
1.5 μL Enzyme Mix (NV) 4
4.0 μL sterile water

(Reaction conditions)
The reaction liquid (25 μL) was reacted in the following temperature cycle. In addition, since fluorescence detection was not performed for the first 5 cycles, the result described Ct value which added 5 cycles.
42 ° C 5min,
95 ° C 30 sec,
95 ° C 5sec-56 ° C 40sec 5 cycles 90 ° C 5sec-56 ° C 40sec 35 cycles (FAM, ROX, Cy5 fluorescence detection)

  The measurement results are shown in FIG. White bar graph is a group of A-treated (10 minutes standing time, 15 minutes centrifugation time) A solution of inactivated Norovirus (GI / GII) diluted 1000 times (N = 3) and 10000 times diluted solution (N = 3) Indicates the average value of Black bars indicate samples treated with B-treated (no standing time, 5 minutes centrifugation time) a 1000-fold diluted solution (N = 3) and a 10000-fold diluted solution (N = 3) of inactivated Norovirus (GI / GII) Indicates the average value of The measurement results for both A and B treatments showed equivalent reactivity at each concentration, and it was found that there is no problem even if the settling time and the centrifugation time are shortened.

  INDUSTRIAL APPLICABILITY The present invention is used in clinical examinations, food hygiene management, examinations for the purpose of identification of breeds in foods, determination of presence or absence of genetic modification, and the like.

SEQ ID No: 1; PCR forward Primer FCV gp2-F.
SEQ ID No: 2; PCR Reverse Primer FCV gp2-R.
SEQ ID No: 3; Probe FCVGP2-P. 5'-end is labeled FAM and 3'-end is labeled BHQ1.

Claims (14)

A method of virus concentration,
(A) adding a coprecipitant to a sample containing virus,
(B) adding a water-soluble monohydric alcohol having 3 or more carbon atoms to a sample to which a coprecipitant has been added, and (c) recovering a precipitate containing a virus from the mixture obtained in step (b),
Methods of virus concentration including:   The method according to claim 1, wherein the water-soluble monohydric alcohol having 3 or more carbon atoms is normal propyl alcohol or isopropyl alcohol.   The water-soluble monohydric alcohol having 3 or more carbon atoms is isopropyl alcohol, and is added to the sample so that the final concentration of isopropyl alcohol is 40 to 60% (v / v) with respect to the sample containing virus. A method according to claim 1, characterized in that.   The method according to claim 1, wherein the coprecipitant is a polysaccharide having glucose as a constituent sugar.   The polysaccharide having glucose as a constituent sugar is selected from the group consisting of glycogen, glycogen azur, starch, soluble starch, starch syrup, amylopectin, amylopectin anthranilate, amylopectin azul, insoluble amylopectin, soluble amylose, and amylose azure 5. The method of claim 4 which is at least one polysaccharide.   The method according to claim 5, wherein the polysaccharide having glucose as a constituent sugar is glycogen or a modified product thereof.   7. The method according to claim 6, wherein glycogen or its modified substance is added to the sample to a final concentration of 40 to 500 ug / mL in step (a).   The method according to claim 1, characterized in that the virus-containing sample of step (a) is a suspension of the harvest product.   The method according to claim 8, wherein the solvent of the suspension of the collected matter by wiping is a solvent selected from the group consisting of sterile water, phosphate buffered saline or saline. A method of detecting a virus, comprising the steps of: (a) adding a coprecipitant to a sample possibly containing the virus;
(B) adding a water-soluble monohydric alcohol having 3 or more carbon atoms to a sample to which a coprecipitant has been added,
(C) recovering the precipitate from the liquid mixture obtained in step (b),
(D) treating the precipitate obtained in step (c) by a nucleic acid recovery method, and (e) subjecting the treated product obtained in step (d) to nucleic acid synthesis,
Virus detection methods, including:   11. The method according to claim 10, wherein the alcohol having 3 or more carbon atoms is normal propyl alcohol or isopropyl alcohol.   The method according to claim 10, wherein the nucleic acid recovery method in step (d) is a step comprising the addition of a strong alkali to the precipitate.   11. The method of claim 10, wherein the nucleic acid synthesis of step (e) comprises a reverse transcription reaction.   The method according to claim 10, wherein the nucleic acid synthesis in step (e) is by PCR.

Images