JP7338134B2 - Virus disinfection effect determination method and antiviral disinfectant selected by the determination method - Google Patents

Description

The present invention (1) concentrates and detects viruses present in the environment (including facilities, equipment, equipment, instruments, etc. in them, as well as food materials and biological materials), and (2) when detected (3) After disinfection, conduct a confirmation test to see if the virus has disappeared. By developing and maintaining the methods for constructing these series of systems and the reagents used for them, we can prevent environmental pollution by viruses (especially pathogenic viruses) and spread viruses to organisms (especially humans) through these. You can increase your defense against infection.

There are many pathogenic viruses that cause disease in living organisms, but the main diseases caused by pathogenic viruses that infect humans and cause serious illness include AIDS, smallpox, polio, viral hepatitis, and Japanese encephalitis. , measles, rabies, trachoma, inguinal lymphoma, and influenza (especially highly pathogenic).

Food poisoning caused by viruses frequently occurs in daily life (especially diet), although it is less serious than the pathogenic viruses described above.

Among the pathogenic viruses that cause food poisoning, norovirus (NV) is the cause of food poisoning that frequently occurs in Japan from winter to early spring. NV is classified into seven gene groups (GI to GVII), of which GI, GII and GIV cause gastroenteritis in humans, but recently GII (especially GII.4 type) is the main one. It has become a popular stock. NV accounts for about 70% of all infectious food poisoning causes, and although 10,000 to 30,000 people are reported to be infected each year, it is estimated that about 100 times that number are actually infected.

NV infection routes are assumed to be (1) sewage → shellfish → people, (2) cooking workers → utensils / ingredients → people, (3) people → people, people → facility environment → people. Although (1) route accounted for the majority, in recent years, (2) and (3) infection cases due to contamination via humans have come to account for the majority.

It is believed that the following characteristics of NV are largely responsible for the spread of NV. Namely, (1) the NV gene body is RNA, which is susceptible to mutation, and immune defense against NV is mediated by the intestinal mucosa; therefore, once infected, immunity is ineffective or does not last long. (2) A large amount of virus is excreted not only in feces but also in vomit (10 ⁵ -10 ⁹ virus/g in patient stool, 10 ³ -10 ⁷ virus/g in patient vomit). (3) Infected individuals continue to shed NV for a long period of time even after symptoms have disappeared (it has been reported that adults shed NV for 3 weeks and children shed more than 1 month). (4) There are many infected people who do not show symptoms (inapparently infected people), but they also excrete NV in their feces in an amount sufficient to infect humans. (5) It is so contagious that half of the people are infected by exposure to 100 to 1,000 NV. (6) It has a long survival period in the environment (especially at low temperatures) (estimated to be about 2 months when stored in a dry state at 4°C and about 1 month when stored at 20°C).

Under such circumstances, the "Manual for Hygiene Management of Mass Cooking Facilities" (March 24, 1997) is intended for cooking facilities (mass cooking facilities) that provide 300 or more meals of the same menu at one time or 750 or more meals per day. Tsukeei-shoku No. 85 Attachment Final revision: In June 2008 revision of the June 16, 2017 Shokuan Notification No. 0610-1), the Ministry of Health, Labor and Welfare announced "NV stool test" Recommendations such as "Refrain from cooking work" were added.

However, NV still accounts for about two-thirds of all food poisoning cases. moreover,
In addition to facilities that directly handle food such as restaurants, caterers, inns, and factories, the outbreak facilities are diverse, including offices, schools, hospitals, sales offices, and homes.

Therefore, in order to prevent NV infection, in addition to the conventional NV stool test (individual test), food
Wide-area (environmental) inspections such as NV inspections and monitoring in locations where NV contamination is suspected, including handling locations, were considered to be extremely effective.

Now, in the virus test in the environment, first, equipment, equipment, instruments, foodstuffs, etc. in places where virus contamination is a concern are impregnated with a buffer solution such as phosphate buffer, physiological saline, or distilled water. Wipe off with a cotton swab or the like.

The wiped virus is suspended in a buffer solution, physiological saline, or distilled water contained in a wiping jig, then mixed with a concentrated solution containing polyethylene glycol (hereinafter referred to as PEG) and centrifuged, or suspended. It is directly concentrated by ultracentrifugation.

To confirm whether the virus is present in the concentrate, directly from the virus or after extracting and purifying its gene (nucleic acid: DNA or RNA), if the viral gene is DNA, the DNA is analyzed by PCR or the like. In the case of RNA, the RNA itself is amplified, or it is converted to DNA by reverse transcription (RT) reaction and then amplified. Amplified products are detected and analyzed in real time by a probe method or the like, or after amplification, a so-called genetic test is performed by melting temperature analysis (MCA), agarose electrophoresis, or the like.

However, with the conventional PEG precipitation method, it is difficult to recover the virus at a high rate and in a stable manner, the work process is complicated, the process takes a long time, and the virus cannot be concentrated easily, quickly, and at a high recovery rate. Ta. In addition, the ultracentrifugation method has large problems in terms of versatility, for example, the apparatus is expensive, the operation requires skill, and the number of specimens that can be processed at one time is small.

Therefore, by optimizing the addition and amount of polysaccharide to the PEG solution and the concentration of the added salt, we developed a method for concentrating viruses in an aqueous solution simply, quickly, and stably with a high recovery rate, and its use. He invented a reagent that does so and filed a patent application (Japanese Patent Application No. 2018-126772).

Viruses collected from the environment at a high rate using the above methods are directly (without nucleic acid extraction/purification) subjected to PCR, etc. when the nucleic acid to be detected is DNA, and reverse transcription (for RNA). After conversion to cDNA by RT) reaction, amplification and detection by PCR etc. has made it possible to detect and monitor viruses existing in the environment at high speed and easily.

Now, when a virus is detected from an object to be inspected, disinfection is required. There are many disinfectants that claim to be effective against viruses, but for viruses that are difficult to culture, related viruses that can be cultured (murine norovirus and feline calicivirus for NV) were used as substitutes. Currently, the culture method is used to determine the effect, and the effect on the actual target is estimated and used (Patent Document 1, Non-Patent Document 1).
However, it is unclear whether this method can inactivate the target virus in the actual field. This is especially true when using disinfectants whose effectiveness is greatly reduced in the presence of organic matter, such as chlorine-based agents such as sodium hypochlorite, which are frequently used as disinfectants against pathogenic microorganisms including viruses. That is. Furthermore, even when using a disinfectant whose disinfection effect has been determined using a target virus, culture takes a long time to determine the effect, and is not suitable for confirming the disinfection effect in the actual field.

From the above, it has been demonstrated that target viruses present in the environment to be disinfected or simulated environments are effective in preventing environmental contamination by viruses and increasing defenses against viral infections of organisms through these. The use of disinfectants and disinfection methods is mandatory. In addition, after disinfection, a rapid and highly efficient virus collection method and a rapid and highly sensitive detection method such as direct genetic testing are combined to rapidly and highly sensitively determine the disinfection effect, further enhancing the defense effect. be able to.

JP 2016-210807 A

Journal of Environmental Infection Vol.31 no.3, 2016

The type, concentration, and method of use of the drug that has a disinfecting effect on the virus present in the test object in the environment are set according to the target virus and the environment in which it exists. In addition, (1) rapid concentration and detection of the target virus present in the test object. (2) If a virus is detected in (1) or if there is a high possibility of virus contamination, disinfect with a disinfectant and disinfection method suitable for the virus and the environment in which it exists. (3) Promptly confirm the disinfection effect. By preparing the methods for constructing these series of systems and the reagents, instruments, and devices used in them, we can prevent environmental pollution by viruses (especially pathogenic viruses) and protect organisms (especially humans) through these. can increase the defense against viral infection of

We believe that a method for highly efficient and stable concentration and recovery of viruses, such as the previously invented virus concentration method (Japanese Patent Application No. 2018-126772), is actually useful in addition to the inspection and monitoring of viral contamination in the environment. , and the development and selection of disinfectants suitable for the target virus and the environment in which it exists. .
That is, the present invention provides a solution in which the target virus is treated with a candidate antiviral disinfectant, or the agent is diluted or diluted to the extent that it does not affect the RNA amplification reaction, reverse transcription (RT) reaction, or DNA amplification reaction such as PCR. To the mixed liquid, add a liquid that can stably concentrate the virus, such as a concentrate containing polyethylene glycol (PEG) and polysaccharides shown in Japanese Patent Application No. 2018-126772, and concentrate the virus by centrifugation. do. After that, gene amplification of the target virus in the concentrated liquid is carried out, and the disinfection effect of the candidate disinfectant is judged.
Examples of diluents include distilled water and various buffer solutions, and examples of neutralizing solutions include pH-neutralizing solutions such as acids and alkalis, as well as chlorine-neutralizing solutions such as sodium thiosulfate.
As the polysaccharide to be added to the PEG solution, there is a high possibility that any polysaccharide that becomes suspended in the PEG solution can be used. Examples include, but are not limited to, glucose-derived amylopectin, curdlan, glycogen, cellulose, α-cellulose, dextrin, N-acetylglucosamine-derived chitin, fructose-derived inulin, and mixtures thereof. It is also possible to use Among the above polysaccharides, glycogen, dextrin, and inulin are recommended, but glycogen is the most recommended.

More specifically, after treating NV with various disinfectant candidates, dilute with distilled water so that the disinfectant does not affect NV genes (do not affect RT-PCR results) or less. The virus was immediately concentrated by the virus concentration method described above. After the sample treatment solution is added to the sediment after centrifugation and heated, direct RT-PCR (hereinafter referred to as direct RT-PCR) is performed without purifying RNA, and a system that detects in real time with a fluorescently labeled probe (hereinafter referred to as disinfection effect determination system) to determine the antiseptic effect of various antiseptics against NV.
Antiviral disinfectant candidates include alcohol-based disinfectants, chlorine-based disinfectants, benzalkonium chloride, glutaraldehyde, potassium peroxysulfate, and metasilicic acid. Sodium, trisodium phosphate, hydrogen peroxide, and heating were tested, but chlorine-based disinfectants and sodium metasilicate were particularly effective in this disinfection effect evaluation system. In addition, this evaluation system uses a method to directly confirm the presence of nucleic acids, and the same amount of NV-derived RT-PCR From the fact that the product was obtained (data not shown), these drugs directly or indirectly affect the nucleic acid inside the virus, such as penetrating the virus and degrading the RNA, or breaking the virus shell and releasing the RNA inside. It is presumed that In addition, due to its presumed mechanism of action and the fact that chlorine-based agents are widely used for disinfection purposes, the agents found in this disinfection effect determination system are effective against viruses in general (especially those with RNA genomes). We believe that it can also be used as a general-purpose disinfectant, such as disinfection and disinfection of microorganisms other than viruses such as bacteria and fungi.

Among the disinfectants that were found to be effective in this disinfection effect determination system, the presence of organic matter in the aqueous solution of chlorine-based disinfectants drastically reduced the virus (RNA) reduction effect (disinfection effect). was also recognized. For example, sodium hypochlorite exhibits a sufficient antiseptic effect against NV even at 0.025% (final concentration) in distilled water, but the effect diminishes as the amount of bovine serum albumin (BSA) added as an organic substance increases. However, in the presence of 5% BSA (final concentration), no effect was observed even with 0.1% (final concentration) sodium hypochlorite.

On the other hand, in the case of sodium metasilicate, the antiseptic effect against NV was shown from about 0.5% (final concentration), but the antiseptic effect was not affected by the presence or absence of BSA addition. Furthermore, it was found that the use of a chlorine-based disinfectant in combination with sodium metasilicate is effective in compensating for the weakening of the disinfecting effect of the chlorine-based disinfectant in the presence of organic matter.
Accordingly, the present invention also provides an antiviral disinfectant selected by the determination system of the present invention. Here, the antiviral disinfectant is characterized by containing an alkali metal silicate.
The alkali metal silicate is represented by the general formula M2O.xSiO2.yH2O (M: alkali metal), and silicates of sodium, potassium and lithium can be used, but sodium metasilicate is preferred. The final concentration of sodium metasilicate added is 0.5% or more, preferably 3.5% or more, and more preferably 11% or more.

In addition, the latest invention to amplify and detect nucleic acids directly from viruses such as disinfectants and disinfection methods suitable for the target virus and the environment in which it exists, virus concentration method and direct (RT)-PCR method By combining methods, it became possible to build a system that protects the environment from contamination by viruses. That is, (1) a rapid and highly efficient virus concentration method and a rapid and highly sensitive nucleic acid amplification/detection method are used to test whether or not the target virus is contaminated with the target virus; If a virus is detected in (1) or if there is a high possibility of contamination with a virus, disinfect with a disinfectant and disinfection method suitable for the virus and the environment in which it exists; (3) Inspection after disinfection By applying the inspection method (1) to the target object and conducting a virus confirmation inspection to determine the disinfection effect, by constructing and operating a series of virus cleaning systems, it is possible to prevent environmental pollution by viruses and protect the environment. It has become possible to greatly reduce the risk of viral infection that occurs through the virus, and it has become possible to greatly contribute to epidemic prevention.

A method for concentrating and recovering viruses at a high rate and in a stable manner, such as the virus concentration method (Japanese Patent Application No. 2018-126772) filed for a patent by the applicant of the present application, is a method for collecting target viruses and the environment in which they exist. We found that it was effective in evaluating suitable disinfectants and disinfection methods, and used it to find disinfectants and methods of use that are expected to be effective. Furthermore, by combining the disinfectant and disinfection method found here with the virus concentration method described above and the method of directly amplifying and detecting the concentrated viral nucleic acid (without nucleic acid extraction/purification), the presence of the virus can be detected. We were able to build a system from confirmation to disinfection and even to determine the effect after disinfection. By implementing this system regularly or as needed at locations where contamination is suspected, it is possible to reduce the chances of environmental contamination by viruses and increase the defense against viral infections of organisms (especially humans) through these. .

Testing and monitoring of viral contamination by combining rapid and high-efficiency virus concentration methods from test objects and rapid and highly sensitive methods for amplifying and detecting nucleic acids in viruses, targeting viruses and the environment in which they exist An example of a system for preventing environmental contamination by viruses is shown, which combines a suitable disinfectant and disinfection method, and a rapid effect evaluation method after disinfection.

なお、表中の濃度表示は「添加量」を示しており、文中の添加と同じ（以下の表同様）。

表２では、同じ評価系で、二酸化塩素のNVに対する消毒効果を示している。BSA非存在下では、0.2%添加（終濃度0.1%）の二酸化塩素で、NVに対して8もしくはそれ以上サイクル分のNV RNAの減少効果を示しているが、終濃度5% BSA存在下（BSA 10%添加）では、0.4%添加（終濃度0.2%）でもその効果はほぼ消失しているなど、次亜塩素酸ナトリウムと同様にBSA添加による消毒効果の減弱が認められる。
［表２］二酸化塩素の消毒効果に及ぼすBSA添加の影響 [real time RT-PCR（duplicateでの平均値）におけるCt値の比較]

表３では、同じ評価系でのクロラミンＴのNVに対する消毒効果を示している。前２者に比べると、BSA添加の影響は少ないようであるが、それでもクロラミンTの2.6%添加（終濃度1.3%）において、BSA非存在下では消毒効果が認められたものの、BSA 終濃度5%存在下（BSA 10%添加）ではその効果が完全に消失しているなど、BSAの添加が消毒効果を減弱していることは明らかである。
［表３］クロラミンＴの消毒効果に及ぼすBSA添加の影響 [real time RT-PCRにおけるCt値（duplicateでの平均値）の比較]

[Example 1]
GI & GII genotype NV added distilled water or 2 μl of BSA aqueous solution (1% and/or 10%), add the same amount of diluted chlorine-based disinfectant solution, let it stand for 5 minutes, and then add distilled water. diluted 100 times with The same amount of glycogen-added PEG solution was added to the diluent, and after standing for 10 minutes, centrifugation (14,000 G) was performed at 20°C for 5 minutes. After the supernatant was aspirated and discarded, the remaining sediment was suspended by adding 8 μl of sample treatment solution, and heat-treated at 85° C. for 3 minutes. After ice-cooling the heat-treated sample, 11 μl of the reaction solution was added, real time RT-PCR was directly performed, and the fluorescence generated by the decomposition of the fluorescent-labeled probe accompanying the amplification was measured. The results were obtained by measuring the number of cycles (Ct value) of fluorescence rise in duplicate and showing the average value. It can be interpreted that the higher the Ct value, the lower the amount of virus-derived nucleic acids (the more the disinfection effect is obtained), and the higher the value is by 3.3, the lower the amount of virus-derived nucleic acids is to about 1/10.
Table 1 shows the results using sodium hypochlorite as the disinfectant. No amplification of NV-derived RNA was observed with the addition of 0.05% sodium hypochlorite (0.025% final concentration) without the addition of BSA, indicating a strong disinfection effect. On the other hand, with the addition of 1% BSA (0.5% final concentration), the addition of 0.2% sodium hypochlorite (0.1% final concentration) reduced the amount of NV-derived RNA for 8 to 9 or more cycles, but 0.1% When added (0.05% final concentration), almost no decrease in NV RNA was observed, and the disinfection effect almost disappeared. When adding 10% BSA (final concentration 5%), adding 0.8% (final concentration 0.4%) sodium hypochlorite is necessary to add a strong disinfection effect, and adding 0.2% (final concentration 0.1%) is necessary. ), the effect is completely lost, and it is verified that the disinfection effect of sodium hypochlorite is greatly attenuated by the addition of BSA, using NV (not an alternative virus).
[Table 1] Effect of BSA addition on the disinfection effect of sodium hypochlorite [comparison of Ct value (average value in duplicate) in real time RT-PCR]

The concentration display in the table indicates the "addition amount", which is the same as the addition in the text (similar to the table below).

Table 2 shows the disinfecting effect of chlorine dioxide on NV using the same scale. In the absence of BSA, 0.2% chlorine dioxide (final concentration 0.1%) reduced NV RNA for 8 or more cycles against NV, but in the presence of 5% final BSA ( BSA 10% addition), the effect is almost lost even with 0.4% addition (final concentration 0.2%), and the attenuation of the disinfection effect due to the addition of BSA is observed as with sodium hypochlorite.
[Table 2] Effect of BSA addition on the disinfection effect of chlorine dioxide [comparison of Ct values in real time RT-PCR (average value in duplicate)]

Table 3 shows the antiseptic effect of chloramine T on NV in the same evaluation system. Compared to the former two, the effect of BSA addition seems to be less. It is clear that the addition of BSA weakens the disinfection effect, such as the effect completely disappearing in the presence of 10% BSA (10% BSA added).
[Table 3] Effect of BSA addition on the disinfection effect of chloramine T [comparison of Ct value (average value in duplicate) in real time RT-PCR]

また、表５ではさらに低濃度でのメタケイ酸ナトリウムのNVに対する消毒効果を検討し、1%添加（終濃度0.5%）でも弱いながらも効果が持続していることを示している。即ち、メタケイ酸ナトリウムなどのアルカリケイ酸塩は、ウイルスが存在している環境の有機物的な汚れに影響されることなく消毒効果を発揮する可能性が高く、極めて使い勝手の良い消毒剤になると判断できる。
［表５］メタケイ酸ナトリウムの消毒効果に及ぼすBSA添加の影響-2 [real time RT-PCRにおけるCt値（duplicateでの平均値）の比較]

[Example 2]
Table 4 shows the results of examining the antiseptic effect of sodium metasilicate on NV using the same evaluation system as in Example 1. In this example, regardless of the presence or absence of BSA addition, the effect of reducing NV RNA for 5 to 9 or more cycles with 2.2% sodium metasilicate addition (final concentration 1.1%) was reduced to 7% concentration addition (final concentration 3.5%). At 22% concentration (11% final concentration), neither GI nor GII RNA was detected at all.
[Table 4] Effect of BSA addition on the disinfection effect of sodium metasilicate [comparison of Ct value (average value in duplicate) in real time RT-PCR]

In addition, Table 5 examines the antiseptic effect of sodium metasilicate on NV at a lower concentration, and shows that the effect persists even with the addition of 1% (final concentration 0.5%), although weak. In other words, alkali silicates such as sodium metasilicate have a high possibility of exhibiting a disinfecting effect without being affected by organic dirt in the environment where the virus exists, and it is judged that it will be an extremely easy-to-use disinfectant. can.
[Table 5] Effect of BSA addition on the disinfection effect of sodium metasilicate-2 [comparison of Ct value (average value in duplicate) in real time RT-PCR]

次亜塩素酸ナトリウムの0.2%添加（終濃度0.1%）ではBSA無添加の場合には強い消毒効果が認められるものの、BSAの添加量が増えるに従いその消毒効果が減弱し、5%BSA存在下（BSA 10%添加）では消毒効果が完全に消失している。そこに、2%（終濃度1%）のメタケイ酸ナトリウムを併用することで、BSA非存在下もしくは低濃度存在下ではメタケイ酸ナトリウム単独使用に比べて高い消毒効果を、高濃度BSA存在下では次亜塩素酸ナトリウムの消毒効果消失を防いでいることを示している。このことから、ウイルスへの消毒効果は顕著ながら有機物の存在でその効果が大きく減弱する塩素系消毒剤とアルカリ金属ケイ酸塩の併用は極めて有用性が高いことは明らかである。 [Example 3]
In the same evaluation system as in Example 1, although a significant disinfection effect was observed against NV in distilled water, the effect was not observed at all in the presence of 5% BSA (10% BSA added). Table 6 shows the effects of the combined use of sodium chlorite and relatively low-concentration sodium metasilicate, in which a clear effect is observed.
[Table 6] Synergistic disinfection effect of sodium hypochlorite and sodium metasilicate [comparison of Ct value (average value in duplicate) in real time RT-PCR]

With the addition of 0.2% sodium hypochlorite (0.1% final concentration), a strong disinfection effect was observed in the absence of BSA. (10% BSA added) completely lost its disinfecting effect. There, by using 2% (final concentration 1%) sodium metasilicate in combination, in the absence of BSA or in the presence of low concentrations, a higher disinfection effect than sodium metasilicate alone is used, and in the presence of high concentrations of BSA, It shows that the disinfection effect of sodium hypochlorite is prevented from disappearing. From this, it is clear that the combined use of a chlorine-based disinfectant and an alkali metal silicate, which has a remarkable disinfecting effect against viruses but whose effect is greatly weakened by the presence of organic matter, is extremely useful.

[Example 4]
(1) A rapid and highly efficient method of concentrating viruses from test objects, (2) A method of rapidly and highly sensitively amplifying and detecting genes in viruses, (3) Target viruses and the environment in which they exist (4) Combining the disinfectant and disinfection method suitable for (4) post-disinfection evaluation method (in combination of (1) and (2)) to prevent environmental contamination by viruses, and organisms (especially humans) through these Fig. 1 shows the defense system against food poisoning caused by NV as an example of a system that enhances defenses against viral infection in .
1. Preparation of wiping jig As the wiping jig, a known swab for wiping inspection can be used.
2. Sample collection by wiping the back of the toilet seat using a wiping jig Regularly (for example, once a week in winter and once a month in other seasons) or occasionally (when contamination is suspected, etc.), the risk of contamination is high Wipe the area (e.g., under the toilet seat) to collect a sample.
3. Confirm the status of NV contamination of the samples collected by wiping in 2, such as genetic testing of the above samples.
[Result judgment]4. If disinfection contamination is found (NV positive judgment), the equipment, fixtures, instruments, etc. in the facility, centering on the contaminated area (toilet seat back and surrounding areas), are disinfected with a disinfectant selected according to the degree of contamination of the object. Disinfect with disinfectant methods. In addition to the above work, we will carry out a stool test mainly on people who have used facilities where NV contamination has been confirmed along with a warning, and if a positive person comes out, refrain from cooking work etc. Prevent the spread of NV infection through food by
In the case of NV-negative judgment, it is unlikely that there is a problem with the current environment, so the status quo is maintained.
5. 6. Specimen collection by wiping the back of the toilet seat using a wiping jig after disinfection. Confirm that NV is below the detection limit (hereafter, disappearance) by swabbing test after disinfection such as genetic test of the above specimen.
[Result judgment] Re-disinfection and re-inspection required
If the NV has not disappeared (NV positive judgment), return to 4 above, change the disinfection method (increase the concentration of the disinfectant, wipe twice, etc.) and disinfect again, and the NV disappears after disinfection. Make sure you are
In the case of NV-negative determination after disinfection, follow up over time in parallel with alerting to completely prevent NV infection.

Claims (1)

A solution containing at least polyethylene glycol and glycogen , which is used to concentrate and recover the virus by centrifugation, is added to the solution in which the target virus having the RNA genome has been treated with an antiviral disinfectant. A method for judging the disinfection effect of a virus, which comprises amplifying the gene of a virus and judging the disinfection effect of a disinfectant.

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