WO2020027133A1 - Composition for sterilization and nucleic acid degradation - Google Patents

Composition for sterilization and nucleic acid degradation Download PDF

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WO2020027133A1
WO2020027133A1 PCT/JP2019/029834 JP2019029834W WO2020027133A1 WO 2020027133 A1 WO2020027133 A1 WO 2020027133A1 JP 2019029834 W JP2019029834 W JP 2019029834W WO 2020027133 A1 WO2020027133 A1 WO 2020027133A1
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formaldehyde
nucleic acid
concentration
sterilization
formic acid
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PCT/JP2019/029834
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French (fr)
Japanese (ja)
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利彦 岡崎
鈴木　康士
島垣　昌明
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株式会社シーライブ
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aliphatically bound aldehyde or keto groups, or thio analogues thereof; Derivatives thereof, e.g. acetals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof

Definitions

  • the present invention relates to a sterilizing / nucleic acid decomposing composition capable of sterilizing bacteria and viruses and decomposing nucleic acids and Lipopolysaccharide (hereinafter, LPS).
  • LPS Lipopolysaccharide
  • nucleic acids which are poorly soluble in water, are unnecessary on solid surfaces such as reaction vessels used and in liquids such as reaction solutions. If the bacteria or cells are unnecessarily mixed into the surface of a solid such as a reaction vessel or a liquid such as a reaction solution, it may have a serious adverse effect on the results of tests and research experiments. May cause. In addition, even though sterilized, residual bacteria and LPS present in cells may have a serious adverse effect on the results of tests and research experiments.
  • a water-absorbing resin described in Patent Document 1 and a germicidal material containing an aqueous formaldehyde solution held by the water-absorbing resin have an excellent germicidal action, and are excellent germicidal materials that can be easily disposed of at low cost. It is described that it can be used as.
  • the sterilizing material described in Patent Document 1 requires an aqueous solution having a formaldehyde concentration of 10 to 50% in order to obtain a sufficient sterilizing effect.
  • High concentrations of formaldehyde are acutely toxic to the human body, mainly irritating to mucous membranes, and vapors are toxic, causing inflammation of the respiratory system, eyes and throat.
  • concentration of formaldehyde was as low as 5% or less, a sufficient bactericidal effect could not be obtained.
  • a sterilization system using a composite gas generated by a catalytic reaction from methanol has a sterilizing power higher than that of ethylene oxide gas (EOG) or ozone which has been frequently used as a gas used for sterilization of medical instruments and the like. It has been confirmed that they have extremely low corrosion resistance and corrosiveness, and because of their excellent permeability and diffusivity, they are currently attracting attention in many fields.
  • EOG ethylene oxide gas
  • Composite gas is a gas that has a strong bactericidal and sterilizing effect generated from methanol by a catalyst.
  • the composite gas has a high permeability, and can be sterilized by permeating the inside of the object to be sterilized even at the atmospheric pressure. Since it is not a mist of contact sterilization, it is excellent in that there is little corrosion of metal and deterioration of plastics (corrosiveness), it does not choose the material of the material to be sterilized, and it is hard to remain in the material to be sterilized (persistence). It has a characteristic, has a wide diffusibility, and can be exposed evenly to every corner, penetrates into small gaps, and can be exposed even in the energized operating state of precision equipment, electronic equipment, etc., and has high utility.
  • the environment temperature at which the effect of nucleic acid decomposition is exerted is set to a body temperature range of 37 ° C., and within a short time of 15 minutes or less, and at a low formaldehyde component concentration, a double helix DNA It has the ability to effectively decompose nucleic acids (disassembled into base chains of 10 base pairs (bp) or less) and can achieve a nucleic acid resolution of 99.99% to 100% as a gas phase nucleic acid decomposition method. On the other hand, the identification and detailed mechanism of the components that cause these effects have not been sufficiently elucidated.
  • LPS is a component of the outer membrane of the cell wall of Gram-negative bacteria and is a substance (glycolipid) composed of lipids and polysaccharides.
  • LPS is an endotoxin (endotoxin) that exerts various biological activities when it acts on cells of other organisms such as humans and animals.
  • the expression of physiological action of LPS is carried out via Toll-like receptor ⁇ TLR ⁇ ⁇ 4 ⁇ (TLR4) present on the cell membrane surface of the host cell.
  • TLR4 Toll-like receptor
  • the method of increasing and / or decreasing the concentration of the immunomodulatory active substance described in Patent Document 4 does not decompose LPS, but increases or decreases the amount by a receptor-mediated adsorption mechanism.
  • LPS is thermally and chemically stable, and cannot be inactivated by an autoclave used for normal sterilization. It is only known that inactivation requires heating at 250 ° C. for 30 minutes or irradiation with radiation such as ⁇ -rays. However, under these conditions, it cannot be applied to equipment made of a material having no heat resistance, and there has not been anything that can be applied to environmental purification that cannot be transported.
  • JP 2013-166729 A Japanese Patent No. 5463378 JP 2001-519817 A JP-T-2003-519503
  • the present invention has been made in view of such circumstances, and enables sterilization and nucleic acid decomposition in a low concentration range of formaldehyde, and sterilization and nucleic acid decomposition capable of exhibiting excellent efficacy in a short time. It is a first object to provide a composition for use, and a second object to decompose LPS.
  • the present inventors have found a specific substance that contributes to sterilization and nucleic acid decomposition by analyzing components in a composite gas, and have completed the present invention by clarifying the mechanism thereof.
  • the inventors of the present application use substantially the same sterilization and nucleic acid decomposition composition, so that the reaction in a liquid state and the reaction in a gas state are longer than the time required for sterilization and nucleic acid decomposition.
  • LPS could be inactivated, but at least Limulus activity could be inactivated, although the necessity of exposure was different.
  • one embodiment of the present invention is a composition for sterilization and nucleic acid decomposition in a liquid state capable of decomposing nucleic acid and sterilizing, which contains at least formaldehyde and formic acid, and has a formaldehyde concentration of 10% by weight or less. And the concentration of formic acid is 1% by weight or less.
  • one embodiment of the present invention may further include methanol, and optionally include derivatives of methanol, formaldehyde, and formic acid.
  • More efficacy can be exhibited by appropriately including methanol, a methanol derivative, a formaldehyde derivative, and a formic acid derivative according to an object to be sterilized and use conditions.
  • one embodiment of the present invention is characterized in that LPS can be further decomposed.
  • Another embodiment of the present invention is a gaseous sterilization / nucleolysis composition capable of decomposing nucleic acids and sterilizing, which contains at least formaldehyde and formic acid.
  • a composite gas containing formaldehyde and formic acid in the gas phase can exhibit excellent nucleic acid resolution in a short time even at a low concentration.
  • another embodiment of the present invention may include methanol, formaldehyde, formic acid, carbon monoxide, carbon dioxide, hydrogen, oxygen and / or a radical thereof.
  • compositions and / or their radical species are optional to contain these compositions and / or their radical species.
  • the concentration of formaldehyde may be 500 ppm or less, and the concentration of formic acid may be 400 ppm or less.
  • LPS in another embodiment of the present invention, LPS can be further decomposed.
  • sterilization and nucleic acid decomposition can be performed in a low concentration range, and excellent effects can be exhibited in a short time.
  • FIG. 1 A) to (E) are diagrams showing the results of DNA resolution when only formaldehyde was used as Comparative Example 1.
  • FIG. 1 A) to (D) are diagrams showing the results of DNA resolution when only formic acid was used as Comparative Example 2.
  • (A) and (E) show formic acid only, (B) and (D) show formaldehyde only, and (C) and (F) show the results of DNA resolution when formaldehyde and formic acid are used in combination as examples.
  • FIG. (A) and (E) show formic acid only, (B) and (D) show formaldehyde only, and (C) and (F) show the results of DNA resolution when formaldehyde and formic acid are used in combination as examples.
  • formaldehyde has a sterilizing effect on microorganisms such as bacteria and fungi, and has been put to practical use in various fields.
  • formaldehyde has long been used in pathology for long-term fixation of living tissues by immersion in formalin, which is an aqueous solution of formaldehyde.
  • techniques for extracting nucleic acids such as DNA and RNA from formalin-fixed tissue have been developed. Its development has been applied to its genetic analysis and has greatly contributed to the advancement of medicine.
  • formaldehyde is also generated endogenously when metabolizing amino acids and xenobiotics, and even those who have not been exposed to formaldehyde have a blood formaldehyde concentration of 2.61 ⁇ 0.14 ⁇ g / g (almost 2.6 ppm) (Initial Risk Assessment Report for Chemical Substances_Independent Administrative Institution Product Evaluation Technology Foundation ⁇ ⁇ Chemical Substance Management Center).
  • a cross-linking reaction of a protein molecule is known as an action of formaldehyde, and an aldehyde group in a formaldehyde molecule binds to an amino group of a protein in a tissue, and further cross-links, thereby impairing the three-dimensional structure of the protein.
  • formalin causes cross-linking of nucleic acids and proteins, so that DNA is susceptible to physical stress and fragmentation occurs.
  • the generated composite gas contains Evaluate the correlation between concentration, reaction time and potency for each of the constituent components individually, and further examine the effect on potency and effect by various mixing ratios, and create a database to reduce the concentration.
  • the present invention has been clarified for the first time for a mechanism (Mode-of-Action) that exerts an effect in a short time and in an extremely short time and optimization conditions.
  • the composition for sterilization and nucleic acid decomposition is a composition for sterilization and nucleic acid decomposition in a liquid state capable of decomposing nucleic acids and sterilizing, and contains at least formaldehyde and formic acid.
  • the concentration of formaldehyde is 10% by weight or less, and the concentration of formic acid is 1% by weight or less.
  • the present invention by mixing formaldehyde and formic acid in the above ratio, highly efficient sterilization and nucleic acid decomposition can be performed even at a low concentration at which each of them does not exert its effect alone.
  • formaldehyde By coexisting formic acid, formaldehyde exhibits sterilization and nucleic acid degrading effects even at a concentration of 10% by weight or less.
  • the concentration of formaldehyde may more preferably be 5% by weight or less (from the viewpoint of the minimum concentration for obtaining the sterilizing and nucleic acid degrading effects in the presence of formic acid).
  • the lower limit of the formaldehyde concentration is approximately 0.1% by weight.
  • the concentration of formic acid to be mixed with formaldehyde is not more than 1% by weight.
  • the lower limit of the concentration of formic acid is also approximately 0.1% by weight.
  • the composition for sterilizing and decomposing nucleic acids according to one embodiment of the present invention is effective for sterilizing bacteria, fungi and viruses and decomposing nucleic acids.
  • DNA and RNA such as bacteria, fungi and viruses can be effectively decomposed in a short time of 5 minutes or less, and in one minute depending on conditions ( 10 bp or less in a base chain), and an extremely high nucleic acid degradation effect can be achieved.
  • composition for sterilization / nucleic acid decomposition may use water such as pure water, alcohols such as methanol and ethanol, other organic solvents, or a mixture thereof as a solvent.
  • the solvent may appropriately contain a buffer for adjusting pH, a preservative, and the like.
  • composition for sterilization and nucleic acid degradation according to one embodiment of the present invention may have the formaldehyde and formic acid concentrations at the time of use as long as they are at the above concentrations. And may be used after being appropriately diluted at the time of use. Alternatively, an embodiment may be used in which each component is divided and stored, and then mixed and used at the time of use.
  • composition for sterilization and nucleic acid degradation further contains methanol, and can optionally contain a derivative of methanol, a derivative of formaldehyde, and a derivative of formic acid.
  • a derivative refers to a compound that has been modified to such an extent that the structure or properties of methanol, formaldehyde, or formic acid are not significantly changed, such as introduction of an arbitrary functional group, oxidation, reduction, or substitution of an atom.
  • the composition for decomposing LPS is a composition in a liquid state capable of decomposing LPS, and contains at least formaldehyde and formic acid, as well as for sterilization and nucleic acid decomposition,
  • the concentration of formaldehyde is 10% by weight or less and the concentration of formic acid is 1% by weight or less.
  • LPS by mixing formaldehyde and formic acid in the above ratio, LPS can be decomposed even at a low concentration at which each of them does not exert its effect alone.
  • formaldehyde exerts an LPS decomposition effect even at a concentration of 10% by weight or less.
  • the concentration of formaldehyde may more preferably be 5% by weight or less (from the viewpoint of the minimum concentration for obtaining the sterilizing and nucleic acid degrading effects in the presence of formic acid).
  • the lower limit of the formaldehyde concentration is approximately 0.03% by weight.
  • the concentration of formic acid to be mixed with formaldehyde is not more than 1% by weight.
  • the lower limit of the concentration of formic acid is also approximately 0.01% by weight.
  • the contact reaction is preferably carried out for about 10 minutes to 2 hours, more preferably for about 15 minutes to 2 hours, whereby LPS in an amount of 100 to 1000 EU (endotoxin unit) per mL is reduced to about half or less. Can be made.
  • the mode of use of the composition for sterilization and nucleic acid decomposition according to one embodiment of the present invention in the liquid phase is not particularly limited, for example, sterilization and nucleic acid decomposition, the composition for sterilization and nucleic acid decomposition or LPS decomposition for the target of LPS decomposition Sterilization, nucleic acid decomposition, and LPS decomposition may be performed by immersion in a solution, or sterilization, nucleic acid decomposition, and LPS decomposition composition may be sprayed on a sterilization, nucleic acid decomposition, and LPS decomposition target to sterilize, nucleic acid decomposition, and LPS decomposition. It may be disassembled.
  • the contact temperature is not particularly limited, and may be room temperature, or may be appropriately heated to about 37 to 50 ° C. and further to about 60 ° C. If the temperature is higher than this, it is necessary to consider the heat resistance of the object to be processed, which is not preferable.
  • the composition for sterilizing and decomposing nucleic acids according to one embodiment of the present invention can be used in a gaseous state. That is, the composition for sterilization and nucleic acid decomposition according to one embodiment of the present invention is a composition for sterilization and nucleic acid decomposition in a gaseous state capable of decomposing and sterilizing nucleic acids, and contains at least formaldehyde and formic acid. .
  • the concentration of formaldehyde is 500 ppm or less
  • the concentration of formic acid is 400 ppm or less.
  • the present inventors have confirmed that when the concentration of formaldehyde is 200 ppm or less and the concentration of formic acid is 100 ppm or less, it effectively acts on microorganisms (BI indicator bacteria) within 5 minutes. .
  • a composite gas containing formaldehyde and formic acid in a predetermined concentration range in a gaseous phase can exhibit excellent sterilization and nucleic acid resolution in a short time even at a low concentration.
  • the composition for decomposing LPS is a composition capable of decomposing LPS in a gaseous state, and contains at least formaldehyde and formic acid as in the case of sterilization and nucleic acid decomposition.
  • the concentration of formaldehyde is 500 ppm or less, and the concentration of formic acid is 400 ppm or less.
  • the contact reaction is preferably performed for about 10 minutes to 2 hours, more preferably for about 15 minutes to 2 hours.
  • LPS in an amount of 100 to 1000 EU (endotoxin unit) per 1 mL can be reduced to about half or less.
  • the method of using the composition for sterilization / nucleic acid decomposition according to one embodiment of the present invention in a gaseous state is not particularly limited as long as it is a composite gas containing formaldehyde and formic acid.
  • the composition for sterilization and nucleic acid decomposition according to an embodiment of the present invention in a liquid state may be vaporized by heating, or formaldehyde and formic acid in a complex gas generated by reacting methanol or the like with a catalyst such as copper. May be set in such a manner that is within a predetermined concentration range.
  • the lower limit of the concentration of formaldehyde and formic acid is not particularly limited, it is approximately 1 ppm.
  • composition for sterilization and nucleic acid decomposition contains methanol, formaldehyde, formic acid, carbon monoxide, carbon dioxide, hydrogen, oxygen, and / or their radicals in the process of converting into a gaseous state. It may be.
  • the mode of use of the composition for sterilization / nucleic acid decomposition according to one embodiment of the present invention in the gas phase is not particularly limited.
  • a sterilization / nucleic acid decomposition target is placed in a closed space such as a chamber, and
  • the sterilizing / nucleic acid decomposing composition according to one embodiment of the present invention can be sterilized / nucleic acid decomposed by filling the vaporized composition.
  • an appropriate humidity 50 to 90 relative humidity
  • sterilization and nucleic acid decomposition may be performed at low humidity (less than 50 relative humidity%). Is also good.
  • the contact temperature is not particularly limited, and may be room temperature, or may be appropriately heated to about 37 to 50 ° C., and further to about 60 ° C. If the temperature is higher than this, it is necessary to consider the heat resistance of the object to be processed, which is not preferable.
  • the composition for sterilization / nucleic acid degradation according to one embodiment of the present invention has an effect alone by combining formaldehyde and formic acid in both the liquid phase and the gas phase. Even in a low-concentration range that does not exert its effect, it has an excellent sterilizing effect and the resolution of nucleic acids (DNA, RNA, etc.) and can exert its effect in a short time.
  • the DNA used for the nucleic acid degradation test was obtained from subcultured HeLa cells (human cervical adenocarcinoma) (American Type Culture Collection: ATCC) according to the manufacturer's protocol of DNeasy Blood & Tissue (Qiagen). The extraction and purification were performed as genomic DNA.
  • the concentration of the purified genomic DNA was measured using Nano-drop (NanoDrop One, Thermo Fisher Scientific), and the dilution concentration was adjusted with UltraPure TM DNase / RNase-Free Distilled Water (DW; Thermo Fisher Scientific).
  • the genomic DNA whose concentration had been adjusted was sonicated using an ultrasonic homogenizer (UR-21P, TOMY) and used as the homogenized DNA sample.
  • Comparative Example 1 DNA resolution using formaldehyde alone
  • the DNA resolution was examined based on the formaldehyde concentration and the reaction time.
  • a 0.5 ml PCR tube was subjected to a preparative adjustment so that each formaldehyde dilution concentration was obtained.
  • a crushed DNA sample was added, and the reaction time was measured with a stopwatch. After the completion of the reaction, the sample was immediately washed and collected using a QIAquick PCR purification kit, and the obtained sample was analyzed using a Bioanalyzer.
  • the DNA degradation effect was determined from the ratio of the measured peak value of the sample to the peak value of the Upper Marker (molecular weight: 17,000) as follows: Classify. Upper Marker peak value 75% or more and 100% or less: Level 1 Upper Marker peak value 50% or more and less than 75%: Level 2 Upper Marker peak value 25% or more and less than 50%: Level 3 Less than 25% of Upper Marker peak value: Level 4
  • FIG. 1 shows the results of DNA resolution using formaldehyde alone.
  • the evaluation results of the DNA resolution using the formaldehyde diluent alone showed no obvious DNA degradation effect in a low concentration range of 0.1% to 5.0% in a reaction time of 1 minute.
  • a formaldehyde diluted solution 10% [FA10%] (hereinafter, “GA” represents formic acid and “FA” represents formaldehyde) is at Level 3 (0.385) at 1 min. (A)), even at 3 min., Level 4 (0.138) (FIG. 1 (B)), and no complete DNA degradation effect was obtained.
  • the concentration was as high as [FA30%]
  • the DNA was completely degraded for 5 min. (FIG. 1 (C)).
  • Comparative Example 2 DNA resolution with formic acid alone
  • the DNA resolution by the concentration of formic acid alone and the reaction time was examined.
  • Dilute formic acid 98.0%, WAKO special grade reagent
  • 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 1.5%, 2.0%, 3.0%, 5.0% formic acid A solution was prepared. Thereafter, the DNA resolution was evaluated in the same manner as in Comparative Example 1.
  • FIG. 2 shows the results of DNA resolution using formic acid alone.
  • the results of the evaluation of the DNA resolution using the formic acid dilution alone showed a slight DNA degradation effect at a 0.1% concentration in a reaction time of 1 minute, and a level 3 (0.296) (FIG. 2 (A)) of DNA at a concentration of 1.0%. No complete degradation effect was obtained, indicating a moderate DNA resolution.
  • a concentration reached a high concentration range of 2.0% or more, a complete DNA degradation effect was observed with a reaction time of 1 minute.
  • a slight (approximately level 1) DNA degradation effect was observed at 0.1% concentration (FIG.
  • Example 1 DNA resolution in a mixture of formaldehyde and formic acid
  • DNA resolution was evaluated using the composition for sterilization and nucleic acid degradation according to one embodiment of the present invention.
  • a concentration range in which formaldehyde and formic acid alone could not obtain a complete DNA degrading effect it was examined whether or not a synergistic effect of a DNA degrading effect could be obtained by mixing them at various concentrations.
  • a synergistic effect of a DNA degrading effect could be obtained by mixing them at various concentrations.
  • Table 2 shows the results of DNA resolution when the formaldehyde and formic acid according to the present invention were used in combination.
  • “ns” indicates that almost no (less than level 1) DNA degrading effect was obtained
  • “1" to “4" indicate the level 1 to level 4 DNA degrading effects
  • “CD” indicates that the DNA degradation effect was almost completely obtained.
  • Example 2 DNA resolution in mixed gas of formaldehyde and formic acid
  • nucleic acid degradation was examined using a mixed gas of formaldehyde and formic acid.
  • Table 3 shows the results of the DNA resolution.
  • the results are shown in Table 4.
  • the amount of LPS in the fourth column from the left in the table is the amount of LPS before decomposition, and the degree of LPS decomposition can be determined by comparison with the amount of LPS after exposure in the second column from the right.
  • Examples 3 to 9 show the results when liquid was used. It can be seen that the amount of each of the LPSs before the decomposition was reduced by about half.
  • Examples 10 to 19 show the results of LPS decomposition when exposed to a mixed gas of formaldehyde and formic acid as shown in Table 4.
  • Examples 10 and 11 were carried out in a state in which an LPS aqueous solution was placed in a 1.5 mL Eppendorf tube and evaporated to dryness in a 58 ° C. drier to deposit LPS in a pellet form on the bottom.
  • the LPS aqueous solution was attached to a PVDF filter (0.4 ⁇ m diameter) and air-dried and used as a sample.
  • the model is a model in which LPS spreads over the entire film and spreads thinner than a pellet-like one. It was clearly shown that the decomposition efficiency was higher than that of the pellet.
  • Example 16 the LPS standard vial (ENDOSAFE (ENDOTOXIN INDICATOR; Catalog # EVV2K, code number 513-87082)) was opened and the cap was removed. And then diluted to a predetermined concentration.
  • Example 19 is similar to Examples 10 and 11, in which LPS in the form of pellets was adhered. The value of the residual amount of LPS when a sample prepared by the same method as in Example 19 was irradiated with 25 kGy of ⁇ -rays was obtained. It was 0.33 EU compared to 0.66 EU, indicating a further decline. By using this method, LPS resolution comparable to that of ⁇ -ray irradiation can be obtained close to the user, which proves to be of great industrial use value.
  • composition for sterilization / nucleic acid decomposition is not limited to those described in the embodiment and each example of the present invention, and various modifications can be made.
  • composition for sterilization / nucleic acid degradation can be used in the fields of advanced medical treatment (cell therapy, gene therapy, regenerative medicine), marine research, aerospace, and crisis management (defense, firefighting). DNA / RNA-free (removal and decontamination of bio-nucleic acid level contamination) in medical care and nursing care, etc. Application to the field is possible.

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Abstract

A purpose of the present invention is to provide a composition for sterilization and nucleic acid degradation that enables sterilization and nucleic acid degradation in a low concentration range, and can exhibit excellent efficacy in a short period of time. Additionally, another purpose of the present invention is to provide a composition that can perform lipopolysaccharide (LPS) degradation. This composition of the present invention is a liquid-state composition for sterilization and nucleic acid degradation capable of nucleic acid degradation and sterilization and contains at least formaldehyde and a formic acid, wherein the concentration of formaldehyde is 10 weight% or lower, and the concentration of formic acid is 1 weight% or lower.

Description

滅菌・核酸分解用組成物Composition for sterilization and nucleic acid decomposition
　本発明は、細菌やウイルス等の滅菌と、核酸やLipopolysaccharide（以下、LPS）を分解することが可能な滅菌・核酸分解用組成物に関する発明である。本出願は、日本国において２０１８年７月３０日に出願された日本特許出願番号特願２０１８－１４２５３０を基礎として優先権を主張するものであり、この出願は参照されることにより、本出願に援用される。 The present invention relates to a sterilizing / nucleic acid decomposing composition capable of sterilizing bacteria and viruses and decomposing nucleic acids and Lipopolysaccharide (hereinafter, LPS). This application claims priority based on Japanese Patent Application No. 2018-142530 filed on July 30, 2018 in Japan, and this application is incorporated herein by reference. Incorporated.
　分子生物学や生化学等に係る試験や研究実験等を行うに当り、水に難溶性の高分子である核酸が、使用する反応容器等の固体の表面や反応液等の液体中に不必要に混入していたり、雑菌や細胞が、反応容器等の固体の表面や反応液等の液体中に不必要に混入していたりすれば、それが試験や研究実験等の結果に重大な悪影響を及ぼすおそれがある。また、滅菌はしたものの残存する雑菌や細胞に存在するLPSが試験や研究実験等の結果に重大な悪影響を及ぼすおそれがある。 When conducting tests or research experiments related to molecular biology or biochemistry, etc., nucleic acids, which are poorly soluble in water, are unnecessary on solid surfaces such as reaction vessels used and in liquids such as reaction solutions. If the bacteria or cells are unnecessarily mixed into the surface of a solid such as a reaction vessel or a liquid such as a reaction solution, it may have a serious adverse effect on the results of tests and research experiments. May cause. In addition, even though sterilized, residual bacteria and LPS present in cells may have a serious adverse effect on the results of tests and research experiments.
　例えば、特許文献１に記載されている吸水樹脂、及び吸水樹脂に保持されたホルムアルデヒド水溶液を含有する殺菌材が、優れた殺菌作用を持ち、且つ安価で簡便に廃棄することができる優れた殺菌材として使用できることが記載されている。 For example, a water-absorbing resin described in Patent Document 1 and a germicidal material containing an aqueous formaldehyde solution held by the water-absorbing resin have an excellent germicidal action, and are excellent germicidal materials that can be easily disposed of at low cost. It is described that it can be used as.
　しかしながら、特許文献１に記載の殺菌材では、十分な殺菌効果を得るためには１０～５０％のホルムアルデヒド濃度の水溶液が必要とされている。高濃度のホルムアルデヒドは、人体に対して粘膜への刺激性を中心とした急性毒性があり、蒸気は呼吸器系、目、のどなどの炎症を引き起こすといった毒性を有するものである。一方で、ホルムアルデヒドが５％以下といった低濃度では十分な殺菌効果を得ることができなかった。 However, the sterilizing material described in Patent Document 1 requires an aqueous solution having a formaldehyde concentration of 10 to 50% in order to obtain a sufficient sterilizing effect. High concentrations of formaldehyde are acutely toxic to the human body, mainly irritating to mucous membranes, and vapors are toxic, causing inflammation of the respiratory system, eyes and throat. On the other hand, if the concentration of formaldehyde was as low as 5% or less, a sufficient bactericidal effect could not be obtained.
　ところで、メタノールから触媒反応により発生する複合ガスを利用した滅菌システムは、これまで医療器具等の滅菌に用いるガスとして多用されていたエチレンオキサイドガス（ＥＯＧ）やオゾン等以上の殺菌力を持ち、残留性、腐食性が極めて少ないことが確認されており、浸透性や拡散性も優れていることから現在多くの分野において注目されている。 By the way, a sterilization system using a composite gas generated by a catalytic reaction from methanol has a sterilizing power higher than that of ethylene oxide gas (EOG) or ozone which has been frequently used as a gas used for sterilization of medical instruments and the like. It has been confirmed that they have extremely low corrosion resistance and corrosiveness, and because of their excellent permeability and diffusivity, they are currently attracting attention in many fields.
　複合ガスとは、メタノールから触媒により生じた強力な殺菌効果及び滅菌効果をもつガスのことである。複合ガスは、浸透性が高く、大気圧のままでも被滅菌物の内部まで浸透して滅菌ができる。接触性殺菌のミストでないことから、金属の腐食やプラスチック等の劣化（腐食性）が少なく、被滅菌物の素材を選ばず、さらに、被滅菌物に残留しにくい（残留性）などの優れた特質があり、拡散性も広く隅々まで満遍なく暴露が可能であり、細かな隙間まで浸透し、精密機器や電子機器等の通電稼動状態においても暴露が可能であり、高い有用性がある。 Composite gas is a gas that has a strong bactericidal and sterilizing effect generated from methanol by a catalyst. The composite gas has a high permeability, and can be sterilized by permeating the inside of the object to be sterilized even at the atmospheric pressure. Since it is not a mist of contact sterilization, it is excellent in that there is little corrosion of metal and deterioration of plastics (corrosiveness), it does not choose the material of the material to be sterilized, and it is hard to remain in the material to be sterilized (persistence). It has a characteristic, has a wide diffusibility, and can be exposed evenly to every corner, penetrates into small gaps, and can be exposed even in the energized operating state of precision equipment, electronic equipment, etc., and has high utility.
　この技術分野に関し、本件の発明者等は、自己反応のための触媒反応温度を一定に保ち、安定した濃度の滅菌ガスを発生させる滅菌・核酸分解処理装置を先に提案している（例えば、特許文献２参照）。この滅菌・核酸分解処理装置によれば、核酸分解の効果効能を発揮する環境温度を３７℃の体温域とし、１５分以内の短時間で、且つ、低いホルムアルデヒド成分濃度において、二重螺旋のＤＮＡ核酸を有効に分解（１０ base pair(bp)以下の塩基鎖へのバラバラ状態）する能力を有し、気相の核酸分解法として核酸分解能９９．９９％～１００％を達成することができる。一方で、これらの効能を引き起こす成分の特定や詳しいメカニズムは未だ十分には解明されていなかった。 In this technical field, the present inventors have previously proposed a sterilization / nucleic acid decomposition treatment apparatus that maintains a constant catalytic reaction temperature for self-reaction and generates a sterilized gas having a stable concentration (for example, See Patent Document 2). According to this sterilization / nucleic acid decomposition treatment device, the environment temperature at which the effect of nucleic acid decomposition is exerted is set to a body temperature range of 37 ° C., and within a short time of 15 minutes or less, and at a low formaldehyde component concentration, a double helix DNA It has the ability to effectively decompose nucleic acids (disassembled into base chains of 10 base pairs (bp) or less) and can achieve a nucleic acid resolution of 99.99% to 100% as a gas phase nucleic acid decomposition method. On the other hand, the identification and detailed mechanism of the components that cause these effects have not been sufficiently elucidated.
　LPSは、グラム陰性菌細胞壁外膜の構成成分であり、脂質及び多糖から構成される物質（糖脂質）である。LPSは内毒素（エンドトキシン）であり、ヒトや動物など他の生物の細胞に作用すると、多彩な生物活性を発現する。LPSの生理作用発現は、宿主細胞の細胞膜表面に存在するToll様受容体 (Toll-like Receptor、TLR) 4 (TLR4) を介して行われる。更に本件の発明者等は、グラム陰性菌細胞壁外膜の構成成分であるLPSをも分解する能力があることを見出した。LPSは細胞壁から容易には遊離せず、細菌が死滅したときなどに細胞が融解・破壊されることで遊離し、それが動物細胞などに作用することで毒性を発揮する。このような性質から、細菌が外に分泌する毒素（＝外毒素）ではなく、分泌されない「菌体内に存在する毒素」、すなわち内毒素とも呼ばれる。 LPS is a component of the outer membrane of the cell wall of Gram-negative bacteria and is a substance (glycolipid) composed of lipids and polysaccharides. LPS is an endotoxin (endotoxin) that exerts various biological activities when it acts on cells of other organisms such as humans and animals. The expression of physiological action of LPS is carried out via Toll-like receptor {TLR} {4} (TLR4) present on the cell membrane surface of the host cell. Furthermore, the present inventors have found that the ability to degrade LPS, which is a component of the outer membrane of the cell wall of Gram-negative bacteria, has been found. LPS is not easily released from the cell wall, but is released when cells are thawed or destroyed when bacteria are killed, and exerts toxicity when it acts on animal cells and the like. Due to such properties, it is also called an endotoxin, which is not a toxin secreted outside by bacteria (= exotoxin) but not a secreted toxin present in the inside of the bacterium.
　LPSの分解に関しては、特許文献３に記載のようにオゾン分解を用いて多糖部分を脱重合する方法が示されてはいるが、これらは多糖類からの糖の切り出し方法で有り、LPSの分解不活化を狙ったものではない。 Regarding the decomposition of LPS, a method of depolymerizing the polysaccharide portion using ozonolysis as described in Patent Document 3 has been described. However, these are methods for cutting out the sugar from the polysaccharide. It is not aimed at inactivation.
　また、特許文献４記載の免疫調節活性物質の濃度を増加及び／または減少させる方法については、LPSを分解するのではなく、受容体を介する吸着機構により量の増減を図るものである。 The method of increasing and / or decreasing the concentration of the immunomodulatory active substance described in Patent Document 4 does not decompose LPS, but increases or decreases the amount by a receptor-mediated adsorption mechanism.
　LPSは熱的・化学的にも安定で、通常の滅菌に用いられるオートクレーブでは不活化することができない。不活化には250℃で30分間の加熱や、γ線等の放射線を照射することを要することが知られているのみである。しかしながら、これらの条件では耐熱性のない材料からなる器材には適用できないし、運搬できない環境浄化の用途にも適用できるものは今まで存在しなかった。 LPS is thermally and chemically stable, and cannot be inactivated by an autoclave used for normal sterilization. It is only known that inactivation requires heating at 250 ° C. for 30 minutes or irradiation with radiation such as γ-rays. However, under these conditions, it cannot be applied to equipment made of a material having no heat resistance, and there has not been anything that can be applied to environmental purification that cannot be transported.
特開２０１３－１６６７２９号公報JP 2013-166729 A 特許第５４６３３７８号公報Japanese Patent No. 5463378 特表２００１－５１９８１７号公報JP 2001-519817 A 特表２００３－５１９５０３号公報JP-T-2003-519503
　本発明は、このような状況を鑑みてなされたものであり、ホルムアルデヒドの低濃度域での滅菌と核酸分解を可能とし、かつ且つ短時間で優れた効能を発揮することのできる滅菌・核酸分解用組成物を提供することを一つ目の目的とし、さらにLPSを分解することを二つ目の目的とする。 The present invention has been made in view of such circumstances, and enables sterilization and nucleic acid decomposition in a low concentration range of formaldehyde, and sterilization and nucleic acid decomposition capable of exhibiting excellent efficacy in a short time. It is a first object to provide a composition for use, and a second object to decompose LPS.
　本発明者らは、複合ガス中の成分分析により滅菌及び核酸分解に寄与する特定の物質を見出し、そのメカニズムを明らかにすることで本発明を完成させた。 (4) The present inventors have found a specific substance that contributes to sterilization and nucleic acid decomposition by analyzing components in a composite gas, and have completed the present invention by clarifying the mechanism thereof.
　また、本出願の発明者らは、ほぼ同じ滅菌、核酸分解組成物を用いることで、液体状態での反応についても、気体状態での反応についても、滅菌、核酸分解に要する時間よりも長時間の曝露が必要である点が異なるものの、LPSを不活化、少なくともリムルス活性を不活化させうることを見出した。 In addition, the inventors of the present application use substantially the same sterilization and nucleic acid decomposition composition, so that the reaction in a liquid state and the reaction in a gas state are longer than the time required for sterilization and nucleic acid decomposition. However, it was found that LPS could be inactivated, but at least Limulus activity could be inactivated, although the necessity of exposure was different.
　すなわち、本発明の一態様は、核酸の分解と、滅菌することが可能な液体状態の滅菌・核酸分解用組成物であって、少なくともホルムアルデヒドとギ酸を含有し、ホルムアルデヒドの濃度が１０重量％以下であり、かつ、ギ酸の濃度が１重量％以下である。 That is, one embodiment of the present invention is a composition for sterilization and nucleic acid decomposition in a liquid state capable of decomposing nucleic acid and sterilizing, which contains at least formaldehyde and formic acid, and has a formaldehyde concentration of 10% by weight or less. And the concentration of formic acid is 1% by weight or less.
　本発明の一態様によれば、ホルムアルデヒドとギ酸を上記割合で混合することにより、それぞれ単独では効能を発揮しないような低濃度であっても高効率な核酸分解を可能とする。 According to one embodiment of the present invention, by mixing formaldehyde and formic acid in the above ratio, highly efficient nucleic acid degradation can be achieved even at a low concentration at which each of them does not exert its effect alone.
　このとき、本発明の一態様では、更に、メタノールを含み、メタノール、ホルムアルデヒド、ギ酸の誘導体を任意に含むとしても良い。 At this time, one embodiment of the present invention may further include methanol, and optionally include derivatives of methanol, formaldehyde, and formic acid.
　滅菌対象物や使用条件に合わせて、メタノールや、メタノール誘導体、ホルムアルデヒド誘導体、ギ酸誘導体を適宜含めることによって、より効能を発揮させることができる。 (4) More efficacy can be exhibited by appropriately including methanol, a methanol derivative, a formaldehyde derivative, and a formic acid derivative according to an object to be sterilized and use conditions.
　また、本発明の一態様では、さらにLPSを分解できることを特徴とする。 Further, one embodiment of the present invention is characterized in that LPS can be further decomposed.
　本発明の他の態様は、核酸の分解と、滅菌することが可能な気体状態の滅菌・核酸分解用組成物であって、少なくともホルムアルデヒドとギ酸を含有する。 の Another embodiment of the present invention is a gaseous sterilization / nucleolysis composition capable of decomposing nucleic acids and sterilizing, which contains at least formaldehyde and formic acid.
　本発明の他の態様によれば、気相中にホルムアルデヒドとギ酸を含む複合ガスは、低濃度であっても短時間で優れた核酸分解能を発揮することができる。 According to another aspect of the present invention, a composite gas containing formaldehyde and formic acid in the gas phase can exhibit excellent nucleic acid resolution in a short time even at a low concentration.
　このとき、本発明の他の態様では、メタノール、ホルムアルデヒド、ギ酸、一酸化炭素、二酸化炭素、水素、酸素及び／又はこれらのラジカルを含むとしても良い。 At this time, another embodiment of the present invention may include methanol, formaldehyde, formic acid, carbon monoxide, carbon dioxide, hydrogen, oxygen and / or a radical thereof.
　これらの組成物及び／又はそのラジカル種を含有するかは任意である。 か It is optional to contain these compositions and / or their radical species.
　また、本発明の他の態様では、ホルムアルデヒドの濃度が５００ｐｐｍ以下であり、かつ、ギ酸の濃度が４００ｐｐｍ以下としてもよい。 In another aspect of the present invention, the concentration of formaldehyde may be 500 ppm or less, and the concentration of formic acid may be 400 ppm or less.
　また、本発明の他の態様では、さらにLPSを分解できる。 In another embodiment of the present invention, LPS can be further decomposed.
　本発明によれば、低濃度域での滅菌と核酸分解を可能とし、かつ短時間で優れた効能を発揮することができる。 According to the present invention, sterilization and nucleic acid decomposition can be performed in a low concentration range, and excellent effects can be exhibited in a short time.
（Ａ）～（Ｅ）は、比較例１として、ホルムアルデヒドのみを使用した場合のＤＮＡ分解能の結果を示す図である。(A) to (E) are diagrams showing the results of DNA resolution when only formaldehyde was used as Comparative Example 1. （Ａ）～（Ｄ）は、比較例２として、ギ酸のみを使用した場合のＤＮＡ分解能の結果を示す図である。(A) to (D) are diagrams showing the results of DNA resolution when only formic acid was used as Comparative Example 2. （Ａ）、（Ｅ）は、ギ酸のみ、（Ｂ）、（Ｄ）は、ホルムアルデヒドのみ、（Ｃ）、（Ｆ）は、実施例としてホルムアルデヒドとギ酸を併用した場合のＤＮＡ分解能の結果を示す図である。(A) and (E) show formic acid only, (B) and (D) show formaldehyde only, and (C) and (F) show the results of DNA resolution when formaldehyde and formic acid are used in combination as examples. FIG. （Ａ）、（Ｅ）は、ギ酸のみ、（Ｂ）、（Ｄ）は、ホルムアルデヒドのみ、（Ｃ）、（Ｆ）は、実施例としてホルムアルデヒドとギ酸を併用した場合のＤＮＡ分解能の結果を示す図である。(A) and (E) show formic acid only, (B) and (D) show formaldehyde only, and (C) and (F) show the results of DNA resolution when formaldehyde and formic acid are used in combination as examples. FIG.
　以下、本発明に係る滅菌・核酸分解用組成物について図面を参照しながら説明する。なお、本発明は以下の例に限定されるものではなく、本発明の要旨を逸脱しない範囲で、任意に変更可能である。 Hereinafter, the composition for sterilization and nucleic acid degradation according to the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following examples, and can be arbitrarily changed without departing from the gist of the present invention.
　上述した通り、ホルムアルデヒドは、細菌及び真菌などの微生物に対する滅菌効果を有しており、様々な分野で実用化されている。一方で、ホルムアルデヒドは病理学においてホルムアルデヒドの水溶液であるホルマリン浸漬による生体組織の長期固定などに古くから用いられて来たが、最近ではホルマリン固定組織からＤＮＡやＲＮＡなどの核酸物を抽出する技術が開発されたことで、その遺伝学的解析に応用され医学の進歩に大きく寄与している。 As described above, formaldehyde has a sterilizing effect on microorganisms such as bacteria and fungi, and has been put to practical use in various fields. On the other hand, formaldehyde has long been used in pathology for long-term fixation of living tissues by immersion in formalin, which is an aqueous solution of formaldehyde. Recently, however, techniques for extracting nucleic acids such as DNA and RNA from formalin-fixed tissue have been developed. Its development has been applied to its genetic analysis and has greatly contributed to the advancement of medicine.
　そこで議論されているのは、いかに上手くＮｉｃｋなどの核酸の損傷のない、遺伝情報が保たれた長鎖の核酸物を抽出することができるかということで、その保存方法や抽出方法に様々な工夫がなされてきた。これらから、ホルムアルデヒドには核酸分解能というよりも保存性能の面から利用されて来たことがわかる。 What is being discussed is how to successfully extract long-chain nucleic acid products that preserve genetic information without damage to nucleic acids such as nicks. Ingenuity has been devised. These results indicate that formaldehyde has been used in terms of storage performance rather than nucleic acid resolution.
　科学文献的な観点では、ホルムアルデヒドはアミノ酸や生体異物を代謝する際、内因的にも生成し、ホルムアルデヒドに暴露されていない人でも、血液中ホルムアルデヒド濃度が2.61 ± 0.14 μg/g（ほぼ2.6 ppm）との報告がある（化学物質の初期リスク評価書＿独立行政法人 製品評価技術基盤機構 化学物質管理センター）。しかし、ホルムアルデヒドの作用として蛋白質分子の架橋反応が知られており、ホルムアルデヒド分子中のアルデヒド基が組織中の蛋白質のアミノ基に結合し、さらに架橋することで蛋白質の立体構造を損なわせる。特に高濃度ではホルマリンは核酸と蛋白質の架橋を引き起こすため、DNAは物理的ストレスを受けやすくなり断片化が生じるとされている。 From the scientific literature point of view, formaldehyde is also generated endogenously when metabolizing amino acids and xenobiotics, and even those who have not been exposed to formaldehyde have a blood formaldehyde concentration of 2.61 ± 0.14 μg / g (almost 2.6 ppm) (Initial Risk Assessment Report for Chemical Substances_Independent Administrative Institution Product Evaluation Technology Foundation 評 価 Chemical Substance Management Center). However, a cross-linking reaction of a protein molecule is known as an action of formaldehyde, and an aldehyde group in a formaldehyde molecule binds to an amino group of a protein in a tissue, and further cross-links, thereby impairing the three-dimensional structure of the protein. Particularly at high concentrations, formalin causes cross-linking of nucleic acids and proteins, so that DNA is susceptible to physical stress and fragmentation occurs.
　一方で、ギ酸に関しては核酸分解能に関しての知見は乏しく、一部でDNAを脱プリン化によるDNA鎖切断の要因のことが触れられているに過ぎない。その具体的な濃度による効果効能についての文献は皆無である。すなわち、ホルムルデヒド及びギ酸共に滅菌の効能についての報告は見られるが、それらによる核酸分解能については未だ詳細な報告はない。特に、ホルムアルデヒド及びギ酸による低濃度域での核酸分解の効果効能については明らかにされていない。 On the other hand, formic acid is poorly known for its ability to degrade nucleic acids, and only mentions the cause of DNA strand breaks by depurinating DNA. There is no literature on the efficacy of specific concentrations. That is, although there are reports on the efficacy of sterilization for both formaldehyde and formic acid, there is no detailed report on the nucleic acid resolution by these. In particular, the effect of nucleic acid degradation in a low concentration range by formaldehyde and formic acid has not been clarified.
　上述したような滅菌システムはその発生複合ガスにより、滅菌と核酸分解を同時に可能とする効能効果を有することを既に報告してきたが、本件では滅菌システムが生み出すこれらの効能に関して、発生複合ガス中に含まれる組成成分分子毎に各々単独に、濃度、反応時間と効能との相関関係の評価を行い、さらにそれらの様々な混合比別に効能効果への影響を検討しデータベース化することで、低濃度域でかつ極めて短時間で効能を発揮するメカニズム（Mode of Action）及び最適化条件を初めて明らかにし本発明に至ったものである。 Although the sterilization system as described above has already been reported to have the effect of enabling simultaneous sterilization and nucleic acid decomposition by the generated composite gas, in this case, regarding the effects produced by the sterilization system, the generated composite gas contains Evaluate the correlation between concentration, reaction time and potency for each of the constituent components individually, and further examine the effect on potency and effect by various mixing ratios, and create a database to reduce the concentration. The present invention has been clarified for the first time for a mechanism (Mode-of-Action) that exerts an effect in a short time and in an extremely short time and optimization conditions.
　すなわち、本発明の一実施形態に係る滅菌・核酸分解用組成物は、核酸の分解と、滅菌することが可能な液体状態の滅菌・核酸分解用組成物であって、少なくともホルムアルデヒドとギ酸を含有し、ホルムアルデヒドの濃度が１０重量％以下であり、かつ、ギ酸の濃度が１重量％以下である。 That is, the composition for sterilization and nucleic acid decomposition according to one embodiment of the present invention is a composition for sterilization and nucleic acid decomposition in a liquid state capable of decomposing nucleic acids and sterilizing, and contains at least formaldehyde and formic acid. The concentration of formaldehyde is 10% by weight or less, and the concentration of formic acid is 1% by weight or less.
　本発明によれば、ホルムアルデヒドとギ酸を上記割合で混合することにより、それぞれ単独では効能を発揮しないような低濃度であっても高効率な滅菌と核酸分解を可能とする。ギ酸を共存させることにより、ホルムアルデヒドは濃度が１０重量％以下でも滅菌と核酸分解効果を発揮するようになる。ホルムアルデヒドの濃度は、より好ましくは（ギ酸の共存下で滅菌と核酸分解効果を得るための最低濃度という観点からは）５重量％以下でも良い。ホルムアルデヒド濃度の下限値はおおよそ０．１重量％である。また、ホルムアルデヒドと混合させるギ酸の濃度は１重量％以下で十分である。ギ酸の濃度の下限値もおおよそ０．１重量％である。 According to the present invention, by mixing formaldehyde and formic acid in the above ratio, highly efficient sterilization and nucleic acid decomposition can be performed even at a low concentration at which each of them does not exert its effect alone. By coexisting formic acid, formaldehyde exhibits sterilization and nucleic acid degrading effects even at a concentration of 10% by weight or less. The concentration of formaldehyde may more preferably be 5% by weight or less (from the viewpoint of the minimum concentration for obtaining the sterilizing and nucleic acid degrading effects in the presence of formic acid). The lower limit of the formaldehyde concentration is approximately 0.1% by weight. Further, the concentration of formic acid to be mixed with formaldehyde is not more than 1% by weight. The lower limit of the concentration of formic acid is also approximately 0.1% by weight.
　本発明の一実施形態に係る滅菌・核酸分解用組成物は、細菌、真菌類及びウイルス等の滅菌と核酸分解に有効である。本発明の一実施形態に係る滅菌・核酸分解用組成物によれば、５分以内の短時間で、条件によっては１分間で、細菌、真菌類及びウイルス等のＤＮＡ及びＲＮＡを有効に分解（１０ｂｐ以下の塩基鎖へのバラバラ状態）し、極めて高い核酸分解効果を奏することができる。 The composition for sterilizing and decomposing nucleic acids according to one embodiment of the present invention is effective for sterilizing bacteria, fungi and viruses and decomposing nucleic acids. According to the composition for sterilization / nucleic acid decomposition according to one embodiment of the present invention, DNA and RNA such as bacteria, fungi and viruses can be effectively decomposed in a short time of 5 minutes or less, and in one minute depending on conditions ( 10 bp or less in a base chain), and an extremely high nucleic acid degradation effect can be achieved.
　本発明の一実施形態に係る滅菌・核酸分解用組成物は、純水等の水や、メタノール、エタノール等のアルコール類、その他の有機溶媒、或いはこれらの混合液等を溶媒として用いても良い。溶媒中には、ｐＨを調整するための緩衝液や、保存料等が適宜含まれていても良い。 The composition for sterilization / nucleic acid decomposition according to one embodiment of the present invention may use water such as pure water, alcohols such as methanol and ethanol, other organic solvents, or a mixture thereof as a solvent. . The solvent may appropriately contain a buffer for adjusting pH, a preservative, and the like.
　また、本発明の一実施形態に係る滅菌・核酸分解用組成物は、使用時にホルムアルデヒド及びギ酸が上記濃度となっていればよいため、例えば、使用前の保管時には、高濃度に濃縮された状態とし、使用時に適宜希釈して用いても良い。或いは、それぞれの成分を分割して保管し、使用時に混合して用いるような態様でも良い。 In addition, the composition for sterilization and nucleic acid degradation according to one embodiment of the present invention may have the formaldehyde and formic acid concentrations at the time of use as long as they are at the above concentrations. And may be used after being appropriately diluted at the time of use. Alternatively, an embodiment may be used in which each component is divided and stored, and then mixed and used at the time of use.
　本発明の一実施形態に係る滅菌・核酸分解用組成物は、更に、メタノールを含み、メタノールの誘導体、ホルムアルデヒドの誘導体、ギ酸の誘導体を任意に含むことができる。なお、ここで、誘導体とは、任意の官能基の導入、酸化、還元、原子の置き換えなど、メタノール、ホルムアルデヒド、又はギ酸の構造や性質を大幅に変えない程度の改変がなされた化合物を言う。 The composition for sterilization and nucleic acid degradation according to one embodiment of the present invention further contains methanol, and can optionally contain a derivative of methanol, a derivative of formaldehyde, and a derivative of formic acid. Here, a derivative refers to a compound that has been modified to such an extent that the structure or properties of methanol, formaldehyde, or formic acid are not significantly changed, such as introduction of an arbitrary functional group, oxidation, reduction, or substitution of an atom.
　一方、本発明の一実施形態に係るLPS分解用組成物は、LPSを分解することが可能な液体状態の組成物であって、滅菌・核酸分解用と同じく、少なくともホルムアルデヒドとギ酸を含有し、ホルムアルデヒドの濃度が１０重量％以下であり、かつ、ギ酸の濃度が１重量％以下である。本発明によれば、ホルムアルデヒドとギ酸を上記割合で混合することにより、それぞれ単独では効能を発揮しないような低濃度であってもLPS分解を可能とする。ギ酸を共存させることにより、ホルムアルデヒドは濃度が１０重量％以下でもLPS分解効果を発揮するようになる。ホルムアルデヒドの濃度は、より好ましくは（ギ酸の共存下で滅菌と核酸分解効果を得るための最低濃度という観点からは）５重量％以下でも良い。ホルムアルデヒド濃度の下限値はおおよそ０．０３重量％である。また、ホルムアルデヒドと混合させるギ酸の濃度は１重量％以下で十分である。ギ酸の濃度の下限値もおおよそ０．０１重量％である。LPS分解用組成物によれば、好ましくは１０分～２時間、より好ましくは１５分から２時間程度接触反応させることで、１ｍLあたり、１００から１０００EU（エンドトキシンユニット）の量のLPSを約半減程度以下にさせることが出来る。 On the other hand, the composition for decomposing LPS according to one embodiment of the present invention is a composition in a liquid state capable of decomposing LPS, and contains at least formaldehyde and formic acid, as well as for sterilization and nucleic acid decomposition, The concentration of formaldehyde is 10% by weight or less and the concentration of formic acid is 1% by weight or less. According to the present invention, by mixing formaldehyde and formic acid in the above ratio, LPS can be decomposed even at a low concentration at which each of them does not exert its effect alone. By coexisting formic acid, formaldehyde exerts an LPS decomposition effect even at a concentration of 10% by weight or less. The concentration of formaldehyde may more preferably be 5% by weight or less (from the viewpoint of the minimum concentration for obtaining the sterilizing and nucleic acid degrading effects in the presence of formic acid). The lower limit of the formaldehyde concentration is approximately 0.03% by weight. Further, the concentration of formic acid to be mixed with formaldehyde is not more than 1% by weight. The lower limit of the concentration of formic acid is also approximately 0.01% by weight. According to the composition for decomposing LPS, the contact reaction is preferably carried out for about 10 minutes to 2 hours, more preferably for about 15 minutes to 2 hours, whereby LPS in an amount of 100 to 1000 EU (endotoxin unit) per mL is reduced to about half or less. Can be made.
　本発明の一実施形態に係る滅菌・核酸分解用組成物の液相での使用態様は特に限定されないが、例えば、滅菌・核酸分解、LPS分解対象物を滅菌・核酸分解もしくはLPS分解用組成物溶液に浸漬することにより滅菌・核酸分解およびLPS分解しても良いし、滅菌・核酸分解、LPS分解対象物に滅菌・核酸分解、LPS分解用組成物を吹きかけるようにして滅菌・核酸分解、LPS分解しても良い。特に滅菌・核酸分解用組成物、LPS分解用組成物をミスト状にして対象物に対して噴霧すると効果的である。滅菌・核酸分解用組成物との接触時間は、５分以内で十分な効果を得ることができる。但し、LPS分解用組成物によるLPS分解については、３０分以上の反応を待った方が良い。また、接触温度も特には限定されず、室温でも良いし、適宜３７～５０℃程度に、さらに６０℃程度に加熱しても良い。これ以上の高温になると、被処理物の耐熱性を考慮する必要が出てくるため、好ましくはない。 The mode of use of the composition for sterilization and nucleic acid decomposition according to one embodiment of the present invention in the liquid phase is not particularly limited, for example, sterilization and nucleic acid decomposition, the composition for sterilization and nucleic acid decomposition or LPS decomposition for the target of LPS decomposition Sterilization, nucleic acid decomposition, and LPS decomposition may be performed by immersion in a solution, or sterilization, nucleic acid decomposition, and LPS decomposition composition may be sprayed on a sterilization, nucleic acid decomposition, and LPS decomposition target to sterilize, nucleic acid decomposition, and LPS decomposition. It may be disassembled. In particular, it is effective to spray the composition for sterilization / nucleic acid decomposition and the composition for LPS decomposition into a mist and spray it onto a target. A sufficient effect can be obtained when the contact time with the composition for sterilization / nucleic acid degradation is within 5 minutes. However, for the LPS decomposition by the composition for LPS decomposition, it is better to wait for the reaction for 30 minutes or more. Also, the contact temperature is not particularly limited, and may be room temperature, or may be appropriately heated to about 37 to 50 ° C. and further to about 60 ° C. If the temperature is higher than this, it is necessary to consider the heat resistance of the object to be processed, which is not preferable.
　また、本発明の一実施形態に係る滅菌・核酸分解用組成物は、気体状態で使用することも可能である。すなわち、本発明の一実施形態に係る滅菌・核酸分解用組成物は、核酸の分解と滅菌することが可能な気体状態の滅菌・核酸分解用組成物であって、少なくともホルムアルデヒドとギ酸を含有する。ホルムアルデヒドとギ酸の好ましい濃度範囲の一例として、ホルムアルデヒドの濃度が５００ｐｐｍ以下であり、かつ、ギ酸の濃度が４００ｐｐｍ以下である。また、ホルムアルデヒドの濃度が２００ｐｐｍ以下であり、かつ、ギ酸の濃度が１００ｐｐｍ以下の時、微生物（ＢＩ指標菌）に対して５分以内で効果的に作用することを発明者らは確認している。 The composition for sterilizing and decomposing nucleic acids according to one embodiment of the present invention can be used in a gaseous state. That is, the composition for sterilization and nucleic acid decomposition according to one embodiment of the present invention is a composition for sterilization and nucleic acid decomposition in a gaseous state capable of decomposing and sterilizing nucleic acids, and contains at least formaldehyde and formic acid. . As an example of a preferable concentration range of formaldehyde and formic acid, the concentration of formaldehyde is 500 ppm or less, and the concentration of formic acid is 400 ppm or less. In addition, the present inventors have confirmed that when the concentration of formaldehyde is 200 ppm or less and the concentration of formic acid is 100 ppm or less, it effectively acts on microorganisms (BI indicator bacteria) within 5 minutes. .
　このように、気体状態においても、気相中にホルムアルデヒドとギ酸を所定の濃度範囲で含む複合ガスは、低濃度であっても短時間で優れた滅菌と核酸分解能を発揮することができる。 As described above, even in a gaseous state, a composite gas containing formaldehyde and formic acid in a predetermined concentration range in a gaseous phase can exhibit excellent sterilization and nucleic acid resolution in a short time even at a low concentration.
　一方、本発明の一実施形態に係るLPS分解用組成物は、気体状態でLPSを分解することが可能な組成物であって、滅菌・核酸分解用と同じく、少なくともホルムアルデヒドとギ酸を含有する。ホルムアルデヒドとギ酸の好ましい濃度範囲の一例として、ホルムアルデヒドの濃度が５００ｐｐｍ以下であり、かつ、ギ酸の濃度が４００ｐｐｍ以下である。また、ホルムアルデヒドの濃度が２００ｐｐｍ以下であり、かつ、ギ酸の濃度が１００ｐｐｍ以下の時、LPS分解用組成物によれば、好ましくは１０分～２時間、より好ましくは１５分から２時間程度接触反応させることで、１ｍLあたり、１００から１０００EU（エンドトキシンユニット）の量のLPSを半減程度以下にさせることが出来る。 On the other hand, the composition for decomposing LPS according to one embodiment of the present invention is a composition capable of decomposing LPS in a gaseous state, and contains at least formaldehyde and formic acid as in the case of sterilization and nucleic acid decomposition. As an example of a preferable concentration range of formaldehyde and formic acid, the concentration of formaldehyde is 500 ppm or less, and the concentration of formic acid is 400 ppm or less. When the concentration of formaldehyde is 200 ppm or less and the concentration of formic acid is 100 ppm or less, according to the composition for decomposing LPS, the contact reaction is preferably performed for about 10 minutes to 2 hours, more preferably for about 15 minutes to 2 hours. Thus, LPS in an amount of 100 to 1000 EU (endotoxin unit) per 1 mL can be reduced to about half or less.
　本発明の一実施形態に係る滅菌・核酸分解用組成物を気体状態で使用する方法は、ホルムアルデヒドとギ酸を含む複合ガスとなるのであれば特に限定されない。例えば、液体状態の本発明の一実施形態に係る滅菌・核酸分解用組成物を加熱により気化させても良いし、メタノール等を銅などの触媒と反応させて発生する複合ガス中のホルムアルデヒドとギ酸が所定の濃度範囲となるように反応条件を設定しても良い。ホルムアルデヒドとギ酸の濃度の下限値は特に限定されるわけではないが、おおむね１ｐｐｍである。 方法 The method of using the composition for sterilization / nucleic acid decomposition according to one embodiment of the present invention in a gaseous state is not particularly limited as long as it is a composite gas containing formaldehyde and formic acid. For example, the composition for sterilization and nucleic acid decomposition according to an embodiment of the present invention in a liquid state may be vaporized by heating, or formaldehyde and formic acid in a complex gas generated by reacting methanol or the like with a catalyst such as copper. May be set in such a manner that is within a predetermined concentration range. Although the lower limit of the concentration of formaldehyde and formic acid is not particularly limited, it is approximately 1 ppm.
　また、本発明の一実施形態に係る滅菌・核酸分解用組成物は、気体状態にする過程で、メタノール、ホルムアルデヒド、ギ酸、一酸化炭素、二酸化炭素、水素、酸素及び／又はこれらのラジカルが含まれていても良い。 Further, the composition for sterilization and nucleic acid decomposition according to one embodiment of the present invention contains methanol, formaldehyde, formic acid, carbon monoxide, carbon dioxide, hydrogen, oxygen, and / or their radicals in the process of converting into a gaseous state. It may be.
　本発明の一実施形態に係る滅菌・核酸分解用組成物の気相での使用態様は特に限定されないが、例えば、チャンバー等の閉じた空間内に滅菌・核酸分解対象物を載置し、本発明の一実施形態に係る滅菌・核酸分解用組成物を気化したものを充満させることにより滅菌・核酸分解することができる。この時、適度な湿度（５０～９０相対湿度％）を加えても良いし、滅菌・核酸分解対象物が水分に弱いときは、低湿度（５０相対湿度％未満）で滅菌・核酸分解しても良い。また、接触温度も特には限定されず、室温でも良いし、適宜３７～５０℃程度、さらには６０℃程度までに加熱しても良い。これ以上の高温になると、被処理物の耐熱性を考慮する必要が出てくるため、好ましくはない。 The mode of use of the composition for sterilization / nucleic acid decomposition according to one embodiment of the present invention in the gas phase is not particularly limited.For example, a sterilization / nucleic acid decomposition target is placed in a closed space such as a chamber, and The sterilizing / nucleic acid decomposing composition according to one embodiment of the present invention can be sterilized / nucleic acid decomposed by filling the vaporized composition. At this time, an appropriate humidity (50 to 90 relative humidity%) may be added, and when the object to be sterilized and decomposed of nucleic acid is weak to moisture, sterilization and nucleic acid decomposition may be performed at low humidity (less than 50 relative humidity%). Is also good. Also, the contact temperature is not particularly limited, and may be room temperature, or may be appropriately heated to about 37 to 50 ° C., and further to about 60 ° C. If the temperature is higher than this, it is necessary to consider the heat resistance of the object to be processed, which is not preferable.
　以上、説明してきたように、本発明の一実施形態に係る滅菌・核酸分解用組成物は、液相状態、気相状態のいずれにおいても、ホルムアルデヒドとギ酸を組み合わせることによって、それぞれ単独では効果を発揮しないような低濃度域においても、優れた滅菌効果と核酸（ＤＮＡ、ＲＮＡ等）の分解能を有し、短時間で効能を発揮することができる。 As described above, the composition for sterilization / nucleic acid degradation according to one embodiment of the present invention has an effect alone by combining formaldehyde and formic acid in both the liquid phase and the gas phase. Even in a low-concentration range that does not exert its effect, it has an excellent sterilizing effect and the resolution of nucleic acids (DNA, RNA, etc.) and can exert its effect in a short time.
　以下に、本発明の実施例及び比較例によって本発明をさらに詳細に説明するが、本発明は、これらの実施例によって何ら限定されるものではない。 本 Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples of the present invention, but the present invention is not limited to these Examples.
［１．ＤＮＡの調整］
　核酸分解の試験に用いるDNAは継代培養したHeLa cells (ヒト子宮頸がん由来の細胞株：human cervical adenocarcinoma)(American Type Culture Collection: ATCC)から、DNeasy Blood & Tissue (Qiagen)のメーカープロトコールに従いgenomic DNAとして抽出精製したものを用いて実施した。 [1. Preparation of DNA]
The DNA used for the nucleic acid degradation test was obtained from subcultured HeLa cells (human cervical adenocarcinoma) (American Type Culture Collection: ATCC) according to the manufacturer's protocol of DNeasy Blood & Tissue (Qiagen). The extraction and purification were performed as genomic DNA.
　精製したgenomic DNAの濃度は, Nano-drop (NanoDrop One, Thermo Fisher Scientific)を用いて測定し、UltraPureTM DNase/RNase-Free Distilled Water (DW ; Thermo Fisher Scientific)にて希釈濃度調整した。希釈濃度調整したgenomic DNAを超音波破砕機 (UR-21P, TOMY) を用いて超音波破砕を行ったものを破砕DNAサンプルとして用いた。 The concentration of the purified genomic DNA was measured using Nano-drop (NanoDrop One, Thermo Fisher Scientific), and the dilution concentration was adjusted with UltraPure ™ DNase / RNase-Free Distilled Water (DW; Thermo Fisher Scientific). The genomic DNA whose concentration had been adjusted was sonicated using an ultrasonic homogenizer (UR-21P, TOMY) and used as the homogenized DNA sample.
　得られた破砕DNAの500ngをそれぞれ用いて以下の実験を行なった。 The following experiments were performed using 500 ng of the obtained crushed DNA.
［２．比較例１：ホルムアルデヒド単独でのＤＮＡ分解能］
　比較例１として、ホルムアルデヒドを単独で使用した場合のDNA分解能につき、ホルムアルデヒド濃度及び反応時間によるDNA分解能の検討を行なった。ホルムアルデヒド（37.0％、WAKO特級試薬）を蒸留水で希釈して、0.1%、0.5%、1.0%、2.0%、3.0%、4.0%、5.0%、10%、20%、30%のホルムアルデヒド溶液を調製した。0.5 ml PCRチューブにそれぞれのホルムアルデヒド希釈濃度になるよう分取調整を行い、最後に破砕DNAサンプルを添加し、ストップウオッチにて反応時間を計測した。反応終了後、速やかにQIAquick PCR purification kitによりサンプルの洗浄・回収を行い、得られたサンプルをBioanalyzerにより解析を行った。 [2. Comparative Example 1: DNA resolution using formaldehyde alone]
As Comparative Example 1, regarding the DNA resolution when formaldehyde was used alone, the DNA resolution was examined based on the formaldehyde concentration and the reaction time. Dilute formaldehyde (37.0%, WAKO special grade reagent) with distilled water to form a 0.1%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 10%, 20%, 30% formaldehyde solution. Prepared. A 0.5 ml PCR tube was subjected to a preparative adjustment so that each formaldehyde dilution concentration was obtained. Finally, a crushed DNA sample was added, and the reaction time was measured with a stopwatch. After the completion of the reaction, the sample was immediately washed and collected using a QIAquick PCR purification kit, and the obtained sample was analyzed using a Bioanalyzer.
　なお、以降の比較例及び実施例において、Bioanalyzerに付与されているマーカーのうち、Upper Marker（分子量17000）のピーク値に対してのサンプルの測定ピーク値の割合からDNA分解効果を以下のように分類する。
Upper Markerピーク値の７５％以上１００％以下：レベル１
Upper Markerピーク値の５０％以上７５％未満 ：レベル２
Upper Markerピーク値の２５％以上５０％未満 ：レベル３
Upper Markerピーク値の２５％未満：レベル４ In the following Comparative Examples and Examples, among the markers provided to Bioanalyzer, the DNA degradation effect was determined from the ratio of the measured peak value of the sample to the peak value of the Upper Marker (molecular weight: 17,000) as follows: Classify.
Upper Marker peak value 75% or more and 100% or less: Level 1
Upper Marker peak value 50% or more and less than 75%: Level 2
Upper Marker peak value 25% or more and less than 50%: Level 3
Less than 25% of Upper Marker peak value: Level 4
（結果）
　ホルムアルデヒド単独でのDNA分解能の結果を図１に示す。ホルムアルデヒド希釈液単独でのDNA分解能の評価結果は、１分の反応時間で0.1%～5.0%の低濃度域では明らかなDNA分解効果を認めなかった。10%以上の高濃度域においても、ホルムアルデヒド希釈溶液10％[FA10%]（以下、「ＧＡ」はギ酸を、「ＦＡ」はホルムアルデヒドを表す。）, 1min.でレベル３(0.385)（図１（Ａ））、3min.でもレベル4 (0.138)（図１（Ｂ））で、DNAの完全分解効果は得られなかった。[FA30%]の高濃度になると、5min.（図１（Ｃ））でDNAの完全分解効果を示した。 (result)
FIG. 1 shows the results of DNA resolution using formaldehyde alone. The evaluation results of the DNA resolution using the formaldehyde diluent alone showed no obvious DNA degradation effect in a low concentration range of 0.1% to 5.0% in a reaction time of 1 minute. Even in a high concentration range of 10% or more, a formaldehyde diluted solution 10% [FA10%] (hereinafter, “GA” represents formic acid and “FA” represents formaldehyde) is at Level 3 (0.385) at 1 min. (A)), even at 3 min., Level 4 (0.138) (FIG. 1 (B)), and no complete DNA degradation effect was obtained. When the concentration was as high as [FA30%], the DNA was completely degraded for 5 min. (FIG. 1 (C)).
　低濃度域として0.1%～5.0%におけるDNA分解能を詳細に調べた結果、３分間の反応時間では0.1～3.0%では明らかなDNA分解効果を認めなかったが、4.0%濃度になると若干のDNA分解効果が認められるようになり、5.0%濃度でようやくレベル3 (0.491)（図１（Ｄ））のDNA分解能を認めた。５分間の反応時間においても、0.1～2.0%では明らかなDNA分解効果を認めなかったが、3.0～4.0%濃度で若干のDNA分解能が見られるようになり、5.0%濃度でレベル３ (0.422)（図１（Ｅ））のDNA分解能を認めた。 As a result of detailed examination of the DNA resolution in the low concentration range from 0.1% to 5.0%, no apparent DNA degradation effect was observed in the reaction time of 3 minutes from 0.1% to 3.0%, but a slight DNA degradation was observed at the concentration of 4.0%. The effect began to be recognized, and DNA resolution of level 3 (0.491) (FIG. 1 (D)) was finally recognized at the 5.0% concentration. Even at a reaction time of 5 minutes, no apparent DNA degradation effect was observed at 0.1 to 2.0%, but a slight DNA resolution was observed at a concentration of 3.0 to 4.0%, and a level of 3 (0.422) was observed at a concentration of 5.0%. The DNA resolution shown in FIG. 1 (E) was observed.
［３．比較例２：ギ酸単独でのＤＮＡ分解能］
　比較例２として、ギ酸単独での濃度、反応時間によるDNA分解能につき検討を行なった。ギ酸（９８．０％、WAKO特級試薬）を蒸留水で希釈して、0.1%、0.2%、0.3%、0.4%、0.5%、1.0%、1.5%、2.0%、3.0%、5.0%のギ酸溶液を調製した。以降は、比較例１と同様にしてDNA分解能を評価した。 [3. Comparative Example 2: DNA resolution with formic acid alone]
As Comparative Example 2, the DNA resolution by the concentration of formic acid alone and the reaction time was examined. Dilute formic acid (98.0%, WAKO special grade reagent) with distilled water, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 1.5%, 2.0%, 3.0%, 5.0% formic acid A solution was prepared. Thereafter, the DNA resolution was evaluated in the same manner as in Comparative Example 1.
（結果）
　ギ酸単独でのDNA分解能の結果を図２に示す。ギ酸希釈液単独でのDNA分解能の評価結果は、１分間の反応時間で0.1%濃度で若干のDNA分解効果がみられ、1.0%でもレベル３(0.296)（図２（Ａ））とDNAの完全分解効果は得られず中程度のDNA分解能を示した。2.0%以上の高濃度域になると１分間の反応時間でDNAの完全分解効果を認めた。３分の反応時間では、0.1%濃度では若干（レベル１程度）（図２（Ｂ））のDNA分解効果がみられ、0.2%濃度でレベル２、0.3～0.5%でレベル３と濃度依存的に中程度のDNA分解効果を示し、1.0%以上になるとDNA完全分解効果を示した（図２（Ｃ））。５分の反応時間では、0.1%濃度では若干（図２（Ｄ））のDNA分解効果がみられ、0.2～0.5%の濃度域ではレベル１～３と濃度依存的に中程度のDNA分解効果を示し、1.0%以上になるとDNA完全分解効果を示した。 (result)
FIG. 2 shows the results of DNA resolution using formic acid alone. The results of the evaluation of the DNA resolution using the formic acid dilution alone showed a slight DNA degradation effect at a 0.1% concentration in a reaction time of 1 minute, and a level 3 (0.296) (FIG. 2 (A)) of DNA at a concentration of 1.0%. No complete degradation effect was obtained, indicating a moderate DNA resolution. When the concentration reached a high concentration range of 2.0% or more, a complete DNA degradation effect was observed with a reaction time of 1 minute. At a reaction time of 3 minutes, a slight (approximately level 1) DNA degradation effect was observed at 0.1% concentration (FIG. 2 (B)), and the concentration was dependent on level 2 at 0.2% concentration and level 3 at 0.3-0.5%. Showed a moderate DNA-degrading effect, and showed a complete DNA-degrading effect at 1.0% or more (FIG. 2C). At a reaction time of 5 minutes, a slight DNA degradation effect was observed at a concentration of 0.1% (FIG. 2 (D)), and a moderate level of DNA degradation effect in a concentration range of 0.2 to 0.5% with levels 1 to 3 in a concentration-dependent manner. When it was 1.0% or more, a complete DNA degradation effect was exhibited.
［４．実施例１：ホルムアルデヒド及びギ酸の混合液におけるDNA分解能］
　実施例では、本発明の一実施形態に係る滅菌・核酸分解用組成物を用いてDNA分解能を評価した。ホルムアルデヒド及びギ酸の単独ではDNA完全分解効果が得られない濃度範囲において、両者を様々な濃度で混合することによりDNA分解効果の相乗効果が得られるかどうかを検討した。その結果、それぞれ単独希釈液では実現できない低濃度、短時間反応であっても、最小濃度混合比でDNA完全分解効果が得られる最適化条件を特定できた。 [4. Example 1: DNA resolution in a mixture of formaldehyde and formic acid]
In Examples, DNA resolution was evaluated using the composition for sterilization and nucleic acid degradation according to one embodiment of the present invention. In a concentration range in which formaldehyde and formic acid alone could not obtain a complete DNA degrading effect, it was examined whether or not a synergistic effect of a DNA degrading effect could be obtained by mixing them at various concentrations. As a result, it was possible to identify the optimum conditions under which the complete DNA degradation effect was obtained at the minimum concentration mixing ratio even at a low concentration and a short reaction which could not be achieved with a single dilution.
　ホルムアルデヒド単独での評価結果（比較例１）で明らかになったように、最終濃度が0.1～4.0%の範囲ではほとんどDNA分解効果を認めなかった。同様に、ギ酸単独（比較例２）でも0.1～0.5%の低濃度域では５分間の反応時間によってもレベル４以上のDNA分解効果は得られなかった。そこで、DNA分解効果を示さないホルムアルデヒド濃度の場合に、低濃度のギ酸を添加することによりDNA分解能に対して相乗効果が得られるか否かの評価を行い、相乗効果を認める場合にはDNA完全分解効果を得るためのホルムアルデヒド及びギ酸の最小濃度混合比を明らかにすることを目的に以下の検討を行った。 As is clear from the evaluation results of formaldehyde alone (Comparative Example 1), almost no DNA degradation effect was observed in the final concentration range of 0.1 to 4.0%. Similarly, even with formic acid alone (Comparative Example 2), in a low concentration range of 0.1 to 0.5%, a DNA degradation effect of level 4 or more was not obtained even with a reaction time of 5 minutes. Therefore, in the case of a formaldehyde concentration that does not show a DNA degradation effect, it was evaluated whether or not a synergistic effect on DNA resolution could be obtained by adding a low concentration of formic acid. The following study was conducted to clarify the minimum concentration mixture ratio of formaldehyde and formic acid for obtaining the decomposition effect.
　ホルムアルデヒド及びギ酸原液をそれぞれ蒸留水で希釈し、それらを様々な希釈濃度に調整した混合液を用いてDNA分解能の評価を行なった。すなわち、比較例１、２の場合と同様に、あらかじめ3倍～200倍に希釈した調整試薬を用意し、0.5mlPCRチューブに表１に示すようにそれぞれの希釈試薬を用いて該当する最終濃度に分取調整を行ない、最後に破砕DNAサンプルを添加し、ストップウオッチにて反応時間を計測した。表１に調整したサンプルの一覧を示す。なお、表１中で、「ＧＡ」はギ酸を、「ＦＡ」はホルムアルデヒドを表す。反応終了後、メーカーのマニュアルに従い速やかにQIAquick PCR purification kitによりサンプルの洗浄・回収を行い、得られたサンプルをBioanalyzerにより解析を行った。 (4) Formaldehyde and formic acid stock solutions were each diluted with distilled water, and the DNA resolution was evaluated using mixed solutions prepared at various dilution concentrations. That is, in the same manner as in Comparative Examples 1 and 2, a preparation reagent diluted 3 to 200 times in advance was prepared, and the corresponding final concentration was adjusted to each of the diluted reagents in a 0.5 ml PCR tube as shown in Table 1. Preparative adjustment was performed, and finally a crushed DNA sample was added, and the reaction time was measured with a stopwatch. Table 1 shows a list of the adjusted samples. In Table 1, “GA” represents formic acid, and “FA” represents formaldehyde. After completion of the reaction, the sample was quickly washed and collected by QIAquick PCR purification kit according to the manufacturer's manual, and the obtained sample was analyzed by Bioanalyzer.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（結果）
　本発明に係るホルムアルデヒドとギ酸を併用した場合のDNA分解能の結果を表２に示す。表２中で、「n.s.」はほとんど（レベル１未満）DNAの分解効果が得られなかったことを表し、「１」～「４」は上記レベル１～レベル４のDNAの分解効果を表し、「CD」はほぼ完全にDNAの分解効果が得られたことを表す。ホルムアルデヒド単独での評価結果で明らかになったように、最終濃度が0.1～4.0%の範囲ではほとんどDNA分解効果を認めなかったのに対し、低濃度のギ酸を添加することで、相乗効果によりDNAの完全分解効果を得ることが分かった。実用化においては、短時間で、残留性の少ない条件下での効能評価による運用基準が優先されることから、以下では、作用時間、濃度の面からDNA完全分解効果の得られる最適化条件の具体例を示す。 (result)
Table 2 shows the results of DNA resolution when the formaldehyde and formic acid according to the present invention were used in combination. In Table 2, "ns" indicates that almost no (less than level 1) DNA degrading effect was obtained, and "1" to "4" indicate the level 1 to level 4 DNA degrading effects, “CD” indicates that the DNA degradation effect was almost completely obtained. As is clear from the evaluation results of formaldehyde alone, almost no DNA degrading effect was observed in the final concentration range of 0.1 to 4.0%, but by adding formic acid at a low concentration, DNA synergistic effect was observed. It was found that a complete decomposition effect was obtained. In practical use, priority is given to operational criteria based on efficacy evaluation under conditions with little persistence in a short period of time.Therefore, below, optimization conditions for obtaining a complete DNA degradation effect in terms of action time and concentration will be described. A specific example will be described.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　まず検討した混合希釈液の範囲で、１分間という短時間でDNA完全分解効果を得るための条件は、[GA 0.4% + FA 4.0%], 1minの反応条件が必要であることが分かった。（図３（Ａ）～（Ｃ）参照、図３（Ａ）がギ酸のみ、（Ｂ）がホルムアルデヒドのみであり、（Ｃ）で両者を併用することにより、DNA完全分解効果が得られていることが分かる。） First, it was found that the reaction conditions of [GA 0.4% + FA 4.0%] and 1 min were required as conditions for obtaining a complete DNA degradation effect in a short time of 1 minute within the range of the mixed diluent studied. (See FIGS. 3 (A) to 3 (C), FIG. 3 (A) shows only formic acid, (B) shows only formaldehyde, and (C) shows a complete DNA degrading effect when used in combination. You can see that.)
　ギ酸の最小濃度[GA 0.1%]希釈液の添加によりDNAの完全分解効果の得られるホルムアルデヒド最小濃度は、FA濃度が３％以下では5min.の反応時間によっても得られず、[FA 4.0%]濃度で5min.の反応時間が必要であることが明らかになった。（図３（Ｄ）～（Ｆ）参照、図３（Ｄ）がホルムアルデヒドのみ、（Ｅ）がギ酸のみであり、（Ｆ）が両者を併用した場合である。） The minimum formaldehyde concentration at which a complete DNA degrading effect can be obtained by adding a dilute solution of formic acid [GA] 0.1%] is not obtained even with a reaction time of 5 min. When the FA concentration is 3% or less, [FA 4.0%] It was found that a reaction time of 5 min. Was required at the concentration. (See FIGS. 3 (D) to (F), FIG. 3 (D) shows only formaldehyde, (E) shows only formic acid, and (F) shows a case using both.)
　ホルムアルデヒドの最小濃度[FA 0.1%]希釈液の添加によりDNAの完全分解効果の得られるギ酸最小濃度は0.4%で、[GA 0.4%]で5min.の反応時間によりDNAの完全分解効果を示すことが明らかになった。さらに濃度を上げ[GA 0.5%]では、さらに短時間 (3min.)でDNAの完全分解効果を示した。（図４（Ａ）～（Ｆ）参照、図４（Ａ）、（Ｅ）がギ酸のみ、（Ｂ）、（Ｄ）がホルムアルデヒドのみであり、（Ｃ）、（Ｆ）が両者を併用した場合である。） Minimum concentration of formaldehyde [FA 0.1%] Addition of a diluent to obtain a complete DNA degrading effect The minimum concentration of formic acid is 0.4%, and [GA 0.4%] shows a complete DNA degrading effect with a reaction time of 5 min. Was revealed. When the concentration was further increased [GA 0.5%], the DNA was completely degraded in a shorter time (3 min.). (See FIGS. 4 (A) to 4 (F), FIGS. 4 (A) and 4 (E) show only formic acid, (B) and (D) show only formaldehyde, and (C) and (F) show a combination of both. Is the case.)
　このように、単剤ではDNA分解能が得られない低濃度域であっても、それらを混合することで相乗効果によって極めて高いDNA分解能を獲得することが可能となることが明らかになった。滅菌・核酸分解処理装置を用いてこの混合液に相当するホルムアルデヒドとギ酸を同時に発生させることが可能であり、これにより優れたDNA分解能を示すシステムであると言える。すなわち、この最適化濃度の複合ガスを同時に生成させることのできるシステムである。 Thus, even in a low concentration range where DNA resolution cannot be obtained with a single agent, it was clarified that by mixing them, it is possible to obtain an extremely high DNA resolution by a synergistic effect. It is possible to simultaneously generate formaldehyde and formic acid corresponding to this mixed solution using a sterilization / nucleic acid decomposition treatment apparatus, which can be said to be a system showing excellent DNA resolution. That is, the system is capable of simultaneously generating the composite gas having the optimized concentration.
［5．実施例2：ホルムアルデヒド及びギ酸の混合ガスにおけるDNA分解能］
　実施例１と同様にして、ホルムアルデヒド及びギ酸の混合ガスを用いて、核酸分解を検討した。DNA分解能の結果を表３に示す。 [Five. Example 2: DNA resolution in mixed gas of formaldehyde and formic acid]
In the same manner as in Example 1, nucleic acid degradation was examined using a mixed gas of formaldehyde and formic acid. Table 3 shows the results of the DNA resolution.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　［６．実施例３～１９：ホルムアルデヒド及びギ酸の混合液及びガスにおけるLPS分解能］
　LPSの標準試料としては、ENDOSAFE(ENDOTOXIN INDICATOR; Catalog #EVV2K, コード番号513-87082)を用い、注射用水（大塚）を用いて規定の濃度に希釈したものを使用して測定した。容器や分注チップはすべてLPSフリーのものを用いた。
　測定は、endosafe-PTSを使い、PTSカートリッジJP(5-0.05EU/mL)を用いて測定した。ホルムアルデヒド、ギ酸は、測定上干渉作用があり、予めリムルスES-IIシングルテストを用い検討し、endosafe-PTS測定時のホルムアルデヒド濃度は０．０３％以下、ギ酸の濃度は０．００３％以下になるよう濃度設定をおこない実施した。 [6. Examples 3 to 19: LPS resolution in mixed solution and gas of formaldehyde and formic acid]
As a standard sample of LPS, measurement was performed using ENDOSAFE (ENDOTOXIN INDICATOR; Catalog # EVV2K, code number 513-87082), which was diluted to a specified concentration with water for injection (Otsuka). LPS-free containers and dispensing tips were used.
The measurement was performed using endosafe-PTS and a PTS cartridge JP (5-0.05EU / mL). Formaldehyde and formic acid have interference effects in the measurement, and are examined in advance using the Limulus ES-II single test. The formaldehyde concentration and the formic acid concentration in endosafe-PTS measurement are 0.03% or less and 0.003% or less, respectively. The density was set as follows.
　結果を表４に示す。表中の左から４列目のLPS量とは、分解前のLPS量のことであり、右から２列目の曝露後LPS量との比較によって、LPS分解の程度が分かる。実施例３～９が液体を用いた際の結果である。それぞれ、分解前のLPS量に対して、約半減したことが分かる。 The results are shown in Table 4. The amount of LPS in the fourth column from the left in the table is the amount of LPS before decomposition, and the degree of LPS decomposition can be determined by comparison with the amount of LPS after exposure in the second column from the right. Examples 3 to 9 show the results when liquid was used. It can be seen that the amount of each of the LPSs before the decomposition was reduced by about half.
　また、実施例１０～１９は、表４のとおりホルムアルデヒドとギ酸の混合気体で暴露させたときのLPS分解結果である。実施例１０，１１は、LPS水溶液を１．５ｍLエッペンドルフチューブに取り、５８℃乾燥機中で蒸発乾固させてLPSをペレット状に底に堆積させた状態で実施した。 Examples 10 to 19 show the results of LPS decomposition when exposed to a mixed gas of formaldehyde and formic acid as shown in Table 4. Examples 10 and 11 were carried out in a state in which an LPS aqueous solution was placed in a 1.5 mL Eppendorf tube and evaporated to dryness in a 58 ° C. drier to deposit LPS in a pellet form on the bottom.
　実施例１２～１５では、LPS水溶液をPVDFフィルター（０．４μm径）に付着させ、風乾したものをサンプルとして用いた。LPSが、膜全体に広がり、ペレット状のものに比べて薄く広がった状態のモデルとした。明らかにペレット状のものに比べ分解効率が上がっていることが示された。おなじ状態でPVDFフィルターに付着させ、乾燥後、混合ガスでの暴露だけを行わず、実施例１２～１５と同様に注射用水を用いて回収した際は、LPS量の設定に対し、６５％程度の回収が出来ていることを確認した。 で は In Examples 12 to 15, the LPS aqueous solution was attached to a PVDF filter (0.4 μm diameter) and air-dried and used as a sample. The model is a model in which LPS spreads over the entire film and spreads thinner than a pellet-like one. It was clearly shown that the decomposition efficiency was higher than that of the pellet. When adhered to a PVDF filter in the same state, dried, and then only exposed to a mixed gas, and collected using water for injection in the same manner as in Examples 12 to 15, when the amount of LPS was set, it was about 65%. Was confirmed to be collected.
　また、実施例１６～１８は、LPS標準バイアル（ENDOSAFE(ENDOTOXIN INDICATOR; Catalog #EVV2K, コード番号513-87082)）を開封しキャップを外した状態でそのまま用いて混合ガスに暴露させ、その後注射用水を用いて所定の濃度に希釈し測定した。実施例１９は、実施例１０，１１と同様でペレット状のLPS付着をさせたものであり、実施例１９と同時に同じ手法で作成したサンプルをγ線２５ｋGy照射したときのLPS残存量の値が０．６６EUであるのに対し、０．３３EUとなり更に低下していることが示された。本方法を用いれば、γ線照射に匹敵するLPS分解能を身近に得られるため、産業利用価値が大きいことが分かった。 In Examples 16 and 18, the LPS standard vial (ENDOSAFE (ENDOTOXIN INDICATOR; Catalog # EVV2K, code number 513-87082)) was opened and the cap was removed. And then diluted to a predetermined concentration. Example 19 is similar to Examples 10 and 11, in which LPS in the form of pellets was adhered. The value of the residual amount of LPS when a sample prepared by the same method as in Example 19 was irradiated with 25 kGy of γ-rays was obtained. It was 0.33 EU compared to 0.66 EU, indicating a further decline. By using this method, LPS resolution comparable to that of γ-ray irradiation can be obtained close to the user, which proves to be of great industrial use value.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
［７．比較例３～４：容器付着混合ガス、ホルムアルデヒドのみによるLPS分解］（表５に記載、表中の左から４列目のLPS量とは、分解前のLPS量のことであり、右から２列目の曝露後LPS量との比較によって、LPS分解の程度が分かる。）
　混合ガスに暴露した際、容器に付着した微量の成分がLPS分解を起こしている可能性があるため、容器のみに気体での暴露をおこなった後、LPSを添加して６０分間反応させた後、残存LPSを測定したところ、分解に対して影響はないことがわかった。また、ギ酸を加えない状況での曝露をおこなったところ、表５に示されているとおり、これもほとんどLPS分解は起こっていないことが分かった。 [7. Comparative Examples 3 and 4: LPS Decomposition Only by Mixed Gas Adhered to Container and Formaldehyde] (Listed in Table 5, the amount of LPS in the fourth column from the left in the table is the amount of LPS before decomposition, and the amount of LPS before decomposition is 2 from the right. Comparison with the post-exposure LPS levels in the rows indicates the extent of LPS degradation.)
When exposed to the mixed gas, trace components adhering to the container may have caused LPS decomposition, so after performing exposure to gas only to the container, adding LPS and reacting for 60 minutes When the residual LPS was measured, it was found that there was no effect on the decomposition. In addition, when exposure was performed in a state in which formic acid was not added, as shown in Table 5, it was found that almost no LPS decomposition occurred.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　なお、上記のように本発明の一実施形態および各実施例について詳細に説明したが、本発明の新規事項および効果から実体的に逸脱しない多くの変形が可能であることは、当業者には、容易に理解できるであろう。したがって、このような変形例は、全て本発明の範囲に含まれるものとする。 Although one embodiment and each example of the present invention have been described in detail as described above, it will be apparent to those skilled in the art that many modifications that do not substantially depart from the novel matter and effects of the present invention are possible. , Will be easy to understand. Therefore, such modified examples are all included in the scope of the present invention.
　例えば、明細書または図面において、少なくとも一度、より広義または同義な異なる用語と共に記載された用語は、明細書または図面のいかなる箇所においても、その異なる用語に置き換えることができる。また、滅菌・核酸分解用組成物の構成も本発明の一実施形態および各実施例で説明したものに限定されず、種々の変形実施が可能である。 For example, in the specification or the drawings, a term described at least once together with a broader or synonymous different term can be replaced with the different term in any part of the specification or the drawing. Further, the configuration of the composition for sterilization / nucleic acid decomposition is not limited to those described in the embodiment and each example of the present invention, and various modifications can be made.
　本発明の一実施形態に係る滅菌・核酸分解用組成物は、高度先端的医療（細胞治療、遺伝子治療、再生医療）分野や海洋研究分野、航空宇宙分野の他、危機管理分野（防衛、消防、警察等）、医療、介護等におけるＤＮＡ・ＲＮＡフリー（バイオ系核酸レベルのコンタミネーションの除去・除染）、LPSフリーを必要とする分野や効果効能レベルのコントロールによって滅菌、殺菌、除菌の分野への適用が可能である。 The composition for sterilization / nucleic acid degradation according to one embodiment of the present invention can be used in the fields of advanced medical treatment (cell therapy, gene therapy, regenerative medicine), marine research, aerospace, and crisis management (defense, firefighting). DNA / RNA-free (removal and decontamination of bio-nucleic acid level contamination) in medical care and nursing care, etc. Application to the field is possible.

Claims (7)

  1. 　核酸の分解と、滅菌することが可能な液体状態の滅菌・核酸分解用組成物であって、
    　少なくともホルムアルデヒドとギ酸を含有し、
    　前記ホルムアルデヒドの濃度が１０重量％以下であり、かつ、前記ギ酸の濃度が１重量％以下である滅菌・核酸分解用組成物。     Decomposition of nucleic acid, a composition for sterilization and nucleic acid decomposition in a liquid state capable of sterilization,
    Contains at least formaldehyde and formic acid,
    A composition for sterilization and nucleic acid decomposition, wherein the concentration of the formaldehyde is 10% by weight or less and the concentration of the formic acid is 1% by weight or less.
  2. 　更に、メタノールを含み、
    　前記メタノール、前記ホルムアルデヒド、前記ギ酸の誘導体を任意に含む請求項１に記載の滅菌・核酸分解用組成物。     In addition, it contains methanol,
    The sterilizing / nucleic acid decomposition composition according to claim 1, wherein the composition optionally contains the methanol, the formaldehyde, and the formic acid derivative.
  3. 　さらにLPSを分解できることを特徴とする請求項１又は請求項２に記載の滅菌・核酸分解用組成物。 (3) The sterilizing / nucleic acid decomposition composition according to (1) or (2), wherein LPS can be further decomposed.
  4. 　核酸の分解と、滅菌することが可能な気体状態の滅菌・核酸分解用組成物であって、少なくともホルムアルデヒドとギ酸を含有する滅菌・核酸分解用組成物。 (4) A sterilizing / nucleic acid decomposing composition in a gaseous state capable of decomposing nucleic acids and sterilizing, wherein the composition contains at least formaldehyde and formic acid.
  5. 　メタノール、前記ホルムアルデヒド、前記ギ酸、一酸化炭素、二酸化炭素、水素、酸素及び／又はこれらのラジカルを含む請求項３に記載の滅菌・核酸分解用組成物。 4. The sterilizing / nucleic acid decomposition composition according to claim 3, wherein the composition contains methanol, the formaldehyde, the formic acid, carbon monoxide, carbon dioxide, hydrogen, oxygen, and / or a radical thereof.
  6. 　ホルムアルデヒドの濃度が５００ｐｐｍ以下でありかつギ酸の濃度が、４００ｐｐｍ以下であることを特徴とする請求項４又は請求項５に記載の滅菌・核酸分解用組成物。 The composition for sterilization and nucleic acid decomposition according to claim 4 or 5, wherein the concentration of formaldehyde is 500 ppm or less and the concentration of formic acid is 400 ppm or less.
  7. 　さらにLPSを分解できることを特徴とする請求項４乃至請求項６記載の滅菌・核酸分解用組成物。 7. The sterilizing / nucleic acid decomposition composition according to claim 4, wherein LPS can be further decomposed.
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