Sterility indicator

Abstract

The present application provides a sterilization indicator which does not require complicated culture operation like BI, does not require fluorescein-luciferase detection like EI, can be stably supplied at low cost, and can accurately measure sterilization strength in a dry state. The sterilization indicator includes a substrate and a sensing member carried by the substrate and sensing the sterilization agent. The sensing member is a material including a fluorescent protein as a constituent element, and uses a genetically recombined fluorescent filament. The genetically recombined fluorescent filament emits fluorescent light of a predetermined wavelength by irradiation of excitation light of a predetermined wavelength. The fluorescent intensity of the sensing member changes in proportion to the exposure intensity of the sterilization agent with respect to the sensing member.

Description

Technical Field

[0001] This invention relates to sterilization indicators used to confirm the sterilization effect in clean rooms, pharmaceutical manufacturing facilities, medical devices, and the like, where sterilization is performed using sterilizing agents. Background Technology

[0002] Maintaining a sterile environment in manufacturing sites for pharmaceuticals or food, or in medical settings such as operating rooms, is crucial. In particular, sterilization (also known as "decontamination," but referred to as "sterilization" below) in pharmaceutical manufacturing workshops, requiring a high level of sterilization validation in accordance with GMP (Good Manufacturing Practice).

[0003] In recent years, hydrogen peroxide has been widely used in the sterilization of sterile rooms and other work areas (hereinafter referred to as "sterilization target rooms"). Hydrogen peroxide has a strong sterilization effect, is inexpensive and readily available, and is effective as an environmentally friendly sterilizing gas that ultimately decomposes into oxygen and water.

[0004] Historically, the mainstream method involves heating and evaporating hydrogen peroxide water inside the sterilization chamber to generate hydrogen peroxide gas. This method is known as "flash sterilization." In this method, hydrogen peroxide water at a concentration of, for example, 30 W / V% to 35 W / V% is supplied from outside the sterilization chamber. This water is then heated using a high-temperature evaporator located inside the chamber, producing hydrogen peroxide gas and water vapor. The air inside the sterilization chamber is then circulated to fill the chamber with hydrogen peroxide gas.

[0005] On the other hand, in recent years, in addition to reducing the amount of hydrogen peroxide water used, shortening the sterilization cycle time, and reducing the residual gas concentration after sterilization as issues related to flash sterilization, a sterilization method focusing on the condensation film of hydrogen peroxide water has also been adopted. This method is called "mist injection sterilization". In this method, room temperature hydrogen peroxide water and compressed air are mixed using a two-fluid nozzle or the like to form a hydrogen peroxide water mist, which is then supplied to the interior of the sterilization chamber. Ultrasonic vibration or the like is used to circulate the fine hydrogen peroxide water mist within the sterilization chamber.

[0006] In these "flash sterilization" and "mist sterilization" methods using hydrogen peroxide water, the concentration of hydrogen peroxide gas in the sterilization chamber is used as a parameter to measure the sterilization effect. However, the parameters for managing sterilization conditions involve many aspects, including the concentration of hydrogen peroxide condensate, hydrogen peroxide gas concentration, temperature, and humidity. Therefore, these parameters and the hydrogen peroxide gas concentration remain as reference values. Thus, it is necessary to confirm the sterilization effect (also known as "sterilization intensity") of the sterilization chamber using an accurate method.

[0007] Therefore, sterilization indicators are placed in key areas inside the sterilization chamber to evaluate the distribution of sterilization intensity. Previous sterilization indicators were called biological indicators (BIs), which are biological markers created using spores of microorganisms resistant to specific sterilization methods. BIs can directly evaluate the lethality of spores after sterilization. However, spore culture of BIs is required after sterilization, which presents problems of time and effort required to obtain evaluation results, and because only positive and negative information can be obtained, a detailed evaluation of sterilization intensity distribution is not possible. Furthermore, there are concerns about residual microbial contamination.

[0008] Therefore, it can also be understood as a type of BI, but as a supplementary method to BI, enzyme indicators (EI: Enzyme Indicator) have been used in recent years. For example, in the following Patent Document 1, a biological indicator using a thermostable adenosine kinase, a phosphorylase, is disclosed (the invention is named BI, but its content can be said to be EI). This EI is a patented technology developed by HPA (Health Protection Agency), an agency of the UK government.

[0009] [Existing Technical Documents]

[0010] [Patent Literature]

[0011] [Patent Document 1] Japanese Patent No. 4774039 Summary of the Invention

[0012] [The problem the invention aims to solve]

[0013] This technology measures residual enzyme activity after sterilization by using luciferase-based luminescence intensity. The inactivation of thermostable adenosine monokinase (tAK) coated on the eI (external incubator) is correlated with the amount of sterilizing agent exposure (volume × time), and the residual enzyme activity of tAK is used to evaluate sterilization intensity. Compared to conventional BI (biological incubation), this method eliminates the need for incubation and allows for determination of sterilization intensity in a shorter time. In recent years, its comparability with BI has been confirmed, and it is gaining wider adoption. Specifically, the distribution of sterilization intensity within the sterilized chamber is evaluated by calculating the Log Spore Reduction (LRD) value based on the logarithmic reduction of bacterial count from the luminescence intensity of the sterilized eI.

[0014] However, the luciferin-luciferase assay using this EI also requires the following steps: ADP (an ATP-producing substrate) is added to the sterilized EI, the reaction is initiated using a wet process, and the luminescence intensity is measured using a luminometer to determine the adenylate kinase activity. While this method is generally used for determining enzyme activity, the coefficient of variation (CV) of the measured values ​​is reportedly around 5%–15%, raising concerns about accuracy. Furthermore, the price and stable supply of enzymes in EIs remain uncertain. Additionally, the complex procedure of measuring the luminescence reaction under wet conditions also presents challenges.

[0015] Therefore, the purpose of this invention is to address the above-mentioned problems by providing a sterilization indicator that does not require the complex culture operations of BI or the wet operations such as luciferase-luciferase detection of EI, can be supplied stably at low cost, and can accurately measure sterilization intensity in a dry state.

[0016] [Methods used to solve problems]

[0017] To address the aforementioned issues, the inventors conducted in-depth research and, focusing on recombinant fluorescent filaments that emit fluorescence in a fibrous state, discovered that the fluorescence intensity of these recombinant fluorescent filaments is related to the amount of sterilizing agent exposed (capacity × time), thus completing this invention.

[0018] That is, according to the description of technical solution 1, the sterilization indicator of the present invention is a sterilization indicator used to confirm the sterilization effect in a sterilized object being sterilized using a sterilizing agent. It is characterized by having a substrate and a sensing element supported by the substrate that senses the sterilizing agent. The sensing element is made of a material with fluorescent protein as a constituent element, and is a material that emits fluorescence of a specified wavelength by irradiation with excitation light of a specified wavelength. The fluorescence intensity of the sensing element varies proportionally with the exposure intensity of the sterilizing agent relative to the sensing element.

[0019] Furthermore, according to the description of technical solution 2, the present invention is the sterilization indicator described in technical solution 1, characterized in that the material with the above-mentioned fluorescent protein as a constituent element is a recombinant fluorescent filament produced by a recombinant silkworm, or a mixture of the recombinant fluorescent filament and other materials.

[0020] Furthermore, according to the description in technical solution 3, the present invention is the sterilization indicator described in technical solution 2, characterized in that the gene recombination fluorescent filament is a fluorescent filament in which the genetic information of one or more fluorescent proteins is integrated through gene recombination operation.

[0021] Furthermore, according to the description in technical solution 4, the present invention is the sterilization indicator described in technical solution 3, characterized in that the aforementioned fluorescent protein is GFP, mAG, and analogs of these fluorescent proteins.

[0022] Furthermore, according to the description of technical solution 5, the present invention is a sterilization indicator as described in any one of technical solutions 2 to 4, characterized in that the sensing component is a component that uses material derived from the recombinant filament in whole or in part, and the material derived from the recombinant filament includes cotton-like, fibrous, membrane-like, filament, non-woven fabric, woven fabric, powder, and cocoon peeling sheet.

[0023] Furthermore, according to technical solution 6, the present invention is the sterilization indicator described in technical solution 5, characterized in that the surface of the sensing component supported by the substrate is covered by a breathable film.

[0024] [Invention Effects]

[0025] Based on the above configuration, the sterilization indicator of the present invention is used to confirm the sterilization effect of an object to be sterilized using a sterilizing agent. Furthermore, the sterilization indicator includes a substrate and a sensing element supported on the substrate that senses the sterilizing agent. The sensing element is made of a fluorescent protein and emits fluorescence of a predetermined wavelength when exposed to excitation light of a predetermined wavelength. Additionally, the fluorescence intensity of the sensing element varies proportionally to the exposure intensity of the sterilizing agent relative to the sensing element.

[0026] Therefore, a sterilization indicator can be provided that does not require the complicated culture operations of BI or the wet operations such as luciferase detection of EI, can be supplied stably at low cost, and can accurately measure the sterilization intensity in a dry state.

[0027] Based on the above composition, the material with fluorescent protein as its constituent element is a recombinant fluorescent filament produced by a recombinant silkworm, or a mixture of the recombinant fluorescent filament and other materials. This allows for a more specific exertion of the aforementioned effects.

[0028] Furthermore, based on the above structure, the gene recombination fluorescent filament is a fluorescent filament that integrates the genetic information of one or more fluorescent proteins through gene recombination. Therefore, it can more specifically exert the aforementioned effects.

[0029] Furthermore, based on the above composition, the fluorescent protein can be GFP, mAG, or analogues of these fluorescent proteins. This allows for a more specific exertion of the aforementioned effects.

[0030] Furthermore, according to the above configuration, the sensing component can be a component that uses a material derived from recombinant genetic fibers, either wholly or partially. This material includes cotton-like, fibrous, membrane-like, filamentous, nonwoven, woven, woven fabric, powder, and cocoon peeling sheets. This allows for a more specific application of the aforementioned effects.

[0031] Furthermore, according to the above configuration, the surface of the sensing component supported by the substrate can be covered with a breathable film. This allows the aforementioned effects to be performed more specifically. Attached Figure Description

[0032] Figure 1 (A) is a top view showing the appearance of the fluorescent filament indicator of this embodiment. Figure 1 (B) is a front view showing the appearance of the fluorescent filament indicator of this embodiment.

[0033] Figure 2 This is a graph showing the change in fluorescence intensity retention (FD%) over FSI-based sterilization time (t).

[0034] Figure 3 This is a graph showing the change in fluorescence intensity index Ln(FD0 / FDt) based on FSI exposure time: t.

[0035] Figure 4 This is a graph showing the change in luminescence intensity (EI-RLUt) based on exposure time (t) at EI.

[0036] Figure 5 This is a graph showing the variation of sterilization intensity: EI-LRDt based on exposure time (t) at EI.

[0037] Figure 6 This is a graph showing the correlation between EI-LRD and FSI's Ln(FD0 / FDt). Detailed Implementation

[0038] First, the "recombinant fluorescent filament" constituting the sensing component of the sterilization indicator of the present invention will be described. The recombinant fluorescent filament (hereinafter referred to as "fluorescent filament") refers to the silk (silk) produced by recombinant silkworms, a material that emits fluorescence of a specified wavelength when irradiated with excitation light of a specified wavelength. This recombinant silkworm is obtained by introducing the gene for a fluorescent protein into the silkworm egg, raising the silkworms from the eggs to adults, and selecting eggs containing the gene for fluorescent protein from the eggs obtained through mating.

[0039] The recombinant silkworm originated from the "luminescent silkworm" first successfully developed in 2000 by the Sericulture and Insect Agricultural Technology Research Institute of the Ministry of Agriculture, Forestry and Fisheries of Japan (now the National Research and Development Agency for Agriculture, Food and Rural Development (NRA)) (T. Tamura, et al.: Nat. Biotechnol. 18, 81 (2000)). Currently, basic and applied research on recombinant silkworms has been conducted using the silkworm genome information deciphered in 2008.

[0040] More than 50 proteins with different excitation and fluorescence wavelengths were identified among the fluorescent proteins introduced into the recombinant silkworm gene. In this invention, GFP (Green Fluorescent Protein), mAG (monomeric Azami-Green), and their analogues can be utilized. For example, EGFP (Enhanced Green Fluorescent Protein) is a mutant of GFP developed to improve fluorescence properties. Furthermore, in this invention, GFP and EGFP are not explicitly distinguished, and the term is used interchangeably with "GFP".

[0041] Furthermore, this invention is not limited to GFP, mAG, and their analogues, but also includes specific substances to be used in the future, enabling the wide utilization of fluorescent proteins. Additionally, the fluorescent protein introduced into the silkworm is not limited to one type; multiple types can be introduced.

[0042] The sterilization indicator of the present invention will now be described in detail through embodiments. It should be noted that the present invention is not limited to the embodiments described below. Furthermore, in this embodiment, the sterilization indicator of the present invention will be referred to as a "fluorescent silk indicator (FSI)".

[0043] First, the sterilizing agent used in this embodiment will be described. In this embodiment, hydrogen peroxide in gaseous or mist form is used as the sterilizing agent. As described above, the inventors focused on fluorescent filaments that emit fluorescence in their fibrous state, and discovered that the fluorescence intensity of these filaments is related to the amount of sterilizing agent exposed (volume × time), thus completing this invention. In this case, hydrogen peroxide is used as the sterilizing agent.

[0044] It is believed that fluorescent proteins integrated into fluorescent filaments are damaged by exposure to hydrogen peroxide, resulting in decreased fluorescence intensity. Therefore, this invention is not limited to hydrogen peroxide and is considered effective in various sterilization methods. It is believed that methods other than hydrogen peroxide, such as moist heat sterilization, autoclaving, gamma ray irradiation, electron beam irradiation, and ethylene oxide gas, are also effective.

[0045] Next, the object to be sterilized will be described. In this invention, manufacturing sites for pharmaceuticals or food, or medical sites such as operating rooms, are considered, and examples include clean rooms, isolators, pharmaceutical manufacturing equipment such as RABS, machinery / instruments introduced into isolators, medical devices, etc., but are not limited to these.

[0046] Next, the fluorescent filament indicator in this embodiment will be described. Figure 1 (A) is a top view showing the appearance of the fluorescent filament indicator of this embodiment. Figure 1 (B) is a front view showing the appearance of the fluorescent filament indicator of this embodiment. Figure 1 In this embodiment, a fluorescent filament indicator 10 has a fluorescent filament 12 attached as a sensing element to a portion of the surface of a film rod 11, which serves as a substrate. The material of the film rod 11 is not particularly limited, but in this embodiment, a polymer film (polyolefin film) resistant to sterilizing agents is used.

[0047] Fluorescent filament 12 is a fluorescent filament produced by the recombinant silkworm. In this embodiment, two types of fluorescent filaments are used. One is a fluorescent filament incorporating the fluorescent protein GFP (hereinafter referred to as "GFP fluorescent filament"), which emits green fluorescence at approximately 509 nm under excitation light of approximately 488 nm. It is derived from the green fluorescent protein of the Victoria multituberculate jellyfish (GFP: Dr. Shimomura et al. won the Nobel Prize in Chemistry in 2008). The other is a fluorescent filament produced by the recombinant silkworm incorporating the fluorescent protein mAG (hereinafter referred to as "mAG fluorescent filament"), which emits green fluorescence at approximately 505 nm under excitation light of approximately 492 nm.

[0048] The shape of the fluorescent filament 12 is not particularly limited, as long as it is made entirely or partially from a material derived from recombinant filaments. The material derived from recombinant filaments includes cotton-like (the shape after the cocoon is spread out), fibrous, membrane-like (the shape after raw silk is dissolved to form a membrane), filament (raw silk or silk filaments), non-woven fabric, fabric, woven material, powder (the shape after the cocoon or filament is crushed), and cocoon peeling sheet (the shape after the cocoon is peeled off in a thin sheet).

[0049] Thus, the fluorescent filament 12 is not limited to 100% fluorescent filament. Within the limits of fluorescence intensity attenuation, it can also be a mixture of other materials. For example, if the fluorescent filament 12 is a fabric, the fluorescent filament can be interwoven with ordinary silk filaments or filaments of other fibers.

[0050] Furthermore, there are no particular limitations on the bonding method between the film rod 11 and the fluorescent filament 12, as long as the bonding method does not affect the sterilizing agent sensitivity of the fluorescent filament 12. However, care must be taken when bonding materials such as cotton or powder, where some may disperse. For example, it is preferable to cover the surface of the cotton or powder with a breathable film that allows the sterilizing agent gas or mist to pass through. As a breathable film, for example, nonwoven fabrics made of high-density polyethylene microfibers, Teckeck (trademark), or polymeric dialysis membranes that selectively allow substances to permeate based on molecular size, such as cellulose acetate membranes or polyethersulfone membranes, which are also used in medical devices, can be used.

[0051] Fabrication of Fluorescent Filament Indicators

[0052] The cocoons of GFP and mAG fluorescent filaments were longitudinally divided into four parts. Using a scalpel, the divided cocoon pieces were peeled off to prepare approximately three fluorescent filament cocoon sheets of each type. The thickness of the fluorescent filament sheets was 0.1–0.4 mm. These sheets were then cut into 1 cm × 1 cm pieces. Next, double-sided tape was used to attach the 1 cm × 1 cm sheet to the front end of a polyolefin film rod (1 cm × 5 cm, 0.5 mm thick) to obtain the respective fluorescent filament indicators (GFP-FSI, mAG-FSI). For the obtained GFP-FSI and mAG-FSI, the fluorescence intensity at an excitation wavelength of 495 nm and a fluorescence wavelength of 510 nm was measured using a spectrophotometer (RF-6000, manufactured by Shimadzu Corporation). This fluorescence intensity (FD0) was used as the fluorescence intensity before the hydrogen peroxide gas exposure test.

[0053] Determination of Sterilization Strength of FSI and EI

[0054] The sterilization strength of the GFP-FSI and mAG-FSI prepared above was measured and compared with that of the previously used EI (manufactured by Protak Scientific). First, the hydrogen peroxide concentration in the chamber was adjusted to approximately 200 ppm using a Biological Indicator-Evaluator Resistometer (BIER, manufactured by Airex Co., Ltd.).

[0055] Next, five tablets each of GFP-FSI, mAG-FSI, and EI were simultaneously added to the chamber of the BIER, where the hydrogen peroxide concentration was adjusted to approximately 200 ppm, and then sterilized by exposing it to hydrogen peroxide gas. It should be noted that exposure tests were conducted with exposure times of 3 minutes, 6 minutes, 9 minutes, 12 minutes, 15 minutes, and 18 minutes. After each exposure test, the sterilized GFP-FSI, mAG-FSI, and EI tablets were removed from the BIER chamber, air-dried, and stored in the dark.

[0056] Next, for the EI after the exposure test, luciferin-luciferase reagent and ADP substrate were added, and the luminescence intensity (EI-RLUt) was measured after t minutes of exposure using a Luminometer (PR2A, Protak Scientific). It should be noted that the luminescence intensity (EI-RLUt) is the average of five EI-RLUt values ​​at each exposure time. Then, using analysis software (ATENA, Protak Scientific), the luminescence intensity (EI-RLUt) was converted to sterilization intensity (EI-LRDt). Additionally, the luminescence intensity (EI-RLU0) before sterilization was measured separately using the EI before the exposure test.

[0057] On the other hand, for GFP-FSI and mAG-FSI after the exposure test, the fluorescence intensity at an excitation wavelength of 495 nm and a fluorescence wavelength of 510 nm was measured using a spectrophotometer (RF-6000, manufactured by Shimadzu Corporation). This fluorescence intensity was recorded as the fluorescence intensity FDt after t minutes of hydrogen peroxide gas exposure. It should be noted that the fluorescence intensity FDt is the average of the FDt values ​​of five samples at each exposure time.

[0058] Changes in fluorescence intensity after exposure test

[0059] According to the following formula (1), the fluorescence intensity retention rates of GFP-FSI and mAG-FSI before each exposure test: FD0 and the fluorescence intensity after exposure time t minutes: FDt are calculated as follows: FD%. FD%=(FDt / FD0)×100·······(1).

[0060] Figure 2 This is a graph showing the change in fluorescence intensity retention (FD%) based on FSI exposure time: t. Figure 2 In the experiment, the fluorescence intensity of GFP-FSI and mAG-FSI decreased over time relative to the exposure time (t).

[0061] Fluorescence Intensity Changes of FSI

[0062] The inventors hypothesize that the change in fluorescence intensity of FSI is caused by denaturation of the fluorescent protein induced by hydrogen peroxide. To evaluate the denaturation reaction, the reaction process was evaluated using the following equation (2):

[0063] Ln(FDt)=Ln(FD0)-kt

[0064] kt=Ln(FD0 / FDt)·············(2)

[0065] Here, Ln: natural logarithm, k: reaction rate constant.

[0066] Figure 3 This is a graph showing the change in fluorescence intensity index Ln(FD0 / FDt) based on FSI exposure time: t. Figure 3 In this study, exposure experiments with hydrogen peroxide gas confirmed that the fluorescence intensity index (Ln(FD0 / FDt)) of GFP-FSI and mAG-FSI changed linearly with respect to exposure time (t). This suggests that the fluorescence intensity of the fluorescent filament, as determined by hydrogen peroxide gas exposure, represents a simple first-stage reaction (protein denaturation) of the fluorescent filament protein. Furthermore, the slope (k) of the fluorescence intensity index (Ln(FD0 / FDt)) changes for GFP-FSI and mAG-FSI is approximately the same, suggesting that the denaturation reaction of fluorescent proteins may be the same for both.

[0067] Changes in the luminescence intensity of EI

[0068] On the other hand, the changes in luciferin-luciferase luminescence intensity caused by exposure to hydrogen peroxide gas at EI were confirmed. Figure 4 This is a graph showing the change in luminescence intensity (EI-RLUt) based on exposure time (t) at EI. Figure 4 In the experiment, an exposure test with hydrogen peroxide gas confirmed that the luminescence intensity of EIt (EI-RLUt) decreased over time relative to the exposure time (t).

[0069] Changes in Sterilization Intensity of EI

[0070] Next, the luminescence intensity (EI-RLUt) was converted to sterilization intensity (EI-LRDt) using analysis software (ATENA). Figure 5 This is a graph showing the variation of sterilization intensity: EI-LRDt based on exposure time (t) at EI. Figure 5 In this study, an exposure test using hydrogen peroxide gas confirmed that the sterilization intensity of EI (EI-LRDt) increases over time relative to the exposure time (t). Furthermore, it was confirmed that when EI-LRD is above 4, a roughly linear change is observed. However, when EI-LRD is less than 4, the deviation of the measured values ​​increases, suggesting a loss of linearity.

[0071] Correlation between sterilization intensity of EI and fluorescence intensity index of FSI

[0072] according to Figure 5As a result, as an evaluation of the sterilization intensity of EI-LRD, in the region where the EI-LRD is considered to be of high reliability (4 or above), Ln(FD0 / FDt) of EI-LRD and FSI are plotted. Figure 6 This is a graph showing the correlation between EI-LRD and FSI's Ln(FD0 / FDt). Figure 6 The study revealed that EI-LRD and Ln(FD0 / FDt) have a roughly linear correlation, independent of the types of GFP-FSI and mAG-FSI.

[0073] From the above, it can be determined that in sterilization using hydrogen peroxide, the change in sterilization intensity over time can be calculated by determining the fluorescence intensity index of FSI: Ln(FD0 / FDt). Furthermore, within the chamber of a sterile isolator, which is the object being sterilized using hydrogen peroxide, the sterilization intensity distribution can be determined based on the value of Ln(FD0 / FDt) of FSI.

[0074] As described above, according to the present invention, a sterilization indicator can be provided that does not require complex culture operations like BI or luciferase detection like EI, can be supplied stably at low cost, and can accurately measure sterilization intensity in a dry state.

[0075] Here, we will explain the homogeneity and stable supply of fluorescent silk. Currently, the production of fluorescent silk is carried out by silkworm farmers in specific regions under the guidance of the National Research and Development Agency for Agriculture, Food and Rural Development (NARDA), forming a complete high-value silk production system. Based on the Cartagena process, the silk is produced in an environment that does not affect the biodiversity of wild organisms.

[0076] Because fluorescent proteins are introduced into the gene, the fluorescent luminescence properties of the fluorescent filaments produced by the recombinant silkworm are homogeneous. Furthermore, through production and batch management of sterilization indicators produced from homogeneous fluorescent filaments, a stable supply of sterilization indicators with confirmed equivalence to BI (Bio-Induced Sterilization) can be achieved.

[0077] [Explanation of reference numerals in the attached figures]

[0078] 10…Fluorescent filament indicator, 11…Thin film rod, 12…Fluorescent filament.

Claims

1. A sterilization indicator used to confirm the sterilization effect in an object being sterilized using a sterilizing agent, characterized in that, It comprises a substrate and a sensing component supported on the substrate that senses the sterilizing agent. The sensing component is made of a fluorescent protein, a material that emits fluorescence of a specified wavelength when exposed to excitation light of a specified wavelength. The fluorescence intensity of the sensing element varies proportionally to the exposure intensity of the sterilizing agent relative to the sensing element.

2. The sterilization indicator according to claim 1, characterized in that, The material with fluorescent protein as a constituent element is a recombinant fluorescent filament produced by recombinant silkworm or a mixture of the recombinant fluorescent filament and other materials.

3. The sterilization indicator according to claim 2, characterized in that, The recombinant fluorescent filament is a filament that integrates the genetic information of one or more fluorescent proteins through gene recombination.

4. The sterilization indicator according to claim 3, characterized in that, The fluorescent proteins are GFP and mAG.

5. The sterilization indicator according to any one of claims 2 to 4, characterized in that, The sensing component is a component, either wholly or partially, made of a material derived from the recombinant filament, including cotton-like, fibrous, membrane-like, filamentous, nonwoven, woven fabric, woven material, powder, or cocoon peeling sheet.

6. The sterilization indicator according to claim 5, characterized in that, The surface of the sensing element supported by the substrate is covered by a breathable film.

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