Sterilization indicator

Abstract

Provided is a sterilization indicator that does not require complicated culture operations such as BI or luciferin-luciferase assays such as EI, can be stably supplied at low cost, and makes it possible to accurately measure sterilization intensity in a dry state. The sterilization indicator comprises a base material and a sensitive member that is supported by the base material and is sensitive to a sterilizing agent. The sensitive member is a material having a fluorescent protein as a constituent element, and employs genetically modified fluorescent silk. This genetically modified fluorescent silk emits fluorescence having a prescribed wavelength in response to irradiation of excitation light having a prescribed wavelength. The fluorescence intensity of the sensitive member changes in proportion to the exposure intensity of the sterilizing agent with respect to the sensitive member.

Description

Sterilization Indicator

[0001] The present invention relates to a sterilization indicator used for confirming the sterilization effect in a clean room, a pharmaceutical manufacturing apparatus, a medical instrument, etc. that are sterilized using a sterilizing agent.

[0002] In a manufacturing site for manufacturing pharmaceuticals or foods, or in a medical site such as an operating room, it is important to maintain the aseptic state of the room and the equipment used. Especially in the sterilization of a clean room, which is a working room for pharmaceutical production (also referred to as "decontamination", hereinafter unified as "sterilization"), it is necessary to complete a high-level sterilization validation in accordance with GMP (Good Manufacturing Practice).

[0003] In recent years, hydrogen peroxide has been widely adopted for sterilizing working rooms such as clean rooms (hereinafter referred to as "sterilization target rooms"). This hydrogen peroxide has a strong sterilization effect, is inexpensive and easily available, and is effective as an environmentally friendly sterilizing gas that finally decomposes into oxygen and water.

[0004] Conventionally, and even currently, the mainstream method is to heat and evaporate hydrogen peroxide water inside the sterilization target room to generate hydrogen peroxide gas. This method is called the "flash evaporation sterilization method". In this method, hydrogen peroxide water of, for example, 30 - 35 W / V% is supplied from the outside to the inside of the sterilization target room and heated by a high-temperature evaporation device provided inside the sterilization target room to generate hydrogen peroxide gas and water vapor. Then, the air inside the sterilization target room is circulated to fill the room with hydrogen peroxide gas.

[0005] On the other hand, in recent years, in addition to reducing the usage amount of hydrogen peroxide water, shortening the sterilization cycle time, and reducing the residual gas concentration after sterilization, which are the problems of the flash evaporation sterilization method, a sterilization method focusing on the condensation film of hydrogen peroxide water has been adopted. This method is called the "mist injection sterilization method". In this method, normal-temperature hydrogen peroxide water and compressed air are made into hydrogen peroxide water mist by a two-fluid nozzle or the like and supplied to the inside of the sterilization target room, and fine hydrogen peroxide mist is circulated inside the sterilization target room using ultrasonic vibration or the like.

[0006] In these "flash evaporation sterilization" and "mist injection sterilization" methods using hydrogen peroxide, the concentration of hydrogen peroxide gas in the room to be sterilized is detected as a parameter of sterilization effectiveness. However, since the parameters for managing sterilization conditions are diverse, including the concentration of the hydrogen peroxide condensate film, hydrogen peroxide gas concentration, temperature, and humidity, various parameters and hydrogen peroxide gas concentrations are treated only as reference values. Therefore, it is necessary to confirm the sterilization effectiveness (also called "sterilization strength") of the room to be sterilized using an accurate method.

[0007] Therefore, sterilization indicators are placed in key locations within the sterilization room to evaluate the distribution of sterilization intensity. Conventional sterilization indicators are called biological indicators (BI), and they utilize biological indicators made using spores of microorganisms resistant to specific sterilization methods. These BIs can directly evaluate the lethality of spores after sterilization. However, this method requires culturing the BI spores after sterilization, which is time-consuming and requires significant effort to obtain evaluation results. Furthermore, it only provides positive or negative information, making it impossible to evaluate the detailed distribution of sterilization intensity. There was also the concern of microbial contamination.

[0008] Therefore, it can be considered a type of biological indicator (BI), but in recent years, enzyme indicators (EIs) have come into use as a method to complement BIs. For example, Patent Document 1 below discloses a biological indicator using heat-stable adenylate kinase, a type of phosphorylation enzyme (although the invention is titled BI, its content can be described as an EI). This EI is a patented technology developed by the Health Protection Agency (HPA), which was an agency of the British government.

[0009] Patent No. 4774039

[0010] This technology measures residual enzyme activity after sterilization by measuring the luminescence intensity using a luciferin-luciferase assay. The inactivation of thermostable adenylate kinase (tAK) coated on an EI correlates with the amount of sterilizing agent exposure (volume × time), and the sterilization strength is evaluated by measuring the residual enzyme activity of tAK. Compared to conventional BI, it eliminates the need for culture procedures and allows for measurement of sterilization strength in a relatively short time. In recent years, its comparative equivalence with BI has been confirmed, and its adoption is progressing. Specifically, the LRD value (Log Spore Reduction) is calculated from the luminescence intensity of the EI after sterilization, based on the logarithmic decrease in bacterial count, and the sterilization strength distribution inside the sterilization target room is evaluated.

[0011] However, this EI luciferin-luciferase assay also requires adding ADP (ATP-producing substrate) to sterilized EI, reacting it in a wet state, and measuring the luminescence intensity with a luminometer to determine the enzyme activity of adenylate kinase. While this method is a common method for measuring enzyme activity, the coefficient of variation (CV value) of the measured values ​​is said to be around 5-15%, raising concerns about measurement accuracy. Furthermore, there are still questions regarding the price and stable supply of EI enzyme. Additionally, the fact that the luminescence reaction is measured in a wet state makes the procedure cumbersome, which is another challenge.

[0012] Therefore, the present invention aims to address the above-mentioned problems by providing a sterilization indicator that does not require complicated culture operations such as BI or wet operations such as luciferin-luciferase assays such as EI, can be supplied stably at low cost, and can accurately measure sterilization strength in a dry state.

[0013] In order to solve the above problems, the inventors of this invention, through diligent research, focused on genetically modified fluorescent silk that emits fluorescence while remaining as fibers, and found that the fluorescence intensity of said genetically modified fluorescent silk correlates with the amount of exposure to the sterilizing agent (volume × time), leading to the completion of the present invention.

[0014] That is, according to claim 1, the sterilization indicator according to the present invention is a sterilization indicator used to confirm the sterilization effect on an object to be sterilized using a sterilizing agent, comprising a base material and a sensitive member supported on the base material that is sensitive to the sterilizing agent, wherein the sensitive member is a material composed of a fluorescent protein and emits fluorescence of a predetermined wavelength when irradiated with excitation light of a predetermined wavelength, and the fluorescence intensity of the sensitive member changes in proportion to the exposure intensity of the sterilizing agent to the sensitive member.

[0015] Furthermore, according to claim 2, the present invention is a sterilization indicator according to claim 1, characterized in that the material comprising the fluorescent protein is genetically modified fluorescent silk produced by genetically modified silkworms, or a mixed material of said genetically modified fluorescent silk and other materials.

[0016] Furthermore, according to claim 3, the present invention is a sterilization indicator according to claim 2, characterized in that the genetically modified fluorescent silk is fluorescent silk into which the genetic information of one or more fluorescent proteins has been incorporated by a genetic modification operation.

[0017] Furthermore, according to claim 4, the present invention is a sterilization indicator according to claim 3, characterized in that the fluorescent protein is GFP, mAG, and analogs thereof.

[0018] Furthermore, according to claim 5, the present invention is a sterilization indicator according to any one of claims 2 to 4, characterized in that the sensitive member is a member that uses all or part of a material derived from genetically modified silk, including cotton-like, fibrous, film-like, yarn-like, nonwoven fabric-like, woven, knitted, powder-like, and cocoon-removing sheets.

[0019] Furthermore, according to claim 6, the present invention is a sterilization indicator according to claim 5, characterized in that the sensitive member supported on the substrate is covered with a breathable film on its surface.

[0020] According to the above configuration, the sterilization indicator according to the present invention is used to confirm the sterilization effect on an object to be sterilized using a sterilizing agent. The sterilization indicator comprises a substrate and a sensitive member supported on the substrate that is sensitive to the sterilizing agent. The sensitive member is a material composed of a fluorescent protein and emits fluorescence of a predetermined wavelength when irradiated with excitation light of a predetermined wavelength. Furthermore, the fluorescence intensity of the sensitive member changes in proportion to the exposure intensity of the sterilizing agent to the sensitive member.

[0021] This makes it possible to provide a sterility indicator that can be supplied stably at low cost without requiring complicated culture procedures like BI or wet procedures like luciferin-luciferase assays like EI, and that can accurately measure sterility strength in a dry state.

[0022] Furthermore, according to the above configuration, the material containing fluorescent proteins is either genetically modified fluorescent silk produced by genetically modified silkworms, or a mixture of such genetically modified fluorescent silk and other materials. This allows the above effects to be exhibited more concretely.

[0023] Furthermore, according to the above configuration, genetically modified fluorescent silk is fluorescent silk into which the genetic information of one or more fluorescent proteins has been incorporated through genetic recombination. This allows the above-mentioned effects to be exhibited more concretely.

[0024] Furthermore, according to the above configuration, the fluorescent protein may be GFP, mAG, or analogs of these fluorescent proteins. This allows the above effects to be exhibited more concretely.

[0025] Furthermore, according to the above configuration, the sensing member may be a member that uses, in whole or in part, a material derived from genetically modified silk, including cotton-like, fibrous, film-like, yarn-like, nonwoven fabric-like, woven, knitted, powder-like, and cocoon-removing sheets. This allows the above effects to be exhibited more concretely.

[0026] Furthermore, according to the above configuration, the sensitive member supported on the substrate may have its surface covered with a breathable film. This allows the above effects to be exhibited more concretely.

[0027] (A) is a plan view and (B) is a front view showing the appearance of the fluorescent silk indicator according to this embodiment. This is a graph showing the change in fluorescence intensity retention rate: FD% with respect to the sterilization time: t of the FSI. This is a graph showing the change in fluorescence intensity index: Ln(FD0 / FDt) with respect to the exposure time: t of the FSI. This is a graph showing the change in emission intensity: EI-RLUt with respect to the exposure time: t of the EI. This is a graph showing the change in sterilization strength: EI-LRDt with respect to the exposure time: t of the EI. This is a graph showing the correlation between EI-LRD and Ln(FD0 / FDt) of the FSI.

[0028] First, the "genetically modified fluorescent silk" that constitutes the sensitive component of the sterilization indicator according to the present invention will be described. Genetically modified fluorescent silk (hereinafter simply referred to as "fluorescent silk") is silk produced by genetically modified silkworms, and is a material that emits fluorescence at a predetermined wavelength when irradiated with excitation light of a predetermined wavelength. These genetically modified silkworms are produced by introducing the gene for a fluorescent protein into silkworm eggs, raising the silkworms that hatch from the eggs to adulthood, mating them, and then selecting and raising eggs from which the gene for the fluorescent protein has been introduced.

[0029] Genetically modified silkworms originated with the "Hikaru Kaiko" (glowing silkworm), first successfully developed in 2000 by the Japan Ministry of Agriculture, Forestry and Fisheries' National Institute of Sericultural and Entomological Sciences (now the National Agriculture and Food Research Organization (NARO)) (T. Tamura, et al.: Nat. Biotechnol. 18, 81 (2000)). Currently, basic and applied research on genetically modified silkworms is being conducted using the silkworm genome information deciphered in 2008.

[0030] More than 50 types of fluorescent proteins with different excitation and emission wavelengths have been identified for use in genetically modified silkworms. In this invention, GFP (Green Fluorescent Protein), mAG (monomeric Azami-Green), and their analogs can be used. For example, EGFP (Enhanced Green Fluorescent Protein) is a variant of GFP and is said to have been developed to improve its fluorescence properties. In this invention, GFP and EGFP are not clearly distinguished and are referred to simply as "GFP".

[0031] Furthermore, the present invention is not limited to GFP, mAG, and their analogues, but can utilize a wide range of fluorescent proteins, including those that may be identified in the future. It should also be noted that the fluorescent proteins introduced into silkworms are not limited to one type, but several types may be introduced.

[0032] The sterilization indicator according to the present invention will be described in detail below with reference to embodiments. However, the present invention is not limited to the embodiments described below. In this embodiment, the sterilization indicator according to the present invention is referred to as a "fluorescent silk indicator (FSI)".

[0033] First, the sterilizing agent used in this embodiment will be described. In this embodiment, gaseous or mist-like hydrogen peroxide is used as the sterilizing agent. As described above, the inventors focused on fluorescent silk, which emits fluorescence while still in its fibrous state, and found that the fluorescence intensity of the fluorescent silk correlates with the exposure amount (volume × time) of the sterilizing agent, leading to the completion of the present invention. At that time, hydrogen peroxide was used as the sterilizing agent.

[0034] It is believed that the fluorescent protein incorporated into the fluorescent silk was damaged by exposure to hydrogen peroxide, resulting in a decrease in fluorescence intensity. Therefore, in this invention, we believe that it is effective not only with hydrogen peroxide but also with various other sterilization methods. In addition to hydrogen peroxide, it is also believed to be effective with, for example, moist heat sterilization, autoclaving, gamma ray irradiation, electron beam irradiation, and ethylene oxide gas.

[0035] Next, we will describe the objects to be sterilized. In this invention, we consider manufacturing sites for pharmaceuticals or food products, or medical settings such as operating rooms. Examples include, but are not limited to, clean rooms, pharmaceutical manufacturing equipment such as isolators and RABS, machinery and equipment introduced inside isolators, and medical instruments.

[0036] Next, the fluorescent silk indicator in this embodiment will be described. Figure 1 shows the appearance of the fluorescent silk indicator according to this embodiment (A) a plan view and (B) a front view. In Figure 1, the fluorescent silk indicator 10 has fluorescent silk 12 as a sensing member attached to a part of the surface of a film stick 11 which serves as a base material. The material of the film stick 11 is not particularly limited, but in this embodiment, a polymer film (polyolefin film) that is resistant to sterilizing agents is used.

[0037] Fluorescent silk 12 is fluorescent silk produced by genetically modified silkworms. In this embodiment, two types of fluorescent silk are used. One is fluorescent silk into which the fluorescent protein GFP has been introduced (hereinafter referred to as "GFP fluorescent silk"), which emits green fluorescence at approximately 509 nm when excited by light at approximately 488 nm, and is derived from the green fluorescent protein (GFP: Dr. Shimomura et al. were awarded the Nobel Prize in Chemistry in 2008) of the jellyfish Aequorea victoria. The other is fluorescent silk produced by genetically modified silkworms into which the fluorescent protein mAG has been introduced (hereinafter referred to as "mAG fluorescent silk"), which emits green fluorescence at approximately 505 nm when excited by light at approximately 492 nm.

[0038] The shape of the fluorescent silk 12 is not particularly limited, and it is acceptable as long as it uses, in whole or in part, materials derived from genetically modified silk, including cotton-like (unraveled cocoons), fibrous, film-like (raw silk dissolved into a film), thread (raw silk or silk thread), nonwoven fabric, woven fabric, knitted fabric, powder (crushed cocoons or thread), and cocoon release sheet (cocoons peeled into thin flakes).

[0039] Thus, the fluorescent silk 12 is not limited to 100% fluorescent silk, and a blend of other materials may be used to the extent that a decrease in fluorescence intensity is observed. For example, if the fluorescent silk 12 is a woven fabric, the fluorescent silk yarn may be interwoven with ordinary silk yarn or yarn of other fibers.

[0040] Furthermore, the method of bonding the film stick 11 and the fluorescent silk 12 is not particularly limited, and any bonding method that does not affect the sterilizer sensitivity of the fluorescent silk 12 should be adopted. Also, care must be taken when bonding the fluorescent silk 12 to materials that may scatter, such as cotton-like or powdery materials. For example, it is preferable to cover the surface of cotton-like or powdery materials with a breathable film that allows the sterilizer gas or mist to pass through. As the breathable film, for example, a nonwoven fabric made of high-density polyethylene ultrafine fibers, which is also used in medical devices, Tyvek (trademark), or a polymer dialysis membrane that selectively allows substances to pass through depending on the molecular size, such as a cellulose acetate membrane or a polyethersulfone membrane, may be used.

[0041] <<Preparation of Fluorescent Silk Indicators>> The cocoons of GFP fluorescent silk and mAG fluorescent silk were each divided into four sections lengthwise. Using a cutter, the divided cocoon sections were peeled off to prepare approximately three fluorescent silk cocoon flakes for each type. The thickness of the fluorescent silk cocoon flakes was 0.1 to 0.4 mm. These flakes were cut into 1 cm x 1 cm sections. Next, these flakes (1 cm x 1 cm) were attached to the tip of a polyolefin film stick (1 cm x 5 cm, 0.5 mm thick) using double-sided tape to obtain fluorescent silk indicators (GFP-FSI, mAG-FSI). For the obtained GFP-FSI and mAG-FSI, the fluorescence intensity at an excitation wavelength of 495 nm and an emission wavelength of 510 nm was measured using a spectrofluorometer (RF-6000, Shimadzu Corporation), and the fluorescence intensity before the hydrogen peroxide gas exposure test (FD0) was set.

[0042] <<Measurement of Sterilization Strength of FSI and EI>>The sterilization strengths of the GFP-FSI and mAG-FSI prepared above were measured and compared with those of the conventionally used EI (manufactured by Protak Scientific). First, the hydrogen peroxide concentration in the chamber was adjusted to about 200 ppm using a biological indicator resistance evaluator (BIER: Biological Indicator-Evaluator Resistometer, manufactured by Airrex Co., Ltd.).

[0043] Next, five sheets each of GFP-FSI, mAG-FSI, and EI were simultaneously placed in the chamber of the BIER with the hydrogen peroxide concentration adjusted to about 200 ppm, and hydrogen peroxide sterilization was performed by exposing them to hydrogen peroxide gas. Exposure tests were conducted with exposure times of 3, 6, 9, 12, 15, and 18 min. After each exposure test, the sterilized GFP-FSI, mAG-FSI, and EI taken out from the chamber of the BIER were air-dried and then stored in the dark.

[0044] Next, for the EI after the exposure test, luciferin-luciferase reagent and ADP substrate were added, and the luminescence intensity at t minutes after exposure: EI-RLUt was measured using a luminometer (PR2A, manufactured by Protak Scientific). The luminescence intensity: EI-RLUt was the average value of five EI-RLUt at each exposure time. Then, using analysis software (ATENA, manufactured by Protak Scientific), the luminescence intensity: EI-RLUt was converted to the sterilization strength: EI-LRDt. In addition, the luminescence intensity before sterilization: EI-RLU0 was measured using EI before the exposure test separately.

[0045] On the other hand, for the 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 spectrofluorophotometer (RF-6000, manufactured by Shimadzu Corporation), and the fluorescence intensity at t minutes after exposure to hydrogen peroxide gas: FDt was obtained. The fluorescence intensity: FDt was the average value of five FDt at each exposure time.

[0046] <<Change in Fluorescence Intensity after Exposure Test>>From the fluorescence intensity before each exposure test of GFP-FSI and mAG-FSI: FD0 and the fluorescence intensity after exposure for t minutes: FDt, the fluorescence intensity survival rate: FD% of GFP-FSI and mAG-FSI was determined by the following formula (1). FD% = (FDt / FD0) × 100......(1)

[0047] Figure 2 is a graph showing the change in the fluorescence intensity survival rate: FD% according to the exposure time: t of FSI. In Figure 2, it was confirmed by the exposure test of hydrogen peroxide gas that the fluorescence intensity: FDt of GFP-FSI and mAG-FSI decreases with time with respect to the exposure time: t.

[0048] <<Change in Fluorescence Intensity of FSI>>Assuming that the change in the fluorescence intensity of FSI is due to the denaturation of the fluorescent protein by hydrogen peroxide, the following formula (2) was used to evaluate the denaturation reaction. Ln(FDt) = Ln(FD0) - kt kt = Ln(FD0 / FDt)......(2) Here, Ln: natural logarithm, k: reaction rate constant. The reaction process was evaluated.

[0049] Figure 3 is a graph showing the change in the fluorescence intensity index: Ln(FD0 / FDt) according to the exposure time: t of FSI. In Figure 3, it was confirmed by the exposure test of hydrogen peroxide gas that the fluorescence intensity index: Ln(FD0 / FDt) of GFP-FSI and mAG-FSI changes linearly with respect to the exposure time: t. From this, it was suggested that the fluorescence intensity of the fluorescent silk by the exposure test of hydrogen peroxide gas is a simple reaction (protein denaturation) at one stage of the fluorescent silk protein. Also, since the slopes: k of the changes in the fluorescence intensity indices: Ln(FD0 / FDt) of GFP-FSI and mAG-FSI are almost the same, it was suggested that the denaturation reaction of the fluorescent protein is similar for both fluorescent proteins.

[0050] ≪Changes in Luminescence Intensity of EI≫ On the other hand, the change in luciferin-luciferase luminescence intensity of EI due to exposure to hydrogen peroxide gas was confirmed. Figure 4 is a graph showing the change in luminescence intensity: EI-RLUt with respect to exposure time: t. In Figure 4, it was confirmed that the luminescence intensity of EIt: EI-RLUt decreased over time with respect to exposure time: t due to the hydrogen peroxide gas exposure test.

[0051] ≪Changes in Sterilization Intensity of EI≫ Next, the luminescence intensity: EI-RLUt was converted to sterilization intensity: EI-LRDt using analysis software (ATENA). Figure 5 is a graph showing the change in sterilization intensity: EI-LRDt with respect to EI exposure time: t. In Figure 5, it was confirmed that the sterilization intensity of EI: EI-LRDt increased over time with respect to exposure time: t in the hydrogen peroxide gas exposure test. Furthermore, it was confirmed that when EI-LRD was 4 or higher, it showed a nearly linear change. However, when EI-LRD was less than 4, it was suggested that the variability of the measured values ​​increased and linearity was lost.

[0052] <<Correlation between EI sterilization strength and FSI fluorescence intensity index>> Based on the results in Figure 5, the EI-LRD and FSI Ln(FD0 / FDt) are plotted in the region where the EI-LRD is 4 or higher, which is considered to be a highly reliable evaluation of the sterilization strength of EI-LRD. Figure 6 is a graph showing the correlation between EI-LRD and FSI Ln(FD0 / FDt). In Figure 6, there is a nearly linear correlation between EI-LRD and Ln(FD0 / FDt), suggesting that it does not depend on the type of GFP-FSI or mAG-FSI.

[0053] From the above, it can be concluded that in sterilization with hydrogen peroxide, it is possible to determine the change in sterilization intensity over time by determining the fluorescence intensity index of FSI: Ln(FD0 / FDt). Furthermore, it is possible to determine the sterilization intensity distribution from the value of FSI's Ln(FD0 / FDt) within the chamber of a sterile isolator, which is the target of sterilization with hydrogen peroxide.

[0054] As described above, the present invention provides a sterilization indicator that does not require complicated culture procedures such as BI or luciferin-luciferase assays such as EI, can be supplied stably at low cost, and can accurately measure sterilization strength in a dry state.

[0055] Here, we will explain the homogeneity and stable supply of fluorescent silk. Currently, under the guidance of the National Agriculture and Food Research Organization (NARO), a system for producing high-value-added silk by sericulture farmers in specific regions has been established, and production is carried out in an environment that does not affect the biodiversity of wildlife, in accordance with the Cartagena Protocol.

[0056] Because fluorescent proteins are introduced into the genes, the fluorescence emission performance of fluorescent silk produced by genetically modified silkworms is uniform. Furthermore, by controlling the production and lot management of sterilization indicators produced from this uniform fluorescent silk, it is possible to stably supply sterilization indicators whose comparative equivalence to BI (Biochemical Indicator) has been confirmed.

[0057] 10...Fluorescent silk indicator, 11...Film stick, 12...Fluorescent silk.

Claims

1. A sterilization indicator used to confirm the sterilization effect on an object to be sterilized using a sterilizing agent, comprising a base material and a sensitive member supported on the base material that is sensitive to the sterilizing agent, wherein the sensitive member is made of a material composed of a fluorescent protein and emits fluorescence of a predetermined wavelength when irradiated with excitation light of a predetermined wavelength, and the fluorescence intensity of the sensitive member changes in proportion to the exposure intensity of the sterilizing agent to the sensitive member.

2. The sterilization indicator according to claim 1, characterized in that the material comprising the fluorescent protein is genetically modified fluorescent silk produced by genetically modified silkworms, or a mixed material of said genetically modified fluorescent silk and other materials.

3. The sterilization indicator according to claim 2, characterized in that the genetically modified fluorescent silk is silk into which the genetic information of one or more fluorescent proteins has been incorporated by a genetic modification operation.

4. The sterilization indicator according to claim 3, characterized in that the fluorescent protein is GFP or mAG.

5. The sterilization indicator according to any one of claims 2 to 4, characterized in that the sensing member is a member that uses all or part of a material derived from genetically modified silk, including cotton-like, fibrous, film-like, yarn-like, nonwoven fabric-like, woven, knitted, powder-like, and cocoon-removing sheets.

6. The sterilization indicator according to claim 5, characterized in that the sensitive member supported on the substrate is covered with a breathable film on its surface.

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