Sterilization indicator

Abstract

This invention provides a sterility indicator that does not require complex culture procedures like BI or luciferin-luciferase assays like EI, can be supplied stably at low cost, and can accurately measure sterility intensity in a dry state. [Solution] The device comprises a base material and a sensitive member supported on the base material that is sensitive to the sterilizing agent. The sensitive member is made of a material whose constituent is a fluorescent protein, and genetically modified fluorescent silk is used. This genetically modified fluorescent silk emits fluorescence at a predetermined wavelength when irradiated with excitation light of a predetermined wavelength. The fluorescence intensity of the sensitive member changes in proportion to the exposure intensity of the sterilizing agent to the sensitive member.

Description

Technical Field

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

Background Art

[0002] In a manufacturing site for manufacturing pharmaceuticals or foods, or a medical site such as an operating room, it is important to maintain the aseptic state of the room and the equipment used. In particular, in the sterilization (also referred to as "decontamination", hereinafter unified as "sterilization") of a clean room, which is a working room for pharmaceutical manufacturing, 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 the sterilization of 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 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, for example, 30 to 35 W / V% hydrogen peroxide water is supplied from the outside to the inside of the sterilization target room, heated by a high-temperature evaporation device provided inside the sterilization target room, and hydrogen peroxide gas and water vapor are generated. 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 addressing the challenges of flash evaporation sterilization, such as reducing the amount of hydrogen peroxide used, shortening the sterilization cycle time, and reducing the residual gas concentration after sterilization, sterilization methods that focus on the condensation film of hydrogen peroxide have been adopted. This method is called the "mist injection sterilization method." In this method, hydrogen peroxide at room temperature and compressed air are mixed into a hydrogen peroxide mist using a two-fluid nozzle or similar device and supplied into the sterilization chamber. The fine hydrogen peroxide mist is then circulated inside the chamber using ultrasonic vibrations or similar methods.

[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 (BIs), 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 considerable 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 complementary method to BIs. For example, Patent Document 1 below describes a biological indicator (the invention is titled BI, but its content can be described as an EI) that uses heat-stable adenylate kinase, a type of phosphorylation enzyme. This EI is a patented technology developed by the Health Protection Agency (HPA), which was an agency of the British government. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Patent No. 4774039 [Overview of the Initiative] [Problems that the invention aims to solve]

[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 (electroluminescent barrier) 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 (biopsy barrier), it eliminates the need for culture procedures and allows for measurement of sterilization strength in a relatively short time. Recently, 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 reduction in bacterial count, and the sterilization strength distribution within the sterilization target room is evaluated.

[0011] However, this luciferin-luciferase assay using EI 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. [Means for solving the problem]

[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 the description in 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, The system comprises a base material and a sensitive member supported on the base material that is sensitive to the sterilizing agent, The sensing member is a material whose constituent element is a fluorescent protein, and which emits fluorescence of a predetermined wavelength when irradiated with excitation light of a predetermined wavelength, The fluorescence intensity of the sensing member changes in proportion to the exposure intensity of the sterilizer to the sensing member.

[0015] Furthermore, according to the description in claim 2, the present invention is a sterilization indicator according to claim 1, The material comprising the aforementioned fluorescent protein is characterized by being 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 the description in claim 3, the present invention is a sterilization indicator according to claim 2, The genetically modified fluorescent silk is characterized by being a fluorescent silk into which the genetic information of one or more fluorescent proteins has been incorporated by genetic modification.

[0017] Furthermore, according to claim 4, the present invention is a sterilization indicator according to claim 3, The fluorescent protein is characterized by being GFP, mAG, and analogs of these fluorescent proteins.

[0018] Furthermore, according to claim 5, the present invention is a sterilization indicator according to any one of claims 2 to 4, The sensing member is characterized by being 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 fabric-like, powder-like, and cocoon-release sheets.

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

[0020] According to the above configuration, the sterilization indicator according to the present invention is used to confirm the sterilization effect in an object to be sterilized that is sterilized using a sterilizing agent. Further, the sterilization indicator includes a base material and a sensing member that is supported by the base material and is sensitive to the sterilizing agent. The sensing member is a material that includes a fluorescent protein as a constituent element, and emits fluorescence of a predetermined wavelength when irradiated with excitation light of a predetermined wavelength. Further, the fluorescence intensity of the sensing member changes in proportion to the exposure intensity of the sterilizing agent to the sensing member.

[0021] This enables the provision of a sterilization indicator that does not require complicated culturing operations such as BI or wet operations such as luciferin-luciferase assays like EI, can be stably supplied at low cost, and can accurately measure the sterilization intensity in a dry state.

[0022] Further, according to the above configuration, the material that includes a fluorescent protein as a constituent element is recombinant fluorescent silk produced by recombinant silkworms or a mixed material of the recombinant fluorescent silk and another material. This allows the above-described effects to be more specifically exhibited.

[0023] Further, according to the above configuration, the recombinant fluorescent silk is fluorescent silk into which the genetic information of one or more fluorescent proteins has been incorporated by recombinant operations. This allows the above-described effects to be more specifically exhibited.

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

[0025] Further, according to the above configuration, the sensing member may be a member that uses, in whole or in part, a material derived from recombinant silk including cotton-like, fibrous, film-like, thread, non-woven fabric, woven fabric, knitted fabric, powder, or cocoon peeling sheet. This allows the above-described effects to be more specifically exhibited.

[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. [Brief explanation of the drawing]

[0027] [Figure 1] (A) is a plan view and (B) is a front view showing the appearance of the fluorescent silk indicator according to this embodiment. [Figure 2] This graph shows the change in fluorescence intensity retention rate (FD%) as the sterilization time (t) of FSI changes. [Figure 3] This graph shows the change in the fluorescence intensity index Ln(FD0 / FDt) as the exposure time (t) of FSI increases. [Figure 4] This graph shows the change in luminescence intensity (EI-RLUt) with respect to exposure time (t) to EI. [Figure 5] This graph shows the change in sterilization strength (EI-LRDt) with respect to exposure time (t) to EI. [Figure 6] This graph shows the correlation between EI-LRD and FSI's Ln(FD0 / FDt). [Modes for carrying out the invention]

[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 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 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 analogues can be used. For example, EGFP (Enhanced Green Fluorescent Protein) is a variant of GFP 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 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 will be 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 of the jellyfish Aequorea victoria (GFP: Dr. Shimomura et al. were awarded the Nobel Prize in Chemistry in 2008). 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 form of 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 sheets (cocoons peeled into thin flakes).

[0039] Thus, 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 fluorescent silk 12 is a woven fabric, fluorescent silk threads may be interwoven with ordinary silk threads or threads 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 a 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] <<Fluorescent Silk Indicator Fabrication>> 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 defined.

[0042] <<Measuring Sterilization Strength using FSI and EI>> The sterilization strength of the GFP-FSI and mAG-FSI prepared as described above was compared with that of the conventionally used EI (Protak Scientific). First, the hydrogen peroxide concentration in the chamber was adjusted to approximately 200 ppm using a Biological Indicator-Evaluator Resistometer (BIER, AIREX Corporation).

[0043] Next, five sheets each of GFP-FSI, mAG-FSI, and EI were simultaneously placed in a BIER chamber with a hydrogen peroxide concentration adjusted to approximately 200 ppm, and sterilized by hydrogen peroxide gas exposure. Exposure tests were conducted with exposure times of 3, 6, 9, 12, 15, and 18 minutes. After each exposure test, the sterilized GFP-FSI, mAG-FSI, and EI were removed from the BIER chamber, air-dried, and stored in a dark place.

[0044] Next, after the exposure test, luciferin-luciferase reagent and ADP substrate were added to the EI samples, and the luminescence intensity (EI-RLUt) was measured at t minutes of exposure using a Luminometer (PR2A, Protak Scientific). The luminescence intensity (EI-RLUt) was calculated as 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). Separately, the pre-sterilization luminescence intensity (EI-RLU0) was measured using the EI samples taken before the exposure test.

[0045] On the other hand, the fluorescence intensity of GFP-FSI and mAG-FSI after the exposure test was measured using a spectrofluorometer (RF-6000, Shimadzu Corporation) at an excitation wavelength of 495 nm and an emission wavelength of 510 nm. The fluorescence intensity (FDt) after t minutes of exposure to hydrogen peroxide gas was defined as the average of the FDt values ​​of five images at each exposure time.

[0046] ≪Changes in fluorescence intensity after exposure testing≫ From the fluorescence intensity before exposure testing (FD0) and the fluorescence intensity after exposure time t minutes (FDt) for GFP-FSI and mAG-FSI, the following formula (1) is used: FD% =(FDt / FD0)×100 (1) The fluorescence intensity retention rate (FD%) for GFP-FSI and mAG-FSI was calculated.

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

[0048] <<Changes in FSI fluorescence intensity>> The inventors of this invention assume that the change in the fluorescence intensity of FSI is due to the denaturation of the fluorescent protein by hydrogen peroxide, and in order to evaluate the denaturation reaction, they use the following equation (2): Ln(FDt) = Ln(FD0) - kt kt=Ln(FD0 / FDt) (2) Here, Ln is the natural logarithm, and k is the reaction rate constant. The reaction process was evaluated.

[0049] Figure 3 is a graph showing the change in fluorescence intensity index: Ln(FD0 / FDt) with respect to FSI exposure time: t. In Figure 3, it was confirmed that the fluorescence intensity index: Ln(FD0 / FDt) of GFP-FSI and mAG-FSI changed linearly with respect to exposure time: t in the hydrogen peroxide gas exposure test. This suggests that the fluorescence intensity of fluorescent silk in the hydrogen peroxide gas exposure test is due to a simple one-step reaction (protein denaturation) of the fluorescent silk protein. Furthermore, since the slope: k of the change in fluorescence intensity index: Ln(FD0 / FDt) for GFP-FSI and mAG-FSI is almost identical, it is possible that the denaturation reaction of fluorescent proteins is similar for both fluorescent proteins.

[0050] ≪Changes in EI light intensity≫ On the other hand, the change in luciferin-luciferase emission intensity was confirmed by exposure of EI to hydrogen peroxide gas. Figure 4 is a graph showing the change in emission intensity:EI-RLUt with respect to exposure time:t. In Figure 4, it was confirmed that the emission 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 strength 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 (EI-LRDt) of EI 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, a nearly linear change was observed. 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 Ln(FD0 / FDt) of EI-LRD and FSI are plotted in the region where EI-LRD is 4 or higher, which is considered to be a highly reliable evaluation of sterilization strength of EI-LRD. Figure 6 is a graph showing the correlation between EI-LRD and Ln(FD0 / FDt) of FSI. 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 hydrogen peroxide sterilization, the change in sterilization intensity over time can be determined by calculating the fluorescence intensity index of FSI: Ln(FD0 / FDt). Furthermore, in the chamber of a sterile isolator, for example, which is the target of hydrogen peroxide sterilization, it is possible to determine the sterilization intensity distribution from the value of FSI Ln(FD0 / FDt).

[0054] As described above, the present invention provides a sterility 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 sterility intensity 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. [Explanation of Symbols]

[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, The system comprises a base material and a sensitive member supported on the base material that is sensitive to the sterilizing agent, The sensing member is a material whose constituent element is a fluorescent protein, and which emits fluorescence of a predetermined wavelength when irradiated with excitation light of a predetermined wavelength, A sterilization indicator characterized in that the fluorescence intensity of the sensing member changes in proportion to the exposure intensity of the sterilizing agent to the sensing 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 the 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-release 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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