Sterilization Indicator

Abstract

To provide a sterilization indicator that does not require a complicated culture procedure such as BI or a luciferin-luciferase assay such as EI, can be stably supplied at low cost, and can accurately measure the sterilization strength in a dry state. [Solution] The device includes a substrate and a sensory member supported on the substrate that is sensitive to the sterilizing agent. The sensory member is a material whose constituent element is a fluorescent protein, and employs genetically modified fluorescent silk. The genetically modified fluorescent silk emits fluorescence of a predetermined wavelength when irradiated with excitation light of a predetermined wavelength. The fluorescence intensity of the sensory member changes in proportion to the intensity of exposure of the sensory member to the sterilizing agent.

Description

[Technical field]

[0001] The present invention relates to a sterilization indicator used to confirm the sterilization effect in clean rooms, pharmaceutical manufacturing equipment, medical instruments, etc., which are sterilized using a sterilizing agent. [Background technology]

[0002] In manufacturing sites where pharmaceuticals or food are manufactured, or in medical sites such as operating rooms, it is important to maintain a sterile state in the room and for the equipment used. In particular, in the sterilization of sterile rooms (also called "decontamination," but hereafter referred to as "sterilization") which are the work rooms used in pharmaceutical manufacturing, it is necessary to complete advanced sterilization validation in accordance with GMP (Good Manufacturing Practice).

[0003] In recent years, hydrogen peroxide has been widely used for sterilization of work rooms such as clean rooms (hereinafter referred to as "rooms to be sterilized"). Hydrogen peroxide has a strong sterilizing effect, is inexpensive and easy to obtain, and is effective as an environmentally friendly sterilizing gas that eventually decomposes into oxygen and water.

[0004] Traditionally, and still today, the mainstream method is to generate hydrogen peroxide gas by heating and evaporating hydrogen peroxide water inside the room to be sterilized. This method is called the "flash evaporation sterilization method." In this method, hydrogen peroxide water, for example, at 30-35 W / V%, is supplied from the outside to the inside of the room to be sterilized, and is heated in a high-temperature evaporator installed inside the room to generate hydrogen peroxide gas and water vapor. Then, the air inside the room to be sterilized is circulated to fill the room with hydrogen peroxide gas.

[0005] Meanwhile, in recent years, in addition to the issues of the flash evaporation sterilization method, such as reducing the amount of hydrogen peroxide used, shortening the sterilization cycle time, and reducing the residual gas concentration after sterilization, a sterilization method that focuses on the condensed film of hydrogen peroxide has been adopted. This method is called the "mist injection sterilization method." In this method, room temperature hydrogen peroxide and compressed air are turned into hydrogen peroxide mist using a two-fluid nozzle or the like, and the hydrogen peroxide mist is supplied to the inside of the room to be sterilized, and the fine hydrogen peroxide mist is circulated inside the room to be sterilized using ultrasonic vibration or the like.

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

[0007] For this reason, sterilization indicators are placed at key locations inside the room to be sterilized to evaluate the distribution of sterilization strength. Conventional sterilization indicators are called biological indicators (BIs) and use biological indicator bodies made from microbial spores that are resistant to specific sterilization methods. These BIs can directly evaluate the lethality rate of spores after sterilization. However, there are problems with this, such as the need to culture the BI spores after sterilization, which requires time and effort to obtain evaluation results, and the fact that only positive or negative information can be obtained, making it impossible to evaluate the detailed distribution of sterilization strength. There is also the problem of lingering concerns about microbial contamination.

[0008] Therefore, while it can be considered a type of BI, in recent years enzyme indicators (EIs) have come to be used as a method to complement BIs. For example, Patent Document 1 below discloses a biological indicator (although the invention is named BI, the content can be considered EI) that uses thermostable 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 UK government. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 4774039 Summary of the Invention [Problem to be solved by the invention]

[0010] This technology measures the remaining enzyme activity after sterilization by luminescence intensity using a luciferin-luciferase assay. The inactivation of thermostable adenylate kinase (tAK) applied to the EI correlates with the amount of exposure to the sterilant (volume x time), and the sterilization strength is evaluated by measuring the remaining enzyme activity of tAK. Compared to conventional BIs, no culture operation is required and the sterilization strength can be measured in a relatively short time. In recent years, its comparative equivalence with BIs has been confirmed and it is becoming more and more popular. Specifically, the LRD value (Log Spore Reduction) based on the logarithmic reduction in the number of bacteria is calculated from the luminescence intensity of the EI after sterilization, and the sterilization strength distribution inside the room to be sterilized is evaluated.

[0011] However, even in the luciferin-luciferase assay of EI, ADP (ATP-producing substrate) is added to EI after sterilization, reacted by wet processing, and the luminescence intensity is measured with a luminometer to measure the enzyme activity of adenylate kinase. Although this method is a common method for measuring enzyme activity, the coefficient of variation (CV value) of the measured value is said to be about 5-15%, and there are issues with the measurement accuracy. In addition, there are currently questions about the price of the EI enzyme and its stable supply. Another issue is that the luminescence reaction is measured in a wet state, making the operation complicated.

[0012] Therefore, the present invention addresses the above-mentioned problems and aims to provide a sterilization indicator that does not require complicated culture procedures such as BI or wet procedures such as luciferin-luciferase assays such as EI, can be supplied steadily at low cost, and allows the sterilization strength to be measured with high accuracy in a dry state. [Means for solving the problem]

[0013] In order to solve the above problems, the inventors conducted extensive research and focused on recombinant fluorescent silk that emits fluorescence as a fiber. They discovered that the fluorescence intensity of the recombinant fluorescent silk correlates with the amount of exposure to a sterilant (volume x time), which led to the completion of the present invention.

[0014] That is, the sterilization indicator according to the present invention comprises, according to claim 1: A sterilization indicator used to confirm the sterilization effect in an object to be sterilized using a sterilizing agent, A substrate and a sensory member that is supported on the substrate and is sensitive to the sterilant, The sensory member is a material having a fluorescent protein as a component, and 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 sterilizing agent to the sensing member.

[0015] According to claim 2, the present invention provides a sterilization indicator according to claim 1, The material having the fluorescent protein as a component is characterized in that it is recombinant fluorescent silk produced by recombinant silkworms, or a mixture of the recombinant fluorescent silk with other materials.

[0016] According to claim 3, the present invention provides a sterilization indicator according to claim 2, The recombinant fluorescent silk is characterized in that it is a fluorescent silk into which genetic information for one or more fluorescent proteins has been incorporated by recombinant gene manipulation.

[0017] According to claim 4, the present invention provides a sterilization indicator according to claim 3, The fluorescent protein is characterized in that it is GFP, mAG, or an analogue of these fluorescent proteins.

[0018] According to claim 5, the present invention provides a sterilization indicator according to any one of claims 2 to 4, The sensory component is characterized in that it is a component that uses in whole or in part materials derived from the genetically modified silk, including cotton-like, fibrous, film-like, thread, nonwoven fabric, woven fabric, knitted fabric, powder, and cocoon peeling sheets.

[0019] According to claim 6, the present invention provides a sterilization indicator according to claim 5, The sensory member supported by the base material is characterized in that its surface is covered with a breathable film. Effect of the Invention

[0020] According to 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. The sterilization indicator includes a substrate and a sensory member supported on the substrate and sensitive to the sterilizing agent. The sensory member is a material containing a fluorescent protein as a component, and emits fluorescence of a predetermined wavelength when irradiated with excitation light of a predetermined wavelength. The fluorescence intensity of the sensory member changes in proportion to the exposure intensity of the sterilizing agent to the sensory member.

[0021] This makes it possible to provide a sterilization indicator that does not require complicated culture procedures such as BI or wet procedures such as luciferin-luciferase assays such as EI, can be supplied steadily at low cost, and can accurately measure the sterilization strength in a dry state.

[0022] In addition, according to the above configuration, the material containing fluorescent protein as a component is recombinant fluorescent silk produced by recombinant silkworms, or a mixture of the recombinant fluorescent silk and other materials, which can more specifically exert the above-mentioned action and effect.

[0023] In addition, according to the above configuration, the genetically modified fluorescent silk is a fluorescent silk into which the genetic information of one or more fluorescent proteins is incorporated by genetic recombination manipulation, which can more specifically exert the above-mentioned action and effect.

[0024] Furthermore, according to the above configuration, the fluorescent protein may be GFP, mAG, or an analogue of these fluorescent proteins, which makes it possible to more specifically exert the above-mentioned effects.

[0025] According to the above configuration, the sensory member may be a member that uses, in whole or in part, a material derived from genetically modified silk, including cotton, fiber, film, thread, nonwoven fabric, woven fabric, knitted fabric, powder, and cocoon peeling sheet, which allows the above-mentioned action and effect to be exhibited more specifically.

[0026] According to the above-mentioned configuration, the surface of the sensor supported by the base material may be covered with a breathable film, thereby making it possible to more specifically exhibit the above-mentioned effects. [Brief description of the drawings]

[0027] [Figure 1] FIG. 2A is a plan view and FIG. 2B is a front view showing the appearance of the fluorescent silk indicator of this embodiment. [Diagram 2] 1 is a graph showing the change in the fluorescence intensity residual rate: FD% depending on the sterilization time: t of the FSI. [Diagram 3] 1 is a graph showing the change in the fluorescence intensity index: Ln(FD0 / FDt) depending on the FSI exposure time: t. [Figure 4] 1 is a graph showing the change in luminescence intensity: EI-RLUt depending on the EI exposure time: t. [Diagram 5] 1 is a graph showing the change in sterilization strength: EI-LRDt depending on the EI exposure time: t. [Figure 6] 1 is a graph showing the correlation between EI-LRD and Ln(FD0 / FDt) of FSI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] First, we will explain the "genetically modified fluorescent silk" that constitutes the sensory member of the sterilization indicator according to the present invention. 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 of a specific wavelength when irradiated with excitation light of a specific wavelength. This genetically modified silkworm is produced by introducing a fluorescent protein gene into silkworm eggs, raising the silkworms that hatched from the eggs until they become adults, mating them, and selecting and raising eggs into which the fluorescent protein gene has been introduced.

[0029] Transgenic silkworms originated from the "Shining Silkworm," which was first successfully developed in 2000 by the Silkworm and Insect Agricultural Technology Institute of the Ministry of Agriculture, Forestry and Fisheries of Japan (now the National Agriculture and Food Research Organization (NARO)) (T. Tamura, et al.: Nat. Biotechnol. 18, 81 (2000)). Currently, basic and applied research on transgenic silkworms is being conducted using the silkworm genome information deciphered in 2008.

[0030] More than 50 types of fluorescent proteins with different excitation and fluorescence wavelengths have been identified for introduction into genetically modified silkworms. In the present invention, GFP (Green Fluorescent Protein), mAG (monomeric Azami-Green) and their analogs can be used. For example, EGFP (Enhanced Green Fluorescent Protein) is a mutant of GFP that was developed to improve its fluorescent properties. In the present invention, GFP and EGFP are referred to as "GFP" without making a clear distinction between them.

[0031] In addition, in the present invention, a wide variety of fluorescent proteins can be used, including those to be identified in the future, without being limited to GFP, mAG, and their analogs. The fluorescent proteins to be introduced into silkworms are not limited to one type, and several types may be introduced.

[0032] The sterilization indicator according to the present invention will be described in detail below with reference to the following embodiments. Note that the present invention is not limited to the following embodiments. In the present embodiments, the sterilization indicator according to the present invention is called a "Fluorescent Silk Indicator (FSI)".

[0033] First, the sterilizing agent used in this embodiment will be described. In this embodiment, hydrogen peroxide in gas or mist form is used as the sterilizing agent. As described above, the inventors focused on fluorescent silk that emits fluorescence as a fiber, and found that the fluorescence intensity of the fluorescent silk correlates with the amount of exposure to the sterilizing agent (volume x time), 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 in the fluorescent silk is damaged by exposure to hydrogen peroxide, causing the fluorescence intensity to attenuate. Therefore, in the present invention, it is not limited to hydrogen peroxide, but is also believed to be effective with various sterilization methods. In addition to hydrogen peroxide, it is also believed to be effective with moist heat sterilization, high-pressure steam sterilization, gamma ray irradiation, electron beam irradiation, ethylene oxide gas, etc.

[0035] Next, the object to be sterilized will be described. In the present invention, consideration is given to manufacturing sites where medicines or foods are manufactured, or medical sites such as operating rooms, and examples thereof include clean rooms, medicine manufacturing equipment such as isolators and RABS, machines and instruments introduced into isolators, medical instruments, etc., but are not limited to these.

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

[0037] The 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 of about 509 nm with excitation light of about 488 nm and is derived from the green fluorescent protein of Aequorea victoria (GFP: for which 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 of about 505 nm with excitation light of about 492 nm.

[0038] The shape of the fluorescent silk 12 is not particularly limited, and may be made in whole or in part from materials derived from genetically modified silk, including cotton (unraveled cocoons), fibers, films (raw silk dissolved into a film), threads (raw silk or silk threads), nonwoven fabrics, woven fabrics, knitted fabrics, powders (crushed cocoons or threads), and cocoon peeling sheets (cocoons peeled off into thin flakes).

[0039] In this way, the fluorescent silk 12 is not limited to 100% fluorescent silk, and may be a mixture of other materials as long as the attenuation of the fluorescent intensity is confirmed. For example, when the fluorescent silk 12 is a woven fabric, the fluorescent silk thread may be interwoven with normal silk thread or thread of other fibers.

[0040] In addition, the method of adhering the film stick 11 and the fluorescent silk 12 is not particularly limited, and any adhering method that does not affect the sensitivity of the fluorescent silk 12 to the sterilizing agent may be adopted. In addition, care must be taken when adhering the fluorescent silk 12, which may be partially scattered, such as in the form of cotton or powder. For example, it is preferable to cover the surface of the cotton or powder with a breathable film that allows the gas or mist of the sterilizing agent 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 their molecular size, such as a cellulose acetate membrane or a polyethersulfone membrane, may be used.

[0041] <Production of fluorescent silk indicator> The GFP fluorescent silk and mAG fluorescent silk cocoons were each divided vertically into four pieces. The divided cocoon pieces were peeled off using a cutter to prepare about three fluorescent silk cocoon slices for each. The thickness of the fluorescent silk cocoon slices was 0.1 to 0.4 mm. These slices were cut into 1 cm x 1 cm pieces. Next, these slices (1 cm x 1 cm) were attached to the tip of a polyolefin film stick (1 cm x 5 cm, thickness 0.5 mm) with double-sided tape to obtain each fluorescent silk indicator (GFP-FSI, mAG-FSI). The obtained GFP-FSI and mAG-FSI were measured for fluorescence intensity at an excitation wavelength of 495 nm and a fluorescence wavelength of 510 nm using a spectrofluorometer (RF-6000, Shimadzu Corporation), and the fluorescence intensity before the hydrogen peroxide gas exposure test was set as FD0.

[0042] <FSI and EI sterilization strength measurement> The sterilization strength of the GFP-FSI and mAG-FSI prepared above was measured and compared with that of the conventionally used EI (Protak Scientific). First, the hydrogen peroxide concentration in the chamber was adjusted to about 200 ppm using a Biological Indicator-Evaluator Resistometer (BIER, Airex Co., Ltd.).

[0043] Next, five sheets each of GFP-FSI, mAG-FSI, and EI were placed in a BIER chamber with a hydrogen peroxide concentration adjusted to about 200 ppm, and sterilized by exposing them to hydrogen peroxide gas. Exposure tests were performed for 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 the dark.

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

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

[0046] <Changes in fluorescence intensity after exposure test> From the fluorescence intensity before the exposure test of GFP-FSI and mAG-FSI: FD0 and the fluorescence intensity after the exposure time of t minutes: FDt, the following formula (1) is obtained: FD% =(FDt / FD0)×100 (1) The remaining fluorescence intensity rates (FD%) of GFP-FSI and mAG-FSI were calculated.

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

[0048] <Changes in FSI fluorescence intensity> The present inventors hypothesized that the change in fluorescence intensity of FSI was due to the denaturation of a fluorescent protein by hydrogen peroxide, and calculated the denaturation reaction by the following formula (2): Ln(FDt) = Ln(FD0) - kt kt=Ln(FD0 / FDt) (2) where Ln is the natural logarithm, k is the 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) with the exposure time: t of FSI. In Figure 3, it was confirmed that the fluorescence intensity index: Ln(FD0 / FDt) of GFP-FSI and mAG-FSI changed linearly with the exposure time: t by the hydrogen peroxide gas exposure test. This suggests that the fluorescence intensity of fluorescent silk is a simple one-step reaction (protein denaturation) of the fluorescent silk protein by the hydrogen peroxide gas exposure test. In addition, since the slope: k of the change in the fluorescence intensity index: Ln(FD0 / FDt) of GFP-FSI and mAG-FSI is almost the same, it is suggested that the denaturation reaction of the fluorescent protein may be similar for both fluorescent proteins.

[0050] <Changes in EI emission intensity> On the other hand, we confirmed the change in luciferin-luciferase luminescence intensity due to the hydrogen peroxide gas exposure test of EI. Figure 4 is a graph showing the change in luminescence intensity: EI-RLUt depending on the EI exposure time: t. In Figure 4, it was confirmed that the luminescence intensity of EIt: EI-RLUt decreased over time with respect to the 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 depending on the EI exposure time: t. In Figure 5, it was confirmed that the EI sterilization intensity: EI-LRDt increases over time with respect to the exposure time: t by the hydrogen peroxide gas exposure test. It was also confirmed that when EI-LRD is 4 or more, it shows an approximately linear change. However, it was suggested that when EI-LRD is less than 4, the measurement values ​​vary greatly and linearity is lost.

[0052] <Correlation between EI sterilization strength and FSI fluorescence intensity index> From the results of Figure 5, EI-LRD and Ln(FD0 / FDt) of FSI are plotted in the range of EI-LRD of 4 or more, which is considered to be a reliable evaluation of the sterilization strength of EI-LRD. Figure 6 is a graph showing the correlation between EI-LRD and Ln(FD0 / FDt) of FSI. Figure 6 suggests that there is an approximately linear correlation between EI-LRD and Ln(FD0 / FDt), and that this is independent of 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 strength over time by calculating the FSI fluorescence intensity index: Ln(FD0 / FDt). In addition, it is possible to determine the sterilization strength distribution from the FSI Ln(FD0 / FDt) value in the chamber of a sterile isolator, which is the target of sterilization with hydrogen peroxide.

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

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

[0056] Since the fluorescent protein is introduced into the gene, the fluorescent silk produced by the transgenic silkworms has a uniform fluorescent performance. Furthermore, the production and lot management of the sterilization indicators produced from the homogeneous fluorescent silk allows for a stable supply of sterilization indicators that have been confirmed to be comparable to BIs. [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 in an object to be sterilized using a sterilizing agent, A substrate and a sensory member that is supported on the substrate and is sensitive to the sterilant, The sensory member is a material having a fluorescent protein as a component, and 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 said sensing member changes in proportion to the intensity of exposure of said sensing member to said sterilizing agent.

2. The sterilization indicator according to claim 1, characterized in that the material having the fluorescent protein as a component is recombinant fluorescent silk produced by recombinant silkworms, or a mixture of the recombinant fluorescent silk and other materials.

3. The sterilization indicator of claim 2, wherein the recombinant fluorescent silk is silk into which genetic information of one or more fluorescent proteins has been incorporated by recombinant gene manipulation.

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

5. The sterilization indicator according to any one of claims 2 to 4, characterized in that the sensory component is a component that uses in whole or in part a material derived from the genetically modified silk, including cotton-like, fibrous, film-like, thread, nonwoven fabric, woven fabric, knitted fabric, powder, and cocoon peeling sheet.

6. 6. The sterilization indicator according to claim 5, wherein the sensing member supported on the substrate has a surface covered with a breathable film.

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