Detection method of microorganisms

Abstract

To provide a detection method of microorganisms capable of detecting microorganisms in a short period and with high accuracy.SOLUTION: A detection method of microorganism has: a separation step of centrifuging a first sample including microorganisms to obtain a second sample in which a component other than the microorganisms is reduced further than the first sample; a detection step of detecting the microorganisms included in the second sample; and a coloring step of coloring the microorganisms or a composite including the microorganisms before or after the separation step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for detecting microorganisms. [Background technology]

[0002] In order to improve the efficiency of testing for microorganisms attached to food, microbial testing methods that can replace the official colony counting method are being considered. Patent Document 1 discloses a method for detecting microorganisms by utilizing color development due to the enzymatic activity of the microorganisms. Patent Document 2 discloses a method in which an antigen labeled with a metal compound is attached to the microorganisms through an antigen-antibody reaction to form a complex, and the complex is detected by an electrochemical method. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2020-129984 [Patent Document 2] Patent Publication No. 2021-181888 Summary of the Invention [Problem to be solved by the invention]

[0004] From the perspective of food hygiene, it is necessary to prevent food from containing microorganisms such as pathogens that can cause food poisoning. The colony count method, an official method for detecting microorganisms attached to food, is known. The colony count method is a culture method in which a sample is cultured and the number of colonies is calculated by visually counting the colonies. The test standard requires that the culture be performed for at least 24 hours without the formation of colonies. This colony count method requires a long culture period and is therefore inefficient. Meanwhile, while sensors for detecting microorganisms can easily and quickly determine the presence or absence of microorganisms, they have limited detection limits. Therefore, they have difficulty detecting trace amounts of microorganisms and are unable to accurately determine microorganisms below the detection limit. Therefore, the present disclosure provides a microorganism detection method that can detect microorganisms in a short period of time with high accuracy. [Means for solving the problem]

[0005] One aspect of the present disclosure provides the following method for detecting a microorganism.

[0006] [1] A separation step of centrifuging a first sample containing microorganisms to obtain a second sample in which components other than the microorganisms have been reduced compared to the first sample; a detection step of detecting the microorganism contained in the second sample, A method for detecting microorganisms, comprising a coloring step of coloring the microorganisms or a complex containing the microorganisms before or after the separation step.

[0007] In the microorganism detection method [1] above, a first sample containing microorganisms is centrifuged in the separation step to obtain a second sample in which the microorganisms are concentrated. Since the detection step detects the microorganisms contained in this second sample, even if the first sample contains only trace amounts of microorganisms, the microorganisms can be detected with high accuracy. Furthermore, since the detection method colorizes the microorganisms or complexes containing the microorganisms in the coloring step, the microorganisms can be detected in a short period of time. Thus, the detection method allows the microorganisms contained in a sample to be detected with high accuracy in a short period of time.

[0008] The method for detecting microorganisms in [1] above may be any one of the following [2] to [9].

[0009] [2] The method for detecting microorganisms according to [1], wherein the coloring step includes obtaining the complex through an antigen-antibody reaction between the microorganism and an antibody. [3] The method for detecting microorganisms according to [1] or [2], wherein in the separation step, the second sample is obtained by collecting the microorganisms on a separation membrane or by precipitating the microorganisms. [4] The method for detecting microorganisms according to any one of [1] to [3], wherein in the detection step, the microorganisms are detected using at least one selected from the group consisting of absorbance measurement and image analysis. [5] A method for detecting microorganisms according to any one of [1] to [4], wherein the coloring step includes Gram-staining the microorganisms contained in the first sample, or coloring the complex containing the microorganisms obtained by an antigen-antibody reaction. [6] The method for detecting microorganisms according to any one of [1] to [5], wherein in the separation step, the microorganisms are collected on a separation membrane to obtain the second sample. [7] The method for detecting microorganisms according to any one of [1] to [6], wherein the detection step detects the microorganisms collected on the separation membrane by imaging. [8] The method for detecting microorganisms according to any one of [1] to [7], further comprising a pre-culture step of culturing the microorganisms before the separation step or the staining step. [9] The method for detecting a microorganism according to any one of [1] to [8], wherein the culture time in the pre-culture step is 4 to 12 hours.

[10] The method for detecting microorganisms according to any one of [1] to [9], wherein the microorganisms include Escherichia coli.

[11] The detection step comprises detecting the complex obtained by an antigen-antibody reaction between the microorganism and an antibody labeled with a first metal compound using electrochemical measurement; and detecting the complex obtained by the antigen-antibody reaction between the microorganism and the antibody by electrochemical measurement in the presence of a sensitizer containing a second metal compound; The method for detecting a microorganism according to any one of [1] to

[10] , comprising at least one of the following:

[0010] The microorganism detection method [2] above generates a complex between the microorganism and the antibody through an antigen-antibody reaction, and the sample solution can be colored by causing the antibody to develop a color. This allows for detection with sufficiently high accuracy even when there are only a small amount of microorganisms. Therefore, microorganisms can be detected with even higher accuracy.

[0011] The above-mentioned method for detecting microorganisms [3] can concentrate microorganisms by collecting them on a separation membrane or by precipitating them. By using such a method, microorganisms can be detected easily and with higher accuracy.

[0012] The microorganism detection method [4] above can detect colored microorganisms by absorbance measurement or image analysis, which allows for detection of microorganisms in a shorter period of time and with higher accuracy.

[0013] The microorganism detection method [5] above allows for easy observation of microorganisms using a digital microscope, etc., by staining the microorganisms. Furthermore, minute amounts of microorganisms can be detected by visual inspection, allowing for even higher accuracy in detecting microorganisms.

[0014] In the microorganism detection method [6] above, for example, a first sample containing microorganisms stained by Gram staining can be collected on a separation membrane in the separation step. In this way, by collecting pre-stained microorganisms on a separation membrane, the microorganisms can be immobilized on the separation membrane while still alive. By attaching this separation membrane to a glass slide, the microorganisms can be observed using a digital microscope or the like. Therefore, there is no need to fix the microorganisms on the glass slide using heat or chemicals, and microorganisms can be detected easily with high reproducibility.

[0015] The microorganism detection method described in [7] above detects microorganisms immobilized on a separation membrane by imaging. Detection by imaging allows for easier detection of microorganisms. Furthermore, minute amounts of microorganisms can be detected by visual inspection, allowing for more accurate detection of microorganisms.

[0016] The microorganism detection method described in [8] above includes a pre-cultivation step in which microorganisms are cultured in advance. By including the pre-cultivation step, even in a sample containing only a small amount of microorganisms, the amount of microorganisms can be increased to a detectable amount. Therefore, even trace amounts of microorganisms can be detected.

[0017] The microorganism detection method described in [9] above can detect minute amounts of microorganisms with high accuracy because the incubation time in the pre-incubation step is 4 to 12 hours. Furthermore, compared to the colony counting method, which requires 24 hours, it can detect microorganisms in a shorter time.

[0018] The above-mentioned microorganism detection method

[10] is targeted at Escherichia coli, and therefore can be suitably used to test foods that may cause food poisoning.

[0019] The method for detecting microorganisms described in

[11] above involves reacting an antibody labeled with a first metal compound with a microorganism to obtain a complex, which is then detected electrochemically, or reacting an antibody with a microorganism to obtain a complex, which is then detected electrochemically in the presence of a sensitizer containing a second metal compound. Such methods enable more accurate detection of microorganisms.

[0020] Another aspect of the present disclosure provides the following method for detecting a microorganism.

[0021]

[12] A pre-culture step of culturing a first sample containing microorganisms to obtain a second sample containing the microorganisms in a larger amount than the first sample; a detection step including at least one of detecting a first complex obtained by an antibody-antigen reaction between the microorganism contained in the second sample and an antibody labeled with a first metal compound using electrochemical measurement, and detecting a second complex obtained by an antigen-antibody reaction between the microorganism and an antibody using electrochemical measurement in the coexistence of a sensitizer containing a second metal compound; A method for detecting microorganisms, comprising:

[0022] The above-mentioned method for detecting microorganisms detects microorganisms grown in a pre-culture step by electrochemical measurement. By including the pre-culture step, even if the amount of microorganisms is small, it is possible to grow the amount of microorganisms to a level that can be detected in the detection step. Therefore, it is possible to detect small amounts of microorganisms. Furthermore, the electrochemical method allows for detection of microorganisms in a short period of time with high accuracy. [Effects of the Invention]

[0023] The present disclosure can provide a method for detecting microorganisms that can detect microorganisms with high accuracy in a short period of time. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 12-1. [Figure 2] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 12-2. [Figure 3] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 12-3. [Figure 4] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 12-4. [Figure 5] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 13-1. [Figure 6] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 13-2. [Figure 7] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 13-3. [Figure 8]1 is a photograph showing the state of Escherichia coli after pre-culture in Example 13-4. [Figure 9] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 14-1. [Figure 10] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 14-2. [Figure 11] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 14-3. [Figure 12] 1 is a photograph showing the state of Escherichia coli after pre-culture in Example 14-4. DETAILED DESCRIPTION OF THE INVENTION

[0025] Hereinafter, embodiments of the present disclosure will be described. However, the following embodiments are merely examples for explaining the present disclosure and are not intended to limit the present disclosure to the following content. The upper or lower limit of a numerical range specified in this disclosure may be replaced with any value shown in the examples. Furthermore, the upper and lower limits individually stated may be arbitrarily combined. The symbol "~" used in a numerical range indicates a numerical range that includes the upper and lower limit. For example, "X~Y" indicates a numerical range of "greater than or equal to X and less than or equal to Y." Unless otherwise specified, the materials or components exemplified in this disclosure can be used alone or in combination of two or more.

[0026] [First embodiment] The microorganism detection method of the first embodiment includes a separation step in which a first sample containing microorganisms is centrifuged to obtain a second sample in which components other than microorganisms are reduced compared to the first sample, and a detection step in which the microorganisms contained in the second sample are detected, and includes a coloring step in which the microorganisms are colored before or after the separation step.

[0027] The microorganisms contained in the first sample may include Escherichia coli. The microorganisms may include various pathogens. The pathogens may be microorganisms such as bacteria or viruses. Examples of the microorganisms include Campylobacter, Salmonella, and Escherichia coli. The Escherichia coli may be pathogenic Escherichia coli (O-157).

[0028] The first sample may be a dilution liquid containing microorganisms. The solvent may be distilled water, phosphate buffered saline (PBS), or a liquid culture medium. The first sample may also contain components other than microorganisms.

[0029] From the viewpoint of detecting minute amounts of microorganisms, the concentration of microorganisms contained in the first sample may be 1 CFU / mL or more, 10 CFU / mL or more, 100 CFU / mL or more, or 1000 CFU / mL or more. From the viewpoint of detecting microorganisms with even higher accuracy, the concentration of microorganisms contained in the first sample may be 10 10 CFU / mL or less, 10 9 CFU / mL or less, 10 8 CFU / mL or less, 10 7 CFU / mL or less, or 10 6 It may be less than CFU / mL.

[0030] In the separation step, the first sample is centrifuged to obtain a second sample in which components other than microorganisms have been reduced compared to the first sample. That is, the other components have been reduced in the second sample, and the microorganisms are more concentrated than in the first sample. This allows for detection of microorganisms in a short period of time with high accuracy. The conditions for centrifugation are not particularly limited as long as they allow for the concentration of microorganisms. Centrifugation can be performed using, for example, a tabletop centrifuge called "Chibitan" (trade name, manufactured by Yamato Scientific Co., Ltd.).

[0031] The staining step is performed before or after the separation step. The principle of staining in the staining step of the present disclosure is not particularly limited. For example, in one example of the staining step, a staining agent may be bound to the microorganisms contained in the sample to stain the microorganisms. In another example of the staining step, a complex containing the microorganisms may be stained by a reaction between an enzyme-labeled antibody that has bound to the microorganisms through an antigen-antibody reaction and a chromogenic substrate.

[0032] When the coloring step includes an antigen-antibody reaction, a complex is formed by an antigen-antibody reaction between the microorganism as an antigen and an antibody labeled with an enzyme. A chromogenic substrate is then added, causing the enzyme contained in the complex to react with the chromogenic substrate, resulting in the complex developing a color. This allows the complex containing the microorganism bound to the antibody to be selectively colored. Therefore, microorganisms can be detected with even higher accuracy. Furthermore, because the antigen-antibody reaction is a simple procedure, microorganisms can be detected in an even shorter time. Alternatively, a complex may be formed before the separation step, and a coloring step may be performed after the separation step in which a chromogenic substrate is added to color the complex.

[0033] From the viewpoint of detecting microorganisms with sufficiently high accuracy, the antibody concentration may be 300 ng / mL or more, or 400 ng / mL or more. Furthermore, from the viewpoint of reducing the amount of antibody used and suppressing costs, the antibody concentration may be 4000 ng / mL or less, 3000 ng / mL or less, or 1000 ng / mL or less.

[0034] The coloring step may be performed by Gram staining. When Gram staining is performed, Victoria blue solution may be used as the staining solution. The coloring solution may be subjected to pretreatment to reduce colored particles.

[0035] In the separation step, microorganisms may be collected on a separation membrane, or if a separation membrane is not used, the microorganisms may be precipitated. When collecting on a separation membrane, for example, the first sample in a spin column can be centrifuged to capture large microorganisms on the separation membrane in the spin column. On the other hand, in a method of precipitating microorganisms, the first sample in a microtube can be centrifuged to precipitate the microorganisms. From the viewpoint of reducing costs, the method of precipitating microorganisms in a microtube is preferred. On the other hand, when detecting trace amounts of microorganisms at approximately 1 to 1000 CFU / mL, a method of collecting microorganisms on a separation membrane and directly observing them under a microscope is preferred. The microorganisms collected in the separation step may be a complex bound to an antibody. As the separation membrane, for example, a commercially available product made of PTFE (polytetrafluoroethylene) with a pore size of 200 nm can be used.

[0036] In the detection step of detecting the microorganisms in the second sample, the colored microorganisms may be detected by absorbance measurement or by image analysis. The absorbance measurement may be performed by dispensing the microorganisms in the second sample onto a microplate, or by measuring each sample individually using an absorbance meter.

[0037] In image analysis, the color intensity can be calculated using an image of the second sample dispensed into the microplate taken from above. Alternatively, the microtube may be photographed from above without using a microplate. The photographing device may be a digital camera, a tablet device, or a smartphone device. A polarizing film may be attached to the lens. Photographing may be performed under indoor lighting, or by irradiating the microplate with transmitted light from below. Photographing may also be performed in a darkroom using a flash. The distance from the subject to the lens may be 5 to 25 cm, 10 to 20 cm, or 12 to 17 cm.

[0038] The detection accuracy can be improved by performing image processing on the captured image. For example, image division processing may be performed. Division processing is a correction in which the color intensity of the lightest colored sample is subtracted from the color intensity of each sample. The lightest colored sample is preferably a sample that does not contain microorganisms. By performing division processing, the influence of coloring due to factors other than microorganisms can be subtracted, and the color intensity when microorganisms are stained can be calculated. This makes it possible to evaluate the difference in color intensity due to differences in the amount of microorganisms.

[0039] The color intensity may be calculated by performing hue correction, which inverts the hue of the image. By performing hue correction, if the second sample is colored yellow, the color intensity (B value) of blue, which is the complementary color of yellow, can be calculated as the color intensity. By performing hue correction, the color contrast between the samples is enhanced, making the color differences clearer. Image analysis such as division processing and hue correction can be performed using, for example, "Image Sensing 2023 (product name)" (manufactured by ARS Corporation). Note that image processing is not limited to division processing, and may also be addition processing, multiplication processing, or normalization processing, or a combination of these processing.

[0040] In the detection step, the microorganisms collected on the separation membrane may be detected by image. When a separation membrane is used in the separation step, the separation membrane can be attached to a slide glass, and the microorganisms adsorbed on the separation membrane can be directly observed using a digital microscope or the like. This allows the microorganisms to be observed in a viable state. Furthermore, because the observation is done visually, it is possible to detect trace amounts of microorganisms, for example, 1 to 1000 CFU / mL.

[0041] When detecting microorganisms using images, the number of microorganisms in the observation area can be calculated by visually counting the number of microorganisms in the observation area. If there are too many microorganisms to count, three sections can be selected from the observation area, the number of microorganisms in the three sections can be counted, and the number can be converted into the number of microorganisms per area of the observation area. The number of microorganisms can be counted visually or by image analysis. Image analysis can be performed using, for example, "Image Sensing 2019 (product name)" (manufactured by ARS Corporation).

[0042] The method for detecting microorganisms may include a pre-culture step of culturing microorganisms before the separation step or the staining step. The culturing may be performed in a liquid medium. By including the pre-culture step, even if the amount of microorganisms in the first sample is small, the number of microorganisms can be increased by culturing, thereby making it possible to further increase the accuracy of detection in the detection step.

[0043] The incubation time in the pre-incubation step may be 4 hours or more, 5 hours or more, or 6 hours or more from the viewpoint of detecting microorganisms with higher accuracy. On the other hand, the incubation time may be 12 hours or less, 10 hours or less, or 8 hours or less from the viewpoint of detecting microorganisms in a shorter period of time. The official colony count test requires an incubation period of 24 hours. Therefore, the incubation time of the pre-incubation is shorter than that of the official method, and microorganisms can be detected in a shorter period of time than the official method.

[0044] The culture temperature in the pre-culture step may be 25 to 40°C, 28 to 38°C, or 32 to 37°C. By keeping the culture temperature within the above range, culture can be promoted, and the period of the pre-culture step can be shortened. Therefore, microorganisms can be detected in an even shorter period of time.

[0045] The detection step may include at least one of detecting a complex (first complex) obtained by an antigen-antibody reaction between a microorganism and an antibody labeled with a metal compound using an electrochemical measurement method, and detecting a complex (second complex) obtained by an antigen-antibody reaction between a microorganism and an antibody using electrochemical measurement in the presence of a sensitizer containing a second metal compound. Electrochemical measurement refers to measuring a characteristic value (resistance, current, or voltage) that changes with the redox reaction of the complex, for example.

[0046] The first metal compound used to label the antibody may be a metal complex. The metal complex may include a transition metal complex in which a ligand is coordinated to a transition metal. The transition metal may include, for example, a transition metal element belonging to groups 5 to 11 or a transition metal element belonging to groups 8 to 10 in the new IUPAC periodic table. Of these, from the viewpoint of sufficiently increasing detection sensitivity, it is preferable to include an iron group element (Fe, Co, Ni), and it is more preferable to include an iron complex. The first metal compound may be one type or a mixture of two or more types.

[0047] An antibody labeled with a first metal compound can be obtained by binding a labeling substance to the antibody. From the viewpoint of smoothly obtaining an antibody labeled with a first metal compound, the labeling substance preferably has, in addition to a metal component, a site (functional group) that reacts with an amino group (NH group), a sulfhydryl group (SH group) in the antibody, or an aldehyde group generated by oxidizing the sugar chain of the antibody. Examples of such functional groups include a maleimide group, an aldehyde group, and a succinimidyl group. The labeling substance may be a single metal compound or a mixture of two or more metal compounds. When the labeling substance is a metal complex (transition metal complex), the ligand may have a maleimide group, an aldehyde group, a succinimidyl group, or the like.

[0048] An example of a labeling substance is N-succinimidyl ferrocenecarboxylate. This iron complex having a succinimidyl group reacts with the amino group of an antibody as shown in the following formula (1). In this way, an antibody modified with a first metal compound is obtained. In the following general formula, R'-NH2 represents the antibody, and the labeling substance reacts with the amino group (-NH2) of the antibody. This results in an antibody labeled with a first metal compound.

[0049] [ka]

[0050] An antibody labeled with a first metal compound is mixed with a microorganism as an antigen to form a complex. Since the complex contains an antibody labeled with a first metal compound, the complex is also labeled with the metal compound. The charge of the metal element that constitutes the metal compound easily changes, for example, through a redox reaction. Therefore, the complex can be easily detected by electrochemical methods.

[0051] For example, when the first metal compound is a ferrocene complex as shown in the above reaction formula, the oxidation number of iron in the ferrocene complex changes due to a redox reaction (for example, Fe 2+ →Fe 3+This allows the presence or absence of the complex and its concentration to be detected with high precision by electrochemical techniques.

[0052] The sensitizer containing the second metal compound may have the effect of amplifying the change in the conductivity of the complex. For example, it may be a sensitizer that has the effect of amplifying the signal (resistance value, current value, or voltage value) detected by an electrochemical method. Such a sensitizer may be a metal complex. The metal complex may be the same as that used in producing the antibody labeled with the first metal compound described above. For example, N-succinimidyl ferrocenecarboxylate in the above formula (1) may be used. By detecting the complex by electrochemical measurement in the coexistence of a sensitizer containing the second metal compound, the complex can be detected more efficiently than when the antibody is labeled with the first metal compound.

[0053] [Second embodiment] The microorganism detection method according to the second embodiment includes at least one of a pre-culture step of culturing a first sample containing microorganisms to obtain a second sample containing more of the microorganisms than the first sample, detecting a first complex obtained by an antigen-antibody reaction between the microorganisms contained in the second sample and an antibody labeled with a metal compound using electrochemical measurement, and detecting a second complex obtained by an antigen-antibody reaction between the microorganisms and an antibody using electrochemical measurement in the presence of a sensitizer containing a second metal compound.

[0054] In this detection method, even if the amount of microorganisms in the first sample is minute, the number of microorganisms can be increased in the pre-culture step. Therefore, microorganisms can be detected with higher accuracy in the detection step. Furthermore, because electrochemical measurement is a simple method, microorganisms can be detected in a short period of time.

[0055] The culture time in the pre-culture step may be 4 hours or more, 5 hours or more, or 6 hours or more from the viewpoint of detecting microorganisms with higher accuracy, while the culture time may be 12 hours or less, 10 hours or less, or 8 hours or less from the viewpoint of detecting microorganisms in a shorter period of time.

[0056] The culture temperature in the pre-culture step may be 20 to 40°C, 28 to 38°C, or 32 to 37°C. By keeping the culture temperature within the above range, culture can be promoted, and the period of the pre-culture step can be shortened. Therefore, microorganisms can be detected in an even shorter period of time.

[0057] The detection step includes at least one of detecting a first complex obtained by mixing an antibody labeled with a metal compound with a specimen containing an antigen (microorganism) and causing an antigen-antibody reaction, using electrochemical measurement, and detecting a second complex obtained by causing an antigen-antibody reaction between the microorganism and the antibody, using electrochemical measurement in the presence of a sensitizer containing a second metal compound. Since antigen-antibody reactions are typically performed in a liquid phase, the first and second complexes produced by the antigen-antibody reaction are contained in a reaction solution. The antibody labeled with the first metal compound is obtained by binding a labeling substance to the antibody, as described above. The antibody labeled with the first metal compound can be prepared in the same manner as in the first embodiment described above. Since the first complex contains an antibody labeled with the first metal compound, the complex is also labeled with the metal compound. The charge of the metal element constituting the first metal compound easily changes, for example, through a redox reaction. Therefore, the first complex can be easily detected by an electrochemical method.

[0058] In the detection step, the second complex obtained by the antigen-antibody reaction between the microorganism and the antibody may be detected by electrochemical measurement in the presence of a sensitizer containing a second metal compound. The sensitizer containing the second metal compound may be the same as that in the first embodiment. Although the second complex is not labeled with a metal compound, the second complex can be easily detected by an electrochemical method by being brought into the presence of a sensitizer containing the second metal compound.

[0059] Electrochemical measurement includes, for example, measuring a characteristic value (resistance value, current value, or voltage value) that changes with the redox reaction of the complex. For example, when the first metal compound and the second metal compound are ferrocene complexes as shown in the above formula (1), the oxidation number of iron in the ferrocene complex changes with the redox reaction (for example, Fe 2+ →Fe 3+ ) By electrochemical measurement that can detect such changes in oxidation number, the presence or absence and concentrations of the first complex and the second complex can be detected with high accuracy.

[0060] Although several embodiments have been described above, the present disclosure is not limited to the above embodiments. [Example]

[0061] The present disclosure will be described in more detail with reference to examples, but the present disclosure is not limited to the following examples.

[0062] (Example 1-1) <Pre-incubation and colony count of the first sample> E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to obtain a first sample containing 1 CFU / mL of E. coli. The E. coli contained in the first sample was pre-cultured at an incubation temperature of 36°C for 6 hours (pre-culture step). The pre-cultured first sample was diluted with phosphate-buffered saline (PBS) at a predetermined dilution ratio. A portion of the diluted first sample was subjected to a colony count test. The diluted first sample was spread on an agar medium in a petri dish and cultured at 36°C for 24 hours. The number of colonies after culture was counted, and multiplied by the dilution ratio to calculate the number of E. coli colonies contained in the first sample after pre-culture. Colony counts were performed on two petri dishes, and the average colony count for the two dishes was calculated. The results are shown in Table 1.

[0063] <Preparation of the second sample using a separation membrane> HRP (Whole Radish Peroxidase)-labeled E. coli antibody (HRP-E. coli antibody) was diluted with distilled water to obtain an antibody dilution solution (antibody concentration: 500 ng / mL). 450 μL of the first sample was dispensed into a microtube, and 50 μL of the antibody dilution solution was added to each tube and stirred for approximately 3 seconds. The tube was then incubated at 37°C for 10 minutes to allow the antigen-antibody reaction to proceed, yielding 500 μL of reaction solution containing the complex. 500 μL of distilled water was dispensed into a spin column with a 200 nm PTFE membrane inside, and the column was placed in a centrifuge (Chibitan) and centrifuged for 1 minute to wash the spin column. 500 μL of the resulting reaction solution was then added to the spin column. A blank spin column containing 500 μL of antibody dilution solution without E. coli was also prepared.

[0064] The spin column containing the reaction mixture and the blank spin column were placed in a centrifuge (Chibitan) and centrifuged for 2 minutes. The filtrate collected at the bottom of the separation membrane was discarded. 500 μL of distilled water was then dispensed again, and the column was placed in a centrifuge (Chibitan) and centrifuged for 2 minutes. All of the liquid above the separation membrane was then drained. In this way, the complex or antibody was collected on the separation membrane inside the spin column, and the second sample was prepared.

[0065] Subsequently, a second sample was prepared in the same manner, except that the antibody concentration in the antibody diluent was set to 1000 ng / mL or 3000 ng / mL.

[0066] <Image analysis> A 300 μL aliquot of a coloring agent prepared by mixing a sodium carbonate / citrate buffer solution containing 1 wt% o-phenyldiamine with 0.3 wt% hydrogen peroxide in a 1:1 mass ratio was dispensed into each second sample to develop color. After stirring at 37°C for 10 minutes, 100 μL of a reaction stop solution (1 M H2SO4) was added to each second sample and stirred. The spin column containing the colored solution was placed in a microtube rack and photographed from above using a tablet device. The photographs were taken under room lighting with a distance of 20 cm from the subject to the lens. Image analysis of the captured images was performed using an "Image Sensing 2023" (Ards Corporation). The lightest-colored sample among multiple samples captured in a single image was used as the reference sample for image analysis. After hue correction, the blue intensity (ΔB value) was calculated as the difference from the blank for each antibody dilution solution. The results are shown in Table 1.

[0067] <Absorbance measurement> After imaging, 200 μL of the second sample was dispensed into microplate wells for each concentration of antibody dilution, and the absorbance at a wavelength of 492 nm was measured using a microplate reader (manufactured by TECAN). The results of the absorbance measurements were evaluated as the difference (Δabsorbance) from the blank for each concentration of antibody dilution. The results are shown in Table 1. When there were two or more antibody concentrations at which the Δabsorbance and ΔB values were greater than 0, it was determined that E. coli could be detected.

[0068] (Example 1-2) The first sample was cultured and colony counted in the same manner as in Example 1-1, except that the pre-culture time in the pre-culture step was set to 7 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 1-1. Image analysis and absorbance measurement were performed in the same manner as in Example 1-1, and the Δabsorbance and ΔB values were calculated.

[0069] (Examples 1-3) A first sample was cultured and colony counted in the same manner as in Example 1-1, except that the pre-culture time in the pre-culture step was changed to 12 hours. A second sample was prepared using the cultured first sample in the same manner as in Example 1-1. Image analysis and absorbance measurement were performed using the obtained second sample in the same manner as in Example 1-1, and the Δabsorbance and ΔB value were calculated.

[0070] Example 2-1 E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to prepare a first sample containing 10 CFU / mL of E. coli. This first sample was cultured using the same procedure as in Example 1-1, and colony counting was performed. Using this first sample, a second sample was prepared by carrying out "preparation of a second sample using a separation membrane" using the same procedure as in Example 1-1. Image analysis and absorbance measurement were performed using the obtained second sample using the same procedure as in Example 1-1. The results are shown in Table 1.

[0071] (Example 2-2) The first sample was cultured and colony counted in the same manner as in Example 2-1, except that the pre-culture time in the pre-culture step was set to 7 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 2-1. Image analysis and absorbance measurement were performed in the same manner as in Example 2-1 using the obtained second sample. The results are shown in Table 1.

[0072] (Example 2-3) The first sample was cultured and colony counted in the same manner as in Example 2-1, except that the pre-culture time in the pre-culture step was 12 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 2-1. Image analysis and absorbance measurement were performed in the same manner as in Example 2-1 using the obtained second sample. The results are shown in Table 1.

[0073] (Examples 2-4) The first sample was cultured and colony counted in the same manner as in Example 2-1, except that the culture temperature in the pre-culture step was 38°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 2-1. Image analysis and absorbance measurement were performed in the same manner as in Example 2-1 using the obtained second sample. The results are shown in Table 1.

[0074] (Examples 2-5) The first sample was cultured and colony counted in the same manner as in Example 2-1, except that the culture temperature in the pre-culture step was 40°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 2-1. Image analysis and absorbance measurement were performed in the same manner as in Example 2-1 using the obtained second sample. The results are shown in Table 1.

[0075] (Example 3-1) E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to prepare a first sample containing 100 CFU / mL of E. coli. This first sample was cultured using the same procedure as in Example 1-1, and colony counting was performed. Using this first sample, a second sample was prepared by carrying out "preparation of a second sample using a separation membrane" using the same procedure as in Example 1-1. Image analysis and absorbance measurement were performed using the obtained second sample using the same procedure as in Example 1-1. The results are shown in Table 1.

[0076] (Example 3-2) The first sample was cultured and colony counted in the same manner as in Example 3-1, except that the pre-culture time in the pre-culture step was set to 7 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 3-1. Image analysis and absorbance measurement were performed in the same manner as in Example 3-1 using the obtained second sample. The results are shown in Table 1.

[0077] (Example 3-3) The first sample was cultured and colony counted in the same manner as in Example 3-1, except that the pre-culture time in the pre-culture step was 12 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 3-1. Image analysis and absorbance measurement were performed in the same manner as in Example 3-1 using the obtained second sample. The results are shown in Table 1.

[0078] (Examples 3-4) The first sample was cultured and colony counted in the same manner as in Example 3-1, except that the pre-culture time in the pre-culture step was set to 5 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 3-1. Image analysis and absorbance measurement were performed in the same manner as in Example 3-1 using the obtained second sample. The results are shown in Table 1.

[0079] (Examples 3-5) The first sample was cultured and colony counted in the same manner as in Example 3-1, except that the pre-culture time in the pre-culture step was 5 hours and the culture temperature was 38°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 3-1. Image analysis and absorbance measurement were performed in the same manner as in Example 3-1 using the obtained second sample. The results are shown in Table 1.

[0080] (Examples 3-6) The first sample was cultured and colony counted in the same manner as in Example 3-1, except that the pre-culture time in the pre-culture step was 5 hours and the culture temperature was 40°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 3-1. Image analysis and absorbance measurement were performed in the same manner as in Example 3-1 using the obtained second sample. The results are shown in Table 1.

[0081] [Table 1]

[0082] As shown in Table 1, in all Examples, there were two or more antibody concentrations at which the Δ absorbance and ΔB values were greater than 0. Therefore, it was confirmed that E. coli can be detected by the Δ absorbance and ΔB value. This indicates that E. coli can be detected with high accuracy by the antigen-antibody reaction. Furthermore, it was confirmed that by performing pre-culture, detection is possible even when the original concentration of microorganisms is very small. In other words, it was confirmed that very small amounts of microorganisms can be detected in a short period of time with high accuracy.

[0083] Example 4-1 <Pre-incubation and colony count of the first sample> The E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to obtain a first sample containing 1 CFU / mL of E. coli. The E. coli contained in the first sample was pre-cultured at an incubation temperature of 36°C for 4 hours (pre-culture step). A portion of the first sample after pre-culture was subjected to a colony count test. A portion of the first sample was diluted at a predetermined dilution factor, spread on an agar medium in a petri dish, and cultured at 36°C for 24 hours. The number of colonies after culture was counted and multiplied by the dilution factor to calculate the number of E. coli colonies contained in the first sample after pre-culture. The colony count was performed on two petri dishes for each sample. The results are shown in Table 2.

[0084] <Preparation of the second sample by precipitation> HRP (Whole Radish Peroxidase)-labeled E. coli antibody (HRP-E. coli antibody) was diluted with distilled water to obtain an antibody dilution solution (antibody concentration: 500 ng / mL). After pre-incubation, the first sample was diluted with PBS to prepare first sample dilutions of 0x (undiluted), 10x, 100x, and 1000x. Each first sample dilution solution was dispensed into a microtube, and the volume of each first sample dilution solution was adjusted to 450 μL. Then, 50 μL of the antibody dilution solution was added to each first sample dilution solution. After stirring for approximately 3 seconds, the mixture was incubated at 37°C for 10 minutes to allow the antigen-antibody reaction to proceed, yielding a reaction solution containing the complex.

[0085] 500 μL of distilled water was added to the reaction solution, and the mixture was stirred using a vortex mixer. The mixture was then placed in a centrifuge (Chibitan) and centrifuged for 1 minute. After centrifugation, the top 80% (800 μL) of the supernatant was carefully removed, taking care not to siphon off any precipitate. 800 μL of distilled water was then added, the mixture was again stirred using a vortex mixer, and centrifuged for 1 minute. After centrifugation, the top 80% (800 μL) of the supernatant was carefully removed, taking care not to siphon off any precipitate. In this way, unreacted antibody was removed, and four second samples were obtained.

[0086] <Absorbance measurement> A 400 μL aliquot of a color developer prepared by mixing a sodium carbonate / citrate buffer solution containing 1 wt% o-phenyldiamine with 0.3 wt% hydrogen peroxide in a 1:1 mass ratio was dispensed into each of the second samples to develop color. After stirring at 37°C for 10 minutes, 200 μL of a reaction stop solution (1 M H2SO4) was added to each of the second samples and stirred. Each sample was then dispensed in 200 μL aliquots into four wells of a microplate for each dilution factor, and the absorbance at a wavelength of 492 nm was measured using a microplate reader (manufactured by TECAN). The absorbance measurement results were evaluated by the difference (Δabsorbance) between the absorbance measurement result at a 1000-fold dilution and the absorbance measurement result at each dilution factor. The correlation coefficient R between the microbial concentration and Δabsorbance was also calculated. 2 The results are shown in Table 2.

[0087] <Image analysis> After measuring the absorbance, the microplate was placed on a piece of white paper and photographed from above using a tablet device. The photographing conditions were indoor lighting, with the distance between the object to be photographed and the lens being 15 cm. Image analysis of the photographed images was carried out using "Image Sensing 2023 (product name)" (manufactured by ARS Corporation). In the image analysis, a division process was carried out using a 1000-fold diluted sample as the reference. After that, hue correction was carried out, and the blue intensity (ΔB value) was calculated as the difference from the 1000-fold diluted sample. In addition, the correlation coefficient R between the microbial concentration and the ΔB value was calculated. 2 The results are shown in Table 2.

[0088] (Example 4-2) The first sample was cultured and colony counted in the same manner as in Example 4-1, except that the pre-culture time in the pre-culture step was 6 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 4-1. Absorbance measurement and image analysis were performed in the same manner as in Example 4-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0089] (Example 4-3) The first sample was cultured and colony counted in the same manner as in Example 4-1, except that the pre-culture time in the pre-culture step was 8 hours. The second sample was prepared using the cultured first sample in the same manner as in Example 4-1. Absorbance measurement and image analysis were performed in the same manner as in Example 4-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0090] (Example 4-4) The first sample was cultured and colony counted in the same manner as in Example 4-1, except that the pre-culture time in the pre-culture step was 12 hours. The second sample was prepared using the cultured first sample in the same manner as in Example 4-1. Absorbance measurement and image analysis were performed in the same manner as in Example 4-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0091] (Example 5-1) E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to prepare a first sample containing 10 CFU / mL of E. coli. This first sample was cultured and colony counted using the same procedure as in Example 4-1. Using this first sample, a second sample was prepared by carrying out "preparation of a second sample by precipitation" using the same procedure as in Example 4-1. Using the obtained second sample, absorbance measurement and image analysis were carried out using the same procedure as in Example 4-1. The results are shown in Table 2.

[0092] (Example 5-2) The first sample was cultured and colony counted in the same manner as in Example 5-1, except that the pre-culture time in the pre-culture step was 6 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 5-1. Absorbance measurement and image analysis were performed in the same manner as in Example 5-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0093] (Example 5-3) The first sample was cultured and colony counted in the same manner as in Example 5-1, except that the pre-culture time in the pre-culture step was 8 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 5-1. Absorbance measurement and image analysis were performed in the same manner as in Example 5-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0094] (Example 5-4) The first sample was cultured and colony counted in the same manner as in Example 5-1, except that the pre-culture time in the pre-culture step was 12 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 5-1. Absorbance measurement and image analysis were performed in the same manner as in Example 5-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0095] Example 6-1 E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to prepare a first sample containing 100 CFU / mL of E. coli. This first sample was cultured and colony counted using the same procedure as in Example 4-1. Using this first sample, a second sample was prepared by carrying out "preparation of a second sample by precipitation" using the same procedure as in Example 4-1. Using the obtained second sample, absorbance measurement and image analysis were carried out using the same procedure as in Example 4-1. The results are shown in Table 2.

[0096] (Example 6-2) The first sample was cultured and colony counted in the same manner as in Example 6-1, except that the pre-culture time in the pre-culture step was 6 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 6-1. Absorbance measurement and image analysis were performed in the same manner as in Example 6-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0097] (Example 6-3) The first sample was cultured and colony counted in the same manner as in Example 6-1, except that the pre-culture time in the pre-culture step was 8 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 6-1. Absorbance measurement and image analysis were performed in the same manner as in Example 6-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0098] (Example 6-4) The first sample was cultured and colony counted in the same manner as in Example 6-1, except that the pre-culture time in the pre-culture step was 12 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 6-1. Absorbance measurement and image analysis were performed in the same manner as in Example 6-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 2.

[0099] (Example 7-1) The first sample was cultured and colony counted in the same manner as in Example 4-2, except that the culture temperature in the pre-culture step was 28°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 4-2. Absorbance measurement and image analysis were performed in the same manner as in Example 4-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0100] (Example 7-2) The first sample was cultured and colony counted in the same manner as in Example 4-2, except that the culture temperature in the pre-culture step was 32°C. The second sample was prepared using the cultured first sample in the same manner as in Example 4-2. Absorbance measurement and image analysis were performed in the same manner as in Example 4-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0101] (Example 7-3) The first sample was cultured and colony counted in the same manner as in Example 4-2, except that the culture temperature in the pre-culture step was 40°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 4-2. Absorbance measurement and image analysis were performed in the same manner as in Example 4-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0102] (Example 8-1) The first sample was cultured and colony counted in the same manner as in Example 5-2, except that the culture temperature in the pre-culture step was 28°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 5-2. Absorbance measurement and image analysis were performed in the same manner as in Example 5-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0103] (Example 8-2) The first sample was cultured and colony counted in the same manner as in Example 5-2, except that the culture temperature in the pre-culture step was 32°C. The second sample was prepared using the cultured first sample in the same manner as in Example 5-2. Absorbance measurement and image analysis were performed in the same manner as in Example 5-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0104] (Example 8-3) The first sample was cultured and colony counted in the same manner as in Example 5-2, except that the culture temperature in the pre-culture step was 40°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 5-2. Absorbance measurement and image analysis were performed in the same manner as in Example 5-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0105] (Example 9-1) The first sample was cultured and colony counted in the same manner as in Example 6-2, except that the culture temperature in the pre-culture step was 28°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 6-2. Absorbance measurement and image analysis were performed in the same manner as in Example 6-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0106] (Example 9-2) The first sample was cultured and colony counted in the same manner as in Example 6-2, except that the culture temperature in the pre-culture step was 32°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 6-2. Absorbance measurement and image analysis were performed in the same manner as in Example 6-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0107] (Example 9-3) The first sample was cultured and colony counted in the same manner as in Example 6-2, except that the culture temperature in the pre-culture step was 40°C. The first sample after culture was used to prepare a second sample in the same manner as in Example 6-2. Absorbance measurement and image analysis were performed in the same manner as in Example 6-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 3.

[0108] [Table 2]

[0109] [Table 3]

[0110] As shown in Tables 2 and 3, in all Examples, the correlation coefficient between Δ absorbance and microbial concentration was 0.8 or higher, indicating a high correlation. Furthermore, the ΔB value also tended to show a high correlation with the microbial concentration. Therefore, it was demonstrated that microorganisms can be detected quickly and with high accuracy based on the Δ absorbance and ΔB value. By detecting microorganisms using both the Δ absorbance and ΔB value, the presence and content of microorganisms can be double-checked, improving the reliability of the measurement results. Furthermore, even in dilute samples with microbial concentrations of approximately 1, 10, or 100 CFU / mL, microorganisms could be detected by performing a pre-incubation step. Therefore, it was demonstrated that microbial detection can be performed with sufficiently high accuracy.

[0111] (Example 10-1) <Pre-incubation and colony count of the first sample> The E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to obtain a first sample containing 1 CFU / mL of E. coli. The E. coli contained in the first sample was pre-cultured at an incubation temperature of 36°C for 12 hours (pre-culture step). A portion of the first sample after pre-culture was subjected to a colony count test. A portion of the first sample was diluted at a predetermined dilution factor, spread on an agar medium in a petri dish, and cultured at 36°C for 24 hours. The number of colonies after culture was counted and multiplied by the dilution factor to calculate the number of E. coli colonies contained in the first sample after pre-culture. The colony count was performed on two petri dishes for each sample. The results are shown in Table 4.

[0112] <Preparation of the second sample using a separation membrane> HRP (Whole Radish Peroxidase)-labeled E. coli antibody (HRP-E. coli antibody) was diluted with distilled water to obtain an antibody dilution solution (antibody concentration: 500 ng / mL). After pre-incubation, the first sample was diluted with PBS to prepare first sample dilutions of 0x (undiluted), 10x, 100x, and 1000x. Each first sample dilution solution was dispensed into a microtube, and the volume of each first sample dilution solution was adjusted to 450 μL. Then, 50 μL of antibody dilution solution was added to each first sample dilution solution. After stirring for approximately 3 seconds, the mixture was incubated at 37°C for 10 minutes to allow the antigen-antibody reaction to proceed, yielding 500 μL of reaction solution containing the complex.

[0113] 500 μL of distilled water was dispensed into a spin column with a PTFE separation membrane with a pore size of 200 nm inside, and the column was placed in a centrifuge (Chibitan) and centrifuged for 1 minute to wash the spin column. The distilled water was then discarded, and 500 μL of the reaction solution was dispensed into the spin column. The column was placed in a centrifuge (Chibitan) and centrifuged for 2 minutes, and the liquid that had seeped to the bottom of the separation membrane was discarded. Another 500 μL of distilled water was dispensed, and the column was placed in a centrifuge (Chibitan) and centrifuged for 2 minutes, and all liquid above and below the separation membrane was discarded. In this way, the complex was collected on the separation membrane inside the spin column, and a second sample was prepared. The complexes from the first sample diluted solution at each dilution ratio were collected on the separation membrane using the same procedure, and four second samples were obtained.

[0114] <Image analysis> A 300 μL aliquot of a coloring agent prepared by mixing a sodium carbonate / citrate buffer solution containing 1 wt% o-phenyldiamine with 0.3 wt% hydrogen peroxide in a 1:1 mass ratio was dispensed into each of the second samples to develop color. After stirring at 37°C for 10 minutes, 100 μL of a reaction stop solution (1 M H2SO4) was added to each second sample and stirred. The spin columns after color development were placed in a microtube rack and photographed from above using a tablet device. The photographs were taken under indoor lighting with a distance of 20 cm from the subject to the lens. Image analysis of the captured images was performed using an "Image Sensing 2023 (product name)" (manufactured by ARS Corporation). A 1000-fold diluted sample was used as the reference for the image analysis. After hue correction, the blue intensity (ΔB value) was calculated as the difference from the 1000-fold diluted sample. The correlation coefficient R between the microbial concentration and the ΔB value was also calculated. 2 The results are shown in Table 4.

[0115] <Absorbance measurement> After photographing, 200 μL of the second sample was dispensed into four wells of a microplate for each dilution factor, and the absorbance at a wavelength of 492 nm was measured using a microplate reader (manufactured by TECAN). The results of the absorbance measurements were evaluated as the difference (Δ absorbance) between the absorbance measurement result of the 1000-fold dilution and the absorbance measurement result at each dilution factor. In addition, the correlation coefficient R between the microbial concentration and Δ absorbance was calculated. 2 The results are shown in Table 4.

[0116] (Example 10-2) The E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to obtain a first sample containing 10 CFU / mL of E. coli. The first sample was cultured and colony counted in the same manner as in Example 10-1, except that the culture time was 8 hours. Furthermore, a second sample was prepared from the cultured first sample in the same manner as in Example 10-1. Image analysis and absorbance measurement were performed using the obtained second sample in the same manner as in Example 10-1. The results are shown in Table 4.

[0117] (Example 10-3) The first sample was cultured and colony counted in the same manner as in Example 10-2, except that the culture time was 12 hours. The second sample was prepared using the first sample after culture in the same manner as in Example 10-2. Absorbance measurement and image analysis were performed in the same manner as in Example 10-2, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 4.

[0118] (Example 10-4) The E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to obtain a first sample containing 100 CFU / mL of E. coli. The first sample was cultured and colony counted in the same manner as in Example 10-1, except that the culture time was 8 hours. Furthermore, a second sample was prepared from the cultured first sample in the same manner as in Example 10-1. Image analysis and absorbance measurement were performed using the obtained second sample in the same manner as in Example 10-1. The results are shown in Table 4.

[0119] (Example 10-5) The first sample was cultured and colony counted in the same manner as in Example 10-1, except that the culture time was 12 hours. The first sample after culture was used to prepare a second sample in the same manner as in Example 10-1. Image analysis and absorbance measurement were performed in the same manner as in Example 10-1, and the Δabsorbance and ΔB values were calculated. The results are shown in Table 4.

[0120] [Table 4]

[0121] As shown in Table 4, it was confirmed that microorganisms could be detected with high accuracy based on the Δ absorbance and ΔB values even when a spin column was used to prepare the second sample.

[0122] Example 11 <Pre-incubation and colony count of the first sample> The E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to obtain a first sample containing 100 CFU / mL of E. coli. The E. coli contained in the first sample was pre-cultured at an incubation temperature of 36°C for 4 hours (pre-culture step). A portion of the first sample after pre-culture was subjected to a colony count test. A portion of the first sample was diluted with phosphate-buffered saline (PBS) at a predetermined dilution ratio, then spread on an agar medium in a petri dish, and cultured at 36°C for 24 hours. The number of colonies after culture was counted, and multiplied by the dilution ratio to calculate the number of E. coli colonies contained in the first sample after pre-culture. The colony count was performed on two petri dishes, and the average was calculated. The results are shown in Table 5.

[0123] <Preparation of the second sample by precipitation> HRP (Whole Radish Peroxidase)-labeled E. coli antibody (HRP-E. coli antibody) was diluted with distilled water to obtain an antibody dilution solution (antibody concentration: 500 ng / mL). After pre-incubation, the first sample was diluted with distilled water to prepare first sample dilutions of 0x (undiluted), 10x, 100x, and 1000x. Each first sample dilution solution was dispensed into a microtube, and the volume of each first sample dilution solution was adjusted to 450 μL. Then, 50 μL of the antibody dilution solution was added to each first sample dilution solution. After stirring for approximately 3 seconds, the mixture was incubated at 37°C for 10 minutes to allow the antigen-antibody reaction to proceed, yielding a reaction solution containing the complex.

[0124] 500 μL of distilled water was added to the reaction solution, and the mixture was stirred using a vortex mixer. The mixture was then placed in a centrifuge (Chibitan) and centrifuged for 1 minute. After centrifugation, the top 80% (800 μL) of the supernatant was carefully removed, taking care not to siphon off any precipitate. 800 μL of distilled water was then added, the mixture was again stirred using a vortex mixer, and centrifuged for 1 minute. After centrifugation, the top 80% (800 μL) of the supernatant was carefully removed, taking care not to siphon off any precipitate. In this way, unreacted antibody was removed, and four second samples were obtained.

[0125] <Absorbance measurement> A 400 μL aliquot of a color developer prepared by mixing a sodium carbonate / citrate buffer solution containing 1 wt% o-phenyldiamine with 0.3 wt% hydrogen peroxide in a 1:1 mass ratio was dispensed into each of the second samples to develop color. After stirring at 37°C for 10 minutes, 200 μL of a reaction stop solution (1 M H2SO4) was added to each of the second samples and stirred. Each sample was then dispensed in 200 μL aliquots into four wells of a microplate for each dilution factor, and the absorbance at a wavelength of 492 nm was measured using a microplate reader (manufactured by TECAN). The absorbance measurement results were evaluated by the difference (Δabsorbance) between the absorbance measurement result at a 1000-fold dilution and the absorbance measurement results at each dilution factor. The correlation coefficient R between the microbial concentration and Δabsorbance was also calculated. 2 The results are shown in Table 5.

[0126] [Comparison of shooting conditions in image analysis and verification without division processing] (Example 11-1) <Image analysis> After measuring the absorbance, the microplate was placed on a piece of white paper and photographed from above using a tablet device. The photographing conditions were indoor lighting, with the distance from the object to the lens set at 15 cm. The photographed images were analyzed using "Image Sensing 2023 (product name)" (manufactured by ARS Corporation). No division processing was performed in the image analysis. After that, hue correction was performed, and the blue intensity (ΔB value) was calculated as the difference from a 1000-fold diluted sample. The correlation coefficient R between the microbial concentration and the ΔB value was also calculated. 2 The results are shown in Table 5.

[0127] (Example 11-2) Image analysis: After measuring absorbance, the microplate was placed on a colony viewer (manufactured by Japan Bacteria Inspection Co., Ltd.), and photographed from above using a tablet device with transmitted light shining from below. The light intensity was set to low, and the distance from the object to the lens was set to 15 cm. Image analysis of the photographed images was carried out using "Image Sensing 2023 (product name)" (manufactured by ARS Corporation). No division processing was performed in the image analysis. After that, hue correction was carried out, and the blue intensity (ΔB value) was calculated as the difference from a 1000-fold diluted sample. The correlation coefficient R between the microbial concentration and ΔB value was also calculated. 2 The results are shown in Table 5.

[0128] (Example 11-3) After measuring the absorbance, the microplate was placed on a piece of white paper and covered with a cardboard box. A hole was made in the top of the cardboard box, and an image was taken from above using a smartphone. The distance from the object to be photographed to the lens was 21 cm. A polarizing film was attached to the lens, and the image was taken with a flash. Image analysis of the photographed image was carried out using "Image Sensing 2023 (product name)" (manufactured by ARS Corporation). No division processing was performed in the image analysis. After that, hue correction was carried out, and the blue intensity (ΔB value) was calculated as the difference from a 1000-fold diluted sample. The correlation coefficient R between the microbial concentration and the ΔB value was also calculated. 2 The results are shown in Table 5.

[0129] [Table 5]

[0130] As shown in Table 5, the ΔB value showed a high correlation coefficient even without division processing. Therefore, it was demonstrated that microorganisms can be detected with high accuracy by image analysis even without division processing. It was also demonstrated that microorganisms can be detected in the same way even when the photographing conditions are changed.

[0131] [Method of detecting microorganisms using Gram staining] (Example 12-1) <Pre-incubation of the first sample> The E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to obtain a first sample containing 1 CFU / mL of E. coli. The E. coli contained in the first sample was pre-cultured at an incubation temperature of 28°C for 6 hours (pre-culture step).

[0132] <Preparation of the second sample using Gram staining and separation membrane> 500 μL of Gram stain solution (Victoria Blue solution, BB solution) was dispensed into a spin column with a 200 nm pore size PTFE membrane inside, and the column was placed in a centrifuge (Chibitan) and centrifuged for 2 minutes. The filtrate was collected and centrifuged for another 2 minutes using a spin column with a 300 kD PTFE membrane inside. The filtrate was collected again and centrifuged for another 2 minutes using a spin column with a 100 kD PTFE membrane inside. This centrifugation was repeated three times to remove colored particles from the stain solution and obtain a purified stain solution. Next, another spin column with a 200 nm pore size PTFE membrane inside was prepared, and 500 μL of the first sample obtained in the pre-culture step was dispensed into the spin column. 100 μL of the purified stain solution described above was then added, the column was placed in a centrifuge (Chibitan), and centrifuged for 10 minutes. Gram-stained E. coli was collected on the membrane to obtain a second sample.

[0133] The separation membrane containing the E. coli was removed from the spin column and attached to a glass slide with double-sided tape. It was then observed and photographed at a magnification of 4000x using a digital microscope (product name: "VHX-8000", manufactured by Keyence Corporation). The photographed image is shown in Figure 1.

[0134] <Calculation of the number of captured bacteria> Three randomly selected points on the separation membrane were photographed, and the number of bacteria in one image (76 μm long x 102 μm wide at a magnification of 4000x) was visually counted and divided by the area of the image to determine the number of E. coli bacteria captured on the separation membrane per unit area (number / mm 2 The number of captured bacteria was calculated for each of the three images (n=3) taken, and the average value was calculated. The results are shown in Table 6.

[0135] If there are too many E. coli bacteria on the image to be counted visually, select any three sections from the image, count the number of bacteria in each section, and divide this by the area of the three sections to determine the number of E. coli bacteria captured by the separation membrane (number of bacteria / mm 2 ) was calculated.

[0136] (Example 12-2) A first sample and a second sample were prepared in the same manner as in Example 12-1, except that the culture temperature in the pre-culture step was set to 32°C. Using the obtained second sample, the number of captured bacteria on the separation membrane was determined in the same manner as in Example 12-1. The photographed images are shown in Figure 2. The results are shown in Table 6.

[0137] (Example 12-3) A first sample and a second sample were prepared in the same manner as in Example 12-1, except that the culture temperature in the pre-culture step was set to 36°C. Using the obtained second sample, the number of bacteria captured on the separation membrane was determined in the same manner as in Example 12-1. The photographed images are shown in Figure 3. The results are shown in Table 6.

[0138] (Example 12-4) A first sample and a second sample were prepared in the same manner as in Example 12-1, except that the culture temperature in the pre-culture step was 40°C. Using the obtained second sample, the number of captured bacteria on the separation membrane was determined in the same manner as in Example 12-1. The photographed images are shown in Figure 4. The results are shown in Table 6.

[0139] (Example 13-1) E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to prepare a first sample containing 10 CFU / mL of E. coli. This first sample was used to prepare a second sample by culturing and Gram staining in the same manner as in Example 12-1, and the number of bacteria captured on the separation membrane was determined. The photographed image is shown in Figure 5. The results are shown in Table 6.

[0140] (Example 13-2) A first sample and a second sample were prepared in the same manner as in Example 13-1, except that the culture temperature in the pre-culture step was set to 32°C. Using the obtained second sample, the number of captured bacteria on the separation membrane was determined in the same manner as in Example 13-1. The photographed images are shown in Figure 6. The results are shown in Table 6.

[0141] (Example 13-3) A first sample and a second sample were prepared in the same manner as in Example 13-1, except that the culture temperature in the pre-culture step was set to 36°C. Using the obtained second sample, the number of captured bacteria on the separation membrane was determined in the same manner as in Example 13-1. The photographed images are shown in Figure 7. The results are shown in Table 6.

[0142] (Example 13-4) A first sample and a second sample were prepared in the same manner as in Example 13-1, except that the culture temperature in the pre-culture step was 40°C. Using the obtained second sample, the number of captured bacteria on the separation membrane was determined in the same manner as in Example 13-1. The photographed images are shown in Figure 8. The results are shown in Table 6.

[0143] (Example 14-1) E. coli was diluted in a liquid medium (NUTRIENT BROTH (trade name), manufactured by OXOID) to prepare a first sample containing 100 CFU / mL of E. coli. This first sample was used to prepare a second sample by culturing and Gram staining in the same manner as in Example 12-1, and the number of bacteria captured on the separation membrane was determined. The photographed image is shown in Figure 9. The results are shown in Table 6.

[0144] (Example 14-2) The first and second samples were prepared in the same manner as in Example 14-1, except that the culture temperature in the pre-culture step was set to 32°C. Using the obtained second sample, the number of captured bacteria on the separation membrane was determined in the same manner as in Example 14-1. The photographed images are shown in Figure 10. The results are shown in Table 6.

[0145] (Example 14-3) The first and second samples were prepared in the same manner as in Example 14-1, except that the culture temperature in the pre-culture step was 36°C. Using the obtained second sample, the number of captured bacteria on the separation membrane was determined in the same manner as in Example 14-1. The photographed images are shown in Figure 11. The results are shown in Table 6.

[0146] (Example 14-4) A first sample and a second sample were prepared in the same manner as in Example 14-1, except that the culture temperature in the pre-culture step was 40°C. Using the obtained second sample, the number of captured bacteria on the separation membrane was determined in the same manner as in Example 14-1. The photographed images are shown in Figure 12. The results are shown in Table 6.

[0147] [Table 6]

[0148] As shown in Table 6 and Figures 1 to 12, it was confirmed that by performing Gram staining, E. coli on the separation membrane could be observed as live bacteria. Furthermore, it was confirmed that by performing pre-culture, even samples with E. coli concentrations of approximately 1 to 100 CFU / mL could be detected with high accuracy. Therefore, it was demonstrated that microorganisms can be detected with higher accuracy in a shorter period of time than the colony counting method, which requires 24 hours. [Industrial Applicability]

[0149] According to the present disclosure, a method for detecting microorganisms is provided that is capable of detecting microorganisms in a short period of time with high accuracy.

Claims

1. a separation step of centrifuging a first sample containing microorganisms to obtain a second sample in which components other than the microorganisms have been reduced compared to the first sample; a detection step of detecting the microorganism contained in the second sample, A method for detecting microorganisms, comprising a coloring step of coloring the microorganisms or a complex containing the microorganisms before or after the separation step.

2. The method for detecting microorganisms according to claim 1 , wherein the coloring step includes obtaining the complex through an antigen-antibody reaction between the microorganism and an antibody.

3. 2. The method for detecting microorganisms according to claim 1, wherein the second sample is obtained in the separation step by collecting the microorganisms on a separation membrane or by precipitating the microorganisms.

4. The method for detecting a microorganism according to claim 1 , wherein the detection step detects the microorganism using at least one method selected from the group consisting of absorbance measurement and image analysis.

5. The method for detecting microorganisms according to claim 1, wherein the coloring step includes Gram-staining the microorganisms contained in the first sample, or coloring the complex containing the microorganisms obtained by an antigen-antibody reaction.

6. The method for detecting microorganisms according to claim 1 , wherein in the separation step, the microorganisms are collected on a separation membrane to obtain the second sample.

7. The method for detecting microorganisms according to claim 6 , wherein the detection step detects the microorganisms collected on the separation membrane by imaging.

8. The method for detecting microorganisms according to any one of claims 1 to 7, further comprising a pre-culture step of culturing the microorganisms before the separating step or the staining step.

9. The method for detecting microorganisms according to claim 8, wherein the culture time in the pre-culture step is 4 to 12 hours.

10. The method for detecting microorganisms according to any one of claims 1 to 7, wherein the microorganisms include Escherichia coli.

11. The detection step includes: detecting the complex obtained by an antigen-antibody reaction between the microorganism and an antibody labeled with a first metal compound using electrochemical measurement; and detecting the complex obtained by the antigen-antibody reaction between the microorganism and the antibody by electrochemical measurement in the presence of a sensitizer containing a second metal compound; The method for detecting microorganisms according to any one of claims 1 to 3, comprising at least one of the following:

12. a pre-incubation step of culturing a first sample containing microorganisms to obtain a second sample containing the microorganisms in a larger amount than the first sample; a detection step including at least one of detecting a first complex obtained by an antibody-antigen reaction between the microorganism contained in the second sample and an antibody labeled with a first metal compound, using electrochemical measurement, and detecting a second complex obtained by an antigen-antibody reaction between the microorganism and an antibody, using electrochemical measurement in the coexistence of a sensitizer containing a second metal compound; A method for detecting microorganisms, comprising:

Images