JP2017085938A - Method of detecting microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect microorganism quickly and highly sensitively necessitating neither a complicated procedure nor an expensive apparatus or consumable.SOLUTION: A method of detecting microorganism comprises: a step of fixing test sample 1a on the surface of a base material 1b in a desorbable manner; a step of covering test sample 1a fixed on the base material surface with a solidifiable liquid material 1c, solidifying the liquid material 1c covering the test sample 1a, thereby fixing the test sample 1a on the surface of the base material 1b; a step of desorbing the liquid material 1c of a solidified state from the base material 1b while the test sample 1a is contained in the solidified liquid material 1c; and a step of labeling the test sample 1a from a face of the base material 1b side of the liquid material 1c in the solidified state by using reagent which labels a microorganism to be detected, and then detecting the microorganism 1f to be detected.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for detecting a microorganism.

  In the management of manufacturing processes in the fields of pharmaceuticals and foods, tests such as confirmation of the number of microorganisms present in the environment and identification of microbial species are important from the viewpoints of hygiene management, investigation of the cause of contamination, and measures against biohazards Is being viewed. Conventionally, culture methods have been generally used in these microbial tests, but the culture methods require sterilization of the culture medium and laboratory equipment, and skill in the operation is required for operation in an aseptic environment. In addition, in the culture method, it takes several days for the microorganisms to grow and form detectable colonies. In addition, in order to identify the detected microorganism species, enrichment culture, selective separation culture, identification It is necessary to perform culture and repeated culture. Therefore, a great amount of labor and time are required until a test result is obtained.

In recent years, as a simple and rapid detection method for microorganisms, a kit using an immunological detection method such as an enzyme immunoassay (ELISA) method or an immunochromatography method, or a gene detection method such as a DNA probe method or a polymerase chain reaction (PCR) method. Has been developed and is commercially available.
However, the ELISA method, immunochromatography method, and DNA probe method have problems in detection sensitivity, and it is considered desirable to use in combination with a culture method such as enrichment culture. In addition, the PCR method may not be applicable depending on the sample because impurities in the sample may inhibit nucleic acid amplification. Furthermore, there is a problem that gene detection methods generally cannot distinguish between live and dead bacteria.

As a method for discriminating between live and dead bacteria, a method of detecting adenosine triphosphate (ATP) using luciferase, which is a luminescent enzyme, is known (for example, Non-Patent Document 1). However, since the ATP measurement method is affected by free ATP derived from food residues and the like, the number of microorganisms cannot be quantified although it serves as an index of cleanliness.
For example, Non-Patent Document 2 discloses EMA-PCR using ethidium monoazide (EMA) that penetrates only damaged cell membranes and covalently binds to nucleic acids by exposure to visible light, as a gene detection method selective for viable bacteria. A law has been proposed. In this method, nucleic acid amplification by PCR is inhibited because EMA modifies the nucleic acid in dead bacteria with cell membrane damage, but nucleic acid is amplified without being affected by EMA in live bacteria without cell membrane damage.

Further, for example, in the technique of Patent Document 1, the application range of a sample is expanded by adding a PCR inhibitory action suppressing substance, and a reaction in a microbial cell is realized. However, the electrophoresis and real-time PCR methods exemplified as detection methods in Patent Document 1 are quick in that they require several hours for post-reaction processing and are relative quantifications that require the creation of a calibration curve. There is a problem in coexistence of stability and quantitativeness.
As a method for detecting a sample derived from a living body with high sensitivity and performing absolute quantification, there are digital analysis techniques represented by digital ELISA and digital PCR (see, for example, Patent Document 2 and Patent Document 3). In this method, a reaction solution containing a sample and a reagent is separated into a large number of microscales and reacted, and a signal such as fluorescence obtained from each microsolution is binarized to determine only the presence or absence of a detection molecule. is there. The digital analysis method is a method for separating the reaction solution into microscales. The emulsion method uses minute droplets (water droplets in oil) formed by dispersing the reaction solution in oil as the reaction field, and glass and resin. It is roughly classified into a microwell system using a minute well formed on a substrate as a reaction field.

Japanese Patent No. 4825313 US Registered Patent No. 6391559 International Publication No. 2013/084678

Supervised by Microbiology, Shinnobu Igo, Takayuki Esaki, Kosuke Takatori, Tetsuaki Tudo (2013) 19. Rudi, K .; Naterstad, K .; , Dromtorp, S .; M.M. and Holo, H .; (2005) Lett. Appl. Microbiol. , Vol. 40, pp. 301-306.

However, since the emulsion type digital analysis requires a device for preparing microdroplets, a temperature control device during reaction, and a detection device called a flow cytometer, expensive equipment is required and the procedure required for testing is required. There is a problem that it becomes complicated. On the other hand, in the digital analysis of the microwell method, a chip or plate having a large number of tens of thousands of microwells with a diameter and height of several μm on the base material is necessary as a consumable, and the test cost is high. Become.
Therefore, an object of the present invention is to provide a rapid and highly sensitive microorganism detection method that does not require complicated procedures and does not require expensive equipment or consumables.

  According to one aspect of the present invention, the step of fixing the test sample to the surface of the base material so as to be removable, and the liquid material capable of solidifying the test sample fixed to the base material surface. A step of solidifying the liquid material covering and covering the test sample to fix the test sample to the surface of the base material; and the solidified liquid material from the base material including the test sample. Using the reagent for labeling the microorganism to be detected and a reagent for labeling the microorganism to be detected, after labeling the test sample from the substrate side surface of the solidified liquid material, the microorganism to be detected is detected. And a step of performing a microorganism detection method.

  According to another aspect of the present invention, the step of fixing the test sample to the surface of the base material and the test sample fixed to the surface of the base material are covered with a liquid material that can be solidified. A step of solidifying a liquid material covering the test sample and fixing the test sample to the surface of the base material; and a reagent for labeling a detection target microorganism is diffused in the liquid material in the solidified state to solidify the liquid sample And a step of detecting the microorganism to be detected from the surface of the liquid material on the side where the reagent is diffused.

  According to one embodiment of the present invention, rapid and highly sensitive detection of microorganisms can be performed at low cost without performing culture that requires complicated procedures or advanced techniques.

It is sectional drawing which shows an example of the detection process of the detection method of the microorganisms which concern on 1st embodiment of this invention. It is sectional drawing which shows an example of the detection process of the detection method of the microorganisms which concern on 2nd embodiment of this invention. It is an example of the microscopic image of the microbead fixed using the UV hardening resin on the PET film | membrane base material. It is an example of the microscopic image of the microbead fixed using the agarose gel on the glass plate. It is an example of the microscope image of the substrate side surface of UV hardening resin which peeled mechanically after solidifying on the agarose gel base material. It is an example of the microscope image of the base-material side surface of UV hardening resin which isolate | separated after fixing the biobead-coated microbead on the agarose gel base material. It is an example of the microscope image of the surface of the base material side after the FITC labeling streptavidin treatment of the UV curable resin detached after fixing the biobead-coated microbeads on the agarose gel base material.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
In the following detailed description, specific specific configurations are set forth in order to provide a thorough understanding of embodiments of the present invention. However, it will be apparent that other embodiments may be practiced without limitation to such specific specific configurations. The following embodiments do not limit the invention according to the claims. In addition, not all the combinations of features described in the embodiments are essential for the solving means of the invention.
Below, 1st embodiment of the detection method of the microorganisms which concern on this invention is described, referring FIG. Fig.1 (a)-(d) is the schematic diagram showing the cross section which shows 1st embodiment of the detection method of the microorganisms which concern on this invention in order of a process.

  First, as a first step, as shown in FIG. 1A, the test sample 1a is fixed on the surface of the substrate 1b. At this time, the test sample 1a fixed on the surface of the base material 1b is fixed reversibly with such an intensity that it can be detached from the surface of the base material 1b. The method of fixing the test sample 1a to the substrate 1b differs depending on the form of the test sample 1a. For example, when the test sample 1a is airborne bacteria, the test sample 1a is captured as falling bacteria by leaving the surface of the base material 1b upward for a certain period of time. Alternatively, airborne samples are collected on the base material 1b by collecting air with an air sampler.

When the test sample 1a is, for example, an adherent bacterium on a solid, a homogenized suspension or swab extract containing the adherent bacterium on the solid is dropped onto the surface of the substrate 1b, and if necessary, a solvent or dispersion medium By volatilizing, the adherent bacteria on the solid as the test sample 1a can be fixed on the surface of the substrate 1b. However, the method is not particularly limited as long as the microorganisms to be detected can be reversibly fixed on the surface of the base material 1b with a detachable strength.
The surface portion of the substrate 1b that comes into contact with the test sample 1a can be used without limitation as long as it is a material that does not destroy microorganism cells and does not prevent microorganisms from being fixed, but glass, metal, resin A thin film made of paper, hydrogel is preferably used. More preferably, a base material having a surface subjected to hydrophilic treatment is used. Examples of the hydrophilic treatment performed to increase the affinity between the target microbial cell and the substrate surface include plasma treatment, corona treatment, UV treatment, or fluorine gas treatment, but the desired effect is obtained. Any processing method can be used without particular limitation.

  Next, as a second step, as shown in FIG. 1B, the test sample 1a on the substrate 1b is fixed so as not to flow with the liquid material 1c that can be solidified by an arbitrary method. . After the liquid material 1c is dropped or applied so as to cover the test sample 1a fixed on the substrate 1b, the liquid material 1c is solidified by applying a solidification process so that the test sample 1a does not flow. To fix. As the liquid material 1c, a resin that can be cured by at least one solidification treatment selected from heating, light irradiation, two-component mixing, drying, and water addition is suitably used. However, when the cell body of the microorganism that is the test sample 1a, that is, the shape of the cell body, can be destroyed by heat or mechanical stress that is added or generated secondaryly when the resin is solidified. Excludes this. In addition, when light irradiation is performed as the solidification treatment, it is desirable to use light having a wavelength that does not fragment the nucleic acid in the test sample, but there is no particular limitation when labeling and detecting substances other than nucleic acid.

  Another liquid material 1c that can be suitably used includes a hydrogel that can lose its fluidity by a solidification treatment selected from at least one of heating, cooling, and addition of multivalent ions. Here, the hydrogel contains a gelling agent in a concentration range capable of exhibiting strength capable of being detached from the substrate 1b while maintaining its shape. As the gelling agent, polysaccharides, polyuronic acids, proteins, and synthetic polymers are preferably used, but are not particularly limited as long as the desired effects can be obtained. However, this is excluded when the shape of the bacterial cell as the test sample can be destroyed by heat or mechanical stress that is added or generated secondaryly when the hydrogel is solidified.

Next, as a third step, as shown in FIG. 1C, the solidified liquid material 1c is detached from the substrate 1b. At this time, the shape of the test sample 1a fixed in the liquid material 1c is maintained, and solidification is performed in a state where a part of the test sample 1a is exposed to the substrate-side surface 1d of the solidified liquid material 1c. The liquid material 1c in a state is detached from the base material 1b.
Examples of the method of detaching the solidified liquid material 1c from the substrate 1b while including the test sample 1a include mechanical peeling and removal by melting or dissolving the substrate 1b. However, as long as it is a method capable of detaching in a state where a part of the test sample 1a is exposed to the outside of the solidified liquid material 1c on the substrate-side surface 1d of the solidified liquid material 1c, It is not limited.

  And as shown in FIG.1 (d), as a 4th process, the microbial cell of the test sample 1a fixed in the solidified liquid material 1c detached | separated from the base material 1b is made into a reaction chamber, a base material. A part of the test sample 1a exposed on the side surface 1d is used as an opening of the reaction chamber, and the test sample 1a is labeled and detected using the reagent 1e that labels the detection target microorganism. That is, on the substrate-side surface 1d of the solidified liquid material 1c, a portion where the fungus bodies as the test sample 1a are exposed becomes an open recess when the test sample 1a is removed. That is, this is equivalent to the formation of a recess in the solidified liquid material 1c, and is equivalent to a state in which the fungus bodies as the test sample 1a are embedded in the recess so as to be partially exposed. . Therefore, the concave portion formed in the portion of the test sample 1a of the solidified liquid material 1c can be regarded as a reaction chamber, and the opening portion of the concave portion can be regarded as an opening portion of the reaction chamber. Therefore, the sample 1a is labeled and detected using the labeling reagent 1e, with the recess formed in the solidified liquid material 1c as the reaction chamber and the opening of the recess as the opening of the reaction chamber.

Specifically, first, the solidified liquid material 1c detached from the substrate 1b in the third step is turned upside down, and the substrate-side surface 1d is set as the upper surface. Next, the labeling reagent 1e is dropped, applied or fed onto the substrate side surface 1d. Then, signal detection of the particles labeled with the labeling reagent 1e in the test sample 1a, that is, the detection target microorganism 1f is performed.
For this signal detection, at least one of optical detection and microscopic observation is preferably used. More preferably, microscopic observation of the substrate side surface 1d and image analysis of imaging data are used.

  Thus, in the method of labeling and detecting the test sample 1a with the labeling reagent 1e, the test sample 1a exists in the reaction chamber formed in the solidified liquid material 1c, and the test sample Since the test sample 1a including the detection target microorganism 1f in 1a emits a signal, the number of signals obtained by microscopic observation of the substrate-side surface 1d and image analysis of the imaging data is the area of the substrate-side surface 1d. This corresponds to the number of test samples 1a including the detection target microorganism 1f among the test samples 1a present in the vicinity, that is, the number of detection target microorganisms 1f existing per area of the substrate-side surface 1d. Therefore, it is possible to perform absolute quantification of the detection target microorganism 1f by counting signals emitted by the treatment of the labeling reagent 1e and measuring the number of detection target microorganisms 1f per area of the substrate-side surface 1d. .

  As the labeling reagent 1e, a reagent that stains nucleic acids derived from the nucleus, nucleoid, mitochondria, or ribosome of the detection target microorganism 1f is preferably used. More preferably, a nucleic acid probe that emits a fluorescent, luminescent, or colored signal by hybridizing with a base sequence of a nucleic acid specific to the detection target microorganism 1f is used. Another labeling reagent 1e that can be suitably used in the present invention is a reagent that stains at least one member of the group consisting of lipid, carbohydrate, and protein of the cell wall, cell membrane, or cytoplasm of the detection target microorganism 1f. It is done. However, as long as it is a reagent that can label a substance derived from the detection target microorganism 1f and can obtain a signal that can be distinguished from other microorganisms contained in the test sample 1a and inanimate particles, it is particularly limited. Not.

Next, a second embodiment of the microorganism detection method according to the present invention will be described with reference to FIG. FIGS. 2A to 2C are schematic views showing cross sections showing a second embodiment of the microorganism detection method according to the present invention in the order of steps.
First, as a first step, as shown in FIG. 2A, the test sample 2a is fixed on the surface of the substrate 2b by an arbitrary method. The fixing method of the test sample 2a to the base material 2b differs depending on the form of the test sample 2a. For example, when the test sample 2a is airborne bacteria, the test sample 2a is captured as falling bacteria by keeping the surface of the base material 2b upward for a certain period of time and leaving it open. Or you may fix the to-be-tested sample 2a to the base material 2b by collecting air with an air sampler.

When the test sample 2a is an adherent on the solid, a homogenized suspension or swab extract containing the test sample 2a that is an adherent on the solid is dropped onto the substrate 2b, and if necessary, a solvent or It can be fixed by volatilizing the dispersion medium. However, the method is not particularly limited as long as the microorganisms to be detected can be retained on the surface of the substrate 2b.
The surface portion of the substrate 2b that comes into contact with the test sample 2a can be used without limitation as long as it is a material that does not destroy microorganism cells and does not prevent microorganisms from being fixed, but glass, metal, resin A thin film made of paper, hydrogel is preferably used. More preferably, a base material having a surface subjected to hydrophilic treatment is used. Examples of the hydrophilic treatment performed to increase the affinity between the target microbial cell and the substrate surface include plasma treatment, corona treatment, UV treatment, or fluorine gas treatment, but the desired effect is obtained. Any processing method can be used without particular limitation.

  Next, as a second step, as shown in FIG. 2B, the test sample 2a on the base material 2b is fixed so as not to flow with the liquid material 2c that can be solidified by an arbitrary method. To do. After dropping or applying the liquid material 2c so as to cover the test sample 2a fixed on the base material 2b, the liquid material 2c is solidified by applying a solidification process and fixed so that the test sample 2a does not flow. . As the liquid material 2c, a resin that can be cured by at least one solidification treatment selected from heating, light irradiation, two-component mixing, drying, or water addition is suitably used. However, the resin as the liquid material 2c is limited to a material that can infiltrate and diffuse a reagent for labeling the detection target microorganism into the resin after the solidification treatment. Furthermore, the resin as the liquid material 2c is added to the solidification process or is generated as a result of heat or mechanical stress. This is excluded if the shape can be destroyed. In addition, when light irradiation is performed as the solidification treatment, it is desirable to use light having a wavelength that does not fragment the nucleic acid in the test sample, but there is no particular limitation when labeling and detecting substances other than nucleic acid.

  Further, another liquid material suitably used in the present invention includes a hydrogel that can lose fluidity by at least one solidification treatment selected from heating, cooling, or addition of multivalent ions. . This hydrogel is limited to a material that is capable of penetrating and diffusing a reagent for labeling the detection target microorganism into the resin after the solidification treatment. As a gelling agent for forming a hydrogel, polysaccharides, polyuronic acids, proteins, and synthetic polymers are preferably used, but are not particularly limited as long as they can obtain desired effects. However, this excludes the case where the shape of the bacterial cell, which is the test sample, can be destroyed by heat or mechanical stress that is added or generated secondaryly during the solidification treatment.

  Then, as a third step, as shown in FIG. 2 (c), a reagent 2e for labeling the detection target microorganism is used with the cells of the test sample 2a fixed in the solidified liquid material 2c as a reaction chamber. Thus, the sample 2a is labeled and detected. That is, in the solidified liquid material 2c, a reaction chamber that accommodates the test sample 2a is formed in a portion where the microbial cells as the test sample 2a are present. Therefore, the test sample 2a is labeled and detected using the labeling reagent 2e, assuming that the test sample 2a is in the reaction chamber formed in the solidified liquid material 2c.

  Specifically, first, the labeling reagent 2e is dropped, applied, or fed so as to cover the solidified liquid material 2c on the substrate 2b. Then, signal detection of the particles labeled with the labeling reagent 2e in the test sample 2a, that is, the detection target microorganism 2f is performed. For signal detection, at least one of optical detection and microscopic observation is preferably used. More preferably, microscopic observation of the solidified liquid material 2c and image analysis of imaging data are used. That is, in FIG.2 (c), the solidified liquid material 2c is observed from the base-material 2b side or the side by which the labeling reagent 2e was apply | coated. Therefore, at least one of the base material 2b and the solidified liquid material 2c needs to be a material through which the sample 2a can be observed, that is, a material having excellent light transmittance and a low refractive index.

  Thus, in the method of labeling and detecting the test sample 2a with the labeling reagent 2e, the test sample 2a exists in the reaction chamber formed in the solidified liquid material 2c, and the test sample Since the test sample 2a including the detection target microorganism 2f out of 2a emits a signal, the number of signals obtained by microscopic observation of the solidified liquid material 2c and image analysis of the imaged data is based on the area of the substrate 2b. This corresponds to the number of test samples 2a including the detection target microorganism 2f among the test samples 2a existing in the sample, that is, the number of detection target microorganisms 2f existing per area of the base material 2b. Therefore, it is possible to perform absolute quantification of the detection target microorganism 2f by counting the signal emitted by the treatment of the labeling reagent 2e and measuring the number of detection target microorganisms 2f existing per area of the base material 2b. .

  As the labeling reagent 2e, a reagent capable of penetrating and diffusing in the solidified liquid material 2c is selected. As the labeling reagent 2e, a reagent that stains nucleic acid derived from the nucleus, nucleoid, mitochondria, or ribosome of the detection target microorganism 2f is preferably used. More preferably, a nucleic acid probe that emits a fluorescent, luminescent, or colored signal by hybridizing with a base sequence of a nucleic acid specific to the detection target microorganism 2f is used. Further, another labeling reagent that can be suitably used in the present invention includes a reagent that stains at least one member of the group consisting of lipid, carbohydrate, and protein of the cell wall, cell membrane, or cytoplasm of the detection target microorganism 2f. . However, the reagent is not particularly limited as long as it is a reagent that can label a substance derived from the microorganism to be detected and can obtain a signal that can be distinguished from the microorganism in the test sample and the inanimate particles.

  Thus, according to the microorganism detection method according to the first and second embodiments, it is possible to perform absolute quantification of microorganisms in a simple process without requiring complicated procedures and advanced techniques, and quickly. In addition, highly sensitive microorganism detection can be performed. Moreover, microorganism detection can be performed without requiring expensive equipment and many consumables.

Hereinafter, examples in the case where microorganism detection is performed using the microorganism detection method in the above embodiment will be described. However, the following examples do not limit the invention according to the claims. Many modifications can be made by those skilled in the art without departing from the spirit of the invention.
<Fixing the test sample>
As shown in FIGS. 1B and 2B, the test samples 1a and 2a on the base materials 1b and 2b are covered with the liquid materials 1c and 2c, and the liquid materials 1c and 2c are solidified. The test samples 1a and 2a were fixed. In this example, for the sake of simplicity, polystyrene microbeads having a diameter of 6 μm were regarded as the cells of microorganisms to be detected and used as test samples 1a and 2a. Furthermore, these test samples 1a and 2a fixed in the liquid materials 1c and 2c were observed using a fluorescence microscope.

(material)
In order to clearly distinguish microbeads that look like microorganisms to be detected from particles such as dust that exist in the air, on the substrate, or in liquid materials, microbeads that contain fluorescent dyes were used. . The fluorescent dye has an excitation wavelength peak at 458 nm and an emission wavelength peak at 540 nm, and can be observed with a fluorescence microscope filter set generally called a fluorescein filter. Commercially available microbeads dispersed in water were used after being dehydrated and dried by centrifugation, supernatant removal and ethanol replacement. A polyethylene terephthalate (PET) film and a glass plate were used as a substrate for fixing the test sample. As the liquid material that can be solidified, UV curable resin and agarose gel were used. The agarose gel used was a 2.0 wt% aqueous solution in which agarose powder was dissolved by heating and stirring so as not to clump.

(Method)
First, the dehydrated and dried microbeads were dropped in an appropriate amount onto each of the PET film as the substrate and the glass plate using a micropipette. Thereafter, an appropriate amount of UV curable resin was dropped so as to cover the PET film-like microbeads, and the dropped UV curable resin was solidified by UV irradiation. Also, an appropriate amount of agarose aqueous solution was dropped so as to cover the microbeads on the glass plate, and the agarose aqueous solution was solidified by cooling to room temperature to form a gel. The UV cured resin on the solidified PET film and the agarose gel on the glass plate were observed with a fluorescence microscope equipped with a fluorescein filter.

(result)
FIG. 3 is a microscopic image of microbeads fixed on a PET film substrate using a UV curable resin, where (a) is a bright field image and (b) is a fluorescent image. In FIG. 3, the microbeads existing outside the region 3a covered with the UV curable resin, that is, the microbeads 3b not covered with the UV curable resin and the microbeads inside the region 3a covered with the UV curable resin. In the bright-field image (FIG. 3 (a)), each particle can be distinguished from the beads, that is, the microbeads 3c covered with the UV curable resin. On the other hand, in the fluorescence image (FIG. 3B), the microbead 3b not covered with the UV curable resin was able to capture the fluorescence signal of each particle of the microbead 3b. For the microbeads 3c covered with the resin, the signal / noise ratio is lowered, making it difficult to determine the shape of the bead particles of the microbeads 3c, or a plurality of microbeads 3c are close to each other. In some cases, the fluorescence signal was regarded as the signal of one huge particle, and the correspondence between the actual number of bead particles and the number of obtained fluorescence signals was not accurate.

  In the fluorescence image (FIG. 3B), since the entire region 3a covered with the UV curable resin emits fluorescence, the autofluorescence of the UV curable resin interferes with the fluorescence signal emitted by the microbeads used. It is thought that it was closed. Therefore, when a UV curable resin is used as the liquid material and detection is performed using a fluorescent labeling reagent, it is preferable to select a UV curable resin having a small interference of autofluorescence with respect to the target fluorescence wavelength. In addition, when there is a test sample that is not covered with a liquid material for fixation, such as the microbeads 3b that are not covered with the UV curable resin in FIG. 3, the manner in which the fluorescence signal is obtained varies. There is concern about the decline. Therefore, it is desirable that the liquid material is dropped so as to completely cover the detection range on the substrate.

FIG. 4 is a microscopic image of microbeads fixed on a glass plate using an agarose gel. (A) is a bright field image and (b) is a fluorescence image. In FIG. 4, the particle 4a has a diameter of about 6 μm and can be observed in both a bright field image (FIG. 4A) and a fluorescence image (FIG. 4B). In the example, it can be seen that the microbead is dropped on the base material in the manner of the detection target microorganism. On the other hand, the particle 4b has a large diameter of 10 μm or more and can be observed in the fluorescent image (FIG. 4B) even though it can be observed in the bright field image (FIG. 4A). There wasn't. Since the diameter of the microbeads is different from that of the microbeads and the fluorescent signal is not emitted, it is assumed that the particles 4b are derived from bubbles generated during gel formation, or particles such as dust existing in the agarose aqueous solution or on the base material. Is done.
As described above, the test sample on the substrate can be fixed with a liquid material that can be solidified by an arbitrary method, and the test sample contained in the solidified liquid material is placed in a microscope. It was shown to be observable.

<Desorption of solidified material>
As shown in FIG.1 (c), the liquid material solidified in order to fix a test sample on the base material was desorbed from the base material. In this example, for the sake of simplicity, polystyrene microbeads having a diameter of 6 μm were used as test specimens by regarding the cells of microorganisms to be detected. Furthermore, these test samples fixed in the liquid material were observed using a fluorescence microscope.

(material)
In order to clearly distinguish microbeads that look like microorganisms to be detected from particles such as dust that exist in the air, on the substrate, or in liquid materials, microbeads that contain fluorescent dyes were used. . The fluorescent dye has an excitation wavelength peak at 458 nm and an emission wavelength peak at 540 nm, and can be observed with a fluorescence microscope filter set generally called a fluorescein filter. The microbeads commercially available in a state of being dispersed in water were used after being dehydrated and dried by centrifugation, supernatant removal, and ethanol replacement. An agarose gel was used as a base material for fixing the test sample. A UV curable resin was used as the liquid material that can be solidified. The agarose gel used was a 2.0 wt% aqueous solution in which agarose powder was dissolved by heating and stirring so as not to clump.

(Method)
Using a gel formed by cooling a 2.0 wt% agarose aqueous solution to room temperature and solidifying, a suitable amount of dried microbeads was dropped onto the substrate using a micropipette. An appropriate amount of UV curable resin was dropped so as to cover the microbeads on the substrate, and the glass plate was placed and adjusted so that the UV curable resin became a film having a uniform thickness. Thereafter, the UV curable resin was sufficiently solidified by UV irradiation, and then mechanically peeled from the base material of the agarose gel. The surface of the peeled UV curable resin on the agarose gel substrate side was observed with a fluorescence microscope equipped with a fluorescein filter.

(result)
FIG. 5 is a microscopic image of the substrate-side surface of the UV curable resin that has been mechanically peeled after solidifying on an agarose gel substrate, (a) is a bright field image, and (b) is a fluorescence image. The particle 5a in FIG. 5 has a diameter of about 6 μm, and was observable in both the bright field image (FIG. 5A) and the fluorescence image (FIG. 5B). In the example, it can be seen that the microbead is dropped on the base material in the manner of the detection target microorganism. Therefore, the test sample existing on the substrate is fixed using a liquid material that can be solidified by any method, and the solidified state is maintained while the test sample is held in the liquid material. It has been shown that it is possible to remove the liquid material from the substrate.
In addition, it is possible to detect specific substances (proteins, quality, lipids, nuclei, nucleoids, mitochondria, liposomes, nucleic acids, etc.) present inside the detection target microorganism (cell) from this experimental example. It was shown that.

<Labeling and detection of test sample>
As shown in FIG. 1 (d), the solidified liquid material detached from the base material is used as a reaction chamber with the liquid material portion corresponding to the cells of the test sample fixed in the solidified liquid material. Using the reagent for labeling the detection target microorganism, the surface portion of the liquid material in which a part of the test sample is exposed on the substrate side surface of the solidified liquid material is used as the opening of the reaction chamber. Went. In this example, for the sake of convenience, polystyrene microbeads having a diameter of 3 μm were used as test specimens, assuming that the microbe bodies of microorganisms to be detected were used.

(material)
As microbeads that were considered as microorganisms to be detected, biobead-coated microbead surfaces were selected in order to confirm the presence or absence of a reaction with a labeling reagent. As the labeling reagent, streptavidin fluorescently labeled with fluorescein isothiocyanate (FITC) was used. Since the labeling reagent has an excitation wavelength peak at 458 nm and an emission wavelength peak at 540 nm, biotin-coated microbeads treated with the labeling reagent can be generally observed with a fluorescence microscope filter set called a fluorescein filter. An agarose gel was used as a base material for fixing the test sample. A UV curable resin was used as the liquid material that can be solidified. The agarose gel used was a 2.0 wt% aqueous solution in which agarose powder was dissolved by heating and stirring so as not to clump.

(Method)
A gel formed by cooling a 2.0 wt% agarose aqueous solution to room temperature and solidifying was used as a base material, and an appropriate amount of microbeads was dropped on the base material using a micropipette. An appropriate amount of UV curable resin was dropped so as to cover the microbeads on the substrate, and the glass plate was placed and adjusted so that the UV curable resin became a film having a uniform thickness. Thereafter, the UV curable resin was sufficiently solidified by UV irradiation, and then mechanically peeled from the base material of the agarose gel. The surface of the peeled UV curable resin on the agarose gel substrate side was observed with a fluorescence microscope equipped with a fluorescein filter. Further, FITC-labeled streptavidin was dropped on the surface of the peeled UV curable resin on the agarose gel substrate side, and incubated at room temperature for 5 minutes. Thereafter, it was washed three times with a sufficient amount of purified water and again subjected to observation with a fluorescence microscope equipped with a fluorescein filter.

(result)
FIG. 6 is a microscopic image of the substrate side surface of the UV curable resin peeled off after fixing biotin-coated microbeads on the agarose gel substrate, (a) is a bright field image, (b) is a fluorescence image. It is. In FIG. 6, the microbead 6a was observable in the bright field image (FIG. 6A), but its presence could not be confirmed in the fluorescence image (FIG. 6B). That is, it was confirmed that the microbeads did not emit a fluorescent signal in an untreated state where no labeling reagent was applied.

  FIG. 7 is a microscopic image of the surface of the substrate side after the treatment with FITC-labeled streptavidin of the UV curable resin peeled after fixing the biobead-coated microbeads on the agarose gel substrate, and (a) is a bright-field image. , (B) are fluorescent images. In FIG. 7, the microbead 7a was observable in both the bright field image (FIG. 7 (a)) and the fluorescence image (FIG. 7 (b)). That is, it was possible to stain untreated biotin-coated microbeads encapsulated in UV curable resin by FITC-labeled streptavidin treatment and detect them with a fluorescence microscope. Therefore, the test sample contained in the solidified liquid material peeled off from the base material has a portion corresponding to the test sample of the solidified liquid material as a reaction chamber and is exposed. It was shown that it can be used for a labeling reaction by labeling the cells as the test sample accommodated in the reaction chamber using the substrate-side surface portion of the substrate as the opening of the reaction chamber.

Moreover, it was shown from this experimental example that it is possible to detect specific substances (proteins, carbohydrates, lipids, etc.) present on the surface of the detection target microorganism (cell).
Although the present invention has been described above with reference to specific embodiments, the present invention is not limited to these descriptions. From the description of the invention, other embodiments of the invention will be apparent to persons skilled in the art, along with various variations of the disclosed embodiments. Therefore, it is to be understood that the claims encompass these modifications and embodiments that fall within the scope and spirit of the present invention.

1a, 2a Test sample 1b, 2b Substrate 1c, 2c Liquid material 1d Substrate side surface of solidified liquid material 1e, 2e Labeling reagent 1f, 2f Detection target microorganism 3a Region covered with UV curable resin 3b UV curing Fluorescent microbeads not covered with resin 3c Fluorescent microbeads covered with UV curable resin 4a Fluorescent microbeads fixed with agarose gel 4b Particles other than fluorescent beads 5a Grasped in UV curable resin exfoliated from substrate Fluorescent beads 6a Biotin-coated beads gripped in the UV curable resin peeled from the base material 7a Biotin-coated beads gripped in the UV curable resin peeled from the base material and fluorescently stained

Claims (11)

Fixing the test sample to the surface of the substrate so as to be removable; and
Covering the test sample fixed on the substrate surface with a liquid material that can be solidified, solidifying the liquid material covering the test sample, and fixing the test sample to the substrate surface When,
Desorbing the solidified liquid material from the substrate in a state including the test sample;
Using the reagent for labeling the detection target microorganism, the step of detecting the detection target microorganism after labeling the test sample from the substrate side surface of the solidified liquid material;
A method for detecting a microorganism, comprising: Fixing the test sample on the substrate surface;
Covering the test sample fixed on the substrate surface with a liquid material that can be solidified, solidifying the liquid material covering the test sample, and fixing the test sample to the substrate surface When,
A step of diffusing a reagent for labeling a microorganism to be detected in the solidified liquid material, and detecting the microorganism to be detected from a surface of the solidified liquid material on which the reagent is diffused;
A method for detecting a microorganism, comprising: The surface of the substrate on which the test sample is fixed is
The method for detecting a microorganism according to claim 1 or 2, comprising any one of glass, metal, resin, paper, and hydrogel. The surface of the substrate on which the test sample is fixed is
The method for detecting a microorganism according to any one of claims 1 to 3, wherein a hydrophilic treatment is performed. The liquid material is
The method for detecting a microorganism according to any one of claims 1 to 4, which is a resin that is cured by any one of heating, light irradiation, two-component mixing, drying, and water addition. . The liquid material is
5. The gel according to claim 1, wherein the dispersion medium is water and is a gel that loses fluidity by performing at least one of heating, cooling, and addition of multivalent ions. The detection method of microorganisms as described.   The method for detecting a microorganism according to claim 6, wherein at least one of a polysaccharide, polyuronic acid, protein, and synthetic polymer is used as the gelling agent. A reagent for labeling the test sample,
The detection of a microorganism according to any one of claims 1 to 7, wherein a nucleic acid derived from any one of a nucleus, a nucleoid, a mitochondrion, and a ribosome of the detection target microorganism is stained. Method. A reagent for labeling the test sample,
The probe according to any one of claims 1 to 8, further comprising a probe that emits a fluorescence, luminescence, or chromogenic signal by hybridizing with a base sequence of a nucleic acid specific to the microorganism to be detected. The detection method of microorganisms as described. A reagent for labeling the test sample,
The method for detecting a microorganism according to any one of claims 1 to 7, wherein at least one of lipids, carbohydrates, and proteins in the cell wall or cell membrane of the detection target microorganism is stained. .   11. The detection target microorganism is detected by at least one of optically detecting a signal of a labeling reaction by the label or observing under a microscope. The method for detecting a microorganism according to Item.