JP2007097551A - Method for detecting microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting microorganisms for rapidly detecting the microorganisms in high accuracy. <P>SOLUTION: The microorganism (e.g., salmonella)is detected by the following method: processes for trapping the microorganism by using a trapping substance to which a ligand or an antibody binding to a constituent component of the microorganism (step 1), washing the trapped microorganism (step 2), extracting DNA from the washed microorganism (Step 3), amplifying the extracted DNA by PCR method (step 4) and detecting the target DNA which is an amplified product (step 5). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microorganism detection method.

In order to prevent food poisoning, it is desired to improve the management of food safety and hygiene. Conventionally, methods for detecting specific microorganisms such as food poisoning bacteria, that is, Salmonella, include culture methods, immunochromatography methods, ELISA methods, immunomagnetic bead methods, DNA detection methods, and improved techniques thereof. (For example, refer to Patent Document 1). In Patent Document 1, a food or food material is cultured under conditions for growing the target microorganism, and the obtained culture is cultured under conditions for selectively growing the target microorganism, and then the target microorganism is extracted from the obtained culture. A technique for detecting a target microorganism using an enzyme activity possessed as an index has been disclosed.
JP2003-33997A (page 2-4)

  However, whichever of the above methods is used, enrichment culture is necessary, and it takes a long time to obtain a detection result of microorganisms such as Salmonella. The time required for detection varies depending on the method and the microorganism to be detected, but each requires 8 hours or more. For this reason, in the case of a food with a short shelf life, it cannot be shipped after waiting for the inspection result.

  Moreover, not only microorganisms (for example, Salmonella) to be detected but also other microorganisms may be mixed in the food. In addition, dead microorganisms of the detection target and eluted DNA may be mixed. For this reason, even when a specific microorganism (for example, Salmonella) is detected by detecting DNA, the possibility of misjudgment by DNA derived from microorganisms other than the microorganism to be detected, dead bacteria, and elution It may not be possible to detect and measure only viable bacteria due to the influence of the DNA present.

  The present invention has been made to solve the above problems, and an object of the present invention is to provide a microorganism detection method for detecting a specific microorganism quickly and with high accuracy.

  In order to solve the above-mentioned problems, the invention described in claim 1 includes a step of capturing a microorganism using a capturing body in which a ligand or an antibody that binds to a constituent component of the microorganism is immobilized, and washing the captured microorganism. And a step of extracting DNA from the washed microorganism, a step of amplifying the extracted DNA by a PCR method, and a step of detecting target DNA as an amplification product.

  According to a second aspect of the present invention, in the microorganism detection method according to the first aspect, the step of washing the captured microorganism includes a buffer solution, a buffer solution containing a polyanion, a buffer solution containing a surfactant, and an interface. The gist is to use a liquid selected from an aqueous activator solution and an aqueous alcohol solution.

The gist of the invention described in claim 3 is the microorganism detection method according to claim 1 or 2, wherein the microorganism to be detected is Salmonella.
The invention according to claim 4 is the microorganism detection method according to claim 3, wherein the gene to be amplified in the step of amplifying the extracted DNA by PCR is fimA, agfA, fliC, invA, invE. , SigD, prt, ompC, ompF, ompA, sipB, sipC, tctC.

  According to a fifth aspect of the present invention, in the microorganism detection method according to any one of the first to fourth aspects, the capture body is a magnetic bead or microorganism on which a ligand or an antibody that binds to a constituent component of the microorganism is immobilized. The gist is any one of a magnetic nanoparticle on which a ligand or an antibody that binds to a component of the above is immobilized and a container in which a ligand or an antibody that binds to a component of a microorganism is immobilized on the inner surface.

  According to a sixth aspect of the present invention, in the microorganism detection method according to any one of the first to fifth aspects, the step of detecting the target DNA that is the amplification product is performed by binding a capture probe to the conductor surface. The target DNA is bound to the capture probe and the reporter probe using the electrode, the enzyme-labeled reporter probe, and the substrate that reacts with the enzyme, and the enzyme labeled with the reporter probe and the enzyme The gist is to detect the target DNA by measuring a current value flowing through the electrode by a reaction with a substrate.

  The invention according to claim 7 is the microorganism detection method according to any one of claims 1 to 5, wherein the step of detecting the target DNA that is the amplification product uses electrophoresis or fluorescence. The gist is to carry out by any one of the methods for detecting the amplification of the target DNA.

  The invention according to claim 8 is the microorganism detection method according to any one of claims 1 to 7, wherein the step of extracting DNA from the washed microorganism is bound to the capture body by a thermal extraction method. The gist is to extract DNA from microorganisms in a state.

  The invention according to claim 9 is the microorganism detection method according to any one of claims 1 to 5, wherein the electrode is formed by coupling a capture probe to the surface of the conductor, the temperature control unit, and the electrode flows. The step of extracting DNA from the washed microorganism using a DNA detection device including a current measurement unit for measuring a current value is performed by adjusting the temperature of the sample solution containing the microorganism in a state of being bound to the capture body. The step of extracting the DNA by the thermal extraction method under the control of the sample and amplifying the extracted DNA by the PCR method is performed by controlling the temperature of the sample solution containing the extracted DNA by the temperature control unit by the PCR method. The step of amplifying the target DNA and detecting the target DNA that is the amplification product uses an enzyme-labeled reporter probe and a substrate that reacts with the enzyme, The temperature of the sample solution containing the amplification product is controlled by the temperature control unit, the target DNA is bound to the capture probe and the reporter probe, and the enzyme labeled with the reporter probe by the current measurement unit The target DNA is detected by measuring the value of the current flowing through the electrode by the reaction with the substrate.

  The invention according to claim 10 is the microorganism detection method according to any one of claims 3 to 9, wherein the component is lipopolysaccharide, and the ligand or antibody that binds to the component is polymyxin B. It is a summary.

(Function)
According to the first aspect of the present invention, a microorganism is captured using a capturing body on which a ligand or an antibody that binds to a constituent component of the microorganism is immobilized, the captured microorganism is washed, and DNA is extracted from the washed microorganism. Then, the extracted DNA is amplified by the PCR method, and the target DNA that is the amplification product is detected to detect the microorganism. For this reason, in the case of detecting microorganisms, since the enrichment culture is not performed, the microorganisms can be detected in a short time. In addition, microorganisms can be selected in two stages: selection by capturing and washing microorganisms using a capture body in which ligands or antibodies that bind to the components of the microorganism are immobilized, and detection of DNA from the captured and washed microorganisms. Therefore, the determination accuracy can be improved.

  According to the invention described in claim 2, the captured microorganism is washed from a buffer, a buffer containing a polyanion, a buffer containing a surfactant, an aqueous surfactant solution, and an aqueous alcohol solution. Use liquid. As a buffer solution containing a polyanion, a buffer solution containing 0.5 to 1.0% of a polyanion is preferably used. Moreover, it is preferable that the density | concentration of surfactant is 0.001-0.1%. For example, when washing is performed using a buffer solution containing a polyanion, contaminants that are positively charged can be easily removed from negatively charged microorganisms.

According to the invention described in claim 3, the detection target microorganism can be Salmonella.
According to the invention described in claim 4, in the step of amplifying the extracted DNA by the PCR method, the genes to be amplified are fimA, agfA, fliC, invA, invE, sigD, prt, ompC, ompF, ompA. , SipB, sipC, tctC can be selected.

  According to the invention described in claim 5, the capturing body includes a magnetic bead on which a ligand or an antibody that binds to a component of a microorganism is immobilized, a magnetic nanoparticle on which a ligand or an antibody that binds to a component of a microorganism is immobilized. In addition, a container in which a ligand or an antibody that binds to a constituent component of a microorganism is immobilized on the inner surface can be used as a capturing body.

  According to the sixth aspect of the present invention, the target DNA is bound to the electrode capture probe formed by binding the capture probe to the surface of the conductor and the enzyme-labeled reporter probe. And the target DNA which is an amplification product is detectable by measuring the electric current value which flows into an electrode by reaction with the enzyme and substrate which were labeled by the reporter probe.

According to the seventh aspect of the present invention, the target DNA that is the amplification product can be detected by the method of detecting amplification of the target DNA using electrophoresis or fluorescence.
According to invention of Claim 8, DNA can be extracted from the microorganisms couple | bonded with the capture body with the heat | fever extraction method.

  According to the ninth aspect of the present invention, there is provided a DNA detection apparatus comprising: an electrode formed by coupling a capture probe to a conductor surface; a temperature control unit; and a current measurement unit that measures a current value flowing through the electrode. Can be used to detect Salmonella. In other words, the temperature of the sample solution containing microorganisms bound to the capture body is controlled to extract DNA by the thermal extraction method, and the temperature of the sample solution containing the extracted DNA is controlled to amplify the target DNA by the PCR method. To do. Then, a target DNA is bound to the capture probe and the reporter probe by using an enzyme-labeled reporter probe and a substrate that reacts with the enzyme to control the temperature of the sample solution containing the amplification product. And target DNA is detected by measuring the electric current value which flows into the said electrode by reaction with the said enzyme and the said substrate labeled on the said reporter probe. Thereby, DNA extraction from the captured and washed microorganisms, amplification by PCR method, and detection of target DNA are performed using a DNA detection apparatus including the electrode, the temperature control unit, and the current measurement unit. Can do.

  According to invention of Claim 10, Salmonella can be captured using the capture body which fix | immobilized polymyxin B couple | bonded with the lipopolysaccharide which is a structural component of Salmonella.

  According to the present invention, a specific microorganism can be detected quickly and with high accuracy.

  Hereinafter, an embodiment embodying the present invention will be described with reference to FIG. In this embodiment, a microorganism to be detected is a Salmonella bacterium, and a microorganism detection method for detecting a specific microorganism (Salmonella bacterium) quickly and accurately will be described. In the present specification, any bacteria belonging to the genus Salmonella are collectively referred to as “Salmonella”.

  As shown in FIG. 1, in this embodiment, first, a microorganism is captured using a capturing body on which a ligand or an antibody that binds to a constituent component of Salmonella that is a microorganism to be detected is immobilized (step S1).

  In this step, the capture body is a magnetic bead or magnetic nanoparticle on which a ligand or an antibody that binds to a constituent component of Salmonella that is a microorganism to be detected is immobilized, or a container on which such a ligand or antibody is immobilized on the inner surface. Used as And the microorganisms couple | bond with this capture body, when the structural component of microorganisms (Salmonella etc.) couple | bonds with the ligand or antibody fixed to this capture body. Then, the microorganisms are collected by collecting the capturing bodies combined with the microorganisms (that is, microorganisms such as Salmonella are captured).

  For example, polymyxin B is used as the ligand or antibody that binds to the constituents of Salmonella. Polymyxin B is known to specifically bind to lipopolysaccharide, which is a component of the outer membrane of Gram-negative bacteria such as Salmonella. In this case, polymyxin B, which is a ligand, binds to lipopolysaccharide, which is a constituent of the outer membrane of Salmonella, so that Salmonella binds to the capturing body. In addition, you may use the capture body which fix | immobilized the antibody couple | bonded with the structural component of Salmonella which is a microorganism of a detection target. In this case, an antigen contained in a constituent component of a microorganism (such as Salmonella) and an antibody immobilized on the capturing body bind to each other, so that the microorganism (such as Salmonella) binds to the capturing body.

  When magnetic beads on which ligands or antibodies are immobilized are used as capture bodies, microorganisms (such as Salmonella) can be collected by collecting the magnetic beads with a magnet. As the magnetic nanoparticles, for example, thermoresponsive magnetic nanoparticles in which a thermoresponsive polymer is immobilized on the surface layer of magnetic nanoparticles are used. Such heat-responsive magnetic nanoparticles rapidly aggregate by changing the temperature of the aqueous solution, and can be separated by a magnet. Microorganisms are collected by immobilizing ligands or antibodies on such magnetic nanoparticles and using them as capture bodies and collecting the magnetic nanoparticles in a state of being bound to the microorganisms.

  Examples of the container in which the ligand or antibody is immobilized on the inner surface include a container in which the ligand or antibody is immobilized on the inner surface of a tube or the like used for PCR processing. When the ligand or antibody is immobilized on the inner surface of the container and used as a capture body, when the reaction solution is removed from the container, the components of the microorganism are bound to the ligand or antibody immobilized on the inner surface of the container. Microorganisms adhere to and remain on the inner surface of the container and can be collected.

Next, the captured microorganisms are washed (step S2). In the washing of the captured microorganisms, for example, a buffer solution, a buffer solution containing a polyanion (eg, PBS), a buffer solution containing a surfactant (eg, PBS-T), an aqueous surfactant solution, an aqueous alcohol solution A liquid selected from among them is used. For example, as a buffer solution containing a polyanion, one containing 0.5 to 1.0% of a polyanion is preferable. As the polyanion, for example, alginic acid, polyacrylic acid, polyaspartic acid and the like can be used. Focusing on the negative charge of microorganisms, the positively charged contaminants can be easily removed by washing with such a buffer solution. The concentration of the surfactant is 0.
It is preferable that it is 001 to 0.1%. As the surfactant, for example, TritonX (Triton X), SDS or the like can be used.

  Next, DNA is extracted from the washed microorganism (step S3). The DNA extraction method in this step is not particularly limited, and for example, a known method such as a heat extraction method can be used.

Then, the extracted DNA is amplified by a PCR method (polymerase chain reaction) (step S4). That is, DNA containing this specific region using two kinds of primers designed to sandwich the specific region of the double-stranded DNA of the microorganism (Salmonella) to be detected by the PCR method or improved technology of the PCR method Amplify the fragment.

Specifically, in the case of detection of Salmonella, for example, DNA fragments are amplified using the following genes characteristic of Salmonella bacteria as target genes (specific regions to be subjected to DNA amplification). That is, in the case of detecting Salmonella, for example, fimA <major type 1 subunit fimbrin>, agfA <fimbrin>, fliC <filament structural protein; flagellin>, invA <invasion protein A (Invasive protein)
>, InvE <invasion protein E (invasive protein)>, sigD <cell invasion protein (
Cell invasive protein)>, prt <paratose synthase (paratose synthase which is a K antigenic determinant of bacterial cells)>, ompC <outer membrane protein precursor C>, ompF <outer membrane protein precursor F ( Cell membrane protein precursor)>, ompA <outer membrane protein precursor A>, sipB <invasion
A gene selected from control protein (invasion control protein), sipC <invasion control protein (invasion control protein)>, tctC <tricarboxylate-binding protein (tricarboxylate-binding protein)> Specific area). In the above description, “target gene (region name) <function>” is shown. In addition, in the detection of Salmonella, the above-described target gene as a specific region to be subjected to DNA amplification is an example, and the specific region to be subjected to DNA amplification is not limited thereto.

A specific example regarding the process for amplification of the DNA fragment containing the specific region by the PCR method will be described later.
Then, specific DNA (target DNA) of the detection target microorganism (Salmonella), which is an amplification product, is detected (step S5). That is, when a detection target microorganism (Salmonella) is captured, a DNA fragment containing a specific region of DNA extracted from this microorganism (Salmonella) is amplified, and this DNA fragment is detected. The method for detecting DNA in this step is not particularly limited, and an electrophoresis method, a method for detecting amplification of target DNA using fluorescence emission, an electrochemical detection method using a DNA probe, and the like can be used.

Detection of DNA by electrophoresis can be performed by a known technique.
An example of a method for detecting the amplification of the target DNA using fluorescence emission is a real-time PCR method. This real-time PCR method is a method for monitoring and analyzing the amount of PCR amplification in real time. That is, when this real-time PCR method is used, the step of amplifying the extracted DNA by the PCR method (step S4) and the step of detecting specific DNA as an amplification product (step S5) are divided into the real-time PCR method. Will be performed. Usually, real-time PCR is monitored by fluorescence. Examples of the fluorescence emission monitoring method include known methods such as an incalator method, a TaqMan probe method, and a cycling probe method.

  In the electrochemical detection method using a DNA probe, target DNA is bound to an electrode capture probe formed by binding a capture probe to a conductor surface and an enzyme-labeled reporter probe, and the enzyme labeled with the reporter probe The target DNA that is the amplification product is detected by measuring the value of the current flowing through the electrode due to the reaction with the substrate. The electrodes used in the electrochemical detection method using such a DNA probe will be described in detail below.

The electrode used in the electrochemical detection method using this DNA probe may be configured by laminating a mediator on a conductor.
As the conductor, any material can be used as long as it is usually used as an electrode. For example, graphite, carbon, carbon fabric, etc .; metals or alloys such as aluminum, copper, gold, platinum, silver, SnO ₂ , In ₂ O ₃ , WO ₃ , TiO _2, etc. A layer or a laminated structure of two or more types can be given. The film thickness, size, shape, and the like of the electrode are not particularly limited, and can be appropriately adjusted depending on the type of mediator, enzyme, and the like used, the performance of the biosensor to be obtained, and the like. For example, a rectangular shape having an area of about 0.1 to ⁵ mm and an area of about 1 to 10000 mm ² is appropriate.

  Any mediator can be used as long as it functions as an electron transfer medium that can exchange electrons between the electrode and the enzyme and does not adversely affect the enzyme reaction. For example, ferrocene, alkali metal ferricyanide (potassium ferricyanide, lithium ferricyanide, sodium ferricyanide, etc.) or alkyl substitutions thereof (methyl substitution, ethyl substitution, propyl substitution, etc.), phenazine methosulfate, p One or more redox organic or inorganic compounds such as benzoquinone, 2,6-dichlorophenolindophenol, methylene blue, potassium β-naphthoquinone-4-sulfonate, phenazine etsulfate, vitamin K, viologen, etc. Combinations are listed. Among them, those that are soluble in water or water-soluble organic solvents (such as lower alcohol) and that are easy to handle and those that have a stable function as an electron transfer medium are preferable. For example, ferrocene, potassium ferricyanide, and the like are preferable. Used.

  The film thickness of the mediator laminated on the conductor is not particularly limited and can be appropriately adjusted depending on the size of the conductor, the type of mediator used, the type of enzyme labeled on the reporter probe, and the like. For example, about 0.1 micrometer-1000 micrometers is mentioned. An appropriate method for laminating the mediator on the conductor can be appropriately selected depending on the type of mediator. For example, a method in which the mediator is dissolved in a solvent that does not inhibit its function, for example, water or a water-soluble organic solvent, and is applied and dried. All of the thin film forming methods known in the art such as a method of dispersing and forming a carrier in a thin film, and an alternating lamination method (Hyundai Kagaku, January 1997, p20-25) can be used. Especially, the alternating lamination method mentioned later is preferable.

  In some electrodes of this DNA measuring apparatus, the above-mentioned mediator is laminated in layers with a polymer carrier. As the polymer carrier that can be used here, any one of those having a charge, being a polymer, and being water-soluble, and preferably satisfying all properties is suitable. The charge may be positive or negative. The polymer carrier includes, for example, various proteins (eg, enzymes, antibodies, receptor proteins, etc.), polypeptides (eg, polylysine, polyaspartic acid, polyglutamic acid, etc.), water-soluble synthetic polymer compounds (eg, polyethyleneimine, Acrylic acid, carboxymethyl cellulose, polystyrene sulfonic acid, polydimethyldiallylammonium chloride, etc.) and natural polymers (alginic acid and its salts, tragacanth gum, etc.).

  The method for laminating the mediator and the like together with the polymer carrier is not particularly limited, and the mediator or the like is dispersed in the polymer carrier, or the polymer carrier is mixed with an appropriate solvent (water, buffer solution, water-soluble organic solvent, etc.). A method of dissolving or dispersing, coating and drying may be used, or an alternating lamination method may be used.

For example, the alternate lamination method of laminating mediators (here, the method of laminating each layer sequentially) can be performed as follows.
First, the conductor surface is activated to be positively or negatively charged. Next, the interaction between the positive and negative charges is made by negatively or positively charging the molecule constituting the mediator, or the polymer carrier in which the mediator is dispersed / bound to the mediator, and bringing it into contact with the conductor surface. The mediator is stacked on the surface of the conductor. If the required amount of mediator can be ensured by such a single positive / negative interaction, only one layer may be stacked as described above, but a larger amount of mediator is desired to be stacked on the conductor surface. The above-mentioned interaction may be performed a plurality of times to laminate a plurality of mediator layers, for example, about 2 to 20 layers. In this case, a carrier having the opposite charge (for example, the above-described polymer carrier) or a mediator or the like is laminated on the laminated mediator, and a molecule constituting the mediator is again transferred to the polymer carrier or the like. By repeating a series of operations of stacking on top of each other, it is possible to realize stacking of multiple layers of mediators.

  A DNA fragment is fixed to this electrode. This DNA fragment is a capture probe for pairing with DNA for detection and / or quantification (hereinafter referred to as “target DNA”) using the complementarity of DNA.

  Then, another DNA fragment (target DNA and reporter probe) is added, and the capture probe, the target DNA, and the reporter probe are bound to the electrode in this order via the mediator, and the target DNA is detected and / or quantified. That is, so-called target DNA hybridization is performed on the electrode. Then, by using an electrode to which the target DNA and the reporter probe are bound, indirect electron exchange via the mediator is measured as, for example, a current value, and thus the target DNA can be detected and / or quantified.

  The target DNA is a DNA fragment for the purpose of detection and / or quantification that is paired with this capture probe using the complementarity of DNA. In the microorganism detection method of the present invention, this target DNA is used after the DNA extracted from the captured and washed microorganism (such as Salmonella) is amplified by the polymerase chain reaction (PCR). The reporter probe is paired with a target DNA by utilizing the complementarity of DNA, and an enzyme is labeled for detection and / or quantification of the target DNA.

  As a method for fixing the capture probe on the electrode, all methods known in the art and methods according to them can be used. For example, a method in which streptavidin is laminated on the electrode, the 5 'end of the capture probe is modified with biotin, and is immobilized by the avidin-biotin interaction, or a method according to this method.

In addition, as a method using a DNA fragment, that is, a DNA probe method and DNA hybridization itself, all methods known in the art and methods according to them can be used. Any commercially available DNA fragment may be used. For example, Salmonella probes (see Rahn K., SA De Grandis, RC Clarke, SA McEwen, JE Galan, C. Ginocchio, R. Curtiss and CL Gyles, Mol. Cell. Probes, 6, 271-279 (1992)) , Takara Biochemicals, Shigella and intestinal invasive Escherichia coli (EIEC) Primer Set INV-1 & 2 for invE gene detection, Shigella and intestinal invasive Escherichia coli (EIEC) ipaH gene detection Primer Set IPA-1 & 2, cholera toxin gene Examples include Primer Set VCT-1 & 2 for detection (all manufactured by Shimadzu Corporation).

  The electrode prepared as described above is usually disposed in a reactor that contains the counter electrode, these electrodes and the counter electrode, and can contain the solution containing the substrate. It is suitable to be used in a biosensor filled with a solution so that both electrodes are almost completely immersed therein. In such a biosensor, by using cyclic voltammetry, for example, a constant potential having a predetermined potential difference is applied to both electrodes, and the current value generated due to the reaction of the enzyme is, for example, limited. Determination can be made by measuring as an oxidation current value. The current value can be detected by a current detection unit such as a potentiostat or an ammeter, for example. In the electrochemical detection method using this DNA probe, DNA is detected by measuring the current value using such a biosensor.

As described above, a specific microorganism (Salmonella) can be detected by sequentially executing the steps S1 to S5.
For example, when Salmonella is detected, when capturing a Salmonella, a Gram-negative bacterium having a lipopolysaccharide that binds to Polymyxin B as a constituent component of the outer membrane is used when a capturing body immobilizing Polymyxin B is used. Be captured. Then, since the captured microorganism is washed, the DNA of this microorganism is extracted and the target DNA is detected through amplification of the DNA by the PCR method, the DNA derived from the captured microorganism (in this case, Gram-negative bacteria) Salmonella DNA is detected only for amplification products amplified from the above.

  In addition, when a capturing body in which an antibody that binds to an antigen that is a constituent component of a microorganism (Salmonella) to be detected is used, only the microorganism having this antigen is captured. Then, the captured microorganism is washed, the DNA of the microorganism is extracted, and the target DNA is detected through amplification of the DNA by the PCR method. In this case, the captured microorganism (in this case, the antibody immobilized on the capturing body) Detection of the DNA of the detection target microorganism (Salmonella) is carried out only for the amplification product amplified from the DNA derived from the microorganism having the antigen that binds to the microorganism.

  As described above, in the microorganism detection method of the present embodiment, selection of the first stage by capturing and washing microorganisms, amplification of DNA extracted from the captured and washed microorganisms, and amplification of target DNA that is an amplification product are performed. A two-stage selection is made, with the selection of the second stage to be detected.

As described above, according to the present embodiment, the following effects can be obtained.
(1) In the above-described embodiment, a microorganism is captured using a capturing body on which a ligand or antibody that binds to a constituent of Salmonella is immobilized, the captured microorganism is washed, and DNA is extracted from the washed microorganism. Salmonella is detected by amplifying the obtained DNA by the PCR method and detecting the target DNA as an amplification product. For this reason, when detecting Salmonella, since enrichment culture is not performed, Salmonella can be detected in a short time. In addition, Salmonella is selected in two steps: selection by capturing and washing microorganisms using a capture body on which ligands or antibodies that bind to Salmonella components are immobilized, and detection of DNA from the captured and washed microorganisms. Therefore, the determination accuracy can be improved. In addition, the microorganism is first selected by capturing and washing the microorganism using a capture body in which a ligand or antibody that binds to a constituent of Salmonella is immobilized, and then immobilized on the capture body in order to detect DNA. The microorganism (Salmonella) to be detected can be detected without being affected by the DNA of a microorganism that does not have a constituent component that binds to the ligand or antibody, or the DNA that is eluted by killed bacteria or killed microorganisms.

(2) In the above-described embodiment, a liquid selected from a buffer solution, a buffer solution containing a polyanion, a buffer solution containing a surfactant, a surfactant aqueous solution, and an alcohol aqueous solution is used for washing the captured microorganism. Use. For example, when washing is performed using a buffer solution containing a polyanion, contaminants that are positively charged can be easily removed from negatively charged microorganisms.

  (3) In the above embodiment, magnetic beads, magnetic nanoparticles, and the inner surface of a container, on which a ligand or antibody that binds to a constituent of Salmonella is immobilized, can be used as a capturing body. For this reason, Salmonella can be easily captured.

  (4) In the above embodiment, target DNA that is an amplification product can be detected by electrophoresis. For this reason, detection of target DNA which is an amplification product can be easily performed at a lower cost.

  (5) In the above embodiment, the target DNA that is an amplification product can be detected by a method of detecting the amplification of the target DNA using fluorescence. For this reason, it is possible to detect the target DNA as an amplification product in a shorter time and with higher sensitivity.

  (6) In the above embodiment, the target DNA is bound to the capture probe of the electrode formed by binding the capture probe to the surface of the conductor and the enzyme-labeled reporter probe, and the reaction between the enzyme labeled on the reporter probe and the substrate is performed. By measuring the value of the current flowing through the electrode, the target DNA that is the amplification product can be detected. Thereby, detection of target DNA which is an amplification product can be performed more simply, simply and with high sensitivity.

  (7) In the said embodiment, DNA can be extracted from the microorganisms couple | bonded with the capture body by the heat | fever extraction method. Thereby, the extraction of DNA from the captured and washed microorganism can be performed by a simple operation.

(Specific example using a DNA detector by an electrochemical detection method using a DNA probe)
Next, a specific example using a DNA detection apparatus based on an electrochemical detection method using a DNA probe in the above-described microorganism detection method will be described with reference to FIGS. This DNA detection apparatus includes an electrode in which a capture probe is coupled to the surface of a conductor, a temperature control unit, and a current measurement unit that measures the value of a current flowing through the electrode. And each process of said step S3-step S4 is performed using this DNA detection apparatus.

  As for the above step S1, for example, magnetic beads or magnetic nanoparticles to which a ligand or an antibody that binds to a constituent component of a microorganism (Salmonella) to be detected is immobilized, or such a ligand or antibody is immobilized on the inner surface. Using the obtained container (for example, a tube), the microorganism is captured using a capturing body on which a ligand or an antibody that binds to a constituent component of the microorganism (Salmonella) to be detected is immobilized. In the step of washing the captured microorganism (step S2), for example, a buffer solution (eg, PBS), a buffer solution containing a polyanion, a buffer solution containing a surfactant (eg, PBS-T), an interface Washing is performed using a liquid selected from an aqueous activator solution and an aqueous alcohol solution.

  Then, in the next step, that is, the step of extracting DNA from the washed microorganism (step S3), extraction of the DNA from the microorganism thus captured and washed is performed using this DNA detection apparatus, Furthermore, using this DNA detector, a step of amplifying the extracted DNA by the PCR method (step S4) and a step of detecting the target DNA that is an amplification product (step S5) are executed.

Next, a DNA detection apparatus will be described.
As shown in FIG. 2, a measurement unit 31 is provided on the upper surface of the DNA detection device 30. The measurement unit 31 includes an electrode unit (biosensor) 32 (see FIG. 3), and an electrode moving knob 33 for moving the electrode unit 32 is provided on the upper surface of the measurement unit 31. ing. The measuring unit 31 has a lid shape and can be opened and closed in the vertical direction.

  In the state where the measurement unit 31 is lifted, the two standby stations 34, the hybrid station 35, and the measurement station 36 are exposed. The standby station 34 is used for retracting the electrode unit 32 when performing PCR. The hybrid station 35 sets the sample tube 101 containing the captured and washed microorganisms, performs DNA thermal extraction and PCR using the extracted DNA, inserts the electrode part 32 after the PCR process, Used to cause hybridization. The measurement station 36 is used to set the determination liquid tube 102 into which the determination liquid has been injected, insert the electrode unit 32 taken out from the hybrid station 35, and measure the current.

  On the upper surface of the DNA detection device 30, a power switch (power SW) 41, a start switch (start SW) 42, and a stop switch (stop SW) 43 are further provided.

  A setting rotary switch (setting rotary SW) 44 and a USB connector 45 are provided at the lower front of the DNA detection device 30. On the upper side of the setting rotary SW 44, there is shown a temperature control pattern indicating the transition of the temperature to be controlled, and switches for setting the temperature and time for each stage of PCR, hybridization, and measurement. Are displayed in association with each other. In the setting rotary SW 44, the setting and input of the temperature and time, the number of PCR cycles, and the measurement voltage in each of these stages are performed. The USB connector 45 is used when connecting to a personal computer for transferring measurement data.

  An LCD display unit 51 is provided on the front upper portion of the DNA detection device 30. Further, a cooling fan motor 52 and a heat exhaust port 53 are respectively provided on the side surfaces on both sides of the DNA detection device 30. Although not shown, a cooling fan motor, a fuse holder, and a power input unit are provided on the back surface of the DNA detection device 30.

Next, the block configuration of the DNA detection device 30 will be described with reference to FIG.
As shown in FIG. 3, the DNA detection device 30 includes a CPU 61. The CPU 61 includes a switch control function (SW control function) 62, a set numerical value conversion function 63, a time control function 64, and a PCR step control function 65. Further, the CPU 61 includes a temperature control function 66, a temperature detection / PID calculation function 67, a temperature control function 68, and a temperature detection / PID calculation function 69.

  The SW control function 62 is a function for starting or interrupting processing by controlling the time control function 64 and the PCR step control function 65, respectively, when the start SW 42 and the stop SW 43 are pressed. The set numerical value conversion function 63 performs data conversion related to the temperature, time, the number of PCR cycles, and the numerical value of the measurement voltage set and input by the setting rotary SW 44, and each set value is converted into a time control function 64 and a PCR step control function. 65, a temperature control function 66, 68, and a function for instructing the measurement voltage generation circuit 91.

The time control function 64 is a function for controlling the processing time of each process and displaying information related to time control on the LCD display unit 51. The PCR step control function 65 is a function for controlling the number of PCR steps and displaying information on the PCR step control on the LCD display unit 51.

  The temperature control function 66 and the temperature detection / PID calculation function 67 are functions for performing temperature control in the PCR block 70 based on temperature set values relating to PCR and hybridization.

  A Peltier element 71 is provided on each side and bottom of the PCR block 70. Further, cooling fans 72 are provided on both side surfaces and the back surface of the PCR block 70. Furthermore, a temperature sensor 73 is provided in the PCR block 70. The PCR block 70 is provided with the hybrid station 35 described above.

  The Peltier element 71 is an element using the Peltier effect, and adjusts the temperature cycle of the PCR block 70. The cooling fan 72 is used for heat dissipation. The temperature sensor 73 measures the temperature of the PCR block 70.

  The temperature control function 66 is a function for outputting a control signal for controlling the temperature of the PCR block 70 to the heating / cooling switching circuit 75. The temperature detection / PID calculation function 67 detects the temperature in the PCR block 70 measured by the temperature sensor 73 and performs PID calculation based on the detected temperature and the set temperature instructed from the temperature control function 66. It is a function. Then, a signal related to the calculated value is output to the current control circuit 74.

  The current control circuit 74 outputs a control current based on the thermal control power supply voltage from the thermal control power supply 60 to the heating / cooling switching circuit 75 in accordance with the signal output by the temperature detection / PID calculation function 67.

  The heating / cooling switching circuit 75 outputs a signal instructing switching between heating / cooling to the Peltier element 71. Then, the temperature cycle of the PCR block 70 is adjusted by the Peltier element 71.

The temperature control function 68 and the temperature detection / PID calculation function 69 are functions for performing temperature control in the measurement unit block 80.
The measuring unit block 80 is heated by the heater 81. A temperature sensor 82 is provided in the measurement unit block 80. The measuring unit block 80 is provided with the measuring station 36 described above.

The temperature control function 68 is a function for instructing the temperature detection / PID calculation function 69 of data related to the set temperature of the measurement unit block 80.
The temperature detection / PID calculation function 69 detects the temperature in the measurement unit block 80 measured by the temperature sensor 82, and performs PID calculation based on the detected temperature and the set temperature instructed from the temperature control function 68. This is a function to be performed. Then, a signal related to the calculated value is output to the current control circuit 83.

  The current control circuit 83 outputs a control current based on the thermal control power supply voltage from the thermal control power supply 60 to the heater 81 in accordance with the signal output by the temperature detection / PID calculation function 69. Then, the temperature of the measurement unit block 80 is adjusted by the heater 81.

  The measurement voltage generation circuit 91 generates a measurement voltage and outputs the measurement voltage to the work current measurement head amplifier 92. The electrode part 32 is connected to the work current measuring head amplifier 92. The electrode unit 32 includes an electrode 32a, a reference electrode 32b, and a counter electrode 32c (see FIG. 5).

  The work current measurement head amplifier 92 outputs the measurement current to the A / D conversion circuit 93. The A / D conversion circuit 93 performs A / D conversion on the measurement current and causes the LCD display unit 51 to display the measurement current.

  In this DNA detection device 30, a temperature control function 66, a temperature detection / PID calculation function 67, a current control circuit 74, a heating / cooling switching circuit 75, a Peltier element 71, a cooling fan 72, and a temperature sensor 73 are included in the claims. The temperature control unit described in 1 is configured. The work current measurement head amplifier 92, the A / D conversion circuit 93, and the measurement voltage generation circuit 91 constitute a current measurement unit described in the claims.

  Next, each process of above-mentioned step S3-step S5 is demonstrated. Here, FIGS. 4 and 5 are used in the description of the temperature control and the electrode movement in the PCR process and the measurement process. FIG. 4 is an explanatory diagram of temperature control in the PCR process and the measurement process. Although the number of PCR repetitions can be changed, FIG. 4 shows an example of temperature transition when the number of PCR repetitions is three. FIG. 5 is an explanatory diagram of electrode movement.

First, the process of extracting DNA from captured and washed microorganisms (step S3) and the process of amplifying DNA by the PCR method (step S4) will be described.
In the extraction of DNA from the captured and washed microorganism, DNA is extracted from the microorganism in a state bound to the capturing body. Specifically, first, a solution containing the microorganisms bound to the capturing body is put into the sample tube 101. Here, for example, a magnetic bead or magnetic nanoparticle to which a ligand or an antibody that binds to a constituent component of a microorganism (Salmonella) to be detected is immobilized is used as a capturing body, and a magnetic bead or magnetic nanoparticle in a state in which the microbe is bound is used. The sample tube 101 is charged. Such a ligand or antibody may be immobilized on the inner surface of the sample tube 101 and used as a capturing body. In this case, the solution in the sample tube 101 is replaced with microorganisms attached to the inner surface of the sample tube 101. In addition, the solution put into the sample tube 101 is prepared like the sample liquid mentioned later here.

  Thermal extraction of DNA is performed using a sample tube 101 containing a solution (sample solution) containing microorganisms bound to the capture body. Here, the extraction of DNA from the captured and washed microorganism is performed by extracting the DNA from the microorganism at a high temperature in the PCR process. This will be described later.

  As shown in FIG. 4, in PCR, temperature cycles of high temperature (TH), medium temperature (TM), and low temperature (TL) are repeated. The temperature and time at each of the high temperature (TH), the intermediate temperature (TM), and the low temperature (TL) in this temperature cycle are set using the setting rotary SW 44.

  Specifically, the high temperature (TH) can be set in the range of 84 to 99 ° C, the intermediate temperature (TM) in the range of 67 to 82 ° C, and the low temperature (TL) in the range of 45 to 60 ° C. As for the maintenance time of each temperature when PCR is repeated, the high temperature maintenance time (tH) is 23 to 38 seconds, the intermediate temperature maintenance time (tM) is 23 to 38 seconds, and the low temperature maintenance time (tL) is 1 to 16 seconds. It can be set.

  The number of PCR repetitions is also set using the setting rotary SW 44. Here, the intermediate temperature-high temperature-low temperature can be set as 1 to 48 times, and the number of repetitions can be set with 3 times as one unit.

The temperature and time for hybridization performed after PCR are also set using the setting rotary SW 44.
Specifically, during hybrid operation, the high temperature (THR) can be set in the range of 84 to 99 ° C., and the intermediate temperature (TMR) can be set in the range of 67 to 82 ° C., respectively. In FIG. 4, a temperature higher than the intermediate temperature (TM) in the PCR cycle is set as the intermediate temperature (TMR) at the time of hybrid. The maintenance time of each temperature at the time of hybrid can be set in the range of 0.5 to 8.0 minutes for the high temperature maintenance time (tHR) and 0 to 150 seconds for the medium temperature maintenance time (tMR).

  Further, the measurement temperature, measurement time, and measurement work voltage at the measurement station 36 are also set using the setting rotary SW 44. The measurement temperature can be set in the range of 30 to 45 ° C. The measurement time can be set in the range of 0.5 minutes to 8.0 minutes. Further, the measured work voltage can be set in the range of 50 to 800 mV.

  As shown in FIG. 4, when the power is turned on, the DNA detection device 30 operates to maintain the warm-up temperature (30 ° C.) for the hybrid station 35. The DNA detection device 30 also operates to maintain the measurement set temperature for the measurement station 36. It should be noted that the start lamp of the start SW 42 blinks until the warm-up temperature and the measurement set temperature are reached (during warm-up), and when the temperature stabilizes at each temperature (when the warm-up is completed), the start lamp is lit stationary. Then, the DNA detection device 30 displays a message “Please set a sample and press the start SW” on the LCD display unit 51.

  Accordingly, as shown in FIG. 5, the user sets the sample tube 101 containing the sample liquid containing the captured and washed microorganisms in the hybrid station 35 and measures the determination liquid tube 102 into which the determination liquid is injected. Set in station 36. The captured microorganism is in a state of being bound to the capturing body as described above.

Here, the sample liquid contains components used in general PCR processing. Specifically, this sample solution contains two kinds of primers, DNA polymerase, dNTP, Mg ²⁺ , and captured and washed microorganisms as constituent components. In addition, although it follows the general case about the density | concentration of each structural component, it needs to change according to reaction conditions. As the determination solution, for example, a solution containing a substrate described later in Tris-HCl buffer is used.

  Then, the user attaches the electrode unit 32 to the measurement unit 31 and lowers the measurement unit 31 on the stations 34, 35, and 36. Then, as in Step 1 of FIG. 5, the electrode moving knob 33 is slid to the position of the standby station 34, and the electrode portion 32 is inserted into the sample tube 101 set in the standby station 34.

  Here, when the start SW 42 is pressed, the DNA detection device 30 controls the temperature cycle in accordance with the set temperature, set time, and number of PCR repetitions. In addition, as shown in FIG. 4, at the time of a start, temperature is raised to high temperature (TH). Then, the temperature cycle is repeated according to the set number of PCR repetitions. Here, at the first high temperature (TH), DNA is extracted from the microorganism and double-stranded DNA is dissociated into single-stranded DNA.

In the PCR repetition, a transformation step at a high temperature (TH), an annealing step at a low temperature (TL), and an extension step at an intermediate temperature (TM) are repeated. In the modification step, double-stranded DNA is dissociated into single-stranded DNA. In the annealing step, two types of primers having sequences complementary to the dissociated DNA partially form double strands by hydrogen bonding. In the extension step, another DNA having phase correction is synthesized by DNA polymerase using each of the two types of primers as the starting point of DNA synthesis.

  In the last cycle of the PCR repetition, the process proceeds to the hybridization process without passing through the high temperature maintenance time (tH). The hybrid high temperature (THR) is maintained for the hybrid high temperature maintenance time (tHR).

  Then, after the hybrid high temperature maintenance time (tHR) ends, the hybrid medium temperature (TMR) is controlled to maintain the hybrid medium temperature maintenance time (tMR), while the buzzer is sounded and the electrode unit 32 is slid to the hybrid station 35. Display to prompt. Specifically, “Please move the electrode to the hybrid station and press the start SW” is displayed on the LCD display unit 51. At this time, the start lamp is blinked, and during this time, the progress of the tMR time is stopped.

Hereinafter, the step (step S4) of detecting the amplification product DNA (target DNA) will be described.
According to the instruction, the user moves the electrode unit 32 to the hybrid station 35 and inserts it into the sample tube 101 as in step 2 of FIG. Here, when the electrode portion 32 is inserted into the sample tube 101, the reporter probe 28c labeled with the enzyme 26 in the freeze-dried state is brought into the sample tube 101 by the supply means provided in the DNA detection device 30. To be added. In the present embodiment, the reporter probe 28c labeled with the enzyme 26 in a freeze-dried state is attached to the surface of the electrode 32a. For this reason, when the electrode part 32 including the electrode 32 a is inserted into the sample tube 101, the reporter probe 28 c labeled with the enzyme 26 is supplied into the sample tube 101.

  As shown in FIG. 7, in the electrode 32a of the electrode section 32, a capture probe 28a that hybridizes complementarily to the target DNA 28b is fixed on the surface of the mediator 22 on the carbon electrode 21 that is a conductor.

When the start SW 42 is pressed, the DNA detection device 30 causes the start lamp of the start SW 42 to be lit stationary and starts the progress of the tMR time.
During this tMR time, as shown in FIG. 7, the capture probe 28a, the single-stranded target DNA 28b, and the reporter probe 28c are annealed. Here, when the DNA to be detected is obtained, the DNA amplified by PCR corresponds to the single-stranded target DNA 28b. Therefore, when the sample solution contains DNA to be detected, the capture probe 28a, the single-stranded target DNA 28b, and the reporter probe 28c are annealed.

  After completion of the tMR time, the DNA detection apparatus 30 shifts the hybrid station 35 to the measurement temperature, and when this measurement temperature is reached, the buzzer sounds again and a display prompting the electrode unit 32 to slide to the measurement station 36 is performed. . Specifically, “Please move the electrode to the measurement station and press the start SW” is displayed on the LCD display unit 51. At this time, the start lamp of the start SW 42 is blinked, and during this time, the progress of the measurement time is stopped. In accordance with this instruction, the user moves the electrode unit 32 to the measurement station 36 as in step 3 of FIG.

  Here, when the DNA to be detected is contained in the sample solution, the capture probe 28a, the target DNA 28b, and the reporter probe 28c are bonded in this order on the electrode 32a of the electrode portion 32. The enzyme 26 is bound to the reporter probe 28c.

When the start SW 42 is pressed, the DNA detection device 30 causes the start lamp to be lit stationary and starts the measurement time.
The measurement of the current value is performed by applying a potential of +500 mV to the electrode 32a of the electrode part 32 with reference to the reference electrode 32b, and measuring the current flowing through the electrode 32a.

  Here, when a potential is applied to the electrode 32a of the electrode part 32, the substrate and the enzyme in the determination solution react, and electrons are exchanged via the mediator. Specifically, as shown in FIG. 7, transfer of electrons generated when the enzyme 26 reacts with the substrate 25 to generate an oxidant substrate 27 is transmitted to the carbon electrode 21 via the mediator 22. And the DNA detection apparatus 30 measures the electric current value of the electric current (limit oxidation current) which flows into the carbon electrode 21 of the electrode 32a.

  Even when only an electrolyte solution is used and no enzyme reaction occurs, a base current is generated. However, the electron transfer in the oxidation-reduction reaction when the enzyme reaction occurs is compared with this base current. And can be measured as a larger value.

  Thus, the presence or concentration of the target DNA can be detected by monitoring the electron transfer between the enzyme and the electrode based on the enzyme reaction, that is, by capturing a series of electron transfer as an electric current. For this reason, it becomes possible to detect DNA by a sandwich hybridization assay that uses a reporter probe for obtaining a signal by further binding to a capture probe for capturing the target DNA and the captured target DNA.

During the measurement, the DNA detection device 30 transmits measurement data in a text file format to the personal computer at a predetermined interval (for example, every second).
During the measurement, a display screen 120 related to the measurement state shown in FIG.

  As shown in FIG. 6, the display screen 120 is provided with a measurement numerical value display column 121 and a measurement result graph display column 122. The measurement numerical value display column 121 displays the measurement tube actual temperature, measurement time setting (minutes), measurement work voltage (mV), measurement remaining time (sec), and measurement actual data (nA). In the measurement result graph display field 122, the measurement result is drawn as a graph. In this graph, the X axis is measurement time, and the Y axis is measurement data (current value).

As described above, according to the present embodiment, in addition to the effects (1) to (3), (6), and (7), the following effects can be obtained.
(8) In the said embodiment, the electrode 32a formed by the capture probe 28a couple | bonding with the surface of a conductor (carbon electrode 21), the temperature control part, and the electric current measurement part which measures the electric current value which flows into the electrode 32a are provided. A specific microorganism (Salmonella) can be detected using the DNA detection apparatus 30. That is, the temperature of the sample solution containing the microorganisms bound to the capturing body is controlled to extract DNA by the thermal extraction method, and the temperature of the sample solution containing the extracted DNA is controlled to amplify the target DNA 28b by the PCR method. To do. Then, the reporter probe 28c labeled with the enzyme 26 and the substrate 25 that reacts with the enzyme 26 are used to control the temperature of the sample solution containing the amplification product, and the target DNA 28b is attached to the capture probe 28a and the reporter probe 28c. Combine. Then, the target DNA 28b is detected by measuring the value of the current flowing through the electrode 32a by the reaction between the enzyme 26 labeled on the reporter probe 28c and the substrate 25. Thereby, DNA extraction from the captured and washed microorganism, amplification by the PCR method, and detection of the target DNA can be performed using the DNA detection device 30. For this reason, the target DNA can be detected more easily, simply and with high sensitivity. Therefore, Salmonella can be detected more easily, simply and with high sensitivity.

In addition, you may change the said embodiment into the following aspects.
-In the said embodiment, although the microorganisms to be detected were Salmonella, the microorganisms detected by the microorganism detection method of this invention are not limited to this. When detecting a specific microorganism, a ligand or an antibody immobilized on a capturing body used when capturing a microorganism is combined with a constituent component of the detection target microorganism according to the microorganism to be detected. To do. In addition, depending on the microorganism to be detected, a DNA region that is a target for amplification of DNA by PCR and detection of target DNA that is an amplification product is characteristic of the microorganism to be detected. Thereby, a specific microorganism can be detected quickly and with high accuracy.

  The microorganism detection method of the present invention can be used for detection of microorganisms in a wide range of fields such as for environmental measurement in addition to the medical field and food field.

Explanatory drawing of the process sequence of the microorganisms detection method of this invention. The perspective view of the DNA detection apparatus used by one Embodiment of this invention. The block diagram which shows the circuit structure of the DNA detection apparatus used by one Embodiment of this invention. Explanatory drawing of temperature control of the DNA detection apparatus used by one Embodiment of this invention. Explanatory drawing of the electrode movement in the DNA detection apparatus used by one Embodiment of this invention. Explanatory drawing of the display screen of the DNA detection apparatus used by one Embodiment of this invention. Explanatory drawing of reaction in the electrode of the DNA detection apparatus used by one Embodiment of this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 21 ... Carbon electrode (conductor), 22 ... Mediator, 25 ... Substrate, 26 ... Enzyme, 27 ... Oxidized substrate, 28a ... Capture probe, 28b ... Target DNA, 28c ... Reporter probe, 30 ... DNA detection apparatus, 32 ... Electrode part (biosensor), 32a ... electrode, 32b ... reference electrode, 32c ... counter electrode.

Claims (10)

  A step of capturing a microorganism using a capture body in which a ligand or an antibody that binds to a component of the microorganism is immobilized; a step of washing the captured microorganism; a step of extracting DNA from the washed microorganism; and the extraction A microorganism detection method comprising a step of amplifying the obtained DNA by a PCR method and a step of detecting a target DNA that is an amplification product.   The step of washing the captured microorganisms uses a liquid selected from a buffer solution, a buffer solution containing a polyanion, a buffer solution containing a surfactant, an aqueous surfactant solution, and an aqueous alcohol solution. The microorganism detection method according to claim 1.   The microorganism detection method according to claim 1 or 2, wherein the microorganism to be detected is Salmonella. Genes to be amplified in the step of amplifying the extracted DNA by PCR method are fimA, agfA, fliC, invA, invE, sigD, prt, ompC, ompF, ompA, sipB, sipC, tctC
The microorganism detection method according to claim 3, wherein the microorganism detection method is selected from the group consisting of:   The capture body includes a magnetic bead on which a ligand or an antibody that binds to a component of a microorganism is immobilized, a magnetic nanoparticle on which a ligand or an antibody that binds to a component of a microorganism is immobilized, and a ligand that binds to a component of a microorganism Or it is any one of the containers which fix | immobilized the antibody to the inner surface, The microorganism detection method as described in any one of Claims 1-4 characterized by the above-mentioned.   The step of detecting the target DNA, which is the amplification product, includes the step of detecting the capture probe using an electrode formed by binding a capture probe to the surface of a conductor, an enzyme-labeled reporter probe, and a substrate that reacts with the enzyme. The target DNA is detected by binding a target DNA to the reporter probe, and measuring a value of a current flowing through the electrode by a reaction between the enzyme labeled on the reporter probe and the substrate. Item 6. The microorganism detection method according to any one of Items 1 to 5.   6. The step of detecting the target DNA as an amplification product is performed by any one of electrophoresis and a method of detecting amplification of the target DNA using fluorescence. The microorganisms detection method as described in any one.   8. The microorganism according to any one of claims 1 to 7, wherein the step of extracting DNA from the washed microorganism comprises extracting DNA from the microorganism in a state bound to the capturing body by a thermal extraction method. Detection method. Using a DNA detection apparatus provided with an electrode in which a capture probe is bonded to the surface of a conductor, a temperature control unit, and a current measurement unit that measures a current value flowing through the electrode,
The step of extracting DNA from the washed microorganism extracts DNA by a thermal extraction method by controlling the temperature of the sample solution containing the microorganism in a state of being bound to the capture body by the temperature control unit,
The step of amplifying the extracted DNA by the PCR method amplifies the target DNA by the PCR method by controlling the temperature of the sample solution containing the extracted DNA by the temperature control unit,
The step of detecting the target DNA that is the amplification product uses an enzyme-labeled reporter probe and a substrate that reacts with the enzyme, and controls the temperature of the sample solution containing the amplification product by the temperature controller, The target DNA is bound to the capture probe and the reporter probe, and the current measurement unit measures the value of the current flowing through the electrode by the reaction with the enzyme labeled with the reporter probe and the substrate. The method for detecting a microorganism according to any one of claims 1 to 5, wherein DNA is detected.   The microorganism detection method according to any one of claims 3 to 9, wherein the constituent component is lipopolysaccharide, and the ligand or antibody that binds to the constituent component is polymyxin B.

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