Apparatus and method

Abstract

Provided is a device provided with: a first filtration unit for filtering a fluid sample with a filter; a culturing unit for culturing using a result product obtained by performing a processing treatment for reducing the planar size of the filter after filtration by the first filtration unit; a detection unit for detecting the nucleic acid of the organism cultured by the culture unit; and a processing unit that performs processing on the filter that has been filtered by the first filtering unit to generate a filter processed product as the resulting product, and that cuts the filter that has been filtered by the first filtering unit to generate the filter processed product.

Description

Technical Field

[0001] The present invention relates to an apparatus and a method. Background Art

[0002] Patent Documents 1 to 5 disclose "trapping microorganisms on a membrane filter with a frame, culturing the microorganisms while maintaining them on the membrane filter with the frame, and performing immobilization, fluorescence detection, hybridization, washing, and observation using a fluorescence microscope", etc. Prior Art Documents Patent Document 1: Japanese Unexamined Patent Application Publication No. 2006-296285 Patent Document 2: Japanese Unexamined Patent Application Publication No. 2007-202553 Patent Document 3: Japanese Unexamined Patent Application Publication No. 2009-034093 Patent Document 4: Japanese Unexamined Patent Application Publication No. 2010-213724 Patent Document 5: Japanese Unexamined Patent Application Publication No. 2009-082153 Patent Document 6: International Publication No. 2019 / 43779 Patent Document 7: Japanese Patent No. 5624487 Patent Document 8: Japanese Patent No. 4857373 Summary of the Invention

[0003] In a first aspect of the present invention, there is provided an apparatus including: a first filtration unit that filters a fluid sample with a filter; a culturing unit that cultures using a resultant obtained by processing the filter that has passed through the first filtration unit to reduce a planar size; and a detection unit that detects nucleic acid of a living organism cultured by the culturing unit.

[0004] In the above apparatus, a processing unit may be further included, and the processing unit performs the processing on the filter that has passed through the first filtration unit to generate a filter processed product as the resultant.

[0005] In the above apparatus including the processing unit, the processing unit may cut the filter that has passed through the first filtration unit to generate the filter processed product.

[0006] In the above-described apparatus including the processing unit, the processing unit may bend the filter after being filtered by the first filter unit to generate a processed filter. Bending the filter may mean folding the filter in such a way that the front and back surfaces come into contact, in other words, bending it 180 degrees, or bending the filter at an angle less than 180 degrees. The bending line of the filter may be preset based on the longitudinal or transverse direction of the filter, or may be random. The planar shape of the culture tank of the culture unit may be smaller than the planar shape of the filter, or smaller than the cross-section of the flow path of the first filter unit. As long as the processed filter can be accommodated in the culture tank along the bottom surface of the culture tank, the planar shape of the processed filter may be the same as the planar shape of the culture tank, or a similar shape smaller than the planar shape of the culture tank.

[0007] In the above-described apparatus including the processing unit, it may further include: a second filter unit that filters the cultured liquid medium using the processed filter; and an extraction unit that extracts nucleic acid from the residue filtered by the second filter unit. The detection unit may detect the nucleic acid extracted by the extraction unit.

[0008] In the above-described apparatus, the extraction unit may accommodate the processed filter used in the filtration by the second filter unit in a sealed container and heat it to extract the nucleic acid of the organism.

[0009] In any of the above-described apparatuses, it may further include a third filter unit that filters the fluid sample with a pre-filter before filtration by the first filter unit, and the mesh of the pre-filter is larger than the mesh of the filter.

[0010] In a second aspect of the present invention, a method is provided, which includes: a first filtration step of filtering a fluid sample with a filter; a culture step of culturing using the resultant obtained by performing a processing for reducing the planar size on the filtered filter; and a detection step of detecting the nucleic acid of the organism cultured in the culture step.

[0011] In addition, the above summary of the invention does not list all of the essential features of the present invention. In addition, sub-combinations of these feature groups can also separately form inventions. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Microbial detection apparatus 1 showing an embodiment. Figure 2 Showing the states of the filter 101 and the processed filter 102 in the first filter unit 12, the processing unit 13, the culture unit 14, the second filter unit 15, and the nucleic acid extraction unit 16. Figure 3 Indicates the operation of the microorganism detection device 1. Explanation of Reference Numerals 1 Microorganism detection device 10 Sampling unit 11 Prefilter unit 12 First filter unit 13 Processing unit 14 Cultivation unit 15 Second filter unit 16 Nucleic acid extraction unit 17 Separation unit 18 Detection unit 19 Judgment unit 100 Prefilter 101 Filter 102 Filter processed product 105 Biochip Detailed Embodiment

[0013] Hereinafter, the present invention will be described by way of embodiments of the invention. However, the following embodiments do not limit the invention recited in the claims. In addition, all combinations of features described in the embodiments are not necessarily essential in the solution of the invention.

[0014] <1. Microorganism Detection Device> Figure 1 Indicates the microorganism detection device 1 of the present embodiment. The microorganism detection device 1 can be an example of a device for detecting microorganisms mixed in food and beverages. The microorganism detection device 1 includes a sampling unit 10, a prefilter unit 11, a first filter unit 12, a processing unit 13, a cultivation unit 14, a second filter unit 15, a nucleic acid extraction unit 16, a separation unit 17, a detection unit 18, and a judgment unit 19.

[0015] As an example, the microorganism to be detected may be selected from the group consisting of the genera Acinetobacter, Actinomyces, Aerococcus, Aeromonas, Alcaligenes, Bacillus, Bacteriodes, Bordetella, Branhamella, Brevibacterium, Campylobacter, Candida, Capnocytophaga, Chromobacterium, Clostridium, Corynebacterium, Cryptococcus, Deinococcus, Enterococcus, Erysipelothrix, Escherichia, Flavobacterium, Gemella, Haemophilus, Klebsiella, Lactobacillus, Lactococcus, Legionella, Leuconostoc, Listeria, Micrococcus, Mycobacterium, Neisseria, Cryptosporidium, Nocardia, Oerskovia, Paracoccus, Pediococcus, Peptostreptococcus, Propionibacterium, Proteus, Pseudomonas, Rahnella, Rhodococcus, Rhodospirillium, Staphylococcus, Streptomyces, Streptococcus, Vibrio, and Yersinia.There are microorganisms that form spores or other such morphological forms under oligotrophic conditions, regardless of the state of the cells caused by such growth conditions.

[0016] <1.1. Sampling section 10> The sampling section 10 extracts samples from food and beverages. The sampling section 10 can extract samples by sucking the beverage, or can extract samples by sucking the beverage and filtering it with a filter (also known as a membrane filter). The sampling section 10 can crush the food and extract samples. The sampling section 10 can make the sample into a fluid sample and supply it to the pre-filter section 11. When the sampling section 10 extracts a sample obtained by crushing food, it can supply it to the pre-filter section 11 after putting the sample into physiological saline or a liquid medium to make it a fluid sample.

[0017] <1.2. Pre-filter section 11> The pre-filter section 11 is an example of a third filtering section. Before filtering with the first filtering section 12, the fluid sample is filtered with a pre-filter 100. The mesh size of the pre-filter 100 (also known as the pore size of the filter) can be larger than the mesh size of the filter 101 described later used in the first filtering section 12. For example, the pre-filter section 11 can perform rough filtering on the fluid sample, and the mesh size of the pre-filter 100 can be larger than 5 μm. The pre-filter 100 can remove impurities other than microorganisms from the fluid sample. The material of the pre-filter 100 is not particularly limited as long as it does not interfere with the filtering in the first filtering section 12 and the culturing in the culturing section 14. As the material of the pre-filter 100, in addition to any of the materials described as the material of the filter 101 described later, cellulose fiber (as an example, ordinary filter paper), polyester, PET (as an example, a polyester mesh, a PET mesh) can be used. The pre-filter section 11 can supply the filtered fluid sample to the first filtering section 12.

[0018] <1.3. First filtering section 12> The first filtering section 12 filters the fluid sample with a filter 101.

[0019] The filter 101 used for filtering preferably has a pore size capable of capturing the microorganism to be detected. For example, the first filtering section 12 can perform precision filtering on the fluid sample, and the mesh size of the filter 101 is preferably 0.45 μm or less. The filter 101 can be a screen filter or a depth filter. In this embodiment, as an example, it is a screen filter. As long as the filter 101 is not washed away by the fluid sample in the first filtering section 12, the planar shape of the filter 101 can be the same as the cross-sectional shape of the flow channel of the first filtering section 12. As an example, it can be circular.

[0020] The material of the filter 101 is not particularly limited as long as it does not hinder the nucleic acid extraction in the nucleic acid extraction unit 16 described later and is difficult to adsorb the extracted nucleic acid. For example, the material of the filter 101 can be polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyethersulfone (PES), cellulose mixed ester, polycarbonate (PC), nylon, polyvinyl chloride (PVC), pure silver, etc. In addition, the so-called "cellulose mixed ester" is a material composed of a mixture of biologically inactive cellulose acetate and cellulose nitrate. The material of the filter 101 is preferably hydrophilic. For example, hydrophilic PTFE, hydrophilic PVDF, hydrophilic PES, and hydrophilic PC are preferred. If the filter 101 is hydrophilic, the nucleic acid extracted in the nucleic acid extraction unit 16 described later is difficult to adsorb to the filter 101. Among them, as the material of the filter 101, hydrophilic PC is more preferred. If the material of the filter 101 is hydrophilic PC, the pore size and pore size distribution are likely to be constant. By using the filter 101 with a constant pore size and pore size distribution, microorganisms can be stably captured. As an index indicating that the material of the filter 101 is hydrophilic, the contact angle can be cited. As the above filter, for example, a filter manufactured by Merck KGaA or a filter manufactured by ADVANTEC Toyo Co., Ltd. can be used. The first filtration unit 12 can supply the filter 101 that captures microorganisms as residues to the processing unit 13.

[0021] <1.4. Processing Unit 13> The processing unit 13 performs a processing for reducing the planar size of the filtered filter 101 of the first filtration unit 12 to generate a filter processed product 102. The processing unit 13 can process the filter 101 supplied from the first filtration unit 12 to generate a filter processed product 102. The processing unit 13 can cut the filtered filter 101 of the first filtration unit 12 to generate a filter processed product 102. On this basis or instead of this, the processing unit 13 can bend the filtered filter 101 of the first filtration unit 12 to generate a filter processed product 102.

[0022] The processing unit 13 can generate the filter processed product 102 into a size that can be placed on the solid culture medium in the culture unit 14 or a size that can be immersed in the liquid culture medium in the culture unit 14. For example, the filter processed product 102 can be generated according to the size of a culture tank (not shown). As an example, the processing unit 13 can cut (i.e., trim) the filter 101 using a mold pre-formed according to the planar shape (e.g., bottom surface shape) of the culture tank, or can bend it. Bending the filter 101 means folding the filter 101 with the front and back surfaces in contact, in other words, the filter 101 can be bent at 180 degrees, or the filter 101 can be bent at an angle less than 180 degrees. The bending line of the filter 101 can be preset based on the longitudinal or transverse direction of the filter 101, or can be random. As an example, the filter 101 can be crumpled to generate the filter processed product 102. The planar shape of the culture tank can be smaller than the planar shape of the filter 101 and smaller than the cross-section of the flow channel of the first filter unit 12. As long as the filter processed product 102 is housed along the bottom surface of the culture tank, the planar shape of the filter processed product 102 can be the same as the planar shape of the culture tank or a similar shape smaller than the planar shape of the culture tank.

[0023] In addition, the processing unit 13 generates the filter processed product 102 in a manner that can be used as a filter in the second filter unit 15, and can also generate the filter processed product 102 according to the cross-sectional shape of the flow channel of the second filter unit 15. In this case, the processing unit 13 can cut (i.e., trim) the filter 101 using a mold pre-formed according to the cross-sectional shape of the flow channel of the second filter unit 15, or can bend it. The cross-sectional shape of the flow channel of the second filter unit 15 can be smaller than the cross-sectional shape of the flow channel of the first filter unit 12 and the planar shape of the culture tank. As long as the filter processed product 102 is not washed away by the fluid sample in the second filter unit 15, the planar shape of the filter processed product 102 can be the same as the cross-sectional shape of the flow channel of the second filter unit 15. As an example, it can be circular.

[0024] The processing unit 13 can process the filter 101 in a state wetted by the liquid culture medium to generate the filter processed product 102, or can cut the dried filter 101 to generate the filter processed product 102. The processing unit 13 can discard the remaining part of the filter 101 that has not been used as the filter processed product 102. The processing unit 13 can supply the generated filter processed product 102 to the culture unit 14.

[0025] <1.5. Culture Unit 14> The culturing unit 14 cultures using the resultant obtained by subjecting the filtered filter 101 to a process of reducing its planar size. The culturing unit 14 can culture using the filter processed product 102 generated by the processing unit 13.

[0026] The culturing unit 14 can culture the microorganisms captured on the filter 101. The culturing unit 14 can culture using any conventionally known method and conditions according to the microorganism to be detected. For example, the culturing unit 14 can culture the sample on a solid medium (i.e., solid-phase culture). As an example, the filter processed product 102 can be placed on the solid medium for culturing. Instead, the culturing unit 14 can also culture the sample in a liquid medium (i.e., liquid-phase culture). As an example, the filter processed product 102 can be immersed in the liquid medium for culturing. The liquid medium can be a solution obtained by dissolving the solid medium.

[0027] Here, since the planar size of the filter processed product 102 is smaller than that of the filter 101, the culture tank of the present embodiment that houses the filter processed product 102 can have a smaller planar shape compared to the culture tank that houses the filter 101 (also referred to as the conventional culture tank). Thus, the culture medium in the culture tank of the present embodiment can have a smaller planar size compared to the culture medium in the conventional culture tank. However, the thickness of the solid culture medium (or the depth of the liquid culture medium) in the culture tank of the present embodiment can be the same as the thickness of the solid culture medium (or the depth of the liquid culture medium) in the conventional culture tank.

[0028] Therefore, the volume of the culture medium in the culture tank of the present embodiment can be smaller compared to the conventional culture tank. On the other hand, the culture tank of the present embodiment can culture the same amount of microorganisms as the conventional culture tank. Thus, the culture tank of the present embodiment can culture microorganisms at a higher concentration compared to the conventional culture tank.

[0029] The culturing unit 14 can supply the cultured liquid culture medium to the second filtering unit 15. When solid-phase culture is performed, the culturing unit 14 can dissolve the solid culture medium and supply it as a liquid culture medium to the second filtering unit 15.

[0030] <1.6. Second Filtering Unit 15> The second filtering unit 15 filters the cultured liquid culture medium using the filter processed product 102. Thereby, the cultured microorganisms are recovered as residues.

[0031] The second filtration unit 15 can supply the recovered microorganisms to the nucleic acid extraction unit 16. In the present embodiment, as an example, the second filtration unit 15 can supply the microorganisms recovered as residues on the filter processed product 102 to the nucleic acid extraction unit 16 for each filter processed product 102.

[0032] <1.7. Nucleic Acid Extraction Unit 16> The nucleic acid extraction unit 16 is an example of an extraction unit, and extracts nucleic acids (as examples, genomic DNA, ribosomal RNA, plasmid DNA, etc.) from the residues filtered by the second filtration unit 15. The nucleic acid extraction unit 16 can accommodate the filter (as an example, the filter processed product 102 in the present embodiment) used by the second filtration unit during filtration into a sealed container (also referred to as a sealed container), heat it, and extract the nucleic acids of the microorganisms. For example, the method described in International Publication No. 2019 / 43779 and Japanese Patent No. 5624487, the so-called dHTP method, can be used to extract nucleic acids.

[0033] The nucleic acid extraction unit 16 can expose the microorganisms as residues to high-temperature conditions in a sealed container for each filter processed product 102, thereby destroying the membrane structure of the microorganisms and making them in a state where nucleic acids can be extracted. The container can be, for example, a bag that can be heat-sealed, or a lock-cap microcentrifuge tube (boil lock tube). The container can be, for example, a plastic tube, a glass test tube, or a microfluidic chip.

[0034] The container can be heated by a heating device (not shown). The heating device can be an oil bath or a heat block, and can heat the inside of the container to a temperature of 200°C. By heating, the pressure inside the container can become above atmospheric pressure.

[0035] At least one cell lysis promoter selected from the group consisting of an alkali, an acid, an enzyme, a surfactant, an oxidation-reduction agent, and a protein denaturant having the ability to dissolve the membrane structure can be added to the container.

[0036] As the above-mentioned alkali, for example, sodium hydroxide (NaOH) or potassium hydroxide (KOH) can be cited. As the above-mentioned acid, for example, hydrochloric acid (HCl) or sulfuric acid (H2SO4) can be cited. As the above-mentioned enzyme, for example, proteolytic enzymes such as Proteinase K or polysaccharide-degrading enzymes such as chitinase, lysozyme, and zymolyase can be cited. The above-mentioned surfactant can be, for example, ionic or non-ionic. As the non-ionic surfactant, for example, octylphenol ethoxylate (C 14 H 22O(C2H4O)n), etc. As octylphenol ethoxylate, for example, Triton X-100 (C 14 H 22 O(C2H4O)n, n = 100), etc. commercially available products can be used.

[0037] In addition, the ionic surfactant can be anionic, cationic, or zwitterionic. As anionic surfactants, for example, sodium dodecylsulfate (SDS), etc. can be cited. As cationic surfactants, for example, cetyltrimethylammonium bromide (CTAB), etc. can be cited. As zwitterionic surfactants, for example, betaine, etc. can be cited. Here, the so-called "betaine" is a general term for compounds that have a positive charge and a negative charge at non-adjacent positions within the same molecule, and do not have a dissociable hydrogen atom bonded to the atom with the positive charge, so that the molecule as a whole does not have a charge. As a representative example of betaine, trimethylglycine can be cited. As the above-mentioned redox agents, for example, hydrogen peroxide water, β-mercaptoethanol, dithiothreitol, etc. can be cited.

[0038] As the above-mentioned protein denaturants, for example, guanidine hydrochloride, urea, etc. can be cited. As the above-mentioned chelating agents, for example, ethylenediaminetetraacetic acid (EDTA), etc. can be cited. Even among the above-mentioned cell lysis promoters, it is preferable to add the above-mentioned surfactant, and more preferably to add either or both of SDS and octylphenol ethoxylate. For example, when nucleic acid extracted is to be detected with high sensitivity, SDS can be used. In contrast, when the extracted nucleic acid is to be used in an enzyme reaction inhibited by SDS, octylphenol ethoxylate, which acts more gently on the membrane structure of microorganisms, can be used.

[0039] A buffer solution can also be contained in the container. As buffer solutions, for example, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), etc. can be cited.

[0040] The nucleic acid extraction unit 16 can extract nucleic acid from the liquid in the container after destroying the membrane structure of the microorganism. The nucleic acid extraction unit 16 can, for example, bind the nucleic acid to magnetic particles by the method described in Japanese Patent No. 4857373 and perform extraction using magnetic force. Instead of this, the nucleic acid extraction unit 16 can also extract nucleic acid by at least one of filtration, centrifugation, and electrophoresis. When extracting nucleic acid by electrophoresis, the nucleic acid can be pre-charged by a known method.

[0041] The nucleic acid extraction unit 16 can supply a fluid sample containing the extracted nucleic acid to the separation unit 17. The nucleic acid extraction unit 16 can supply the fluid sample after nucleic acid amplification to the separation unit 17. The nucleic acid extraction unit 16 can extract the nucleic acid after amplification in a liquid in which the membrane structure of the microorganism has been destroyed, or can perform amplification after extracting the nucleic acid. The nucleic acid extraction unit 16 can amplify the nucleic acid by the method of PCR (polymerase chain reaction).

[0042] <1.8. Separation unit 17> The separation unit 17 separates the nucleic acid of the microorganism to be detected from the nucleic acid in the fluid sample. The separation unit 17 can separate the nucleic acid inherent to the microorganism to be detected (also referred to as the target nucleic acid) from the nucleic acid of the microorganism to be detected. The separation unit 17 can use the biochip 105 to separate the target nucleic acid.

[0043] The biochip 105 can have a plurality of probes that hybridize with corresponding base sequences. Among the plurality of probes, at least a part of the probes can have a base sequence complementary to at least a part of the base sequence of the target nucleic acid and can specifically hybridize with the nucleic acid of the corresponding base sequence. Among the plurality of probes, at least a part of the probes can have the same base sequence.

[0044] Each probe can be pre-fixed at an inherent position within the biochip 105. In the present embodiment, as an example, the biochip 105 can have a plurality of probes on the inner side surfaces of two relatively arranged transparent substrates. The biochip 105 can be provided with an injection port for injecting a fluid sample and an outlet port for discharging the fluid sample inside.

[0045] A label that emits a signal detectable by the subsequent detection unit 18 in a hybridized state can be pre-attached to at least one of the probe of the biochip 105 having a complementary base sequence and the nucleic acid in the fluid sample. For example, the label can be attached to the probe, and a signal can be emitted according to the hybridization of the probe and the nucleic acid. Instead, the label can be attached to the nucleic acid in the fluid sample, and a signal is emitted regardless of whether the nucleic acid and the probe are hybridized. In this case, after injecting the fluid sample into the biochip 105, the nucleic acid that has not hybridized with the probe can be removed from the biochip 105, and then the signal can be detected. As the label, a fluorescent dye, a radioactive isotope, a paramagnetic isotope, an enzyme, etc. can be used. In the present embodiment, as an example, the label can be a fluorescent dye and can be attached to the probe.

[0046] <1.9. Detection unit 18> The detection unit 18 detects the nucleic acid of the microorganism after being cultured by the culturing unit 14. The detection unit 18 can detect the nucleic acid extracted by the nucleic acid extraction unit 16. The detection unit 18 can detect the nucleic acid by detecting the labels in the biochip 105. The detection unit 18 can be disposed opposite to the biochip 105 and can detect the labels in the biochip 105 through the transparent substrate of the biochip 105. The detection unit 18 can detect the signal emitted from the label attached to either the hybridized probe or the nucleic acid. The detection unit 18 can supply information indicating the position of the detected label and the intensity of the signal emitted from the label (as an example, the fluorescence intensity in the present embodiment) to the determination unit 19.

[0047] <1.10. Determination unit 19> The determination unit 19 determines whether the nucleic acid of the microorganism to be detected in the fluid sample injected into the biochip 105 exceeds the reference value, in other words, determines whether the fluid sample is positive or negative. The determination unit 19 can make a determination based on the information supplied from the detection unit 18. For example, the determination unit 19 can make a determination based on the detection position of the label detected by the detection unit 18. The determination unit 19 can externally output the determination result indicating whether the fluid sample is positive or negative.

[0048] The reference value can represent the number of nucleic acids and can be the number of labels detected by the detection unit 18 as the target nucleic acid. In the present embodiment, as an example, the reference value can be 0. However, the reference value can also represent the proportion of nucleic acids and can be the proportion of the labels detected by the detection unit 18 as the target nucleic acid among the number of labels detected by the detection unit 18.

[0049] According to the above microorganism detection device 1, using the resultant obtained by processing the filter 101 after filtration by the first filtration unit 12 to reduce the planar size for culturing can reduce the area of the culture medium. Thus, the microorganism can be cultured at a high concentration with a narrow culture medium, and therefore the detection accuracy of the nucleic acid can be improved.

[0050] In addition, the filter 101 after filtration is processed by the processing unit 13 to generate a filter processed product 102. Therefore, compared with the case of generating the filter processed product 102 from the filter 101 outside the microorganism detection device 1, the efficiency from filtration by the first filtration unit 12 to culturing by the culturing unit 14 can be made higher.

[0051] In addition, since the filter 101 after being filtered by the first filter unit 12 is cut to produce the filter processed product 102, it is possible to easily produce the filter processed product 102 having a smaller planar size than the filter 101. On this basis or instead of this, the filtered filter 101 is bent to produce the filter processed product 102, and thus it is possible to easily produce the filter processed product 102 having a smaller planar size than the filter 101.

[0052] In addition, the filter processed product 102 generated from the filter 101 after filtering the liquid sample is used to filter the cultured liquid medium. Therefore, compared with the case where different filters are used for filtering the liquid sample and filtering the liquid medium respectively, the usage amount of the filter can be reduced.

[0053] In addition, since the filter processed product 102 after filtering the cultured liquid medium is housed in a sealed container and heated to extract the nucleic acid of the microorganism, it is possible to save the time and labor for separating the microorganism from the filter processed product 102, and it is possible to easily obtain the nucleic acid of the microorganism in the liquid sample.

[0054] In addition, before filtering by the first filter unit 12, the fluid sample is filtered with a pre-filter 100 having a larger mesh size than the filter 101, so that impurities in the fluid sample can be removed and cultured.

[0055] <2. Filter 101 and Filter Processed Product 102> Figure 2 Shows the states of the filter 101 and the filter processed product 102 in the first filter unit 12, the processing unit 13, the culturing unit 14, the second filter unit 15, and the nucleic acid extraction unit 16. In addition, the white circular marks in the figure represent the microorganisms to be detected. In addition, the white arrow marks in the figure represent the processing flow in the microorganism detection device 1.

[0056] The filter 101 is used for filtering the fluid sample in the first filter unit 12. After capturing the microorganism (refer to the upper left part in the figure), it is subjected to processing by the processing unit 13 to become the filter processed product 102 (refer to the upper central part in the figure). Then, the filter processed product 102 is used for culturing in the culturing unit 14 (refer to the upper right part in the figure) and for filtering the liquid medium in the second filter unit 15 to capture the microorganism (refer to the lower central part in the figure). Moreover, the filter processed product 102 is housed in a sealed container in the nucleic acid extraction unit 16 and heated (refer to the lower right part in the figure) to extract the nucleic acid of the microorganism contained in the sample.

[0057] <3. Operation of Microorganism Detection Device 1> Figure 3Indicates the operation of the microorganism detection device 1. The microorganism detection device 1 determines whether the microorganisms to be detected in each sample exceed the reference value by performing the processes of steps S11 to S29.

[0058] In step S11, the sampling unit 10 extracts a sample from food or beverage. The sampling unit 10 can generate a fluid sample from the sample.

[0059] In step S13, the pre-filtering unit 11 filters the fluid sample with the pre-filter 100. Thereby, impurities in the fluid sample can be removed.

[0060] In step S15, the first filtering unit 12 filters the fluid sample with the filter 101. Thereby, the microorganisms to be detected can be captured as residues.

[0061] In step S17, the processing unit 13 performs a processing for reducing the planar size of the filtered filter 101 to generate a filter processed product 102. The microorganisms captured by the filter 101 in step S15 can adhere to the filter processed product 102. In addition, the processing unit 13 can be cleaned after the generation of the filter processed product 102. Thereby, it is possible to prevent the microorganisms adhering to the filter 101 from remaining in the processing unit 13.

[0062] In step S19, the culturing unit 14 performs culturing using the resultant obtained by performing a processing for reducing the planar size of the filtered filter 101. In the present embodiment, as an example, the culturing unit 14 can perform culturing using the filter processed product 102. The microorganisms can be captured on the filter processed product 102. Thereby, the microorganisms captured on the filter processed product 102 are cultured.

[0063] In step S21, the second filtering unit 15 filters the cultured liquid medium using the filter processed product 102. Thereby, the microorganisms cultured in step S19 can be captured. In step S21, the liquid medium filtered by the filter processed product 102 can be the liquid medium impregnated with the filter processed product 102 in step S19.

[0064] In step S23, the nucleic acid extraction unit 16 extracts the nucleic acid of the microorganisms contained in the liquid medium from the residue filtered by the second filtering unit 15. As an example, the nucleic acid extraction unit 16 can accommodate the microorganisms as residues together with the filter processed product 102 in a container, and after destroying the membrane structure of the microorganisms using the so-called dHTP method, extract the nucleic acid by filtration, centrifugation, etc.

[0065] In step S25, the separation unit 17 separates the nucleic acid of the microorganism to be detected from the nucleic acids in the fluid sample. As an example, the separation unit 17 can hybridize the nucleic acid of the microorganism to be detected contained in the fluid sample with the probes of the biochip 105 for separation.

[0066] In step S27, the detection unit 18 detects the nucleic acid of the cultured microorganism. The detection unit 18 can detect the nucleic acid by detecting the labels in the biochip 105. As an example, the detection unit 18 can detect the signal emitted from the label attached to at least one of the probes of the biochip 105 and the nucleic acid in the fluid sample.

[0067] In step S29, the determination unit 19 determines, based on the information supplied from the detection unit 18, whether the nucleic acid of the microorganism to be detected in the fluid sample injected into the biochip 105 exceeds a reference value, in other words, determines whether the fluid sample is positive or negative.

[0068] <4. Modified Example> In addition, in the above-described embodiment, it is illustrated that the microorganism detection device 1 includes the sampling unit 10, the pre-filter unit 11, the processing unit 13, the second filter unit 15, the nucleic acid extraction unit 16, the separation unit 17, and the determination unit 19, but any of these may not be provided. For example, when the microorganism detection device 1 does not include the processing unit 13, the filter processed product 102 can be generated from the filtered filter 101 by an external device of the microorganism detection device 1 or by an operator. In addition, when the microorganism detection device 1 does not include the second filter unit 15 and the nucleic acid extraction unit 16, the detection unit 18 can detect the nucleic acid of the microorganism cultured on the solid medium by the culturing unit 14. In this case, as an example, the separation unit 17 can bring a permeability imparting composition containing polyethyleneimine and at least one alcohol into contact with the filter processed product 102 and the microorganism on the solid medium, fix the microorganism to the filter processed product 102 with a crosslinking agent, and then allow the molecule containing the label and hybridized with the target nucleic acid to penetrate into the interior of the membrane structure of the microorganism. Thereby, the nucleic acid of the cultured microorganism can be detected. As a method for detecting such nucleic acid, for example, the methods described in Patent Documents 2 to 4 above can be used.

[0069] In addition, it is illustrated that the second filter unit 15 filters the liquid medium using the filter processed product 102 generated from the filter 101, but the liquid medium can also be filtered using another filter different from the filter processed product 102. The other filter can be the same filter as the filter 101.

[0070] In addition, it has been described that the processing unit 13 performs at least one of cutting or bending on the filter 101 to reduce the planar size, but the planar size can also be reduced by other processes. As an example, the processing unit 13 can maintain a state capable of culturing the microorganisms captured on the filter 101 and dissolve at least a part of the filter 101 to reduce the planar size. In this case, the culturing unit 14 can use the dissolution solution obtained by dissolving the filter 101 for culturing.

[0071] In addition, it has been described that the determination unit 19 determines whether the nucleic acid of the microorganism to be detected is contained in the sample, but it can also be determined whether the nucleic acid of an organism other than the microorganism is contained in the sample. In this case, the culturing unit 14 can culture the cells of the organism to be detected, and the second filtering unit 15 can recover the cultured cells. The organism to be detected can be an animal, an insect, a plant, a mycoplasma, a virus, etc. The organism to be detected can be one kind or a plurality of kinds.

[0072] As described above, the present invention has been described using the embodiments, but the technical scope of the present invention is not limited to the scope described in the above embodiments. Those skilled in the art can obviously make various changes or improvements to the above embodiments. It is obvious from the description of the claims that the embodiments to which such changes or improvements are applied can also be included in the technical scope of the present invention.

[0073] It should be noted that the execution order of each process such as the actions, sequences, steps, and stages in the devices, systems, programs, and methods shown in the claims, the specification, and the drawings can be implemented in any order as long as it is not specifically expressed as "earlier", "before", etc., and the output of the previous process is not used in the subsequent process. For the flow of actions in the claims, the specification, and the drawings, descriptions such as "first," "next," etc. are used for easy understanding, but it does not necessarily mean that they must be implemented in that order.

Claims

1. A device, characterized in that: have: A first filtering section, filtering the fluid sample using a filter; a culture section for culturing using a product obtained by processing the filter having been filtered through the first filtration section to reduce its plane size; as well as The detection unit detects nucleic acid of the organism cultured in the culture unit.

2. The device according to claim 1, characterized in that The apparatus further includes a processing unit configured to perform the processing on the filter after filtration by the first filtering unit to generate a processed filter product as the resultant product.

3. The device according to claim 2, characterized in that The processing unit cuts the filter after filtering by the first filtering unit to generate the filter processed product.

4. The device according to claim 2, characterized in that The processing unit bends the filter after filtering by the first filtering unit to generate the processed filter product.

5. The device according to claim 2, characterized in that Also available: a second filtering unit, filtering the cultured liquid culture medium using the filter processed product; and an extraction unit for extracting nucleic acid from the residue filtered by the second filtering unit, The detection unit detects the nucleic acid extracted by the extraction unit.

6. The device according to claim 5, characterized in that The extraction unit stores the filter processed product used for filtration by the second filtration unit in a sealed container and heats the product to extract nucleic acid from the organism.

7. The device according to claim 1, characterized in that The invention further comprises a third filter unit, which filters the fluid sample by using a pre-filter before filtering through the first filter unit, wherein the mesh of the pre-filter is larger than the mesh of the filter.

8. A method, characterized in that include: A first filtering step, filtering the fluid sample with a filter; a culturing step, using the resultant obtained by subjecting the filter after filtration to a treatment to reduce the plane size, for culturing; as well as The detection step is to detect the nucleic acid of the organism cultured in the culture step.

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