Apparatus and method

Abstract

To provide apparatus and method for extracting nucleic acid of organisms.SOLUTION: An apparatus comprises: a first filtration unit that filters a fluid sample with a first filter; a culture unit that immerses the first filter after filtration by the first filtration unit in a liquid medium to perform culture; a second filtration unit that filters the liquid medium after culture with a partial region of the first filter; an extraction unit that extracts nucleic acids of organisms contained in the liquid medium from the residue after filtration by the second filtration unit; and a production unit that cuts out the partial region of the first filter immersed in the liquid medium to produce a second filter, the second filtration unit filtering the liquid medium after culture with the second filter.SELECTED DRAWING: Figure 1

Description

Technical Field

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

Background Art

[0002] Patent Documents 1 to 5 describe "collecting microorganisms on a membrane filter with an outer frame, culturing, immobilizing, hybridizing with a fluorescent probe, washing, and observing with a fluorescence microscope while keeping the microorganisms on the membrane filter with an outer frame", etc. [Prior Art Documents] [Patent Documents] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-296285 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-202553 [Patent Document 3] Japanese Patent Application Laid-Open No. 2009-034093 [Patent Document 4] Japanese Patent Application Laid-Open No. 2010-213724 [Patent Document 5] Japanese Patent Application Laid-Open No. 2009-082153 [Patent Document 6] International Publication No. WO2019 / 43779 [Patent Document 7] U.S. Patent No. 5,624,487 [Patent Document 8] U.S. Patent No. 4,857,373

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 first filter; a culture unit that immerses the first filter after filtration by the first filtration unit in a liquid medium for culturing; a second filtration unit that filters the liquid medium after culturing in a partial region of the first filter; and an extraction unit that extracts nucleic acids of organisms contained in the liquid medium from residues after filtration by the second filtration unit.

[0004] In the above apparatus, it may further include a generation unit that cuts out the partial region of the first filter immersed in the liquid medium to generate a second filter. The second filtration unit may filter the liquid medium after culturing with the second filter.

[0005] In any of the above devices, the cross-sectional shape of the flow path in the second filtration unit may be smaller than the cross-sectional shape of the flow path in the first filtration unit.

[0006] In any of the above devices, the extraction unit may extract nucleic acids of organisms by heating the second filter that has filtered the liquid medium after cultivation, while accommodating the second filter in a sealed container.

[0007] In any of the above devices, a third filtration unit that filters a fluid sample with a pre-filter having a larger mesh size than the first filter may be further provided before filtration by the first filtration unit.

[0008] In a second aspect of the present invention, there is provided a method including: a first filtration step of filtering a fluid sample with a first filter; a cultivation step of immersing the first filter after filtration in the first filtration step in a liquid medium for cultivation; a second filtration step of filtering the liquid medium after cultivation in a partial region of the first filter; and an extraction step of extracting nucleic acids of organisms contained in the liquid medium from the residue after filtration in the second filtration step.

[0009] Note that the above summary of the invention does not list all the necessary features of the present invention. Also, sub-combinations of these feature groups can also be inventions.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Embodiments for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described through embodiments of the invention. However, the following embodiments do not limit the invention according to the claims. Also, not all combinations of features described in the embodiments are essential for the solution means of the invention.

[0012] <1. Microbial detection device> FIG. 1 shows a microbial detection device 1 according to the present embodiment. The microbial detection device 1 may be an example of a device and detects microorganisms mixed in food and beverages. The microbial detection device 1 includes a sampling unit 10, a pre-filtering unit 11, a first filtering unit 12, a culturing unit 13, a generating unit 14, a second filtering unit 15, a nucleic acid extraction unit 16, a separation unit 17, a detection unit 18, and a determination unit 19.

[0013] The microorganisms to be detected are, as an example, Acintobacter species, Actinomyces species, Aerococcus species, Aeromonas species, Alclaigenes species, Bacillus species, Bacteriodes species, Bordetella species, Branhamella species, Brevibacterium species, Campylobacter species, Candida species, Capnocytophaga species, Chromobacterium species, Clostridium species, Corynebacterium species, Cryptococcus species, Deinococcus species, Enterococcus species, Erysipelothrix species, Escherichia species, Flavobacterium species, Gemella species, Haemophilus species, Klebsiella species, Lactobacillus species, Lactococcus species, Legionella species, Leuconostoc species, Listeria species, Micrococcus species, Mycobacterium species, Neisseria species, Cryptosporidium species, Nocardia species, Oerskovia species, Paracoccus species, Pediococcus species, Peptostreptococcus species, Propionibacterium species, Proteus species, Pseudomonas species, Rahnella species, Rhodococcus species,It can be selected from the group consisting of Rhodospirillium species, Staphlococcus species, Streptomyces species, Streptococcus species, Vibrio species, and Yersinia species. There are microorganisms that take forms such as spores and cysts in oligotrophic states, regardless of the cell state due to such growth conditions.

[0014] <1.1. Sampling unit 10> The sampling unit 10 extracts samples from foods and beverages. The sampling unit 10 may extract a sample by sucking a beverage, or may extract a sample by sucking a beverage and filtering it with a filter (also referred to as a membrane filter). The sampling unit 10 may crush a food to extract a sample. The sampling unit 10 may supply the sample as a fluid sample to the pre-filtering unit 11. When the sampling unit 10 extracts a sample obtained by crushing a food, the sampling unit 10 may put the sample into physiological saline or a liquid medium and supply it as a fluid sample to the pre-filtering unit 11.

[0015] <1.2. Pre-filtering unit 11> The pre-filtering unit 11 is an example of the third filtering unit, and filters the fluid sample with a pre-filter 100 before filtering by the first filtering unit 12. The pre-filter 100 may have a larger mesh (also referred to as the pore size of the filter) than the first filter 101 described later used in the first filtering unit 12. For example, the pre-filtering unit 11 may roughly filter the fluid sample, and the mesh of the pre-filter 100 may be larger than 10 μm. The pre-filter 100 may remove contaminants other than microorganisms from the fluid sample. The material of the pre-filter 100 is not particularly limited as long as it does not inhibit the filtering in the first filtering unit 12 or the culturing in the culturing unit 13. As the material of the pre-filter 100, any of the materials described as the material of the first filter 101 described later may be used. The pre-filtering unit 11 may supply the filtered fluid sample to the first filtering unit 12.

[0016] <1.3. First filtering unit 12> The first filtration unit 12 filters the fluid sample with the first filter 101.

[0017] The first filter 101 used for filtration preferably has a pore size capable of capturing the microorganism to be detected. For example, the first filtration unit 12 may perform microfiltration on the fluid sample, and the mesh of the first filter 101 is preferably 0.45 μm or less. The first filter 101 may be a screen filter or a depth filter, but in this embodiment, it is a screen filter as an example. As long as the first filter 101 is not flowed by the fluid sample in the first filtration unit 12, the planar shape of the first filter 101 may be the same as the cross-sectional shape of the flow path of the first filtration unit 12, and may be circular as an example.

[0018] The material of the first filter 101 is not particularly limited as long as it does not inhibit nucleic acid extraction in the nucleic acid extraction unit 16 described later and the extracted nucleic acid is not easily adsorbed. For example, the material of the first filter 101 may be polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyethersulfone (PES), cellulose mixed ester, polycarbonate (PC), nylon, polyvinyl chloride (PVC), pure silver, etc. Note that "cellulose mixed ester" is a material composed of a mixture of cellulose acetate and cellulose nitrate that is biologically inert. The material of the first filter 101 is preferably hydrophilic. For example, hydrophilic PTFE, hydrophilic PVDF, hydrophilic PES, and hydrophilic PC are preferred. When the first filter 101 is hydrophilic, the nucleic acid extracted in the nucleic acid extraction unit 16 described later is not easily adsorbed to the first filter 101. Among them, hydrophilic PC is more preferred as the material of the first filter 101. When the material of the first filter 101 is hydrophilic PC, the pore size and pore size distribution are likely to be constant. By using the first filter 101 with a constant pore size and pore size distribution, microorganisms can be stably collected. One indicator indicating that the material of the first filter 101 is hydrophilic is the contact angle. As the above filter, for example, a filter manufactured by Merck KGaA (Millipore Corporation) or a filter manufactured by Advantec Toyo Co., Ltd. can be used. The first filtration unit 12 may supply the first filter 101 that has captured microorganisms as residues to the culture unit 13.

[0019] <1.4. Culture Unit 13> The culture unit 13 immerses the first filter 101 after filtration by the first filtration unit 12 in a liquid medium for culturing. The culture unit 13 may culture the microorganisms captured by the first filter 101. The culture unit 13 may perform culturing by any conventionally known method and conditions according to the microorganism to be detected. The liquid medium may be a solution in which a solid medium is dissolved.

[0020] The culture unit 13 may supply the liquid medium after culturing to the second filtration unit 15. The culture unit 13 may supply the first filter 101 immersed in the liquid medium to the generation unit 14. As an example, the culture unit 13 may take out the first filter 101 from the liquid medium after the completion of culturing and supply it to the generation unit 14, or may take out the first filter 101 from the liquid medium during the period from the start to the completion of culturing and supply it to the generation unit 14.

[0021] <1.5. Generation unit 14> The generation unit 14 cuts out a partial region of the first filter 101 immersed in the liquid medium to generate the second filter 102. The generation unit 14 may generate the second filter 102 from the first filter 101 supplied from the culture unit 13. The generation unit 14 may cut the first filter 101 in a state wet with the liquid medium to generate the second filter 102, or may cut the first filter 101 after drying it to generate the second filter 102. The generation unit 14 may discard the remaining portion of the first filter 101 that was not used as the second filter 102. When the first filter 101 is taken out from the liquid medium and supplied to the generation unit 14 during the period from the start to the completion of culturing by the culture unit 13, the generation unit 14 may return the remaining portion of the supplied first filter 101 that was not used as the second filter 102 to the culture unit 13 and immerse it again in the liquid medium.

[0022] The generation unit 14 may generate the second filter 102 in accordance with the cross-sectional shape of the flow path of the second filtration unit 15. As an example, the generation unit 14 may generate the second filter 102 by cutting the first filter 101 using a mold formed in advance in accordance with the cross-sectional shape of the flow path of the second filtration unit 15. The cross-sectional shape of the flow path in the second filtration unit 15 may be smaller than the cross-sectional shape of the flow path in the first filtration unit 12 and may be smaller than the planar shape of the first filter 101. As long as the second filter 102 is not flowed by the fluid sample in the second filtration unit 15, the planar shape of the second filter 102 may be the same as the cross-sectional shape of the flow path of the second filtration unit 15 and may be circular, for example. The generation unit 14 may supply the generated second filter 102 to the second filtration unit 15.

[0023] <1.6. Second Filtration Unit 15> The second filtration unit 15 filters the liquid medium after culturing in a partial area of the first filter 101. Thereby, the cultured microorganisms are recovered as residues. The second filtration unit 15 may filter the liquid medium after culturing with the second filter 102 generated by the generation unit 14.

[0024] The second filtration unit 15 may supply the recovered microorganisms to the nucleic acid extraction unit 16. In this embodiment, as an example, the second filtration unit 15 may supply the microorganisms recovered as residues on the second filter 102 to the nucleic acid extraction unit 16 together with the second filter 102.

[0025] <1.7. Nucleic Acid Extraction Unit 16> The nucleic acid extraction unit 16 extracts nucleic acids (such as genomic DNA, ribosomal RNA, plasmid DNA, etc.) of microorganisms contained in the liquid medium from the residues after filtration by the second filtration unit 15. The nucleic acid extraction unit 16 may accommodate the second filter 102 that has filtered the liquid medium after culturing in a sealed container (also referred to as a sealed vessel) and heat it to extract the nucleic acids of the microorganisms. For example, the method described in International Publication No. WO2019 / 43779 or Japanese Patent No. 5624487, so-called dHTP method, may be used to extract the nucleic acids.

[0026] The nucleic acid extraction unit 16 may expose the microorganisms as residues together with the second filter 102 to high-temperature conditions in a sealed container to destroy the membrane structure of the microorganisms and make the nucleic acids extractable. The container may be, for example, a heat-sealable bag, or a boil-lock tube. The container may be, for example, a plastic tube, a glass test tube, or a microfluidic chip.

[0027] The container may be heated by heating means (not shown). The heating means may be an oil bath or a heat block, and the inside of the container may be heated to a temperature up to 200°C. The pressure inside the container may be equal to or higher than atmospheric pressure by heating.

[0028] At least one cell lysis promoter selected from the group consisting of an alkali, an acid, an enzyme, a surfactant, a redox agent, and a protein denaturant having the ability to dissolve the membrane structure may be added to the container.

[0029] Examples of the above alkali include sodium hydroxide (NaOH) or potassium hydroxide (KOH). Examples of the above acid include hydrochloric acid (HCl) or sulfuric acid (H2SO4). Examples of the above enzyme include proteolytic enzymes such as Proteinase K, or polysaccharide-degrading enzymes such as chitinase, lysozyme, and zymolyase. The above surfactant may be, for example, ionic or non-ionic. Examples of the non-ionic surfactant include octylphenol ethoxylate (C14H22O(C2H4O)n). As octylphenol ethoxylate, commercially available products such as Triton X-100 (C14H22O(C2H4O)n, n = 100) manufactured by SIGMA can be used.

[0030] Also, the ionic surfactant may be anionic, cationic, or zwitterionic. Examples of the anionic surfactant include sodium dodecyl sulfate (SDS). Examples of the cationic surfactant include cetyltrimethylammonium bromide (CTAB). Examples of the zwitterionic surfactant include betaine. Here, "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 a dissociable hydrogen atom is not bonded to the atom having the positive charge, and the molecule as a whole has no charge. A representative example of betaine is trimethylglycine. Examples of the above redox agent include hydrogen peroxide solution, β-mercaptoethanol, dithiothreitol, and the like.

[0031] Examples of the protein denaturant include guanidine hydrochloride, urea, and the like. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA). Among the above-described cell lysis promoters, it is preferable to add the surfactant, and it is more preferable to add either one or both of SDS and octylphenol ethoxylate. For example, when it is desired to detect the extracted nucleic acid with high sensitivity, SDS may be used. On the other hand, when the extracted nucleic acid is used for an enzyme reaction inhibited by SDS, octylphenol ethoxylate, which acts more gently on the membrane structure of microorganisms than SDS, may be used.

[0032] The container may further contain a buffer solution. Examples of the buffer solution include tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl).

[0033] After the membrane structure of the microorganism is destroyed, the nucleic acid extraction unit 16 may extract the nucleic acid from the liquid in the container. The nucleic acid extraction unit 16 may extract the nucleic acid by binding the nucleic acid to magnetic fine particles and extracting it magnetically, for example, by the method described in Japanese Patent No. 4857373. Alternatively, the nucleic acid extraction unit 16 may extract the nucleic acid by at least one of filtration, centrifugation, and electrophoresis. When extracting the nucleic acid by electrophoresis, the nucleic acid may be charged in a known manner in advance.

[0034] The nucleic acid extraction unit 16 may supply the fluid sample containing the extracted nucleic acid to the separation unit 17. The nucleic acid extraction unit 16 may supply the fluid sample in which the nucleic acid is amplified to the separation unit 17. The nucleic acid extraction unit 16 may amplify the nucleic acid in the liquid in a state where the membrane structure of the microorganism is destroyed and then extract the nucleic acid, or may extract the nucleic acid and then amplify it. The nucleic acid extraction unit 16 may amplify the nucleic acid by the method of polymerase chain reaction (PCR).

[0035] <1.8. Separation Unit 17> The separation unit 17 separates the nucleic acid of the microorganism to be detected from among the nucleic acids in the fluid sample. The separation unit 17 may separate the nucleic acid specific to the microorganism to be detected (also referred to as the target nucleic acid) from among the nucleic acids of the microorganism to be detected. The separation unit 17 may separate the target nucleic acid using the biochip 105.

[0036] The biochip 105 may have a plurality of probes that hybridize with corresponding base sequences. At least some of the plurality of probes may have a base sequence complementary to at least a part of the base sequence of the target nucleic acid and may specifically hybridize with the nucleic acid of the corresponding base sequence. At least some of the plurality of probes may have the same base sequence.

[0037] Each probe may be pre-fixed at a unique position within the biochip 105. In this embodiment, as an example, the biochip 105 may have a plurality of probes on the inner surfaces of two opposed transparent substrates. The biochip 105 may have an injection port for injecting the fluid sample and a discharge port for discharging the internal fluid sample.

[0038] At least one of the probe of the biochip 105 having a complementary base sequence and the nucleic acid in the fluid sample may be pre-attached with a label that emits a signal detectable by the detection unit 18 described later in a hybridized state. For example, the label may be attached to the probe and may emit a signal in response to the probe and the nucleic acid hybridizing. Alternatively, the label may be attached to the nucleic acid in the fluid sample and may emit a signal regardless of whether the nucleic acid hybridizes with the probe. In this case, after injecting the fluid sample into the biochip 105, the nucleic acid that did not hybridize with the probe may be removed from the biochip 105, and then the signal may be detected. As the label, a fluorescent dye, a radioisotope, a paramagnetic isotope, an enzyme, etc. can be used. In this embodiment, as an example, the label may be a fluorescent dye and may be attached to the probe.

[0039] <1.9. Detection unit 18> The detection unit 18 detects the labels within the biochip 105. The detection unit 18 may be disposed opposite to the biochip 105 and may detect the labels within the biochip 105 through the transparent substrate of the biochip 105. The detection unit 18 may detect a signal emitted from a label attached to either the hybridized probe or the nucleic acid. The detection unit 18 may supply information indicating the position of the detected label and the intensity of the signal emitted from the label (fluorescence intensity in this embodiment as an example) to the determination unit 19.

[0040] <1.10. Determination unit 19> The determination unit 19 determines whether the nucleic acid of the microorganism to be detected is contained in the fluid sample injected into the biochip 105 more than the reference value, in other words, whether the fluid sample is positive or negative. The determination unit 19 may make a determination based on the information supplied from the detection unit 18. For example, the determination unit 19 may make a determination according to the detection position of the label by the detection unit 18. The determination unit 19 may externally output a determination result indicating whether the fluid sample is positive or negative.

[0041] The reference value may indicate the number of nucleic acids and may be the number of labels detected by the detection unit 18 as the target nucleic acid. In this embodiment, as an example, the reference value may be 0. However, the reference value may also indicate the ratio of nucleic acids and may be the ratio 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.

[0042] According to the above microorganism detection device 1, since organisms can be extracted from the liquid medium after culturing using a partial region of the first filter 101 for filtering organisms contained in the fluid sample, the amount of filter used can be reduced as compared with the case of using separate filters before and after culturing.

[0043] In addition, since a part of the region of the first filter 101 is cut out to generate the second filter 102 of the second filtration unit 15 that filters the liquid medium, the second filter 102 that is compatible with the second filtration unit 15 can be generated from the first filter 101 used in the first filtration unit 12. Therefore, filtration can be surely performed in the second filtration unit 15.

[0044] In addition, since the cross-sectional shape of the flow path in the second filtration unit 15 is smaller than the cross-sectional shape of the flow path in the first filtration unit 12, filtration can be surely performed in the second filtration unit 15 using a part of the region of the first filter 101.

[0045] In addition, since the second filter 102 that has filtered the liquid medium after culturing is housed in a sealed container and heated to extract the nucleic acid of the microorganism, the labor of separating the microorganism from the second filter 102 is saved, and the nucleic acid of the microorganism in the liquid sample can be easily obtained.

[0046] In addition, before filtration by the first filtration unit 12, since the fluid sample is filtered by the pre-filter 100 having a larger mesh size than the first filter 101, contaminants in the fluid sample can be removed and culturing can be performed.

[0047] <2. The First Filter 101 and the Second Filter 102> FIG. 2 shows the states of the first filter 101 and the second filter 102 in the first filtration unit 12, the culturing unit 13, the generating unit 14, the second filtration unit 15, and the nucleic acid extraction unit 16. Note that the white circular symbols in the figure indicate the microorganisms to be detected. Also, the white arrow symbols in the figure indicate the flow of the process in the microorganism detection device 1.

[0048] The first filter 101 is used for filtering a fluid sample in the first filtration unit 12 to capture microorganisms (see the upper left part in the figure), and then is immersed in a liquid medium in the culturing unit 13 (see the upper middle part in the figure). Next, after the first filter 101 is processed by the generating unit 14 to become the second filter 102 (see the upper right part in the figure), it is used for filtering the liquid medium in the second filtration unit 15 to capture microorganisms (see the lower middle part in the figure). Then, when the second filter 102 is housed in a sealed container and heated in the nucleic acid extraction unit 16 (see the lower right part in the figure), nucleic acids of the microorganisms contained in the sample are extracted.

[0049] <3. Operation of the microorganism detection device 1> FIG. 3 shows the operation of the microorganism detection device 1. The microorganism detection device 1 determines whether or not each sample contains a microorganism to be detected more than a reference value by performing the processes of steps S11 to S29.

[0050] In step S11, the sampling unit 10 extracts a sample from food or beverage. The sampling unit 10 may generate a fluid sample from the sample.

[0051] In step S13, the pre-filtration unit 11 filters the fluid sample with the pre-filter 100. Thereby, contaminants in the fluid sample may be removed.

[0052] In step S15, the first filtration unit 12 filters the fluid sample with the first filter 101. Thereby, the microorganism to be detected may be captured as a residue.

[0053] In step S17, the culturing unit 13 immerses the filtered first filter 101 in a liquid medium for culturing. Microorganisms may be captured on the first filter 101. Thereby, the microorganisms captured on the first filter 101 are cultured.

[0054] In step S19, the generation unit 14 cuts out a partial region of the first filter 101 immersed in the liquid medium to generate the second filter 102. Microorganisms captured by the first filter 101 in step S15 and / or microorganisms that have grown in step S17 may be attached to the second filter 102. Note that the generation unit 14 may be washed after the generation of the second filter 102. This prevents microorganisms attached to the first filter 101 from remaining in the generation unit 14.

[0055] In step S21, the second filtration unit 15 filters the liquid medium after cultivation with a partial region of the first filter 101 (the second filter 102 as an example in this embodiment). Thereby, the microorganisms cultured in step S17 may be captured. The liquid medium filtered by the second filter 102 in step S21 may be the liquid medium in which the first filter 101 serving as the source of the second filter 102 was immersed in step S17.

[0056] In step S23, the nucleic acid extraction unit 16 extracts the nucleic acid of the microorganisms contained in the liquid medium from the residue after filtration by the second filtration unit 15. As an example, the nucleic acid extraction unit 16 may store the microorganisms as the residue together with the second filter 102 in a container, break the membrane structure of the microorganisms using the so-called dHTP method, and then extract the nucleic acid by filtration, centrifugation, or the like.

[0057] In step S25, the separation unit 17 separates the nucleic acid of the microorganisms to be detected from the nucleic acids in the fluid sample. As an example, the separation unit 17 may separate the nucleic acid of the microorganisms to be detected contained in the fluid sample by hybridizing it with the probe of the biochip 105.

[0058] In step S27, the detection unit 18 detects the label in the biochip 105. As an example, the detection unit 18 may detect a signal emitted from a label attached to at least one of the probe of the biochip 105 and the nucleic acid in the fluid sample.

[0059] In step S29, the determination unit 19 determines, based on the information supplied from the detection unit 18, whether or not the nucleic acid of the microorganism to be detected is contained in the fluid sample injected into the biochip 105 in an amount greater than the reference value, in other words, whether the fluid sample is positive or negative.

[0060] <4. Modification Example> In the above embodiment, the microorganism detection device 1 has been described as including the sampling unit 10, the pre-filtering unit 11, the generation unit 14, the separation unit 17, the detection unit 18, and the determination unit 19. However, it may be configured not to include any of these components. For example, when the microorganism detection device 1 does not include the generation unit 14, the second filtering unit 15 may filter the liquid medium in a partial region by covering the entire cross-section of the flow path of the second filtering unit 15 with a partial region of the first filter 101. As an example, the second filtering unit 15 may be provided with tubular connection parts that are in contact with each other along the flow direction of the liquid medium, and by sandwiching the first filter 101 between these connection parts, the entire cross-section of the flow path of the second filtering unit 15 may be covered with a partial region of the first filter 101.

[0061] Also, the generation unit 14 has been described as generating the second filter 102 from the first filter 101 that is used for filtering the liquid sample and immersed in the liquid medium. However, the second filter 102 may be generated from the first filter 101 that has not been immersed in the liquid medium after being used for filtering the liquid sample. In this case, the culturing unit 13 may immerse the remaining portion of the first filter 101 that has not been used as the second filter 102 in the liquid medium for culturing.

[0062] Also, the determination unit 19 has been described as determining whether or not the nucleic acid of the microorganism to be detected is contained in the sample. However, it may also determine whether or not the nucleic acid of an organism other than a microorganism is contained in the sample. In this case, the culturing unit 13 may culture the cells of the organism to be detected, and the second filtering unit 15 may collect the cultured cells. The organism to be detected may be an animal, an insect, a plant, mycoplasma, a virus, or the like. The organism to be detected may be of one type or multiple types.

[0063] As described above, the present invention has been described using embodiments. However, the technical scope of the present invention is not limited to the scope described in the above embodiments. It is obvious to those skilled in the art that various changes or improvements can be made to the above embodiments. It is clear from the description of the claims that forms with such changes or improvements can also be included in the technical scope of the present invention.

[0064] It should be noted that the execution order of each process such as operations, procedures, steps, and stages in the apparatus, system, program, and method shown in the claims, the specification, and the drawings is not explicitly stated as "before" or "preceding" etc., and can be realized in any order unless the output of the previous process is used in the subsequent process. Regarding the operation flow in the claims, the specification, and the drawings, even if it is described using "first," "next," etc. for convenience, it does not mean that it is essential to implement in this order.

Explanation of Reference Numerals

[0065] 1 Microbial detection device 10 Sampling unit 11 Prefiltration unit 12 First filtration unit 13 Cultivation unit 14 Generation unit 15 Second filtration unit 16 Nucleic acid extraction unit 17 Separation unit 18 Detection unit 19 Determination unit 100 Prefilter 101 First filter 102 Second filter 105 Biochip

Claims

1. A first filtration unit that filters a fluid sample with a first filter; A culture unit that immerses the first filter after filtration by the first filtration unit in a liquid medium and performs culturing; A second filtration unit that filters the liquid medium after culturing in a partial region of the first filter; An extraction unit that extracts nucleic acids of organisms contained in the liquid medium from the residue after filtration by the second filtration unit; An apparatus comprising the above.

2. The apparatus further comprises a generation unit that cuts out the partial region of the first filter immersed in the liquid medium to generate a second filter, wherein the second filtration unit filters the liquid medium after culturing with the second filter, according to Claim 1.

3. The apparatus according to Claim 1, wherein a cross-sectional shape of a flow path in the second filtration unit is smaller than a cross-sectional shape of a flow path in the first filtration unit.

4. The apparatus according to Claim 2, wherein the extraction unit extracts nucleic acids of organisms by accommodating the second filter that has filtered the liquid medium after culturing in a sealed container and heating it.

5. The apparatus according to Claim 1, further comprising a third filtration unit that filters the fluid sample with a pre-filter having a larger mesh size than the first filter before filtration by the first filtration unit.

6. A first filtration step of filtering a fluid sample with a first filter; A culture step of immersing the first filter after filtration by the first filtration step in a liquid medium and performing culturing; A second filtration step of filtering the liquid medium after culturing in a partial region of the first filter; An extraction step of extracting nucleic acids of organisms contained in the liquid medium from the residue after filtration by the second filtration step; A method comprising the above.

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