Apparatus and method

Abstract

To provide a microorganism detection apparatus and a detection method.SOLUTION: An apparatus comprises: a first filtration unit that filters a fluid sample through a filter; a culture unit that performs culture using a resultant product obtained by processing the filter after filtration by the first filtration unit to reduce its planar size; a detection unit that detects nucleic acids of an organism cultured by the culture unit; a processing unit that performs the processing on the filter after filtration by the first filtration unit to generate a filter-processed product as the resultant product, the processing unit cutting the filter after filtration by the first filtration unit to generate the filter-processed product.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 retaining the microorganisms on the membrane filter with the outer frame", etc. [Prior Art Documents] [Patent 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] Patent No. 5624487 [Patent Document 7] 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 performs culturing using a resultant obtained by performing a processing for reducing a planar size on the filter after filtration by the first filtration unit; and a detection unit that detects nucleic acid of an organism cultured by the culturing unit.

[0004] In the above apparatus, a processing unit that performs the processing on the filter after filtration by the first filtration unit to generate a processed filter as the resultant may be further provided.

[0005] In the above-described apparatus including the processing unit, the processing unit may cut the filter after filtration by the first filtration unit to generate a processed filter product.

[0006] In the above-described apparatus including the processing unit, the processing unit may bend the filter after filtration by the first filtration unit to generate a processed filter product.

[0007] In the above-described apparatus including the processing unit, the apparatus may further include a second filtration unit that filters a liquid culture medium after culturing using the processed filter product, and an extraction unit that extracts nucleic acid from the residue after filtration by the second filtration unit. The detection unit may detect the nucleic acid extracted by the extraction unit.

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

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

[0010] 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 filter; a culturing step of culturing using a resultant product obtained by performing a processing operation to reduce a planar size of the filter after filtration; and a detection step of detecting nucleic acid of an organism cultured in the culturing step.

[0011] 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

[0012]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0013] 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.

[0014] <1. Microorganism Detection Device> FIG. 1 shows a microorganism detection device 1 according to the present embodiment. The microorganism detection device 1 may be an example of a device and detects microorganisms mixed in foods and beverages. The microorganism detection device 1 includes a sampling unit 10, a pre-filtration unit 11, a first filtration unit 12, a processing unit 13, a culture unit 14, a second filtration unit 15, a nucleic acid extraction unit 16, a separation unit 17, a detection unit 18, and a determination unit 19.

[0015] The microorganisms to be detected include, as an example, 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,It can be selected from the group consisting of the genus Rhodospirillium, the genus Staphylococcus, the genus Streptomyces, the genus Streptococcus, the genus Vibrio, and the genus Yersinia. There are microorganisms that take forms such as spores and cysts in an oligotrophic state, regardless of the state of the cells due to such a growth state.

[0016] <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.

[0017] <1.2. Pre-filtering unit 11> The pre-filtering unit 11 is an example of the third filtering unit, and filters a 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 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 5 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 and the culturing in the culturing unit 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 (ordinary filter paper as an example), polyester, or PET (mesh made of polyester or mesh made of PET as an example) may be used. The pre-filtering unit 11 may supply the filtered fluid sample to the first filtering unit 12.

[0018] <1.3. First Filtration Unit 12> The first filtration unit 12 filters the fluid sample with the filter 101.

[0019] The 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 filter 101 is preferably 0.45 μm or less. The 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 filter 101 is not washed by the fluid sample in the first filtration unit 12, the planar shape of the 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.

[0020] The material of the 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 hardly adsorbs to it. For example, the material of the 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 filter 101 is preferably hydrophilic. For example, hydrophilic PTFE, hydrophilic PVDF, hydrophilic PES, and hydrophilic PC are preferred. When the filter 101 is hydrophilic, the nucleic acid extracted in the nucleic acid extraction unit 16 described later hardly adsorbs to the filter 101. Among these, hydrophilic PC is more preferred as the material of the filter 101. When 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 collected. One index indicating that the material of the filter 101 is hydrophilic is the contact angle. 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 may supply the filter 101 that has captured microorganisms as residues to the processing unit 13.

[0021] <1.4. Processing Unit 13> The processing unit 13 performs a processing operation to reduce the planar size of the filter 101 after filtration by the first filtration unit 12 to generate a processed filter 102. The processing unit 13 may process the filter 101 supplied from the first filtration unit 12 to generate a processed filter 102. The processing unit 13 may cut the filter 101 after filtration by the first filtration unit 12 to generate a processed filter 102. In addition to, or instead of, this, the processing unit 13 may bend the filter 101 after filtration by the first filtration unit 12 to generate a processed filter 102.

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

[0023] In addition to this, the processing unit 13 may generate the filter processed product 102 so that it can be used as a filter in the second filtration unit 15, and may generate the filter processed product 102 according to the cross-sectional shape of the flow path in the second filtration unit 15. In this case, the processing unit 13 may cut (i.e., trim) the filter 101 using a mold pre-formed according to the cross-sectional shape of the flow path in the second filtration unit 15, or may bend it. 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 the planar shape of the culture tank. As long as the filter processed product 102 is not flowed by the fluid sample in the second filtration unit 15, the planar shape of the filter processed product 102 may be the same as the cross-sectional shape of the flow path in the second filtration unit 15 and may be circular, for example.

[0024] The processing unit 13 may process the filter 101 in a state wet with the liquid medium to generate a processed filter 102, or may dry the filter 101 and then cut it to generate a processed filter 102. The processing unit 13 may discard the remaining portion of the filter 101 that was not used as the processed filter 102. The processing unit 13 may supply the generated processed filter 102 to the culturing unit 14.

[0025] <1.5. Culturing Unit 14> The culturing unit 14 performs culturing using the resultant product obtained by performing a processing operation that reduces the planar size of the filter 101, which is generated from the filtered filter 101. The culturing unit 14 may perform culturing using the processed filter 102 generated by the processing unit 13.

[0026] The culturing unit 14 may culture the microorganisms captured by the filter 101. The culturing unit 14 may perform culturing using any conventionally known method and conditions according to the microorganism to be detected. For example, the culturing unit 14 may culture the sample on a solid medium (i.e., solid-phase culturing), and as an example, may place the processed filter 102 on the solid medium and perform culturing. Alternatively, the culturing unit 14 may culture the sample in a liquid medium (i.e., liquid-phase culturing), and as an example, may immerse the processed filter 102 in the liquid medium and perform culturing. The liquid medium may be a solution in which the solid medium is dissolved.

[0027] Here, since the planar size of the processed filter 102 is smaller than that of the filter 101, the culture tank of the present embodiment that houses the processed filter 102 may have a smaller planar shape compared to the culture tank that houses the filter 101 (also referred to as a conventional culture tank). As a result, the culture medium in the culture tank according to the present embodiment may have a smaller planar dimension 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 according to the present embodiment may 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, in the culture tank according to the present embodiment, the volume of the culture medium may be smaller than that of the conventional culture tank. On the other hand, in the culture tank according to the present embodiment, it may be possible to culture the same amount of microorganisms as in the conventional culture tank. Thereby, in the culture tank according to the present embodiment, microorganisms may be cultured at a higher concentration than in the conventional culture tank.

[0029] The culturing unit 14 may supply the liquid culture medium after culturing to the second filtering unit 15. When solid-phase culturing is performed, the culturing unit 14 may 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 liquid culture medium after culturing using the filter-processed product 102. Thereby, the cultured microorganisms are recovered as residues.

[0031] The second filtering unit 15 may supply the recovered microorganisms to the nucleic acid extraction unit 16. In the present embodiment, as an example, the second filtering unit 15 may supply the microorganisms recovered as residues on the filter-processed product 102 to the nucleic acid extraction unit 16 together with the 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 (such as genomic DNA, ribosomal RNA, plasmid DNA, etc. as an example) from the residues after filtration by the second filtering unit 15. The nucleic acid extraction unit 16 may extract the nucleic acids of microorganisms by accommodating the filter (in this embodiment, the filter-processed product 102 as an example) used for filtration by the second filtering unit in a sealed container (also referred to as a sealed container) and heating it, and may extract nucleic acids using, for example, the methods described in International Publication No. 2019 / 43779 or Japanese Patent No. 5624487, so-called dHTP method.

[0033] The nucleic acid extraction unit 16 may break the membrane structure of microorganisms by exposing the microorganisms as residues together with the filter product 102 to high temperature conditions in a sealed container, and make the nucleic acids in an extractable state. 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.

[0034] The container may be heated by heating means (not shown). The heating means may be an oil bath or a heat block, and the interior 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 the atmospheric pressure due to heating.

[0035] 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 into the container.

[0036] Examples of the above-mentioned alkali include sodium hydroxide (NaOH), potassium hydroxide (KOH), etc. Examples of the above-mentioned acid include hydrochloric acid (HCl), sulfuric acid (H2SO4), etc. Examples of the above-mentioned enzyme include proteolytic enzymes such as Proteinase K, or polysaccharide-degrading enzymes such as chitinase, lysozyme, zymolyase, etc. The above-mentioned 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 TritonX-100 (C14H22O(C2H4O)n, n = 100) manufactured by SIGMA can be used.

[0037] In addition, the ionic surfactant may be anionic, cationic, or zwitterionic. Examples of anionic surfactants include sodium dodecyl sulfate (SDS). Examples of cationic surfactants include cetyltrimethylammonium bromide (CTAB). Examples of zwitterionic surfactants 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 the hydrogen atom dissociable from the atom with the positive charge is not bonded, and the molecule as a whole has no charge. A representative example of betaine is trimethylglycine. Examples of the above reducing agent include hydrogen peroxide solution, β-mercaptoethanol, dithiothreitol, etc.

[0038] Examples of the above protein denaturant include guanidine hydrochloride, urea, etc. Examples of the above chelating agent include ethylenediaminetetraacetic acid (EDTA), etc. Among the above cell lysis accelerators described above, it is preferable to add the above 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 in an enzyme reaction inhibited by SDS, octylphenol ethoxylate, which acts more gently on the membrane structure of microorganisms than SDS, may be used.

[0039] The container may further contain a buffer solution. Examples of the buffer solution include tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), etc.

[0040] After the membrane structure of the microorganism is disrupted, the nucleic acid extraction unit 16 may extract nucleic acid from the liquid in the container. The nucleic acid extraction unit 16 may bind the nucleic acid to magnetic microparticles and extract it magnetically, for example, by the method described in Japanese Patent No. 4857373. Alternatively, the nucleic acid extraction unit 16 may extract nucleic acid by at least one of filtration, centrifugation, and electrophoresis. When extracting nucleic acid by electrophoresis, the nucleic acid may be charged in advance by a known method.

[0041] The nucleic acid extraction unit 16 may supply a fluid sample containing the extracted nucleic acid to the separation unit 17. The nucleic acid extraction unit 16 may supply a fluid sample in which the nucleic acid has been 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 disrupted 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 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 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 the nucleic acid of the microorganism to be detected. The separation unit 17 may use the biochip 105 to separate the target nucleic acid.

[0043] 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.

[0044] 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 a fluid sample and a discharge port for discharging the internal fluid sample.

[0045] 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-labeled with a label that emits a signal detectable by the detection unit 18 described later in the hybridized state. For example, the label may be attached to the probe and may emit a signal in response to the hybridization of the probe and the nucleic acid. 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.

[0046] <1.9. Detection Unit 18> The detection unit 18 detects the nucleic acid of the microorganism cultured by the culture unit 14. The detection unit 18 may detect the nucleic acid extracted by the nucleic acid extraction unit 16. The detection unit 18 may detect the nucleic acid by detecting the label in the biochip 105. The detection unit 18 may be disposed opposite to the biochip 105 and may detect the label in the biochip 105 through the transparent substrate of the biochip 105. The detection unit 18 may detect the signal emitted from the 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 (fluorescent intensity in this embodiment as an example) 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 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. 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 output an external determination result indicating whether the fluid sample is positive or negative.

[0048] 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 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.

[0049] According to the above microorganism detection device 1, by using, for culturing, the resultant of the processed product with a reduced planar size with respect to the filter 101 after filtration by the first filtration unit 12, the area of the culture medium can be reduced. Thereby, since the microorganism can be cultured at a high concentration in a narrow culture medium, the detection accuracy of the nucleic acid can be improved.

[0050] Further, the filter 101 after filtration is processed by the processing unit 13 to generate a filter processed product 102. Therefore, compared with the case where the filter processed product 102 is generated from the filter 101 outside the microorganism detection device 1, the efficiency from the filtration by the first filtration unit 12 to the culture by the culture unit 14 can be improved.

[0051] Further, since the filter 101 after filtration by the first filtration unit 12 is cut to generate the filter processed product 102, the filter processed product 102 having a planar size smaller than that of the filter 101 can be easily generated. In addition to this, or instead of this, since the filter 101 after filtration is bent to generate the filter processed product 102, the filter processed product 102 having a planar size smaller than that of the filter 101 can be easily generated.

[0052] In addition, since the liquid medium after culturing is filtered using the filter-processed product 102 generated from the filter 101 that has filtered the liquid sample, the amount of filters used can be reduced as compared with the case of using separate filters for the filtration of the liquid sample and the filtration of the liquid medium.

[0053] In addition, since the filter-processed product 102 obtained by filtering the liquid medium after culturing is housed in a sealed container and heated to extract the nucleic acid of the microorganism, the trouble of separating the microorganism from the filter-processed product 102 is saved, and the nucleic acid of the microorganism in the liquid sample can be easily obtained.

[0054] In addition, before filtration by the first filtration unit 12, since the fluid sample is filtered with the pre-filter 100 having a larger mesh size than the filter 101, contaminants in the fluid sample can be removed and culturing can be performed.

[0055] <2. Filter 101 and Filter-Processed Product 102> FIG. 2 shows the states of the filter 101 and the filter-processed product 102 in the first filtration unit 12, the processing unit 13, the culturing 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 processing in the microorganism detection device 1.

[0056] The filter 101 is used for filtering the fluid sample in the first filtration unit 12 to capture microorganisms (see the upper left part in the figure), and then is subjected to a processing treatment by the processing unit 13 to become the filter-processed product 102 (see the upper right part in the figure). Next, the filter-processed product 102 is used for culturing in the culturing unit 14 (see the upper center part in the figure), and is used for filtering the liquid medium in the second filtration unit 15 to capture microorganisms (see the lower center part in the figure). Then, when the filter-processed product 102 is housed in a sealed container and heated in the nucleic acid extraction unit 16 (see the lower right part in the figure), the nucleic acid of the microorganisms contained in the sample is extracted.

[0057] <3. Operation of Microorganism Detection Device 1> FIG. 3 shows the operation of the microorganism detection device 1. The microorganism detection device 1 determines whether or not the microorganism to be detected is contained in each sample at a reference value or more 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 may 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, contaminants in the fluid sample may be removed.

[0060] In step S15, the first filtering unit 12 filters the fluid sample with the filter 101. Thereby, the microorganism to be detected may be captured as a residue.

[0061] In step S17, the processing unit 13 performs a processing to reduce the planar size of the filtered filter 101 to generate a filter processed product 102. The microorganism captured by the filter 101 in step S15 may adhere to the filter processed product 102. Note that the processing unit 13 may be washed after the generation of the filter processed product 102. Thereby, it is prevented that the microorganism adhering to the filter 101 remains in the processing unit 13.

[0062] In step S19, the culturing unit 14 performs culturing using the resultant product obtained by performing a processing to reduce the planar size of the filter 101 generated from the filtered filter 101. In the present embodiment, as an example, the culturing unit 14 may perform culturing using the filter processed product 102. The filter processed product 102 may capture microorganisms. Thereby, the microorganisms captured by the filter processed product 102 are cultured.

[0063] In step S21, the second filtration unit 15 filters the liquid medium after culturing using the processed filter 102. Thereby, the microorganisms cultured in step S19 may be captured. The liquid medium filtered by the processed filter 102 in step S21 may be the liquid medium in which the processed filter 102 was immersed 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 after filtration by the second filtration unit 15. As an example, the nucleic acid extraction unit 16 may store the microorganisms as residues together with the processed 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.

[0065] In step S25, the separation unit 17 separates the nucleic acid of the microorganisms to be detected among 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 with the probes of the biochip 105.

[0066] In step S27, the detection unit 18 detects the nucleic acid of the cultured microorganisms. The detection unit 18 may detect the nucleic acid by detecting 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 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 microorganisms 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.

[0068] <4. Modification Example> In the above-described embodiment, the microorganism detection device 1 has been described as including the sampling unit 10, the pre-filtering unit 11, the processing unit 13, the second filtering unit 15, the nucleic acid extraction unit 16, the separation unit 17, and the determination unit 19. However, it may not include any of these. For example, when the microorganism detection device 1 does not include the processing unit 13, the filter-processed product 102 may be generated from the post-filtering filter 101 by an external device of the microorganism detection device 1 or an operator. Further, when the microorganism detection device 1 does not include the second filtering unit 15 and the nucleic acid extraction unit 16, the detection unit 18 may 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 contacts the filter-processed product 102 and the microorganism on the solid medium with a permeability-imparting composition containing polyethyleneimine and at least one alcohol, fixes the microorganism to the filter-processed product 102 with a cross-linking agent, and then allows a molecule containing a label and hybridizing with the target nucleic acid to permeate 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 nucleic acids in this way, for example, the methods described in Patent Documents 2 to 4 above can be used.

[0069] Also, although the second filtering unit 15 has been described as filtering the liquid medium using the filter-processed product 102 generated from the filter 101, the liquid medium may be filtered using another filter different from the filter-processed product 102. The other filter may be the same filter as the filter 101.

[0070] Also, although the processing unit 13 has been described as performing at least one of cutting or bending on the filter 101 to reduce the planar size, the planar size may be reduced by other processes. As an example, the processing unit 13 may reduce the planar size by dissolving at least a part of the filter 101 while keeping the microorganism captured by the filter 101 in a culturable state. In this case, the culturing unit 14 may perform culturing using the lysate in which the filter 101 has been dissolved.

[0071] Also, although 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, it may determine whether or not the nucleic acid of an organism other than the microorganism is contained in the sample. In this case, the culturing unit 14 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 one type or a plurality of types.

[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. 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 the forms with such changes or improvements 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 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 "earlier" or "preceding" etc., and unless the output of the previous process is used in the subsequent process, it can be realized in any order. 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

[0074] 1 Microorganism detection device 10 Sampling unit 11 Prefiltering unit 12 First filtering unit 13 Processing unit 14 Culturing unit 15 Second filtering unit 16 Nucleic acid extraction unit 17 Separation unit 18 Detection unit 19 Determination unit 100 Prefilter 101 Filter 102 Filter processed product 105 Biochip

Claims

1. a first filtration unit that filters a fluid sample with a filter; a culture unit that performs culturing using a resultant product obtained by performing a processing for reducing a planar size on the filter after filtration by the first filtration unit; a detection unit that detects nucleic acid of an organism cultured by the culture unit; An apparatus comprising:

2. The apparatus according to claim 1, further comprising a processing unit that performs the processing on the filter after filtration by the first filtration unit to generate a filter processed product as the resultant product.

3. The apparatus according to claim 2, wherein the processing unit cuts the filter after filtration by the first filtration unit to generate the filter processed product.

4. The apparatus according to claim 2, wherein the processing unit bends the filter after filtration by the first filtration unit to generate the filter processed product.

5. a second filtration unit that filters a liquid medium after culturing using the filter processed product; an extraction unit that extracts nucleic acid from a residue after filtration by the second filtration unit; further comprising: The apparatus according to claim 2, wherein the detection unit detects the nucleic acid extracted by the extraction unit.

6. The apparatus according to claim 5, wherein the extraction unit accommodates the filter processed product used for filtration by the second filtration unit in a sealed container and heats it to extract nucleic acid of an organism.

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

8. a first filtration step of filtering a fluid sample with a filter; a culture step of performing culturing using a resultant product obtained by performing a processing for reducing a planar size on the filter after filtration; a detection step of detecting nucleic acid of an organism cultured by the culture step; A method comprising:

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