Filter suction holder, nucleic acid extraction system, and nucleic acid extraction method

Abstract

The filter suction holder (310) can be inserted into a reaction tube for extracting nucleic acid from a sample, together with a filter for collecting the sample by filtering the sample including the sample having nucleic acid, the filter suction holder (310) having a housing part (311). The accommodating part (311) is provided with an inner wall surface (312) which is in close contact with the surface of the filter and adsorbs the filter.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of Japanese Patent Application No. 2023 - 217112 filed on December 22, 2023, and the entire contents of the prior application disclosed therein are incorporated herein by reference for reference purposes. Technical field

[0003] The present invention relates to a filter suction holder, a nucleic acid extraction system, and a nucleic acid extraction method. Background art

[0004] Nucleic acid extraction methods are disclosed in Patent Document 1 and Patent Document 2.

[0005] In current nucleic acid extraction methods, a sample is filtered using a membrane filter to recover microorganisms in the sample onto the surface of the membrane filter. Moreover, the membrane filter is blocked using sterilized forceps and inserted into a reaction tube to accommodate the membrane filter in the reaction tube. Then, a surfactant is added to the reaction tube and the reaction tube is heated to a temperature greater than or equal to the boiling point, thereby forming a high - temperature and high - pressure state to extract nucleic acid from the microorganisms.

[0006] Patent Document 1: WO 2019 / 043779

[0007] Patent Document 2: Japanese Unexamined Patent Application Publication No. 2012 - 157265 Summary of the invention

[0008] Regarding the operation of recovering the filtered filter and inserting it into the reaction tube, it is necessary to handle a very thin filter using forceps, so it is very cumbersome. Moreover, in order to extract nucleic acid, it is necessary to immerse the filter in a reaction solution containing a surfactant, but the wet filter easily adheres to the inner wall of the reaction tube, so it is difficult to insert the filter to the bottom of the reaction tube.

[0009] An object of the present invention is to easily insert a filter into a reaction tube.

[0010] Hereinafter, a filter suction holder, a nucleic acid extraction system, and a nucleic acid extraction method according to several embodiments will be described.

[0011] [1] A filter suction holder that can be inserted into a reaction tube for extracting nucleic acid from a specimen together with a filter that captures the specimen by filtering a sample containing the specimen having nucleic acid, wherein,

[0012] The filter suction holder has a housing portion for housing the filter, and the housing portion has an inner wall surface that is in close contact with the surface of the filter and adsorbs the filter.

[0013] By using such a filter attracting and holding member, it is easy to insert the filter into the reaction tube.

[0014] [2] The filter attracting and holding member according to [1], wherein

[0015] The housing portion has a hollow conical or frustoconical shape, and the bottom surface of the cone or the frustum is open so that the inner wall surface is exposed, and it is inserted into the reaction tube from the end of the smaller diameter of the cone or the frustum.

[0016] By using such a filter attracting and holding member, it is easy to insert the filter to the bottom of the reaction tube.

[0017] [3] The filter attracting and holding member according to [1] or [2], wherein

[0018] The housing portion has a heat resistance of at least 180 °C.

[0019] Regarding such a filter attracting and holding member, accidental deterioration during heating can be prevented.

[0020] [4] The filter attracting and holding member according to any one of [1] to [3], wherein

[0021] The housing portion is formed of a material that melts when a constant heat is applied.

[0022] By using such a filter attracting and holding member, the filter attracting and holding member melts during nucleic acid extraction, and the contact area between the filter and the reaction solution containing a surfactant increases, improving the reaction efficiency.

[0023] [5] The filter attracting and holding member according to any one of [1] to [4], wherein

[0024] The filter attracting and holding member further has a surfactant fixedly attached to the surface of the housing portion.

[0025] By using such a filter attracting and holding member, the operation of adding the surfactant required for nucleic acid extraction to the reaction tube can be simplified.

[0026] [6] A nucleic acid extraction system, wherein

[0027] The nucleic acid extraction system has:

[0028] [1] to [5] The filter attracting and holding member according to any one of them; and

[0029] A reaction tube that can accommodate the filter attracting and holding member for each filter.

[0030] Regarding such a nucleic acid extraction system, it is easy to insert a filter into a reaction tube.

[0031] [7] A nucleic acid extraction method, wherein,

[0032] The nucleic acid extraction method includes the following steps:

[0033] Filtering a sample including a specimen having nucleic acid by using a filter to capture the specimen;

[0034] Accommodating the filter in a housing portion of a filter suction holder having an inner wall surface that is in close contact with the surface of the filter and adsorbs the filter;

[0035] Inserting the filter suction holder into a reaction tube for each said filter; and

[0036] Extracting the nucleic acid from the specimen by using the reaction tube.

[0037] Regarding such a nucleic acid extraction method, it is easy to insert a filter into a reaction tube.

[0038] Effects of the Invention

[0039] According to the present invention, it is easy to insert a filter into a reaction tube. Description of the Drawings

[0040] Figure 1 is a diagram showing the structure of a nucleic acid analysis system according to an embodiment of the present invention.

[0041] Figure 2 is a diagram showing an example of a filter and forceps according to an embodiment of the present invention.

[0042] Figure 3 is a diagram showing an example of a filter suction holder according to an embodiment of the present invention.

[0043] Figure 4 is a diagram showing an example of a reaction tube according to an embodiment of the present invention.

[0044] Figure 5 is a flowchart showing a nucleic acid extraction method according to an embodiment of the present invention. Detailed Embodiments

[0045] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0046] In each figure, the same or corresponding parts are denoted by the same reference numerals. In the description of each embodiment, the same or corresponding parts are appropriately omitted or simplified.

[0047] Refer to Figure 1The structure of the nucleic acid analysis system 100 according to this embodiment will be described.

[0048] The nucleic acid analysis system 100 includes a specimen recovery system 200, a nucleic acid extraction system 300, a mixing reaction system 400, and a detection system 500.

[0049] The specimen recovery system 200 is a system for recovering the specimen contained in the sample 110 from the sample 110.

[0050] The specimen is, for example, bacteria, fungi, viruses, or other microorganisms. In the case of performing an inspection on a beverage as an object, the sample 110 is the manufactured beverage. Alternatively, the sample 110 can be the water used for manufacturing the beverage. Alternatively, the sample 110 can be the liquid generated during the manufacturing process of the beverage. In order to check the presence or absence or degree of contamination of fungi in the manufacturing environment, when recovering fungi from a cotton swab by vibrating the cotton swab that has wiped the environment in a liquid or vibrating the liquid into which the cotton swab is inserted, the sample 110 can be this liquid.

[0051] The specimen recovery system 200 has Figure 2 the filter 210 shown.

[0052] For example, the sample 110 is pressurized or depressurized and filtered using the filter 210 for recovery. When recovering bacteria or fungi, the pore size of the filter 210 is, for example, 0.45 mm or 0.22 mm.

[0053] Specifically, the filter 210 is a membrane filter. The diameter of the filter 210 is, for example, 13 mm. The filter 210 only needs to be of a size that can be inserted into the reaction tube 320 described later. The shape of the filter 210 is, for example, circular. The material of the filter 210 is, for example, MCE, PVDF, PES, or PC. "MCE" is an abbreviation for mixed cellulose ester. "PVDF" is an abbreviation for polyvinylidene fluoride. "PES" is an abbreviation for polyethersulfone. "PC" is an abbreviation for polycarbonate.

[0054] The nucleic acid extraction system 300 is a system for extracting the nucleic acid possessed by the specimen from the specimen recovered by the specimen recovery system 200.

[0055] The nucleic acid extraction system 300 has Figure 3 the filter suction holder 310 shown, and Figure 4 the reaction tube 320 shown.

[0056] After recovering the specimen using the filter 210, usingFigure 2 The forceps 220 shown move the filter 210 toward the filter suction holder 310. The filter suction holder 310 sucks and holds the filter 210. The filter suction holder 310 is inserted into the reaction tube 320 for each filter 210. In the present embodiment, instead of directly inserting the filter 210 into the reaction tube 320 using the forceps 220, the filter 210 is inserted into the reaction tube 320 after being housed in the filter suction holder 310. Therefore, the filter suction holder 310 can prevent the filter 210 from falling. The filter suction holder 310 can fix the filter 210, so it is easy to place the filter 210 into the reaction tube 320. Since the filter suction holder 310 does not cause the filter 210 to closely adhere to the inner wall of the reaction tube 320, it is easy to insert the filter 210 to the bottom of the reaction tube 320. The filter suction holder 310 is used once, so the risk of contamination can also be reduced.

[0057] Vacuum forceps can be used instead of the forceps 220. By using the vacuum forceps, without the operation of clogging the filter 210 using the forceps 220, the filter 210 can be recovered by a simple operation. Thus, it is not affected by the skills of the operator, and the filter 210 can be reliably recovered.

[0058] After the filter 210 filters the sample 110 to capture the specimen, the filter suction holder 310 is inserted into the reaction tube 320 together with the filter 210. Therefore, the filter suction holder 310 is sized to be insertable into the reaction tube 320.

[0059] The filter suction holder 310 has a housing portion 311 for housing the filter 210. The housing portion 311 has an inner wall surface 312 that closely adheres to the surface of the filter 210 and sucks and holds the filter 210.

[0060] In the present embodiment, the housing portion 311 has a hollow conical or frustoconical shape. The bottom surface of the conical or frustoconical shape of the housing portion 311 is open to expose the inner wall surface 312. The housing portion 311 has: a first end 313 corresponding to the bottom surface of the cone or frustum; and a second end 314 corresponding to the vertex of the cone or the opposite bottom surface of the frustum. The housing portion 311 is inserted into the reaction tube 320 from the second end 314 where the diameter of the cone or frustum is smaller among the first end 313 and the second end 314.

[0061] In the present embodiment, regarding the housing portion 311, not only the bottom surface of the conical or frustoconical shape is open, but also the vertex of the cone or the opposite bottom surface of the frustum is open. That is, the housing portion 311 has openings formed at both the first end 313 and the second end 314.

[0062] Preferably, the housing portion 311 has a heat resistance of at least 180°C. For example, the housing portion 311 may be formed of PES.

[0063] The housing portion 311 may be formed of a material that melts when a constant heat is applied. For example, the housing portion 311 may be formed of a thermally decomposable polymer. When the housing portion 311 melts during nucleic acid extraction, the area of contact between the filter 210 and the reaction solution containing a surfactant is increased, and an improvement in reaction efficiency can be expected.

[0064] The filter attracting and holding member 310 may also have a surfactant fixedly attached to the surface of the housing portion 311. For example, by previously drying and fixedly attaching a reaction solution containing a surfactant used in nucleic acid extraction to the housing portion 311, the operation of adding the reaction solution can be simplified.

[0065] The reaction tube 320 has a structure capable of housing the filter attracting and holding member 310 for each filter 210. As the reaction tube 320, a usual PCR tube can be used. "PCR" is an abbreviation of polymerase chain reaction. The capacity of the reaction tube 320 is, for example, 0.2 mL. The method of extracting nucleic acid from a specimen using the reaction tube 320 is the same as known methods such as those disclosed in Patent Document 1 or Patent Document 2 except for the use of the filter attracting and holding member 310, and thus the description thereof is omitted.

[0066] A liquid containing another nucleic acid B that reacts with the extracted nucleic acid can be mixed in the sample 110 from which the nucleic acid has been extracted. Nucleic acid B may be a nucleic acid having a site that exhibits fluorescence, luminescence, or extinction under specific conditions for detection using the detection system 500.

[0067] The mixing reaction system 400 is a system for performing the mixing reaction of the sample 110.

[0068] The sample 110 is heated to, for example, 60°C and stirred using the mixing reaction system 400 to react with the nucleic acid that matches the aforementioned nucleic acid B. For example, a site that exhibits fluorescence, luminescence, or extinction under specific conditions given to nucleic acid B reacts with the nucleic acid in the sample 110, thereby exhibiting fluorescence, luminescence, or extinction. Nucleic acid B is designed by structural design to react with a specific nucleic acid, so that nucleic acid B reacts only with the nucleic acid structure possessed by a specific microorganism. Therefore, by using a specific nucleic acid B in the treatment of the mixing reaction system 400, the fluorescence, luminescence, or extinction given to nucleic acid B can be exhibited only when the sample 110 contains a specific microorganism.

[0069] The detection system 500 is a system for detecting the presence and degree of fluorescence, luminescence, or extinction caused by the reaction of nucleic acid B in the processed sample 110 in the hybrid reaction system 400.

[0070] For example, the detection can be performed by the following method, that is, exciting the fluorescence effect imparted to nucleic acid B by using excitation laser and manifested by the reaction with the nucleic acid in the sample 110, and detecting the excited fluorescence by using a high-sensitivity camera. Or, the detection can be performed by the following method, that is, detecting the luminescence effect imparted to nucleic acid B and manifested by the reaction with the nucleic acid in the sample 110 by using a high-sensitivity camera. Or, the detection can be performed by the following method, that is, regarding the extinction effect imparted to nucleic acid B and manifested by the reaction with the nucleic acid in the sample 110, detecting the degree of extinction of the fluorescence or luminescence imparted to the vicinity of the site imparted with the extinction effect by using a high-sensitivity camera.

[0071] By using this nucleic acid analysis system 100, it is possible to analyze whether the sample 110 contains a specific microorganism or its concentration.

[0072] Refer to Figure 5 The nucleic acid extraction method according to this embodiment will be described.

[0073] The nucleic acid extraction method according to this embodiment includes a trapping step S1, a housing step S2, an insertion step S3, and an extraction step S4. The trapping step S1 is a step of filtering and trapping the specimen in the sample 110 containing the specimen having nucleic acid by using the filter 210. The housing step S2 is a step of housing the filter 210 in the housing portion 311 of the filter attracting and holding member 310 having an inner wall surface 312 that is in close contact with the surface of the filter 210 and adsorbs the filter 210. The insertion step S3 is a step of inserting the filter attracting and holding member 310 into the reaction tube 320 for each filter 210. The extraction step S4 is a step of extracting nucleic acid from the specimen by using the reaction tube 320.

[0074] By using this nucleic acid extraction method, it is easy to insert the filter 210 into the reaction tube 320.

[0075] The present invention is not limited to the above embodiments. Modifications can be made without departing from the gist of the present invention.

[0076] Explanation of reference numerals

[0077] 100 Nucleic acid analysis system

[0078] 110 Sample

[0079] 200 Specimen recovery system

[0080] 210 Filter

[0081] 220 Tweezers

[0082] 300 Nucleic Acid Extraction System

[0083] 310 Filter Suction Holder

[0084] 311 Receiving Portion

[0085] 312 Inner Wall Surface

[0086] 313 First End

[0087] 314 Second End

[0088] 320 Reaction Tube

[0089] 400 Mixing and Reaction System

[0090] 500 Detection System

Claims

1. A filter suction holder capable of being inserted into a reaction tube for extracting nucleic acid from a sample containing a sample having nucleic acid together with a filter for capturing the sample by filtering the sample, wherein: The filter suction holder has a housing portion for housing the filter, and the housing portion has an inner wall surface that is in close contact with the surface of the filter and adsorbs the filter.

2. The filter suction holder according to claim 1, wherein: The housing portion has a hollow cone or truncated cone shape, the bottom surface of the cone or truncated cone is open to expose the inner wall surface, and is inserted into the reaction tube from the end of the cone or truncated cone with a smaller diameter.

3. The filter attracting and retaining member according to claim 1, wherein: The housing portion has heat resistance of at least 180° C.

4. The filter attracting and retaining member according to claim 1, wherein: The housing portion is formed of a material that melts if constant heat is applied thereto.

5. The filter attracting and retaining member according to claim 1, wherein: The filter suction and holding member further includes a surfactant fixedly attached to the housing portion.

6. A nucleic acid extraction system, wherein: The nucleic acid extraction system comprises: The filter suction holder according to any one of claims 1 to 5; as well as A reaction tube is capable of accommodating the filter suction holder for each of the filters.

7. A method for extracting nucleic acid, wherein: The nucleic acid extraction method comprises the following steps: Using a filter to filter a sample including a specimen having nucleic acid to capture the specimen; The filter is housed in a housing portion of a filter suction holder having an inner wall surface that is in close contact with the surface of the filter and adsorbs the filter; inserting the filter suction holder into a reaction tube for each of the filters; as well as The nucleic acid is extracted from the sample using the reaction tube.

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