JP2017018053A - Nucleic acid extraction kit, nucleic acid extraction method, and nucleic acid extraction apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid extraction kit capable of loading a large amount of a sample when treating a sample having a low genomic DNA concentration such as serum.SOLUTION: The nucleic acid extraction kit comprises: an extraction filter cartridge 160 having an extraction filter 162 for separating and extracting nucleic acid from a biological sample; a first container 1 including a reagent well 120 for storing at least a reagent, a waste liquid well 131 for containing a waste liquid, and a recovery well 140 for recovering the extracted nucleic acid, and having an outer periphery provided with a wall surface higher than a projection toward the bottom of the reagent well; and a second container 2, which is separated from the first container, including a reagent well 120 for storing at least a reagent and a sample well 110 for introducing a biological sample, and having an outer periphery provided with a wall surface higher than a projection toward the bottom of the reagent well, where the extraction filter cartridge is formed so that it can be carried on the waste liquid well and the recovery well in the first container.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a nucleic acid extraction kit, a nucleic acid extraction method, and a nucleic acid extraction apparatus, and more particularly, a pretreatment technique related to a method and apparatus for concentrating and purifying a nucleic acid extracted from a biological sample in a gene analysis method and performing a gene analysis process, The present invention relates to a nucleic acid extraction kit that makes it possible to dispose of a reagent supply source and waste consumables generated by sample processing in a pretreatment step without being contaminated, and a technique suitable for use in a nucleic acid extraction method using the kit.

  In recent years, attempts have been made to introduce genetic analysis to medical sites. These are tests and analyzes performed on nucleic acids collected from biological samples. Biological samples include blood, serum, plasma, semen, lymph fluid and other body fluids, various tissues such as skin, hair, muscle tissue, feces, urine and other excreta It shows the sample originating in. These tests may enable early detection of diseases and risk of onset, infectious diseases, malignant tumors, and the like.

  Extraction of nucleic acids from biological samples is an important process in the fields of genetic engineering and clinical testing. For example, when blood or tissue is collected from a human body or the like and tested, it is necessary to extract and purify only nucleic acid from a biological sample such as blood. In order to separate proteins and nucleic acids from other components, a biological sample is physically and chemically dissolved, and then the proteins and lipids are decomposed by extraction to release the nucleic acids. At this time, the process of separating and purifying proteins and nucleic acids tends to cause sample contamination. As a standard method of extraction, an organic solvent is often used. However, this toxicity, the labor of waste disposal, the labor of centrifugation, the contamination of the sample and the infection from the sample are problematic.

  The BOOM method is known as a nucleic acid extraction / recovery method. The BOOM method is a nucleic acid extraction technique in which a chaotropic reagent and solid phase silica are combined, and utilizes the fact that nucleic acid is adsorbed on the silica surface in the presence of chaotropic ions.

  In the conventional method, the destruction / release process and the extraction / separation process are performed separately, and the separation and extraction of nucleic acids cannot be performed consistently as a pretreatment. There is always the possibility of infection, possible contamination of the sample, possible sample misplacement, and possible contamination between samples (cross-contamination). Furthermore, since these operations require manpower, it is inefficient and cumbersome, and the possibility of infection of pathogens to workers is also conceivable.

  For example, an apparatus using a solid phase extraction filter includes a nucleic acid extraction apparatus disclosed in Patent Document 1. In Patent Document 1, in order to simultaneously process a plurality of samples, an integrated column in which a number of columns are two-dimensionally arranged is used. The integrated column (extraction filter cartridge) is hermetically installed in the upper opening of the vacuum chamber to form a vacuum chamber, and solid phase extraction is performed by a so-called suction method. When nucleic acid is extracted, a series of steps including adsorption of the nucleic acid onto the carrier, washing of the carrier, and attachment / detachment / recovery of the nucleic acid from the carrier must be performed.

  In addition, a method has been proposed in which a container (well) containing a reagent for extracting nucleic acid, a recovery liquid container, and a waste liquid container are separately attached to the apparatus (Patent Document 2). Since the nucleic acid extraction apparatus and cartridge disclosed in Patent Document 2 can be processed simply by adding a sample to the machine without performing complicated reagent dispensing operations, a plurality of samples can be processed in large quantities. Is suitable.

  However, in the technique disclosed in the above-mentioned patent document, it takes time to prepare these extraction filter cartridges, recovery tubes, waste liquid tubes, and the like, and sufficient time is required even if they are installed in a machine. Furthermore, in these apparatuses, the influence of contamination to the surroundings can be considered before and after the operation of the apparatus. In other words, in the nucleic acid extraction step, it is necessary to remove a large number of integrated columns or containers in the apparatus and remove them after use, so there is a high possibility that the waste liquid will scatter due to vibration, etc. There is a risk of contaminating the extracted sample. Also, when it is discarded, there is a possibility that the waste liquid tube set in the apparatus may be contaminated by overturning or deviation.

  In addition, a technique for solving the problems such as the complexity and the fall of waste has been proposed (Patent Document 3). In the nucleic acid extraction apparatus and cartridge disclosed in Patent Document 3, a waste liquid reservoir, a recovery liquid reservoir, a sample reservoir, a processing reagent, a nucleic acid extraction filter, and a sample are all mounted in the same container.

JP 2002-506384 A Japanese Patent No. 3635645 Japanese Patent No. 4911264

  However, in the technique disclosed in the above-mentioned patent document, in order to mount everything, the size of each part has to be reduced, so that a large amount of sample cannot be mounted. For example, a solution such as serum or plasma When processing a sample with a low concentration of genomic DNA, a large amount of sample is required, but not all can be loaded.

  The present invention has been made in view of the above circumstances, and in a nucleic acid extraction apparatus for extracting nucleic acid from a sample, sample contamination is prevented, and preparation and disposal of complicated reagents before and after nucleic acid extraction are performed quickly and easily. Furthermore, an object of the present invention is to provide a nucleic acid extraction cartridge, a nucleic acid extraction apparatus, and a nucleic acid extraction method capable of automatically extracting nucleic acids from a large amount of samples.

  As described above, in the present invention, in order to be able to process a large amount of sample, a cartridge on which reagents, waste liquid, and a nucleic acid separation filter are mounted and a cartridge that stores a sample and performs heat treatment are separately provided. By using these cartridges, advance preparation is unnecessary, and a quick and simple nucleic acid extraction method and simple and safe disposal can be realized.

The nucleic acid extraction kit of the present invention includes an extraction filter for separating and extracting nucleic acid from a biological sample, and an extraction filter cartridge including a support filter member laminated on at least one surface of the extraction filter,
A reagent well for storing at least a reagent, a waste well for containing waste liquid, a recovery well for recovering the extracted nucleic acid, and a protective film covering a portion of each well forming surface where the reagent well is formed, and on the outer periphery. A first container provided with a wall surface higher than the protrusion to the bottom of the reagent well;
A second container that is separated from the first container and includes at least a reagent well for storing a reagent, a sample well for introducing a biological sample, and a wall surface higher than the protrusion of the reagent well to the bottom of the outer periphery. When,
Have
The extraction filter cartridge is formed to be supported on the waste well and the recovery well in the first container.
In the nucleic acid extraction kit of the present invention, the wall surface of the first container can be provided with a fixing and alignment mechanism for the nucleic acid extraction device.
In the nucleic acid extraction kit of the present invention, it is preferable that the opening surface of the reagent well of the first container is formed on the same surface and the opening surface of the reagent well of the second container is formed on the same surface.
In the nucleic acid extraction kit of the present invention, a protective film that covers the well forming surface is provided on the well forming surface of the first container and the second container, and the film can be penetrated by a dispensing tip that sucks and discharges the reagent. Can be formed.
In the nucleic acid extraction kit of the present invention, the protective film covering the well forming surface can have a water vapor barrier property.
The nucleic acid extraction kit of the present invention may be provided so that the protective film in the second container does not cover the sample well.
In the nucleic acid extraction kit of the present invention, the first container has a plurality of the reagent wells extending in the front-rear direction, the waste wells are connected to the front-rear position of the reagent well, The dimension in the width direction intersecting with the front-rear direction may be set larger than the width dimension of the reagent well.
The nucleic acid extraction kit of the present invention may be provided in the first container so that the waste liquid well is located on both sides in the width direction of the recovery well.
In the nucleic acid extraction kit of the present invention, the first container may be provided with dilution stirring wells so that the waste wells are located on both sides in the width direction.
The nucleic acid extraction method of the present invention is a nucleic acid extraction method using any of the nucleic acid extraction kits described above,
Mixing a biological sample and a reagent to elute the nucleic acid;
Adding the liquid eluting the nucleic acid to the extraction filter cartridge carried on the waste well;
Discarding the solution containing impurities leaving the nucleic acid from the solution on the extraction filter cartridge in the waste well;
Having the extraction filter cartridge supported on the recovery well and recovering the extracted nucleic acid,
The nucleic acid extraction can be used by penetrating the protective film.
In the nucleic acid extraction method of the present invention, the instrument used for penetrating the protective film can be a dispensing tip for sucking and discharging a solution.
A nucleic acid extraction apparatus of the present invention comprises any one of the nucleic acid extraction kits described above,
Extraction filter pressurizing means for allowing liquid to pass through the extraction filter cartridge;
Means for moving the extraction filter cartridge; and dispensing means for moving reagents and sample liquid by suction and discharge;
Can have.
In the nucleic acid extraction apparatus of the present invention, the extraction filter pressurizing means may have a pressurized air supply mechanism that supplies pressurized air from above the extraction filter cartridge and allows liquid to pass through the extraction filter.
In the nucleic acid extraction apparatus of the present invention, the dispensing means may have a dispensing tip comprising a dispensing tip inert equipped with a suction and discharge mechanism and a dispensing tip detachable at the tip of the dispensing tip inert. it can.

The nucleic acid extraction kit of the present invention includes an extraction filter for separating and extracting nucleic acid from a biological sample, and an extraction filter cartridge including a support filter member laminated on at least one surface of the extraction filter,
A reagent well for storing at least a reagent, a waste well for containing waste liquid, a recovery well for recovering the extracted nucleic acid, and a protective film covering a portion of each well forming surface where the reagent well is formed, and on the outer periphery. A first container provided with a wall surface higher than the protrusion to the bottom of the reagent well;
A second container that is separated from the first container and includes at least a reagent well for storing a reagent, a sample well for introducing a biological sample, and a wall surface higher than the protrusion of the reagent well to the bottom of the outer periphery. When,
Have
By setting the extraction filter cartridge to be supported on the waste well and the recovery well in the first container, a nucleic acid extraction kit in which a biological sample is added to the sample well is set in the nucleic acid extraction device, It is possible to automate the nucleic acid extraction process.

  The nucleic acid extraction kit of the present invention includes a fixing and positioning mechanism for the nucleic acid extraction device on the wall surface of the first container, so that the nucleic acid extraction kit is set in the automated nucleic acid extraction device and the nucleic acid extraction process is performed. It becomes possible.

  In the nucleic acid extraction kit of the present invention, the opening surface of the reagent well of the first container is formed on the same surface, and the opening surface of the reagent well of the second container is formed on the same surface, A protective film can be provided on the well opening surface.

  In the nucleic acid extraction kit of the present invention, a protective film that covers the well forming surface is provided on the well forming surface of the first container and the second container, and the film can be penetrated by a dispensing tip that sucks and discharges the reagent. By being formed (breakable), it is possible to break through the protective film when sucking the liquid contained in each well with the dispensing tip, thereby preventing the occurrence of contamination and contamination Can do.

  In the nucleic acid extraction kit of the present invention, the protective film covering the well forming surface has a water vapor barrier property, so that the accuracy of the nucleic acid extraction process can be achieved.

  In the nucleic acid extraction kit of the present invention, the protective film in the second container is provided so as not to cover the sample well, so that before setting the nucleic acid extraction kit in the nucleic acid extraction apparatus, and in other wells Before opening the protective film, it is possible to add a biological sample to the sample well, and to prevent contamination and contamination to other wells.

  In the nucleic acid extraction kit of the present invention, the first container has a plurality of the reagent wells extending in the front-rear direction, the waste wells are connected to the front-rear position of the reagent well, By setting the dimension in the width direction intersecting with the front-rear direction to be larger than the width dimension of the reagent well, the nucleic acid dispensing process can be completed in a state where the total amount of the waste liquid is stored in the waste liquid well. After extraction, no waste liquid remains in the apparatus, and waste liquid treatment can be simplified. Therefore, the possibility of being contaminated with waste liquid can be sufficiently reduced.

  The nucleic acid extraction kit according to the present invention moves the extraction filter cartridge between the reagent well and the recovery well in the first container so that the waste well is located on both sides in the width direction of the recovery well. In this case, it is possible to reduce the distance straddling the waste liquid well and increase the capacity of the waste liquid well.

  In the nucleic acid extraction kit of the present invention, in the first container, the extraction filter cartridge is provided between the reagent well and the dilution agitation well by providing the dilution agitation well so that the waste well is located on both sides in the width direction. When moving, it is possible to reduce the distance straddling the waste liquid well and to increase the capacity of the waste well.

The nucleic acid extraction method of the present invention is a nucleic acid extraction method using any of the nucleic acid extraction kits described above,
Mixing a biological sample and a reagent to elute the nucleic acid;
Adding the liquid eluting the nucleic acid to the extraction filter cartridge carried on the waste well;
Discarding the solution containing impurities leaving the nucleic acid from the solution on the extraction filter cartridge in the waste well;
Having the extraction filter cartridge supported on the recovery well and recovering the extracted nucleic acid,
By using the protective film while penetrating it during nucleic acid extraction, nucleic acid extraction can be automated.

  In the nucleic acid extraction method of the present invention, the instrument used for penetrating the protective film is a dispensing tip that sucks and discharges the solution, so that nucleic acid extraction can be performed automatically.

A nucleic acid extraction apparatus of the present invention comprises any one of the nucleic acid extraction kits described above,
Extraction filter pressurizing means for allowing liquid to pass through the extraction filter cartridge;
Means for moving the extraction filter cartridge; and dispensing means for moving reagents and sample liquid by suction and discharge;
Thus, nucleic acid extraction can be performed automatically.

  In the nucleic acid extraction apparatus of the present invention, the extraction filter pressurizing means has a pressurized air supply mechanism for supplying pressurized air from above the extraction filter cartridge and allowing liquid to pass through the extraction filter, thereby performing nucleic acid extraction. It becomes possible to carry out by automating.

  In the nucleic acid extraction apparatus of the present invention, the dispensing means has a dispensing tip comprising a dispensing tip inert equipped with a suction and discharge mechanism and a dispensing tip detachable at the tip of the dispensing tip inert. The nucleic acid extraction can be automated.

  According to the present invention, an extraction filter for extracting a nucleic acid is directly supported on a reagent container so that the extraction can be performed, thereby simplifying an instrument used for one nucleic acid extraction operation and putting it on a human hand. Work became easier. In addition, by providing waste wells to discard reagents in the container, preparation of reagents when using an automatic nucleic acid extractor and waste liquid treatment after nucleic acid recovery are simplified, and nucleic acid extraction can be performed quickly and easily without contamination. It became.

  Furthermore, according to the nucleic acid extraction apparatus of the present invention, preparation of reagents when using an automatic nucleic acid extractor and waste liquid treatment after nucleic acid recovery are simplified, and nucleic acid extraction can be performed quickly and easily without contamination. More specifically, a cartridge equipped with an extraction filter is arranged directly on the waste well and the recovery well, and the separation and extraction operations can be performed by the above-described components, thereby allowing the solution of the extraction filter to be pressurized and extracted. Contamination due to overturning of filter members was eliminated, and simplification of extraction work was realized.

It is a perspective view which shows the 1st container and 2nd container in 1st Embodiment of the nucleic acid extraction kit which concerns on this invention. It is a schematic plan view which shows the 1st container and 2nd container in 1st Embodiment of the nucleic acid extraction kit which concerns on this invention. It is a schematic plan view which shows the 1st container in 1st Embodiment of the nucleic acid extraction kit which concerns on this invention. It is a schematic plan view which shows the 1st container in 1st Embodiment of the nucleic acid extraction kit which concerns on this invention. It is arrow sectional drawing in FIG. It is the schematic which shows the extraction filter cartridge in 1st Embodiment of the nucleic acid extraction kit which concerns on this invention. It is the schematic which shows 1st Embodiment of the nucleic acid extraction apparatus which concerns on this invention. It is a flowchart which extracts a nucleic acid from a sample using the nucleic acid extraction method in 1st Embodiment of the nucleic acid extraction method which concerns on this invention. It is a perspective view at the time of apparatus operation | movement in 1st Embodiment of the nucleic acid extraction method which concerns on this invention. It is a perspective view at the time of apparatus operation | movement in 1st Embodiment of the nucleic acid extraction method which concerns on this invention. It is a perspective view at the time of apparatus operation | movement in 1st Embodiment of the nucleic acid extraction method which concerns on this invention. It is a perspective view at the time of apparatus operation | movement in 1st Embodiment of the nucleic acid extraction method which concerns on this invention. It is a perspective view at the time of apparatus operation | movement in 1st Embodiment of the nucleic acid extraction method which concerns on this invention. It is a perspective view at the time of apparatus operation | movement in 1st Embodiment of the nucleic acid extraction method which concerns on this invention. It is a perspective view at the time of apparatus operation | movement in 1st Embodiment of the nucleic acid extraction method which concerns on this invention. It is a perspective view at the time of apparatus operation | movement in 1st Embodiment of the nucleic acid extraction method which concerns on this invention.

Hereinafter, a first embodiment of a nucleic acid extraction kit, a nucleic acid extraction method, and a nucleic acid extraction apparatus according to the present invention will be described with reference to the drawings.
FIG. 1 is a perspective view showing a nucleic acid extraction kit in the present embodiment, FIG. 2 is a plan view showing each well forming surface of the nucleic acid extraction kit in the present embodiment, and FIG. 3 is a nucleic acid in the present embodiment. FIG. 4 is a plan view showing the first container of the extraction kit, FIG. 4 is a plan view showing the first container of the nucleic acid extraction kit in the present embodiment, and FIG. 5 is a cross-sectional view taken along the arrow in FIG. 6 is a schematic view showing an extraction filter cartridge according to the present embodiment. In the figure, reference numeral 100 denotes a nucleic acid extraction kit.

1. Nucleic Acid Extraction Device The nucleic acid extraction kit 100 according to the present embodiment automatically inserts a biological sample 110a into the sample well 110 of the second container 2 and sets it in a nucleic acid extraction device (not shown) as described later. Thus, nucleic acid extraction work can be performed.

  As shown in FIG. 1, the nucleic acid extraction kit 100 according to this embodiment includes a first container 1 and a second container 2 as reagent cartridge main bodies, and an extraction filter independent of the first container 1 and the second container 2. And a cartridge 160.

As shown in FIG. 2, the first container 1 according to the present embodiment collects the reagent well 120 on the base material constituting the container, the waste liquid well 131 that discards unnecessary solutions separated during extraction, and the extracted nucleic acid. It has a recovery well 140 and a dilution stirring well 150 for diluting the recovered nucleic acid.
The reagent well 120 of the first container 1 contains a washing solution for washing away unnecessary solutions, an elution solution for eluting nucleic acid from the extraction filter cartridge 160, a diluting solution for diluting the solution from which nucleic acid is eluted, and the like. In addition, the solution accommodated in a well is not restricted to these.

As shown in FIG. 2, the second container 2 has a sample well 110 capable of accommodating a large amount of biological sample 110 a and a reagent well 120 </ b> B capable of accommodating a large amount.
The sample well 110 of the second container 2 is designed to have a shape that can contact a temperature control unit (not shown) of the nucleic acid extraction device. In the lysate well 122 as the reagent well 120B of the second container 2, an enzyme and lysate for thawing a biological substance such as a cell membrane are accommodated. The solution stored in the well is not limited to this.

  The nucleic acid extraction kit 100 according to the present embodiment can be fixed to a nucleic acid extraction apparatus 400 as shown in FIG. 7, and the extraction operation using the dispensing tip 200 can be mechanically performed.

  Hereinafter, each component will be described. Note that the apparatus and each component of the present invention are not limited to the configuration shown in the drawings, and can be combined with other components as long as the effects of the present invention are not hindered.

<Reagent cartridge body>
The shape of the 1st container 1 should just be able to be fixed and used for a nucleic acid extraction apparatus. As a fixing method, for example, as shown in FIG. 4, it is possible to fix the position in the left-right direction by providing a tab 5 on the reagent cartridge and hooking it on a position fixing means (not shown) on the apparatus side. Further, it is possible to provide the stage front alignment unit 6 and the stage rear alignment unit 7 and to perform the alignment by hooking on a projection (not shown) on the apparatus side, but this is not restrictive. The stage front alignment unit 6 and the stage rear alignment unit 7 constitute a fixing and alignment mechanism.

  As shown in FIGS. 2 and 3, the first container 1 can be provided with a region where reagent wells 120 are arranged, a region where waste wells 131 are formed, and a region where recovery wells 140 are formed.

  As shown in FIG. 2, the first container 1 includes, as reagent wells 120, wells 121, 124, 125, regions in which the wells 127, 128, 126, and 129 are arranged in two rows in the front-rear direction, and two wells A region in which the adsorbing liquid well 123 having a larger width in the left-right direction than the wells 121, 124, 125, 127, 128, 126, and 129 is arranged in a rectangular parallelepiped shape. .

  In the region of the reagent well 120 of the first container 1, as shown in FIGS. 2 to 5, the wells 123, 121, 124, 125, 126, 127 are formed so that the opening surfaces are the same surface. . Further, the opening surfaces of these wells 123, 121, 124, 125, 126, 127 and the waste liquid well 131 are formed on the same surface.

  In addition, in the hole portion 129 adjacent to the well 126, as shown in FIG. 1, the dispensing tip 201a is temporarily placed without being covered with the protective film 3 to be described later, or the inside of the dispensing tip 201a for wiping off the dispensing tip 201a. A sponge or the like can also be arranged.

  As shown in FIG. 2, the adsorbing liquid well 123 has a width dimension in the left-right direction larger than that of each of the wells 121, 124, 125, 126, 127, and 129. The amount of adsorbed liquid to be stored can be increased to several tens of ml.

  Further, the first container 1 has a rectangular parallelepiped shape in which the waste well 131 having a width wider than the region of the reagent well 120 is formed in succession to the adsorbent well 123, with respect to the region of the reagent well 120. It can connect to the front-back direction position shown by the up-down direction in FIGS. The area of the waste liquid well 131 is set so that the horizontal dimension is more than double that of the reagent well 120 area.

  By providing the waste liquid well 131 in the wide rectangular parallelepiped region in the first container 1, the amount of waste liquid to be stored can be dramatically increased. For this reason, for example, even when a sample with a low genomic DNA concentration in a solution such as serum or plasma is processed, a necessary amount of liquid up to, for example, about several tens of ml can be discharged.

The wide rectangular parallelepiped region in the first container 1 in which the waste liquid well 131 is formed has a recovery well 140, a dilution stirring well 150, a cartridge support (waste liquid extraction filter cartridge installation position) 130, and a stage front alignment. The part 6 can be formed.
The recovery well 140, the dilution stirring well 150, and the cartridge support part (waste liquid extraction filter cartridge installation position) 130 are lower than the opening surface of the reagent well 120, but higher than the liquid level when the waste liquid is accommodated in the waste liquid well 131. The extraction filter cartridge 160 can be carried at the position.

  The waste well 131 is connected to the region of the reagent well 120, but the recovery well 140, the dilution stirring well 150, and the front-rear direction are arranged in the same position in the width direction as the width of the reagent well 120.

  Further, the lowest bottom portion 140b in the recovery well 140 and the lowest bottom portion 150b in the dilution stirring well 150 are the lowest bottom portions 127b, 128b, 126b, etc. of the wells 127, 128, 126 in the reagent well 120 as shown in FIG. It is provided so as to be positioned in the same straight line in the front-rear direction as 129b. As a result, as will be described later, the movement of the dispensing tip 201a between the recovery well 140 and each of the wells 127, 128, 126, and 129 is shortened, and the dispensing tip 201a is diluted with the dilution stirring well. It is set to shorten the movement when moving between 150 and each of the wells 127, 128, and 126.

  Here, the lowest bottom portions 140b, 150b, 127b, 128b, 126b, and 129b of the wells are shrunk toward the position where the bottoms in the recovery well 140, dilution stirring well 150, wells 127, 128, 126, and 129 are lowest. In order to allow the liquid to be stored to be sucked by the dispensing tip without any residue, as shown in FIG. 5, the inner diameters of the lowest bottom portions 140b, 150b, 127b, 128b, 126b, and 129b are used. It is set to be slightly larger than the outer diameter of the tip of the dispensing tip. In each well, the diameter is preferably reduced from the top opening to the lowest bottom so that there is no unevenness.

  To place the extraction filter cartridge 160 on the opposite side of the recovery well 140 from the dilution stirring well 150, that is, on the waste well 131 on the reagent well 120 side of the recovery well 140 when the recovery of the nucleic acid is completed. The extraction filter cartridge installation part 132 which becomes the extraction filter cartridge installation position 132 is provided. On the extraction filter cartridge installation part 132, the extraction filter cartridge 160 can be detachably fixed as shown by a broken line in FIG.

  In addition, as shown in FIG. 5, the first container 1 is provided with a wall surface 1a or legs higher than the protrusions of the bottoms 127a, 128a, 126a, etc. of each well on the outer periphery thereof, whereby the first container 1 is self-supporting. A structure is desirable. By adopting such a structure, stability is increased when the user temporarily puts it during operation. Further, the wall surface 1 a can be provided with a fixing mechanism for the apparatus as the above-described claw 5 and stage rearward alignment unit 7.

As shown in FIG. 3, the waste well extraction filter cartridge installation position 130 has a shape capable of carrying the extraction filter cartridge 160. On the cartridge support portion 130, as shown in FIG. 160 can be detachably fixed.
The shape that can be supported in the present invention means that the extraction filter cartridge 160 is fixed when it is supported so that it cannot fall over. For example, the outer shape of the extraction filter cartridge 160 and the shape of the opening of the cartridge support part 130 (or a part thereof) can be matched to form a supportable shape.
Similarly, the recovery well 140 has a shape capable of carrying the extraction filter cartridge 160 as shown in FIG. 3, and the extraction filter cartridge 160 can be attached to and detached from the well 140 as shown in FIG. Can be fixed to.

  As shown in FIGS. 2, 4, and 5, the waste well extraction filter cartridge installation position 130 and the extraction filter cartridge installation section 132 hold the lower peripheral position when the extraction filter cartridge 160 is placed. As will be described later, the lower side of the discharge portion 165 which is the lower end of the extraction filter cartridge 160 shown in FIG. 6 is set to a position facing the waste liquid well 131, and the discharge portion 165 can be discharged to the waste liquid well 131.

  In the first container 1 as the reagent cartridge main body, the waste liquid well 131 and the recovery well 140 are adjacent to each other as shown in FIGS. This is because the extraction filter cartridge 160 is moved from the waste liquid well 131 to the recovery well 140 in the extraction step, as will be described later. By minimizing the distance of this movement, contamination on the container can be prevented.

  Further, in the first container 1 as the reagent cartridge main body, the regions where the waste liquid well 131 and the reagent well 120 are formed are preferably adjacent to each other as shown in FIGS. This is because the reagent well 120 is mainly dispensed with the extraction filter cartridge 160 on the waste well 131. Adopting this position configuration is expected to prevent contamination and shorten work time.

  As shown in FIG. 2, the recovery well 140 in the first container 1 serving as the reagent cartridge main body also plays a role for storing the extraction filter cartridge 160. Since the extraction filter cartridge 160 is supported in the recovery well 140 in advance, it can be set as the nucleic acid extraction kit 100 in the nucleic acid extraction apparatus 400, so that handling is easy.

  As shown in FIGS. 1 and 2, the second container 2 as the reagent cartridge main body is formed as a separate body from the first container 1, and its height dimension is higher than that of the first container 1. The amount of the biological sample 110a that is set and accommodated in the sample well 110, and the enzyme and lysate that dissolves a biological substance such as a cell membrane, which are accommodated in the lysate well 122, can be increased. For this reason, even when a sample with a low genomic DNA concentration in a solution such as serum or plasma is processed, it is possible to use a necessary amount of liquid, for example, up to about several tens of ml.

  The opening surfaces of the wells 110 and 122 of the second container 2 are formed on the same surface as shown in FIGS. In addition, the opening surfaces of the wells 110 and 122 are set so that the height positions are higher than the opening surfaces of the wells 120 in the first container 1.

  The material for forming the first container 1 and the second container 2 as the reagent cartridge main body is not particularly limited as long as it does not affect the sample, the reagent, etc., and in particular, any one of polypropylene, polycarbonate, and acrylic is used. If the resin material containing is used, favorable visible light permeability can be ensured and the state of the solution can be confirmed. As the polypropylene, homopolypropylene or a random copolymer of polypolypropylene and polyethylene can be used. As the acrylic, polymethyl methacrylate or a copolymer of methyl methacrylate and other monomers such as methacrylic acid ester, acrylic acid ester and styrene can be used. Moreover, when using these resin materials, heat resistance and intensity | strength can also be ensured.

  As a method for forming the first container 1 and the second container 2, in the case of a resin material, various resin molding methods such as injection molding and vacuum molding, mechanical cutting, and the like can be used.

<Extraction filter cartridge>
The nucleic acid extraction kit 100 of this embodiment includes an extraction filter cartridge 160 that includes an extraction filter for separating and extracting nucleic acids from the biological sample 110a. As shown in FIG. 6, the extraction filter cartridge 160 has an extraction filter 162 fixed to at least the extraction filter cartridge holder 161.

As shown in FIG. 6, in the extraction filter cartridge 160, an extraction filter 162 for adsorbing nucleic acids is held in a substantially horizontal direction at the center of a cylindrical extraction filter cartridge holder 161 having an open top. A support filter 163 is arranged immediately below the extraction filter 162 in parallel with the extraction filter 162 for the purpose of preventing deformation of the extraction filter 162.
The extraction filter cartridge holder 161 is formed such that the lower part of the extraction filter cartridge holder 161 is reduced in a funnel shape from the support filter 163, and a discharge part 165 on the nozzle is formed to protrude to a predetermined length at the center of the lower end.
As shown in FIG. 6, the extraction filter 162 is provided with a cylindrical filter holder 164 so as to be in contact with the peripheral portion of the upper surface which is the filtration surface.

A sample solution, a cleaning solution, and a recovery solution, which will be described later, are dispensed into the extraction filter cartridge holder 161 from the upper opening, pressurized air is introduced from the pressurizing mechanism 300 through the upper opening, and each solution is discharged through the extraction filter 162. It is discharged from the part 165.
Depending on the installation position of the extraction filter cartridge 160, the liquid discharged from the discharge unit 165 flows down to either the waste liquid well 131 or the recovery well 140 and is discharged or recovered.

The extraction filter 162 is formed by forming a porous material that adsorbs nucleic acids into a film shape. The porous material of the extraction filter 162 is configured to adsorb and hold nucleic acids when washed with a washing solution, and to weaken and separate the nucleic acid adsorption force when collected with a collecting solution.
The porous material in the present embodiment includes a material in which fibrous materials such as glass wool are overlapped. The material of the filter is not particularly limited as long as it can adsorb a biological substance in the presence of an organic substance, but it is preferable to use a porous material in which a hydroxyl group is introduced as a hydrophilic group.
As the extraction filter cartridge holder 161, a material such as a resin can be used as in the case of the first container 1 and the second container 2 described above, and a similar forming method can be employed.

Further, the support filter 163 may be stacked below the extraction filter 162. The extraction filter 162 may bend due to pressurization by the pressurization mechanism, and there is a possibility that the solution leaks from an undesired portion. However, by laminating the support filter member 163 having relatively high rigidity, Can be prevented.
As a method for preparing the support filter 163, it is preferable to use a filter prepared by baking and solidifying resin particles, but the present invention is not limited to this, and a substance that affects the reaction is not eluted and the solution can be filtered. What is necessary is just to have a hole. The material is not particularly limited as long as it can be formed porous so that the solution can pass through the filter.

  As shown in FIG. 6, the filter retainer 164 comes into contact with the peripheral edge of the upper surface, which is the filtration surface of the extraction filter 162, and is generated by friction generated between the inner wall surface of the extraction filter cartridge holder 161 and the outer peripheral surface of the filter retainer 164. By being supported in the extraction filter cartridge holder 161, the extraction filter 162 held by the end of the filter holder 164 is prevented from being lifted. In view of the above-described support mode, it is desirable that the outer diameter of the filter retainer 164 and the inner diameter of the extraction filter cartridge holder 161 are substantially the same.

From the viewpoint of securing the filtration surface of the extraction filter 162 as large as possible, the area of the end surface of the filter holder 164 is preferably as small as possible, and is preferably 30% or less of the area of the filtration surface of the extraction filter 162.
However, in order to hold the extraction filter 162 while suppressing the lifting, the filter holder 164 needs to have a certain rigidity, so the thickness of the filter holder 164 needs to be 0.2 mm or more, and 0.3 mm or more. It is preferable that

  The height (dimension in the axial direction) of the filter holder 164 is set to be not less than half the height of the side surface of the extraction filter cartridge holder 161 and not more than the height of the side surface of the extraction filter cartridge holder 161. Preferably, the height is the same as the height of the portion or slightly lower than the height of the side surface portion of the extraction filter cartridge holder 161. If the height of the filter holder 164 is smaller than this, the contact area with the extraction filter cartridge holder 161 becomes small, and the frictional force for holding the extraction filter 162 becomes insufficient. Also. If the height of the side surface of the extraction filter cartridge holder 161 is exceeded, the filter retainer 164 protrudes from the upper surface opening of the extraction filter cartridge holder 161, which hinders the execution of each step in the process such as nucleic acid extraction.

  In the step of purifying and extracting nucleic acid, a lysate, a washing solution, and the like are dispensed into the extraction filter cartridge 160. Even when the maximum amount is dispensed, the liquid level remains in the filter holder 164 in the extraction filter cartridge 160. It is preferable that the height of the filter holder 164 is set so as not to be higher. This is because if the liquid level is higher than the filter holder 164, the liquid may enter the gap between the outer peripheral surface of the filter holder 164 and the inner surface of the extraction filter cartridge holder 161, and it is difficult to apply pressure due to the air layer, and clogging may occur. This is because it becomes higher. When such a situation occurs, the drying process is insufficiently dried, and ethanol in the cleaning liquid used for cleaning the extraction filter 162 remains. As a result, when the nucleic acid is recovered from the extraction filter 162, ethanol is mixed into the recovered nucleic acid, and the occurrence rate of defects related to the reaction using the recovered nucleic acid is increased.

  The material for forming the filter retainer 164 is not limited as long as it does not dissolve in the solvent used for washing or the like and does not affect the sample, the reagent, etc. In particular, like the extraction filter cartridge holder 161, polypropylene, It is preferable to use a resin material containing either polycarbonate or acrylic. As for the forming method, various methods such as molding and cutting may be used.

  As a method for inserting the support filter 163, the extraction filter 162, and the filter retainer 164 into the extraction filter cartridge holder 161, a known assembly robot or manufacturing method can be used, and the support filter 163 and the extraction filter 162 are inserted into the extraction filter cartridge holder 161. Any method can be used as long as the layers can be stacked so that the filter holders 164 are horizontal in the order of the filter holders 164.

<Protective member>
As shown in FIG. 1, the nucleic acid extraction kit 100 according to the present embodiment is preferably provided with a protective member on the first container 1 and the second container 2 in order to prevent contamination and contamination. In the present embodiment, the first container 1 is sealed with the first container sealing film 3 and the second container 2 is sealed with the second container sealing film 4 with respect to the opening end surface of the reagent well 120. . The first container sealing film 3 and the second container sealing film 4 may cover a plurality of reagent wells with a single seal, or cover the entire region where the reagent wells are formed with a single seal. You may do it.

  Furthermore, the 1st container sealing film 3 and the 2nd container sealing film 4 use the thing of the intensity | strength which can penetrate at the front-end | tip (dispensing pipette tip) of the dispensing tip 200 used in the case of dispensing shown in FIG. Thus, the first container sealing film 3 and the second container sealing film 4 can be penetrated through the dispensing chip 200 to collect reagents from the wells and perform nucleic acid extraction work. There is no need to peel 3 and the second container sealing film 4 in advance, and there is no concern about contamination of reagents and the like.

As the first container sealing film 3 and the second container sealing film 4, films having no influence on the sample and the reagent can be arbitrarily used. For example, as the plastic film, various polypropylenes, polyethylene terephthalate, Polyester such as naphthalate or metal thin film such as aluminum can be used.
In order to seal each reagent well 120, the first container sealing film 3 and the second container sealing film 4 are bonded so as to be in a sealed state at the mouth portion of each reagent well 120. When the first container sealing film 3 and the second container sealing film 4 are plastic films, they can be sealed with heat, laser welding or the like.

Moreover, if what has transparency is used for the 1st container sealing film 3 and the 2nd container sealing film 4, after heat welding, there exists an advantage that sealing performance can be identified by seeing a welding surface visually. “Having transparency” in the film is sufficient if at least a part of the visible light region is transmitted so that the weldability can be visually recognized. In addition, the structure of the 1st container sealing film 3 and the 2nd container sealing film 4 is good also as a laminated structure. For example, a plastic film may be exposed on one side of the film as a welding surface, and an inorganic material such as SiO ₂ may be laminated on the other side to improve the sealing performance.

<Dispensing tip>
As with the nucleic acid extraction kit 100, it is desirable that the dispensing chip 200 used for dispensing reagents is discarded every time nucleic acid is extracted, in order to prevent contamination. Therefore, in the nucleic acid extraction apparatus 400 of the present embodiment, as shown in FIG. 7, the dispensing tip inert 210 having a liquid suction / discharge function and the dispensing tip 200 detachable from the dispensing tip inert 210 are separated. Thus, the dispensing tip 200 can be discarded.

  Dispensing chips 201a to 201d are preferably installed in the apparatus in a state of being stored in a dispensing chip rack 202 that can store the dispensing chips 201a to 201d used in one nucleic acid extraction operation. By adopting such a configuration, the dispensing tips 201a to 201d can be discarded together with the dispensing tip rack 202 after the work is completed, so that the disposal work is easy.

  It is desirable that the dispensing tip rack 202 has a wall surface or a leg that is higher than the protrusion of the bottom of the dispensing tip storage portion on the outer periphery, and thereby has a self-supporting structure. By adopting such a structure, stability can be increased when the user temporarily places the device during operation, and contamination of the biological sample due to overturning can be prevented. Further, the dispensing tip rack 202 is provided with a lid (not shown) in the dispensing tip storage portion, thereby preventing contamination and contamination of foreign substances before use, and dispensing tips 201a to 201d after extraction. Contamination due to scattering of the solution adhering to can be prevented.

<Nucleic acid extraction device>
FIG. 7 is a perspective view showing components of the nucleic acid extraction apparatus 400 in which the nucleic acid extraction kit 100 of the present embodiment is installed.
As shown in FIG. 7, the nucleic acid extraction apparatus 400 of the present invention includes a pressurizing mechanism 300 for allowing a sample liquid to pass through at least a sample solution through an extraction filter, an extraction filter cartridge moving means 310, and liquids of reagents and samples. A dispensing tip inert 210 and a dispensing tip 200 are provided as dispensing means for moving.

  The nucleic acid extraction kit 100 can be used by being installed on a detachable fixing base. The nucleic acid extraction apparatus 400 of the present embodiment can be installed as a single container with the extraction filter cartridge 160 mounted on the first container 1 in which the wells are grouped together, and can automate operations after cartridge installation. The extraction process can be simplified. At this time, the second container 2 is installed on a fixed base of the nucleic acid extraction apparatus 400 separately from the first container 1. As a result, when the liquid stored in the well 110 or the well 122 of the second container 2 having a large capacity is heated, the first container 1 is prevented from being excessively heated. it can.

The nucleic acid extraction apparatus of the present embodiment is not limited to the form of the nucleic acid extraction kit 100 as described above. The nucleic acid extraction apparatus of the present embodiment places the extraction filter cartridge 160 on the waste well 131 and the recovery well 140. By directly arranging and enabling the dispensing operation, it is possible to eliminate the contamination caused by the scattering of the extraction filter pressurizing solution and the fall of the extraction filter member, thereby simplifying the extraction operation.
Hereinafter, each component in the nucleic acid extraction apparatus of this embodiment is demonstrated in detail.

  As the extraction filter pressurizing means, a pressurizing mechanism 300 in which an electronic regulator is connected to the downstream side of the air pump is used, and a gas such as air or nitrogen is pressurized and sent from above the extraction filter cartridge 160 by the pressurizing mechanism 300. Is done. Thus, a part of the solution introduced into the extraction filter cartridge 160 is passed through the extraction filter 162 and is a component for performing separation. Here, since the extraction filter pressurizing means includes an electronic control regulator, it is possible to arbitrarily set the applied pressure of the pressurized air.

The extraction filter cartridge moving mechanism 310 moves the extraction filter cartridge 160 from the initial position onto the waste well extraction filter cartridge installation position 130 or the recovery well 140 and installs it.
In the nucleic acid extraction apparatus 400 of the present embodiment, as shown in FIG. 7, an extraction filter moving mechanism 310 supported by a jaw formed on the extraction filter cartridge 160 is provided below the pressurizing mechanism 300. The elevating mechanism provided therewith allows the pressurizing process to be performed simply by lowering the pressurizing mechanism 300 after the movement.

  A dispensing pipette can be used as a dispensing means for dispensing the reagents and sample liquid stored in the well. In the nucleic acid extraction apparatus 400 of this embodiment, as shown in FIG. 7, the dispensing means are a dispensing tip inert 210 having a liquid suction / discharge mechanism, and a dispensing tip that is a detachable dispensing pipette tip. 201a-d.

  In the nucleic acid extraction apparatus 400 of this embodiment, by providing a plurality of dispensing tips 201a to 201d, it is possible to replace the dispensing tips for each solution characteristic and perform nucleic acid extraction work with one dispensing tip inert 210. It is. Also in this case, it is preferable that the dispensing tip inert 210 is moved onto the dispensing tips 201a to 201d to be mechanically detachable. Dispensing chips 201 a to 201 d can be housed in a dedicated dispensing chip rack 202 and installed in the apparatus simultaneously or separately with the nucleic acid extraction kit 100.

  In the nucleic acid extraction apparatus 400 of this embodiment, the pressurization mechanism 300, the extraction filter moving mechanism 310, and the dispensing tip inert 210 each have moving means, thereby automating the extraction process after the nucleic acid extraction kit 100 is installed. Can do. These moving means may use a common moving mechanism, or may be provided as individual moving mechanisms.

  Further, a pedestal (not shown) for installing the first container 1 and the second container 2 in the nucleic acid extraction apparatus 400 can be provided with a heat block. It is also possible to promote a series of extraction steps by heating some wells such as the wells 121 and 127 of the first container 1 or the well 110 of the second container by the heat block.

2. Nucleic Acid Extraction Method Hereinafter, the nucleic acid extraction method in the present embodiment will be described.
FIG. 8 is a flowchart showing the nucleic acid extraction method in the present embodiment, FIG. 9 is a perspective view showing the apparatus operation in the nucleic acid extraction method in the present embodiment, and FIG. 10 is the nucleic acid extraction method in the present embodiment. FIG. 11 is a perspective view showing the operation of the apparatus in the nucleic acid extraction method of the present embodiment. FIG. 12 shows the operation of the apparatus in the nucleic acid extraction method of the present embodiment. FIG. 13 is a perspective view showing the apparatus operation in the nucleic acid extraction method of the present embodiment, and FIG. 14 is a perspective view showing the apparatus operation of the nucleic acid extraction method in the present embodiment. 15 is a perspective view showing the operation of the apparatus in the nucleic acid extraction method of the present embodiment, and FIG. 16 shows the operation of the apparatus in the nucleic acid extraction method of the present embodiment. It is a to perspective view.

In the nucleic acid extraction method according to the present embodiment, as shown in FIG. 8, nucleic acid extraction is performed by steps S1 to S15.
In this embodiment, nucleic acid extraction from serum will be described as an example of a nucleic acid extraction method.

  In addition, as biological samples, various body fluids such as blood, plasma, serum, semen, and lymph, various tissues such as skin, hair, and muscle tissue, various excrements such as stool and urine, and samples derived therefrom are also in the same process. Nucleic acid extraction can be performed with The basic configuration of the nucleic acid extraction method is the same as the BOOM method that is generally used. The BOOM method is a nucleic acid extraction technique in which a chaotropic reagent and silica or the like are combined, and utilizes the fact that nucleic acid is adsorbed on the silica surface in the presence of chaotropic ions.

First, the collected biological sample 110 a (in this case, serum) is injected into the sample well 110 of the second container 2 and set in the nucleic acid extraction device 400.
Next, the first container 1 and the dispensing tip 200 are set in the nucleic acid extraction apparatus 400, and the apparatus starts automatic operation. The following operations can be automatically performed by electronically driving and controlling.
9 to 16, the first container sealing film 3 and the second container sealing film 4 are omitted for easy understanding of the operation.

<Nucleic acid extraction process>
In step S1, the extraction filter cartridge 160 is transferred to the upper portion of the waste well extraction filter cartridge installation position 130 by the extraction filter cartridge moving mechanism 310, as shown in FIG.

  Next, in step S2, as shown in FIG. 10, the proteolytic enzyme 121a is aspirated from the enzyme well 121 of the first container 1 using the dispensing tip 201a, and as shown in FIG. The proteolytic enzyme 121a is discharged and stirred into the biological sample 110a dispensed into the sample well 110.

  Next, as step S3, as shown in FIG. 12, the lysate 122a is aspirated from the lysate well 122, dispensed into the sample well 110, and stirred with the mixture to elute the nucleic acid.

Next, as step S4, as shown in FIG. 13, the adsorbing liquid 123a containing the chaotropic salt is sucked from the adsorbing liquid well 123, dispensed into the sample well 110 and added, and sucked using the dispensing tip 201a. Mix and stir repeatedly. Thereby, the nucleic acid can be adsorbed to the extraction filter 162 shown in FIG.
When performing these operations, although not shown in FIG. 13, the first container sealing film 3 and the second container sealing film 4 are each broken and dispensed by the dispensing tip 201a. Since the sealing property of the reagent can be maintained, the extraction operation can be realized without causing contamination or deterioration.

  As shown in FIGS. 10 and 12, a proteolytic enzyme 121a, a lysis solution 122a, and an adsorption solution 123a are sequentially dispensed into the sample well 110 into which the biological sample 110a has been dispensed by the dispensing chip 201a. In the sample well 110, the proteolytic enzyme 121a, the lysing solution 122a, and the biological sample 110a are mixed by repeating suction and discharge using the dispensing tip 201a, and the biological material can be dissolved.

  Thereafter, in step S5, as shown in FIG. 14, the solution is dispensed to the extraction filter cartridge 160 installed at the waste well extraction filter cartridge installation position 130, and the nucleic acid is adsorbed to the extraction filter 162.

  Next, as step S6, as shown in FIG. 15, the pressurizing mechanism 300 of the nucleic acid extraction apparatus 400 moves onto the extraction filter cartridge 160, and then descends, and the mixed liquid dispensed by the pressurized air is removed. Extrude. At this time, the nucleic acid is adsorbed by the extraction filter 162 shown in FIG. 6 and the impurities (unnecessary substances) are discarded in the waste liquid well 131.

  At that time, it is preferable that the pressurizing mechanism 300 removes all the solution from the extraction filter 162 shown in FIG. 6, and it is necessary to apply a pressure larger than the pressure for passing the filter. However, there is a very high possibility that this large pressure will cause the solution to scatter and foam, contaminating the inside of the apparatus and the working environment. Therefore, it is preferable to apply a pressure that does not allow the solution to completely pass through the extraction filter 162. That is, in the air pressurization step S6, a part of the solution containing the impurity remains in the vicinity of the discharge portion 165 of the extraction filter cartridge 160. By repeating the nucleic acid washing step described later, Can be removed.

<Nucleic acid washing process>
In the cleaning process of the extraction filter 162 to which the nucleic acid has been adsorbed, as shown in FIG. 16, the cleaning liquid (1) 124a stored in the well 124 is sucked by the dispensing tip 201b and added to the extraction filter cartridge 160 as shown in FIG. .
Next, as step S <b> 8, the impurity that has not been adsorbed and bound to the extraction filter 162 is pushed out to the waste liquid well 131 by the pressurizing mechanism 300 using pressurized air.
Furthermore, the adsorbed nucleic acid is subjected to the washing liquid (2) 125a housed in the well 125 and the washing liquid (3) 126a housed in the well 126 in the same manner as the well 124 shown in FIG. Washed multiple times.
In the case where a plurality of pipetting pipettes 201b to 201d are provided, they may be replaced when different reagents are used in the cleaning process or the following recovery process.

<Recovery process>
The recovery well 140 is used for recovering the nucleic acid.
First, as step S13, the extraction filter cartridge 160 is moved to the upper part of the recovery well 140 by the extraction filter cartridge moving mechanism 310 so that it can be recovered in the recovery well 140, which is a clean well, as shown in FIG.
Next, as step S14, the eluate 127a in the eluate well 127 is dispensed into the extraction filter cartridge 160 by the dispensing tip 201c. Thereafter, in step S15, the pressurized solution is supplied by the pressurizing mechanism 300 to elute the genome solution into the recovery well 140.

  When the extraction filter cartridge 160 is installed on the recovery well 140 when the recovery of the nucleic acid is completed, it is necessary to move it because the next operation cannot be performed. Specifically, after using the waste liquid well 131, the extraction filter cartridge 160 is moved to the extraction filter cartridge installation position 132 using the extraction filter cartridge moving mechanism 310, as indicated by a broken line in FIG. As a result, the genomic solution can be aspirated from the recovery well 140.

<Dilution process>
When performing a molecular biological reaction represented by PCR, the genome solution collected in the collection well 140 needs to be diluted. Specifically, the diluted solution 128a dispensed in the diluted solution well 128 and the genome solution are mixed. Examples of the mixing method include a method in which the diluent 128a is added to the collected genome solution and a method in which the genome solution is aspirated and added to the diluent well 128. The above two methods include the genome solution and the diluent. Since the dispersion of the liquid amount of 128a affects the dilution rate, it is not preferable. Therefore, by providing the dilution stirring well 150 and dispensing the genomic solution and the diluent 128a into the dilution stirring well 150, the dilution can be realized with good reproducibility.

  According to this embodiment, as described above, the nucleic acid extraction kit 100 includes the first container 1, the second container 2, and the extraction filter cartridge 160, so that the biological sample 110a is sampled. After adding to the well 110 and setting the nucleic acid extraction kit 100 in the nucleic acid extraction apparatus 400, the nucleic acid extraction operation can be completely automated. In addition, since the waste liquid well 131 is formed wider in the first container 1 than the other well regions, no waste liquid remains in the apparatus after nucleic acid extraction, and the waste liquid treatment can be simplified. Therefore, the possibility of being contaminated with waste liquid can be sufficiently reduced.

  Furthermore, since the waste liquid well 131 is formed wider than the other well regions in the first container 1, the amount of liquid necessary for processing is increased when nucleic acid is extracted from a sample having a low nucleic acid concentration such as serum. Even if it increases, it is possible to sufficiently cope with it, and it is possible to perform an automatic processing operation as it is using the conventional nucleic acid extraction apparatus 400. At the same time, the second container 2 is separated from the first container 1 as a separate body, and the height dimension of the second container 2 is set higher than the height dimension of the first container 1, so that the liquid to be stored The amount can be dramatically increased, and even when the amount of liquid necessary for the processing is increased, it can be sufficiently handled.

  Moreover, since at least a part of the wells formed inside the first container 1 and the second container 2 are protected by a film, the reagents are integrated, so that they are easy to handle. Furthermore, since it is integrated with the film, it can be safely discarded.

DESCRIPTION OF SYMBOLS 1 ... First container 2 ... Second container 3 ... First container sealing film 4 ... Second container sealing film 5 ... Claw 6 ... Stage alignment (front)
7… Stage alignment (rear)
DESCRIPTION OF SYMBOLS 100 ... Nucleic acid extraction kit 110 ... Sample well 110a ... Biological sample 120 ... Reagent well 121 ... Enzyme well 121a ... Proteolytic enzyme 122 ... Lysis solution well 122a ... Dissolution solution 123 ... Adsorption solution well 123a ... Adsorption solution 124 ... Washing solution 1 well 124a ... Cleaning liquid 1
125 ... Washing liquid 2 well 125a ... Washing liquid 2
126 ... Washing liquid 3 well 126a ... Washing liquid 3
127 ... Eluate well 127a ... Eluate 128 ... Diluent well 128a ... Diluent 129 ... Wipe part 130 ... Cartridge support part (Waste liquid well extraction filter cartridge installation position)
131 ... Waste liquid well 132 ... Extraction filter cartridge installation position after using waste liquid well 140 ... Recovery well 150 ... Dilution stirring well 160 ... Extraction filter cartridge 161 ... Extraction filter cartridge holder 162 ... Extraction filter 163 ... Support filter 164 ... Filter holder 165 ... Discharge Part 200 ... Dispensing tip 201a ... Dispensing tip 1
201b ... dispensing tip 2
201c: Dispensing tip 3
201d: Dispensing tip 4
202 ... Dispensing tip rack 210 ... Dispensing tip inert 300 ... Pressurizing mechanism 310 ... Extraction filter cartridge moving mechanism 400 ... Nucleic acid extraction device

Claims (14)

An extraction filter cartridge comprising an extraction filter for separating and extracting nucleic acid from a biological sample and a support filter member laminated on at least one surface of the extraction filter;
A reagent well for storing at least a reagent, a waste well for containing waste liquid, a recovery well for recovering the extracted nucleic acid, and a protective film covering a portion of each well forming surface where the reagent well is formed, and on the outer periphery. A first container provided with a wall surface higher than the protrusion to the bottom of the reagent well;
A second container that is separated from the first container and includes at least a reagent well for storing a reagent, a sample well for introducing a biological sample, and a wall surface higher than the protrusion of the reagent well to the bottom of the outer periphery. When,
Have
The nucleic acid extraction kit, wherein the extraction filter cartridge is formed so as to be supported on the waste well and the recovery well in the first container.   The nucleic acid extraction kit according to claim 1, wherein the wall surface of the first container is provided with a fixing and alignment mechanism for the nucleic acid extraction device.   The opening surface of the reagent well of the first container is formed on the same surface, and the opening surface of the reagent well of the second container is formed on the same surface. Nucleic acid extraction kit.   The well forming surface of the first container and the second container is provided with a protective film that covers the well forming surface, and the film is formed to be penetrable by a dispensing tip that sucks and discharges the reagent. The nucleic acid extraction kit according to any one of claims 1 to 3.   The nucleic acid extraction kit according to claim 4, wherein the protective film covering the well forming surface has a water vapor barrier property.   The nucleic acid extraction kit according to claim 4 or 5, wherein the protective film in the second container is provided so as not to cover the sample well.   In the first container, a plurality of the reagent wells extend in the front-rear direction, the waste liquid wells are connected to the front-rear position of the reagent well, and the width direction intersects with the front-rear direction in the waste liquid wells The nucleic acid extraction kit according to any one of claims 1 to 3, wherein the dimension in is set larger than the width dimension of the reagent well.   The nucleic acid extraction kit according to claim 7, wherein the first container is provided so that the waste well is located on both sides in the width direction of the recovery well.   The nucleic acid extraction kit according to claim 7 or 8, wherein the first container is provided with dilution stirring wells so that the waste wells are located on both sides in the width direction. A nucleic acid extraction method using the nucleic acid extraction kit according to any one of claims 1 to 9,
Mixing a biological sample and a reagent to elute the nucleic acid;
Adding the liquid eluting the nucleic acid to the extraction filter cartridge carried on the waste well;
Discarding the solution containing impurities leaving the nucleic acid from the solution on the extraction filter cartridge in the waste well;
Having the extraction filter cartridge supported on the recovery well and recovering the extracted nucleic acid,
A nucleic acid extraction method, wherein the protective film is used by penetrating the nucleic acid during nucleic acid extraction.   The nucleic acid extraction method according to claim 10, wherein the instrument used for penetrating the protective film is a dispensing tip that sucks and discharges a solution. A nucleic acid extraction kit according to any one of claims 1 to 9,
Extraction filter pressurizing means for allowing liquid to pass through the extraction filter cartridge;
Means for moving the extraction filter cartridge; and dispensing means for moving reagents and sample liquid by suction and discharge;
A nucleic acid extraction apparatus comprising:   13. The nucleic acid extraction apparatus according to claim 12, wherein the extraction filter pressurizing unit includes a pressurized air supply mechanism that supplies pressurized air from above the extraction filter cartridge and allows liquid to pass through the extraction filter. . 13. The dispensing means has a dispensing tip comprising a dispensing tip inert having a suction and discharge mechanism and a dispensing tip detachable at the tip of the dispensing tip inert. Nucleic acid extraction apparatus.

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