JP2021127169A - Reagent kit, reagent storage method, reagent mixing method, and analyzing method - Google Patents

Abstract

To separately store a plurality of reagents in a sealed state and to easily mix them in the sealed state.SOLUTION: A reagent kit according to an embodiment includes: a filter; a container which has a first space and a second space sectioned with the filter; an encapsulation member which is contained in the first space and has a first liquid reagent encapsulated; and a second reagent which is contained in the second space.SELECTED DRAWING: Figure 1

Description

The embodiments disclosed herein and in the drawings relate to reagent kits, reagent storage methods, reagent mixing methods and analytical methods.

In recent years, the field of molecular biology has developed remarkably and has been applied to medical technology. Among them, the technique of genetically engineering or immunologically detecting a target substance such as a nucleic acid or a protein in a biological sample is one of the most important methods for detecting a cancer or an infectious disease.

Many kinds of reagents such as enzymes, fluorescent labeling reagents or antibodies are used to detect the target substance. When handling a plurality of reagents and samples in this way, complicated operations are required to prevent deterioration, measurement, contamination, etc. during storage of the reagents. In addition, there are many cases where the human body is adversely affected depending on the type of reagent or sample. Therefore, there is a demand for simplification of reagent handling operations in clinical settings where the handling of a plurality of reagents is required, as well as in the manufacturing industry or experiments.

Japanese Patent Publication No. 2004-508541

One of the problems to be solved by the embodiments disclosed in the present specification and the drawings is to separate and store a plurality of reagents in a closed state and easily mix them in a closed state. However, the problems to be solved by the embodiments disclosed in the present specification and the drawings are not limited to the above problems. It is also possible to position the problem corresponding to each effect of each configuration shown in the embodiment described later as another problem.

The reagent kit according to the embodiment includes a filter, a container having a first space and a second space partitioned by the filter, an encapsulating member containing the first liquid reagent contained in the first space, and a first. Includes a second reagent housed in two spaces.

FIG. 1 is an exploded perspective view showing the reagent kit of the first embodiment. FIG. 2 is a flowchart showing a method for manufacturing the reagent kit of the first embodiment. FIG. 3 is a flowchart showing the reagent storage method of the first embodiment. FIG. 4 is a flowchart showing the reagent mixing method of the first embodiment. FIG. 5 is a front view showing the reagent kit of the first embodiment and the mixing step using the reagent kit. FIG. 6 is a front view showing the reagent kit of the second embodiment and the mixing step using the reagent kit. FIG. 7 is a front view showing the reagent kit of the second embodiment. FIG. 8 is a flowchart showing an example of the analysis method of the third embodiment. FIG. 9 is a flowchart showing an example of the analysis method of the third embodiment. FIG. 10 is a front view showing a detection step using the reagent kit. FIG. 11 is a plan view showing the reagent kit of the fourth embodiment. FIG. 12 is a side view showing the reagent kit of the fourth embodiment.

Hereinafter, embodiments of the reagent kit, the method for producing the reagent kit, the method for storing the reagent using the reagent kit, the method for mixing the reagent, and the method for analysis will be described in detail with reference to the drawings.

The reagent kit of the embodiment includes a filter, a container having a first space and a second space partitioned by the filter, an encapsulation member containing the first liquid reagent contained in the first space, and a second space. Includes a second reagent housed in. As will be described in detail later, in one use example of the reagent kit, the encapsulating member is broken to release the first reagent in the encapsulating member into the first space, and the first reagent is moved to the second space through a filter. Mixes the first reagent and the second reagent.

(First Embodiment)
-Reagent Kit As shown in FIG. 1, the reagent kit 1 of the first embodiment includes a container 2. The container 2 includes, for example, a filter column 3 and a closed column 4.

The filter column 3 is a first side wall portion 3a having a cylindrical shape, a filter 3b provided so as to close an opening at one end of the first side wall portion 3a, and an opening at the other end of the first side wall portion 3a. It includes an opening 3c and a first flange 3d that surrounds the first opening 3c. The filter column 3 has, for example, a bottomed cylindrical shape with the filter 3b as the bottom.

The filter 3b is, for example, a porous membrane that does not allow solids to pass through but allows gases and liquids to pass through. The material of the filter 3b is, for example, a non-woven fabric made of polypropylene fiber, polyethylene fiber, polyester fiber, nylon fiber or the like. The pore size of the holes of the filter 3b is preferably, for example, 1 to 100 μm. By using such a filter, it is possible to pass through the encapsulation member with a centrifugal force g of a simple tabletop centrifuge.

The first side wall portion 3a may be made of the same material as the filter 3b, and the first side wall portion 3a and the filter 3b may be integrally formed of one porous film.

Alternatively, the first side wall portion 3a may be made of, for example, a resin such as polypropylene. In that case, the filter 3b is fixed to one end of the first side wall portion 3a by, for example, adhesion, heat fusion, O-ring, or the like. A commercially available spin column or the like may be used as the filter column 3.

An encapsulation member 5 in which the first reagent 6 is encapsulated is housed inside the filter column 3. The encapsulating member 5 is, for example, a hollow spherical shape.

The material of the encapsulating member 5 is preferably polypropylene, polyethylene, polyvinyl chloride, natural rubber, synthetic rubber, or the like. The size and thickness of the encapsulating member 5 can be destroyed, for example, by physical stimuli from the outside, but at other times it is selected according to its material so that it has the strength to maintain the morphology of the hollow body. .. For example, the measurement of the strength of the encapsulating member 5 can be determined by examining whether or not the encapsulating member 5 is destroyed by a destructive means described later, for example, centrifugation or pressure from the outside. The strength of the encapsulating member 5 is preferably such that it is destroyed by a centrifugal force of, for example, about 800 to 3000 g.

The first reagent 6 is a liquid. The type of the first reagent 6 is not limited, and any liquid reagent can be used. The first reagent 6 may be a composition in which a plurality of types of reagents are mixed. The first reagent 6 is, for example, a liquid containing a reagent used in the nucleic acid amplification method.

The particle size of the encapsulating member 5 may be a size that can be accommodated in the filter column 3 to be used. The particle size is not limited, but is preferably 1 to 8 mm, for example. The amount of the first reagent 6 to be sealed in one sealing member 5 is selected according to the use of the reagent kit 1, the type of the first reagent 6, and the particle size of the sealing member 5, and is not limited. However, for example, it is preferably 1 to 200 μL.

The filter column 3 may contain a plurality of encapsulation members 5 in which the first reagent 6 is encapsulated. These encapsulation members 5 may contain the same type of first reagent 6 or may contain different types of first reagent 6. For example, it is possible to enclose two liquid reagents that are not suitable for mixing and storing in separate encapsulation members 5 and accommodate them in one filter column 3.

The closing column 4 has a cylindrical second side wall portion 4a, a bottom portion 4b provided so as to close an opening at one end of the second side wall portion 4a, and a second opening which is an opening at the other end of the second side wall portion 4a. A portion 4c, a second flange 4d surrounding the second opening 4c, and a lid 4e are provided. The second side wall portion 4a may have a tapered portion whose diameter is reduced toward the bottom portion 4b. The lid 4e is provided with, for example, a disk-shaped plug 4f having a diameter slightly smaller than the inner diameter of the second side wall portion 4a, and is inserted into the closing column 4 to airtightly close the closing column 4. As the closed column 4, a commercially available tube such as a micro tube or a PCR tube can be used.

The closed column 4 contains the second reagent 7. The second reagent 7 may be liquid or solid, and may be in any state such as powder, gel or cream. The second reagent 7 is preferably, for example, a dry solid reagent. The type of the second reagent 7 is not limited, but is, for example, a reagent used in the nucleic acid amplification method.

FIG. 1 shows the reagent kit 1 in a state where the lid 4e is opened for convenience, but it is preferable that the reagent kit 1 is provided in a state where the lid 4e is closed and the inside of the container 2 is sealed. A cross-sectional view of the reagent kit 1 with the lid 4e closed cut in parallel with the longitudinal direction of the container 2 is shown in FIG. 5 (a).

As shown in FIG. 5A, the filter column 3 is suspended inside the closed column 4. The filter column 3 can also be removed from the closed column 4. With this configuration, the internal space of the container 2 is partitioned into a first space 8 inside the filter column 3 and a second space 9 inside the closed column 4 with the filter 3b as a sakai.

In a further embodiment, the first side wall portion 3a and the second side wall portion 4a do not have to be cylindrical, and may have a square tubular shape or the like.

-Method of manufacturing the reagent kit The method of manufacturing the reagent kit 1 of the embodiment includes, for example, the following steps shown in the schematic flowchart of FIG.
Encapsulation step (S1) of encapsulating the first reagent 6 which is a liquid in the encapsulation member 5.
The first storage step (S2), in which the encapsulation member 5 containing the first reagent 6 is housed in the first space 8 of the container 2 having the first space 8 and the second space 9 partitioned by the filter 3b.
A second accommodating step (S3) of accommodating the second reagent 7 in the second space 9 and a sealing step (S4) of sealing the container to obtain a reagent kit.

Hereinafter, an example of the method for producing the reagent kit 1 shown in FIG. 1 will be described.

First, the first reagent 6 and the material of the encapsulation member 5 are prepared, and the first reagent 6 is encapsulated in the encapsulation member 5 (encapsulation step (S1)). The encapsulation step (S1) can be performed by a general method of encapsulating a liquid in a hollow body. For example, is manufactured by a method of dropping the first reagent 6 onto the material of the sheet-shaped encapsulation member 5 to wrap the first reagent 6, or a method of injecting the first reagent 6 into the encapsulation member 5 formed into a capsule shape. be able to.

The encapsulation step (S1) is performed by using, for example, a chemical method, a physicochemical method, a mechanical / physical method, or the like. Such a method can be carried out by, for example, an orifice method, a vibration nozzle method, a Centrifugal extrusion method, an interfacial inorganic reaction method, or the like.

Next, the container 2 is prepared. A desired number of encapsulating members 5 containing the first reagent 6 are contained in the filter column 3 of the container 2 (first accommodating step (S2)). The number of encapsulation members 5 to be accommodated is adjusted to include, for example, the amount of the first reagent 6 required to perform one analysis using the reagent kit 1. When a plurality of encapsulating members 5 including a plurality of types of first reagents 6 are accommodated, the number of each reagent is adjusted so as to be contained in a desired ratio.

Next, the second reagent 7 is stored in the closed column 4 (second space 9) of the container 2 (second storage step (S3)). The storage of the second reagent 7 may be carried out by a method according to the type of the second reagent 7.

Next, the filter column 3 is inserted into the closed column 4 from the filter 3b side and suspended in the closed column 4. For example, the entire filter column 3 is inserted into the closed column 4. The filter 3b is fixed so as to be located near the middle between the bottom 4b of the closed column 4 and the second opening 4c.

For example, the second flange 4d of the closed column 4 is deformed by pushing the first flange 3d of the filter column 3, and the repulsive force fixes the filter column 3 at a desired position. Alternatively, the filter column 3 may be fixed by a repulsive force between the first side wall portion 3a of the filter column 3 and the second side wall portion 4a of the closed column 4. Alternatively, the filter column 3 may be fixed by adhering the first opening 3c of the filter column 3 to the inside of the second opening 4c.

Next, the lid 4e is closed to seal both the first space 8 and the second space 9 of the container 2 (sealing step (S4)).

In a further embodiment, the lid 4e may have a cap shape in which a male screw is processed on the inner peripheral surface. In that case, a female screw is machined at least on the outside of the second flange 4d. For example, when the filter column 3 is inserted, the first flange 3d overlaps the second flange 4d, and the filter column 3 is suspended without falling into the closed column 4. Next, the cap-shaped lid 4e is put on the first flange 3d. At this time, the lid 4e has a depth that overlaps with the second flange 4d. Then, by rotating the lid 4e, the screws of the lid 4e and the second flange 4d are engaged with each other to seal the closing column 4. The lid 4e may further include a disc-shaped plug 4f and may be inserted into the filter column 3.

The reagent kit 1 is obtained by the above steps. From the encapsulation step (S1) to the sealing step (S4), each procedure is preferably performed aseptically. Each of the above steps may be performed automatically by the device.

-Reagent Preservation Method According to the embodiment, a method of separating and preserving the first reagent and the second reagent using the reagent kit 1 is also provided. In the reagent storage method according to the embodiment, for example, as shown in FIG. 3, a storage step (S5) for storing the reagent kit 1 is further performed after the reagent kit 1 is manufactured by the encapsulation step (S1) to the sealing step (S4). include.

The storage step (S5) may be performed, for example, at room temperature or lower. The normal temperature is, for example, 5 to 35 ° C. The storage step (S5) may include storage at a refrigerating temperature, and is more preferably performed at, for example, 1 to 35 ° C. The temperature condition is preferably a condition in which the reagent is stored in a state where its function is not lost, depending on the type and state of the first reagent 6 and the second reagent 7. For example, the range of appropriate temperature conditions can be expanded by adding a preservative to the first reagent 6 and the second reagent 7, or by keeping the state in a state where it can be easily stored. For example, the second reagent 7 is preferably a freeze-dried solid.

According to the reagent kit 1 and the reagent storage method of the first embodiment, the first reagent and the second reagent can be stored in a sealed state in a separated state in one container 2. By sealing, storage conditions can be relaxed. Therefore, for example, a plurality of reagents that are difficult to coexist can be stored at one time at room temperature or the like without deterioration, and the management of the reagents is convenient. The plurality of reagents that are difficult to coexist are, for example, reagents in different states, for example, a liquid (first reagent 6) and a solid (second reagent 7).

-Reagent Mixing Method According to a further embodiment, a reagent mixing method using the reagent kit 1 is provided. As shown in FIG. 4, the reagent mixing method includes a preparation step (S6) for preparing a reagent kit, breaking the encapsulating member 5 to release the first reagent 6 into the first space 8, and passing through the filter 3b. The first reagent 6 is moved to the second space 9, and the mixing step (S7) of mixing the first reagent 6 and the second reagent 7 is included.

First, in the preparation step (S6), any of the above reagent kits is prepared. The reagent kit 1 may be manufactured by another person or may be manufactured by itself.

Next, the mixing step (S7) is performed. The mixing step (S7) is performed, for example, by applying an external stimulus to the encapsulating member 5. The external stimulus is, for example, centrifugal force and / or pressure.

For example, when the reagent kit 1 of FIG. 1 is used, an external stimulus can be given by performing centrifugation. For example, when the reagent kit 1 shown in FIG. 5A is centrifuged, the encapsulating member 5 is destroyed by the centrifugal force g as shown in FIG. 5B, and the first reagent 6 is outside the encapsulating member 5. And further move to the second space 9 via the filter 3b. As a result, the first reagent 6 is mixed with the second reagent 7 in the second space 9, and the mixture 10 is obtained. On the other hand, since the broken encapsulation member 5 is solid, it does not pass through the filter 3b and remains in the first space 8.

Centrifugal treatment is performed by, for example, setting the reagent kit 1 in a centrifuge device and centrifuging. The centrifugal treatment is preferably performed with a centrifugal force g such that the sealing member 5 is destroyed, for example, 800 to 3000 g. Here, the centrifugal force g is a relative centrifugal force (RCF), and is determined by the rotation radius r and the rotation speed rpm of the centrifugal force.

After the mixing step (S7), the mixture 10 may be further stirred. Stirring can be performed, for example, by vortexing, inversion mixing, spin-down, or the like.

The mixing step (S7) and, if necessary, stirring can be performed, for example, with the lid 4e of the container 2 closed, that is, in a closed state.

According to the reagent mixing method described above, the first reagent 6 and the second reagent 7 can be mixed in a sealed state by a simple operation such as centrifugation. Further, according to the reagent kit 1 of the embodiment, since the reagent is weighed and contained, the trouble of weighing and dispensing the reagent is omitted, and the mixing operation becomes easier. In addition, since it is not necessary to provide a hole for discharging air in the container by using the centrifuge, the airtightness of the container can be further improved.

Further, by performing the centrifugal treatment, a centrifugal force g is applied to the entire reagent, and the first reagent 6 adhering to the first side wall portion 3a and the like and the second reagent 7 adhering to the second side wall portion 4a and the like are also directed toward the bottom 4b. Due to the movement, the amount of reagent remaining immiscible in the mixture can be reduced. Therefore, a mixture having the intended composition can be obtained.

In particular, reagents in different states, such as a liquid first reagent and a solid second reagent, can be stably stored in the same container 2 to prevent deterioration, and they can be easily mixed. Therefore, it is possible to easily obtain the mixture 10 in a good state with little deterioration.

In a further embodiment, the container may have a first space and a second space partitioned by a filter, and may not necessarily include the filter column 3 and the closed column 4 as shown in FIG. .. For example, a mixing step may be performed by crushing and compressing the first space from the outside using a flexible container having a first space and a second space partitioned by a filter.

(Second Embodiment)
In a second embodiment, a reagent kit is provided in which the container 2 further comprises means for breaking the encapsulation member 5 and moving the first reagent 6 to the second space 9 via the filter 3b.

The means is, for example, a plunger. As shown in FIG. 6A, the reagent kit 11 includes a plunger 12 on the lid 4e of the container 2. The plunger 12 includes a rod-shaped shaft portion 13, a pressurizer 14 attached to one end of the shaft portion 13, and a pressurizer 15 attached to the other end of the shaft portion 13. The shaft portion 13 penetrates the lid 4e perpendicularly to the surface direction of the lid 4e, the pressurizer 14 is arranged outside the container 2, and the pressurizer 15 is arranged in the first space 8. The pressurizer 14 and the pressurizer 15 are, for example, disk-shaped. It is preferable that the diameter of the presser 15 in the surface direction is substantially the same as the inner diameter of the first side wall portion 3a.

The plunger 12 is attached so as to be movable up and down in the longitudinal direction of the shaft portion 13. At the time of manufacturing the reagent kit 11, the plunger 12 is installed so as to provide a gap between the presser 15 and the filter 3b so that the encapsulating member 5 containing the first reagent 6 can exist. Other configurations are the same as those of the reagent kit 1 of the first embodiment.

In the reagent storage method using the reagent kit 11, the plunger 12 is stored so as not to move from the position at the time of manufacture.

In the reagent mixing method using the reagent kit 11, in the mixing step (S7), as shown in FIG. 6B, the pressurizer 14 of the plunger 12 is pushed in the direction of the filter 3b. As a result, the presser 15 presses the encapsulating member 5 against the filter 3b. As a result, the encapsulating member 5 is destroyed, and the first reagent 6 moves to the second space 9 via the filter 3b.

According to the reagent kit 11, the reagents can be mixed by a simple procedure of pushing the plunger 12 without using a centrifuge. Therefore, it is possible to obtain the mixture 10 regardless of the area and equipment conditions.

In a further embodiment, the means is a protrusion on the filter. As shown in FIG. 7, the reagent kit 21 has a protrusion 22 on the first space 8 side of the filter 3b. The tip of the protrusion 22 is sharp.

The dimensions and density of the protrusions 22 are selected according to the material and strength of the encapsulating member 5. The protrusion 22 may have a structure that does not destroy the encapsulating member 5 just by touching the encapsulating member 5, but destroys the encapsulating member 5 when pressed against the filter 3b.

The filter 3b having the protrusions 22 can be manufactured, for example, by coating the filter with a triangular pyramid made of plastic having a side of about 0.2 to 1 mm. Alternatively, a commercially available filter having protrusions may be used.

In the reagent mixing method using the reagent kit 11 including the protrusion 22, the encapsulating member 5 bursts by moving the encapsulating member 5 in the direction of the protrusion 22 and piercing the protrusion 22. For example, the encapsulating member 5 can be moved in the direction of the protrusion 22 by centrifugation or using the plunger 12 of the second embodiment. By using the protrusion 22, it is possible to rupture the encapsulating member 5 with a weaker external stimulus.

(Third Embodiment)
In a third embodiment, the reagent kit is used as an analytical method for amplifying and detecting nucleic acids in a sample.

In the reagent kit according to the third embodiment, at least one of the first reagent 6 and the second reagent 7 contains a reagent used in the nucleic acid amplification method. The nucleic acid amplification method includes, for example, a method involving a temperature change such as a PCR method, and an isothermal amplification method such as the LAMP method. Alternatively, a reverse transcription reaction may be carried out before amplification.

Reagents used for nucleic acid amplification include, for example, amplification enzymes (polymerases), reverse transcriptases, primer sets, deoxynucleoside triphosphates (dNTPs), salts, surfactants, thickeners, buffers for pH adjustment, etc. Includes ions and the like or combinations thereof. The combination and concentration of reagents are selected according to the nucleic acid amplification method used.

For example, a reagent that is preferably liquid is preferably contained in the first reagent 6, and a reagent that may be provided as a solid is preferably contained in the second reagent 7. In one embodiment, the second reagent 7 comprises a dried enzyme, dNTP, primer and the first reagent 6 comprises another reagent. However, the composition of each reagent is not limited to this. For example, it is preferable that the combination of the first reagent 6 and the second reagent 7 contains a set of reagents necessary for amplification.

As the container, any of the first embodiment, the second embodiment, or the fourth embodiment described later may be used.

As shown in FIG. 8, the analysis method according to the third embodiment includes the following steps:
Preparation step for preparing the reagent kit (S6),
Mixing step (S7), in which the encapsulating member 5 is broken, the first reagent 6 is moved to the second space through a filter, and the first reagent and the second reagent are mixed.
A sample addition step (S8) in which a sample is added to a container before or after the mixing step, and a nucleic acid amplification step (S9) in which the obtained sample mixture is maintained under nucleic acid amplification conditions after the mixing step and the sample addition step.

The mixing step (S7) can be performed by any of the methods described above. For example, the mixture 10 obtained by the mixing step has a composition capable of nucleic acid amplification by adding a sample.

The sample added in the sample addition step (S8) is, for example, an analysis target that may contain nucleic acid, and is preferably a body fluid obtained from a living body, for example. Body fluids include, for example, blood, serum, blood cells, plasma, interstitial fluid, urine, stool, sweat, saliva, oral mucosa, sputum, lymph, spinal fluid, tears, breast milk, amniotic fluid, semen, cell extracts, Amniotic fluid, etc. Alternatively, the sample may be cells, tissues, microorganisms, for example, bacteria, fungi or viruses, a solution containing them, a culture of these, or a culture supernatant thereof. The living body is, for example, a mammal and is preferably a human. Alternatively, the living body may be a bird, a reptile, an amphibian, a fish, or the like.

The sample may be an environment-derived sample such as soil, river water, seawater or water and sewage, a beverage or food-derived sample, or the like. Alternatively, the sample may be a liquid or the like containing an artificially synthesized nucleic acid.

As a method for collecting a sample, a general method selected according to the type of sample may be used. The collected sample may be used as it is, or may have been subjected to treatments such as homogenization, dilution or concentration, drug addition, and nucleic acid extraction.

The sample is added, for example, to the first space 8 or the second space 9 before the mixing step (S7). By adding to the first space 8, the sample can be filtered at the same time in the mixing step (S7). Alternatively, the sample may be added to the mixture 10 after the mixing step (S7).

The addition may be carried out by a general method depending on the type of sample, and is carried out by using, for example, a pipette, a dropper or a syringe. For example, the lid 4e of the container 2 may be aseptically opened to add the sample into the container 2, and then the lid 4e may be closed to reseal the container 2. Alternatively, it is also possible to add the sample in a closed system using the reagent kit 31 according to the fourth embodiment described later.

Next, the obtained sample mixture is maintained under nucleic acid amplification conditions (nucleic acid amplification step (S9)). The nucleic acid amplification condition is, for example, a temperature condition at which a nucleic acid amplification reaction occurs. When an amplification method involving a temperature change such as a PCR method is used, the container 2 is placed in a thermal cycler or the like, and the sample mixture is subjected to an appropriate temperature cycle. When an isothermal amplification method such as the LAMP method is used, the container 2 is installed in an incubator or the like and maintained at an appropriate temperature.

According to this analysis method, since the reagent kit of the embodiment is used, a mixture capable of nucleic acid amplification can be prepared easily and without contamination. Further, the nucleic acid amplification step can be performed with the sample mixture contained in the container 2, and the nucleic acid amplification can be easily performed.

According to a further embodiment, the analytical method comprises a detection step (S10) of detecting an optical signal from the sample mixture during or after the nucleic acid amplification step (S9), as shown in FIG.

The detection step (S10) can be performed while the sample mixture is contained in the container 2. In this case, the portion of the container 2 corresponding to the second space 9, for example, the material of the second side wall portion 4a of the closed column 4 is a light transmissive material. The light-transmitting material is, for example, polypropylene or the like. With this configuration, it is possible to irradiate light from the outside of the container 2 and detect an optical signal on the outside of the container 2.

The optical signal is, for example, fluorescence. In this case, a dye that fluoresces as the amplification product increases may be contained in the first reagent 6 or the second reagent 7, or may be added to the sample mixture before the nucleic acid amplification step (S9). good. Alternatively, the optical signal may be, for example, turbidity or absorbance (eg, absorption, scattering, reflection intensity when the mixture 10 is irradiated with light).

As shown in FIG. 10, the detection step (S10) is performed using, for example, a light source 23 installed outside the container 2 and an optical sensor 24.

The light source 23 is arranged so as to irradiate the mixture 10 in the second space 9 with light. The light source 23 is, for example, an LED or the like. When the optical signal is fluorescent, the light source 23 irradiates fluorescent excitation light.

The optical sensor 24 is arranged, for example, so as to face the bottom portion 4b. However, the position of the optical sensor 24 may be any position as long as it can detect the optical signal from the mixture 10 housed in the second space 9. The optical sensor 24 is a general sensor capable of detecting the presence or absence or amount of an optical signal, for example, a fluorescence sensor, a spectrophotometer, or the like.

With such a configuration, an optical signal from the amplification product produced in the sample mixture can be obtained. For example, the optical signal increases or decreases as the amplification product increases. Alternatively, as the number of amplification products increases, the time required for obtaining an optical signal becomes faster. Therefore, the presence or absence or amount of the target nucleic acid may be determined from the presence or absence or amount of the optical signal or the time until the signal is obtained.

With such a configuration, according to the analysis method of the embodiment, the target nucleic acid existing in the second space 9 is amplified by using the container 2 as it is by a simple procedure of adding the sample to the container 2 containing the mixture 10. And can be detected.

Alternatively, any one of the mixing step (S7), the addition step (S8), the nucleic acid amplification step (S9), and the detection step (S10) is performed using the container 2, and then the mixture is transferred to another container. , A later step may be performed. In this case, since the mixing step (S7) is performed using the reagent kit according to the embodiment, a mixture containing the nucleic acid amplification reagent can be easily obtained.

In a further embodiment, the first reagent 6 and the second reagent 7 do not have to contain all of the reagents required for nucleic acid amplification. For example, additional necessary reagents may be added before the nucleic acid amplification step (S9).

(Fourth Embodiment)
In a fourth embodiment, a reagent kit further comprising a third space is provided. As shown in FIG. 11A, the container 32 of the reagent kit 31 according to the fourth embodiment has a tubular second accommodating portion 34 and a first accommodating portion 33 communicating with one end of the second accommodating portion 34. And a third accommodating portion 35 communicating with the other end of the second accommodating portion 34. A filter 37 is arranged on one end side of the second accommodating portion 34 so as to close the inner cross section thereof. The first accommodating portion 33 and the third accommodating portion 35 are made of a flexible material such as polyethylene or polyvinyl chloride.

In the reagent kit 31, the inside of the first storage portion 33 is the first space 8, the inside of the second storage portion 34 is the second space 9, and the inside of the third storage portion 35 is the third space 36. The first space 8 and the second space 9 are separated by a filter 37.

The first accommodating portion 33 accommodates the encapsulating member 5 containing the first reagent 6. The second reagent 7 is housed inside the second storage unit 34. For example, reagents and the like are not stored in the third storage unit 35. The third accommodating portion 35 is provided with, for example, an injection port 38, from which a sample can be injected later, for example, by a syringe, a pipette, or the like. The inlet 38 includes, for example, a lid (not shown) that seals the third space 36.

In the reagent mixing method using the reagent kit 31 according to the fourth embodiment, for example, as shown in FIG. 11B, pressure N is applied from the outside of the first storage unit 33 to compress the first storage unit 33. .. The compression is preferably performed by, for example, applying pressure N in the longitudinal direction of the second accommodating portion 34 (hereinafter, referred to as “vertical direction”) in the direction toward the second accommodating portion 34. As a result, the first reagent 6 is released from the encapsulation member 5 and moves into the second accommodating portion 34.

The sample is injected into the third containment section 35 before or after compression of the first containment section 33. The sample is, for example, the one described above.

Next, the third accommodating portion 35 is compressed. The compression is preferably performed by, for example, applying a pressure N to the third accommodating portion 35 in the direction toward the second accommodating portion 34 in the vertical direction. As a result, the sample in the third accommodating portion 35 also moves into the second accommodating portion 34.

An exhaust port for removing air from the container 32 at the time of compression may be provided at any position of the container 32. The exhaust port has, for example, a check valve structure so that outside air cannot enter the container 32.

As a result of the compression of both storage portions, the first reagent 6, the second reagent 7, and the sample can be mixed in the second storage portion 34.

The reagent kit 31 can also be used in the nucleic acid amplification method as in the third embodiment. In that case, the first reagent 6 and / or the second reagent 7 contains a nucleic acid amplification reagent. For example, nucleic acid amplification can be performed by heating the container 32 after the above mixing.

By using the second accommodating portion 34 as a light transmitting material, it is possible to detect an optical signal from the mixture 10. In the detection, for example, a light source 23 is used to irradiate one side surface of the second accommodating portion 34 with light, and an optical sensor 24 installed on the other side surface side detects an optical signal.

For example, the compression, detection, and / or temperature control of the reagent kit 31 may be automatically performed by the apparatus. For example, such a device comprises a reagent kit attachment for installing the reagent kit 31. The reagent kit mounting portion includes two pressors for compressing the first accommodating portion 33 and the third accommodating portion 35, a heater for controlling the temperature of the second accommodating portion 34, and a light source for irradiating the second accommodating portion 34 with light. 23, according to the optical sensor 24 that detects the optical signal from the second accommodating unit 34, the parameters and programs for controlling these, the storage unit that stores the detection result of the optical signal, and the parameters and programs of the storage unit. It is equipped with a processor or the like that controls these and processes optical signals.

The compression direction does not have to be the vertical direction. FIG. 12A is a side view of the reagent kit 31 shown in FIG. 11A. For example, as shown in FIG. 12B, the first accommodating portion 33 and the third accommodating portion 35 may be compressed from the side, that is, from the lateral direction.

In a further embodiment, the third storage unit 35 may contain a third reagent instead of the sample. Further, a plurality of additional accommodating portions (spaces) communicating with the second accommodating portion 34 may be provided.

Further, in a further embodiment, the container of any of the first and second embodiments may be provided with a third space that communicates with the second space 9, and further, if necessary, communicates with the second space 9. There may be a plurality of other spaces to be provided.

According to the reagent kit 31 having such a third space 36, the sample can be added in a sealed state.

According to at least one embodiment described above, a plurality of reagents can be separated and stored in a closed state, and can be easily mixed in a closed state.

Although some embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other embodiments, and various omissions, replacements, changes, and combinations of embodiments can be made without departing from the gist of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, as well as in the scope of the invention described in the claims and the equivalent scope thereof.

1, 11, 21, 31 ... Reagent kits, 2, 32 ... Containers, 3b, 37 ... Filters,
5 ... Encapsulation member, 6 ... 1st reagent, 7 ... 2nd reagent, 8 ... 1st space, 9 ... 2nd space,
10 ... mixture, 12 ... plunger, 22 ... protrusion, 36 ... third space

Claims (10)

A filter, a container having a first space and a second space partitioned by the filter, and a container.
An encapsulation member containing a first liquid reagent contained in the first space, and an encapsulation member.
A reagent kit containing a second reagent housed in the second space. The reagent kit according to claim 1, wherein the container is further provided with a plunger that pushes the encapsulating member against the filter to break it in the first space, or a protrusion provided on the first space side of the filter. The reagent kit according to claim 1 or 2, wherein a plurality of the encapsulating members containing first reagents of different types are housed in the first space. The reagent kit according to any one of claims 1 to 3, wherein the first reagent and / or the second reagent contains a reagent used in a nucleic acid amplification method. The reagent kit according to any one of claims 1 to 4, further comprising a third space communicating with the second space. It is a method of separating and storing the first reagent and the second reagent, which are liquids.
Encapsulation step of encapsulating the first reagent in an encapsulation member,
A first storage step of accommodating the encapsulation member containing the first reagent in the first space of a container having a first space and a second space partitioned by a filter.
A second accommodating step of accommodating the second reagent in the second space,
A reagent storage method comprising a sealing step of sealing the container to obtain a reagent kit and a storage step of storing the reagent kit. A method of mixing the first reagent and the second reagent.
The encapsulating member containing the first liquid reagent contained in the first space of the container including the first space and the second space partitioned by the filter, and the second reagent housed in the second space are provided. A step of preparing a reagent kit containing the reagent, and a mixing step of breaking the encapsulating member, moving the first reagent to the second space through the filter, and mixing the first reagent and the second reagent. Reagent mixing method including. The mixing step involves centrifuging the container, pressing the encapsulating member against the filter in the first space using a plunger, and using the filter provided with a protrusion on the first space side to the protrusion. The method according to claim 7, wherein the encapsulating member is pressed and / or the first space is crushed from the outside of the container. An analytical method for detecting nucleic acids in a sample.
An encapsulating member containing a first liquid reagent contained in the first space of a container containing a first space and a second space partitioned by a filter, and a second reagent housed in the second space are provided. The process of preparing the reagent kit including
A mixing step of breaking the encapsulating member, moving the first reagent to the second space through the filter, and mixing the first reagent and the second reagent.
It comprises a sample addition step of adding the sample to the container before or after the mixing step, and a nucleic acid amplification step of maintaining the obtained sample mixture under nucleic acid amplification conditions after the mixing step and the sample addition step.
The first reagent and / or the second reagent contains a reagent used in a nucleic acid amplification method.
Analytical method. The method of claim 9, further comprising a detection step of detecting an optical signal from the sample mixture during or after the nucleic acid amplification step.

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