JP2018171028A - Nucleic acid separator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid separation apparatus capable of automating a pretreatment step such as a dissolution treatment which has been conventionally performed manually. SOLUTION: A nucleic acid separation device injects a stirring mechanism for stirring and dissolving a sample and a reagent injected into a processing tube, and the sample liquid for separating and recovering the nucleic acid from a sample liquid containing the nucleic acid after the dissolution treatment. A pressurized gas supply mechanism that supplies pressurized gas to the filter cartridge, a dispensing mechanism that injects a sample, reagent, or sample liquid after dissolution treatment into the processing tube and filter cartridge, and a pressurized gas supply mechanism and dispensing mechanism. The dispensing mechanism is provided with a moving mechanism for moving the liquid, and the dispensing mechanism is capable of sucking and discharging the liquid through the dispensing tip and discharging the liquid from the nozzle. It is equipped with a liquid supply mechanism. [Selection diagram] Fig. 2

Description

  The present invention relates to a nucleic acid separation apparatus that automatically performs from a pretreatment step of a sample solution containing nucleic acid to a nucleic acid extraction step.

  In recent years, techniques for extracting and analyzing nucleic acids (mainly DNA) that store genetic information of life from human blood, animal and plant tissues, and the like have been widely used. In order to extract DNA from blood, tissue, etc., various dedicated reagents and devices are used.

  Recently, the nucleic acid extraction process has been automated (see, for example, Patent Document 1). On the other hand, in the so-called pretreatment process such as adding a reagent to blood or tissue and stirring, it is necessary to handle a large number of reagents. In addition, the pretreatment process includes various operations such as reagent addition, stirring, and pressurization, and automation has not progressed yet and has been performed manually.

Japanese Patent No. 36335645

  When handling blood, tissues, etc., it is necessary to avoid infection due to contamination or scattering, and it has been desired to automate as much as possible by reducing manual work.

  However, in the pretreatment step, it is necessary to handle the above-described plurality of reagents. In addition, the pretreatment process includes many steps for dissolution treatment such as a reagent addition step, a stirring step, and a heating step, and it has been difficult to automate.

  An object of the present invention is to provide an automatic nucleic acid separation apparatus capable of automating a pretreatment process such as a lysis treatment which has been conventionally performed manually.

The nucleic acid separation device according to the present invention comprises a stirring mechanism for stirring and dissolving the sample and reagent injected into the processing tube;
A pressurized gas supply mechanism for supplying a pressurized gas to the filter cartridge into which the sample solution has been injected in order to separate and recover the nucleic acid from the sample solution containing the nucleic acid after the lysis treatment;
A dispensing mechanism for injecting the sample, reagent, or sample solution after dissolution treatment into the processing tube and the filter cartridge;
A moving mechanism for moving the pressurized gas supply mechanism and the dispensing mechanism;
With
The dispensing mechanism is
A suction and discharge mechanism capable of attaching and detaching a dispensing tip and capable of sucking and discharging a liquid through the dispensing tip;
A liquid supply mechanism for discharging liquid from the nozzle;
Is provided.

  The nucleic acid separation apparatus according to the present invention can automate a plurality of steps such as dispensing and pressurization, and can automate not only the nucleic acid extraction step but also a pretreatment step that has been conventionally performed manually.

FIG. 2 is a perspective schematic plan view showing a planar configuration when the top plate of the nucleic acid separation device according to Embodiment 1 is seen through. It is a see-through | perspective schematic front view which shows the structure at the time of seeing through the front panel of the nucleic acid separation apparatus of FIG. 1A. 1 is a block diagram showing a configuration of a nucleic acid separation device according to Embodiment 1. FIG. It is a top view which shows the whole structure of the dispensing chip | tip / reagent set part of the nucleic acid separation apparatus of FIG. FIG. 2 is a plan view illustrating a configuration in a reagent container tray of a dispensing chip / reagent setting unit of the nucleic acid separation device of FIG. 1. It is a top view which shows the structure of the sample setting part of the nucleic acid separation apparatus of FIG. It is a figure which shows the example of the sample container arrange | positioned in the sample setting part of FIG. 4A. It is a top view which shows the structure of the heating / stirring part of the nucleic acid separation apparatus of FIG. FIG. 5B is a front view of FIG. 5A. It is a perspective view which shows the structure of the process tube arrange | positioned at the heating / stirring part of FIG. 5A. It is a top view which shows a mode that a process tube is stirred in a heating / stirring part. It is a front view of the heating / stirring part which heats a process tube. It is a top view which shows the structure of the nucleic acid extraction part of the nucleic acid separation apparatus of FIG. It is a front view of FIG. 8A. It is a schematic perspective view which shows the structure of the filter cartridge arrange | positioned at the nucleic acid extraction part of FIG. 8A. It is a schematic perspective view which shows the structure of the collection tube arrange | positioned at the nucleic acid extraction part of FIG. 8A. It is a top view which shows a mode that a filter cartridge is moved on the collection tube for nucleic acid collection in order to isolate | separate a nucleic acid in a nucleic acid extraction part. It is a front view of FIG. 9A. It is a schematic perspective view which shows the structure of the dispensing pressurization unit of the nucleic acid separation apparatus of FIG. FIG. 10B is a partial view showing a liquid supply nozzle of the dispensing pressurization unit of FIG. 10A. FIG. 10B is a partial view showing a pressure head of the dispensing pressure unit of FIG. 10A. FIG. 10B is a partial view showing a suction / discharge nozzle of the dispensing pressurization unit of FIG. 10A. (A) is a top view of a dispensing pressure unit, (b) is a side view of the dispensing pressure unit, and (c) is a front view of the dispensing pressure unit. (A) is a top view which shows the liquid supply nozzle in a dispensing pressurization unit, (b) is a side view of a dispensing pressurization unit, (c) is a front view of a dispensing pressurization unit. (D) is a schematic diagram showing a tube pump and a reagent bottle connected to a liquid supply nozzle. (A) is a top view which shows the pressurization head in a dispensing pressure unit, (b) is a side view of a dispensing pressure unit, (c) is a front view of a dispensing pressure unit. (D) is the schematic which shows the piping connected to the pressurization head. 3 is a flowchart showing each step of the nucleic acid separation method according to Embodiment 1. (A) is a schematic perspective view which shows the reagent aspiration of reagent addition process S01 of FIG. 14, (b) is a schematic perspective view which shows dispensing of the reagent to the processing tube. (A) is a schematic perspective view which shows the blood suction of blood addition process S02 of FIG. 14, (b) is a schematic perspective view which shows dispensing to the processing tube of blood. (A) is a schematic perspective view which shows reagent aspiration of reagent addition process S03 of FIG. 14, (b) is a schematic perspective view which shows dispensing to the processing tube of a reagent. It is a schematic perspective view which shows a mode that the process tube is stirred in stirring process S04 of FIG. It is a schematic perspective view which shows a mode that a process tube is heated in heating process S05 of FIG. (A) is a schematic perspective view which shows reagent aspiration of reagent addition process S06 of FIG. 14, (b) is a schematic perspective view which shows dispensing to the processing tube of a reagent. It is a schematic perspective view which shows a mode that the process tube is stirred in stirring process S07 of FIG. (A) is a schematic perspective view which shows suction from the processing tube of the liquid transfer process S08 of FIG. 14, (b) is a schematic perspective view which shows dispensing to a filter cartridge. It is a schematic perspective view which shows a mode that the inside of a filter cartridge is pressurized in pressurization process S09 of FIG. FIG. 15 is a schematic perspective view showing how a cleaning reagent is dispensed into a filter cartridge in the reagent addition step S10 of FIG. It is a schematic perspective view which shows a mode that the inside of a filter cartridge is pressurized in pressurization process S11 of FIG. FIG. 15 is a schematic perspective view showing how a cleaning reagent is dispensed into a filter cartridge in the reagent addition step S12 of FIG. It is a schematic perspective view which shows a mode that the inside of a filter cartridge is pressurized in pressurization process S13 of FIG. FIG. 15 is a schematic perspective view showing a state in which a cleaning reagent is dispensed into the filter cartridge in the reagent addition step S14 of FIG. It is a schematic perspective view which shows a mode that the inside of a filter cartridge is pressurized in pressurization process S15 of FIG. (A) is a schematic perspective view which shows the reagent (collection liquid) suction | attraction of reagent addition process S16 of FIG. 14, (b) is a schematic perspective view which shows dispensing of the reagent (collection liquid) to the filter cartridge. is there. It is a schematic perspective view which shows a mode that the inside of a filter cartridge is pressurized in pressurization process S17 of FIG.

The nucleic acid separation device according to the first aspect includes a stirring mechanism that stirs and dissolves the sample and reagent injected into the processing tube;
A pressurized gas supply mechanism for supplying a pressurized gas to the filter cartridge into which the sample solution has been injected in order to separate and recover the nucleic acid from the sample solution containing the nucleic acid after the lysis treatment;
A dispensing mechanism for injecting the sample, reagent, or sample solution after dissolution treatment into the processing tube and the filter cartridge;
A moving mechanism for moving the pressurized gas supply mechanism and the dispensing mechanism;
With
The dispensing mechanism is
A suction and discharge mechanism capable of attaching and detaching a dispensing tip and capable of sucking and discharging a liquid through the dispensing tip;
A liquid supply mechanism for discharging liquid from the nozzle;
Is provided.

The nucleic acid separation device according to a second aspect is the nucleic acid separation apparatus according to the first aspect, wherein the suction and discharge mechanism sucks or discharges gas in the dispensing tip and sucks or discharges liquid in the dispensing tip. And
A first dispensing tip mounting portion having an O-ring having a first diameter at the tip and capable of attaching and detaching the first dispensing tip having the first diameter, and coaxially with the O-ring having the first diameter from the first diameter. A second dispensing tip mounting portion that has an O-ring having a large second diameter and that can be attached to and detached from the second dispensing tip having the second diameter.

  Hereinafter, a nucleic acid separation apparatus according to an embodiment will be described with reference to the accompanying drawings. In the drawings, substantially the same members are denoted by the same reference numerals.

(Embodiment 1)
FIG. 1A is a perspective schematic plan view showing a planar configuration when the top plate of the nucleic acid separation device 10 according to Embodiment 1 is seen through. FIG. 1B is a perspective schematic front view showing a configuration when the front panel of the automatic nucleic acid separation device 10 of FIG. 1A is seen through. FIG. 2 is a block diagram illustrating a configuration of the nucleic acid separation device 10 according to the first embodiment.
The nucleic acid separation device 10 includes a dispensing chip / reagent setting unit 11, a sample setting unit 12, a heating / stirring unit 13, a nucleic acid extraction unit 14, a dispensing pressurizing unit 15, a control unit 16, It has.
Further, as shown in FIGS. 1A, 1B, and 2, the nucleic acid separation apparatus 10 includes a heating / stirring unit 13 that includes a stirring mechanism 51 that stirs and dissolves the processing tube 53 into which the sample and the reagent are injected, In order to separate and recover the nucleic acid from the sample solution containing the nucleic acid after the lysis treatment, the pressurized gas supply mechanism 23 that supplies the pressurized gas to the filter cartridge 63 into which the sample solution has been injected, and the sample to the processing tube 53 and the filter cartridge 63 , Dispensing mechanisms 21 and 22 for injecting a reagent or sample solution after dissolution treatment, and a pressurized gas supply mechanism 23 and a moving mechanism 24 for moving the dispensing mechanisms 21 and 22. This dispensing mechanism has a dispensing tip 32 detachable, a suction and discharge mechanism 22 capable of sucking and discharging liquid via the dispensing tip 32, and a liquid supply mechanism 21 for directly discharging liquid from a nozzle. .
In addition, arrangement | positioning of FIG. 1A and FIG. 1B is an illustration, Comprising: Arrangement | positioning of each member is not limited to these. The dispensing tip / reagent setting unit 11, the sample setting unit 12, the heating / stirring unit 13, and the nucleic acid extraction unit 14 can be placed at any location within a range that can be moved by the dispensing pressurizing unit 15. You may arrange.

  The nucleic acid separation apparatus 10 includes dispensing mechanisms 21 and 22 that inject a sample solution and a reagent through a dispensing chip 32, and a pressurized gas supply mechanism 23 that supplies a pressurized gas. Thereby, a plurality of steps such as dispensing and pressurization can be automated, and not only the nucleic acid extraction step but also a pretreatment step which has been conventionally performed manually can be automated.

  Below, each structural member of this nucleic acid separation apparatus 10 is demonstrated.

<Dispensing tip / reagent set part>
FIG. 3A is a plan view showing the overall configuration of the dispensing tip / reagent setting unit 11 of the nucleic acid separation device 10 of FIG. FIG. 3B is a plan view for explaining the configuration in the reagent container tray 35 of the dispensing tip / reagent setting unit 11 of the nucleic acid separation device 10 of FIG.

  The dispensing tips 32a, 32b, 32c, and 32d are stored in the dispensing tip racks 31a, 31b, and 31c, respectively. The dispensing tips 32a, 32b, 32c, and 32d may have different capacities. For example, the dispensing tips 32a and 32c may be 1.2 ml, and the dispensing tips 32b and 32d may be 10 ml. In addition, a dispensing tip ejecting portion 33 is provided as a place where a dispensing tip that is no longer needed is removed from the dispensing pressure unit 15. The removed dispensing tip 32 is discarded in the dispensing tip disposal box 34. The dispensing tip disposal box 34 may be provided at the lower part of the apparatus, for example.

  Reagent containers 36 a, 36 b, 36 c, 36 d, and 36 e and a reagent waste container 37 are provided on a reagent container tray 35. Examples of the reagent include a pretreatment enzyme used in the pretreatment step, a lysis solution and ethanol, and a washing solution and a recovery solution used in the nucleic acid extraction step. For example, the pretreatment enzyme may be placed in the reagent container 36a, the solution may be placed in the reagent container 36b, ethanol may be placed in the reagent container 36c, and the recovered liquid may be placed in the reagent container 36d. In addition, what reagent should be put in which reagent container may be appropriately set in consideration of convenience. Since the cleaning liquid is used in a large amount, a reagent bottle 36f may be separately provided at the lower part of the apparatus as shown in FIG.

<Sample set part>
FIG. 4A is a plan view showing the configuration of the sample setting unit 12 of the nucleic acid separation device 10 of FIG. FIG. 4B is a diagram illustrating an example of sample containers 42a and 42b arranged in the sample setting unit 12 of FIG. 4A. The sample container 42 is, for example, a blood collection tube, and contains a sample from which a nucleic acid is extracted. The sample container 42 is stored in the sample racks 41a, 41b, 41c. As shown in FIG. 4A, a plurality of sample containers 42 are held in a row in the sample racks 41a, 41b, 41c. Here, eight sample containers 42 are held in each sample rack. In the example of FIG. 4A, a total of 24 sample containers 42 can be stored.

Sample rack presence / absence detection sensors 43a, 43b, and 43c for the sample racks 41a, 41b, and 41c are provided at the rear ends of the setting directions 44 of the sample racks 41a, 41b, and 41c, respectively. The sample rack presence / absence detection sensors 43a, 43b, and 43c detect whether the sample racks 41a, 41b, and 41c are set. In addition, each sample rack 41a, 41b, 41c may be provided with a shape that engages with the rear end of each corresponding lane. As a result, the sample racks 41a, 41b, and 41c are not engaged at the rear end in the non-corresponding lanes, so that it is possible to suppress mistakes that are inserted into the wrong lanes.
Further, as shown in FIG. 4B, sample containers 42a and 42b having various shapes can be used as the sample container 42.

<Sample>
In the diagnostic field, for example, in the diagnostic field, whole blood collected as a specimen, plasma, serum, urine, feces, semen, saliva and other body fluids, plants (or a part thereof), animals (or a part thereof) Or solutions prepared from biological materials such as lysates and homogenates thereof.

<Warming / stirring section>
FIG. 5A is a plan view showing the configuration of the heating / stirring unit 13 of the nucleic acid separation device 10 of FIG. FIG. 5B is a front view of FIG. 5A. FIG. 5C is a perspective view showing the structure of the processing tube 53 arranged in the heating / stirring unit 13 of FIG. 5A. FIG. 6 is a plan view showing how the processing tube 53 is stirred in the heating / stirring unit 13. FIG. 7 is a front view of the heating / stirring unit 13 for heating the processing tube.

  In the heating / stirring unit 13, the liquid in the processing tube 53 can be stirred and heated. The heating / stirring unit 13 is provided with a processing tube rack 52 for storing the processing tube 53 in the stirring unit 51. In the example of FIG. 5A, eight processing tubes 53 are arranged in one row, arranged in three rows, and have 24 processing tubes 53 as a whole. Each processing tube 53 corresponds to a sample in one sample container 42. That is, in each processing tube 53, the sample of the corresponding sample container 42 is dissolved.

  Further, as shown in FIG. 5C, the processing tube 53 has a thin neck portion 54, a thick trunk portion, and a conical bottom portion 55. By forming a “return” between the thin neck portion 54 and the thick body portion, it is possible to suppress scattering due to the rising of the liquid even when stirring with the upper surface opened. Further, by making the bottom part conical, the remaining liquid during suction by the dispensing tip 32 can be reduced.

  As shown in FIG. 6, at the time of stirring, the sample liquid in each processing tube 53 is stirred by rotating the entire processing tube rack 52 eccentrically by the stirring unit 51. The stirring unit 51 corresponds to a stirring mechanism. Agitation is, for example, 1500 rpm or less in terms of rotational speed, and the amplitude is, for example, from 1 mm to 10 mm. Moreover, stirring time is 240 seconds or less, for example.

  As shown in FIG. 7, at the time of heating, each processing tube 53 can be heated by the heater 58 below the processing tube rack 52. The heating is, for example, a temperature of 20 ° C. or more and 60 ° C. or less where the pretreatment enzyme and the lysis solution work well.

<Nucleic acid extraction part>
FIG. 8A is a plan view showing the configuration of the nucleic acid extraction unit 14 of the nucleic acid separation device 10 of FIG. FIG. 8B is a front view of FIG. 8A. FIG. 8C is a schematic perspective view showing the configuration of the filter cartridge 63 arranged in the nucleic acid extraction unit 14 of FIG. 8A. FIG. 8D is a schematic perspective view showing the configuration of the collection tube 66 arranged in the nucleic acid extraction unit 14 of FIG. 8A. FIG. 9A is a plan view showing how the filter cartridge 63 is moved onto the collection tube 66 for nucleic acid recovery in order to separate nucleic acids in the nucleic acid extraction unit 14. FIG. 9B is a front view of FIG. 9A.

  The nucleic acid extraction unit 14 includes filter cartridge racks 61a, 61b, and 61c that store the filter cartridge 63, and collection tube racks 62a, 62b, and 62c that store the collection tube 66. Eight filter cartridges 63 are stored in the filter cartridge racks 61a, 61b, and 61c, respectively. Eight collection tubes 66 are stored in each of the collection tube racks 62a, 62b, and 62c. In the example of FIG. 8A, 24 filter cartridges 63 and a collection tube 66 are stored. Further, the pair of filter cartridges 63 and the collection tube 66 correspond to one sample container 42 and one processing tube 53. That is, the sample solution containing the nucleic acid dissolved in the corresponding processing tube 53 is dispensed into the collection tube 66. Finally, for each collection tube 66, the nucleic acid contained in the sample in the corresponding one sample container 42 is collected.

  As shown in FIG. 8A, filter cartridge racks 61a, 61b, 61c and collection tube racks 62a, 62b, 62c are alternately arranged. The filter cartridge rack 61a and the collection tube rack 62a correspond to each other. Similarly, the filter cartridge rack 61b and the collection tube rack 62b correspond to each other, and the filter cartridge rack 61c and the collection tube rack 62c correspond to each other.

  The filter cartridge racks 61a, 61b, 61c may be provided with shapes that engage with each other at the rear end portion for each corresponding lane. As a result, the filter cartridge racks 61a, 61b, and 61c do not engage at the rear ends of the non-corresponding lanes, so that it is possible to suppress mistakes in inserting into the wrong lanes. Similarly, the collection tube racks 62a, 62b, and 62c may be provided with shapes that engage with each other at the rear end for each corresponding lane. As a result, the collection tube racks 62a, 62b, and 62c do not engage at the rear end in the non-corresponding lanes, so that it is possible to suppress mistakes in inserting into the wrong lanes.

  Further, as shown in FIG. 8B, a waist tube 65 is connected below the filter cartridge 63 in the filter cartridge racks 61a, 61b, 61c. On the other hand, in the collection tube racks 62a, 62b, and 62c, only the collection tube 66 is set until the nucleic acid extraction step is performed.

<Filter>
As shown in FIG. 8C, the filter cartridge 63 has a filter 64. The filter 64 (nucleic acid-adsorbing porous membrane) is a porous membrane that adsorbs nucleic acids by an interaction that does not involve ionic bonds. The filter 64 is configured to retain adsorption with a nucleic acid when washed with a washing liquid and weaken the adsorption force of the nucleic acid when collected with a collecting liquid. More preferably, the filter 64 is a porous film having a hydroxyl group as a hydrophilic group, and the material forming the porous film itself treats or coats the porous film having a hydroxyl group or the material forming the porous film. This means a porous membrane into which a hydroxyl group has been introduced. The material for forming the porous film may be either an organic material or an inorganic material, but it is preferable to use an organic material such as an organic polymer in terms of ease of processing. The filter 64 is, for example, nylon, polysulfone, polyethersulfone, polycarbonate, polyacrylate, acrylate copolymer, polyurethane, polyamide, polyvinyl chloride, polyfluorocarbonate, polytetrafluoroethylene, polyvinylidene difluoride, polyethylene tetrafluoro. Ethylene copolymer salt, polybenzimidazole, polyethylene chlorotrifluoroethylene copolymer salt, polyimide, polyphenylene sulfide, cellulose, cellulose mixed ester, nitrocellulose, acetylcellulose, polyacrylonitrile, polyacrylonitrile copolymer, nitrocellulose, polypropylene And / or a porous membrane containing polyester.

  Examples of the porous film made of an organic material having a hydroxyl group include surface saponified products of acetyl cellulose described in JP-A No. 2003-128691. As acetyl cellulose, any of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose may be used, but triacetyl cellulose is particularly preferable. In this case, the amount (density) of hydroxyl groups on the solid surface can be controlled by the degree of saponification treatment (saponification degree). In order to increase the nucleic acid separation efficiency, it is preferable that the amount (density) of hydroxyl groups is large. For example, in the case of acetylcellulose such as triacetylcellulose, the saponification rate is preferably about 5% or more, and more preferably 10% or more. In order to increase the surface area, the porous membrane of acetylcellulose is saponified. The amount (density) of the spatial hydroxyl group can be controlled by a combination of the degree of saponification treatment (saponification degree) and the pore diameter of the porous membrane. In this case, the porous film may be a front and back symmetric porous film, but a front and back asymmetric porous film can be preferably used.

  Other examples of porous membranes of organic materials having hydroxyl groups include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid, polyoxyethylene, acetylcellulose, acetyl Examples thereof include a porous film formed of a mixture of acetylcelluloses having different values, but a porous film of an organic material having a polysaccharide structure can be preferably used. In particular, a porous membrane of an organic polymer composed of a mixture of acetyl celluloses having different acetyl values can be preferably used. A mixture of triacetyl cellulose and diacetyl cellulose, a mixture of triacetyl cellulose and monoacetyl cellulose, and triacetyl cellulose. A mixture of diacetyl cellulose and monoacetyl cellulose and a mixture of diacetyl cellulose and monoacetyl cellulose can be preferably used.

  Examples of the filter 64 include a porous membrane made of an organic material obtained by saponifying a mixture of acetylcelluloses having different acetyl values. For example, a saponified product of each acetylcellulose mixture described above is preferably used. it can. In the saponification treatment, acetylcellulose is brought into contact with a saponification treatment solution (for example, sodium hydroxide). Regenerated cellulose is introduced into the portion of acetylcellulose in contact with the saponification treatment solution, and a hydroxyl group is introduced. In order to increase the nucleic acid separation efficiency, it is preferable that the number of introduced hydroxyl groups is larger. For example, the saponification rate is preferably about 5% or more, more preferably about 10% or more.

  As the filter 64, a porous membrane of regenerated cellulose can be preferably used. Regenerated cellulose is obtained by saponifying the solid surface or the whole of acetyl cellulose by saponification treatment, and is different from the original cellulose in terms of crystal state and the like.

  As another filter 64, a porous film containing a silica compound as a porous film which is an inorganic material having a hydrophilic group, for example, a glass filter can be exemplified. Further, a porous silica thin film as described in Japanese Patent Publication No. 3058342 can be given. This porous silica thin film is an amphiphilic substance by spreading a developing solution of a cationic type amphiphilic substance having a bilayer-forming ability on a substrate and then removing the solvent from the liquid film on the substrate. The multilayer bilayer thin film can be prepared by bringing it into contact with a solution containing a silica compound, and then extracting and removing the multilayer bilayer thin film.

  As the filter 64, a porous film having a thickness of 10 μm or more and 500 μm or less, preferably 50 μm or more and 250 μm or less can be used. In addition, a porous film having a minimum pore diameter of 0.22 μm or more, more preferably 0.5 μm or more can be used. A porous membrane having a ratio of the maximum pore diameter to the minimum pore diameter of 2 or more, more preferably 5 or more can be used. A porous film having a porosity of 50% to 95%, more preferably 65% to 80% can be used.

  Further, the number of filters 64 used may be one, but a plurality of filters may be used. The plurality of filters 64 may be the same or different. The filter 64 may be a combination of an inorganic material filter and an organic material filter. For example, a combination of a glass filter and a porous membrane of regenerated cellulose can be mentioned. The plurality of filters may be a combination of an inorganic material filter and an organic material non-nucleic acid-adsorbing porous membrane, for example, a combination of a glass filter and a nylon or polysulfone porous membrane. be able to.

<Collection tube>
As shown in FIG. 8D, the collection tube 66 may include a lid that can close the upper surface. The collection tube 66 collects the nucleic acid together with the collection solution.

<About the movement of the filter cartridge>
As shown in FIG. 9B, it is necessary to move the filter cartridge 63 from the filter cartridge racks 61a, 61b, 61c to the collection tube racks 62a, 62b, 62c before collecting the nucleic acid.
For example, the filter cartridge 63 is moved in the following three stages.
1) First, the filter cartridge 63 is moved up by the filter cartridge racks 61a, 61b, 61c.
2) Next, the filter cartridge 63 is moved laterally from the filter cartridge racks 61a, 61b, 61c to the collection tube racks 62a, 62b, 62c.
3) Next, the filter cartridge 63 is moved downward from above the collection tube racks 62a, 62b, 62c.
As described above, the filter cartridge 63 can be moved from the filter cartridge racks 61a, 61b, 61c to the collection tube racks 62a, 62b, 62c. The filter cartridge 63 side may be fixed, and the collection tube 66 and waist tube 65 side may be moved.

<Dispensing pressure unit>
FIG. 10A is a schematic perspective view showing the configuration of the dispensing pressurization unit 15 of the nucleic acid separation device 10 of FIG. FIG. 10B is a partial view showing the liquid supply nozzles 21a and 21b of the dispensing pressurization unit 15 of FIG. 10A. FIG. 10C is a partial view showing the pressure heads 23a and 23b of the dispensing pressure unit 15 of FIG. 10A. FIG. 10D is a partial view showing the suction / discharge nozzles 22a and 22b of the dispensing pressurization unit 15 of FIG. 10A. 11A is a plan view of the dispensing pressurizing unit 15, FIG. 11B is a side view of the dispensing pressurizing unit 15, and FIG. 11C is a dispensing. 2 is a front view of a pressure unit 15. FIG.

  The dispensing and pressurizing unit 15 is configured to supply a pressurized gas into the dispensing mechanism 21a, 21b, 22a, and 22b that supplies the liquid and the container 63 with respect to the container (processing tube 53, filter cartridge 63) whose vertical upper surface is opened. The pressurized gas supply mechanisms 23a and 23b to be supplied and the moving mechanism 24 for moving the whole vertically above the containers 53 and 63 are provided. The dispensing mechanism also includes suction and discharge mechanisms 22a and 22b and liquid supply mechanisms 21a and 21b that discharge liquid directly from the nozzles. The suction and discharge mechanisms 22a and 22b are detachable from the dispensing tips 32a and 32b. The suction and discharge mechanisms 22a and 22b discharge the liquid such as the sample liquid and the reagent to the containers 53 and 63 through the dispensing tips 32a and 32b, and the liquid from the container 53 is sucked into the dispensing tips 32a and 32b. In addition, liquid can be supplied to the containers 53 and 63 by the liquid supply mechanisms 21a and 21b.

  The dispensing pressure unit 15 may be provided with a workpiece detection sensor 28. The work detection sensor 28 detects whether or not a predetermined number of sample containers 42, dispensing tips 32, reagent containers 36, processing tubes 53, filter cartridges 63, collection tubes 66, and the like are set at predetermined positions. As the work detection sensor 28, a photoelectric sensor or the like combining a CMOS sensor and a laser light source can be used. The work detection sensor 28 can be moved to the vicinity of the upper part of each work to be detected by the moving mechanism 24 to detect the presence or absence of each work. Each workpiece is preferably checked by the workpiece detection sensor before the start of the pretreatment process.

The dispensing pressurizing unit 15 includes two sets of dispensing mechanisms 21a, 21b, 22a, 22b and pressurized gas supply mechanisms 23a, 23b, but is not limited to two sets and may be one set. . Alternatively, three or more sets may be included. Moreover, the dispensing mechanisms 21a and 22a and the pressurized gas supply mechanism 23a, and the dispensing mechanisms 21b and 22b and the pressurized gas supply mechanism 23b may be separately movable in the z-axis direction. Thus, since each dispensing mechanism etc. can move independently in the z-axis direction, individual dispensing operations are possible. For example, if the dispensing mechanism has two sets and the sample containers 42 to be processed are an odd number, first, two sets are dispensed, and the last remaining one of the sample containers is one It is possible to operate by operating only the dispensing mechanism. When the dispensing mechanism is not independent, all the dispensing mechanisms move in the same manner, so that complicated processing is required for mounting dispensing tips.
Moreover, as shown to FIG. 10A, you may provide the saucer 25 in a dispensing pressurization unit. The tray 25 receives a liquid such as a sample or a reagent dropped from the tip of the dispensing tip 32 when the dispensing pressure unit 15 is moved. The tray 25 is movable in the x-axis direction, and is stored in a position that does not interfere with the dispensing operation during the dispensing operation by the dispensing mechanism 22 (see FIG. 11C).
Moreover, each said member which comprises the dispensing pressurization unit 15 does not comprise one unit, and may be provided separately.

<Suction and discharge mechanism>
The suction and discharge mechanisms (pipettes) 22a and 22b do not dispense the cleaning liquid etc. directly through the pipe connected to the bottle having the cleaning liquid etc., but inject the liquid through the dispensing tip 32 attached to the tip. It is characterized by doing. Conventionally, there has been a problem that it is necessary to prepare a large number of dispensing mechanisms for each type of liquid such as cleaning liquid and recovery liquid. Further, there is a method in which one discharge nozzle is provided at the tip and the liquid to be discharged is switched by a switching valve or the like. However, in this case, in order to avoid mixing at the time of switching, it is necessary to perform cleaning with the liquid after switching, and wasteful liquid is discarded. On the other hand, according to the suction and discharge mechanisms 22a and 22b, the dispensing tips 32 can be replaced for each liquid to be dispensed. That is, various liquids can be dispensed substantially by a pair of suction and discharge mechanisms 22a and 22b. Further, the gas in the dispensing tip 32 can be sucked or discharged, and the liquid can be sucked or discharged into the dispensing tip 32.

  Further, as shown in FIG. 10D, the suction and discharge mechanisms 22a and 22b have, for example, an O-ring having a first diameter at the tip, and a first dispensing tip in which a first dispensing tip 32a having a first diameter can be attached and detached. A second dispensing tip mounting portion 27a having a tip mounting portion 26a, 26b and an O-ring having a second diameter larger than the first diameter on the inner side of the tip, and the second dispensing tip 32b having the second diameter being removable; 27b. Thus, two types of dispensing tips 32a and 32b having different capacities can be mounted by one suction and discharge mechanism 22a and 22b. Thereby, an appropriate dispensing tip can be properly used according to the dispensing amount.

<Liquid supply mechanism>
12A is a plan view showing liquid supply nozzles 21a and 21b that discharge liquid in the dispensing pressurizing unit 15, and FIG. 12B is a side view of the dispensing pressurizing unit 15. FIG. FIG. 12C is a front view of the dispensing pressurizing unit 15, and FIG. 12D shows the tube pump 71 and the reagent bottle 36f connected to the liquid supply nozzles (liquid supply mechanisms) 21a and 21b. FIG. The liquid supply nozzles 21a and 21b supply a reagent (for example, a cleaning liquid) from the reagent bottle 36f by the tube pump 71. A single supply amount by the liquid supply nozzles 21a and 21b is, for example, 12 ml or less and may be supplied in units of 0.25 ml.

<Pressurizing head (pressurized gas supply mechanism)>
13A is a plan view showing the pressure heads 23a and 23b in the dispensing pressurizing unit 15, and FIG. 13B is a side view of the dispensing pressurizing unit 15. FIG. (C) is a front view of the dispensing pressurizing unit 15, and (d) of FIG. 13 is a schematic diagram showing piping connected to the pressurizing heads 23a and 23b.
The pressurizing heads 23a and 23b are connected to air filters 81a and 81b provided in the ventilation ports via pulse motors 82a and 82b, pressurizing pumps 83a and 83b, and pressure sensors 86a and 86b, respectively. The pressure setting range is, for example, 30 kPa or more and 160 kPa or less. For example, air or nitrogen can be used as the pressurized gas.
The pressure heads 23a and 23b are arranged so as not to interfere with the operations of the suction and discharge mechanisms 22a and 22b and the liquid supply nozzles 21a and 21b.

<Movement mechanism>
The moving mechanism 24 can move the entire dispensing pressure unit 15 vertically above the containers 53 and 63. For example, as shown in FIGS. 1A and 1B, an X-axis actuator 24a, a Y-axis actuator 24b, and a Z-axis actuator 24c can be used as the moving mechanism 24.

<Control unit>
By the control unit 16, a dispensing tip / reagent setting unit 11, a sample setting unit 12, a heating / stirring unit 13, a nucleic acid extraction unit 14, and a dispensing pressurizing unit 15 that constitute the nucleic acid separation device 10 are arranged. Control. The control unit 16 may be a computer, for example. The computer only needs to include physical components such as the CPU 1, the memory 2, the storage unit 3, the display unit 4, the input unit 5, and the interface 6.

<Nucleic acid separation method>
FIG. 14 is a flowchart showing each step of the nucleic acid separation method according to the first embodiment. In FIG. 14, the steps from the reagent addition step (S01) to the stirring step (S07) are pretreatment (dissolution treatment) steps, and the steps from the liquid transfer step (S08) to the pressurization step (S17) are nucleic acid extraction (separation and recovery) steps. It is.
(1) The reagent (pretreatment enzyme) is aspirated from the reagent container 36 to the dispensing tip 32 (FIG. 15 (a)) and dispensed into the processing tube 53 (FIG. 15 (b)) (reagent addition step S01).
(2) The sample (whole blood) is sucked into the dispensing tip 32 from the sample container (blood collection tube, 42) (FIG. 16 (a)) and dispensed into the processing tube 53 (FIG. 16 (b)) (sample addition) Step S02).
(3) The reagent (dissolved solution) is aspirated from the reagent container 36 to the dispensing tip 32 (FIG. 17A) and dispensed to the processing tube 53 (FIG. 17B) (reagent addition step S03).
(4) The processing tube 53 is stirred (FIG. 18) (stirring step S04). The processing tube 53 is agitated at a motor speed of 1500 rpm or less. The amplitude is 1 mm or more and 10 mm or less.
(5) The processing tube 53 is heated (FIG. 19) (heating step S05). Heating is performed in a temperature range of 20 ° C. or more and 60 ° C. or less, for example.

The “lysis treatment” is a treatment with an aqueous solution containing a reagent (eg, a solution containing a chaotropic salt or a guanidine salt, a surfactant and a proteolytic enzyme) that dissolves a cell membrane and a nuclear membrane to solubilize a nucleic acid. When the target sample is whole blood, in order to prevent non-specific adsorption and clogging to the filter 64, the red blood cells and various proteins are decomposed and reduced in molecular weight, and the white blood cells and nuclei are used to solubilize the nucleic acid to be extracted. Dissolve the membrane. Examples of the “water-soluble organic solvent” include ethanol, isopropanol, propanol and the like, and ethanol is particularly preferable. The concentration of the water-soluble organic solvent is preferably 5% by weight or more and 90% by weight or less, and more preferably 20% by weight or more and 60% by weight or less. The concentration of ethanol added is particularly preferably as high as possible without causing aggregates.
By dissolving the whole blood or a specimen containing cells or viruses, a water-soluble organic solvent is added to a solution in which nucleic acids are dispersed in the liquid.
(6) The reagent (ethanol) is aspirated from the reagent container 36 to the dispensing tip 32 (FIG. 20 (a)) and dispensed into the processing tube 53 (FIG. 20 (b)) (reagent addition step S06).
(7) The processing tube 53 is stirred (FIG. 21) (stirring step S07). The processing tube 53 is agitated at a motor speed of 1500 rpm or less. The amplitude is 1 mm or more and 10 mm or less.

(8) The sample solution containing the nucleic acid dissolved from the processing tube 53 is sucked into the dispensing tip 32 (FIG. 22A) and dispensed into the filter cartridge 63 (FIG. 22B) (transfer process) S08). As a result, the sample solution containing the nucleic acid subjected to the lysis treatment can be injected into the filter cartridge 63.
(9) The filter cartridge 63 is filtered under pressure (FIG. 23) (pressurizing step S09). The pressure filtration is performed by introducing pressurized air into the filter cartridge 63 and pressurizing. In this case, the inside of the filter cartridge 63 is kept sealed. As a result, the sample solution is passed through the filter 64 and the nucleic acid is adsorbed by the filter 64. The liquid portion that has passed through the filter 64 is discharged through a waist tube 65 disposed below the filter cartridge 63. When all the sample liquid passes through the filter 64, the pressure drops below the liquid discharge completion pressure, and the end of extraction is detected in each filter cartridge 63 by each pressure sensor 86a, 86b. Thereafter, for example, the pressure heads 23a and 23b are moved upward to release the sealed state, and the pressure process is terminated.
(10) The reagent (cleaning liquid) is dispensed into the filter cartridge 63 from the liquid supply nozzles 21a and 21b (FIG. 24) (reagent addition step S10).

<Cleaning liquid>
The “washing liquid” has a function of washing out impurities in the sample liquid adhering to the filter 64 together with the nucleic acid, and has a composition for removing the impurities without removing the nucleic acid. The “cleaning liquid” includes, for example, an aqueous solution containing a main agent, a buffering agent, and, if necessary, a surfactant. The main agent is methanol, ethanol, isopropanol, n-isopropanol, butanol, acetone or the like. Further, the main agent is about 10% by weight or more and 100% by weight or less, preferably 20% by weight or more and 100% by weight or less, more preferably 40% by weight or more and 80% by weight or less in the aqueous solution.

(11) The filter cartridge 63 is filtered under pressure (FIG. 25) (pressurizing step S11). The pressure filtration is performed by introducing pressurized air into the filter cartridge 63 and pressurizing. As a result, other impurities are washed away while the nucleic acid is retained in the filter 64. The liquid part such as the cleaning liquid that has passed through the filter 64 is discharged through a waist tube 65 connected to the filter cartridge 63.
(12) The reagent (cleaning liquid) is dispensed into the filter cartridge 63 from the liquid supply nozzles 21a and 21b (FIG. 26) (reagent addition step S12).
(13) The filter cartridge 63 is filtered under pressure (FIG. 27) (pressurizing step S13).
(14) Reagent (cleaning liquid) is dispensed into the filter cartridge 63 from the liquid supply nozzles 21a and 21b (FIG. 28) (reagent addition step S14).
(15) The filter cartridge 63 is filtered under pressure (FIG. 29) (pressurizing step S15). The combination of the reagent addition step S10, the pressurization step S11, the reagent addition step S12, the pressurization step S13, the reagent addition step S14, and the pressurization step S15 is repeated three times here. In addition, it is not restricted to said example, What is necessary is just to perform at least 1 set of reagent addition process and pressurization process.

(16) The filter cartridge 63 is moved from the filter cartridge racks 61a, 61b, 61c to the collection tube racks 62a, 62b, 62c (FIGS. 9A and 9B). For example, as indicated by an arrow 67 in FIG. 9B, the filter cartridge 63 is moved upward. Next, as indicated by an arrow 68, the filter cartridge 63 is moved laterally onto the collection tube 66. Next, the filter cartridge 63 is moved downward as indicated by an arrow 69, and the filter cartridge 63 is disposed on the collection tube 66. The filter cartridges 63 are sequentially moved in the same manner, and the filter cartridges 63 are arranged on the collection tubes 66, respectively.
(17) The reagent (recovered solution) is aspirated from the reagent container 36 to the dispensing tip 32 (FIG. 30 (a)) and dispensed to the filter cartridge 63 (FIG. 30 (b)) (reagent addition step S16).

<Recovered liquid>
The “recovered solution” preferably has a low salt concentration, and in particular, a solution having a salt concentration of 0.5 M or less, such as purified distilled water, TE buffer, or the like is used. The pH of the recovered liquid is preferably pH 2 or more and pH 11 or less, more preferably pH 5 or more and pH 9 or less.

(18) The filter cartridge 63 is filtered under pressure (FIG. 31) (pressurizing step S17). The pressure filtration is performed by introducing pressurized air into the filter cartridge 63 and pressurizing. The binding force between the filter 64 and the nucleic acid is weakened by the recovery liquid, the nucleic acid adsorbed on the filter 64 is released, and the nucleic acid can be recovered in the collection tube 66 together with the recovery liquid. Thereafter, the collection tube 66 may be capped as necessary.
As described above, the recovery solution containing the nucleic acid can be recovered. The nucleic acid in the collection tube 66 is subjected to the following nucleic acid analysis process.

  According to the nucleic acid separation device 10 and the nucleic acid separation method, the plurality of sample containers 42 are sequentially performed sequentially from the pretreatment process to the nucleic acid extraction process for each sample container. Therefore, it is not necessary to perform a conventional manual pretreatment process.

  In this embodiment, a plurality of sets of sample containers 42, processing tubes 53, and filter cartridges 63 are provided, but the present invention is not limited to this. For example, the nucleic acid separation apparatus 10 according to the first embodiment can be applied to a set of sample containers 42, processing tubes 53, and filter cartridges 63.

  Further, the nucleic acid separation apparatus 10 may include a UV irradiation unit (not shown) that sterilizes the apparatus by irradiating ultraviolet rays. The UV irradiation unit may be configured to irradiate ultraviolet rays while moving in the apparatus, for example, in the xy direction. Thereby, the inside of the apparatus can be kept clean.

  It should be noted that the present disclosure includes appropriately combining any of the various embodiments and / or examples described above, and each of the embodiments and / or examples. The effect which an Example has can be show | played.

  According to the nucleic acid separation device of the present invention, a dispensing mechanism for dispensing a sample, a reagent, or a sample solution after dissolution treatment via a dispensing chip, and a pressurized gas supply mechanism for supplying pressurized gas, Prepare. Thereby, a plurality of steps such as dispensing and pressurization can be automated, and not only the nucleic acid extraction step but also a pretreatment step which has been conventionally performed manually can be automated.

DESCRIPTION OF SYMBOLS 10 Nucleic acid separator 11 Dispensing chip / reagent setting part 12 Sample setting part 13 Heating / stirring part 14 Nucleic acid extraction part 15 Dispensing pressurization unit 16 Control part 21, 21a, 21b Reagent addition nozzle (liquid supply mechanism)
22, 22a, 22b Suction / discharge nozzle (suction and discharge mechanism)
23, 23a, 23b Pressurizing head (pressurized gas supply mechanism)
24, 24a, 24b, 24c Moving mechanism 25 Trays 26a, 26b First dispensing tip mounting portion 27a, 27b Second dispensing tip mounting portion 28 Work detection sensor 31a, 31b, 31c Dispensing tip rack 32, 32a, 32b, 32c, 32d Dispensing tip 33 Dispensing tip ejector 34 Dispensing tip disposal box 35 Reagent container trays 36, 36a, 36b, 36c, 36d, 36e, 36f Reagent containers 41a, 41b, 41c Sample racks 42, 42a, 42b Sample container (blood collector)
43a, 43b, 43c Sample rack presence / absence detection sensor 44 Sample rack setting direction 51 Stirring mechanism 52 Processing tube rack 53 Processing tube (lysate tube)
54 Neck 55 Bottom 56 Rotation direction 58 Heater 61a, 61b, 61c Filter cartridge rack 62a, 62b, 62c Collection tube rack 63 Filter cartridge 64 Filter 65 West tube 66 Collection tube 67 Upward movement 68 Lateral movement 69 Downward movement 71 Tube pump 72 Piping 81a, 81b Air filters 82a, 82b Pulse motors 83a, 83b Pressure pumps 86a, 86b Pressure sensors

Claims (2)

A stirring mechanism for stirring and dissolving the sample and reagent injected into the processing tube;
A pressurized gas supply mechanism for supplying a pressurized gas to the filter cartridge into which the sample solution has been injected in order to separate and recover the nucleic acid from the sample solution containing the nucleic acid after the lysis treatment;
A dispensing mechanism for injecting the sample, reagent, or sample solution after dissolution treatment into the processing tube and the filter cartridge;
A moving mechanism for moving the pressurized gas supply mechanism and the dispensing mechanism;
With
The dispensing mechanism is
A suction and discharge mechanism capable of attaching and detaching a dispensing tip and capable of sucking and discharging a liquid through the dispensing tip;
A liquid supply mechanism for discharging liquid from the nozzle;
A nucleic acid separation apparatus comprising: The suction and discharge mechanism sucks or discharges gas in the dispensing tip, and sucks or discharges liquid in the dispensing tip,
A first dispensing tip mounting portion having an O-ring having a first diameter at the tip and capable of attaching and detaching the first dispensing tip having the first diameter, and coaxially with the O-ring having the first diameter from the first diameter. 2. The nucleic acid separation device according to claim 1, further comprising: a second dispensing tip mounting portion having an O-ring having a large second diameter and capable of attaching and detaching the second dispensing tip having the second diameter.

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