JP4573563B2 - Nucleic acid extraction equipment - Google Patents

Description

  The present invention provides a device for quickly and easily extracting a nucleic acid from a sample containing nucleic acid and attaching the extracted nucleic acid to a carrier, and without requiring an operation for separating the nucleic acid attached to the carrier from the carrier. The present invention relates to an apparatus that can be accommodated in a container for performing nucleic acid analysis (for example, nucleic acid amplification).

  Nucleic acid extraction is an important and indispensable operational step in analyzing the target nucleic acid in the field of handling genes such as clinical, bacterial or food. The technique for extracting nucleic acid from a sample containing nucleic acid has been known for a long time, such as phenol / chloroform extraction method, alkali dissolution method or guanidine method. However, a sample containing nucleic acid and a reagent are contained in a container, and the container Is then centrifuged using a centrifuge and the supernatant is removed using a pipette, filter paper, or the like to extract nucleic acid (see Patent Document 1). Here, the sample containing nucleic acid is, for example, endogenous nucleic acid such as whole blood, serum or plasma, urine, feces, tissue, saliva or semen, foreign nucleic acid such as microorganisms including parasites, bacteria or viruses, Samples containing nucleic acids contained in cell cultures, foods or drinking water contaminated with microorganisms, genetically modified plants, and the like are included.

  However, such a conventional nucleic acid extraction technique requires a complicated operation by an operator and takes a long time for extraction, requires a special device such as a centrifuge, and requires a large space for nucleic acid extraction. Has the problem.

  In recent years, methods for recovering nucleic acids have been constantly sought, and in order to use the extracted nucleic acid for nucleic acid amplification, for example, 1) First, impurities in the sample containing the nucleic acid are removed by a desired method, and 2) the nucleic acid is solid-phased. 3) The nucleic acid is extracted and purified by means such as attaching and immobilizing the nucleic acid on a solid phase, in which the nucleic acid is recovered from the carrier in a liquid phase. Technology has been proposed.

  However, such a technique has a problem that the operation of the operator is complicated and the operation time is long because the process of removing the nucleic acid from the carrier is included.

  In view of such problems of the prior art, nucleic acid can be extracted quickly without using a centrifuge that is not complicated and time-consuming, and a specific substance in a sample can be analyzed quickly. It has been desired to provide a device that can be used.

  In addition, a technology that allows the sample that can be attached to the sample to be moved to multiple processing steps in the container in order to quickly extract a specific substance from the sample, and to analyze the specific substance in the sample quickly. Has been developed. According to these apparatuses, it is possible to perform analysis work of a specific substance regardless of the place, and it is expected to be used as a POC (Point of Care) apparatus (see Patent Document 2 and Patent Document 3).

JP-A-2-283279 Japanese translation of PCT publication No. 2003-501673 JP-T-2001-524681

  However, in the techniques disclosed in Patent Document 2 and Patent Document 3, when the carrier to which the sample is attached is taken out from the inside of the apparatus body, the carrier to which the sample is attached is taken out to the outside of the apparatus body in an exposed state. Depending on the type of sample, there is a problem that it is necessary to pay particular attention to the handling of the carrier.

  Therefore, the present invention enables a carrier with a nucleic acid attached to be accommodated in a container within the apparatus main body without separating the nucleic acid from the carrier to which the nucleic acid is attached, and a complicated operation or large size. Alternatively, an object of the present invention is to provide a nucleic acid extraction apparatus capable of extracting and purifying nucleic acid from a sample containing nucleic acid easily and quickly without requiring a special apparatus.

The nucleic acid extraction apparatus of the present invention includes (1) a sample supply unit that supplies a sample containing nucleic acid to a carrier for holding nucleic acid, and a first processing unit that performs insolubilization processing on nucleic acid contained in the sample attached to the carrier And a second processing unit for removing components other than the nucleic acid attached to the carrier, and a container mounting part for detachably supporting the container in which the carrier is accommodated at a carrier accommodation position. And an internal space that has a first guide surface and communicates with each of the opening of the container supported by the sample supply unit, the first processing unit, the second processing unit, and the container mounting unit. (2) the carrier is detachably supported, and the carrier is supported by the container mounting portion from the sample supply section in the internal space of the body. Move along the first guide surface And (3) carrier separating means for separating the carrier moved to the opening of the container by the carrier moving means and separating the carrier from the carrier moving means and storing the carrier in the container. It is equipped with. In the present invention, the container mounting portion is movably supported on the main body so that the container detachably supported by the container mounting portion can be moved from the carrier receiving position to the carrier taking-out position. A second guide surface is formed. Moreover, the lid | cover which can be opened and closed is attached to the said opening part of the said container.
And the said container supported by the said container attaching part so that attachment or detachment was possible,
After the opening is set at the carrier receiving position in a state of being closed by the lid, the lid is opened at the opening of the carrier moving means that has moved in the internal space, and the opening is After communicating with the internal space, containing the carrier separated from the carrier moving means by the carrier separating means;
When the container mounting portion is moved along the second guide surface, the lid in the opened state is moved by the closing means of the main body when the container mounting portion is moved from the carrier receiving position to the carrier taking-out position. It is designed to be closed.

  The nucleic acid extraction apparatus of the present invention is characterized in that a reagent for nucleic acid amplification is stored in the container in advance.

  In the nucleic acid extraction apparatus of the present invention, the nucleic acid amplification reagent is a nucleic acid amplification reagent used in the LAMP method.

  According to the present invention, since the carrier holding the nucleic acid itself is accommodated in the container in the main body, the carrier to which the nucleic acid is attached is not taken out of the main body in an exposed state, and the carrier is handled. Becomes easier. Moreover, according to the present invention, since the carrier to which the nucleic acid is attached is accommodated in the container, nucleic acid amplification or the like can be performed in the container, and the nucleic acid held on the carrier is separated from the carrier. There is no need. As a result, according to the present invention, a complicated operation and a large-sized or special apparatus for separating the nucleic acid from the carrier are not required, and the nucleic acid can be easily and quickly extracted and purified from the sample containing the nucleic acid. .

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  1 to 3 are diagrams showing a nucleic acid extraction apparatus 1 according to an embodiment of the present invention. Among these, FIG. 1 is a front view of the nucleic acid extraction apparatus 1, and is a front view of the nucleic acid extraction apparatus 1 in which a sample supply container, a processing agent supply container, and a cleaning agent supply container, which will be described later, are omitted. FIG. 2 is a plan view of the nucleic acid extraction apparatus 1, in which a sample supply container, a processing agent supply container, and a cleaning agent supply container, which will be described later, are omitted. FIG. 3 is a cross-sectional view of the nucleic acid extraction apparatus 1 cut along the line AA in FIG.

[Outline of nucleic acid extraction apparatus]
As shown in these drawings, in the nucleic acid extraction apparatus 1 according to the present embodiment, the first slider (carrier moving means) 4 is movably engaged in the internal space 3 of the main body 2 made of resin material. It has become. Here, as shown in detail in FIGS. 4 to 6, the first slider 4 is attached to the front end side of the first slider 4 so that a substantially disc-shaped carrier 5 (made of filter paper, membrane filter or nonwoven fabric) can be attached and detached. ing. Moreover, the 2nd slider (container attaching part) 6 is attached to the resin-made main body 2 so that sliding is possible. Here, the second slider 6 detachably supports the container 8 in which the carrier 5 separated from the first slider 4 by the pressing rod (carrier separating means) 7 is accommodated.

[Main body of nucleic acid extraction device]
In the main body 2, the sample supply unit 10, the first processing unit 11, the second processing unit 12, the third processing unit 13, and the carrier separation unit 14 are arranged in order from the left side in FIG. Among these, first, the sample supply unit 10 supplies a sample containing a nucleic acid to the carrier 5 on the tip side of the first slider 4. Next, the first processing unit 11 drops the nucleic acid coprecipitation agent and the insolubilizing agent on the carrier 5 on the distal end side of the first slider 4. Here, the nucleic acid coprecipitation agent generates a large insolubilized product by aggregating the insolubilized nucleic acid, and is supported on the surface of the carrier. It serves to prevent nucleic acids present in the material from passing through the filter paper. Next, the second processing unit 12 drops the cleaning liquid onto the carrier 5 on the tip side of the first slider 4. Next, the third processing unit 13 removes excess liquid components of the sample, nucleic acid coprecipitation agent, insolubilizing agent, and washing liquid supplied or dropped onto the carrier 5 on the tip side of the first slider 4 by the absorber 15 (filter paper, membrane). Filter, cotton, non-woven fabric, sponge, etc.). The carrier separating unit 14 separates the carrier 5 from the first slider 4 by pushing down the carrier 5 supported on the distal end side of the first slider 4 with the pressing rod 7, and the separated carrier 5 is used for nucleic acid amplification. In the container 8 (see FIG. 8G).

Here, as shown in FIGS. 8E to 8I, the container 8 has a lid 17 attached via a hinge portion, and is configured so that the lid 17 can be opened and closed at the opening 16. The second slider 6 slidably supported by the main body 2 is supported (set) so that it can be attached and detached with the lid 17 closed. The lid 17 of the container 8 is pushed and opened by an open lid 18 formed at the tip of the first slider 4, and the carrier 5 supported by the first slider 4 is directly above the opening 16 ( carrier accommodation). Position ) (see FIGS. 8 (f) and 8 (g)).

  9 to 10 are views showing a state in which the main body 2 is disassembled. As shown in FIG. 9, the main body 2 overlaps and fixes a first main body 20 positioned on the lower side and a second main body 21 positioned on the upper side of the first main body 20. The second main body 21 is configured to be integrated by tightening and fixing with a screw (not shown) (see FIGS. 1 to 3).

(First body)
Among the main bodies 2, the first main body 20 is a slide guide for slidably supporting the plate-like first slider 4 on the upper surface 22 side as shown in FIGS. 3 and 9A and 9B. A recess 23 is formed. The slide guide recess 23 opens on one side surface (left side surface in FIG. 9) 24 of the first main body 20 and is substantially constant up to the vicinity of the other side surface (right side surface in the drawing) 25 of the first main body 20. It is formed with a width and a substantially constant depth. And when the 2nd main body 21 was piled up on the upper part of this 1st main body 20, the upper part of the 1st main body 20 and the recessed part 23 for slide guides is obstruct | occluded by the mating surface (lower surface) 26 of the 2nd main body 21, An internal space 3 is formed in the main body 2. Here, in the first slider 4, any one of the concave side surfaces 23 a and 23 b and the concave bottom surface 23 c of the slide guide concave portion 23 or the lower surface 26 of the second main body 21 serves as the first guide surface, and the first guide surface thereof. It moves in the interior space 3 along.

  Moreover, as shown in FIG.3 and FIG.9, the 1st main body 20 has the absorber accommodation hole 27 corresponding to each of the sample supply part 10 and the 1st-3rd process parts 11-13 in a mating surface (upper surface 22). It is formed so as to open to the side. Each of the absorber accommodation holes 27 has a cubic shape with a substantially square plane, and accommodates the absorber 15 capable of absorbing a liquid sample or reagent therein. A groove 28 having a shape substantially similar to the shape of the opening edge of the absorber housing hole 27 is formed in a region surrounding the opening edge of the absorber housing hole 27 of the first main body 20. The groove 28 is for preventing the sample and the reagent from being mixed between the adjacent absorber receiving holes 27, and flows through the internal space 3 (the surface of the slide guide recess 23). Function to dampen the.

  Further, as shown in FIG. 9, the first main body 20 opens to the other side surface (right side surface in FIG. 9) 25 side and extends to the vicinity of the absorber housing hole 27 of the third processing unit 13. The guide groove 30 guides the movement of the second slider 6. And as shown in FIG. 9, the notch part 32 for attaching slider holding | maintenance board 31a, 31b is formed in the lower surface side of the part in which the guide groove 30 of the 1st main body 20 was formed. The guide groove 30 slidably contacts the first guide groove portion 35 that slidably accommodates the protrusions 34 on both side surfaces 33 of the second slider 6 shown in FIG. 12 and the both side surfaces 33 above the protrusions 34 of the second slider 6. A second guide groove 36.

  Further, as shown in FIGS. 9A and 9C, the first main body 20 is a stopper that engages a stopper protrusion 37 of the second main body 21 at the substantially center in the width direction of the right end portion in the drawing. A protrusion engaging groove 38 is formed, and the stopper protrusion engaging groove 38 communicates with the second guide groove portion 36. The groove width of the stopper projection engaging groove 38 is smaller than the width dimension of the slide guide recess 23 and smaller than the groove width of the second guide groove portion 36.

(Second slider)
FIG. 12 shows the second slider 6 made of a resin material that is movably attached to the guide groove 30 of the first main body 20.

  The second slider 6 has a substantially rectangular planar shape. Both side surfaces 33 of the second slider 6 are slidably engaged with the second guide groove 36 of the first main body 20, and can be slid into the first guide groove 35 of the first main body 20. The protrusion 34 engaged with the is integrally formed like a rail along the longitudinal direction (sliding direction). The protrusion 34 has a rectangular cross-sectional shape, and can smoothly move in the first guide groove 35 while being supported by the slider pressing plates 31a and 31b from below.

  Further, the second slider 6 includes an operation portion 40 extending downward from one end side thereof, and the side surface shape is substantially L-shaped (see FIG. 12B). The operation unit 40 is formed to have a length that protrudes below the first main body 20, and can be grasped by the operator with a fingertip when the second slider 6 is attached to the first main body 20. 6 can be easily slid with respect to the first main body 20.

  Further, the second slider 6 has a container accommodation hole 41 penetrating in the thickness direction on the other end side (left end portion side in the figure). Then, a hinge connecting the container 8 and the lid 17 is engaged with the opening edge on the upper surface side of the container accommodation hole 41 to align the direction of the lid 17 of the container 8 and prevent the container 8 from rotating. The positioning groove 42 is formed. A container (plastic tube) 8 for nucleic acid amplification provided with a lid 17 that can be opened and closed in the opening 16 is detachably accommodated in the container accommodation hole 41 of the second slider 6.

  Further, a stopper groove 44 slidably engaged with the stopper projection 37 of the second body 21 is provided on the upper surface (surface facing the second body) 43 side of the second slider 6 at one end (FIG. 12A). To the vicinity of the positioning groove 42. The second slider 6 slides in the pull-out direction with respect to the first main body 20, and when the container 8 accommodated in the container accommodation hole 41 moves to a predetermined take-out position, the end 44a of the stopper groove 44 becomes the second main body. 21, the slide movement is stopped (see FIGS. 3 and 8 (i)).

  Further, the second slider 6 has a stopper hole 45 penetrating in the thickness direction on the end 44 a side of the stopper groove 44. The stopper hole 45 is set at a position where the container 8 supported by the second slider 6 accommodates the carrier 5 (a position corresponding to the carrier separating portion 14, hereinafter simply referred to as a carrier accommodation position (14)). In this state, the tip of the pressing rod 7 held by the second main body 21 is engaged (see FIG. 3). In a state where the tip of the pressing rod 7 is engaged with the stopper hole 45, the second slider 6 can no longer slide with respect to the first main body 20 and the second main body 21, and the container 8 can be moved at the tip of the first slider 4. The container 8 is not displaced from the carrier accommodation position (14) by the pressing force of the first slider 4 when the lid 17 is to be opened.

The second slider 6 moves in the guide groove 30 with the surface of the member facing either one of the side surfaces 33, the outer surface of the protrusion 34, the upper surface 43, or the stopper groove 44 as the second guide surface. Can do.

(Slider holding plate)
As shown in FIG. 11, the slider pressing plates 31 a and 31 b are plate-like bodies having a substantially rectangular plane shape, and a pair of left and right symmetrical shapes are used. As shown in FIGS. 3 and 9, the slider pressing plates 31 a and 31 b are notched portions 32 on the back surface side of the first main body 20 and screws (not shown) at positions on both sides of the first guide groove portion 35. And is engaged with the side surface 33 of the second slider 6 with a gap, and supports the protrusion 34 of the second slider 6 so as to be slidable from below. As shown in FIG. 11, a plurality of screw engagement holes 46 are formed, and the slider pressing plates 31 a and 31 b are not shown for positioning and fixing with respect to the first main body 20 and the second main body 21. A positioning hole 47 into which the positioning pin is inserted is formed.

(Second body)
FIG. 10 shows a second main body 21 made of a resin material.

  The second main body 21 is formed with a hole 50 for accommodating the sample supply container 48 at a position corresponding to the sample supply unit 10, and further, a bottom surface 50 a of the hole 50 from the bottom surface (surface facing the first main body). ) A sample supply hole 51 penetrating up to 26 is formed. The second main body 21 has a hole 53 for accommodating the processing agent supply container 52 at a position corresponding to the first processing unit 11, and the processing agent supply penetrating from the bottom surface 53 a to the lower surface 26 of the hole 53. A hole 54 is formed. The second main body 21 has a hole 56 for accommodating the cleaning liquid supply container 55 at a position corresponding to the second processing unit 12, and a cleaning liquid supply hole 57 penetrating from the bottom surface 56 a to the lower surface 26 of the hole 56. Is formed. Further, the second main body 21 is formed with an absorber moving hole 60 through which the absorber 58 shown in FIG. 13 protrudes toward the lower surface 26 at a position corresponding to the third processing unit 13. Yes. In addition, the second body 21 is provided with a recess 61 at a position corresponding to the carrier separating portion 14 that allows the lid 17 of the container 8 supported by the second slider 6 to be opened and closed. A pressing rod insertion hole 63 that penetrates from the upper surface 62 to the recess 61 is formed at a position corresponding to the carrier separation portion 14 of the second main body 21.

  Further, the second main body 21 includes an inclined wall 64 that is inclined from the ceiling surface 61 a of the recess 61 toward the pulling-out direction of the second slider 6 (right side in the figure), and is a container supported by the second slider 6. When the container 8 is moved from the carrier accommodation position (14) to a position corresponding to the carrier removal portion 66 (hereinafter, abbreviated as the carrier removal position (66)), the lid 17 of the opened container 8 is moved by the inclined wall 64. Push it down to close it. Therefore, the inclined wall 64 formed in the second main body 21 functions as a closing means for closing the lid 17 of the container 8. Note that a groove 65 extending in the pulling direction of the second slider 6 is formed at a substantially central portion in the width direction of the inclined wall 64. The groove 65 of the inclined wall 64 closes while the lid 17 is displaced in a direction perpendicular to the pulling-out direction of the second slider 6 when the container 8 with the lid 17 opened is moved from the carrier accommodation position to the carrier removal position. In order to prevent this, it is engaged with the substantially hemispherical head of the lid 17 to regulate the closing direction of the lid 17 so that the lid 17 reliably closes the opening of the container 8.

  The second main body 21 is formed at a position adjacent to the pressing rod insertion hole 63 so that a pressing rod receiving hole 67 for storing the pressing rod 7 penetrates from the upper surface 62 to the inclined wall 64. When the pressing rod 7 is received in the pressing rod receiving hole 67, the shaft portion 7a on the distal end side of the pressing rod 7 is engaged with the stopper hole 45 of the second slider 6 that supports the container 8 at the carrier receiving position (14). The second slider 6 can be locked by the pressing rod 7 (see FIG. 3).

  Further, the second main body 21 has a carrier take-out portion 66 formed at a position adjacent to the recess 61, and a container detachment hole 68 large enough to allow the container 8 to be taken in and out is formed in the carrier take-out portion 66. Yes. Then, by cutting out the upper side of the container detachment hole 68 to one side, a container takeout space 70 is formed on the upper side of the container detachment hole 68, and a grip for grasping the container 8 in the container takeout space 70. Tools and operator's fingers can be inserted. Accordingly, the container 8 supported by the second slider 6 and moved to the carrier removal position (66) can be easily taken out from the container detachment hole 68.

  Further, the second main body 21 is on the lower end side (the right end portion in FIG. 10) of the second slider 6 and next to the container detachment hole 68 (on the right side in FIG. 10 (c)). A protruding stopper projection 37 is integrally formed. The stopper protrusion 37 has a rectangular parallelepiped shape, and is slidably engaged with the stopper protrusion engaging groove 38 of the first main body 20 and slidably engaged with the stopper groove 44 of the second slider 6. It has become. When the container 8 supported by the second slider 6 moves to the carrier take-out position (66), the stopper projection 37 abuts on the end 44a of the stopper groove 44 of the second slider 6 and pulls out the second slider 6. The movement in the direction is prevented, and the container 8 is positioned at the carrier removal position (66).

  In addition, annular grooves 71 to 73 are formed on the lower surface 26 of the second main body 21 so as to surround the sample supply hole 51, the processing agent supply hole 54, and the cleaning liquid supply hole 57, respectively. The annular grooves 71 to 73 mix the sample on the adjacent sample supply hole 51 side with the treatment agent on the treatment agent supply hole 54 side, or mix the treatment agent on the treatment agent supply hole 54 side with the cleaning liquid supply hole 57 side. It is formed in order to prevent the cleaning liquid from being mixed, and exhibits a function of blocking liquid flowing along the surface in the internal space 3.

  In addition, a groove 81 that accommodates a protrusion 80 formed at the lower end of the head 78 of the absorber operating rod 77 shown in FIG. 13 is formed in the upper surface side opening of the absorber moving hole 60 of the second main body 21. Yes. By accommodating the protrusion 80 of the head portion 78 of the absorber operation rod 77 in the groove 81, the absorber 58 attached to the tip of the absorber operation rod 77 is moved by the dead weight of the absorber operation rod 77 in the first slider 4. It moves to the set position where it abuts the upper carrier 5 and its surroundings and absorbs excess liquid. Further, by removing the protrusion 80 of the head portion 78 of the absorber operating rod 77 from the groove 81 and supporting it by the upper surface 62 of the second main body 21, the absorber 58 can be held at the retracted position separated from the carrier 5. It is like that.

[First slider of nucleic acid extraction apparatus]
4 to 5 are diagrams showing the first slider 4.

  As shown in these drawings, the first slider 4 is movable along the first guide surface in the internal space 3 formed in the overlapped portion of the first main body 20 and the second main body 21. A plate-shaped body made of a resin material accommodated in the container, having a substantially rectangular planar shape, and is formed with an opening portion 18 for opening the lid 17 of the container 8 by notching one end side (tip end side) thereof. Yes. The opening portion 18 of the first slider 4 is cut out in a rectangular shape in a width dimension so that the lower portion of the lid 17 of the container 8 can be accommodated at the center in the width direction at one end of the first slider 4. A joint groove 82 is formed, and an inclined portion 82c is formed by obliquely cutting from the upper surface 4a of the side surface deepest portion 82a of the container engagement groove 82 toward the lower surface 4b of the side surface side tip portion 82b. The side end portion 82b of the container 82 is thinned, and the thickness of the side surface side of the container engaging groove 82 is gradually increased from the side end portion 82b of the container engaging groove 82 toward the deepest side portion 82a. . As a result, after the side end portion 82b of the container engaging groove 82 is inserted between the lid 17 and the opening 16 of the container 8, and the first slider 4 is further pushed into the internal space 3, the container engagement is achieved. The lid 17 is gradually pushed open by the inclined portion 82c on the side surface side where the thickness of the groove 82 gradually increases (see FIG. 8F).

  On the other end side (rear end side) of the first slider 4, a thick grip portion 83 that can be gripped by an operator is formed. The grip portion 83 is an open end of the internal space 3 of the main body 2. By striking the part, the sliding movement of the first slider 4 is prevented, the lid 17 of the container 8 is fully opened, and the opening 16 of the container 8 is completely opened (see FIG. 8F).

  Further, the first slider 4 is formed with a carrier support portion 84 that removably supports the substantially disc-shaped carrier 5 in the vicinity of the container engaging groove 82 and in the center in the width direction. The carrier support portion 84 is formed with a carrier detachment hole 85 having substantially the same diameter as the outer periphery of the carrier 5, and a plurality of carrier support protrusions 86 project from the circumferential direction of the inner wall surface of the carrier detachment hole 85. Yes. A presser plate engagement hole 87 having a diameter larger than that of the carrier separation hole 85 is formed in the upper portion of the carrier separation hole 85. In the present embodiment, as described above, the carrier 5 includes a filter paper, a membrane filter or a non-woven fabric, as long as it can hold the nucleic acid coprecipitate and the giant insolubilized nucleic acid on the surface of the carrier. -The material is not limited. Note that it is preferable to use filter paper as the carrier 5 from the viewpoint of ease of handling.

  In the carrier support portion 84 of the first slider 4, first, after the carrier 5 is placed on the carrier support protrusion 86, the pressing plate 88 shown in FIG. 14 is lightly press-fitted into the pressing plate engaging hole 87, The carrier 5 is sandwiched between the carrier support protrusion 86 and the pressing plate 88. As shown in detail in FIG. 14, the presser plate 88 has a substantially hollow disk shape, and a hole 90 having substantially the same diameter as the outer diameter of the carrier 5 is formed. Therefore, a sample, a reagent, or the like is dropped onto the carrier 5 supported by the first slider 4 through the hole 90 of the pressing plate 88. Further, when the carrier 5 supported by the first slider 4 is pushed down by the shaft portion 7a of the pressing rod 7 through the hole 90 of the holding plate 88, the carrier 5 is slightly deformed and detached from the carrier supporting projection 86, and the carrier is detached. The hole 85 is dropped.

  Further, the first slider 4 has a spring action portion 93 provided with a protrusion 92 protruding from the side surface 91 on one side surface 91 on the tip side. The spring action portion 93 includes a slit 94 that can be retracted to a position where the projection 92 does not protrude from the side surface 91 when the projection 92 is pushed, and the thin portion 95 on which the projection 92 is formed is elastically deformed. Spring action.

  When the first slider 4 is pushed into the internal space 3 of the main body 2, the protrusions 92 of the spring acting portion 93 are sequentially engaged with the positioning grooves 96 formed on the concave side surface 23 a of the slide guide concave portion 23, The carrier 5 is positioned with respect to the sample supply unit 10, the first processing unit 11, the second processing unit 12, the third processing unit 13, and the carrier separation unit 14. When the projection 92 of the spring action portion 93 of the first slider 4 is engaged with the positioning groove 96 of the slide guide recess 23, a click sound is generated and the resistance force against the movement of the first slider 4 changes. Can experience that the carrier 5 is positioned at each of the sample supply unit 10, the first processing unit 11, the second processing unit 12, the third processing unit 13, and the carrier separation unit 14.

  In addition, as shown in FIG. 3, the processing agent supply container 52 which dripped a nucleic acid coprecipitation agent and an insolubilizing agent is accommodated in the hole 53 of the 1st process part 11. As shown in FIG. Further, the second processing unit 12 accommodates a cleaning agent supply container 55 for dropping the cleaning liquid.

[Use condition of nucleic acid extraction equipment]
7 to 8 are diagrams for explaining a usage state of the nucleic acid extraction apparatus 1 according to the present embodiment.

  First, FIG. 7A shows a state in which the container 8 accommodated in the container accommodation hole 41 of the second slider 6 is arranged at the carrier accommodation position (14), and the second slider 6 is locked by the pressing rod 7. Show. The leading end side of the first slider 4 is inserted into the internal space 3 of the main body 2, and the carrier 5 supported on the leading end side of the first slider 4 is positioned in the sample supply unit 10.

  Next, as shown in FIG. 7B, the sample containing the nucleic acid accommodated in the sample supply container 48 is dropped from the sample supply container 48 onto the carrier 5. As a result, the sample containing nucleic acid adheres to the carrier 5. In the present specification, the term “attachment” refers to a state in which a nucleic acid or a nucleic acid insolubilized product is simply held on the carrier 5, and does not actively use the chemical interaction caused by the binding between the nucleic acid and the carrier 5. Is the meaning. The surplus sample that has passed through the carrier 5 is absorbed by the absorber 15.

  Next, as shown in FIG. 7C, when the sample dropping from the sample supply container 48 to the carrier is completed, the first slider 4 is further pushed into the internal space 3 of the main body 2, and the carrier 5 is moved to the first processing section. 11 is moved. In the first processing unit 11, a predetermined amount of the nucleic acid coprecipitation agent and the insolubilizing agent are dropped onto the carrier 5 from the processing agent supply container 52. Here, the surplus nucleic acid coprecipitation agent and the insolubilizing agent that have passed through the carrier 5 are absorbed by the absorber 15.

  Next, as shown in FIG. 7D, the first slider 4 is further pushed into the internal space 3 of the main body 2, and the carrier 5 is moved to the second processing unit 12. In the second processing unit 12, the cleaning liquid is dropped from the cleaning agent supply container 55 onto the carrier 5. Components other than the excess washing solution that has passed through the carrier 5 and the insolubilized nucleic acid removed by the washing solution are absorbed by the absorber 15.

  Next, as shown in FIG. 8E, the first slider 4 is further pushed into the internal space 3 of the main body 2, and the carrier 5 is moved to the third processing unit 13. Here, the absorber operating rod 77 is pushed down, and the absorber 58 attached to the tip of the absorber operating rod 77 is pressed around the carrier 5 and the periphery of the carrier 5, and the carrier 5 and the absorber 15 below the carrier 5 The excess liquid adhering to the carrier 5 and its periphery is absorbed by the absorber 58 and the absorber 15.

  Next, as shown in FIG. 8 (f), the absorber operating rod 77 is lifted to the retracted position, and the first slider 4 is further pushed into the internal space 3 of the main body 2. At this time, the lid 17 of the container 8 is opened by the open lid 18 at the tip of the first slider 4, and the carrier 5 moves to a position directly above the opening 16 of the container 8 (carrier accommodation position (14)).

  Next, as shown in FIG. 8G, when the pressure rod 7 is replaced with the pressure rod insertion hole 63 from the pressure rod housing hole 67 and the pressure rod 7 is pushed down, the tip of the shaft portion 7 a of the pressure rod 7 becomes the carrier 5. Is separated from the first slider 4 and the carrier 5 with the nucleic acid attached is dropped into the container 8 containing the reagent for nucleic acid amplification. Here, as in this operation step, the carrier 5 to which the insoluble nucleic acid is attached after being subjected to the washing treatment is accommodated in the container 8 already containing the reagent for nucleic acid amplification, so that the insolubilized nucleic acid attached to the carrier 5 is converted into the nucleic acid. It elutes in the buffer used in the amplification reaction, and can be used for the nucleic acid amplification reaction immediately thereafter.

  Next, as shown in FIG. 8 (h), the pressing rod 7 is extracted from the main body 2, and the second slider 6 is slid from the carrier accommodation position (14) toward the carrier removal position (66). At this time, the lid 17 of the opened container 8 is closed by the inclined wall 64.

  Next, as shown in FIG. 8 (i), when the second slider 6 is slid in the pulling direction and the container 8 whose lid 17 is completely closed by the inclined wall 64 is moved to the carrier removal position (66), 2 The movement of the slider 6 is blocked by the stopper projection 37, and the container 8 supported by the second slider 6 is taken out from the container detachment hole 68. The extracted container 8 accommodates the carrier 5 with the nucleic acid attached therein and is used for the nucleic acid amplification reaction. The nucleic acid amplification reaction was performed by fluorescence detection with a fluorescence device iCycler (manufactured by Biorad).

  As described above, according to the present embodiment, the nucleic acid attached to the carrier 5 is amplified by bringing the carrier 5 together with the nucleic acid amplification buffer directly into contact with the nucleic acid amplification buffer. Can be omitted. In addition, according to the present embodiment, the entire amount of nucleic acid can be recovered particularly in a sample that can be collected only in a very small amount, and even a sample having a low nucleic acid content can be concentrated and recovered in the carrier 5, so that a small amount This nucleic acid can be subjected to an amplification reaction without loss.

  Here, the nucleic acid coprecipitation agent used in the present invention has high affinity with nucleic acid and can encapsulate insolubilized nucleic acid greatly, and starches such as amylopectin and glycogen are preferable. Examples of insolubilizers include lower alcohols such as ethanol, isopropyl alcohol, and butyl alcohol. However, activity inhibitors such as sodium citrate that can suppress nuclease activity, which is a nucleic acid degrading enzyme, and cell membranes are destroyed. In addition, the combined use of a protein denaturing agent such as guanidine for denaturing the protein in the complex can improve the nucleic acid recovery efficiency. Further, the reagent used in the washing step is for dissolving and washing away the denatured protein while carrying the insolubilized nucleic acid, and alcohol having a concentration lower than that used as the insolubilizing agent is used.

The reagent for nucleic acid amplification used in the present invention is, for example, a PCR (polymerase chain reaction) method, which is the most common method for in vitro nucleic acid amplification, and a LAMP (Loop-Mediated Isothermal).
Amplification) method (Patent No. 3313358, etc.), SDA (Strand Displacement Amplification) method (Japanese Patent Publication No. 7-114718, etc.), NASBA (Nucleic)
Examples include amplification reagents used in the Acid Sequence Based Amplification) method (Japanese Patent No. 2650159), and a reagent for LAMP that can easily carry out the detection of amplification products from nucleic acid amplification is preferred.

  The LAMP method is called a loop-mediated isothermal amplification method, and anneals its 3 ′ end to a template nucleotide to serve as a starting point for complementary strand synthesis, and by combining primers that anneal to the loop formed at this time, This is a nucleic acid amplification method that enables an isothermal complementary strand synthesis reaction.

  The LAMP method consists of 4 types of oligonucleotide primers that recognize base sequences of a total of 6 regions of the target nucleic acid, that is, 2 types of inner primer (FI primer and RI primer) and 2 types of outer primer (F3 primer and R3 primer), strand displacement Using a nucleic acid synthase having a synthetic activity and a substrate, a highly specific gene amplification reaction proceeds promptly and isothermally without requiring a heat denaturation step. Furthermore, by using a primer (loop primer) complementary to the base sequence of the single-stranded loop on the 5 ′ end side of the dumbbell structure formed during the amplification reaction, the starting point of nucleic acid synthesis is increased, and the reaction time is increased. It is also possible to shorten and improve the detection sensitivity (WO02 / 24902).

  The detection of the nucleic acid amplification product by the LAMP method can be detected by using the electrophoresis method or the fluorescent intercalator method as in the PCR method (Japanese Patent Laid-Open No. 2001-242169). In addition, the detection method (WO01 / 83817) using the insoluble magnesium pyrophosphate, which is an amplification by-product taking advantage of the characteristics of the LAMP method, as an index, and the detection method (WO01 / 83817), a metal ion and a metal indicator There is also a detection method (Japanese Patent Application No. 2003-57342) using coloration and fluorescence accompanying chelation as indicators. In the detection method using turbidity, precipitation, or fluorescence as an indicator, the presence / absence of amplification can be visually confirmed even from the outside of the reaction vessel. Therefore, unnecessary carriers after the nucleic acid is eluted in the buffer are particularly removed. Even if it is not, it can be left in the reaction solution as it is. If the nucleic acid extraction apparatus of the present embodiment described above is used, it becomes possible to efficiently detect a nucleic acid amplification product by the LAMP method.

  According to the nucleic acid extraction apparatus 1 according to the present embodiment configured as described above, the carrier 5 itself holding the nucleic acid is accommodated in the container 8 in the main body 2, and thus the carrier 5 to which the nucleic acid is attached. Is not taken out of the main body 2 in the exposed state (in the atmosphere), there is no risk of environmental contamination due to nucleic acid adhering to the carrier 5 or impurities in the air adhering to the nucleic acid adhering to the carrier, Handling of the carrier 5 is facilitated. Moreover, according to the nucleic acid extraction apparatus 1 according to the present embodiment, since the carrier 5 to which the nucleic acid is attached is accommodated in the container 8, nucleic acid amplification or the like can be performed in the container 8, There is no need to separate the nucleic acid held on the carrier 5 from the carrier 5. As a result, according to the present embodiment, a complicated operation and a large-sized or special apparatus for separating the nucleic acid from the carrier 5 are not required, and the nucleic acid can be easily and quickly extracted and purified from the sample containing the nucleic acid. It becomes possible.

  Moreover, in this Embodiment, although the nucleic acid extraction apparatus 1 does not need to be all transparent, it is preferable that at least the part which requires visibility is formed with a transparent material. In this way, the operator can visually recognize the processing process, and the work becomes efficient and the work is ensured.

  Hereinafter, nucleic acid extraction using the nucleic acid extraction apparatus 1 according to the present invention will be specifically described with reference to Examples. The following description is based on the usage state of the nucleic acid extraction apparatus 1 shown in FIGS.

  First, in the sample supply unit 10, 1 μL of bovine embryo cultured cell suspension (100 cells / μL) is used as a sample containing nucleic acid, and 1 μL of this bovine embryo cultured cell suspension is used as a carrier (qualitative filter paper No. 1 (ADVANTEC). ))).

  Next, in the 1st process part 11, 1 microliters of nucleic acid coprecipitation agents and 500 microliters of insolubilizing agents were dripped. Here, a 40 mg / mL amylopectin solution was used as the nucleic acid coprecipitation agent. As the insolubilizing agent, a mixed solution containing 60% isopropanol, 2.4 mol / L guanidine, 0.5% N-lauroyl sarcosine sodium and 15 mmol / L sodium citrate was used.

  Next, in the 2nd process part 12, 100 microliters of washing | cleaning liquids were dripped at the support | carrier, and the support | carrier was wash | cleaned. Here, a mixed solution of 40% isopropanol and 200 mmol / L potassium chloride was used as the cleaning solution.

  Next, in the third processing unit 13, the insoluble liquid component contained in the carrier was absorbed and removed by the absorber 58 and the absorber 15.

  Next, in the carrier separation unit 14, the carrier 5 was separated from the first slider 4 by the pressing rod 7, and the separated carrier 5 was accommodated in the container 8 into which the nucleic acid amplification reagent was injected.

  In addition, what carried out the same operation in the nucleic acid extraction apparatus 1 using distilled water instead of the said cow embryo cultured cell suspension was made into the negative control.

  Next, the container 8 containing the carrier is taken out from the carrier taking-out part 66, and the container 8 is attached to a fluorescence device iCycler (manufactured by Biorad), and nucleic acid amplification (63 ° C., 30 minutes) and the progress of nucleic acid amplification was confirmed in real time by measuring the fluorescence intensity.

Next, nucleic acid amplification using the LAMP method will be specifically described with reference to examples.
[Reaction solution composition]
Reaction solution composition for LAMP (in 25 μL)
20 mM Tris-HCl (pH 8.8)
10 mM KCl
10 mM (NH4) 2SO4
8mM MgSO4
80nM Oxazole Yellow 0.1% Tween20
0.8M Betaine
1.4 mM dNTPs
8U Bst DNA polymerase (New England Biolab)
1.6 μM FI primer 1.6 μM RI primer 0.2 μM F3 primer 0.2 μM R3 primer 0.8 μM Loop primer F
0.8μM Loop Primer R

The six primers in the reaction solution composition were designed as follows based on the base sequence of the bull specific base sequence plasmid S4.
・ FA primer
5'-AGCTATGTGG CATGTGGGAT CCTTCCCTGG
AAATGTTTAA GTG-3 '(SEQ ID NO: 1)
・ RA primer
5'-TAAAGCCAGA CACAGAGGTC ACTTTTGCTT
CTCTTTCCTG CTTC-3 '(SEQ ID NO: 2)
・ F3 primer
5'-AGCCAAGAAG TGGATGAATC-3 '(SEQ ID NO: 3)
・ R3 primer
5'-GCAGTGCATT TCCTCCTC-3 '(SEQ ID NO: 4)
・ Loop Primer F
5'-GGGATGGAAA CTGTGCAT-3 '(SEQ ID NO: 5)
・ Loop Primer R
5'-ATTGCATGTG GAAGAACTGT AG-3 '(SEQ ID NO: 6)

To the reaction solution for LAMP, 6 × 10 ⁻²⁰ mol of bovine genomic DNA was added as a template for LAMP reaction, and amplification reaction by LAMP method was performed at 63 ° C. for 30 minutes. The amplification reaction was carried out by fluorescence detection with a fluorescence device iCycler (Biorad).

  As a result, as shown in FIG. 15, it was confirmed that the fluorescence intensity began to increase when the reaction time passed 14 minutes, and that the fluorescence intensity increased rapidly after the reaction time passed 16 minutes. Thereby, it was confirmed that a nucleic acid can be extracted by using the nucleic acid extraction apparatus 1 of the present invention. In FIG. 15, since no amplification was observed in the negative control (reaction curve with 0 cells), it was confirmed that the filter paper carrier that had undergone a series of treatments did not induce non-specific amplification reaction.

It is a front view of the nucleic acid extraction apparatus which concerns on embodiment of this invention, and abbreviate | omits and shows a sample supply container, a processing agent supply container, and a washing | cleaning agent supply container. It is a top view of the nucleic acid extraction apparatus which concerns on embodiment of this invention, and is a top view which abbreviate | omits and shows a sample supply container, a processing agent supply container, and a washing | cleaning agent supply container. It is a longitudinal cross-sectional view of the front side of the nucleic acid extraction device according to the embodiment of the present invention, and is a cross-sectional view of the nucleic acid extraction device shown cut along the line AA in FIG. It is a figure which shows the 1st slider which concerns on embodiment of this invention. (A) is a top view of a 1st slider, (b) is a side view of a 1st slider. It is sectional drawing of the 1st slider cut | disconnected and shown along the BB line of FIG. It is a top view of the 1st slider which shows the state with which the support | carrier was mounted | worn. It is a figure which shows the operation state of the nucleic acid extraction apparatus which concerns on embodiment of this invention, and is a figure which shows operation state (a)-(d). It is a figure which shows the operation state of the nucleic acid extraction apparatus which concerns on embodiment of this invention, and is a figure which shows operation state (e)-(i). It is a figure which shows a 1st main body. (A) is a top view of a 1st main body, (b) is sectional drawing cut | disconnected and shown along the CC line of (a), (c) is a back view of a 1st main body. It is a figure which shows a 2nd main body. (A) is a top view of a 2nd main body, (b) is a side view of a 2nd main body, (c) is sectional drawing cut | disconnected and shown along the DD line of (a), (d) is 2nd It is a reverse view of a main body. It is a figure which shows a pair of slider pressing board. (A) is a plan view of a slider pressing plate disposed on one side of the guide groove, (b) is a cross-sectional view taken along line EE of (a), and (c) is a cross-sectional view of (a). The right side view of the slider pressing plate, (d) is a plan view of the slider pressing plate disposed on the other side of the guide groove, (e) is a cross-sectional view cut along the FF line of (d), (F) is a right side view of the slider pressing plate of (d). It is a figure which shows a 2nd slider. (A) is a top view of a 2nd slider, (b) is sectional drawing cut | disconnected and shown along the GG line of (a), (c) is a back view of a 2nd slider, (d) is (a) It is a right side view of the second slider. It is a figure which shows an absorber operating rod. (A) is the front view of an absorber operation rod, (b) is the figure (back view) which looked at the absorber operation rod from the absorber side. It is a figure which shows a pressing plate. (A) is a top view of a pressing board, (b) is a longitudinal cross-sectional view of a pressing board. It is an amplification reaction curve by fluorescence in the direct method.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Nucleic acid extraction apparatus, 2 ... Main body, 3 ... Internal space, 4 ... 1st slider (carrier moving means), 5 ... Carrier, 6 ... 2nd slider (container attaching part), 7 ... Press rod (carrier separating means), 8 ... container, 10 ... sample supply unit, 11 ... first processing unit (processing unit), 12 ... second processing unit (processing unit), 14 ... carrier separating unit (Carrier receiving position), 16 ... opening, 17 ... lid, 18 ... open lid, 64 ... inclined wall (lid closing means), 66 ... carrier takeout part (carrier takeout position)

Claims (3)

(1) A sample supply unit for supplying a sample containing nucleic acid to a carrier for holding nucleic acid, a first processing unit for insolubilizing nucleic acid contained in a sample attached to the carrier, and attached to the carrier A second processing section for removing components other than nucleic acids, and a container mounting section for removably supporting a container in which the carrier is accommodated in a carrier accommodation position, and
An internal space that has a first guide surface and communicates with each of the opening of the container supported by the sample supply unit, the first processing unit, the second processing unit, and the container mounting unit is formed. Become
The body,
(2) The carrier is detachably supported, and the first guide from the sample supply part to the opening of the container supported by the container mounting part in the internal space of the main body. Carrier moving means formed to be movable along the surface;
(3) Carrier separating means for separating the carrier moved to the opening of the container by the carrier moving means and separating the carrier from the carrier moving means and storing it in the container;
A nucleic acid extraction apparatus comprising:
A second guide for movably supporting the container mounting portion on the main body so that the container detachably supported by the container mounting portion can be moved from the carrier receiving position to the carrier take-out position. A surface is formed,
A lid that can be opened and closed is attached to the opening of the container,
The container detachably supported by the container mounting portion is
After the opening is set at the carrier accommodating position in a state of being closed by the lid, the lid is opened by the opening lid of the carrier moving means that has moved in the internal space, and the opening is After communicating with the internal space, containing the carrier separated from the carrier moving means by the carrier separating means;
When the container mounting portion is moved along the second guide surface, the lid in the opened state is moved by the closing means of the main body when the container mounting portion is moved from the carrier receiving position to the carrier take-out position. Closed,
A nucleic acid extraction apparatus characterized by the above. The nucleic acid extraction apparatus according to claim 1, wherein a reagent for nucleic acid amplification is stored in the container in advance . The nucleic acid extraction apparatus according to claim 1 or 2 , wherein the nucleic acid amplification reagent is a nucleic acid amplification reagent used in the LAMP method .

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