JP2015139398A - Apparatus and method for nucleic acid extraction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compact, inexpensive, and easily handled apparatus for nucleic acid extraction that prevents exposure to pathogens in operators and a method for nucleic acid extraction.SOLUTION: An apparatus for nucleic acid extraction comprises separation means equipped with a container for a sample and a filter, pressurizing means connected to the separation means, recovering means that can be connected to the separation means, dispensing means that dispenses the sample, driving means that drives the dispensing means, and control means that controls the pressurizing means and the driving and pressurizing means. The control means dispenses the sample into the separation means by driving the dispensing means and then pressurizes the separation means with the pressurizing means to separate the sample and a nucleic acid.

Description

  The present invention relates to a nucleic acid extraction apparatus and a nucleic acid extraction method for extracting a nucleic acid from a specimen and preparing a solution containing the nucleic acid.

  Conventionally, as a method for extracting nucleic acid (deoxyribonucleic acid: DNA (deoxyribonucleic acid) or ribonucleic acid: RNA (ribonucleic acid)), separation using magnetic beads (see, for example, Patent Document 1) There is known a method of separating with a focus (for example, refer to Patent Document 2, hereinafter referred to as extraction column centrifugation).

Special table 2009-509549 gazette JP 2009-131264 A

  However, the separation method using magnetic beads has a problem of high cost and low nucleic acid recovery rate in the case of a highly viscous specimen such as sputum.

  In contrast, the extraction column centrifugation method is a method in which nucleic acid is finally extracted by immobilizing nucleic acid in the extraction column and removing residual liquid or the like by centrifugal force. For this reason, although it is easy to separate nucleic acids even for highly viscous specimens, a centrifuge is essential, and the size and complexity of the apparatus cannot be avoided. Further, since the apparatus is expensive, there is a limit to reducing the extraction cost.

  The nucleic acid extracted by the above method is analyzed to identify pathogens in the cocoon. Specifically, a patient's nucleic acid extracted by extraction column centrifugation or the like is dispensed into a plurality of containers, and, for example, a plurality of different primer solutions (predetermined primers, PCR reagents, enzymes and A solution prepared with a buffer solution or the like is dispensed to each, and then a PCR reaction is performed and analyzed to identify a pathogen.

  However, conventionally, the dispensing operation of the nucleic acid and each primer solution has been performed manually, and it has been necessary to dispense a plurality of primer solutions for each patient. In such a method, mistakes are likely to occur due to repetition of simple work, and there is a risk that a pathogen is infected to the worker. In addition, the dispensing work is simple for each individual work, but it is necessary to change the tip for each primer solution and patient, and the number of dispensing is increased, so the whole work is complicated and time-consuming work. . For this reason, problems such as forgetting to replace the chip and contamination due to mixing of the primer (solution) occurred.

  An object of the present invention is to provide a nucleic acid extraction apparatus and a nucleic acid extraction method that are small and inexpensive, suppress exposure of a pathogen to an operator, and are easy to operate.

  The present invention solves the above problems by the following means.

  (1) The present invention provides a nucleic acid extraction apparatus for extracting nucleic acid from a specimen, the separation means including a container for storing the specimen and a filter, a pressurizing means connected to the separation means, and the separation means Recovery means that can be connected, dispensing means for dispensing the sample, driving means for driving the dispensing means, control means for controlling the pressurizing means, the driving means, and the pressurizing means, And the control means drives the dispensing means to dispense the sample to the separation means, and pressurizes the separation means by the pressurizing means to separate the sample and the nucleic acid. A nucleic acid extraction apparatus.

  (2) Further, the present invention relates to the above invention, wherein the pressurizing unit pressurizes the separating unit to a pressure exceeding a set pressure threshold, and then holds the separating unit under the pressure for a predetermined time, Thereafter, the pressure increasing / decreasing cycle for depressurization is performed a plurality of times for the one specimen dispensed to the separation means.

  (3) Further, the present invention relates to the above invention, wherein the pressurizing unit is configured such that the pressure applied to the separating unit does not fall below the set pressure threshold even after the predetermined time has elapsed. The separation means is depressurized.

  (4) In addition, the present invention relates to the above-mentioned invention, and an error notification that notifies an error when the pressure applied to the separation means does not fall below the set pressure threshold even after the predetermined time has elapsed. It has a means, It is characterized by the above-mentioned.

  (5) In addition, the present invention relates to the above-mentioned invention, wherein the additional pressurization cycle is executed when the pressure applied to the separation means does not fall below the set pressure threshold. It is.

  (6) In addition, the present invention relates to the above-mentioned invention, and further includes another collecting means corresponding to the collecting means, and a primer holding means for holding a primer solution, and the dispensing means is configured by the control means. When driven, the separated nucleic acid present in the collecting means and the primer solution held by the primer holding means are dispensed to the other collecting means.

  (7) Further, in the present invention, in relation to the above-described invention, a plurality of the other collecting means and the primer holding means are provided for one collecting means, and each primer holding means holds a different primer solution. It is characterized by that.

  (8) Further, the present invention relates to the above-mentioned invention, and further comprises a chip exchanging means for automatically exchanging the chips of the dispensing means.

  (9) In addition, the present invention relates to the above invention, wherein the separation means is a solid phase extraction column.

  (10) The present invention also relates to a nucleic acid extraction method for extracting nucleic acid from a specimen, the step of dispensing the specimen into a separation means having a container for storing the specimen and a filter, and a plurality of the separation means. A nucleic acid extraction method comprising the steps of separating the nucleic acid from the specimen by repressurization and collecting the effluent discharged from the separation means and extracting the nucleic acid.

  (11) In addition, the present invention relates to the above-mentioned invention, and after pressurizing the separation means with a pressure exceeding a set pressure threshold for one specimen dispensed to the separation means, the separation means is A pressurizing / depressurizing cycle for holding for a predetermined time under pressure and then depressurizing is executed a plurality of times.

  (12) Further, the present invention relates to the above-described invention, and when the pressure applied to the separation means does not fall below the set pressure threshold even after the predetermined time has elapsed, the separation means is decompressed. It is characterized by that.

  (13) Further, the present invention relates to the above-mentioned invention, wherein the separation means is a solid phase extraction column.

  (14) Further, the present invention relates to the above invention, and further comprises a step of adding a primer solution to the extracted nucleic acid.

  ADVANTAGE OF THE INVENTION According to this invention, the nucleic acid extraction apparatus and nucleic acid extraction method which are small and cheap, suppress the exposure of the pathogen to an operator, and are easy to operate can be provided.

It is an external appearance perspective view of the nucleic acid extraction apparatus which concerns on this embodiment. It is the schematic which extracts and shows the principal part of the nucleic acid extraction apparatus of this embodiment. It is a top view of the base in the nucleic acid extraction apparatus of this embodiment. It is a figure which shows the nucleic acid extraction part of this embodiment, (a) It is a front view, (b) It is a side view. It is a general | schematic side surface which shows operation | movement of the nucleic acid extraction part of this embodiment. It is a figure which shows a dispensing part, (a) It is a front view, (b) It is a side view. It is a protocol figure which shows an example of the nucleic acid extraction method using the nucleic acid extraction apparatus of this embodiment. It is a figure explaining the error detection mechanism of a pressurization part, (a) It is a figure which shows the state at the time of normal, (b) It is a figure of the state which detected the error by column clogging. It is an example of the screen transition displayed on a display part. It is an example of the screen transition displayed on a display part. It is the result of having identified the pathogen by the nucleic acid extraction method using the extraction column centrifugation method which concerns on a prior art, and the nucleic acid extraction method of this embodiment. It is the figure which compared the nucleic acid extraction method using the extraction column centrifugation method which concerns on a prior art, and the nucleic acid extraction method of this embodiment.

  Hereinafter, a nucleic acid extraction apparatus and a nucleic acid extraction method according to embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is an external perspective view of a nucleic acid extraction apparatus 10 according to this embodiment. The nucleic acid extraction apparatus 10 includes a housing 1, a display unit 2, a nucleic acid extraction unit 3, a primer processing unit 4, a dispensing unit 5, and a control unit CT. In FIG. 1, the control unit CT is shown outside the nucleic acid extraction device 10 for convenience of explanation, but the control unit 10 is provided inside the nucleic acid extraction device 10.

  The housing 1 is provided with a front door 11. The front door 11 can be opened and closed in a vertical direction by a slider (not shown) provided at the left and right ends, for example.

  The display unit 2 is a touch panel that displays predetermined information and enables input, and is provided on the front surface of the housing 1 (for example, below the front door 11).

  A base 12 inside the housing 1 is provided with a nucleic acid extraction unit 3 and a primer processing unit 4. A dispensing portion 5 is suspended from the ceiling portion of the housing 1 so as to be movable in the vertical and horizontal directions in the housing 1. The dispensing unit 5 includes a plurality of dispensing cylinders 50, a cylinder holding unit 51 that holds these, and a drive unit 52.

  The nucleic acid extraction unit 3 includes a pressure unit 31, a separation unit 32, and a moving mechanism (not shown here). The pressurizing unit 31 pressurizes the separating unit 32, thereby separating and extracting nucleic acids contained in the specimen. The specimen of this embodiment is, for example, a patient's sputum, and the nucleic acid to be extracted is, for example, DNA in the sputum (including a pathogen).

  The primer processing unit 4 includes a PCR tube holding unit 41 that holds a tube for primer solution dispensing (hereinafter referred to as a PCR (polymerase chain reaction) tube), and a primer holding unit 42 that holds a plurality of primer solutions. Yes. In the primer processing unit 4, the nucleic acid extracted by the nucleic acid extraction unit 3 is dispensed into the PCR tubes, and various primer solutions are dispensed into the PCR tubes. At this time, the primer solution already contains predetermined primers, PCR reagents, enzymes, buffer solutions and the like necessary for PCR.

  FIG. 2 is a schematic view showing a part of the pressurizing unit 31 and the separating unit 32 in the present embodiment. The separation unit 32 includes a solid phase extraction column 321 and a nucleic acid recovery tube 324. The solid-phase extraction column 321 includes, for example, a filter (membrane) F made of glass fiber, charged resin, or the like in the column CM, and separates the sample and the nucleic acid by using the adsorptivity of the nucleic acid to the filter F. To do. That is, a nucleic acid having a hydrophilic property is adsorbed on the filter F in a highly hydrophobic solution, and proteins and lipids having a hydrophobic property are likely to flow off as they are.

  The pressurizing unit 31 includes a pressurizing column cap 311, a pressurizing tube 312, a pressurizing syringe 313, a pressure sensor 315, and the like. The pressurizing column cap 311 seals the upper part of the solid phase extraction column 321. One end of the pressure tube 312 is connected to the pressure column cap 311, and the other end of the pressure tube 312 is connected to the pressure syringe 313. The pressurizing syringe 313 includes a cylindrical syringe 313 a and a movable plunger 313 b, and pressurizes the solid phase extraction column 321 through the pressurizing tube 312. For example, a pressure sensor 315 is attached to the pressurizing tube 312 so that the pressure of the solid phase extraction column 321 (pressure applied to the solid phase extraction column 321) can be measured.

  In the nucleic acid extraction unit 3 of the present embodiment, a sample such as sputum is dispensed into the column CM of the solid phase extraction column 321 and pressurized by the pressurization unit 31, so that the nucleic acid containing the pathogen is adsorbed to the filter F. The waste liquid passes through the filter F and flows out of the solid phase extraction column 321. The waste liquid may be discharged to a waste liquid tube or may be discharged to a waste liquid tank. Thereafter, the solid phase extraction column 321 is placed on the nucleic acid recovery tube 324, and when the nucleic acid recovery solution or distilled water is dispensed and pressurized to the solid phase extraction column 321, the nucleic acid adsorbed on the filter F Is recovered in the recovery tube 324.

  The nucleic acid extraction apparatus 10 also dispenses the sample to the solid phase extraction column 321, pressurization by the pressurizing unit 31, dispenses the extracted nucleic acid to the PCR tube, and further adds the primer solution to the PCR tube All of the above can be automated and processed continuously.

  3-6 is a figure which shows the structure in the nucleic acid extraction apparatus 10, and FIG. 3 is a top view of the base 12. FIG. FIG. 4 is a diagram illustrating the nucleic acid extraction unit 3 extracted, in which FIG. 4 (a) is a front view and FIG. 4 (b) is a side view. FIG. 5 is an external side view showing how the nucleic acid extraction unit 3 operates. FIG. 6 is a view showing the dispensing unit 5 extracted, FIG. 6 (a) is a front view, and FIG. 6 (b) is a side view.

  As shown in FIG. 3, the nucleic acid extraction unit 3 and the primer processing unit 4 are provided on the same surface of the base 12.

  The separation unit 32 of the nucleic acid extraction unit 3 includes a solid phase extraction column 321, a column holding unit 322 that holds a plurality of (here, four) solid phase extraction columns 321 side by side, a waste liquid tank 323, and a collection tube 324. And a collection tube holding part 325 for holding the collection tube 324. The pressurizing unit 31 includes a pressurizing column cap 311, a cap holding unit 316 that holds the pressurizing column cap 311, and a pressurizing tube, a pressurizing syringe, a pressure sensor, and the like (not shown) (see FIG. 2). . In addition, the nucleic acid extraction unit 3 moves the cap holding unit 316 in the vertical direction (perpendicular to the base 12) and the column moving unit 322 in the vertical direction and the column holding unit 322. And a second moving mechanism 34 that moves the collection tube holding portion 325 in the front-rear direction (horizontal with respect to the base 12).

  The solid phase extraction column 321 can be moved below the pressurizing column cap 311 by the second moving mechanism 34 and is sealed by the pressurizing column cap 311.

  The waste liquid tank 323 is a tank that collects the waste liquid discharged from the solid phase extraction column 321 and can be moved below the solid phase extraction column 321 by the second moving mechanism 34. The recovery tube 324 is a tube for recovering the separated nucleic acid, and can be moved below the solid phase extraction column 321 by the second moving mechanism 34 at a timing different from that of the waste liquid tank 323.

  Here, as an example, the column holding unit 322 can hold four solid-phase extraction columns 321 at a time, and correspondingly, a pressurizing column cap 311, a collection tube 324, and a sample tube 20 (20 </ b> A, 20 </ b> B). , 20C, 20D) are also provided. That is, the nucleic acid extraction work for four types of samples (samples for four people) at a time can be processed in parallel.

  The primer processing unit 4 includes a PCR tube holding unit 41 and a primer holding unit 42. A plurality of (for example, 12 to 16) PCR tubes 43 held by the PCR tube holding unit 41 are prepared and arranged in a matrix to constitute one block B. . That is, here, four blocks (B1 to B4) of PCR tube holding portions 41 are arranged corresponding to four specimens. The primer holding unit 42 includes a plurality (for example, 16) of primer tubes 44, and these are arranged in a matrix to hold a plurality of types (for example, 16 types) of different primer solutions. The number of PCR tubes 43 in one block corresponds to the number of primer tubes 44 of the primer holding unit 42 or is more than that.

  The base 12 includes chip racks (chip holders) 6 (6a, 6b) that hold detachable chips 54 in a matrix at the tip of a dispensing cylinder (not shown here) of the dispensing unit. . For example, the chip includes a 1000 μL chip 541 and a 20 μL chip 542. The chip rack 6a holds 1000 μL chips, and the chip rack 6b holds 20 μL chips.

  Further, a waste chip storage 8 for storing waste chips, a reagent tank 9, and a sample storage 21 for arranging and holding a plurality of sample tubes 20 are provided. The specimen place 21 has a stirring function and a temperature adjustment function.

  The nucleic acid extraction unit 3 will be further described with reference to FIG. In the same figure, the pressurization part 31 and the isolation | separation part 32 have shown the state which exists in an initial position. Moreover, in the figure (b), the right is made into the front (front) side, and the left is made into the back (back) side.

  As shown in FIGS. 4A and 4B, at the initial position, the cap holding portion 316 and the pressurizing column caps 311 (311A, 311B, 311C, 311D) are arranged in the vertical (perpendicular to the base 12) direction. ) In the upper stage, column holding section 322 and each solid phase extraction column 321 (321A, 321B, 321C, 3221D) in the middle stage, collection tube holding section 325, each collection tube 324 (324A, 324B, 324C, 324D). And the waste liquid tank 323 located in the back | inner part (rear part) side of the tube 324 for collection | recovery is arrange | positioned in the lower stage.

  Further, as shown in FIG. 5B, in the initial position, the cap holding part 316 and the pressurizing column cap 311 are located at the rearmost part (the innermost part) in the front-rear (horizontal) positional relationship, and the collection tube 324 is provided. The recovery tube holding part 325 is arranged at the foremost part (operator side), and the column holding part 322, the solid phase extraction column 321 and the waste liquid tank 323 are arranged at the middle part between the innermost part and the foremost part. At the initial position, the upper part of the solid phase extraction column 321 is opened, and the upper part of the collection tube 324 is also opened. Note that the cap holding portion 316 is rotatable in the direction of the broken line arrow about the rotation shaft 316C.

  The first moving mechanism 33 moves the cap holding part 316 in the vertical direction (perpendicular to the base 12). The range of movement of the cap holder 316 is the distance V. The second moving mechanism 34 moves the column holding unit 322 in the vertical direction. The range of movement of the column holding unit 322 is the distance V2. That is, in this example, while the first moving mechanism 33 moves the cap holding part 316 downward from the initial position to the distance V1 (V1 <V), the column holding part 322 is stopped, but the cap holding part 316 is stopped. When the movement amount exceeds the distance V1, the first moving mechanism 33 moves the cap holding portion 316, and the second moving mechanism 34 moves the column holding portion 322 downward from the initial position. The amount of movement of both is the distance V2. That is, in the vertical direction, there are a case where the cap holding part 316 moves alone and a case where the cap holding part 316 and the column holding part 322 move synchronously.

  The second moving mechanism 34 also moves the column holding part 322 in the front-rear direction (horizontal with respect to the base 12). The range of movement of the column holding unit 322 is the distance H1. The second moving mechanism 34 further moves the collection tube holding portion 325 in the front-rear direction. The range of movement of the collection tube holder 325 is a distance H. That is, the second moving mechanism 34 integrally moves the column holding part 322 and the collection tube holding part 325 from the initial position to the distance H1 backward. When the distance H1 is exceeded, the movement of the column holding part 322 stops, but the collection tube holding part 325 can move backward even if the distance H1 is exceeded. The moving amount of the collection tube holder 325 after the distance H1 is exceeded is the distance H2. That is, the second moving mechanism 34 moves the collection tube holding portion 325 in two steps in the horizontal direction.

  FIG. 5 shows a mode of movement of the pressurizing column cap 311, the solid phase extraction column 321, the waste liquid tank 323, and the recovery tube 324 by the first moving mechanism 33 and the second moving mechanism 34. It is a corresponding side surface schematic diagram. In the figure, the first moving mechanism 33, the second moving mechanism 34, the cap holding part 316, and the column holding part 322 are not shown. In the figure, the right is the front (front) side and the left is the rear (back) side.

  In the initial position shown in FIG. 5 (a), the pressurizing column cap 311 is located at the upper and rearmost part, the solid phase extraction column 321 is located at the middle and middle part, and the recovery tube 324 is located at the lower and foremost part. To do. The waste liquid tank 323 is located in the lower stage, behind the collection tube 324 and directly below the solid phase extraction column 321. In the first movement state, the solid phase extraction column 321 and the waste liquid tank 323 move rearward by the second movement mechanism 34 (not shown here). These movement amounts are the distance H1.

  As a result, the solid phase extraction column 321 is positioned immediately below the pressurizing column cap 311 and the waste liquid tank 323 is positioned directly below the solid phase extraction column 321 as shown in FIG. The collection tube 324 also moves rearward (distance H1) as the waste liquid tank 323 moves, but the upper portion of the collection tube 324 is open as shown in the figure. The movement of the solid phase extraction column 321 and the waste liquid tank 323 may or may not be simultaneous. As a result of the rearward movement at a distance H1, the vertical axis centers of the pressurizing column cap 311 and the solid phase extraction column 321 substantially coincide with each other in the vertical direction as shown in FIG. What is necessary is just to come to be located directly under these.

  In the second movement state, the pressurizing column cap 311 is moved downward by the first movement mechanism 33 (not shown here). When the amount of movement of the pressurizing column cap 311 reaches the distance V1, it comes into contact with the upper end of the solid phase extraction column 321 located in the middle stage (FIG. 3C).

  As shown in FIG. 4D, the first moving mechanism 33 further moves the pressurizing column cap 311 downward, and the second moving mechanism 34 (not shown) moves the solid-phase extraction column 321 accordingly. Move downward. These movement amounts are the distance V2. That is, the solid-phase extraction column 321 moves the distance V2 while being sealed by the pressurizing column cap 311.

  Thereafter, for example, only the movement in the vertical direction returns to the initial position (FIG. 5B), and then transitions to the third movement state. In the third movement state, the collection tube 324 (and the waste liquid tank 323) is moved rearward by the second movement mechanism 34 (not shown). These movement amounts are the distance H2.

  As a result, as shown in FIG. 5E, the solid phase extraction column 321 is positioned immediately below the pressurizing column cap 311 and the recovery tube 324 is positioned directly below the solid phase extraction column 321. The waste liquid tank 323 also moves rearward (distance H2) as the collection tube 324 moves, but the upper part of the waste liquid tank 323 is open as shown in the figure. The solid phase extraction column 321 and the waste liquid tank 323 (recovery tube 324) may return to the initial position shown in FIG. 5A, in which case the solid phase extraction column 321 moves backward (the amount of movement is The distance H1) and the collection tube 324 also move rearward (the amount of movement is distance H1 + H2 = H). These movements may be performed simultaneously or not simultaneously. That is, the solid-phase extraction column 321 moves backward (the amount of movement is the distance H1), and the collection tube 324 also moves backward (the amount of movement is the distance H from the initial position, or the distance H2 from the position of the second movement state). As a result, as shown in FIG. 5E, the vertical axis centers of the pressurizing column cap 311, the solid phase extraction column 13, and the collection tube 324 may be substantially aligned in the vertical direction.

  In the fourth moving state, the pressurizing column cap 311 is moved downward by the first moving mechanism 33 (not shown here). When the amount of movement of the pressurizing column cap 311 reaches the distance V1, it comes into contact with the upper end of the solid-phase extraction column 321 located in the middle stage (f).

  The first moving mechanism 33 further moves the pressurizing column cap 311 downward, and accordingly, the second moving mechanism 34 (not shown) moves the solid-phase extraction column 321 downward. These movement amounts are the distance V2. That is, the solid-phase extraction column 321 is moved by the distance V2 in a state of being sealed by the pressurizing column cap 311 and the lower end of the solid-phase extraction column 321 is included in the recovery tube 324 ((g) in the figure). .

  As an example, the cap holding part 316 is biased toward the initial position (upward) by biasing means (for example, a coil spring), and the column holding part 322 is also biased toward the initial position (upward). It is biased by means (for example, a coil spring or the like). Therefore, by pressing the first moving mechanism 33 and the second moving mechanism 34 downward against the urging force, the cap holding part 316 and the column holding part 322 move downward, and when the pressing is released, the cap The holding unit 316 and the column holding unit 322 return to the initial positions. However, the present invention is not limited to this, and the motor may be returned to the initial position. Further, the horizontal movement of the second moving mechanism 34 is performed by a motor or the like.

  Next, the dispensing unit 5 will be described with reference to FIG.

  The dispensing unit 5 includes a cylinder holding unit 51 that holds a plurality of (for example, eight) dispensing cylinders 50 and a driving unit 52. The drive unit 52 includes a first drive unit 521 that individually moves the plurality of dispensing cylinders 50 and a second drive unit 522 that moves the dispensing unit 5 integrally. In this example, the cylinder holding part 51 holds four dispensing cylinders 501 for 1000 μL on the front side (front side) and four dispensing cylinders 502 for 20 μL on the rear side (back side). Show.

  The dispensing unit 5 is freely moved and controlled in a three-dimensional space according to a sequence control by a computer along a guide rail provided on the ceiling of the housing 1 shown in FIG. Further, each of the dispensing cylinders 50 of the dispensing unit 5 can be moved in the vertical direction or the front-rear direction with respect to the dispensing unit 5 individually or simultaneously. Dispensable tip 54 is attached to the tip of dispensing cylinder 50. That is, a 1000 μL tip 541 is attached to the 1000 μL dispensing cylinder 501, and a 20 μL tip 542 is attached to the 20 μL dispensing cylinder 502. The tip 54 can be engaged by being pushed into the tip portion of the dispensing cylinder 50, and can be easily detached when the tip 54 and the dispensing cylinder 50 are pulled apart in the opposite direction.

  Referring to FIG. 3, when attaching the tip 54 to the dispensing cylinder 50, the dispensing cylinder 50 is inserted into the hole of the tip rack 6. Thereby, the chip | tip 54 accommodated in the chip | tip rack 6 is pushed into the front-end | tip of the dispensing cylinder 50, and is attached. On the other hand, when the tip 54 is detached, the dispensing cylinder 50 is inserted into the waste tip storage area 8. The waste chip storage 8 is provided with a separation part 81 in which a large hole part 81b having a diameter larger than the diameter of the chip 54 and a small hole part 81s having a diameter smaller than the diameter of the chip 54 are continuous (see FIG. 3). When the dispensing cylinder 50 is inserted into the hole portion 81b and the dispensing cylinder 50 is moved up to the small hole portion 81s to raise the dispensing cylinder 50, the small hole portion 81s restricts the tip 54 from being lifted up. Detach from the tip of the cylinder 50. The detachment portion 81 is also provided with two sizes corresponding to the sizes of the chips 541 and 542. In this way, the chip 54 can be automatically attached and detached.

  A pressing portion 55 is provided at the rear end portion of the cylinder holding portion 51 to press down the pressurizing column cap 311.

  As described above, in the nucleic acid extraction apparatus 10, the driving unit 52 of the nucleic acid extraction unit 3 and the dispensing unit 5 moves the nucleic acid extraction unit 3 and the dispensing unit 5 to a predetermined position by the nucleic acid extraction processing program. Nucleic acid extraction processing for extracting nucleic acid from the solution and processing for preparing a solution containing the nucleic acid can be performed.

  That is, the control unit CT drives the dispensing unit 5 to dispense the sample to the solid phase extraction column 321 and pressurizes the solid phase extraction column 321 in the nucleic acid extraction unit 3 to separate the sample and the nucleic acid. Further, the control unit CT drives the dispensing unit 5 to dispense the separated nucleic acids into the PCR tubes 43 of each block, and further dispenses various primer solutions into each PCR tube 43. Note that a fluorescent lamp, a germicidal lamp, and the like are also provided inside the nucleic acid extraction apparatus 10.

  Next, the procedure of the nucleic acid extraction process executed by the nucleic acid extraction apparatus 10 of this embodiment will be described with reference to the protocol diagram of FIG. The numbers in parentheses in FIG. 7 indicate processing steps. Further, Step P1 and Step P2 are pre-processing steps performed manually by the operator, and the steps after Step 1 are automatically executed by the nucleic acid extraction apparatus 10. In this example, the case where the operator performs steps P1 and P2 manually will be described as an example. However, by incorporating these into the program, the nucleic acid extraction apparatus 10 can automatically execute them.

  Note that the protocol diagram of FIG. 7 shows an example of nucleic acid extraction processing when nucleic acid is extracted from a patient's eyelid. Moreover, the code | symbol of each structure of the nucleic acid extraction apparatus 10 in the following description shall show each structure shown in FIGS.

  <Pretreatment process>

  In Step P1, 23 μL of protease is dispensed into four specimen tubes 20 (20A to 20D) for proteolysis (digestion treatment). The sample tube 20 is prepared for each patient, for example (see FIG. 3).

  In Step P2, 200 μL of sample is dispensed into each sample tube 20. The specimen is a sputum of patients A to D, and the sputum of patient A in the sample tube 20A, patient B in the specimen tube 20B, patient C in the specimen tube 20C, and patient D in the specimen tube 20D, respectively. Noted. The sample tube 20 is placed on the sample place 21 of the nucleic acid extraction apparatus 10.

  Further, the chip rack 6 (6a, 6b) in which the chips are sufficiently accommodated is placed, a new solid-phase extraction column 321 is placed in the column holder 322, and a new collection tube 324 is collected in the collection tube holder 325. In addition, new PCR tubes 43 are placed on the PCR tube holder 41, respectively. In addition, a reagent tank 9 and a primer solution (predetermined primer, PCR reagent, enzyme, buffer solution, dNTP (a mixture of four types of deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP)) and water The primer tube 44 containing the prepared solution is placed at a predetermined position of the nucleic acid extraction apparatus 10.

  <Nucleic acid extraction process (automatic operation by the control unit CT)>

  In step 1, the nucleic acid extraction apparatus 10 is initialized. The control unit CT moves the pressurizing unit 31 and the separating unit 32 to the initial positions shown in FIG. The control unit CT drives the second drive unit 522 to move the dispensing unit 5 to the initial position. The initial position of the dispensing unit 5 is, for example, above the tip rack 6a.

  The control unit CT drives the first drive unit 521 to lower the dispensing unit 5 and insert the tip of each dispensing cylinder 501 (501A, 501B, 501C, 501D) into the tip rack 6a, and dispense. A 1000 μL tip (first tip 541) is attached to the tip of the cylinder 501 (see FIG. 3). Hereinafter, although explanation about the 1st drive part 521 and the 2nd drive part 522 is omitted, when moving dispensing part 5, the 2nd drive part 522 is driven and dispensing cylinder 50 is collectively or each. When moving to, the first drive unit 521 is driven.

  Thereafter, the control unit CT moves the dispensing unit 5 to a predetermined position on the reagent tank 9 and sucks 200 μL of the buffer AL into each of the dispensing cylinders 501.

  The control unit CT moves the dispensing unit 5 to the sample place 21 and disposes each dispensing cylinder 501 (501A, 501B, 501C, 501D) above each sample tube 20A to 20D. Then, each dispensing cylinder 501 is lowered, and the entire amount of the buffer AL is discharged to each of the sample tubes 20A to 20D. Thereafter, the control unit CT moves the dispensing unit 5 to the disposal tip storage site 8, lowers each dispensing cylinder 501 to detach and discard the first tip 541, and then moves the dispensing unit 5 to the initial position. To do.

  In step 2, the mixture of the buffer AL and the sample is stirred for 15 seconds for each of the sample tubes 20A to 20D. In the subsequent step 3, these are incubated (cultured) at a temperature of 56 ° C. for a predetermined time (10 minutes to 120 minutes, preferably 10 minutes to overnight). As described above, the specimen storage 21 has a stirring function and a temperature control function.

  In step 4, a new tip 54 is mounted on each dispensing cylinder 501 (tips are replaced). That is, the control unit CT moves the dispensing unit 5 onto the tip rack 6a, lowers it, and attaches the second tip 541 (1000 μL tip) to each dispensing cylinder 501. Thereafter, the control unit CT moves the dispensing unit 5 onto the reagent tank 9, sucks 200 μL of ethanol, and moves the dispensing unit 5 above the sample tubes 20A to 20D. The controller CT discharges the entire amount of ethanol to each of the sample tubes 20A to 20D. Thereafter, the control unit CT moves the dispensing unit 5 to the disposal tip storage 8 and lowers each dispensing cylinder 501 to remove and discard the second tip 541, and then put the dispensing unit 5 to the initial position. Moving. In Step 5, the control unit CT stirs the sample tubes 20A to 20D for 15 seconds.

  Next, in Step 6, the control unit CT moves the dispensing unit 5 onto the tip rack 6a, lowers it, and attaches a new third tip 541 (1000 μL tip) to each dispensing cylinder 501. The dispensing unit 5 is moved above the sample tubes 20A to 20D. The control unit CT descends the dispensing unit 5 and sucks the mixed liquid in the sample tubes 20A to 20D by each dispensing cylinder 501. Thereafter, the control unit CT moves the dispensing unit 5 above the solid phase extraction column 321, and discharges the entire amount of the liquid mixture to each of the solid phase extraction columns 321 (321A, 321B, 321C, 321D). Thereafter, the control unit CT moves the dispensing unit 5 to the disposal tip storage 8 and lowers the dispensing cylinder 501 to disengage and discard the third tip 541.

  Next, the control unit CT is located immediately below each pressurizing column cap 311 (311A, 311B, 311C, 311D) to which each solid-phase extraction column 321 corresponds, and the waste liquid tank 323 is located below the solid-phase extraction column 321. Thus, the column holding part 322 and the collection tube holding part 325 are moved rearward (first movement state shown in FIGS. 5A and 5B). Then, the dispensing unit 5 is moved down above the cap holding unit 316 and then lowered, and the cap holding unit 316 is moved downward by the pressing unit 55 of the dispensing unit 5. As a result, the pressurizing column cap 311 descends and comes into contact with the corresponding solid-phase extraction column 321 (second moving state shown in FIGS. 5B and 5C). When the control unit CT further lowers the dispensing unit 5, both move further downward with the solid-phase extraction column 321 sealed by the pressurizing column cap 311 (FIG. 5D).

  In Step 7, the control unit CT controls the pressurizing syringe 313 of the pressurizing unit 31 to apply a predetermined air pressure to each of the sealed solid-phase extraction columns 321 to increase the pressure to a pressure exceeding the set pressure threshold. First pressurization is performed. Thereafter, the control unit CT stops pressurization (increase in pressure) and maintains the application state of pressure exceeding the set pressure threshold for the solid phase extraction column 321 for a predetermined time. By pressurizing and stopping the pressurization for a predetermined time, each solid phase extraction column 321 has at least a part of the nucleic acid adsorbed on the filter, and the waste liquid is discharged (eluted) into the waste liquid tank 323. As a result, the pressure applied to each solid phase extraction column 321 is reduced (natural pressure reduction) within a predetermined time and falls below the set pressure threshold. When the control unit CT releases the pressure of the pressurizing unit 31, the pressurizing column caps 311 and the solid phase extraction columns 321 are raised. Even after the solid-phase extraction column 321 stops at the initial position, the pressurizing column cap 311 further rises, returns to the initial position, and opens above the solid-phase extraction column 321. Thereby, the solid phase extraction column 321 is opened under atmospheric pressure, and the pressure applied to the solid phase extraction column 321 is reduced. In this embodiment, “pressurization” means a state in which the pressure is increased, “stopping pressurization” means a state in which the increase (change) in pressure is stopped, and “decompression” means the pressure. The state that is decreasing. In addition, “depressurization” of the present embodiment will be described by taking as an example a case where the solid-phase extraction column 321 is opened under atmospheric pressure to return from the pressure exceeding the set pressure threshold to atmospheric pressure (decrease the pressure). The pressure reduction includes a case where the pressure is reduced from the pressure exceeding the set pressure threshold value by suction instead of (or in addition to) opening to the atmospheric pressure.

  In step 8, the control unit CT again moves the cap holding unit 316 downward by the pressing unit 55 of the dispensing unit 5. As a result, the pressure column cap 311 is lowered, and the solid-phase extraction column 321 is sealed. In this state, the pressurizing syringe 313 of the pressurizing unit 31 is controlled to apply a predetermined air pressure (increase the pressure) to each of the sealed solid-phase extraction columns 321 to perform the second pressurization. The control part CT maintains the application state of the pressure set exceeding the set pressure threshold for a predetermined time (stops pressurization (increase in pressure)) (FIGS. 5B to 5D).

  The pressurization settings (pressurization (air supply) speed, air supply amount) of the second pressurization are equivalent to the pressurization settings of the first pressurization as an example, and the time for maintaining the pressurization (acceleration) The pressure stop time) is also equivalent to the first pressurization. In the first pressurization, the entire amount of the mixed solution containing the specimen cannot be eluted, and the nucleic acid may remain in the specimen. However, in the present embodiment, by performing the second pressurization (and the second pressurization stop), even if the liquid mixture containing the specimen remains in each solid phase extraction column 321, all The nucleic acid in the specimen is eluted and adsorbed on the filter, and the waste liquid is discharged to the waste liquid tank 323. Thereby, the pressure applied to each solid phase extraction column 321 is reduced (natural reduced pressure). When the control unit CT releases the pressing of the pressurizing unit 31, the pressurizing column cap 311 and the solid phase extraction column 321 rise. Even after the solid-phase extraction column 321 stops at the initial position, the pressurizing column cap 311 further rises and returns to the initial position, and the upper part of the solid-phase extraction column 321 is opened, so that the solid-phase extraction column 321 is under atmospheric pressure. Opened and the pressure applied to the solid phase extraction column 321 decreases. Thereafter, the control unit CT returns the dispensing unit 5, the pressurizing unit 31, and the separating unit 32 to the initial positions.

  Thus, in this embodiment, after pressurizing each solid-phase extraction column 321 to a pressure exceeding the set pressure threshold, the pressurization is stopped and each solid-phase extraction column 321 is kept at a pressure exceeding the set pressure threshold for a predetermined time. A nucleic acid is extracted from a specimen by performing a pressurization / depressurization cycle that is held and then decompressed, dispensed to each solid-phase extraction column 321 and transferred multiple times (here, twice). is there. By performing the first pressurization and the second pressurization, the entire amount of the sample in each solid phase extraction column 321 can be surely eluted, and the nucleic acid in the sample can be adsorbed to the filter F.

  In Step 9, the control unit CT moves the dispensing unit 5 onto the tip rack 6a, lowers it, and attaches the fourth tip 541 (1000 μL tip) to each dispensing cylinder 501. Thereafter, the control unit CT moves the dispensing unit 5 onto the reagent tank 9 and sucks 500 μL (750 μL) of the buffer AW1 (cleaning solution), and then moves the dispensing unit 5 onto the column holding unit 322, The entire amount of the buffer AW1 is discharged to the extraction column 321. Thereafter, the control unit CT moves the dispensing unit 5 to the disposal tip storage area 8, lowers the dispensing cylinder 501 to detach the fourth tip 541 and discards it, and then moves the dispensing unit 5 to the initial position. (See FIG. 3).

  In Step 10, the control unit CT determines that the solid phase extraction column 321 is positioned immediately below the pressurizing column cap 311, and the column holding unit 322 and the recovery tube are positioned so that the waste liquid tank 323 is positioned below the solid phase extraction column 321. The holding part 325 is moved backward (FIG. 5B). Then, the dispensing unit 5 is moved down above the cap holding unit 316 and then lowered, and the cap holding unit 316 is moved downward by the pressing unit 55 of the dispensing unit 5. As a result, the pressurizing column cap 311 is lowered, and the solid-phase extraction column 321 is sealed, and both further move downward (FIGS. 5C and 5D).

  Then, the control unit CT controls the pressurizing syringe 313 of the pressurizing unit 31 to apply a predetermined air pressure (increase the pressure) to each of the sealed solid-phase extraction columns 321 to perform the third pressurization. The filter of each solid phase extraction column 321 is washed. The nucleic acid is washed by the buffer AW1 while adsorbed on the filter, and the washing liquid is discharged to the waste liquid tank 323 (FIG. 5 (d)). Thereafter, the control unit CT releases the pressure of the pressurizing unit 31 and raises the pressurizing column cap 311 to open the upper side of the solid phase extraction column 321. The cap holding part 316 returns to the initial position.

  In step 11, the control unit CT moves the dispensing unit 5 onto the tip rack 6 a, lowers it, and attaches the fifth tip 541 (1000 μL tip) to each dispensing cylinder 501. Thereafter, the control unit CT moves the dispensing unit 5 onto the reagent tank 9, sucks 500 μL (750 μL) of the buffer AW2 (cleaning solution), moves the dispensing unit 5 onto the column holding unit 322, and extracts each solid phase. The entire amount of the buffer AW2 is discharged to the column 321. Buffer AW1 and buffer AW2 are cleaning liquids having different alcohol concentrations. Thereafter, the control unit CT moves the dispensing unit 5 to the disposal tip storage site 8, lowers each dispensing cylinder 501, removes and discards the fifth tip 541, and moves the dispensing unit 5 to the initial position. (See FIG. 3).

  In step 12, the control unit CT determines that the solid phase extraction column 321 is positioned immediately below the pressurizing column cap 311 and the waste tank 323 is positioned below the solid phase extraction column 321, and the column holding unit 322 and the recovery tube. The holding part 325 is moved backward (FIG. 5B). Then, the dispensing unit 5 is moved down above the cap holding unit 316 and then lowered, and the cap holding unit 316 is moved downward by the pressing unit 55 of the dispensing unit 5. As a result, the pressurizing column cap 311 is lowered, and the solid-phase extraction column 321 is sealed, and both further move downward (FIGS. 5C and 5D).

  Then, the control unit CT controls the pressurization syringe 313 of the pressurization unit 31 to apply a predetermined air pressure (increase the pressure) to each of the sealed solid-phase extraction columns 321 to perform the fourth pressurization. The filter of each solid phase extraction column 321 is washed. The nucleic acid is washed by the buffer AW2 while adsorbed on the filter, and the washing liquid is discharged to the waste liquid tank 323. Thereafter, the control unit CT releases the pressure of the pressurizing unit 31 and raises the pressurizing column cap 311 to open the upper side of the solid phase extraction column 321. The cap holding part 316 returns to the initial position.

  In step 13, the control unit CT determines that the solid-phase extraction column 321 is positioned immediately below the pressurizing column cap 311 and the four recovery tubes 324 (324A, 324B, 324C, 324D) correspond to the respective solid-phase extraction columns 321. The column holding part 322 and the collection tube holding part 325 are moved rearward so as to be positioned immediately below (fourth movement state shown in FIGS. 5D and 5F).

  In step 14, the control unit CT moves the dispensing unit 5 onto the tip rack 6 a and lowers it to mount the sixth tip 541 (1000 μL tip) on each dispensing cylinder 501. Is moved onto the reagent tank 9 and 500 μL of buffer AE (recovery solution) or distilled water is sucked. Subsequently, the control unit CT moves the dispensing cylinder 501 onto each solid phase extraction column 321, and discharges the entire amount of the solution for recovery or distilled water to each. Thereafter, the solid phase extraction column 321 is allowed to stand at room temperature for 1 to 5 minutes. The control unit CT moves the dispensing unit 5 to the disposal tip storage area 8, lowers the dispensing cylinder 501 to disengage and discard the sixth tip 541, and moves the dispensing unit 5 to the initial position.

  In step 15, the dispensing unit 5 is moved down above the cap holding unit 316 and then lowered, and the cap holding unit 316 is moved downward by the pressing unit 55 of the dispensing unit 5. As a result, the pressurizing column cap 311 is lowered and the solid-phase extraction column 321 is sealed, and both of them move further downward. The pressurizing column cap 311 seals the solid-phase extraction column 321 (FIG. 5 ( e) (f)).

  Then, the control unit CT controls the pressurization syringe 313 of the pressurization unit 31 to apply a predetermined air pressure (increase the pressure) to each of the sealed solid-phase extraction columns 321 to perform the fifth pressurization. Do. Thereby, the nucleic acid adsorbed on the filter of each solid-phase extraction column 321 is discharged together with the buffer AE or distilled water to the corresponding collection tube 324, and 500 μL of the nucleic acid purification solution is obtained (FIG. 5 (g)).

  Control part CT cancels the press of pressurization part 31, and moves separation part 32 and dispensing part 5 to an initial position.

  <Primer solution dispensing process (automatic operation by the control unit CT)>

  In the subsequent steps, a primer solution is dispensed into the obtained nucleic acid purification solution.

  That is, in Step 16, the control unit CT moves the dispensing unit 5 onto the tip rack 6b, lowers it, and attaches the seventh tip 542 (20 μL tip) to each dispensing cylinder 502 (FIG. 2). reference). Then, the control unit CT moves the dispensing unit 5 above each collection tube 324 (324A, 324B, 324C, 324D) that is open at the top. The control unit CT sucks the nucleic acid purification solution from each collection tube 324 and discharges it to the PCR tube 43. The nucleic acid purification solution in the collection tube 324A is discharged to all the PCR tubes 43 in the block B1, the nucleic acid purification solution in the collection tube 324B is discharged to all the PCR tubes 43 in the block B2, and the nucleic acid purification solution in the collection tube 324C. Is discharged to all the PCR tubes 43 in the block B3, and the nucleic acid purification solution in the recovery tube 324D is discharged to all the PCR tubes 43 in the block B4. In this example, different samples of nucleic acids are extracted from the blocks B1, B2, B3, and B4.

  Thereafter, various primer solutions are dispensed into the PCR tubes 43 of the blocks B1 to B4. That is, the control unit CT moves the dispensing unit 5 to the disposal tip storage site 8, lowers the dispensing cylinder 501 to remove and discard the seventh tip 542, and places the dispensing unit 5 on the tip rack 6b. For example, only the dispensing cylinder 502A at the left end is lowered and the eighth tip 542 (20 μL tip) is attached. Then, the dispensing unit 5 is moved above the primer holding unit 42, the primer solution (first primer solution) is sucked by the dispensing cylinder 502A, and discharged to the first PCR tube 43 of the block B1. Thereafter, the eighth tip 542 (20 μL tip) of the dispensing cylinder 502A at the left end is discarded, and the ninth tip 542 (20 μL tip) is mounted. Then, the dispensing unit 5 is moved above the primer holding unit 42, the next primer solution (second primer solution) is sucked by the dispensing cylinder 502A, and discharged to the second PCR tube 43 of the block B1. Thereafter, the tip of the dispensing cylinder 502A is replaced for each primer solution, and a plurality of primer solutions (for example, 16 types of primer solutions) in the primer holding unit 42 are dispensed with the block B1, that is, the patient A nucleic acid purification solution. Dispense into 16 PCR tubes 43. The same processing is performed for blocks B2 to B4. In addition, since there may be one kind of specimen, in this embodiment, the case where the primer solution is dispensed for each block has been described as an example, but the primer solution may be dispensed in any order.

  When the automatic operation is completed as described above, a mixed solution in which 16 types of primer solutions are dispensed into the nucleic acid purification solution for each patient (specimen) is prepared for four patients.

  After the nucleic acid extraction apparatus 10 is stopped, the operator takes out the primer holding unit, amplifies it in a real-time PCR process, and measures absorbance at 230 nm, 260 nm, and 280 nm.

  Thus, according to the present embodiment, after the operator dispenses the sample of the protease into the sample tube prepared for each sample, the nucleic acid extraction apparatus from the nucleic acid extraction step to the primer solution dispensing step is performed. 10 enables automatic continuous processing. As described above, the pre-processing steps of steps P1 and P2 can be automated.

  In FIG. 7, step 1 and subsequent steps are automatically controlled by the nucleic acid extraction apparatus 10. However, when the incubation process in step 3 is for a long time (for example, 2 hours or more), the apparatus operating rate decreases. Therefore, it is good also as a program which controls automatically after Step 4 shown with a broken line.

  As an example, this embodiment is suitable for pretreatment of the HIRA-TAN (Human cell-controlled Identification of the Respiratory Agent from TAN (痰)) method described in Japanese Patent No. 4665203. The HIRA-TAN method is a rapid diagnostic method used for diagnosis of respiratory infections. Since the HIRA-TAN method is a determination method for identifying pathogens using quantitative PCR amplification reactions specific to various pneumococcus and human cells, the operation for each primer solution, that is, the type of bacteria to be identified It is necessary to prepare as many primer solutions and PCR solutions as possible, and to dispense specimens (patients). In this method, for example, when 16 types of primer solutions exist, 16 PCR tubes are prepared for one patient, the primer solution dispensing operation needs 16 times, and the sample dispensing operation requires 16 times. In addition to complicated operations, problems such as errors due to repeated simple operations, occurrence of contamination, and infection to the operator arise.

  In this embodiment, by automating these operations, it is possible to avoid mistakes in complex operations, avoid errors due to contamination, reduce the infection and burden on the operator, and thus reduce the cost of judgment. it can.

  In addition, since a centrifuge is not required in the nucleic acid extraction step, the apparatus can be reduced in size and cost. Furthermore, in the nucleic acid extraction process, since the operation is complicated, many steps are required, and time is required, it has been a part that is desired to be automated. According to this embodiment, the operation time is shortened and the reproducibility of the results is improved by automation Can be realized.

  <Detection of column clogging>

  As described above, in the present embodiment, nucleic acid is extracted by pressurizing the solid-phase extraction column 321, but column clogging can be detected by monitoring the pressure of the solid-phase extraction column 321. .

  FIG. 8 is a diagram for explaining the error detection function of the pressurizing unit 31. FIG. 8A is a diagram illustrating a normal state, and FIG. 8B is a diagram illustrating a state in which an error due to column clogging is detected. FIG.

  As shown in FIG. 8A, when the nucleic acid extraction unit 3 (pressurization unit 31) is operating normally, a predetermined first time from the start of pressurization (pressure increase) of the pressurization unit 31 Within T1, the set pressure threshold is exceeded and the maximum pressure is reached. Then, when the pressurization is stopped and a predetermined second time T2 elapses in this state, the sample of the solid phase extraction column 321 is eluted, and at the same time, the inside of the solid phase extraction column 321 is decompressed (natural decompression), Within the third time T3, the pressure falls below the set pressure threshold.

  However, when the solid phase extraction column 321 is clogged (filter clogged), such as when the viscosity of the specimen is particularly high, as shown in FIG. The pressure in the phase extraction column 321 may not be reduced (natural pressure reduction) and may not fall below the set pressure threshold.

  In the present embodiment, the pressure sensor 315 of the pressurizing unit 31 ensures that the pressure in the solid phase extraction column 321 does not fall below the set pressure threshold within the second time T2 (the pressure exceeding the set pressure threshold is If the time applied to the extraction column 321 exceeds the second time T2), the operation of the nucleic acid extraction apparatus 10 is temporarily stopped, and the sealed state of the solid-phase extraction column 321 by the pressurizing unit 31 is released. Return to atmospheric pressure. In that case, an error display is displayed on the display unit or the like (and voice may be output) to notify that the column is clogged. After notification of the error, an additional pressure increase / decrease cycle in steps 7 to 8 is automatically executed. After notification of the error, the operation of the nucleic acid extraction apparatus 10 is stopped, and the additional pressurization / decompression cycle in steps 7 to 8 is executed on condition that the operator cancels the error (for example, operation of the release button). You may do it.

  In the conventional extraction column centrifugation method, after completion of the centrifugation process for a predetermined time, an operator visually checks the discharged amount of waste liquid in the tube to determine whether the column is clogged. In this case, unless each centrifugation operation is completed, column clogging cannot be determined, which causes an increase in operation time. On the other hand, in this embodiment, the general elution time (second time T2) has elapsed after the solid-phase extraction column 321 has been pressurized (increase in pressure) and stopped (held under a constant pressure). However, if the pressure does not fall below the set pressure threshold, it can be immediately determined that the column is clogged, so that the work time when the column is clogged can be greatly reduced.

  The pressurizing speed and holding time by the pressurizing unit, the second time T2 when determining that the column is clogged, the set pressure threshold value, and the number of treatments can be appropriately selected depending on the type and amount of the specimen. For example, the solid-phase extraction column 321 is pressurized (increase in pressure), and stopped / pressurized (maintained under a constant pressure), and subjected to a depressurization process (release to atmospheric pressure) five times, and the fifth column pressure Judgment of column clogging may be performed. Further, when the amount of specimen is small or large, the elution time (second time T2) may be set short or long.

  9 and 10 are examples of screen transitions displayed on the display unit.

  The display unit 2 is provided with various switches and keys. By operating these switches, various control conditions can be input and programs can be changed. The nucleic acid extraction apparatus 10 of this embodiment can be operated as a stand-alone.

  Control conditions can also be input from the display unit 2 to the control unit CT, whereby data and programs in the data storage unit inside the control unit CT can be rewritten and changed. The program is executed according to the control conditions input from the display unit 2, and the procedure of the nucleic acid extraction process is executed.

  Further, the display unit 2 can display work procedures, work currently being executed, and the like. Various parameter settings are also possible. Examples of parameter settings include filtration settings for solid phase extraction column 321 (setting of air supply volume, air supply speed, limit pressure, etc. for each pressurizing step) and stirring settings (stirring time, stirring speed, acceleration, etc.) Setting). Also, a normal end display that informs you that the automatic operation has been completed normally, such as an error display that informs you that the automatic operation has been terminated due to a chip shortage, an emergency stop display that informs you that the operation is being stopped due to some abnormality, etc. Also do. Further, for example, during the pressurization process, the filtration pressure current graph can be displayed by switching the display of the display unit. The filtration pressure current graph displays the pressurization state in real time by a sensor that monitors the pressurization sensor of the pressurization unit.

  FIG. 11 shows an example of a conventional method in which a nucleic acid (here, DNA) is prepared by extraction column centrifugation to identify a pathogen, and a nucleic acid (here, DNA) is prepared using the nucleic acid extraction apparatus 10 of the present embodiment. It is the figure which summarized the result of the experiment which identified the pathogen. The left of the table is the result of the conventional method, and the right is the result of the automatic preparation of the present embodiment. In the conventional method, a centrifuge is used, so there are cases where it is called a centrifugal method. In this embodiment, compressed air is sent to the solid phase extraction column.

  In the conventional method (centrifugation method), a plurality of specimens are manually dispensed for nucleic acid extraction, DNA is extracted using a centrifuge, and each specimen is manually added to the DNA purification solution of each specimen. A plurality of primer solutions were dispensed into and subjected to PCR analysis. On the other hand, in the pumping method using the nucleic acid extraction apparatus 10 according to the present embodiment, as described above, the automation processing was performed on the plurality of samples. Note that the steps from Step P1 to Step 3 in FIG.

  In addition, for identification of pathogens, both the conventional method and the present embodiment employ the HIRA-TAN method for distinguishing the pathogenic pathogens of acute respiratory infection. Briefly explaining the HIRA-TAN method, the pathogen to be differentiated is an airway colonizing pathogen, and firstly in a nucleic acid (for example, DNA) extracted from a specimen containing the target respiratory tract secretion (for example, a patient's sputum). Then, the copy number of the gene derived from the target cell is measured, and the copy number of the gene derived from the pathogen in the extracted DNA is measured. And the relative number of the copy number of the gene derived from a pathogen with respect to the copy number of the gene derived from the object cell is calculated. This process is performed for a plurality of objects, and a relative number is obtained for each object. Then, by comparing the relative number of each subject with the clinical diagnosis result of each subject, the lower limit of the relative number of positive pathogenic pathogens in clinical diagnosis; and / or the relative negative of pathogenic pathogens in clinical diagnosis This is a technique for determining whether or not a pathogen is an infectious pathogen based on the upper limit of the number as a discrimination reference value and based on the discrimination reference value and the relative number of gene copies.

In the HIRA-TAN method, the relative copy number of a gene (pathogen) in a specimen is represented by the cycle number (Ct value: Cycle threshold) when a certain fluorescence intensity is reached in PCR analysis. Then, a relative amount of pathogen cells ΔCt has a human cell with an internal control as shown in FIG. 11, Ct value of human cells as a control (comparison) from _{(Ct human),} Ct value of a certain pathogen _{(Ct pathogen} ) Is subtracted (ie,-(Ct _pathogen -Ct _human ). Here, pathogens in cases of pulmonary inflammation are identified, and the differential cutoff value (Positive cutoff value) of these pathogens is LytA> -4, P.aer> -2, KPNE> -4, HINF> -1, and MCAT> 0.

  As a result, of all the samples, only the sample 305 indicated by the broken line is determined to be different, and the correct answer of the pumping method of the present embodiment compared to the centrifugal method is assumed when the determination result of the conventional method is assumed to be a correct answer. The rate (match rate) was 95%. However, in the sample 305, ΔCt by the centrifugal method is negative at −4.03, and ΔCt by the pumping method is positive at −3.87. In this case, the discrimination reference value of the pathogen LytA is -4, which are all close to the discrimination reference value and are difficult to determine. However, in the HIRA-TAN method, ΔCt is based on past subject statistical analysis and necessarily includes an error, so a result closer to a positive value is actually more preferable. Considering these, it can be said that ΔCt (−4.03) of the centrifugation method is a false positive. Therefore, it can be said that the pumping method of the present embodiment in which this is determined as positive is superior.

  FIG. 12 is also a diagram showing a comparison between the centrifugal method of the conventional method and the pumping method of the present embodiment, and is a result of identification of the pathogen.

  In addition, in the conventional method and the preparation method of this embodiment, said step P1-3 is performed in common.

  The number of patients (specimens) is 50, and the number of fixed pathogens to be treated and the number of non-fixed pathogens to be treated are the same in both the conventional method and the present embodiment.

Further, the average DNA purity (A260 nm / A280 nm) based on the light absorption rate (absorbance) was 1.76 in the conventional method, and 1.73 in the present embodiment, which is almost the same value. This value is 1.8, which indicates that the DNA purity is good. Further, the average human cell copy number (Ct _human value, Ct value in the figure) was 24.05 in the conventional method, and 24.11 in the present embodiment.

  Also from this result, this embodiment shows that the number of patients to be treated is completely consistent with the conventional method (of course, patients are also consistent), and the average DNA purity and human cell copy number (Ct value) are also approximately the same. Even so, the accuracy of the preparation method of this embodiment could be demonstrated.

  Furthermore, the average time for reporting results from nucleic acid extraction (time (h) per specimen) was 3 hours in this embodiment, compared with 4 hours for the conventional method. Here, 2 hours of the average time from the nucleic acid extraction to the result report is a time required for the enzyme reaction of the specimen, and is an essential (common) time for both. Therefore, when compared with the average time excluding this time, the conventional method is 2 hours, whereas in this embodiment, it is 1 hour. According to this embodiment, the determination time is halved compared to the conventional method. The remarkable effect that it was possible to appear.

  In addition, in this embodiment, the exposure of the pathogen to the operator is small, the reproducibility can be improved, and the determination cost due to the release of the operator's work time can be greatly reduced.

  In addition, since a centrifuge is not required, the apparatus can be reduced in size and cost.

  Although the present invention has been described in detail with the embodiment, the specific configuration is not limited to this embodiment, and even if there is a design change or the like without departing from the gist of the present invention, it is within the scope of the present invention. It is included.

  That is, in the above embodiment, the case where the nucleic acid extraction unit 3 and the primer processing unit 4 are housed in one nucleic acid extraction device 10 has been described as an example, but a nucleic acid extraction device having only a nucleic acid extraction unit may be used. . Alternatively, the nucleic acid extraction unit 3 and the primer processing unit 4 may be separate, and the nucleic acid extraction device may be a production line so that continuous processing is possible. The PCR fluorescence analyzer may also be included in the nucleic acid extraction apparatus 10 as a single unit.

  In the above-described embodiment, the preprocessing steps (steps P1 and P2) can be automatically controlled from step 1 by incorporating them into the automatic program.

  Various operations can be performed by rewriting the automatic control program. For example, in the above embodiment, the case where four samples are simultaneously processed has been described as an example, but simultaneous processing of one to three samples may be performed. In addition, depending on the purpose, only the nucleic acid extraction process can be automated, and the primer solution dispensing can be held manually or vice versa, and can be selected and implemented by the operator as appropriate. It is also possible.

  In addition, regarding the pressure reduction in the pressurization / decompression cycle of the solid phase extraction column 321, the pressure column cap 311 is opened, and the solid phase extraction column 321 is opened to the atmospheric pressure, thereby returning the pressure from the pressure exceeding the set pressure threshold to the atmospheric pressure. The case of (decompressing) has been described as an example. However, the present invention is not limited to this, and instead of (or in addition to) opening the solid-phase extraction column 321 under atmospheric pressure, the nucleic acid extraction unit 3 is provided with a suction means ( For example, a suction device or the like may be provided, and the pressure may be decreased from the pressure exceeding the set pressure threshold by pulling (suctioning) the solid-phase extraction column 321.

  Further, the decompression in the detection of column clogging is not limited to the case where the decompression is performed by opening the pressurizing column cap 311 and opening the solid phase extraction column 321 under the atmospheric pressure, but the solid phase extraction column 321 is under the atmospheric pressure. Instead of (or in addition to) opening to the set pressure threshold, the nucleic acid extraction unit 3 is provided with a suction means (for example, a suction device), and the solid-phase extraction column 321 is suctioned (sucked). The pressure may be reduced from a pressure exceeding.

  Moreover, although the said Example demonstrated to the case where DNA in a sample was extracted as a nucleic acid as an example, it is applicable not only to this but RNA extraction. In the above embodiment, the extraction of DNA used for the pretreatment for identifying pathogens from the patient's sputum has been described as an example. However, the present invention is not limited to this. For example, extraction of DNA (or RNA) in blood, The present invention can be widely applied to preparation of DNA (or RNA).

  The present invention can be used, for example, in the case of preparing a nucleic acid as a pretreatment for identifying a pathogen by analyzing a nucleic acid.

DESCRIPTION OF SYMBOLS 1 Housing 2 Display part 3 Nucleic acid extraction part 4 Primer process part 5 Dispensing part 6 Chip rack 8 Waste chip place 9 Reagent tank 10 Nucleic acid extraction apparatus 20 Sample tube 31 Pressurization part 32 Separation part 33 1st moving mechanism 34 2nd Movement mechanism 41 Tube holding part 42 Primer holding part 50 Dispensing cylinder 54 Tip 311 Pressurizing column cap 312 Pressurizing tube 313 Pressurizing syringe 313a Syringe 313b Plunger 315 Pressure sensor 316 Cap holding part 321 Solid phase extraction column 323 Waste liquid tank 324 Recovery Tube 325 recovery tube holder 521 first drive unit 522 second drive unit

Claims (14)

A nucleic acid extraction apparatus for extracting nucleic acid from a specimen,
Separation means comprising a container for storing the specimen and a filter;
Pressurizing means connected to the separating means;
A recovery means that can be connected to the separation means;
A dispensing means for dispensing the specimen;
Drive means for driving the dispensing means;
Control means for controlling the pressure means, the drive means and the pressure means;
With
The control means includes
Driving the dispensing means to dispense the sample into the separating means;
Pressurizing the separating means by the pressurizing means to separate the sample and the nucleic acid;
A nucleic acid extraction apparatus characterized by the above. The pressurizing means is
For the one sample dispensed to the separation means, the pressurizing / depressurization cycle in which the separation means is pressurized to a pressure exceeding a set pressure threshold, the separation means is held under the pressure for a predetermined time, and then the pressure is reduced Run multiple times,
The nucleic acid extraction apparatus according to claim 1. The pressurizing means depressurizes the separating means when the pressure applied to the separating means does not fall below the set pressure threshold even after the lapse of the predetermined time.
The nucleic acid extraction apparatus according to claim 2. Error notification means for notifying an error when the pressure applied to the separation means does not fall below the set pressure threshold even after the lapse of the predetermined time;
The nucleic acid extraction apparatus according to claim 2 or claim 3, wherein If the pressure applied to the separation means does not fall below the set pressure threshold, perform an additional pressurization cycle;
The nucleic acid extraction apparatus according to claim 4. Other recovery means corresponding to the recovery means;
A primer holding means for holding the primer solution,
The control means drives the dispensing means to dispense the separated nucleic acid present in the collecting means and the primer solution held in the primer holding means to the other collecting means.
The nucleic acid extraction apparatus according to any one of claims 1 to 5, wherein A plurality of the other collecting means and the primer holding means are provided for one collecting means,
Each primer holding means holds different primer solutions.
The nucleic acid extraction apparatus according to claim 6. Comprising a tip changing means for automatically changing the tip of the dispensing means;
The nucleic acid extraction apparatus according to any one of claims 1 to 7, wherein The separation means is a solid phase extraction column;
The nucleic acid extraction apparatus according to any one of claims 1 to 8, wherein A nucleic acid extraction method for extracting nucleic acid from a specimen,
Dispensing the sample into a separating means comprising a container for storing the sample and a filter;
Separating the nucleic acid from the specimen by pressurizing the separation means multiple times;
Recovering the effluent discharged from the separation means and extracting the nucleic acid;
A nucleic acid extraction method comprising: For one specimen dispensed to the separation means, after pressurizing the separation means at a pressure exceeding a set pressure threshold, the separation means is held under the pressure for a predetermined time, and then the pressure increasing / decreasing cycle for reducing the pressure is performed. Run multiple times,
The nucleic acid extraction method according to claim 10. If the pressure applied to the separation means does not fall below the set pressure threshold even after the predetermined time has elapsed, the separation means is depressurized.
The nucleic acid extraction method according to claim 11. The separation means is a solid phase extraction column;
The nucleic acid extraction method according to any one of claims 10 to 12, wherein: Adding a primer solution to the extracted nucleic acid,
The nucleic acid extraction method according to any one of claims 10 to 13, wherein