JP2015077079A - Biological sample crushing device and extraction measurement system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a crusher capable of effectively crushing a sample related to a living body and collecting crushed material of the sample related to a living body.SOLUTION: A crusher 100 of a sample related to a living body comprises: a container 10 housing a solution comprising a magnetic crushed particles A and a sample B related to a living body; a shaking portion 20 shaking the container 10; a magnetic force applying portion 40 applying magnetic force to the magnetic crushed particles A; and a dispensation mechanism 500 moving the solution to the inside or outside the container 10. By shaking the container 10 by the shaking portion 20, the magnetic crushed particles A are collided with the sample B related to a living body and the sample B related to a living body is crushed. In a state that the magnetic force applying portion 40 causes the magnetic crushed particles A to be attracted to the container 10, the dispensation mechanism 500 separates crushed material of the sample B related to a living body from the magnetic crushed particles A.

Description

  The present invention relates to a crushing apparatus that crushes a biological sample using magnetic crushing particles, and an extraction measurement system that extracts and measures nucleic acid or the like from the crushed biological sample.

  Conventionally, in order to analyze biologically related substances such as cells and viruses, as a pretreatment, this has been crushed by ultrasonic vibration or mechanically crushed.

  Patent Document 1 proposes a method for extracting nucleic acids by crushing a sample such as bacteria using amorphous zirconia powder. In this method, the sample and the container containing the zirconia powder are stirred by hand to crush the sample. The zirconia powder is a fine particle having a major axis of 32 μm or less. When collecting the crushed sample, the zirconia powder is sedimented by centrifugation, and the supernatant containing the crushed sample is collected.

  Patent Document 2 describes a cell crushing device that crushes biological cells using magnetic spheres or metal spheres. This cell disruption device disrupts cells by applying vibration to the magnetic beads and metal spheres by changing the magnetic force of an external electromagnet with respect to the fine magnetic beads and metal spheres in the sample container.

Japanese Patent No. 4797602 (Tosoh Corporation) Japanese Patent Laid-Open No. 2003-000226

  Conventionally, when crushing a sample by ultrasonic vibration, the container is vibrated with a horn of an ultrasonic vibrator with the solution containing the sample placed in the container, and the sample in the solution is pulverized with ultrasonic waves. . During the pulverization, the nucleic acid contained in the sample may be damaged by ultrasonic cavitation (cavity phenomenon) transmitted in the solution. For example, it is a case where M. tuberculosis is crushed. Since the cell wall of M. tuberculosis is relatively hard, stronger ultrasound is required to crush M. tuberculosis in the solution and extract the nucleic acid. When such ultrasonic waves are added to a solution containing Mycobacterium tuberculosis, the nucleic acid of Mycobacterium tuberculosis is destroyed by cavitation. As a result, a nucleic acid fragment cannot be maintained, and there is a possibility that a nucleic acid having a length suitable for measurement cannot be extracted. The occurrence of cavitation depends on a plurality of conditions such as the frequency and amplitude of the ultrasonic wave, the shape of the container, and the amount of the solution. Therefore, it is difficult to automatically adjust the crushing conditions of the sample by ultrasonic waves while preventing the occurrence of cavitation, and it has been difficult to automate the crushing process.

  In the method of Patent Document 1, when crushing a sample, it is necessary to stir the container by hand, and it is difficult to control the crushing conditions uniformly. In addition, the sample could not be efficiently crushed, and the crushing process could not be automated. Furthermore, in the method of Patent Document 1, since the zirconia powder to be used is small, the supernatant by centrifugation is collected, which is cumbersome and has a drawback that a crushed sample not contained in the supernatant cannot be collected. It was happening.

  In the apparatus of Patent Document 2, the mass of magnetic beads and metal spheres vibrated by an external magnetic force in the sample container is relatively small, so that there is a possibility that sufficient kinetic energy cannot be secured for collision with the sample and crushing.

  An object of the present invention is to provide a crushing apparatus that can efficiently crush a biological sample and easily recover a crushed material of the biological sample. Another object of the present invention is to provide an extraction measurement system capable of executing a series of operations such as crushing, extraction, and analysis of biological materials.

As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that a relatively heavy heavy magnetic fragment and a biological sample are housed in a container and shaken to efficiently perform biological related. The sample was successfully crushed and the present invention was completed.
That is, the present invention is as follows.

  (1) A container that contains a solution containing a biological sample and magnetic crushed particles, a shaking unit that shakes the container, a magnetic force applying unit that applies magnetic force to the magnetic crushed particles, and in the container or outside the container And a dispensing mechanism for moving the solution to the biologically relevant sample crushing device, wherein the shaking unit shakes the container so that the magnetically crushed particles collide with the biologically relevant sample and In the crushing device, the sample is crushed, and the dispensing mechanism separates the biologically-related sample crushed from the magnetic crushed particles in a state where the magnetic force imparting unit fixes the magnetic crushed particles. is there.

  (2) The crushing device according to (1), wherein the shaking part includes an eccentric cam. (3) The crushing apparatus according to (1) or (2), further including a buffer unit that buffers shaking of the container by the shaking unit. (4) The magnetic force imparting portion is a permanent magnet capable of imparting a magnetic force to the magnetic crushed particles by moving to the vicinity of the container, (1) to (3), Is a crushing apparatus. (5) The magnetic force imparting section is an electromagnet that is disposed in the vicinity of the container and is capable of imparting a magnetic force to the magnetic crushed particles, according to any one of (1) to (3). It is a crushing device. (6) The crushing apparatus according to any one of (1) to (5), wherein a seal capable of being pierced is provided in the opening of the container.

  (7) The crushing apparatus according to any one of (1) to (6), wherein the magnetic crushing particles include an acute angle portion. (8) The crushing apparatus according to any one of (1) to (7), wherein the magnetic crushing particles are a ferromagnetic metal. (9) The crushing device according to any one of (1) to (8), wherein the magnetic crushing particles are formed by wire-cutting a metal. (10) The crushing apparatus according to any one of (1) to (8), wherein the biological sample is biological tissue, food, protein, sputum, pus, cells, or bacteria. is there. (11) Any one of (1) to (10), comprising a control unit that automatically performs operations of the shaking unit, the magnetic force applying unit, and the dispensing mechanism according to a predetermined operation. A crushing apparatus according to claim 1.

  (12) The crushing device according to any one of (1) to (11), an extraction unit that extracts nucleic acid from the biological sample crushed by the crushing device, and a nucleic acid obtained from the extraction unit An extraction measurement system comprising: an amplification measurement unit that amplifies and measures the signal. (13) The extraction according to (12), wherein the control unit automatically performs crushing of the biological sample and extraction, amplification, and measurement of the nucleic acid according to a predetermined operation. It is a measurement system. (14) The dispensing mechanism includes a magnet that adsorbs the magnetic particles in a state in which the solution containing the magnetic particles that bind to the nucleic acid is sucked, and is described in (12) or (13) Extraction measurement system.

  (15) Any one of (12) to (14), wherein nucleic acid extraction in the extraction unit and / or nucleic acid amplification in the amplification measurement unit is performed using the dispensing mechanism. The extraction measurement system according to item. (16) The extraction measurement system according to any one of (12) to (15), wherein the amplification of the nucleic acid in the amplification measurement unit is emulsion PCR. (17) The extraction measurement system according to (16), wherein the crushing apparatus performs emulsion formation and / or emulsion breaking.

  (18) A crushing method for crushing a biological sample using the crushing device according to (1) to (11), the step of storing a solution containing the biological sample and magnetic crush particles in the container, The step of crushing the biological sample by shaking the container using the shaking unit, and the crushing particles are crushed using the dispensing mechanism in a state where the crushing particles are fixed in the vessel. And a step of separating the biological sample. (19) The crushing method according to (18), wherein the biological sample is crushed using an acute angle portion formed in the magnetic crushed particles. (20) The crushing method according to (18) or (19), wherein the biological sample is biological tissue, food, protein, sputum, pus, cells, or bacteria.

  (21) A method for extracting and measuring a nucleic acid contained in a biological prefectural substance, the step of crushing the biological sample using the crushing device according to any one of (1) to (11), A method comprising: extracting a nucleic acid from the crushed biological sample, amplifying the extracted nucleic acid, and measuring the amplified nucleic acid. (22) The method according to (21), wherein the nucleic acid amplification is performed by emulsion PCR. (23) The method according to (22), wherein the crushing apparatus performs emulsion formation and / or emulsion breaking.

  In the crushing apparatus of the present invention, the biologically related sample can be crushed using the magnetically crushed particles by shaking the container, and at the same time, the crushed material of the biologically related sample can be easily and purely separated from the magnetically crushed particles. it can. In the extraction measurement system of the present invention, a biological sample can be crushed, and nucleic acids contained in the crushed biological sample can be extracted, amplified, and measured.

It is a block diagram which shows the crushing apparatus of the biological sample which concerns on embodiment of this invention. It is a perspective view which shows the magnetic crushing particle used for the biological body related sample crushing apparatus of FIG. It is a perspective view which shows the cutting process process of the magnetic crushing particle | grains of FIG. It is a block diagram which shows the vibration operation | movement of the crushing apparatus of FIG. It is a block diagram which shows the state which attracts | sucks a crushed material from the crushing apparatus of FIG. It is a top view of the extraction measurement system concerning the embodiment of the present invention.

  A biological-related sample crushing apparatus and a biological-related sample crushed material extraction measurement system according to an embodiment of the present invention will be described with reference to the drawings. In each figure, the same parts are denoted by the same reference numerals, and the description of the same parts is omitted.

(Crushing device for biological samples)
A biological-related sample crushing apparatus 100 according to an embodiment of the present invention will be described with reference to FIGS. The crushing device 100 includes a container 10 that contains a solution, a shaking unit 20 that shakes or vibrates the container 10, a buffer unit 30 that buffers shaking of the container 10, and a magnetic force applying unit that changes the magnetic force applied to the container 10. 40 and a dispensing mechanism 500 (FIG. 5).

  The container 10 accommodates therein the magnetic crushed particles A, the biological sample B which is a sample to be crushed, and the solution. The shape of the container 10 can be a thing with a wide bottom side, for example, a flask shape so that the movement, stirring, and impact of the magnetically crushed particles A are increased when the container 10 is shaken. The opening 11 of the container 10 can be sealed with an aluminum sealing member 12. The seal member 12 can be perforated by a syringe or the like.

  The shaking unit 20 includes a motor 22, a rotating shaft 22a of the motor 22, an eccentric cam 23 that rotates together with the rotating shaft 22a, and a first contact member 24 that shakes together with the container 10. The shaking part 20 is not limited to the structure that shakes linearly as shown in FIG. 1, and the container 10 may be shaken using a vortex mixer or the like.

  The buffer portion 30 is a second contact member 31 that shakes integrally with the container 10, and a pressing member 32 such as a spring that is connected to the second contact member 31 at one end and presses the second contact member 31 toward the container 10. And a fixing member 34 for fixing the other end of the pressing member 32. The magnetic force application unit 40 can be a permanent magnet, and includes a magnet moving mechanism (not shown) for moving the magnet with respect to the container 10. The magnetic force application unit 40 may be an electromagnet that is disposed in the vicinity of the container 10 and can apply a magnetic force to the magnetic crushed particles A.

  The shaking motion of the container 10 is preferably a linear motion or a simple motion such as vortex with respect to a total amount of 100 μl to 500 μl of the biological sample B contained in the solution. The shaking cycle of the container 10 can be, for example, 1000 rpm to 10,000 rpm, and the shaking amplitude of the container 10 can be, for example, 5 mm to 20 mm.

  The crushing apparatus 100 also includes a control unit that automatically controls the operations of the magnetic force application unit 40, the motor 22 of the shaking unit 20, and the dispensing mechanism 500. The first contact member 24 and the second contact member 31 are elastic members such as rubber and are in contact with the outer surface of the container 10.

  The biological sample B includes, for example, biological tissue, food, protein, sputum, pus, bacteria (bacteria) such as tuberculosis bacteria and methicillin-resistant Staphylococcus aureus (MRSA), or viruses, but is not limited thereto. The crushing apparatus of this embodiment physically crushes these objects such as cell walls, cell membranes, proteins, and capsids (shells).

  The magnetic crushed particles A can have a diameter (major axis) of preferably 0.1 mm to 1.0 mm. In addition, as shown in FIG. 2, the magnetic crushed particles A are provided with sharp corners a such as sharp end faces and protrusions. The acute angle part a has a crushing effect on the object to be crushed. The magnetic crushed particles A are assumed to have a mass sufficient to crush the material to be crushed. For example, the mass of the magnetically crushed particles A can be preferably 6 μg to 6 mg.

  As shown in FIG. 3, the magnetic crushed particles A can be prepared by cutting a superferromagnetic metal wire 60 such as iron by electric discharge machining such as wire cutting. The acute angle part a can be formed in the magnetically crushed particles depending on the angle of the wire cut with respect to the wire. The diameter of the wire can be preferably 0.1 mm to 1.0 mm, more preferably 0.3 mm to 0.5 mm.

  Next, operation | movement of the crushing apparatus 100 of this embodiment is demonstrated. First, the magnetically crushed particles A, the biological sample B, and the solution are charged into the container 10 using the dispensing mechanism 500 or another mechanism. Next, the eccentric cam 23 rotates in synchronization with the rotation of the motor 22, and the container 10 moves in the right direction indicated by the arrow from the position in FIG. After the eccentric cam 23 rotates and the container 10 reaches the position shown in FIG. 4, when the eccentric cam 23 further rotates, the container 10 moves from the position shown in FIG. Return to position 1. Therefore, when the eccentric cam 23 is continuously rotated, the container 10 is shaken (oscillated) from side to side. As the container 10 is shaken, the magnetically crushed particles A repeatedly collide with the living body related sample B in the solution of the container 10 to break the living body related sample B. After the container 10 is shaken for a predetermined time (eg, about 3 to 7 minutes, preferably about 5 minutes), the shake is stopped. With the shaking stopped, the magnetic force application unit 40 is brought closer to the container 10.

  When the magnetic force application unit 40 approaches the container 10 as shown in FIG. 5, the magnetic crushed particles A that are ferromagnetic materials are adsorbed (fixed) to the inner surface of the container 10 by the magnetic force of the magnetic force application unit 40. With the magnetic crushed particles A adsorbed on the inner surface of the container 10, the pipette tip 510 of the dispensing mechanism 500 is moved into the container 10 through the opening 11 of the container 10, and the solution and the crushed biological sample are inhaled. By doing so, the crushed material of the biological sample can be easily and purely separated from the magnetic crushed particles A.

(Extraction measurement system)
An embodiment of a biological sample extraction measurement system 1000 according to the present invention will be described with reference to FIG. The present extraction measurement system 1000 automatically and consistently performs disruption of biological samples and extraction, amplification, and measurement of nucleic acids from biological samples.

  The extraction measurement system 1000 includes a plurality of processing lines 1100-1500. The processing line of the extraction measurement system 1000 includes the crushing apparatus 100, the extraction unit 200, the amplification measurement unit 300, and the dispensing mechanism 500 shown in FIG. FIG. 6 illustrates an extraction measurement system 1000 including five processing lines. However, the number of processing lines is not limited to six, and the extraction measurement system may include one or a plurality of processing lines. .

  The dispensing mechanism 500 includes a pipette tip 510 for sucking and discharging a solution, a magnet 520 disposed in the vicinity of the pipette tip 510, a magnet moving mechanism for moving the magnet 520 relative to the pipette tip 510, and a pipette tip 510. And a pipette tip moving mechanism for moving the magnet 520 on the processing line in a direction perpendicular to and parallel to the processing line.

  The dispensing mechanism 500 can arbitrarily move a solution, a reagent, and a sample between the crushing device 100, the extraction unit 200, and the amplification measurement unit 300 using a pipette tip moving mechanism. The dispensing mechanism 500 draws a solution containing fine magnetic particles (beads having a diameter of several μm) into the pipette tip 510, and moves the magnet 520 closer to the pipette tip 510, thereby causing magnetic particles on the inner surface of the pipette tip 510. Can be adsorbed. Magnetic particles can directly or indirectly adsorb samples such as nucleic acids and proteins contained in crushed material of biological samples.

  The extraction unit 200 is a cartridge including a plurality of wells, and extracts and purifies nucleic acid exposed from a crushed biological sample using magnetic particles. The amplification measurement unit 300 is a cartridge including a plurality of wells, and includes a measurement device that amplifies the nucleic acid and the like extracted and purified by the extraction unit 200 by polymerase chain reaction (PCR) and measures the amplified nucleic acid and the like. . The PCR can be real-time PCR and further emulsion PCR. As a result, large-capacity PCR can be realized.

  The extraction measurement system 1000 includes a control unit such as a computer, so that the crushing operation of the crushing apparatus 100, the magnetic force application operation of the magnetic force application unit 40, the suction / discharge operation of the dispensing mechanism 500, and the movement operation of the pipette tip 510 with respect to the processing line. The operation of the magnet moving mechanism of the magnet 520 and the temperature control operation and measurement operation of the amplification measuring unit 300 can be automatically controlled according to a predetermined procedure.

  The crushing apparatus 100 can also be used for emulsion PCR processing. For example, a bead is obtained by moving a bead to which a nucleic acid or the like (DNA), which is a crushed material of a biological sample, is bonded and a solution for emulsion to the container 10 of the crushing apparatus 100 using a dispensing mechanism 500 and shaking and stirring. A water-in-oil emulsion containing can be formed in the container 10. The emulsion PCR can be executed by moving this emulsion to the amplification measuring unit 300 by the dispensing mechanism 500. Further, after the emulsion PCR is completed, the emulsion solution containing the amplified DNA is moved to the container 10 of the crushing apparatus 100 using the dispensing mechanism 500. Furthermore, by breaking a breaking solution such as isopropanol into the container 10 using the dispensing mechanism 500 and vibrating the crushing device 100, the emulsion can be broken (called braking).

A magnetic crushed particles B biological sample 10 container 20 shaking unit 22 motor 23 eccentric cam 24 first contact member 30 buffer unit 31 second contact member 32 pressing member 34 fixing member 40 magnetic force applying unit 100 crushing device 200 extraction unit 300 amplification measurement 500 Dispensing mechanism 1000 Extraction measurement system

Claims (23)

A container containing a solution containing a biological sample and magnetic crushed particles, a shaking part for shaking the container, a magnetic force applying part for applying a magnetic force to the magnetic crushed particles, and the solution inside or outside the container A biologically relevant sample crushing device comprising a dispensing mechanism for moving
When the shaker shakes the container, the magnetically crushed particles collide with the biological sample, and crush the biological sample.
The crushing apparatus, wherein the dispensing mechanism separates the biologically-related sample crushed from the magnetic crushed particles in a state where the magnetic force imparting unit fixes the magnetic crushed particles.   The crushing apparatus according to claim 1, wherein the shaking portion includes an eccentric cam.   The crushing apparatus according to claim 1, further comprising a buffer unit that buffers shaking of the container by the shaking unit.   The said magnetic force provision part is a permanent magnet which can provide a magnetic force to the said magnetic crushing particle | grains by moving to the vicinity of the said container, The crushing apparatus as described in any one of Claims 1-3 characterized by the above-mentioned. .   The crushing apparatus according to any one of claims 1 to 3, wherein the magnetic force application unit is an electromagnet arranged near the container and capable of applying a magnetic force to the magnetic crushing particles.   The crushing apparatus according to any one of claims 1 to 5, wherein a seal capable of being pierced is provided in the opening of the container.   The said magnetic crushing particle is equipped with an acute angle part, The crushing apparatus as described in any one of Claims 1-6 characterized by the above-mentioned.   The crushing apparatus according to any one of claims 1 to 7, wherein the magnetic crushing particles are a ferromagnetic metal.   The crushing apparatus according to any one of claims 1 to 8, wherein the magnetic crushing particles are formed by wire-cutting a metal.   The crushing apparatus according to any one of claims 1 to 8, wherein the biological sample is biological tissue, food, protein, sputum, pus, cells, or bacteria.   The control part which performs automatically the operation | movement of the said shaking part, the said magnetic force provision part, and the said dispensing mechanism according to predetermined operation | movement is provided, It is characterized by the above-mentioned. Crushing equipment.   The crushing device according to any one of claims 1 to 11, an extraction unit for extracting nucleic acid from the biological sample crushed by the crushing device, and amplifying and measuring the nucleic acid obtained from the extraction unit. An extraction measurement system comprising an amplification measurement unit.   The extraction measurement system according to claim 12, wherein the control unit automatically performs crushing of the biological sample and extraction, amplification, and measurement of the nucleic acid according to a predetermined operation.   The extraction measurement system according to claim 12 or 13, wherein the dispensing mechanism includes a magnet that adsorbs the magnetic particles in a state in which a solution containing the magnetic particles that bind to the nucleic acid is sucked.   The extraction according to any one of claims 12 to 14, wherein nucleic acid extraction in the extraction unit and / or nucleic acid amplification in the amplification measurement unit is performed using the dispensing mechanism. Measuring system.   The extraction measurement system according to any one of claims 12 to 15, wherein the nucleic acid amplification in the amplification measurement unit is emulsion PCR.   The extraction measurement system according to claim 16, wherein the crushing apparatus performs emulsion formation and / or emulsion breaking. A crushing method for crushing a biological sample using the crushing device according to claim 1,
Storing a solution containing a biological sample and magnetic crushed particles in the container;
Crushing the biological sample by shaking the container using the shaking unit;
A step of separating the biologically-related sample crushed using the dispensing mechanism in a state where the magnetic force imparting portion fixes the crushed particles in the container.   The crushing method according to claim 18, wherein the biological sample is crushed using an acute angle portion formed in the magnetic crushed particles.   The crushing method according to claim 18 or 19, wherein the biological sample is biological tissue, food, protein, sputum, pus, cells, or bacteria. A method for extracting and measuring nucleic acids contained in a living body related substance,
A step of crushing the biological sample using the crushing device according to any one of claims 1 to 11,
Extracting nucleic acid from the crushed biological sample;
Amplifying the extracted nucleic acid;
Measuring the amplified nucleic acid.   The method according to claim 21, wherein the nucleic acid amplification is performed by emulsion PCR.   The method according to claim 22, wherein the crushing apparatus performs emulsion formation and / or emulsion breaking.