JP2005125311A - Chemical reactor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chemical reactor, improved in its usage, being applied to a nucleic-acid amplification apparatus especially using a polymerase chain reaction(PCR:Polymerase Chain Reaction) method. <P>SOLUTION: While a vessel is uniformly rotatably arrayed in a casing, the inside of the casing is divided, toward a rotational direction of the vessel, to spaces set up to have a plural of determined temperatures and determined sizes and a plural of sample holders, that have held each sample in the vessel, are arranged to be held toward the rotational direction. In particular, spaces, set up to have a plurality of determined temperatures and determined sizes, are consisted of a thermal denaturation space set up such that sample holders pass at a temperature and duration that a thermal denaturation of the nucleic acid is made to carry out, an annealing space set up such that sample holders pass at temperature and duration that an annealing of a primer is made to carry out, and a synthesizing space set up such that sample holders pass at temperature and duration that a synthesizing of the nucleic acid is made to carry out. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a chemical reaction apparatus, and in particular, to a nucleic acid amplification apparatus using a polymerase chain reaction (PCR) method.

  With the remarkable development of genetic engineering in recent years, a plurality of types of chemical reaction devices represented by nucleic acid amplification devices using the polymerase chain reaction method have been provided.

  As such a chemical reaction apparatus, for example, a single specimen container in which a plurality of accommodating parts for accommodating a specimen are integrally arranged in a lattice shape, such as PRISM7900HT manufactured by Applied Biosystems, A plurality of specimens stored in the specimen container are simultaneously heated or cooled to cause a chemical reaction.

In addition, as a conventional chemical reaction apparatus, a plurality of specimens are sequentially moved inside a spiral glass tube, whereby the specimens are sequentially heated or cooled to perform a chemical reaction (for example, Patent Documents). 1), for example, a Roche Light Cycler DX400, which heats or cools a plurality of glass tubes for containing a specimen using air as a heat medium to simultaneously carry out a chemical reaction of a plurality of specimens. Are known.
JP-A-6-30776

  However, the above-described conventional chemical reaction apparatus has the following problems.

  That is, in the case where a chemical reaction is performed by simultaneously heating or cooling a plurality of specimens, when the chemical reaction apparatus is operated using a specimen container containing a plurality of specimens, the next chemical reaction is completed. The chemical reaction apparatus could not be used for this sample. In addition, once the apparatus is operated, only a part of the samples cannot be taken out. Furthermore, since a plurality of specimens are simultaneously heated or cooled using a single heating device or cooling device, it is difficult to rapidly heat or cool the specimens themselves, and wasted time required for temperature changes. As a result, it took a long time for the chemical reaction to occur. Moreover, unnecessary and undesirable side reactions have occurred due to the specimen being exposed to a temperature other than the predetermined temperature.

  Moreover, even if the chemical reaction is performed by sequentially heating or cooling the specimen in the glass tube, once the apparatus is operated, only a part of the specimen cannot be removed. Furthermore, since the specimen is heated or cooled via the glass tube, it is difficult to rapidly heat or cool the specimen itself due to the thermal conductivity of the glass tube, resulting in wasted time required for temperature changes. As a result, it took a long time to perform the chemical reaction.

  As described above, the conventional chemical reaction apparatus is inconvenient and uncomfortable for the user, and has a high possibility of causing a poor reaction in which a side reaction product is mixed into the reaction product.

  Therefore, in the present invention according to claim 1, the container is disposed in the casing so as to be capable of rotating at a constant speed, and the inside of the casing is set to a plurality of predetermined temperatures and predetermined sizes in the rotation direction of the container. There is provided a chemical reaction device characterized in that a plurality of sample containers, each of which is partitioned into a space and contains individual samples in the container, are arranged side by side at an arbitrary angle with respect to the rotation direction.

  Further, in the present invention according to claim 2, in the present invention according to claim 1, the sample container is configured to store the sample between two adjacent thin plates.

  Further, in the present invention according to claim 3, in the present invention according to claim 1 or 2, the container is formed in an annular shape, and a heat source is disposed in the container, and the heat source is supplied to the specimen container. It was decided to blow warm air at a predetermined temperature.

  Further, in the present invention according to claim 4, in the present invention according to claim 1 or 2, the container is formed in an annular shape, a liquid is accommodated in the container, and the liquid is set to a predetermined temperature. Decided to control.

  Further, in the present invention according to claim 5, in the present invention according to any one of claims 1 to 4, the temperature set in the plurality of predetermined temperatures and predetermined sizes is a temperature at which nucleic acid is thermally denatured. And a heat-denaturing space set so that the specimen container passes through at a time, a temperature allowing annealing of the primer and an annealing space set so as to pass through the specimen container at time, a temperature allowing synthesis of the nucleic acid, and It was decided that it would consist of a synthetic space that was set so that the specimen container would pass by time.

  And in this invention, there exists an effect described below.

  That is, in the present invention according to claim 1, the container is disposed in the casing so as to be capable of rotating at a constant speed, and the inside of the casing is set to a plurality of predetermined temperatures and predetermined sizes in the rotation direction of the container. Since a plurality of specimen containers containing individual specimens are arranged in a space and arranged in the rotational direction, the specimens can be arbitrarily selected even after the operation of the apparatus is once started. Since the container can be taken out or newly inserted, the usability of the chemical reaction apparatus can be improved.

  Further, in the present invention according to claim 2, since the specimen container is configured such that the specimen is accommodated between two adjacent thin plates, the specimen can be rapidly heated or cooled. Since a useless time for increasing or decreasing the temperature can be reduced, the time required for the chemical reaction can be shortened, and unnecessary and undesirable side reactions can be reduced.

  Further, in the present invention according to claim 3, since the container is formed in an annular shape, a heat source is disposed in the container, and hot air of a predetermined temperature is blown from the heat source toward the specimen container. The specimen can be uniformly heated or cooled, which can also reduce the time required for the chemical reaction and reduce unnecessary and undesirable side reactions.

  In the present invention according to claim 4, the container is formed in an annular shape, the liquid is accommodated in the container, and the liquid is controlled at a predetermined temperature. It can be cooled, and this can also reduce the time required for the chemical reaction and reduce unnecessary and undesirable side reactions.

  Further, in the present invention according to claim 4, a heat-denaturing space that is set so that the specimen container passes through a space that is set to a plurality of predetermined temperatures and a predetermined size at a temperature and time for performing heat denaturation of nucleic acids; It consists of an annealing space that is set so that the sample container can pass at the temperature and time for primer annealing, and a synthesis space that is set so that the sample container can pass at temperature and time for performing nucleic acid synthesis. Therefore, nucleic acid amplification can be performed using the polymerase chain reaction method.

  In the chemical reaction device according to the present invention, the container is disposed in the casing so that the container can be continuously rotated at a constant speed or intermittently and repeatedly rotated and stopped at a constant amount, and the casing is rotated in the direction of rotation of the container. A plurality of specimen containers containing individual specimens in the container, arranged side by side at an arbitrary angle with respect to the rotation direction. is there.

  Moreover, the specimen container is configured to accommodate the specimen between two adjacent thin plates.

  Further, in the present invention, the container may be formed in an annular shape, a heat source may be provided in the container, and warm air of a predetermined temperature may be blown from the heat source toward the specimen container. It may be formed in an annular shape, and a liquid may be accommodated in the container, and the liquid may be controlled to a predetermined temperature by a thermostat or the like.

  Furthermore, a heat denaturation space set so that the specimen container passes through a plurality of spaces set at a predetermined temperature and a predetermined size at a temperature and time at which the nucleic acid undergoes heat denaturation, and a temperature and time at which the primer annealing is performed. The annealing space set so that the sample container can pass through and the synthesis space set so that the sample container can pass at the temperature and time at which the nucleic acid is synthesized can also be configured.

  Hereinafter, a specific structure of the chemical reaction apparatus according to the present invention will be described with reference to the drawings.

  As shown in FIGS. 1 and 2, the chemical reaction apparatus 1 has a motor 3 attached to a lower portion of a cylindrical casing 2, and a base end of a support body 5 extending radially on a drive shaft 4 of the motor 3. An annular container 6 is attached to the upper end of the support 5. Thereby, the chemical reaction apparatus 1 arrange | positions the container 6 inside the casing 2 so that rotation is possible.

  Further, the chemical reaction apparatus 1 has a support column 8 attached to the center of the ceiling wall 7 of the casing 2, and a disk-shaped bottom wall 9 is attached to the lower end of the support column 8. The three partition walls 10, 11, 12 are radially formed from the rotational center position of the container 6 in the radial direction to partition the inside of the casing 2 into three spaces 13, 14, 15; Heat sources 16, 17, and 18 for maintaining a predetermined temperature are disposed in the spaces 13, 14, and 15, respectively. In the figure, 19 is a fan provided in each heat source 16, 17, 18 when air is used as a heat medium, and 20 is a suction port formed through the bottom wall 9.

  In addition, the chemical reaction apparatus 1 has a temperature sensor 21 attached to the inside upper portions of the spaces 13, 14, and 15, and the temperature sensor 21 is connected to the controller 22, and the temperature of the heat sources 16, 17, and 18 is determined by the controller 22. To control.

  Further, the chemical reaction apparatus 1 has a carry-in mechanism 23 and a carry-out mechanism 24 attached to the upper part of the casing 2, and a measurement mechanism 25 is attached to the carry-out mechanism 24.

  Next, the specific structure of the container 6 will be described. As shown in FIGS. 1 to 3, the container 6 has a plurality of substantially U-shapes that are linearly extended in a direction (radial direction) perpendicular to the rotation direction. A pair of upper and lower connecting walls 27, 28 and a bottom wall 29 having a substantially L-shaped cross section are attached to the inside of the partition wall 26, while a pair of upper and lower connecting walls 30, 31 and a substantially L-shaped cross section are disposed outside the partition wall 26. The bottom wall 32 is installed. Here, the container 6 has an inner bottom wall 29 connected to the support 5. However, it is not essential that the directions of the plurality of substantially U-shaped partition walls 26 be orthogonal to the rotation direction of the container 6, and the partition walls 26 can be installed at an arbitrary angle.

  In addition, the container 6 forms an accommodating portion 34 for accommodating a specimen container 33 (described later) between the partition walls 26 and 26 adjacent to each other in the rotation direction (circumferential direction). A protrusion 35 is formed on the partition wall 26 to be firmly held in the housing portion 34.

  Next, the specific structure of the specimen container 33 will be described. As shown in FIGS. 3 to 4, the specimen container 33 is formed between a metal thin plate 36 and a transparent glass or resin thin plate 37. In addition, a sample storage space 38 for storing the sample is formed, an opening 39 for injecting the sample is formed above the sample storage space 38, and a resin-made material is formed around the metal thin plate 36. A frame 40 is attached. Here, the inner surface of the metal thin plate 36 is mirror-finished.

  Next, the structure of the spaces 13, 14, 15 formed in the casing 2 will be described.

  In the chemical reaction apparatus 1, the casing 2 has an angle formed by the first partition wall 10 and the second partition wall 11 of 72 degrees, and the angle formed by the second partition wall 11 and the third partition wall 12. The angle formed by the third partition wall 12 and the first partition wall 10 is 216 degrees.

  Moreover, the chemical reaction apparatus 1 is set so that the container 6 rotates once in 60 seconds by the motor 3, and the temperature of the heat source 16 is controlled so that the internal temperature of the first space 13 becomes 94 ° C. The temperature of the heat source 17 is controlled so that the internal temperature of the second space 14 becomes 54 ° C., and the temperature of the heat source 18 is controlled so that the internal temperature of the third space 15 becomes 72 ° C.

  Thereby, in the chemical reaction apparatus 1, the specimen container 33 passes through the first space 13 at 94 ° C. and 10 seconds, passes through the second space 14 at 54 ° C. and 12 seconds, and passes through the third space 15. It passes through at 72 ° C and 36 seconds.

  By setting the temperature and size of each space 13, 14, 15 in this way, in the chemical reaction device 1, the temperature (about 94 ° C.) and time (about 94 ° C.) at which the first space 13 performs thermal denaturation of nucleic acids. 12 seconds), the heat denaturation space is set so that the sample container 33 can pass through, and the second space 14 has a temperature (about 54 ° C.) and a time (about 12 seconds) at which primer annealing is performed. 33 is set to pass through the sample container 33 at a temperature (about 72 ° C.) and a time (about 36 seconds) at which the nucleic acid is synthesized. Thus, a nucleic acid amplification apparatus using a polymerase chain reaction (PCR) method is formed.

  The chemical reaction apparatus 1 measures the amplification amount of the nucleic acid by a fluorescence reaction using a fluorescence microscope type measurement mechanism 25.

  That is, as shown in FIG. 6, the measurement mechanism 25 irradiates the sample in the sample container 33 with light having a predetermined wavelength from the light source 41 through the lens 42, the optical filter 43, the dichroic mirror 44, and the lens 45. Thus, the fluorescence generated in the specimen enters the solid-state image sensor (CCD) 46 via the lens 45, the dichroic mirror 44, the optical filter 47, and the lens 48, and is converted into an electric signal by the solid-state image sensor 46. ing.

  The chemical reaction apparatus 1 is configured as described above, and performs a chemical reaction as described below. In the following description, a case where the sample is subjected to the polymerase chain reaction will be described.

  First, the chemical reaction apparatus 1 controls the heat sources 16, 17, and 18 with the controller 22 so that the temperature inside each space 13, 14, and 15 becomes a predetermined temperature.

  Here, the temperature of the heat source 16 is controlled so that the internal temperature of the first space 13 becomes 94 ° C., the temperature of the heat source 17 is controlled so that the internal temperature of the second space 14 becomes 54 ° C., and The temperature of the heat source 18 is controlled so that the internal temperature of the third space 15 is 72 ° C.

  Next, the chemical reaction apparatus 1 drives the motor 3 so that the container 6 rotates at a predetermined constant speed inside the casing 2. The chemical reaction apparatus 1 is not limited to the case of continuously rotating at a constant speed, and may be rotated while repeating a predetermined amount of rotation and stop.

  Here, the container 6 is rotated once in the casing 2 in 60 seconds, whereby the specimen container 33 passes through the first space 13 in 12 seconds and passes through the second space 14 in 12 seconds. And pass through the third space 15 in 36 seconds.

  Next, the specimen containers 33 are carried one by one into the empty accommodation section 34 of the container 6 using the carry-in mechanism 23.

As a result, the sample accommodated in the sample container 33 rotates inside the casing 2 together with the container 6 and passes through the first space 13 at 94 ° C. for 12 seconds. , Primer annealing is performed by passing through the second space 14 at 54 ° C. and 12 seconds,
Thereafter, the nucleic acid is synthesized by passing through the third space 15 at 72 ° C. for 36 seconds.

  Next, the sample containers 33 rotated by an appropriate number of times are taken out one by one using the carry-out mechanism 24 and measured by the measurement mechanism 25. If necessary, the sample containers 33 are again placed in the container 34 of the container 6. Carry in.

  As described above, the chemical reaction apparatus 1 performs the chemical reaction by repeatedly rotating the specimen container 33.

  As described above, the above-described chemical reaction apparatus 1 arranges the container 6 inside the casing 2 so as to be capable of rotating at a constant speed or repeating a predetermined amount of rotation and stopping, and the casing 2. The interior is partitioned into a plurality of spaces 13, 14, and 15 that are set to a predetermined temperature and a predetermined size in the rotation direction of the container 6, and a plurality of sample containers 33 that store individual samples in the container 6 are rotated. Housed side by side in the direction.

  Therefore, after the operation of the chemical reaction apparatus 1 is started, the specimen container 33 can be carried out from the container 6 at an arbitrary timing, or a new specimen container 33 can be carried into the container 6. Usability of the chemical reaction apparatus 1 can be improved, and discomfort to the user can be prevented.

  Further, the chemical reaction apparatus 1 is configured such that the specimen container 33 accommodates the specimen between the two adjacent thin plates 36 and 37.

  Therefore, the specimen can be rapidly heated or cooled, so that a wasteful time for temperature rise or fall can be reduced, and the time required for the chemical reaction can be shortened, as shown in FIG. In addition, generation of unnecessary and undesirable side reaction products can be reduced.

  In FIG. 7, M is a standard substance for estimating the molecular weight of a reaction product, and A is an existing apparatus in which a reaction specimen is placed in a resin container using an aluminum block whose temperature is controlled by a Peltier element. B is a reaction product in an existing apparatus in which air is used as a heat medium and a reaction sample is put in a glass tube, and C is a case where air is used as a heat medium in the chemical reaction apparatus according to the present invention. The reaction product D is a reaction product when oil is used as a heat medium in the chemical reaction apparatus according to the present invention. Main Band indicates a desired reaction product, and Sub Band indicates a side reaction product. .

  Further, the chemical reaction apparatus 1 forms the container 6 in an annular shape, and arranges heat sources 16, 17, 18 at the center of rotation of the container 6, and directs the heat source 16, 17, 18 from the sample container 33 to the specimen container 33. The hot air is blown.

  Therefore, since the specimen can be heated or cooled quickly and uniformly, the time required for the chemical reaction can be shortened.

  In addition, the chemical reaction apparatus 1 has a heat denaturation space set so that the sample container 33 passes at a temperature and time at which nucleic acid is thermally denatured, and a sample container 33 at a temperature and time at which primer annealing is performed. An annealing space that is set to pass through and a synthesis space that is set to pass through the specimen container at a temperature and time for performing nucleic acid synthesis are provided.

  Therefore, nucleic acid amplification using the polymerase chain reaction method can be performed using the chemical reaction apparatus 1.

1 is a plan sectional view showing a chemical reaction device according to the present invention. FIG. The elements on larger scale which show a container. The top view which shows a sample container. II sectional drawing of FIG. Explanatory drawing which shows a measurement mechanism. The photograph which shows the result of the electrophoresis which shows the production | generation of a reaction product and a side reaction product.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Chemical reaction apparatus 2 Casing 3 Motor 4 Drive shaft 5 Support body 6 Container
10, 11, 12 Partition wall
13,14,15 space
16,17,18 Heat source
19 fans
21 Temperature sensor
22 Controller
23 Loading mechanism
24 Unloading mechanism
25 Measuring mechanism
33 Sample container
34 Enclosure
36,37 sheet
38 Specimen storage space M Standard material for estimating the molecular weight of the reaction product A A reaction product in an existing apparatus in which a reaction specimen is placed in a resin container using an aluminum block whose temperature is controlled by a Peltier element B Air The reaction product in the existing apparatus in which the reaction specimen is put in the glass tube with the heat medium as the heat medium C The reaction product in the case where the air is the heat medium in the chemical reaction apparatus according to the present invention D The oil in the chemical reaction apparatus according to the present invention Product when heat is used as heat medium Main Band Expected reaction product Sub Band Side reaction product

Claims (5)

  A container is disposed in the casing so as to be capable of rotating at a constant speed, and the inside of the casing is partitioned into a plurality of spaces set to a predetermined temperature and a predetermined size in the rotation direction of the container. A chemical reaction apparatus characterized in that a plurality of specimen containers containing the specimens are accommodated at an arbitrary angle with respect to the rotation direction.   The chemical reaction apparatus according to claim 1, wherein the sample container is configured to store a sample between two adjacent thin plates.   3. The container according to claim 1, wherein the container is formed in an annular shape, a heat source is disposed in the container, and hot air having a predetermined temperature is blown from the heat source toward the specimen container. Chemical reactor.   The chemical reaction apparatus according to claim 1 or 2, wherein the container is formed in an annular shape, a liquid is accommodated in the container, and the liquid is controlled at a predetermined temperature.   The plurality of spaces set to the predetermined temperature and the predetermined size are the heat denaturation space set so that the specimen container passes at the temperature and time for performing the heat denaturation of the nucleic acid, and the temperature and time for performing the primer annealing. The annealing space set so that the sample container can pass through, and the synthesis space set so that the sample container can pass at the temperature and time for synthesizing the nucleic acid. Item 5. The chemical reaction device according to any one of Items 4 to 6.

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