JP3070134B2 - Incubation device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an incubation apparatus which can be used for, for example, a gene amplification reaction (PCR reaction).

[0002]

2. Description of the Related Art For example, in order to carry out a PCR reaction, it is necessary to repeat a cycle of sequentially heating and cooling a reaction vessel filled with a sample solution at different temperatures a plurality of times. Conventionally,
The incubation apparatus used for such a PCR reaction had a single heating / cooling block for heating / cooling the reaction vessel. Then, the block is heated and cooled to a predetermined temperature by a heater or a cooler, thereby heating and cooling the reaction vessel inserted into the block, and then the temperature of the heating and cooling block is adjusted to a different predetermined temperature. By repeating the operation
A CR reaction was being performed.

[0003]

In a conventional incubation apparatus, a single block is used for heating and cooling the reaction vessel. Therefore, in order to adjust the reaction vessel to different temperatures, the heating vessel must be heated each time. It is necessary to control the temperature of the cooling block, and waste time and power are required.

As a material of the reaction vessel, a synthetic resin material or the like is used because it does not react with the reaction reagent and the preparation is easy. However, the sample solution is heated and cooled to a target temperature because of poor thermal conductivity. However, there is a problem that unnecessary time and power are required more than necessary.

An object of the present invention is to solve the above-mentioned problems of the prior art.

[0006]

A feature of the incubation apparatus of the present invention is that it comprises a plurality of reaction vessel heating / cooling media which are previously adjusted to different temperatures, and each heating / cooling medium is independent of each other. And a plurality of blocks that are brought into contact with the reaction vessel in a certain order and that are preliminarily temperature-adjusted to different temperatures and parallel in one horizontal direction as the reaction vessel heating / cooling medium. A plurality of concave portions corresponding to the predetermined number of repetitions of the temperature cycle are provided so as to be arranged in parallel along a horizontal direction perpendicular to the block parallel direction, and the reaction vessel and the block are arranged along the block parallel direction. Means for relative movement, means for relatively moving the reaction vessel and the block along the recess parallel direction at every parallel pitch of the recess, The lies in that a means for relatively moving said reaction vessel and said block vertically so as to be out in the recess. The incubation apparatus of the present invention includes a plurality of reaction vessel heating / cooling media which have been previously adjusted to different temperatures, and each heating / cooling medium is independently contacted with the reaction vessel in a certain order, It is preferable that the reaction vessel is formed from a material in which a metal thin film is covered with a synthetic resin.

[0007]

In order to heat and cool the reaction vessel by the incubation apparatus according to the present invention, first, the respective heating and cooling media are adjusted to different temperatures in advance. The specific temperature is determined according to the type of reaction. Next, the reaction vessel is sequentially heated and cooled by each heating and cooling medium. Thus, the temperature of the reaction vessel is adjusted to a different temperature according to the heating / cooling order of each heating / cooling medium. The order of heating and cooling the reaction vessel by each heating and cooling medium is constant according to the type of reaction. Also, instead of heating and cooling a plurality of reaction vessels at once with each heating and cooling medium, the reaction vessels can be heated and cooled one by one and the reaction can be sequentially advanced at each repetition of a predetermined temperature cycle.

Since the reaction vessel is formed from a material in which a metal thin film is coated with a synthetic resin, the reaction vessel has a high thermal conductivity and does not react with the reaction reagent.

[0009]

Embodiments of the present invention will be described below with reference to the drawings.

The incubation apparatus 1 shown in FIGS. 1 and 2 includes a plurality of heating and cooling media. In this embodiment, the heating / cooling medium includes three blocks 2a, 2b, 2
c. Each block 2a, 2b, 2c
In FIG. 1, heaters or cooling elements (not shown) are built in, and each block 2a, 2b, 2c is built in by heaters or cooling elements.
Are adjusted to different temperatures. Each of the blocks 2a, 2b, and 2c has a plurality of recesses 3 having upward openings.
a, 3b and 3c are formed. These recesses 3a, 3
b, 3c are arranged in parallel in the left-right direction in FIG.

As shown in FIG. 3, blocks 2a, 2b,
The reaction vessel 4 is inserted into the recesses 3a, 3b, and 3c by moving the 2c upward, whereby the reaction vessel 4 is heated and cooled. In addition, each block 2
a, 2b and 2c are capable of heating and cooling the reaction vessel 4 independently of one another in a certain order.

That is, the blocks 2a, 2b, and 2c are respectively mounted on the carriage 14 via the guide rails 5 so as to be movable up and down.
Driven up and down by a, 6b and 6c. The trolley 14
Is movable in the left-right direction on the rail 15 in FIG. 1 by the fluid pressure cylinder 7. The reaction vessel 4 is shown in FIG.
Is provided with a belt conveyor 8 that conveys from left to right, and the reaction vessel 4 is held by the belt conveyor 8 above the blocks 2a, 2b, and 2c. That is, as shown in FIG.
a has concave portions 3a, 3b of blocks 2a, 2b, 2c;
The holding holes 9 are formed at the same pitch as the formation pitch of 3c, and the reaction vessel 4 is formed with a flange 4a. Thereby, the reaction container 4 is held by the belt conveyor 8 by inserting the reaction container 4 into the holding hole 9 and making the flange 4a contact the upper surface of the belt 8a.

Using the above incubation apparatus 1, P
The operation when performing a CR reaction will be described. In addition, PCR
As shown in FIG. 6, the temperature cycle in the reaction is such that the sample filled in the reaction vessel is first heat-denatured at 94 ° C. for 1 minute and then denatured at 55 ° C. for 1 minute. Anneal, then 72 ° C
The DNA is synthesized by maintaining the temperature for 1 minute. This temperature cycle is to be repeated 16 times. In this case, the concave portion 3 formed in each block 2a, 2b, 2c
The numbers a, 3b and 3c are equal to the number of repetitions of the temperature cycle and are set to 16.

First, the temperature of each of the blocks 2a, 2b and 2c is previously adjusted to 94 ° C., 54 ° C. and 72 ° C., respectively. In addition, the carriage 2 is moved by the fluid pressure cylinder 7 to position the block 2 a, the temperature of which has been adjusted to 94 ° C., below the belt 8 a of the belt conveyor 8. Further, the concave portion 3 located at the rear end of the belt conveyor 8 in the transport direction of the reaction container 4.
At a position above “a”, the reaction container 4 filled with the sample solution is held by the belt conveyor 8.

From this state, as shown in FIG.
The block 2a whose temperature has been adjusted to 4 ° C.
The sample is moved upward by a, and the reaction vessel 4 is inserted into the recess 3a and heated, whereby the sample is thermally denatured. Next, the block 2a is lowered, and the carriage 4 is moved, whereby the block 2b whose temperature has been adjusted to 55 ° C. is located below the reaction vessel 4. Thereafter, the block 2b whose temperature has been adjusted to 55 ° C. is moved upward, and the reaction vessel 4 is inserted into the concave portion 3b and cooled to maintain a constant temperature.
Anneal the primer of the sample. next,
By lowering the block 2b and moving the cart 4,
Block 2c temperature-controlled to 72 ° C. below reaction vessel 4
Position. Thereafter, the block 2c whose temperature has been adjusted to 72 ° C. is moved upward, and the reaction vessel 4 is placed in the recess 3c.
Is inserted and heated to maintain a constant temperature, whereby the DNA strand of the sample is synthesized. Next, the block 2c is lowered, and the carriage 4 is moved, whereby the block 2a whose temperature has been adjusted to 94 ° C. is located below the reaction vessel 4. In addition, the belt conveyor 8 is moved forward by one pitch of the recesses 3a, 3b, and 3c in the transport direction, and a new reaction container 4 filled with the sample solution is placed in a belt above the recess 3a located at the rear end in the transport direction. It is held by the conveyor 8. Thereafter, the heating and cooling of the reaction vessel 4 by the blocks 2a, 2b and 2c is performed in the same manner as described above.

By repeating the above operation 16 times, the processing of the sample inside the reaction vessel 4 in which the reaction has been performed first is completed, and the reaction vessel 4 is removed from the belt conveyor 8. This removal may be performed manually by an operator or may be performed using a manipulator.

According to the above embodiment, the PCR reaction can be performed by using the blocks 2a, 2b, and 2c which are adjusted to different temperatures, and by performing the sequential processing instead of the batch processing. It can be greatly reduced compared to the conventional case.

It is preferable that the operation of the incubation apparatus 1 is controlled by, for example, a sequence controller, and the control program is changed so as to be able to cope with reactions of different temperature cycles.

In order to prevent the reaction solution from evaporating during the treatment, a mineral oil may be placed on the reaction solution or a lid may be welded to the reaction container 4. By filling the insides of the recesses 3a, 3b, 3c of the blocks 2a, 2b, 2c with mineral oil, the reaction solution and the block 2 are filled.
The heat conduction between a, 2b, and 2c may be improved.

A second embodiment of the present invention will be described with reference to FIGS.

The incubation device 51 shown in FIG.
Has a holder 53 for holding the reaction vessel 52 and three tanks 54a, 54b, 54c. Each tank 54
Silicon oil is stored in a, 54b and 54c as a medium for heating and cooling the reaction vessel 52. A heater or a cooling element (not shown) for heating each of the tanks 54a, 54b and 54c is provided.
The silicon oil stored in b, 54c is adjusted to different temperatures. Each tank 54a, 54b, 54c
Outlet pipes 55a, 55b for extracting silicon oil from
On the way of 55c, on-off valves 56a, 56b, 56
c is provided. Outlet pipes 55a, 55b, 55c
Are connected to each other to form a single supply pipe 57. This supply pipe 57 is connected to the holder 53.

The holder 53 is a silicon oil storage layer having an upward opening, and stores the silicon oil supplied from the supply pipe 57. To discharge the stored oil, a pipe 58 is inserted into a discharge hole 53a formed in the holder 53.
Is connected, and a pump 59 is provided in the middle of the pipe 58. The pipe 58 branches off and the tank 5
4a, 54b, and 54c. A branch pipe 58a from this pipe 58 to each tank 54a, 54b, 54c,
On the way between 58b and 58c, on-off valves 59a and 59, respectively.
b, 59c are provided. In addition, it is preferable to provide a heater and a cooler for controlling the temperature of the silicon oil stored inside the holder 53.

As shown in FIGS. 9 and 10, the reaction vessel 52 is formed of a material in which a metal thin film 60 is covered with a resin 61, and has a large number of upwardly-opening concave portions 62 for containing a reaction solution. As the metal thin film 60, a material having good thermal conductivity such as aluminum is used. Also,
As the synthetic resin 61 that covers the metal thin film 60, a resin that does not react with a reaction reagent such as Teflon is used.
The number of the concave portions 62 is 96 in this embodiment. The reaction container 52 covers the upward opening of the holder 53 so that the reaction container 52 is held by the holder 53. A plurality of columns 53b for supporting the reaction vessel 52 are provided inside the holder 53 at intervals.

As shown in FIG. 8, a reaction vessel 52 comprises a thin metal film 60 made of aluminum or the like and a synthetic resin 61 made of Teflon or the like.
Is covered with a lid 63 made of a material covered with the. The lid 63 and the reaction container 52 are bonded with an adhesive or the like. Further, in order to press the lid 63 against the reaction vessel 52 and keep the temperature, the lid 63 is sandwiched between a plate 64 having a built-in heater therein and the holder 53.

The operation when a PCR reaction is carried out using the incubation device 51 will be described. In addition, PC
The temperature cycle of the R reaction is the same as the condition shown in FIG.

First, the reaction vessel 52 is filled with a sample solution, sealed with a lid 63, and then held so as to sandwich the reaction vessel 52 between a holder 53 and a plate 64. Further, the temperature of the silicone oil stored in the tanks 54a, 54b, 54c is previously adjusted to 94 ° C., 54 ° C., 72 ° C., respectively.

Next, by opening the on-off valve 56a, the silicone oil stored in the tank 54a and adjusted to a temperature of 94 ° C. is supplied through the supply pipe 57 into the holder 53. This supply is performed by the opening 53 c provided in the holder 53.
Until the silicone oil overflows from
After the supply, the on-off valve 56a is closed. At this time, the temperature of the holder 53 is preferably adjusted to 94 ° C. by a heater. As a result, the reaction vessel 52 is heated by the silicone oil whose temperature has been adjusted to 94 ° C., and the sample is thermally denatured.

Next, the on-off valve 59a is opened and the pump 5
By driving the nozzle 9, the silicone oil in the holder 53 is returned to the tank 54a, and after the return, the on-off valve 59a is closed and the pump 59 is stopped.

Next, the on-off valve 56b is opened, and silicone oil whose temperature has been adjusted to 55 ° C. is supplied into the holder 53 through the supply pipe 57. This supply is performed until the silicone oil overflows from the opening 53c, and after the supply, the on-off valve 56b is closed. At this time, it is preferable to cool the holder 53 to 55 ° C. with a cooler. This allows
The reaction vessel 52 is cooled with 55 ° C. silicone oil to anneal the primer of the sample.

Next, the on-off valve 59b is opened and the pump 5
9 to drive the silicon oil in the holder 53 into the tank 5
After the reflux, the on-off valve 59a is closed and the pump 59 is stopped.

Next, the opening / closing valve 56c is opened, and silicon oil whose temperature has been adjusted to 72 ° C. is supplied into the holder 53 through the supply pipe. This supply is performed until the silicone oil overflows from the opening 53c, and after the supply, the on-off valve 56c is closed. At this time, it is preferable to adjust the temperature of the holder 53 to 72 ° C. by a heater. As a result, the reaction vessel 52 is heated by the silicon oil to synthesize a DNA chain. Thereafter, by opening the on-off valve 59c and driving the pump 59, the silicon oil is stored in the tank 5.
After the reflux, the on-off valve 59c is closed and the pump 59 is stopped.

As described above, one temperature cycle is completed. The reaction is completed by performing this temperature cycle 16 times.

According to the second embodiment, since the reaction vessel 52 can be heated and cooled by the silicone oil adjusted to different temperatures, wasteful time can be reduced as compared with the conventional case. Moreover, since the reaction container 52 is formed of a material in which the metal thin film 60 is covered with the synthetic resin, the reaction container 52 has good thermal conductivity and does not react with the reaction reagent. Moreover, since it can be easily manufactured, it is usually preferable as a disposable reaction container.

As shown in FIG. 11, a band-shaped material in which a metal thin film is coated with a synthetic resin is wound on a reel 67, and the material fed from the reel is formed into a reaction vessel 52 using dies 65 and 66. May be incorporated into the incubation device.

[0035]

According to the incubation apparatus of the present invention, the reaction vessels can be heated and cooled in a certain order by using heating and cooling media adjusted to different temperatures, and the sample filled in the reaction vessel can be heated and cooled. Can be shortened and the power can be minimized. In addition, by sequentially proceeding the reaction one by one in a plurality of reaction vessels, consistency with pretreatment before the reaction, detection processing after the reaction, and the like, which must be sequentially performed in a time series, is improved. Can be put out. In addition, by forming the reaction vessel from a material in which a metal thin film is coated with a synthetic resin, the reaction vessel can be made to have good thermal conductivity and not react with the reaction reagent, thereby reducing processing time and wasting power. can do.

[Brief description of the drawings]

FIG. 1 is a front view of an incubation apparatus according to a first embodiment of the present invention.

FIG. 2 is a side view of the incubation apparatus according to the first embodiment of the present invention.

FIG. 3 is a side view of the heating / cooling medium according to the first embodiment of the present invention.

FIG. 4 is a partial plan view of the incubation device according to the first embodiment of the present invention.

FIG. 5 is a diagram illustrating the operation of the incubation device according to the first embodiment of the present invention.

FIG. 6 is a diagram showing temperature conditions of a PCR reaction.

FIG. 7 is an explanatory diagram of a configuration of an incubation apparatus according to a second embodiment of the present invention.

FIG. 8 is a partial cross-sectional view of an incubation device according to a second embodiment of the present invention.

FIG. 9 is a sectional view of a reaction vessel according to a second embodiment of the present invention.

FIG. 10 is a plan view of a reaction vessel according to a second embodiment of the present invention.

FIG. 11 is a configuration explanatory view of a reaction vessel manufacturing apparatus according to a second embodiment of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Incubation apparatus 2a, 2b, 2c Block (heating / cooling medium) 4 Reaction vessel 51 Incubation apparatus 52 Reaction vessel 60 Metal thin film 61 Synthetic resin

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-3-262499 (JP, A) JP-A-4-325080 (JP, A) International Publication 90/5023 (WO, A1) West German Patent Application Publication 3889482 (DE, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12M 1/38 C12M 1/00 B01L ⁷ /00-7/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. An incubation apparatus comprising a plurality of reaction vessel heating / cooling media preliminarily adjusted to different temperatures, wherein each heating / cooling medium is independently contacted with the reaction vessel in a certain order. The reaction vessel heating / cooling medium includes a plurality of blocks that are previously temperature-controlled to different temperatures and are arranged in parallel in one horizontal direction. Each block has a predetermined number of repetitions of a temperature cycle. Means for moving the reaction vessel and the block relative to each other along the block parallel direction, and a plurality of recesses provided parallel to each other along a horizontal direction perpendicular to the block parallel direction. Means for relatively moving the reaction vessel and the block along the parallel pitch of the concave portion along with, so that the reaction container can be taken in and out of the concave portion. Means for vertically moving said reaction vessel and said block relative to each other. 2. A heating and cooling medium comprising a plurality of reaction vessel heating / cooling media which have been previously adjusted to different temperatures, wherein each heating / cooling medium is independently contacted with the reaction vessel in a certain order, 2. The incubation apparatus according to claim 1, wherein is formed from a material obtained by coating a metal thin film with a synthetic resin.