JP2001074756A - Pretreatment device of specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve the pretreatment device of a specimen that can accurately judge the deterioration of a solid phase in a nozzle that brings a sample into contact. SOLUTION: In the pretreatment device, pressure is reduced by a syringe 32 for sucking the mixing liquid of a specimen and a combination accelerator into the inside of a nucleic acid capture chip 31. After fixed time from a point when the reduction of the pressure is started, the pressure in piping 35 is measured. When a pressure value is outside a specific pressure range r1, entire liquid in the chip 31 is discharged, the chip 31 is removed from a nozzle 39, and the new chip 31 is connected to the nozzle 39. On the other hand, when the pressure value is within the range r1, it is judged that a solid phase 38 in the chip 31 is normal, and suction and discharge are repeated for a specific number of times for bringing the solid phase into contact with the mixing liquid only when the pressure value of the piping 35 exists within a specific pressure range r2 while the operation of the syringe 32 is stopped, and at the same time the pressure value is within a pressure range r3 after fixed time t3 from a point when the operation of the syringe 32 is stopped.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample pretreatment device, and more particularly to a sample pretreatment device for recovering nucleic acid from a nucleic acid-containing substance.

[0002]

2. Description of the Related Art Advances in molecular biology have led to the development of a number of gene-related technologies, and the isolation and identification of many disease-causing genes by these technologies. as a result,
In the field of medical treatment, molecular biology techniques have been introduced into diagnosis or testing methods, enabling diagnosis that has been impossible in the past, and significantly shortening the number of testing days.

[0003] This rapid advance is mainly due to the nucleic acid amplification method, in particular, the PCR method (polymerase chai).
n reaction: polymerase chain reaction, Sai
kiet al. , Science, 239, 4
87-391 (1988)).

[0004] The PCR method can amplify a nucleic acid in a solution in a sequence-specific manner. For example, a virus which is present only in a trace amount in serum can be detected by amplifying the nucleic acid which is the gene of the virus. Can be proved indirectly.

[0005] However, there are some problems when this PCR method is used for daily inspection in a clinical setting. In particular, the importance of nucleic acid extraction and purification steps in sample pretreatment has been pointed out (Oshima et al., JJCLA,
22 (2), 145-150 (1997)). this is,
This is due to the effect of an inhibitor that could not be removed during nucleic acid purification. As the inhibitor, hemoglobin in blood, a surfactant used in the extraction step, and the like are known.

[0006] In addition, the extraction step requires a complicated operation using a manual technique and a great deal of labor by a skilled person.
Therefore, this is an obstacle to introducing a new genetic test into a laboratory in a hospital, and automation of this process is eagerly desired.

[0007] In addition, in institutions conducting molecular biological research, plasmid DNA is frequently used as a material for performing gene recombination operations. Similarly, automation of the process of extracting and purifying nucleic acids is desired.

[0008] A known method for recovering a nucleic acid from a biological sample containing the nucleic acid in a highly purified state that does not contain an inhibitor is to react a surfactant with the biological sample in the presence of a protease. Nucleic acid is released, mixed with phenol (and chloroform), centrifuged to separate the aqueous and organic phases several times, and then the nucleic acid is recovered from the aqueous phase in the form of a precipitate with alcohol. Are known.

However, this preparation method uses an organic solvent such as phenol which is a deleterious substance in the process, or requires a centrifugal separation step, so that the container is taken in and out of the rotor of the centrifuge or after centrifugation. There is a problem that automation such as fractionation of a solution is very difficult.

In addition to the above-described method using aqueous / organic phase separation, a method for recovering DNA from agarose gel by utilizing the property of nucleic acid binding to the glass surface in the presence of a chaotropic substance (B) .Vogelstein
and D. Gillespie, Proc. Nat
l. Acad. Sci. USA, 76 (2), 615-
619 (1979)).

In this method, the use of phenol which is a deleterious substance can be avoided, but the separation of the glass and the solution after the nucleic acid is bound to the glass surface requires a centrifugation step.
As with the aqueous / organic phase separation described above, automation of the centrifugation step becomes a problem.

As a prior art for avoiding the centrifugation step, B / F (bond form / free for) in an immunoassay apparatus utilizing an antigen-antibody reaction is known.
m) Separation techniques are applicable.

A technique suitable for application to the present technique is a technique in which a sample probe chip has a constriction and a resin in which an antibody is physically or chemically bound between the constrictions is filled. Exists (JP-A-63-88)
No. 456).

In this prior art, the use of a solid phase containing silica instead of the resin to which the antibody is bound can be applied to the recovery of nucleic acids, but the solid phase is enclosed between the constrictions. Therefore, it becomes very difficult to form the sample probe tip.

For this reason, FIG. 7 or FIG.
A nucleic acid capturing chip 31 having a shape as shown in FIG. 1 is prepared, and combined with a movable nozzle for sucking and discharging a liquid is suitable for nucleic acid recovery.

That is, the sample containing nucleic acid is applied to the solid phase 38 or 4
It is reciprocated several times through 1 and is captured by the solid phase 38 or 41. Reference numeral 40 denotes a holding material for holding the solid phase 38 in the nucleic acid capturing chip 31.

[0017]

As described above, in the technique for recovering nucleic acids, a sample containing nucleic acids is immobilized on a solid phase 38.
Alternatively, the solid phase 38 or 41 needs to be reciprocated several times through the solid phase 38 or 41, and the quality of the solid phase 38 or 41 greatly affects the amount of nucleic acid recovered. For this reason, it is very convenient if the quality of the solid phase 38 or 41 of the nucleic acid capturing chip 31 can be automatically determined during the nucleic acid recovery processing.

Thus, it is conceivable to use the technology described in Japanese Patent Application Laid-Open No. 2-184762 or Japanese Patent Application Laid-Open No. 6-27120. In other words, the techniques described in these publications determine whether or not the suction port of the nozzle that suctions the sample is in a closed state, for example, by determining the pressure in the nozzle after a certain period of time has elapsed since the start of the nozzle suction operation. To detect
If the pressure change rate is equal to or more than a predetermined value, it is determined that the suction port of the nozzle is in a closed state.

However, the technique described in these publications is a technique for judging a closed state of the tip of a nozzle in a state where the tip does not have a solid phase or the like in a normal state. Even if it is applied as it is to the technique for judging the quality of the solid phase which is arranged in advance at the front end portion, it is difficult to make an accurate judgment.

That is, in the case of the nucleic acid capturing chip, since the prerequisite is that the sample passes through the solid phase, the pressure change in the chip from the start of aspirating the sample to the aspirating of a sufficient amount of the sample is as described above. The change in pressure of the nozzle described in the gazette is not equivalent. In the case of a nucleic acid capture chip, even if a predetermined pressure value or a rate of change in pressure is satisfied after a certain time from the start of aspirating the sample, it is necessary to further aspirate the sample and pass through the solid phase. It is also conceivable that the solid phase degrades before a sufficient sample is aspirated. In such a case, it is difficult to determine the deterioration of the solid phase by the technique described in the above-mentioned publication.

Therefore, it has been desired to realize a technique capable of accurately determining the deterioration of the solid phase, which is provided at the tip portion thereof, through which the sample passes.

An object of the present invention is to realize a sample pretreatment apparatus which can accurately judge deterioration of a solid phase, which is provided in a nozzle and comes into contact with a sample.

[0023]

To achieve the above object, the present invention is configured as follows. (1) A pipe in which a solid phase in contact with a liquid sample is installed, a pressure source, and a liquid is suctioned and discharged to the pipe via the solid phase by a pressure change due to the operation of the pressure source. In a sample pretreatment device that performs sample pretreatment, a pressure detection unit that detects a pressure change in the pipe,
The pressure value in the pipe at the time of starting the operation of the pressure source is within a first pressure range, and the pressure value in the pipe at the time of stopping the operation of the pressure source is within a second pressure range. There is provided a determination means for determining that the solid phase is normal at one time.

(2) Preferably, in the above (1),
A chip is provided at the tip of the pipe, and the solid phase is disposed on the chip.

(3) Preferably, in the above (1) or (2), means is provided for displaying a warning based on the state of the solid phase determined by the determining means.

(4) Preferably, the above (1),
In (2) and (3), the solid phase has a function of capturing a specific component in a specimen.

(5) Preferably, in the above (4), the solid phase is a substance containing silica, and the specific component to be captured is a nucleic acid.

(6) Preferably, the above (3),
In (4) and (5), a chip is provided at the tip of the pipe, the solid phase is disposed on the chip, and the chip can be replaced for each sample.

(7) Preferably, the above (1),
In (2), (3), (4), (5), and (6), a chip was placed at the tip of the pipe, the solid phase was placed on the chip, and the chip was attached to the pipe. In this state, it is brought into contact with a specific solution, and the inside of the pipe is depressurized by the above-mentioned pressure source. It has a function to judge.

(8) Preferably, the above (6),
In (7), a function is provided in which the state of the solid phase is determined before the chip comes into contact with the sample, and when the solid phase is determined to be defective, the function is automatically replaced with a new chip.

(9) In the above (7), a function is used in which, when a sample is used for the specific solution and the solid phase is judged to be defective, the sample is discharged and then automatically replaced with a new chip. Prepare.

When a sample is sucked into the pipe, the pressure in the pipe is measured after a lapse of a predetermined time from the start of pressure generation,
If the pressure is within the first pressure range, the pressure in the pipe is measured during the time when the pressure generation is stopped. If the pressure is within the second pressure range, the solid phase in the nozzle is determined to be normal, and The suction and discharge are repeated a number of times to bring the solid phase into contact with the sample.

Therefore, it is possible to realize a sample pretreatment device which can accurately determine the deterioration of the solid phase in the nozzle, which comes into contact with the sample, in the nozzle.

[0034]

Embodiments of the present invention will be described below with reference to the accompanying drawings. FIG. 1 is a schematic diagram showing the principle configuration of a sample pretreatment device 100 according to an embodiment of the present invention, FIG. 2 is an external view of the sample pretreatment device, FIG. 3 is a plan view of the device, and FIG. FIG. 5 is an operation control block diagram of the apparatus, FIG. 5 is an explanatory view of an operation of attaching a chip to a nozzle, FIG. 6 is an explanatory view of an operation of removing a chip from a nozzle, and FIG.

The syringes (pressure generating sources) 10 and 32 can independently and automatically control the suction and discharge of the liquid. The syringes 10 and 32 are independently connected to nozzles 36 and 39 via a pipe 42, a nozzle holder 17, a pipe 35, and a nozzle holder 34, respectively. Further, the dispensing nozzle 36 and the movable nozzle 39 for sucking and discharging the liquid are fixed to the nozzle holders 17 and 34, respectively. The nozzle holders 17 and 34 are respectively attached to the arms 16 and 33 in the Y direction (the arms 16 and 33 in FIG. 2).
2) and a Z-direction (a direction orthogonal to the longitudinal direction of the arms 16 and 33 in FIG. 2).

The arms 16 and 33 are independently moved in the X direction (directions orthogonal to both the Y direction and the Z direction).
Can be moved, and a part can be overlapped in the X direction by giving a difference between the positions in the Z direction. Thus, the nozzle can be moved to the main part in the plane of the apparatus by a combination of the operations of the nozzle holders 17 and 34 and the arms 16 and 33.

The nozzle holder 34 and the syringe 32
A pressure sensor 101 is attached to a pipe 35 between the two. Then, the pipe 35 is detected by the pressure sensor 101.
The pressure change in the inside is converted into an electric signal. The pressure change converted into the electric signal is amplified by the amplifier 102 and
(PC) 60 is taken in as pressure change information,
It is processed. Reference numeral 65 denotes a mechanism control unit. The mechanism control unit 65 receives a command from the PC 60 and controls the nozzle holder 3.
4. Control the operation of the syringe 32 and the like.

A dispensing tip can be stored in the dispensing tip holder 14 (FIGS. 2 and 3), and a total of three identical tip holders (14a, 14b, 14c) can be installed on the apparatus. Further, 48 reaction containers 24 can be installed in the reaction container rack 23, and 48 purified product storage containers 26 can be installed in the purified product rack 25.

Further, on the apparatus, a washing liquid bottle 19, an eluent liquid bottle 20, a diluent liquid bottle 21, and a binding promoter bottle 22 can be installed one by one. And 48 dispensing chips 31 can be installed in the nucleic acid capturing chip rack 30.

By controlling the operation of the arm 16 and the nozzle holder 17, the tip holder 14 (14a,
4b, 14c) After moving the nozzle 36 above the target dispensing tip 15 above, the nozzle holder 17 is moved downward.
Is moved to bring a predetermined position of the dispensing tip 15 into contact with the nozzle 36. Thereby, the dispensing tip 15 can be automatically attached to the tip of the nozzle 36. The same control is performed for the nozzle 39, the nozzle holder 34, the arm 3
3, the nucleic acid capturing chip 31 can be attached to the tip of the nozzle 39.

After the operation of the arm 16 and the nozzle holder 17 is controlled, the nozzle 36 is moved to a position just above the tip punch 27, and the joint between the nozzle 36 and the dispensing tip 15 is pulled by the nozzle holder 17. The nozzle 36 is moved so as to be lower than the nozzle 27, and further, the nozzle 36 is moved in the direction of the chip punch 27.

Thereafter, a part of the nozzle 39 is removed
The dispensing tip 15 can be automatically removed from the nozzle 36 by moving the nozzle holder 17 upward while contacting the nozzle 7. That is, the state is as shown in FIG.

By performing the same control on the nozzle 39, the nozzle holder 34, and the arm 33, the nucleic acid capturing chip 31 can be removed from the nozzle 39. In addition, it is possible to separately collect waste chips by installing the chip remover 27 according to the type of chip to be used.

The liquid receivers 11 and 28 can receive the liquid discharged from the nozzles 36 and 39 and function at their respective home positions, and the received liquid is sent as waste liquid. The cleaning unit 18 discharges the running water to discharge the nozzle holder 1.
Dispensing tip 15 attached to nozzle 7 via nozzle 36
Can be washed.

The nucleic acid capture chip 31 shown in FIG. 7 is prepared such that holding members 40 are provided above and below the tip of the chip, and a state in which the solid phase 38 is sealed by these two holding members 40 can be maintained. ing. This nucleic acid capturing chip 31
Can be stored in the nucleic acid capture chip rack 30 on the device shown in FIG.

Here, the holding material 40 has a corrosion resistance and a passage property with respect to a liquid and a gas to be used, and has a solid phase 3
Any substance can be used as long as it can block the passage of No. 8, but since non-specific adsorption of proteins and nucleic acids affects the purification degree and yield of the nucleic acid solution, it is desirable to use a substance with low non-specific adsorption.

In this case, polypropylene is used as the holding material 40, and use of an adhesive for fixing inside the chip 31 may cause impairment of water permeation and air permeability due to immersion inside the solid phase 38. Since it was not preferable, it was installed by press fitting. As the solid phase 38, any substance containing silicon oxide, such as glass particles, quartz particles, quartz filter paper, quartz wool, or crushed or processed products thereof can be used. Quartz particles having a particle size larger than the gap of the material 40 were used.

The nucleic acid capturing chip 31 shown in FIG.
Is arranged such that a solid phase 41 having an outer diameter equivalent to a part of the inner diameter of the chip 31 is arranged, and the solid phase 41 can be held in the chip 31. Then, the chip 31 shown in FIG. 8 can be stored in the nucleic acid capturing chip rack 30 on the device shown in FIG.

The shape of the solid phase 41 is not limited as long as the solid phase 41 is a silicon oxide-containing substance having water permeability and gas permeability and can be held inside the chip 31 by the solid phase 41 alone. However, the use of an adhesive is not preferable because it may impair water permeability and air permeability due to immersion in the solid phase 41 or cause chemical and physical modification of the solid phase surface.

Here, a substance (quartz sintered body) sintered to a state in which quartz particles can be passed through and vented and having an outer diameter equivalent to a part of the inside of the chip 31 is used as a solid phase 41.
The nucleic acid capturing chip 31 was used by being pressed from above. When the solid phase 41 has a high possibility of crushing during use or transportation, or has a possibility of jumping out of the chip 31 during transportation or the like, similarly to the chip 31 shown in FIG. It may take the form of being sandwiched between.

Next, referring to FIG.
Will be described. In FIG. 4, the operation of the syringes 10 and 32 is controlled by the PC 60 controlling the stepping motors 71 and 72 by the mechanism control unit 65, respectively. In addition, the nozzle holders 17, 3
Operation 4 is controlled by the PC 60 controlling the stepping motors 73 and 74 by the mechanism control unit 65, respectively. The operation of the arms 16 and 33 is controlled by the PC 60 controlling the AC servomotors 75 and 76 by the mechanism control unit 65, respectively.

The operator's command is supplied to the PC 60 via the keyboard 61. Also, under the control of the PC 60,
Various information is displayed on the CRT 62. For example, when it is determined that the solid phase is defective, an indication to that effect is displayed on the CRT 62.

Next, the nucleic acid of the specimen having the nucleic acid is captured,
The operation for collecting will be described. By controlling the operation of the arm 16 and the nozzle holder 17, the dispensing tip 15 is attached to the nozzle 36 by a predetermined operation. Thereafter, by controlling the operation of the arm 16, the nozzle holder 17, and the syringe 10, the binding promoter bottle 2 is controlled.
Aspirating a predetermined amount of the binding promoter from 2 into the dispensing tip 15;
Further, a predetermined amount of air is sucked. And dispensing tip 1
5 is moved to the washing table 18 and the outer wall of the dispensing tip 15 is washed with running water.

After washing the outer wall of the dispensing tip 15, the nozzle holder 17 is moved to the position of the predetermined specimen 13 on the specimen rack 12, and the operation of the syringe 10 is controlled so that a predetermined amount of the specimen is dispensed. Is sucked. Then, after aspirating the sample from the dispensing tip 15, the nozzle holder 17 is moved onto the reaction container rack 23, and the entire amount of the sample in the dispensing tip 15 is discharged to a predetermined reaction container 24 of the rack 23.

After the sample is discharged into the reaction container 24, the sample in the reaction container 24 is further suctioned and discharged by the dispensing tip 15 to mix the sample and the binding promoter. After the mixture of the sample and the binding promoter, the nozzle holder 17 is moved to the position of the tip punch 27, and the dispensing tip 15 is removed from the nozzle 36 by the above-described predetermined operation.

By controlling the operation of the arm 33 and the nozzle holder 34, the nucleic acid capturing chip 31
Is attached by a predetermined operation. Thereafter, the nozzle holder 34 is moved to the reaction container 24 on the reaction container rack 23 containing the mixture (mixture of the sample and the binding promoter). Then, by controlling the operation of the syringe 32, the mixed solution is sucked into the nucleic acid capturing chip 31.

At this time, the solid phase 3 in the nucleic acid capturing chip 31
If 8 or 41 is defective, it will be difficult to sufficiently capture nucleic acids. Therefore, the quality of the solid phase 38 or 41 is determined as follows.
The determination is made when the mixed solution is sucked into the nucleic acid capturing chip 31. The determination of the quality of the solid phase is performed by the PC 60. In addition, the determination of the elapsed time described later is also performed by the PC.
60 is what it does.

The determination of the quality of the solid phase 38 or 41 will be described below. FIG. 9 shows a result of temporally detecting a pressure change in the pipe 35 when the mixed solution is sucked into the nucleic acid capturing chip 31.

In FIG. 9, the horizontal axis represents time, and time 0
Indicates a time at which the pressure in the pipe 35 is started to suction the mixed liquid by the operation of the syringe 32. The vertical axis indicates a pressure change in the flow path (the pipe 35).

After a lapse of a certain time t1 from the start of the pressure reduction (when the solid phases 38 and 41 are normal, the time when the pressure in the pipe 35 becomes close to a stable value, which is about 0.15 in the example of FIG. 9).
In s), the pressure in the pipe 35 is measured. The measured pressure value is within a predetermined pressure range r1 (about -0.02 in the example of FIG. 9).
5 to -0.06 kgf / cm ² ), and if it is out of the range, after discharging all the liquid sucked into the nucleic acid capturing chip 31, the nucleic acid capturing chip 31 is moved from the nozzle 39. Remove and connect the new nucleic acid capture chip 31 to the nozzle 39. Then, the solution once sucked and discharged by the nucleic acid capturing chip 31 is sucked again, and the pressure in the pipe 35 is measured after a lapse of a certain time t1 from the start of the pressure reduction.

If the pressure value in the pipe 35 is within the pressure range r1, the operation of the syringe 32 is stopped at time t2 (FIG. 9).
At about 5 s in the example, the pressure in the pipe 35 is measured. It is determined whether or not the measured pressure value is within a predetermined pressure range r2 (about -0.04 to -0.075 kgf / cm ^{2 in} the example of FIG. 9). After all the liquid sucked into the capture chip 31 is discharged, the nucleic acid capture chip 31 is removed from the nozzle 39, and a new nucleic acid capture chip 31 is connected to the nozzle 39. Then, the solution once sucked and discharged by the nucleic acid capturing chip 31 is sucked again, and the pressure in the pipe 35 is measured after a lapse of a certain time t1 from the start of the pressure reduction.

When the pressure range r1 at the time t1 is satisfied and the pressure range r2 at the time t2 is satisfied, a certain time t3 (from t2 in the example of FIG. 9) from the time when the operation of the syringe 32 is stopped. After about 1 s), the pressure in the pipe 35 is measured. Then, the measured pressure value falls within the pressure range r3 (about 0.02 to-
0.01 kgf / cm ² ) or not.

If the measured pressure value is not within the pressure range r3, all the liquid sucked into the nucleic acid capturing chip 31 is discharged, and then the nucleic acid capturing chip 31 is removed from the nozzle 39,
The new nucleic acid capturing chip 31 is connected to the nozzle 39. Then, the solution once sucked and discharged by the nucleic acid capturing chip 31 is sucked again, and the above-described quality judgment of the solid phases 38 and 41 is executed.

If the measured pressure value is within the pressure range r3, the solid phases 38 and 41 in the nucleic acid capturing chip 31 are determined to be normal, and the suction and discharge are repeated a predetermined number of times.
Contact 1 with the mixture.

That is, after the elapse of a certain time t1 from the start of the pressure reduction for suction, the time t when the operation of the syringe 32 is stopped
2. Measure the pressure value in the pipe 35 after a lapse of a certain time t3 from the time point t2, and determine whether or not the pressure value in the flow path expected from the viscosity of the sucked mixed liquid is within the range of individual difference, When all the ranges r1, r2, and r3 are satisfied, it is determined that the solid phases 38 and 41 are normal.

As described above, by determining the solid phases 38 and 41, the quality of the solid phases 38 and 41 is accurately determined, and a sufficient amount of the mixed solution is reciprocally passed through the normal solid phases 38 and 41. To capture nucleic acids. In some cases, the determination after the elapse of the time t3 from the time t2 may be omitted.

After determining that the solid phases 38 and 41 are normal and repeating the suction and discharge a predetermined number of times, the mixed solution in the reaction container 24 is sucked into the nucleic acid capturing chip 31 and then the arm 33 and the
The tip 31 is moved to the waste liquid port 29 by controlling the operation of the nozzle holder 34. Then, the mixed liquid in the nucleic acid capturing chip 31 is discharged to the waste liquid port 29 by controlling the syringe 32. After discharging the mixed liquid to the waste liquid port 29, the tip of the chip 3 is transferred to the liquid receiver 28 by controlling the operation of the arm 33 and the nozzle holder 34.
Move one.

After the dispensing tip 15 is attached to the nozzle 36 by controlling the operation of the arm 16 and the nozzle holder 17, the cleaning liquid bottle 1 is controlled by controlling the operation of the arm 16, the nozzle holder 17 and the syringe 10.
A predetermined amount of the washing liquid is sucked from 9. Then, the nozzle holder 17 is operated to discharge the cleaning liquid onto a predetermined reaction vessel 24 on the reaction vessel rack 23.

After the discharge of the cleaning liquid, the operation of the arm 16 and the nozzle holder 17 is controlled to move the nozzle holder 17 to the position of the tip punch 27, and the dispensing tip 15 is removed from the nozzle 36 by a predetermined operation.

After the nozzle holder 17 moves, the arm 3
3. By controlling the operation of the nozzle holder 34,
The nucleic acid capturing chip 31 is moved to a predetermined reaction container 24 on the reaction container rack 23 containing the cleaning liquid, and the cleaning liquid is sucked into the nucleic acid capturing chip 31 by the operation of the syringe 32. After the suction of the cleaning liquid, the suction and discharge are repeated a predetermined number of times by the operation of the syringe 32, and the solid phase 38 is cleaned with the cleaning liquid.

After the suction and discharge have been repeated a predetermined number of times, the washing solution in the reaction vessel 24 is sucked into the nucleic acid capturing chip 31, and the operation of the arm 33 and the nozzle holder 34 is controlled so that the nucleic acid capturing chip 31 is removed. It moves to the waste liquid port 29 and discharges the cleaning liquid in the nucleic acid capturing chip 31 by the operation of the syringe 32. After the ejection of the washing liquid, the nucleic acid capturing chip 31 is moved to the liquid receiver 28 by the operations of the arm 33 and the nozzle holder 34.

If necessary, the nucleic acid washing step is repeated a predetermined number of times. In this case, the nucleic acid washing step may be repeated as it is, but a plurality of washings may be dispensed with the dispensing tip 15.
And discharge the required amount to the reaction container 24. And
After discharging the cleaning liquid, the cleaning liquid is moved to the position of the liquid receiver 11, and after the cleaning operation of the nucleic acid capturing chip 31, the required amount of the cleaning liquid is discharged again to the reaction container 24 by the dispensing tip 15, thereby efficiently performing the nucleic acid cleaning step. Can be repeated.

Next, by controlling the operation of the arm 16 and the nozzle holder 17, the dispensing tip 1
After attaching 5, the arm 16 and the nozzle holder 17,
Then, a predetermined amount of the eluent is sucked from the eluent bottle 20 by the operation of the syringe 10, and the eluant is discharged from the nozzle holder 17 to a predetermined reaction container 24 on the reaction container rack 23.

After the discharge of the eluent, the operation of the arm 16 and the nozzle holder 17 is controlled to move the nozzle holder 17 to the position of the tip punch 27, and the dispensing tip 15 is removed from the nozzle 36 by a predetermined operation.

After the movement of the nozzle holder 17, the arm 3
3. By controlling the operation of the nozzle holder 34,
The nucleic acid capturing chip 31 is moved to a predetermined reaction container 24 on the reaction container rack 23 containing the eluent, and the eluate is sucked into the nucleic acid capturing chip 31 by the operation of the syringe 32. After suction of the eluent, suction and discharge are repeated a predetermined number of times by the operation of the syringe 32 to bring the solid phase 38 into contact with the eluent.

After the suction and discharge are repeated a predetermined number of times, the eluate in the reaction vessel 24 is sucked into the nucleic acid capturing chip 31, and then the operation of the arm 33 and the nozzle holder 34 is controlled, thereby making it possible to remove the purified product rack. The nucleic acid capturing chip 31 is moved to a predetermined refined product storage container 26 stored in the container 25, and the eluate in the nucleic acid capturing chip 31 is discharged into the storage container 26 by the operation of the syringe 32. After discharging the eluent, arm 3
3. The nucleic acid capturing chip 31 is moved to the liquid receiver 28 by the operation of the nozzle holder 34.

The elution step is repeated a predetermined number of times, if necessary. At this time, the above-mentioned elution step may be repeated as it is, but a plurality of eluents are sucked into the dispensing tip 15 and the required amount is discharged to the reaction vessel 24. After discharging the eluent, the dispensing tip 15 is moved to the position of the liquid receiver 11, and after the suction / discharge operation of the nucleic acid capturing chip 31, the required amount of the eluent is again discharged to the reaction container 24 by the dispensing tip 15. Thereby, the above-mentioned elution step can be efficiently repeated.

After the above steps are completed, the operation of the arm 33 and the nozzle holder 34 is controlled to move the nozzle holder 34 to the position of the tip punch 27, and the nucleic acid capturing chip 31 is removed from the nozzle 39 by a predetermined operation.

As described above, according to one embodiment of the present invention, when the mixed solution is sucked into the nucleic acid capturing chip 31, the pressure in the pipe 35 is measured after a lapse of a certain time t1 from the start of the pressure reduction. It is determined whether or not the pressure is within a predetermined pressure range r1. If the pressure is out of the predetermined range, all the liquid sucked into the nucleic acid capturing chip 31 is discharged. Is connected to the nozzle 39. If the pressure value in the pipe 35 is within the pressure range r1, at a time t2 when the operation of the syringe 32 is stopped, the pressure in the pipe 35 is measured, and it is determined whether or not the pressure is in a predetermined pressure range r2. If it is within the range, the pressure in the pipe 35 is measured after a certain period of time t3 has elapsed since the operation of the syringe 32 was stopped. Then, it is determined whether or not the measured pressure value is within the pressure range r3. If the measured pressure value is within the pressure range r3, the solid phases 38 and 41 in the nucleic acid capturing chip 31 are determined to be normal, and the suction and discharge are repeated a predetermined number of times.
41 is brought into contact with the mixture.

Therefore, it is possible to realize a sample pretreatment device that can accurately determine the deterioration of the solid phase in the nozzle, which comes into contact with the sample, in the nozzle.

In the above example, the syringe 3
Although the sample and the like are sucked into the nozzle by reducing the pressure in the pipe 35 by 2, the sample and the like can be supplied into the nozzle by pressurizing the sample and the like. In this case, the quality of the solid phase may be determined by detecting a change in pressurization in the pipe 35 (the pressure curve may be a pressure curve obtained by inverting the pressure curve in FIG. 9 to the positive side).

The present invention is applicable to any material containing a nucleic acid. In particular, clinical samples such as whole blood, serum, sputum, and urine, or biological samples such as cultured cells and cultured bacteria can be used. A suitable sample or a nucleic acid held in a gel after electrophoresis is suitable.

The present invention is also effective for a reaction product such as a DNA amplification enzyme or a material containing a partially purified nucleic acid. The nucleic acid referred to here is a double-stranded, single-stranded, or partially double-stranded, or a DN having a single-stranded structure.
A (deoxyribonucleic acid) or RNA (ribonucleic acid)
It is.

As the solid phase in the present invention, any substance containing silicon oxide, such as glass particles, silica powder, quartz filter paper, quartz wool, or a crushed product thereof or diatomaceous earth can be used. In order to prevent the solid phase from coming out of the chip, the inner diameter of the tip is smaller than the inner diameter of the connection part to the pipe of the chip, and the solid phase has an outer diameter larger than the inner diameter of the tip. Let Alternatively, a holding material having a smaller hole diameter than the outer diameter of the solid phase is placed near the tip in the chip. Alternatively, sintering is performed in such a shape that the solid phase itself can be fitted into a part of the chip and that the liquid can pass through the inside. The above-described treatment is required, and a known processing technique can be used for this.

Also, by increasing the contact probability between the nucleic acid and the solid phase, the binding efficiency can be improved and the working time can be shortened. Therefore, the dispensing mechanism sucks and discharges the solution between the chip and the container. It is desirable to perform the operation multiple times.

[0086]

According to the present invention, when a sample is sucked into the nozzle, the pressure in the pipe is measured after a lapse of a predetermined time from the start of pressure generation, and if the pressure is within the first pressure range, furthermore, At the time when the pressure generation is stopped, the pressure in the pipe is measured. If the pressure is within the second pressure range, the solid phase in the nozzle is determined to be normal, and suction and discharge are repeated a predetermined number of times. And contact.

Accordingly, it is possible to realize a sample pretreatment device which can accurately determine the deterioration of the solid phase in the nozzle, which is brought into contact with the sample, in the nozzle.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the principle configuration of a sample pretreatment apparatus according to an embodiment of the present invention.

FIG. 2 is an external view of the pretreatment device shown in FIG.

FIG. 3 is a plan view of the pretreatment device shown in FIG.

FIG. 4 is an operation control block diagram of the preprocessing device shown in FIG. 1;

FIG. 5 is an explanatory view of an operation of attaching a chip to a nozzle.

FIG. 6 is an explanatory diagram of an operation of removing a chip from a nozzle.

FIG. 7 is a schematic configuration diagram of an example of a nucleic acid capturing chip.

FIG. 8 is a schematic configuration diagram of another example of the nucleic acid capturing chip.

FIG. 9 is a diagram of a pressure change in a nozzle or a pipe.

[Explanation of symbols]

 10, 32 Syringe 12 Sample rack 15 Dispensing tip 16, 33 Arm 17, 34 Nozzle holder 19 Washing liquid bottle 20 Eluent bottle 22 Binding promoter bottle 24 Reaction container 26 Purified product storage container 27 Chip removal 31 Nucleic acid capture chip 36 minutes Injection nozzle 38 Quartz particle 39 Movable nozzle for sucking and discharging liquid 40 Holding material 41 Quartz sintered body 60 Personal computer 62 CRT 65 Mechanism control unit 100 Sample pretreatment device 101 Pressure sensor 102 Signal amplifier

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) G01N 35/06 G F-term (Reference) 2G045 BB03 BB14 CB30 DA80 2G058 BA08 CB15 EA02 ED02 ED33 ED35 FA04 FB03 FB05 FB07 FB12 GB10 4B024A AA19 CA01 CA11 4B029 AA07 AA23 BB20 CC01 FA15

Claims (5)

[Claims] 1. A pipe in which a solid phase in contact with a liquid sample is installed, a pressure source, and a suction and discharge of a liquid to the pipe via the solid phase by a pressure change due to the operation of the pressure source. In the sample pre-processing device for performing pre-processing of the sample, a pressure detecting means for detecting a pressure change in the pipe, a pressure value in the pipe at the start of operation of the pressure source is a first pressure Determining means for determining that the solid phase is normal when the pressure value in the pipe is within a range and the pressure value in the pipe when the operation of the pressure generating source is stopped is within a second pressure range. Characteristic sample pretreatment device. 2. The sample pretreatment apparatus according to claim 1, wherein the solid phase has a function of capturing a specific component in the sample, and the solid phase is a substance containing silica. A sample pretreatment device, wherein the component is a nucleic acid. 3. A sample pretreatment apparatus according to claim 2, wherein a chip is provided at a tip portion of said pipe, said solid phase is disposed on said chip, and said chip is exchangeable for each sample. A sample pretreatment device characterized by the above-mentioned. 4. The sample pretreatment apparatus according to claim 3, wherein the state of the solid phase is determined before the chip and the sample come into contact with each other. A sample pretreatment device having a function of exchanging for a new chip. 5. The sample pretreatment apparatus according to claim 1, wherein a tip is provided at a tip of the pipe, and the solid phase is disposed on the tip. Then, the chip is brought into contact with a sample in a state of being attached to the pipe, the inside of the pipe is depressurized by the pressure source, and the determination means detects a change in pressure in the pipe after the pressure source is activated with time. A sample pre-processing device having a function of automatically exchanging a new chip after ejecting the sample when the solid phase is determined to be defective.