JP2011234693A - Nucleic acid examination apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid examination apparatus which can prevent unintended amplification reaction with a simple mechanism while achieving space saving.SOLUTION: The nucleic acid examination apparatus detecting fluorescence intensity or turbidity by amplifying a sample nucleic acid using a reagent includes: a dispensing chip for dispensing the reagent and the sample nucleic acid to a reaction vessel; a plugging mechanism for covering the dispensing chip; a stirring mechanism for stirring the reagent and the sample nucleic acid after plugging; a temperature controlling part for controlling the temperature at which the reaction vessel requires for amplifying the sample nucleic acid; a heating block 14 for deactivating enzyme; and a chip disposal box 11.

Description

  The present invention relates to a nucleic acid test apparatus that amplifies and detects a target nucleic acid.

  Nucleic acid amplification technology that amplifies and detects a small amount of target nucleic acid is used as a method for infectious diseases and genetic testing. Among them, PCR (Polymerase chain reaction) is a method of denaturation of double-stranded template DNA into single strand, annealing of primer into single-stranded template DNA, extension of complementary strand from primer, or denaturation and extension. It is widely known as a method of amplifying a target nucleic acid exponentially by repeating these two steps.

  This method has problems such as complicated temperature control and long reaction time. In order to solve the problem, a nucleic acid amplification method capable of amplification at constant temperature has been developed. For example, NASBA (Nucleic Acid Sequence-Based Amplification) method (Non-patent Document 1), TMA (transcription-mediated amplification) method (Non-patent Document 2), TRC (Transcription-reverse transcription concerted) method (Patent Document 1), LAMP The (loop-mediated isothermal amplification) method (Patent Document 2), the SMAP (Smart Amplification) method (Non-Patent Document 3), and the like are known as main methods. Detection is generally performed by adding a primer that binds to the target nucleic acid, a fluorescent dye having a specific sequence, a fluorescent dye that directly intercalates with DNA, and so on. Some substances are detected by turbidity or fluorescence (Patent Document 3).

  The isothermal amplification method has advantages such that it can be measured with a simple incubator and a detector, and results can be obtained faster than the PCR method. However, on the other hand, the feature has a risk of causing cross contamination at room temperature. Here, cross-contamination means that one sample mixes with another sample, and in genetic testing, the target nucleic acid is amplified exponentially, so when amplification products are mixed into other samples, Even if it is a very small amount, it may cause a false determination. In particular, in a constant temperature amplification reaction, amplification can be performed at a constant temperature in an environment where reagents, nucleic acids, and enzymes including primers are mixed, and thus amplification is started even at room temperature. Genetic testing by isothermal amplification has traditionally been performed manually, and has been addressed by laboratory technicians with full attention to cross contamination.

  For example, in an assay such as the LAMP method or the SMAP method, a primer solution and an enzyme are prepared on ice. Subsequently, the heat-denatured nucleic acid extract is added, and after closing, the mixture is stirred by tapping or vortexing. Thereafter, detection is performed while amplifying with a detector maintained at a constant temperature around 60 ° C. Since the reagent is prepared at a low temperature, amplification does not occur in a region controlled at a low temperature even if there is a splash liquid. Primer solution, enzyme, and / or dispensing tip containing either nucleic acid, or quickly sealed by an inspector before starting nucleic acid amplification.

  In the assay such as NASBA method, the reagent is prepared at room temperature. First, the heat-denatured nucleic acid extract is added to the primer solution, which is then closed and heated to 41 ° C. In the meantime, add the enzyme separately to the back of the cap, replace the cap of the reaction vessel, close the cap, and then remove the enzyme with a centrifuge. Thereafter, stirring is performed, and detection is performed while amplifying with a detector maintained at 41 ° C. In this method, since the dispensing tip for dispensing the enzyme does not come into contact with the solution containing the primer or nucleic acid, the risk of cross-contamination in the dispensing tip can be avoided. However, these operations are complicated, require skill, and have a great mental burden on the inspector. Therefore, there is an increasing demand for an automated system for genetic testing to reduce the burden on the inspector.

  Examples of conventional systems for automating genetic testing include AmpriPrep (Roche) and OSNA fully automated genetic testing equipment (Sysmex). AmpriPrep (Roche) installed a separate waste box in the consumable box in order to prevent scattering of the dispensing tips, and avoided cross-contamination by disposing it in a separate sealed space. In addition, the OSNA method fully automated genetic tester (Sysmex) dispenses the chip by discarding the chip in a small-capacity waste bag and disposing it by the inspector in a short cycle, so that the waste chip does not take a long time. Cross contamination caused by chips was avoided.

JP 2002-327130 A JP 2002-330796 A JP 2004-283161 A

Compton. J. Et Al. Nature, 350, 91- (1991) Takakura S et. Al. J Clin Microbiol. Pp.5435-9 (2005) Mitani.Y. Et Al. Nature Methods, pp.257-262 (2007)

  In the genetic test apparatus, the nucleic acid concentration after amplification is much higher than the initial nucleic acid concentration, and thus cross contamination of the amplified product may lead to erroneous determination. The cause of cross-contamination is considered to be a dispensing mechanism, stirring mechanism, carry-over from a reaction vessel, etc., and for this measure, in genetic testing, disposable dispensing tips, disposable reactions Generally, a container is used and stirring is performed in a non-contact manner. However, even in such a case, if the tip after dispensing is left for a long time, the problem remains that it is amplified at room temperature and affects the test results of other samples. Specifically, there is a problem that scattering of the reaction liquid when discarding the dispensed chips is mixed in another container and affects the result.

  In addition, if the inside of the dispensing tip to which any or all of the primer solution, nucleic acid, or enzyme is attached, or if the tip to which each was dispensed touches at the disposal site, the amplification reaction starts and affects other samples. There is a possibility to give.

  By the way, the nucleic acid amplification reaction may actually start before the measured values of fluorescence intensity and turbidity increase. Here, the TRC method will be described as an example. The TRC method is a genetic test method targeting RNA, and is an analysis method in which an amplification reaction proceeds at an isothermal temperature of around 40 ° C. and the reaction is completed in 10 to 60 minutes. First, a specific primer binds to a target RNA, cDNA is generated by the action of reverse transcriptase, and RNA is synthesized by the action of T7 RNA polymerase. In this method, RNA amplification is confirmed in about 5 to 10 minutes in a high concentration sample, but it is generated at an earlier stage than the intermediate cDNA. Since a large amount of RNA is synthesized from one cDNA, the risk of cross-contamination of cDNA is equivalent to the risk of cross-contamination of amplified RNA, and causes misjudgment.

  In the method of preparing a reagent at a low temperature such as the LAMP method and the SMAP method, the amplification is controlled by confining the working area in the area controlled at a low temperature even if there is a splash liquid, and the reaction solution is touched. A possible dispensing chip avoids cross-contamination of amplification products by having the examiner quickly seal and DNA degradation before starting nucleic acid amplification. However, in an automated device, when discarding the dispensing tip after dispensing, the remaining liquid splatters and contaminates the device, and the dispensing tip is left in the disposal box, causing unnecessary amplification. It was a problem that could happen. Furthermore, there is a problem that it cannot be applied to an analytical method such as NASBA method, TRC method, or TMA method that affects the amplification reaction by preparing a reagent at a low temperature. Therefore, the NASBA method, the TRC method, and the TMA method conventionally employ a method in which an enzyme is added to the back of the cap and then added with a centrifuge after closing the cap. In this method, when the reagent, nucleic acid, and enzyme are mixed, the stopper is closed, and the dispensing chip does not start amplification because the reagent, nucleic acid, and enzyme are not mixed. However, this method is complicated and time-consuming, and if it is equipped with a centrifuge and a special cap closing mechanism, the apparatus becomes complicated and expensive.

  In addition, the conventional genetic testing system that avoids cross-contamination by discarding dispensing tips into individual containers requires disposal boxes as many as the number of dispensing tips, saving space and consuming a large amount of dispensing tips. Realization of high throughput was difficult. In addition, when trying to achieve high throughput with an inspection system that requires the inspector to replace the waste bag in a short cycle by reducing the capacity of the waste bag, the user is required to dispose of the waste frequently, thus reducing the merit of automation. There was a problem of being.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a nucleic acid test apparatus capable of preventing an unintended amplification reaction with a simple mechanism and saving space. Can do.

  As a result of diligent studies to solve the above problems, it was noted that the largest risk of cross-contamination in the nucleic acid test apparatus was an unintended amplification reaction of the scattering liquid containing the enzyme. Provided.

  As described above, by providing a heating unit that deactivates the enzyme, the enzyme adhering to the dispensing tip can be deactivated, preventing the unintended amplification, which is the greatest risk of cross-contamination. it can.

The typical example of the automatic analyzer concerning embodiment of this invention. 1 is a schematic view of a dispensing mechanism equipped in an automatic analyzer according to an embodiment of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS The heating block integrated type chip disposal box schematic equipped with the automatic analyzer concerning embodiment of this invention. The expansion example of the chip | tip waste box integrated with a heating block with which the automatic analyzer concerning embodiment of this invention is equipped. BRIEF DESCRIPTION OF THE DRAWINGS The heating block independent type chip disposal box schematic equipped with the automatic analyzer concerning embodiment of this invention.

  Hereinafter, the best mode for carrying out the present invention will be described below with reference to the drawings. However, the present invention mainly includes a dispensing mechanism, a container transport mechanism, a capping mechanism, a non-contact stirring mechanism, a detection mechanism, and a chip disposal box. I have.

  First, FIG. 1 is a schematic diagram of a configuration of a nucleic acid test apparatus according to the present invention. The nucleic acid test apparatus according to the present embodiment mainly includes a dispensing mechanism and an arm (container transport mechanism) 1 equipped with a container holding mechanism for transporting the container, a capping mechanism 10, a chip disposal box 11, and a non-contact stirring mechanism 12. , A detector (detection mechanism) 13. Among these, the dispensing mechanism dispenses the primer solution containing the fluorescent dye, the nucleic acid extract, and the enzyme into the reaction container, and discards the dispensed chip 6 after the reaction in the chip disposal box 11. The reaction container into which the reaction solution (primer solution, nucleic acid extract, and enzyme) has been dispensed is closed by the closing mechanism 10, stirred after the closing by the non-contact stirring mechanism 12, and detected by the detection mechanism 13. The detection mechanism 13 has a function of controlling to a constant temperature, and is a measuring instrument capable of performing nucleic acid amplification and detection at the same time. The detection mechanism 13 detects the fluorescence intensity at regular intervals and records the change over time of the measurement value. As the detection mechanism 13, for example, a detector capable of detecting fluorescence independently of each other around a disk-shaped carousel may be provided. In this case, either one or both of the carousel side and the detector side may be rotated. Further, as the closing mechanism 10, for example, a mechanism that pushes a spherical lid into the container may be employed.

  The nucleic acid test apparatus further includes a dispensing chip erection rack 2, a reagent erection rack 7, a reaction container erection rack 8, and a sample erection rack 9.

  Next, the structure of the dispensing mechanism and the dispensing method of the gene testing apparatus (nucleic acid testing apparatus) according to the present invention will be described with reference to FIG. The dispensing mechanism includes a stepping motor 4 as a drive source, a ball screw 5 that converts rotational motion into linear motion, a syringe unit 3 that performs suction / discharge, and a dispensing tip 6.

  By using the stepping motor 4 as a drive source and via the ball screw 5, the rotational motion is converted into a linear motion. The reagent is aspirated and discharged by moving the plunger attached to the drive unit up and down. In a dispensing mechanism used in genetic testing (nucleic acid testing), a dispensing tip 6 is attached to the tip, and reagents and samples are dispensed. The reagent here refers to a primer solution or an enzyme, and the sample refers to a nucleic acid extract. The dispensing tip after the reagent or sample is discharged is discarded in the tip disposal box 11 and temporarily stored in the apparatus.

  An embodiment of the present invention will be described in detail with reference to FIG.

  The heating block integrated chip disposal box 11 shown in FIG. 3 is deformed by heat and pressure while pressing the tip of the chip against the heating block 14. That is, the dispensing tip 6 that has finished the dispensing operation is carried to the tip disposal box 11 by the arm 1 shown in FIG. 1 and inserted into an openable / closable hole (opening) 15 on the heating block 14.

  When the presence or absence of the tip is confirmed in conjunction with the movement of the arm 1 in the Z direction, the hole diameter contracts and the dispensing tip is pressed against the heating block. Thereafter, the openable / closable hole 15 is opened and dropped into the chip disposal box connected below. The dispensing tip whose residual liquid at the tip is deformed by heat and pressure can be discarded without splashing the residual liquid. Moreover, by deform | transforming a front-end | tip in this way, a front-end | tip is crushed and it can also prevent that a residual liquid leaks from a front-end | tip after discarding. Furthermore, heating at a high temperature inactivates the enzyme when it remains at the tip of the dispensing tip 6, and causes unnecessary amplification reaction in the waste tip and between the dispensing tips 6. Can be prevented. That is, the dispensing tip 6 can be quickly and safely discarded by performing a series of operations from the processing for avoiding residual liquid splattering to disposal.

  The chip discard box integrated with the heating block is not limited to FIG. FIG. 4 shows a modification of the heating block integrated chip disposal box. The dispensing tip 6 that has finished the dispensing operation is carried to the tip disposal box 11 by the arm 1 shown in FIG. The ball screw 17 connected to the stepping motor 16 converts a rotational force into a lateral force and moves the piston 18 in the lateral direction. The heating block 14 connected to the piston 18 applies heat and pressure to the dispensing tip 6, sandwiched from both sides, and deformed at the tip of the dispensing tip 6, the same processing for avoiding residual liquid scattering as described above was performed. It is discarded afterwards. Further, when the remaining solution contains an enzyme, the enzyme is deactivated at a high temperature and discarded.

  Another embodiment for carrying out the present invention will be described with reference to FIG. That is, the heating block and the chip disposal box may be installed independently. In this case, the basic configuration of the nucleic acid test apparatus as shown in FIG. 1 is maintained. That is, the genetic test apparatus according to the present invention includes a dispensing mechanism, a container transport mechanism, a capping mechanism, a chip disposal box, a non-contact stirring mechanism, and a detection mechanism, and is a high-temperature heat treatment for preventing scattering and inactivating the enzyme when discarding the chip. Is to do. When the primer solution, nucleic acid, and enzyme are dispensed, the dispensing mechanism shown in FIG. 2 moves to the heating block 14 as shown in FIG. The dispensing tip after the reaction is pressed against the heating block 14 and is deformed by applying heat and pressure. That is, the tip of the dispensing tip is crushed. Then, it is discarded in a chip disposal box 11 installed separately. As a result, when the chip is discarded, the remaining liquid can be prevented from scattering, and the enzyme can be deactivated to prevent unnecessary amplification.

  Here, the heating block 14 shown in FIG. 5 has been described as having the same hole as that shown in FIG. 3, but is not limited to this, as shown in FIG. 4, or a simple block shape without a hole. It may be a thing. Even a simple block having no holes can be prevented from scattering and enzyme deactivation by pressing the dispensing tip and crushing the tip.

  In this way, disposal of the dispensing tip after avoiding splattering of the dispensing tip and enzyme deactivation is not directly related to the reaction, so it can be applied to all of a plurality of different analysis methods and is a simple device. Systemization can be realized while maintaining the configuration. That is, a plurality of assays with different testing methods can be measured with one apparatus without changing the analysis procedure.

  The above nucleic acid analyzer reduces the risk of splashing of the reaction solution, inactivates the enzyme remaining at the tip of the dispensing tip, prevents contamination between samples, and does not change the procedure directly related to the reaction In addition, while maintaining a simple apparatus configuration, it is possible to provide a gene automation apparatus corresponding to all analysis methods, thereby greatly reducing the burden on the examiner.

  In recent years, in the field of genetic testing, a method for isothermal amplification of genes, which can obtain results quickly with a simple apparatus, has become widespread. However, there are various procedures in the isothermal amplification method, and some of them are complicated and require skill, and there is a demand for the development of an autogenous apparatus for isothermal gene testing that can be analyzed with a simple apparatus configuration. However, it is difficult to realize a high processing capacity that saves space and consumes a large amount of dispensing tips by using a procedure that corresponds to all analysis methods and discarding dispensing tips in individual containers for each sample. there were. In addition, there is a problem that the merit of automation is impaired in an inspection system in which the capacity of the waste bag is reduced and the inspector is required to replace the waste bag in a short cycle. On the other hand, in the present invention, it is possible to provide a genetic test apparatus characterized by having a function of preventing contamination between samples that can be applied to all isothermal amplification methods while maintaining a simple structure. it can. However, the present invention does not prevent application to the PCR nucleic acid amplification method.

1 Arm 2 Dispensing tip mounting rack 3 Syringe units 4, 16 Stepping motor 5, 17 Ball screw 6 Dispensing tip 7 Reagent mounting rack 8 Reaction vessel mounting rack 9 Sample mounting rack 10 Closure mechanism 11 Chip disposal box 12 Non-contact stirring mechanism 13 Detection mechanism 14 Heating block 15 Openable / closable hole 18 Piston

Claims (13)

In a nucleic acid test apparatus for amplifying a sample nucleic acid using a reagent to detect fluorescence intensity or turbidity,
Dispensing tip for dispensing reagent and sample nucleic acid into reaction container; Closing mechanism for covering dispensing tip; Stirring mechanism for stirring reagent and sample in reaction container after closing; and reaction container amplifying sample nucleic acid A nucleic acid test apparatus comprising a temperature control unit that controls to a temperature necessary for the heating, a heating unit that deactivates the enzyme, and a dispensing chip disposal unit. In claim 1,
The nucleic acid test apparatus, wherein the heating unit is driven to press against the dispensing tip. In claim 1,
The nucleic acid test apparatus, wherein the dispensing chip is driven so as to be pressed against the heating unit. In claim 1,
The said heating part is provided with the opening part, The opening of this opening part can be opened and closed freely, The nucleic acid test | inspection apparatus characterized by the above-mentioned. In claim 1,
The said heating part consists of a some member, These members operate | move so that a dispensing chip | tip may be inserted | pinched, The nucleic acid test | inspection apparatus characterized by the above-mentioned. In claim 5,
A nucleic acid test apparatus comprising a stepping motor and a ball screw that linearly moves by rotation of the stepping motor, and each member of the heating unit is operated to sandwich the dispensing chip by the movement of the ball screw. In claim 1,
A nucleic acid test apparatus comprising the dispensing tip discarding unit below the heating unit. In claim 1,
A nucleic acid test apparatus comprising the dispensing tip discarding unit adjacent to the heating unit. In claim 1,
A nucleic acid test apparatus comprising a syringe unit capable of detaching the dispensing tip at the tip. In claim 4,
A nucleic acid test apparatus, wherein the size of the opening changes in conjunction with the vertical movement of the dispensing tip. In a nucleic acid test apparatus for amplifying a sample nucleic acid using a reagent to detect fluorescence intensity or turbidity,
A nucleic acid test apparatus characterized in that an enzyme is inactivated after dispensing a reagent and a sample nucleic acid into a reaction container using a dispensing chip. In a nucleic acid test apparatus for amplifying a sample nucleic acid using a reagent to detect fluorescence intensity or turbidity,
A nucleic acid test apparatus characterized by crushing a tip of a dispensing chip after dispensing a reagent and a sample nucleic acid into a reaction container using the dispensing chip. In a nucleic acid test apparatus for amplifying a sample nucleic acid using a reagent to detect fluorescence intensity or turbidity,
A nucleic acid test apparatus, wherein after dispensing a reagent and a sample nucleic acid into a reaction container using a dispensing chip, the tip of the dispensing chip is crushed while inactivating the enzyme.

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