JP4071907B2 - Automatic nucleic acid testing equipment - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an automatic nucleic acid test apparatus. More specifically, the present invention relates to an automatic nucleic acid test apparatus in which the steps of extracting a nucleic acid component from a sample, amplifying the extracted nucleic acid component, and detecting the amplified nucleic acid are all automated.
[0002]
[Prior art]
Complementary binding between nucleic acids is highly specific compared to antigen-antibody reactions, so technologies that specifically amplify and detect desired nucleic acids are useful for genetic disease testing and forensic evaluation. Very useful.
[0003]
The main steps of such tests are (i) extraction of nucleic acid components from a sample, (ii) amplification of nucleic acids, and (iii) detection of amplified nucleic acids. All these steps can be performed automatically. Nucleic acid testing devices are already on the market.
[0004]
However, the current automatic nucleic acid test apparatus has the following problems in each of the above steps.
[0005]
First, as a nucleic acid extraction operation, in the case of manual operation, it is most common to perform centrifugation after extraction with an organic solvent such as ethanol, and the automatic gene analyzer disclosed in JP-A-6-327476 is an operation of this kind. Is adopted.
[0006]
However, in order to extract nucleic acid from a sample by such an operation, it is necessary to install a mechanism for opening and closing the lid of the sample container in addition to the sample mixing means and the centrifuge means. Inevitable. In addition, since the sample container needs to be exchanged with the centrifugal treatment, it is difficult to perform continuous treatment.
[0007]
On the other hand, JP-A-9-9292 and JP-A-8-62224 disclose separation and extraction methods using non-specific adsorption of nucleic acids on the glass surface as new nucleic acid extraction steps suitable for automatic nucleic acid testing devices. ing.
[0008]
In this method, a protein-degrading substance is added to a sample in advance, and then glass-coated ferrite magnet beads are added. Since the nucleic acid component is adsorbed on the glass portion of the bead, only the nucleic acid component can be extracted by sucking the bead onto the magnet.
[0009]
However, since this method also requires a washing operation and an extraction operation, a means for that is required, and the extraction / separation process takes 10 to 12 minutes. Also, with this method, it is difficult to extract nucleic acids from mL-level specimens. Therefore, the method is not perfect for application to an apparatus capable of examining a large number of specimens in a short time. Moreover, since a separate container is required for each specimen, it is difficult to perform continuous processing.
[0010]
Next, as a point to be improved in the amplification step, adjustment of the liquid temperature when performing PCR can be mentioned. When nucleic acids with a high GC content are used in this process, nucleic acid denaturation does not occur at 93 ° C, but nucleic acid denaturation may occur at 94 ° C. It becomes.
[0011]
In this regard, in the conventional PCR reaction apparatus, the PCR reaction solution and the sample are indirectly stored by inserting and inserting the vertically long cup-shaped container to which the PCR reaction solution and the sample are added into the heated heat block. Therefore, there is a drawback that a time lag occurs in the heating, and accurate temperature control is difficult.
[0012]
Finally, in the PCR amplification product detection step, there are still problems such as operation time and the problem that the staining solution gives to the environment.
[0013]
For example, the above Japanese Patent Laid-Open No. 6-327476 discloses an automatic gene analyzer incorporating a simple gel electrophoresis apparatus for separating and detecting PCR products. However, detection using electrophoresis not only requires at least about 60 minutes, but the number of samples that can be handled in one operation is limited. Therefore, an automatic nucleic acid test apparatus using gel electrophoresis is not suitable for use in a clinical test in which a large number of specimens must be analyzed in a short time. In addition, various dyes used for staining and labeling, such as fluorescent dyes, have genetic toxicity, so care must be taken when handling them as waste.
[0014]
[Problems to be solved by the invention]
The present invention has been made to solve the above-mentioned problems existing in the prior art, and (i) extraction of nucleic acid components from a sample, (ii) amplification of the extracted nucleic acid, and (iii) detection of the amplified nucleic acid. An object of the present invention is to provide an automatic nucleic acid test apparatus improved so that at least one of them is suitable for automatic operation.
[0015]
[Means for Solving the Problems]
In order to solve the above problems, the present invention provides:
A nucleic acid extraction means capable of continuously extracting nucleic acid components from a plurality of samples containing nucleic acid;
A nucleic acid amplification means for specifically amplifying a desired nucleic acid that can be contained in the nucleic acid component extracted by the nucleic acid extraction means;
A nucleic acid detection means for detecting the desired nucleic acid amplified by the nucleic acid amplification means;
An automatic nucleic acid test apparatus comprising:
[0016]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is an automated system improved so that at least one of the steps consisting of (i) extraction of nucleic acid components from a sample, (ii) amplification of extracted nucleic acid, and (iii) detection of amplified nucleic acid is suitable for automatic operation. A nucleic acid test apparatus is provided.
[0017]
Therefore, according to the apparatus of the present invention, it is possible to inspect a large number of samples in a short time as compared with the conventional similar apparatus. More specifically, 96 samples can be examined in 5-6 hours, which is approximately one third of the time required for a conventional device.
[0018]
In order to achieve this effect, the apparatus of the present invention uses free flow electrophoresis or capillary electrophoresis as a nucleic acid extraction means, a heating body integrated nucleic acid amplification container as a PCR amplification container, and elliptical polarization analysis as a nucleic acid detection means. To do. In the apparatus of the present invention, one or more of these nucleic acid extraction means, PCR amplification containers, and nucleic acid detection means suitable for automatically testing a large number of specimens can be used in combination. These nucleic acid extraction means, PCR amplification container, and nucleic acid detection means are preferably used simultaneously.
[0019]
Hereinafter, the present invention will be described in more detail by way of examples, but is not intended to limit the scope of the present invention described in the claims in any way.
[0020]
[Example 1]
FIG. 1 is a top view of the inside of the automatic nucleic acid test apparatus of the present embodiment. Hereinafter, the automatic nucleic acid test apparatus of the present invention will be described in detail with reference to FIG.
[0021]
The automatic nucleic acid test apparatus 101 can automatically perform all steps of nucleic acid extraction / amplification / detection, and each of the processes includes a nucleic acid extraction means 111 and a PCR reaction apparatus disposed inside the automatic nucleic acid test apparatus 101, respectively. 119 and the nucleic acid detection means 132 are performed in this order.
[0022]
Therefore, according to the automatic nucleic acid test apparatus 101, an inspector can perform a nucleic acid test simply by starting the automatic nucleic acid test apparatus 101 after setting a sample tube to which a sample has been added to a sample stand (not shown). it can.
[0023]
When the automatic nucleic acid test apparatus 101 is started, the sample is inhaled by an inhaler (not shown) and then transferred to the nucleic acid extraction means 111.
[0024]
The nucleic acid extraction means 11 can be any extraction means including the extraction means disclosed in JP-A-6-327476, JP-A-9-19292, and JP-A-8-62224. Therefore, it is highly preferable to use free flow electrophoresis or capillary electrophoresis.
[0025]
In general, capillary electrophoresis is suitable when the sample is small, and free flow electrophoresis may be suitable when the number of nucleic acids of interest in the specimen is small (less than 1000).
[0026]
Free-flow electrophoresis typically has the structure shown in FIG. 2 and is known per se (see JP-A-10-318982). The element for free-flow electrophoresis is a pair of glass plates 201a and 201b having a thickness of 0.1 to 5 mm, preferably 0.5 to 2 mm, and silicon plates 202a and 202b sandwiched between the two glass plates. It consists of electrodes 206a (anode) and 206b (cathode). Since the flat hollow gap is formed by the glass plates 201a and 201b and the silicon plates 202a and 202b, when the sample solution is injected from the sample inlet 205, the sample is transferred from the buffer inlet 203 to the buffer outlet 204. Flows in the direction of the arrow in the figure.
[0027]
Since the electrodes 206a and 206b form an electric field, a substance having a negative charge such as a nucleic acid migrates while moving in the direction of the anode 206a. Therefore, if the effluent is collected on the right side of the buffer outlet 204 in the figure, the nucleic acid component can be extracted.
[0028]
The distance between the two glass plates 201a and 201b is preferably 5 μm to 150 μm so that the sample and the buffer solution do not diffuse and mix with each other. It is preferable to use platinum or gold which is not easily corroded for the electrode. When the distance between the glass plates 201a and 201b is narrow, the applied voltage can be increased to several kV, but can be set as appropriate.
[0029]
Various buffers are known as buffers that can be used for free-flow electrophoresis. Phosphate buffer, acetate buffer, glycine-tris (hydroxymethyl) aminomethane buffer, tris (hydroxymethyl) amino Methane buffers are included, but are not limited to these.
[0030]
The size of the free-flow electrophoretic element can be 1-30 cm in length (flow direction), 1-30 cm in width, preferably 2-10 cm in length, 2-10 cm in width, most preferably 3-5 cm in length, 3-5 cm in width . As the size of the free flow electrophoresis, an appropriate size may be selected according to the resolution, the volume of the sample, the separation time, and the like. The standard separation time when extracting nucleic acid components from a sample of about 1 mL using free flow electrophoresis is generally within 1 minute, and can be as short as 30 seconds by selecting good conditions. Nucleic acid components can be extracted in a very short time and continuously.
[0031]
Furthermore, free flow electrophoresis can be loaded with a large amount of sample. This is a particularly useful feature when the sample contains only a small amount of desired nucleic acid (one molecule in some cases) and it is necessary to use a large amount of sample for the nucleic acid extraction means.
[0032]
Capillary electrophoresis can be loaded with a very small amount of sample, which is effective for examining a very small amount of sample. An element for capillary electrophoresis connects a pair of electrodes (anode and cathode) to a hollow elongated cylindrical pipe that generates a capillary force, and a sample inflow side and a sample outflow side of the pipe to give an electric charge. Thus, the nucleic acid component is moved from the injection side to the outflow side, and within about 2 minutes, it is possible to perform separation within 1 minute by shortening the suitable conditions, in particular, the capillary length (Japanese Patent Laid-Open No. Hei 9- 89840 etc.). Therefore, preferably, the nucleic acid extraction means 11 is provided with both a free flow electrophoresis and a capillary electrophoresis apparatus so as to selectively inject a sample and apply a voltage, thereby extracting according to the amount of the sample. Can be performed. Such an instruction for selective nucleic acid extraction is designated by an operator by means of an input means provided on the user interface 136, or an appropriate reading means provided by providing a barcode encoding the sample amount or ID on the outer wall of the sample cup. It is only necessary to automatically instruct input.
[0033]
The nucleic acid-containing fraction extracted by the nucleic acid extraction means 111 is dropped from the outlet of the element or sucked by an appropriate suction means so as to be collected in the sample cup for each specimen. As the sample cup, the one stored in the sample cup stocker 115 is used. Therefore, in order to extract the nucleic acid-containing fraction extracted by the nucleic acid extraction means 111, an operation of transporting the sample cup to a predetermined position is automatically performed at the latest by the time of extracting the nucleic acid-containing fraction. Since the nucleic acid is negatively charged as a whole, when free flow electrophoresis is used as the nucleic acid extraction means 111, the sample cup is located on the right side of the buffer solution outlet 204 (the side where the anode 206a is present). It is placed at an appropriate position.
[0034]
The sample cup is transported by the cup transport hand 106. The cup transport hand 106 is attached to a robot arm 104 engaged with a robot arm 103 that can move in the X-axis direction in the drawing along the robot arm 102. Furthermore, the cup transport hand 106 can freely move on the robot arm 105 extending in the Z-axis direction (perpendicular to the paper surface). Therefore, the cup transport hand 106 can be moved to an arbitrary position on the XY plane in the drawing determined by the positions where the robot arm 103 and the robot arm 104 exist. If the robot arm 104 is then lowered in the Z-axis direction, the sample cup disposed below the vertical position of the robot arms 102 to 104 can be gripped.
[0035]
The robot arms 103 and 104 are provided with a PCR module transport hand 108 and a reaction tray transport hand 109 in addition to the cup transport hand 106, and the equipment and reagents described below are separated from a certain point. All of the various operations for moving to the point are performed with these hands. However, various robot arms may be changed so that the belt conveyor can carry a long distance or a long time by only transferring the same container, module, tray, etc. to the belt conveyor.
[0036]
After the nucleic acid-containing fraction is extracted into the sample cup, the sample cup is again held by the cup transport hand 106 and stored in the sample storage hole formed on the upper surface of the sample concentrating heat block 112.
[0037]
Normally, samples are placed in all sample storage holes. cup Until the sample is stored, the nucleic acid extraction operation described above and the sample to the heat block 112 for sample concentration are performed. cup The above-described operation including the storage of the above is repeated (in FIG. 1, the operation is repeated eight times).
[0038]
The sample concentration heat block 112 can concentrate the nucleic acid-containing fraction by employing an appropriate concentration technique such as heating the nucleic acid-containing fraction in the sample cup to evaporate the liquid component. The sample concentrating heat block 112 is preferably made of a material having good thermal conductivity, such as a metal, and is a general heat block used for this type of purpose. Although heating temperature can be set to arbitrary temperature, the temperature range of 60-100 degreeC is preferable.
[0039]
Concentration of the sample need only be performed if it is desired to reduce the sample volume for use in the subsequent PCR amplification step, so this step can be omitted. However, if free flow electrophoresis is used as the nucleic acid extraction means 111, a concentration operation will often be required.
[0040]
The nucleic acid-containing fraction, preferably the concentrated nucleic acid-containing fraction, is sucked into a pipette means (not shown) by the action of the pump 107. A pipette tip housed in a pipette tip container 123 is automatically attached to the pipette means. Pipette tips can be those commonly used.
[0041]
After the nucleic acid-containing fraction is removed by suction, the sample cup stored in the heat block 112 is gripped by the cup transport hand 106 and discarded in the disposal box 113.
[0042]
The pipette means is moved by the robot arms 102 to 104 to a container juxtaposed with the PCR reagent cup stocker 116, and the nucleic acid-containing fraction, preferably a concentrated nucleic acid-containing fraction is discharged into the container.
[0043]
The PCR amplification reagent 114 is dispensed into the container after or before the nucleic acid-containing fraction is discharged. If the PCR amplification reagent 114 is dispensed in advance before discharging the nucleic acid-containing fraction, the test time can be shortened. The PCR amplification reagent 114 is aspirated from the pipette by the action of the pump 110 and is discharged into a container juxtaposed with the PCR reagent cup stocker 116. The pipette tips are exchanged for each operation and for each sample, and the used pipette tips are discarded in the disposal box 113.
[0044]
The PCR amplification reagent 114 contains substances necessary for PCR such as a primer having a predetermined sequence, a heat-resistant polymerase such as Taq polymerase, deoxynucleotide triphosphates (dTTP, dATP, dCTP, dGTP), and a buffer solution. . Since many variations of PCR have been developed, the PCR amplification reagent 114 can contain two or more kinds of various reagents according to the PCR technique to be applied.
[0045]
The nucleic acid-containing fraction mixed with the PCR amplification reagent 114 (hereinafter referred to as a PCR mixed solution) is injected into a sample inlet on the PCR module 120 previously placed on the PCR reaction device 119. Injection into the PCR module 120 is also performed by the pump 110 and the pipette means.
[0046]
Hereinafter, a preferred structure of the PCR module 120 and a sample injection operation to the PCR module 120 will be described with reference to FIGS.
[0047]
A preferred PCR module 120 is shown in FIG. 3 (top view), FIG. 4 (cross-sectional view), first substrate of the structure shown in FIG. 5 (enlarged view of FIG. 4) and FIG. 6 (top view). Having a second substrate of the structure.
[0048]
As shown in FIG. 4, the first substrate includes a smooth substrate 301 on which at least one reaction cell 302 for injecting a PCR mixture is formed, and a surface on which the reaction cell 302 is formed. A resin film 303 adhered to the opposing surface is provided.
[0049]
As a material of the substrate 301, a silicon wafer having a molding method established in semiconductor engineering can be used.
[0050]
When a silicon wafer is used as the material of the substrate 301, the reaction cell 302 can be formed by etching to have a smooth bottom surface. In order to improve the etching accuracy and to make the depth of each reaction cell uniform, and to prevent variation in the temperature of the PCR mixture in each reaction cell, the reaction cell of both surfaces of the silicon wafer should be It is preferable to dope boron atoms into the non-molded surface.
[0051]
The reaction cell 302 may have any shape and size in accordance with the above-described configuration, but preferably has a flat bottom surface and a substantially flat space. Further, since the volume of the PCR mixture is usually 100 μL or less, for example, when the shape is rectangular as shown in the figure, it is preferable that one side is 2 to 50 mm and the depth is 0.1 to 1.0 mm.
[0052]
3 and 4 show a substrate with eight reaction cells 302, the number of reaction cells may be any number greater than or equal to one. It would be preferable to increase the number of reaction cells because a large number of specimens can be measured simultaneously.
[0053]
As illustrated in FIG. 5, the heating body 304 and the temperature detection unit 5 are embedded in the resin film 303. When the PCR module having such a structure is used, the bottom of the reaction cell is flat, so that the heat transfer efficiency is good. In addition, since the heating body and the temperature detection unit are integrally disposed at the bottom of the reaction cell, and the PCR mixture in the reaction cell 302 is directly heated, the temperature of the PCR mixture can be adjusted strictly. Since the success or failure of PCR can be greatly affected by the reaction temperature, heating by a PCR module with an embedded heating element provides better results compared to the mode of indirectly heating the PCR module via a heat block. . Thus, nucleic acid amplification can be performed in a short time by using a reaction cell with improved heating efficiency. Further, by forming a plurality of separate reaction cells 302, the same or different samples can be processed simultaneously.
[0054]
Since the strength of the substrate 301 is significantly reduced in the recess where the reaction cell 302 is formed, the resin film 303 also plays an extremely important role of giving strength to the recess of the substrate 1. Therefore, the material of the resin film 303 must be a material having strong strength, water-insoluble, and heat resistance in a temperature range of at least 0 to 100 ° C. Specifically, it may be a fluororesin such as Teflon, a polyimide resin, a polycarbonate, a polypropylene resin, a vinyl chloride resin, or the like.
[0055]
The thickness of the resin film 303 is preferably 1 μm or more and 200 μm or less so that both strength and thermal conductivity are satisfied.
[0056]
The heating element 304 can be made of a material having a certain level of electrical resistance, such as silicon carbide, titanium, nickel-chromium alloy or the like. A part of the heating body 304 communicates with the bottom surface of the resin film 304, forms an electrical contact for energization, and the PCR mixture in the reaction cell 302 is heated by energization.
[0057]
The temperature detection unit 305 may be made of a metal whose resistivity changes linearly in a temperature range of 0 to 100 ° C., such as platinum or titanium. A part of the temperature detection unit 305 is also communicated with the bottom surface of the resin film 303 and forms an electrical contact for energization.
[0058]
As shown in FIG. 6, a second substrate 402 provided with a sample inlet / outlet port 401 is bonded onto the first substrate. The sample inlet / outlet port 401 is arranged in a pair so as to correspond to both ends of each reaction cell 404 of the first substrate 403, and is a small hole for injecting and discharging the PCR mixture into each reaction cell. is there. The second substrate 302 is configured to be airtight so as to prevent the PCR mixed solution in the reaction cell from evaporating.
[0059]
A PCR reaction tank 501 formed by bonding a first substrate and a second substrate is placed and fixed on a substrate member 502 as shown in FIG. 7 and covered with a frame member 503. The frame member 503 is provided with a pair of sample inlets 504 that can be airtightly connected to the pair of sample inlets / outlets 401 shown in FIG. Monitoring in the reaction vessel is possible as appropriate.
[0060]
In the automatic nucleic acid test apparatus, the tip of a pipette is inserted into the sample inlet 504 in FIG. 7, and the PCR mixture is added to the reaction cell 404 shown in FIG. 6 through the sample inlet 504.
[0061]
A plurality of PCR modules 120 are stacked in the PCR module stocker 121, and are conveyed onto the PCR reaction device 119 by the PCR hand 108.
[0062]
After the PCR module 120 is transported onto the PCR reaction device 119, the PCR mixture in the container accommodated in the PCR reagent cup stocker 116 is dispensed to each reaction cell inside the PCR module 120. The dispensing is also performed by the pipette means. After inserting the tip of the pipette tip attached to the pipette means into the sample inlet (see FIG. 7) arranged on the PCR module 120, the PCR mixture is discharged. The discharged PCR mixed solution reaches the reaction cell inside the PCR module 120. The volume of the PCR mixture is preferably 100 μL or less. Here, the reaction cell after PCR has no contamination when it is newly replaced rather than reused by washing, and an accurate inspection can be performed. Therefore, the advantage of using the exchangeable module as described above is great.
[0063]
The PCR reaction device 121 is a normal PCR having a function of energizing the heating body of the PCR module 120 to heat the PCR mixture in each reaction cell of the PCR module 120 and a function of cooling the heated PCR mixture. It is an amplification device.
[0064]
The PCR reaction apparatus 121 has a structure in which appropriate positioning means and heating means are provided, or a common frame is provided so that a conventional module (for example, see JP-A-10-117764) can be mounted and used. So it has excellent versatility.
[0065]
A PCR reaction cycle typically includes a repetition of a heat denaturation step, an annealing step, and an extension reaction step, and heating and cooling are performed by the PCR reaction device 121 so that the temperature is suitable for each step. These operations are well known to those skilled in the art, and the settings of temperature, reaction time, and number of cycles can be appropriately selected by the inspector. The cooling method may be any method including a compressor method and a Peltier method.
[0066]
After the PCR reaction is completed, the PCR mixed solution containing the amplified nucleic acid is transferred to a sample cup set in the heat block 112 for concentration by pipette means, and a denaturer reaction is performed. To the sample cup, before or after adding the PCR mixed solution, and preferably before adding the PCR mixed solution, the denaturer solution stored in the detainer solution storage unit 124 is added. The denaturer solution is a well-known nucleic acid denaturing solution containing a guanidinium salt, urea, a surfactant and the like.
[0067]
The heat block 112 for concentration is preferably heated to 90 to 95 ° C., and the amplified nucleic acid is denatured to become a single strand. Heating may be performed for about 3 to 5 minutes.
[0068]
In the nucleic acid amplification step, if an asymmetric PCR reaction, that is, a PCR reaction in which only one of the primer pairs is excessively added, the denaturer step can be omitted.
[0069]
The PCR mixture solution subjected to such denaturation treatment is transferred to each sample tube previously stored in the hybridization reagent cup stocker 118 by the pump 107 and pipette means. A hybridization reagent 117 is previously added to the sample tube by pipetting. The tip attached to the pipette means is discarded in the disposal box 113.
[0070]
The hybridization reagent 117 may be a solution containing 5 × SSC (NaCl, sodium citrate mixed solution), 10% SDS, 10 × Denhardt).
[0071]
After adding the hybridization reagent 117, the PCR mixture is dispensed on each reaction element 125 placed in the reaction tray 126 set in the reaction unit 128 for each sample. The volume of the PCR mixture to be dispensed is typically several tens of μL.
[0072]
The reaction element 125 is placed by the robot arm 105 in a reaction element accommodation hole 802 (FIG. 8) formed on at least one surface of the reaction tray 126 for accommodating the reaction element 125.
[0073]
As the reaction tray 126, it is preferable to use a tray having a structure shown in FIGS.
[0074]
FIG. 8 is a top view of a reaction tray 801 having a preferred structure. The reaction tray 801 is formed of a member having good heat conductivity, such as aluminum, brass, copper, or the like, and eight reaction element accommodation holes 802 are provided on one surface of the reaction tray 801. The number of reaction element accommodation holes 802 may be one or more.
[0075]
9 is an enlarged top view of the reaction element housing hole 802, and FIG. 10 is a cross-sectional view taken along line AA ′ of FIG.
[0076]
The reaction element storage hole 802 includes an inclined portion 803 that widens in the direction of the opening, a vertical portion 804 that is provided continuously below the inclined portion 803, a protrusion 805 that is disposed on the bottom surface, and a reaction element storage hole 802. A recess 806 formed at one corner is provided.
[0077]
The vertical portion 804 is sized to fit the size of the small reaction element so that the reaction element to be accommodated does not move. Positioning when inserting / removing the robot arm 105 is difficult. Therefore, the reaction element accommodation hole 802 includes an inclined portion 803 as a space for inserting the robot arm 105. If there is no inclined portion 803, the size of the opening is almost the same as that of the reaction element, so that it is very difficult to put the reaction element into and out of the reaction element accommodation hole 802. The size of the opening of the inclined portion 803 can be any size that allows the robot arm 105 holding the reaction element to be inserted.
[0078]
The reaction element is placed on the protrusion 805 inside the reaction element housing hole 802. Since liquid such as a sample and a cleaning solution is added to the reaction element storage hole 802, if there is no protrusion 805, the liquid infiltrates between the bottom surface of the reaction element storage hole 802 and the reaction element, and it is difficult to take out the reaction element. It becomes. The heights of the protrusions are preferably the same so that the reaction element can be stably placed, and two or more, preferably four or more are preferably provided, and the positions of the protrusions 805 are reaction and measurement. It is preferable to provide in a region where the process is performed, for example, in a region avoiding the central portion.
[0079]
The indented portion 806 has substantially the same depth as the bottom surface of the reaction element housing hole 802 and a depth lower than the projection 805, and the nozzle 130 attached to the cleaning / drying device 129 is placed in an optimum position. The space extends sufficiently in the diagonal direction of the reaction element housing hole 802 to the extent that it can be moved. That is, if the indented portion 806 exists, the nozzle 130 can be moved to the inside of the indented portion 806, so that the area in which the nozzle 130 can move is increased, and the cleaning liquid or the like can be discharged or sucked to an optimum position. it can. One or more indentations 806 may be provided, or may be provided at the periphery of the opening.
[0080]
It should be noted that the three small indentations other than the indentation 806 in FIG. 9 are provided in advance for precisely forming the reaction element accommodation hole 802 and are not essential parts depending on the material and manufacturing method.
[0081]
The reaction tray 126 is stored in a reaction tray storage box before the reaction, and is moved to the reaction unit 128 by the robot arm 104 during the reaction.
[0082]
The reaction element 125 placed in the reaction element accommodation hole 802 on the reaction tray 126 has a desired nucleic acid solid-phased at a high density, and is generally called a DNA chip or a microarray.
[0083]
The material of the reaction element may be any material that has a flat surface shape and is suitable for fine processing. Therefore, the material of the reaction element may be glass, silicon single crystal plate, amorphous such as quartz and pyrex, metal such as aluminum and stainless steel, plastic such as fluororesin and polypropylene. A silicon single crystal plate is most preferable because it can be easily processed by a known semiconductor process technology.
[0084]
The reaction element 125 is preferably rectangular from the viewpoint of ease of processing, but may be any shape such as a polygon such as a rhombus, a hexagon, and an octagon, a circle, and an ellipse.
[0085]
The reaction element 125 may have any size, but when the shape is a rectangle, the long side is preferably 0.1 to 15 cm and the short side is preferably 0.1 to 15 cm. The thickness of the reaction element 125 may be 0.01 to 0.5 cm.
[0086]
Methods for immobilizing nucleic acid molecules on the reaction element 125 are known and disclosed in, for example, Science 251: 767-773 (1991).
[0087]
The outline of the method is as follows:
(1) By performing a silane treatment on a silicon substrate, amino groups are formed on the substrate, and a photoprotective molecule is bonded to each amino group.
(2) By using a mask, selectively irradiate the desired position with ultraviolet light, remove the photoprotective molecule at the desired position, and expose only the amino group at the position.
(3) A desired nucleobase having a photoprotective molecule linked thereto is bound only to the exposed amino group.
(4) The steps (2) and (3) are repeated to elongate the nucleic acid molecule.
[0088]
Other improved methods for immobilizing nucleic acid molecules are also disclosed in International Publication No. WO93 / 09668 (Japanese Patent Publication No. 7-506561) and International Publication No. WO99 / 11754, which are known to those skilled in the art. Any method can be used to immobilize nucleic acid molecules.
[0089]
Generally, 1cm ² In this reaction element 125, thousands or more kinds of nucleic acid molecules can be immobilized. Therefore, if the reaction element 125 is used, multiple items of inspection can be performed simultaneously.
[0090]
A substance other than a nucleic acid may be immobilized on the reaction element 125 instead of the nucleic acid or together with the nucleic acid. Substances other than nucleic acids include proteins including antibodies. If an antibody is immobilized on the reaction element 125, an immunological test can be performed.
[0091]
When a reaction element is used, a multi-item test can be performed in a short time. Therefore, it is highly preferable to apply the reaction element to the automatic nucleic acid test apparatus of the present invention, but a method without using a reaction element is also possible. Such methods include, but are not limited to, plasmon resonance detection methods in addition to classical gel electrophoresis.
[0092]
The reaction between the nucleic acid solid-phased on the reaction element 125 and the nucleic acid to be amplified in the PCR mixed solution to which the hybridization reagent 117 is added is a reaction / washing / drying device 129 provided with a heater (not shown) below. Done on. Although the standard of heating temperature and reaction time is about 40-50 degreeC and 30-120 minutes, respectively, what is necessary is just to set suitably according to the base composition of a desired nucleic acid.
[0093]
A standard tray used for the purpose of calibration is stored in the standard product storage unit 131. By performing calibration, the thickness, density change, etc. of the reaction element after hybridization can be quantified from the ellipsometric measurement (reflection intensity) result.
[0094]
After performing the hybridization reaction, the reaction tray 126 is subsequently washed and dried by the reaction / washing / drying device 129. The nozzle 130 includes a cleaning liquid discharge nozzle for discharging a cleaning liquid onto the reaction element 125, a sample suction nozzle for sucking a PCR mixed liquid mixed with the cleaning liquid, and an air blowing nozzle for drying the reaction element 125. obtain. These nozzles are general nozzles used for the purpose of automatic ejection and automatic suction, and may be, for example, a thin metal. Switching between the nozzles is performed by, for example, an electromagnetic valve.
[0095]
After completion of the hybridization reaction, washing, and drying, the reaction element 125 is moved to the nucleic acid detection means 132, and the reaction tray 126 is transferred to the reaction tray collection unit 127 and collected.
[0096]
Since the reaction element 125 is fragile and must be accurately attached to the nucleic acid detection means 132, the reaction element 125 is preferably moved by an air suction mechanism (not shown) attached to the robot arm 105. . The air suction mechanism uses a negative pressure generated by the air suction means by bringing a suction port connected to an air suction means such as an air pump close to the periphery of the reaction element (ie, a portion other than the detection region). Thus, the reaction element 125 can be sucked into the suction port. The reaction element 125 is moved to a predetermined part of the nucleic acid detection unit 132 while being sucked into the suction hole.
[0097]
The nucleic acid detection means 132 may be any detection means including a fluorescence detection means, a colorimeter, and a chemiluminescence detection means, but does not require any fluorescent label operation or the like in the PCR amplification process, and measures multiple items simultaneously. Therefore, it is very preferable to detect by ellipsometry.
[0098]
Here, “elliptical polarization analysis” means analyzing the optical characteristics of the film, and more specifically, measuring the film pressure and refracted light. Elliptical ellipsometry is known per se and is typically used for the purpose of measuring semiconductor membrane pressure, but has never been used in the field of nucleic acid analysis.
[0099]
By using ellipsometry, it is possible to detect a change in thickness (1 mm or more) or a change in density in the part where the hybrid is formed, and the detection operation is completed in about 1 minute.
[0100]
The elliptical ellipsometer that can be used includes, but is not limited to, a comparative ellipsometer or a folded optical path type comparative ellipsometer.
[0101]
As the ellipsometer, a structure referring to JP-A-61-258104, JP-A-5-203564, etc. can be employed.
[0102]
After the detection by the nucleic acid detection means 132, the used reaction element 125 is discarded in the reaction element disposal box 133 (directly below the robot arm 103, the dotted line), and the reaction tray 126 is placed in the used reaction tray storage box 134. Moved.
[0103]
The measurement result of the nucleic acid detection means 132 is processed by the information processing means 135 such as a computer and then output by an output device (printer, preferably a display screen) provided in the user interface 136.
[0104]
The above process is an outline of the process of automatically inspecting a set of samples (8 samples in FIG. 1), but many modifications are made to the extraction / amplification / detection means within the scope of the subject matter of the present invention. It will be apparent to those skilled in the art that changes can be made. For example, a plurality of free flow electrophoresis elements and / or capillary electrophoresis elements as nucleic acid extraction means may be provided side by side to increase the processing capability.
[0105]
After the inspection of one set of samples is completed, the inspection of the next set of samples can continue. In order to shorten the inspection time, it is also useful to start the inspection of the next set of samples before the inspection of one set of samples is completed.
[0106]
【The invention's effect】
According to the automatic nucleic acid test apparatus of the present invention, it is possible to perform nucleic acid tests on a large number of specimens accurately in a short time.
[Brief description of the drawings]
FIG. 1 is a top view of the inside of an automatic nucleic acid test apparatus of the present invention.
FIG. 2 is a diagram showing free flow electrophoresis that can be used in the automatic nucleic acid test apparatus of the present invention.
FIG. 3 is a top view of a substrate constituting a PCR module that can be used in the automatic nucleic acid test apparatus of the present invention.
FIG. 4 is a side view of a substrate constituting a PCR module that can be used in the automatic nucleic acid test apparatus of the present invention.
FIG. 5 is a side view of a substrate constituting a PCR module in which a heating body and a temperature detection unit are embedded.
FIG. 6 is a diagram showing a configuration of a PCR module of the present invention.
FIG. 7 shows a PCR module of the present invention.
FIG. 8 is a view showing a reaction tray having a plurality of reaction element storage holes.
FIG. 9 is an enlarged top view of the reaction element housing hole.
FIG. 10 is an enlarged side view of a reaction element housing hole.

Claims (7)

A nucleic acid extraction means that is capable of continuously extracting nucleic acid components from a plurality of samples containing nucleic acids via an outlet, and is free-flow electrophoresis and / or capillary electrophoresis ;
To collect desired nucleic acid that can be contained in the nucleic acid component extracted by the nucleic acid extraction means from the outlet to a predetermined sample cup, and dispense the collected nucleic acid into a reaction cell for specific amplification. Nucleic acid amplification means of
A nucleic acid detection means for detecting the desired nucleic acid amplified by the nucleic acid amplification means;
An automatic nucleic acid testing device equipped with   The heat block for condensing the solution containing the nucleic acid component extracted by the nucleic acid extraction means and / or denaturing the desired nucleic acid amplified by the amplification means. Automatic nucleic acid testing device.   2. The automatic nucleic acid test apparatus according to claim 1, wherein the nucleic acid amplification means further comprises a storage unit having a plurality of reaction cells and a transport unit for exchanging the storage unit.   The nucleic acid amplification means is a PCR reaction apparatus for heating and cooling a PCR solution of the nucleic acid component, a heat-resistant polymerase, and a primer having a partial sequence in the desired nucleic acid. 2. The automatic nucleic acid test apparatus according to 1.   2. The automatic nucleic acid test apparatus according to claim 1, wherein the nucleic acid detection means is ellipsometry.   5. The automatic nucleic acid test method using the automatic nucleic acid test apparatus according to claim 4, wherein a container having a heating body is used as a container for containing the PCR solution.   In the automatic nucleic acid test method using the automatic nucleic acid test apparatus according to any one of claims 1 to 5, the nucleic acid group containing a nucleic acid complementary to the desired nucleic acid is amplified to a solid phase reaction element. An automated nucleic acid test method comprising: hybridizing the desired nucleic acid, and detecting hybridization of the desired nucleic acid by the detection means.

Images