JP2004012290A - Genetic diagnostic device - Google Patents

Abstract

Provided is a gene diagnosis apparatus that does not require a quantitative operation required for nucleic acid purification to amplification.
Kind Code: A1 A centrifugal force is used to send an amplification solution to a nucleic acid binding member using a rotatable device having a nucleic acid adsorption member and an amplification solution holding container holding an amplification solution, and elute nucleic acids. I do. Alternatively, amplification is performed on the nucleic acid binding member together with elution.
[Effect] Since the step of quantifying the purified nucleic acid and the amplification solution can be omitted, the operation time is shortened and the structure of the gene diagnosis apparatus is simplified.
[Selection] Fig. 22

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a genetic diagnostic apparatus for extracting nucleic acids in a sample and analyzing the extracted nucleic acids.
[0002]
[Prior art]
As an extraction apparatus for extracting a specific chemical substance such as a nucleic acid from a sample containing a plurality of chemical substances, Japanese Patent Publication No. Hei 8-501321 describes a method for purifying and separating a nucleic acid mixture by chromatography. In this method, a nucleic acid mixture is adsorbed onto an inorganic substrate such as silica gel from an aqueous adsorption solution containing a high-concentration salt, then washed with a washing solution, and the nucleic acid is eluted with a solution containing a low-concentration salt. Silica gel is fixed in a cylindrical hollow column, and a solution of the nucleic acid mixture to be separated is poured, and the solution is passed through an inorganic substrate by suction or centrifugation.
[0003]
WO 00/78455 also describes a microstructure and a method for inspection using amplification. In this apparatus, the DNA mixture is passed through a glass filter as an inorganic substrate, and then passed through a washing solution and an eluent, using the method for purifying and separating a nucleic acid mixture described in JP-A-8-501321. Only have recovered. The glass filter is provided on a rotatable structure, and reagents such as a washing liquid and an eluent are held in respective reagent reservoirs in the same structure. Each reagent flows by the centrifugal force generated by the rotation of the structure, and the reagent passes through the glass filter by opening a valve provided in a fine flow path connecting each reagent reservoir and the glass filter.
[0004]
As a chemical analyzer for extracting and analyzing a specific chemical substance such as a nucleic acid from a sample containing a plurality of chemical substances, JP-A-2001-527220 describes an integrated fluid operation cartridge. In this apparatus, reagents such as a lysis solution, a washing solution, and an eluent, and a capture component for capturing nucleic acids are provided inside the integrated cartridge, and after a sample containing nucleic acids is injected into the cartridge, the sample and the eluent are mixed. And pass the capture component, further pass the wash component through the capture component, further pass the eluent through the capture component, contact the eluate after passing through the capture component with the PCR reagent and into the reaction chamber And shedding.
[0005]
JP-T-2001-502793 discloses an apparatus and a method for chemical analysis. In this device, a chamber, a flow path, a reservoir, and an analysis cell are provided inside a disk-shaped member, a blood sample is injected into a centrifugal chamber, blood cells and serum are centrifuged, and the serum is coated with beads coated with a reagent. Only the serum is flowed into the reaction chamber, the solution for washing is then flowed into the reaction chamber, the elution solution is further flowed into the reaction chamber, and the elution solution is moved from the reaction chamber to a plurality of analysis cells. There are a plurality of analysis cells, which are dispensed along a certain time series. The inlet of the analysis cell is on the rotation center side.
[0006]
[Problems to be solved by the invention]
After the nucleic acid is eluted, it is necessary to perform amplification by adding a certain amount of the amplification solution to an eluate containing a certain amount of the nucleic acid for amplification. At the time of detection, in the conventional method, it is necessary to mix the solution containing the purified nucleic acid and the amplification solution at a fixed ratio, and therefore, the test result may be reflected in the skill of the inspector. In addition, in the process of shifting from nucleic acid purification to amplification, it is necessary to quantify a solution and an amplification solution containing the purified nucleic acid, which requires an operation time and a complicated process for automation. Furthermore, since a part of the purified nucleic acid is lost by quantification, it is necessary to have a genetic diagnostic device that can perform nucleic acid purification, amplification, and detection consistently and can proceed to the amplification process without quantifying the purified nucleic acid. There was sex.
[0007]
In the purification separation method described in Japanese Patent Publication No. Hei 8-501321, which is a conventional technique, a nucleic acid mixture is poured into a cylindrical hollow column to which silica gel is fixed, and the nucleic acid mixture is passed through the silica gel using centrifugal force. Only the nucleic acid is recovered by passing a plurality of reagents thereafter, but there is no disclosure of a method of injecting each reagent into a hollow column or a method of recovering a washing solution and an eluate passed through silica gel.
[0008]
In the method for eluting nucleic acid described in JP-T-2001-527220, it is described that nucleic acid is eluted with a PCR reagent. However, elution operation with other amplification reagents and amplification of nucleic acid on a nucleic acid capture component are described. Is not disclosed.
In the structure described in JP-T-2001-527220, a flow control device (a valve, a shunt accelerator, a fluid diode) provided in a microchannel connecting each reagent reservoir and a nucleic acid capturing component is opened, and each reagent is forcibly forced. Flows through a fluid power source and passes through the nucleic acid capture component. The flow direction of the reagent is controlled by the flow control device. In the structure described in WO 00/78455, when a valve provided in a fine flow path connecting each reagent reservoir and a glass filter is opened, each reagent flows under the action of centrifugal force and passes through the glass filter. The flow direction of the reagent is controlled by opening a valve. The flow control devices used in the first prior art are costly and unsuitable for disposable devices. In the second conventional technique, when the flow path is switched, the switched flow path remains open, so that the liquid accumulated at the tip of the switched flow path flows backward due to vibration or the like. However, there is a possibility that the switched flow path will be contaminated.
[0009]
In the method of dispensing a sample into a plurality of test containers described in JP-A-2001-502793, since the load direction applied to the sample and the direction in which the sample flows into the detection unit are the same, the air in the detection unit is detected without escaping. May be disturbed at the time. Also, it is not suitable for the purpose of simultaneously dispensing test reagents.
An object of the present invention is to provide a genetic diagnostic apparatus for extracting a nucleic acid in a sample and analyzing the extracted nucleic acid by solving the above problems.
[0010]
[Means for Solving the Problems]
The above problem can be solved by providing a gene diagnosis apparatus capable of amplifying a nucleic acid on a nucleic acid adsorbing member. That is, first, the gene diagnosis apparatus of the present invention includes a nucleic acid adsorbing member for capturing a nucleic acid in a sample, and a plurality of reagent holding units for separately holding a plurality of reagents, and the nucleic acid adsorbing member and the reagent holding unit In the gene diagnostic apparatus having a flow path for connecting the parts inside the structure, the nucleic acid adsorbing member also serves as a nucleic acid amplifying part.
[0011]
Secondly, the gene diagnosis apparatus of the present invention includes a rotatably supported structure, a nucleic acid adsorbing member that captures nucleic acids in a sample, and a plurality of reagent holding units for separately holding a plurality of reagents. In the gene diagnostic apparatus provided with a flow path for connecting the nucleic acid adsorbing member and the reagent holding section inside the structure, the nucleic acid adsorbing member also serves as nucleic acid amplification.
[0012]
Alternatively, the problem can be solved by providing a temperature control mechanism for controlling the temperature of the nucleic acid adsorbing member and providing a gene diagnosis apparatus capable of supplying an amplification solution to the nucleic acid adsorbing member. That is, thirdly, the gene diagnosis apparatus of the present invention includes a rotatably supported structure, a nucleic acid adsorbing member for capturing nucleic acids in a sample, and a plurality of reagent holding units for separately holding a plurality of reagents. And a temperature control mechanism for controlling the temperature of the nucleic acid adsorbing member, wherein the nucleic acid adsorbing member is provided with a flow path for connecting the nucleic acid adsorbing member and the reagent holding section inside the structure. Is supplied with an amplification solution.
[0013]
Alternatively, the problem can be solved by providing a temperature control mechanism for controlling the temperature of the amplification section and providing a gene diagnosis apparatus capable of supplying an amplification solution to the nucleic acid adsorption member. Fourth, the gene diagnostic apparatus of the present invention includes a rotatably supported structure, a nucleic acid adsorbing member for capturing nucleic acid in a sample, and a plurality of reagent holding units for separately holding a plurality of reagents. In a gene diagnostic apparatus comprising an amplification unit and a flow path for connecting the nucleic acid adsorption member and the reagent holding unit inside the structure, a temperature control mechanism for controlling the temperature of the amplification unit is provided, The amplification liquid is supplied to the nucleic acid adsorption member.
[0014]
In particular, in the third or fourth gene diagnosis apparatus, downstream of the nucleic acid binding member, the first flow path for the flow of the reagent before the amplification solution is supplied to the nucleic acid adsorption member, and passes through the nucleic acid adsorption member It is preferable that a second flow path through which the amplified solution flows is provided, and a cross-sectional area of a part of the first flow path is reduced. Here, it is preferable that the nucleic acid binding member and a part of the narrow flow path of the first flow path face the center of rotation.
[0015]
Further, in the third or fourth gene diagnosis apparatus, a detection unit or an amplification unit is provided downstream of the nucleic acid binding site, and a part of the flow path connecting the nucleic acid binding site and the detection unit or the amplification unit is oriented in the rotation center direction. Preferably it is peeled.
In any one of the above, it is desirable to have a means for injecting a sealing material into a part of the first flow path or a part of the flow path facing the center of rotation.
[0016]
Further, in the above, a reservoir is provided on the flow path connecting the detection unit and the amplification unit, and the width of the flow path connecting the reservoir and the detection unit is smaller than the minimum width of the reservoir, and the reservoir is It is preferable to have a ventilation hole.
Furthermore, in the above description, it is preferable that there are a plurality of the detection units, the width of the flow path on the inner diameter side of the detection unit is smaller than the minimum width of the detection unit, and the flow paths on the inner diameter side of the detection unit are connected.
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
With reference to FIGS. 1 to 51, an embodiment of a gene analyzer using an extraction device according to the present invention will be described.
FIG. 1 is an overall configuration diagram of a gene analyzer according to the present invention. The gene analyzer 1 includes a holding disk 12 rotatably supported by a motor 11, a plurality of fan-shaped analysis disks 2 positioned by protrusions 121 on the holding disk 12, and a punch for controlling the flow of liquid. 13, two optical devices for heating and detection, namely an upper optical device 14 and a lower optical device 15, and a position detector 16. The holding disk 12 has a holding disk optical window 122 for the lower optical device 15.
[0018]
FIG. 2 is a sectional view of a set of the analysis disk 2. The analysis disk 2 is configured by joining an upper cover 20 (FIG. 3), a flow path 30 (FIG. 4), and a lower cover 40 (FIG. 5). FIG. 6 shows the correspondence between the upper cover of the analysis disk and the flow path. The upper cover 20 includes a sample inlet 210, a plurality of reagent inlets 220, 230, 240, 250, 260, 290, 1210, 1220, 1230, and a plurality of air holes 202, 221, 231, 241, 251, 261, 271. , 281, 291, 1211 and 1212. The lower cover 40 has a positioning hole 460 and a flow path optical window 490. The analysis disk 2 is positioned by fitting the positioning holes 460 to the protrusions 121 of the holding disk 12.
[0019]
FIG. 4 shows a configuration diagram of the flow path unit 30. The embodiment of the flow path unit shown in FIG. 4 constitutes a flow path for extracting nucleic acid contained in virus in serum and analyzing the serum after separating serum from whole blood.
The operation of extracting and analyzing viral nucleic acids when whole blood is used as a sample will be described below. The flow of the extraction and analysis operation is shown in FIGS.
[0020]
The operator passes the reagent from the upper cover 20 of the analysis disk 2 through each of the reagent inlets 220, 230, 240, 250, 260, 290, 1210, 1220, and 1230 to each of the reagent containers 320 and 330, which are the reagent holding units in the present invention. , 340, 350, 360, 390, 1311, 1321, and 1331, and cover them. After injecting the reagent into a required number of analysis disks according to the number of analysis, the analysis disks are mounted on the holding disk 12.
[0021]
Next, whole blood collected by a vacuum blood collection tube or the like is injected into the sample container 310 from the sample injection port 210. After the whole blood is injected, the holding disk 12 is rotated by the motor 11. The whole blood injected into the sample container 310 flows to the outer peripheral side by the action of the centrifugal force generated by the rotation of the holding disk 12.
[0022]
The flow state of the whole blood is shown in FIGS. The serum storage container 313 described below is located immediately below the serum holding unit 312, and the serum storage container 313 and the serum holding unit 312 communicate with each other through the serum outlet 315. FIGS. , The serum storage container 313 is shown separated from the serum holding unit 312. The vertical positional relationship of the whole blood flowing portion is shown in a vertical sectional view of the analysis disk (FIGS. 11 to 14). 11 to 14 also show a reagent container, a mixing unit, and a waste liquid container described later. FIG. 15 shows a top view of the analysis disk.
[0023]
As shown in FIGS. 7 and 11, the injected whole blood 316 is held in the sample container 310. When the holding disk 12 rotates (FIGS. 8 and 12), the whole blood 316 flows into the blood cell separating unit 311 by centrifugal force. A separating agent 314 has been injected into the blood cell separating unit 311 in advance. The specific gravity of the separating agent 314 is adjusted to be larger than serum and smaller than blood cells. When the blood cell component and the serum gradually separate due to the difference in specific gravity as the rotation speed of the holding disk 12 increases, the separating agent 314 is located between the blood cell 317 and the serum 318 to separate the blood cell 317 and the serum 318 ( 9 and 13). When the rotation of the motor 11 is stopped, only the serum flows out from the serum holding part 312 to the serum storage container 313 directly below through the serum outlet 315 (FIGS. 10 and 14).
[0024]
During the above-described series of serum separation operations, the vent holes 221, 231, 241, 251, 261, 271, 281, 291, 1211 and 1212 of the reagent containers in the upper cover (FIG. 3) are covered and air is introduced. There is no state. Although each reagent tends to flow out of the outer periphery of the reagent container due to the centrifugal force, the pressure in the reagent container decreases because air does not enter the container, and the reagent cannot flow out in proportion to the centrifugal force. However, when the rotation speed increases and the centrifugal force increases, the pressure in the reagent container gradually decreases, and when the pressure falls below the saturated vapor pressure of the reagent, bubbles are generated. Therefore, as shown in FIG. 15, a flow path structure (return flow paths 322, 332, 342, 352, 362, 372, 372) is used to temporarily return the reagent flowing out from the outer circumference of each reagent container to the inner circumference. Further, it suppresses the pressure drop in the reagent container and prevents the generation of bubbles. As described above, during the serum separation operation shown in FIGS. 4 and 5, each reagent (the reagent 321 in FIGS. 11 to 14) does not flow while being held in the reagent container 320.
[0025]
When the serum separation operation is completed and the serum 318 is stored in the serum storage container 313, the punch 13 then makes a hole in the lid of the vent hole above each reagent container, rotates the motor 11, and centrifuges each reagent. Fluidize with force.
[0026]
The operation after the end of the serum separation will be described below.
FIG. 18 shows a partial detailed view of the downstream side of the nucleic acid binding member 301. FIG. 15 shows a positional relationship of each hole of the upper cover 20 with respect to the flow path unit 30.
A lysis solution for dissolving the virus membrane protein in the serum is dispensed into the lysis solution container 320 (FIG. 13). When the motor 11 is rotated after the perforator 13 makes a hole in the lid of the solution vent 221, the solution is centrifugally moved to cause the solution to flow from the solution container 320 through the solution return channel 322 to the mixing container 380. Flow into At this time, the serum in the serum storage container 313 also flows by the action of the centrifugal force and flows into the mixing container 380.
[0027]
16 and 17 show how the serum and the lysing solution flow into the mixing container 380. The mixing container 380 communicates with the serum storage container 313 through the serum outflow channel 319, communicates with the lysis solution storage container 320 through the lysate outflow channel 329, and flows through the mixing container vent 383 via the mixing container vent 383. 3) communicates with the outside. When the serum 318 and the lysis solution 321 flow into the mixing vessel 380, the mixed solution 381 is temporarily returned to the inner peripheral side by the flow path structure (mixed solution return flow path 382). Once held in the mixing container 380 (FIG. 16), the serum and the lysis solution are sufficiently mixed in the mixing container 380. As shown in FIG. 17, when the amount of the mixed liquid in the mixing container 380 increases and exceeds the innermost peripheral side of the mixed liquid return flow path 382, the mixed liquid 381 flows from the mixed liquid return flow path 382 to the mixed liquid outflow flow path 389. After that, it flows out to the nucleic acid binding member 301 (FIG. 4).
[0028]
The lysing solution functions to dissolve the membrane from the virus or bacteria in the serum to elute the nucleic acid, and further promotes the adsorption of the nucleic acid to the nucleic acid binding member 301. As such a reagent, guanidine hydrochloride may be used for elution and adsorption of DNA, and guanidine thiocyanate may be used for RNA, and a porous material such as quartz or glass or a fiber filter may be used as a nucleic acid binding member.
[0029]
When the mixture of the lysis solution and the serum passes through the nucleic acid binding member in this way, the nucleic acid is adsorbed on the nucleic acid binding member, and the liquid flows into the eluate recovery container 390.
Next, after stopping the motor 11 and making a hole in the lid of the first cleaning liquid ventilation hole 231 for supplying air to the first cleaning liquid container 330 with the drilling machine 13, when the motor 11 is rotated again, the centrifugal force is reduced. By the action, the first cleaning liquid flows from the first cleaning liquid container 330 through the first cleaning liquid return flow path 332 to the nucleic acid binding member 301, and cleans unnecessary components such as proteins attached to the nucleic acid binding member 301. As the first cleaning liquid, for example, the above-described solution or a solution in which the salt concentration of the solution is reduced may be used.
[0030]
The waste liquid after the washing flows out to the waste liquid storage container 302 via the eluent liquid recovery container 390 as in the above-described mixed liquid.
The same cleaning operation is repeated a plurality of times. For example, in the embodiment of FIG. 4, after the first cleaning liquid, in the state where the motor is stopped, the lid of the second cleaning liquid ventilation hole 241 for supplying air to the second cleaning liquid container 340 by the perforator 13 is opened, and the motor is again turned on. By rotating 11, unnecessary components such as salts attached to the nucleic acid binding member 301 are washed. As the second cleaning solution, for example, ethanol or an aqueous ethanol solution may be used.
[0031]
Similarly, a hole is made in the lid of the third washing liquid vent 251 for supplying air to the third washing liquid container 350, and unnecessary components such as salts attached to the nucleic acid binding member 301 are washed again. As the third cleaning liquid, for example, ethanol or an aqueous ethanol solution may be used.
After the nucleic acid binding member 301 is thus washed and brought into a state where only the nucleic acid is adsorbed, the process proceeds to the nucleic acid amplification step.
[0032]
(Example 1)
Hereinafter, a case where the nucleic acid is amplified on the nucleic acid adsorption member will be described.
FIG. 19 shows a flow state around the nucleic acid binding member 301.
After the motor 11 is stopped, a hole is made in the lid of the sealant holding container vent 271 for supplying air to the sealant holding container 370. The motor 11 is rotated again. The sealant flows into the flow path 1371 and closes the flow path 1371 (FIG. 20). Because of the tapered flow path 1381 (FIG. 21), it stops in the middle of the sealant flow path 1371. It is desirable to use silicone grease or an adhesive as the sealant.
[0033]
After the motor 11 is stopped, a hole is made in the lid of the vent hole 261 for the amplifying solution holding container for supplying air to the amplifying solution holding container 360 by the perforator 13, and the motor 11 is rotated at a low speed so that the amplifying solution is transferred to the nucleic acid binding member 301. And centrifugal force and penetration (FIG. 22). When the motor 11 is stopped, the nucleic acid binding site 301 preferably has a structure shown in FIG. FIG. 23 is a sectional view taken along line aa ′ of FIG.
[0034]
The amplification solution is a reagent for amplifying and detecting a nucleic acid, and contains a primer, a nucleic acid synthetase, deoxyribotriphosphate, a buffer, a metal ion, and the like necessary for amplification. Depending on the method of amplification or detection, it may contain a fluorescent dye, a primer modified with a fluorescent dye or biotin, or deoxyribotriphosphate modified with a fluorescent dye.
[0035]
The motor 11 is stopped, and the nucleic acid binding member 301 is heated to a temperature suitable for the amplification method. For example, in the case of the constant temperature amplification method and the LAMP method, 55-65 ° C. is desirable. Since the heat capacity of the nucleic acid binding portion is large, the amplification method is preferably a constant temperature amplification method in which the number of temperature changes during amplification is small or not.
[0036]
After the amplification is completed, a hole is made in the lid of the detection container vent 281 for bleeding air from the amplification solution holding container 304 with the perforator 13, and the motor 11 is rotated again. Flows through the flow path 1361, the flow path 1372, and the flow path 1374 into the detection container 304 (FIG. 24). Since the detection container 1310 has the detection liquid holding container 1311 protruding to the outer peripheral side, the detection liquid 1314 previously stored in the detection container 1311 does not flow out during rotation (FIG. 25). Since the detection solution waste container vent hole 1212 for removing air from the amplification solution waste container 1315 is closed, the amplification solution does not flow into the detection solution waste container 1315.
[0037]
When the motor 11 is stopped, a hole is opened in the lid of the additional liquid holding container ventilation hole 291 for supplying air to the additional liquid holding container 390, and the motor 11 is rotated again, the additional liquid passes through the flow paths 1361, 13721374, The amplification solution in the amplification solution holding container 304 is pushed up to the detection containers 1310, 1320, and 1330 (FIG. 26). Water or the like is used as the additional liquid. In particular, when mixing of the amplification solution and the additional solution becomes a problem, it is preferable to use a hydrophobic solvent, for example, a mineral oil for PCR. The pushed-up amplification solution is mixed with the detection solution held in the detection containers 1311, 1321, and 1331. The amount of the additional liquid is set so as not to flow into the micro channels 1312, 1322, and 1332.
[0038]
In order to prevent the detection solution in one detection container from being mixed into another detection container, a mixture of the amplification solution and the additional solution filling the amplification solution holding container 304 connecting the detection containers 1310, 1320, and 1330 is used. remove. The motor 11 is stopped, and a hole is formed in the lid of the amplification liquid holding container vent hole 1211 for supplying air to the amplification liquid holding container 304, and the lid of the detection liquid waste liquid container vent hole 1212 for letting air escape from the detection liquid waste container 1315. Perforate. When the motor 11 is rotated again, the mixture of the amplification liquid and the additional liquid stored in the amplification liquid holding container 304 flows through the flow path 1312 and the flow path 1313 to the detection liquid waste container 1315 (FIG. 27). The mixed solution of the amplification solution and the detection solution stored in the detection containers 1310, 1320, and 1330 does not flow out of the microchannels 1312, 1313, 1322, 1323, 1332, and 1333 due to the surface tension (FIG. 28).
[0039]
The upper optical device 14 is moved downward and the lower optical device 15 is moved above the detection containers 1310, 1320, and 1330. FIG. 28 shows a state of the measurement. The upper optical device 14 irradiates light, and the amount of light emitted or absorbed is measured by the lower optical device 15. When measuring the amount of emitted light, a filter for blocking the excitation light is attached in front of the light receiving device (not shown).
[0040]
At the time of perforation, heating, and detection, it is necessary to stop the holding disk 12 at a predetermined position. As shown in FIG. 29, the holding disk 12 is provided with positioning projections 17, the position detector 16 detects the rotation position of the holding disk, and the controller 18 rotates the motor 11 and the punch 13 to rotate and Motion, rotation, irradiation and detection of the upper optical device 14 and the lower optical device 15 are controlled.
[0041]
For example, FIG. 30 shows the operation timing of the drilling machine 13. The holding disk 12 lowers the rotation speed after the end of the flow of whole blood or each reagent, and maintains the low-speed rotation for positioning. When the position detector 16 detects the positioning projection 17, the holding disk 12 is stopped, and the punch 13 is lowered to make a hole in the lid of the vent hole of each reagent storage container, and then rise again. The post-perforation holding disk 12 rotates at such a low speed that the reagent does not flow out of the reagent storage container after the perforation is completed, and rotates at the position of the next analysis disk, that is, 60 degrees when six analysis disks are mounted. Stop and repeat the same perforation operation. Where the analysis disk is mounted may be determined by, for example, irradiating light from the flow path optical window 490 with the lower optical device and examining the reflected light. After all the analysis disks have been pierced, the holding disk rotates at high speed to allow the reagent to flow.
[0042]
According to the present embodiment, in the process of shifting from the nucleic acid purification step to the amplification step, it is not necessary to quantify the purified nucleic acid, so that the nucleic acid purified by quantification can be lost or the operation required for quantification can be omitted. The measurement can be performed with higher accuracy and in a shorter time. Further, in order to amplify a nucleic acid at a nucleic acid binding site, it is possible to amplify a larger amount of nucleic acid than passing an eluent through the nucleic acid binding site and amplifying the eluted nucleic acid.
[0043]
An object of the above embodiment is to eliminate the necessity of quantifying a purified nucleic acid during the transition from the nucleic acid purification step to the amplification step.
When the step of purifying nucleic acid and the step of amplifying are separated as in the conventional method, the step of quantifying the purified product is indispensable to connect the two steps. In this example, the quantitative operation was made unnecessary by connecting the two steps.
[0044]
According to the present embodiment, the switching of the flow path is performed by closing the old flow path, so that the old flow path and the waste liquid stored in the old flow path reversely flow due to vibration or the like and contaminate the switched new flow path. To prevent this, the reliability of the measurement is improved. Since the flow path is closed with an inexpensive sealant, a less expensive device can be provided.
[0045]
According to this embodiment, since the sample to be detected flows into the detection container from the direction opposite to the load direction, the air in the detection container can be completely pushed out of the detection container. The measurement can be performed with higher accuracy and higher sensitivity.
[0046]
(Example 2)
Hereinafter, an embodiment in which the nucleic acid is eluted by the nucleic acid adsorbing member and then the nucleic acid amplification process is performed in another downstream vessel will be described.
The analysis disk 2 is configured by joining the upper cover 20 (FIG. 34), the flow path 30 (FIG. 35), and the lower cover 40 (FIG. 36). The upper cover 20 includes a sample inlet 210, a plurality of reagent inlets 220, 230, 240, 250, 260, 290, 1210, 1220, 1230, and a plurality of air holes 202, 221, 231, 241, 251, 261, 271. , 281, 291, 1211 and 1212. The lower cover 40 has a positioning hole 460 and a flow path optical window 490. The analysis disk 2 is positioned by fitting the positioning holes 460 to the protrusions 121 of the holding disk 12.
[0047]
FIG. 35 shows a configuration diagram of the flow path unit 30. The embodiment of the channel section shown in FIG. 35 constitutes a channel for separating a serum from whole blood, and then extracting and analyzing a nucleic acid contained in a virus in the serum.
FIG. 38 shows a partial detail view on the downstream side of the nucleic acid binding member 301. FIG. 37 shows the positional relationship of each hole of the upper cover 20 with respect to the flow path unit 30.
The process from serum separation to binding of the nucleic acid to the nucleic acid binding site 301 and washing of the nucleic acid binding site 301 (FIG. 31) is the same, and will not be described. The flow from the nucleic acid amplification step to the detection step is shown in FIGS.
[0048]
FIG. 39 shows a flow state around the nucleic acid binding member 301. While the motor is stopped, a hole is made in the lid of the amplification container vent hole 1241 for supplying air to the amplification container 1340 by the drilling machine 13. The motor 11 is again rotated to flow the anti-mixing liquid into the waste liquid storage container 302 (FIG. 40). The anti-contamination liquid is for preventing the dissolving liquid and the cleaning liquids 1, 2, and 3 from flowing into the detection container, and has an effect of cleaning the flow paths 1472 and 1471 during the flow of the liquid. Water or the like is used as the mixing prevention liquid.
[0049]
Next, with the motor stopped, a hole is made in the lid of the ventilation hole 271 for supplying air to the sealing liquid holding container 370. The motor 11 is rotated again. The sealing liquid flows into the flow channel 1471 and closes the flow channel 1471 (FIG. 41). Since the flow path 1371 has a tapered shape (FIG. 42), it stops in the middle of the sealant flow path 1371. It is desirable to use silicone grease or an adhesive as the sealant.
[0050]
After the motor 11 is stopped, a hole is made in the lid of the vent hole 261 of the amplifying solution holding container for supplying air to the amplifying solution holding container 360 by the perforator 13, and the amplifying solution flows through the nucleic acid binding member 301. Since the amplification vessel vent 1261 for allowing air to escape from the amplification vessel 1340 has a hole and the flow path 1471 is closed, the amplification solution passes through the flow path 1461, passes through the nucleic acid binding member 301, and passes through the flow path. 1472, flows through the microchannel 1476 and flows into the amplification container 1340 (FIG. 43). Detection vessel vent 281 for allowing air to escape from the detection vessels 1310, 1320, 1330, amplification solution holding vessel vent 1211 for allowing air to escape from the amplification fluid holding vessel, and detection liquid disposal for allowing air to escape from the detection liquid disposal vessel Since the container vents 1212 are closed, the amplification liquid does not flow into the amplification liquid holding container 304 and the amplification liquid waste container 303. The nucleic acid adsorbed on the nucleic acid binding member 301 elutes into the amplification solution. The remaining amplification liquid fills the amplification container 1340 and flows into the amplification liquid waste container 1350.
[0051]
The amplification solution is a reagent for amplifying and detecting a nucleic acid, and contains a primer, a nucleic acid synthetase, deoxyribotriphosphate, a buffer, a metal ion, and the like necessary for amplification. Depending on the method of amplification or detection, it may contain a fluorescent dye, a primer modified with a fluorescent dye or biotin, or deoxyribotriphosphate modified with a fluorescent dye.
[0052]
After the motor is stopped, no air enters the amplification container 1340 from the micro flow channel 1476 and the micro flow channel 1477 due to the surface tension of the amplification liquid (FIG. 44). The amplification container 1340 filled with the amplification solution is heated and amplified to a temperature according to the amplification method by the optical device 14 also serving as an optical measurement device. For example, in the case of the constant temperature amplification method or the LAMP method, the temperature is raised from 55 ° C to 65 ° C.
[0053]
When a hole is made in the lid of the detection container vent hole 281 for allowing the air to escape from the detection containers 1310, 1320, and 1330, and the motor 11 is rotated again, the amplification liquid in the amplification container 1340 is centrifugally moved to generate a fine flow path. It flows into the amplification liquid holding container 304 through the channel 1476 and the channel 1475 (FIG. 45). Since the detection container 1310 has the detection liquid holding container 1311 protruding to the outer peripheral side, the detection liquid put in the detection liquid holding container 1311 in advance does not flow out during rotation (FIG. 46).
[0054]
When the motor 11 is stopped, a hole is opened in the lid of the additional liquid holding container ventilation hole 291 for supplying air to the additional liquid holding container, and the motor 11 is rotated again, the additional liquid passes through the flow paths 1475 and 1474. Then, the amplification solution in the amplification solution holding container 304 is pushed up to the detection containers 1310, 1320, and 1330 (FIG. 47). The pushed-up amplification liquid is mixed with the detection liquid held in the detection liquid holding containers 1311, 1321, and 1331. The amount of the additional liquid is set so as not to flow into the minute channels 1412, 1422, and 1432. Water or the like is used as the additional liquid. If contamination with the amplification solution is a problem, use a hydrophobic solvent. For example, mineral oil used in PCR is desirable.
[0055]
In order to prevent the detection solution in one detection container from being mixed into another detection container, a mixture of the amplification solution and the additional solution filling the amplification solution holding container 304 connecting the detection containers 1310, 1320, and 1330 is used. remove. The motor 11 is stopped, and a hole is formed in the lid of the amplification liquid holding container vent hole 1211 for supplying air to the amplification liquid holding container 304, and the lid of the detection liquid waste liquid container vent hole 1212 for letting air escape from the detection liquid waste container 1315. Perforate. When the motor 11 is rotated again, the mixture of the amplification liquid and the additional liquid stored in the amplification liquid holding container 304 flows through the flow paths 1474 and 1473 and flows into the detection liquid waste container 1315 (FIG. 48). The mixed solution of the amplification solution and the detection solution stored in the detection containers 1310, 1320, and 1330 does not flow out of the microchannels 1312, 1313, 1322, 1323, 1332, and 1333 due to the surface tension (FIG. 49).
[0056]
The upper optical device 14 is moved downward and the lower optical device 15 is moved above the detection containers 1310, 1320, and 1330. FIG. 14k shows a state of the measurement. The upper optical device 14 irradiates light, and the amount of light emitted or absorbed is measured by the lower optical device 15. When measuring the amount of emitted light, a filter for blocking the excitation light is attached in front of the light receiving device (not shown).
[0057]
According to the present embodiment, in the process of shifting from the nucleic acid purification step to the amplification step, it is not necessary to quantify the purified nucleic acid, so that the nucleic acid purified by quantification can be lost or the operation required for quantification can be omitted. The measurement can be performed with higher accuracy and in a shorter time. Further, by newly providing a site for amplifying a nucleic acid, amplification with a very high degree of purification becomes possible.
[0058]
An object of the above embodiment is to eliminate the necessity of quantifying a purified nucleic acid during the transition from the nucleic acid purification step to the amplification step.
When the step of purifying nucleic acid and the step of amplifying are separated as in the conventional method, the step of quantifying the purified product is indispensable to connect the two steps. In this example, the quantitative operation was made unnecessary by connecting the two steps.
[0059]
According to the present embodiment, the switching of the flow path is performed by closing the old flow path, so that the old flow path and the waste liquid stored in the old flow path reversely flow due to vibration or the like and contaminate the switched new flow path. To prevent this, the reliability of the measurement is improved. Since the flow path is closed with an inexpensive sealant, a less expensive device can be provided. In addition, since it is possible to make the nucleic acid purification, amplification, and detection sites independent, each step is prevented from hindering other steps, so that more sensitive and accurate detection can be achieved. Will be possible.
[0060]
According to this embodiment, since the sample to be detected flows into the detection container from the direction opposite to the load direction, the air in the detection container can be completely pushed out of the detection container. Can be measured.
[0061]
【The invention's effect】
According to the present invention, the elution step in the purification step is included in the amplification step, which leads to a reduction in time and simplification of the operation. Further, by performing the amplification process at the nucleic acid binding portion, it is expected that the recovery amount of the nucleic acid is increased as compared with the conventional method. Since the flow path is switched by closing the flow path, the structure is simplified, and the flow path is prevented from being contaminated by the backflow of the waste liquid to the switched flow path. When the reagent flows into the detection unit or amplification unit, the internal air is pushed out, so that amplification and detection are not hindered. Is possible.
[Brief description of the drawings]
FIG. 1 is an overall configuration diagram of a gene analyzer according to an embodiment of the present invention.
FIG. 2 is a sectional view of a set of analysis disks according to an embodiment of the present invention.
FIG. 3 is a configuration diagram of an upper cover of an analysis disk according to an embodiment of the present invention.
FIG. 4 is a configuration diagram of a channel portion of an analysis disk according to an embodiment of the present invention.
FIG. 5 is a configuration diagram of a lower cover of an analysis disk according to an embodiment of the present invention.
FIG. 6 is a diagram illustrating a correspondence between an upper cover of an analysis disk and a flow path according to an embodiment of the present invention.
FIG. 7 is a diagram illustrating the operation of the blood cell separation unit according to the embodiment of the present invention.
FIG. 8 is a diagram illustrating the operation of the blood cell separation unit according to the embodiment of the present invention.
FIG. 9 is a diagram illustrating the operation of the blood cell separation unit according to the embodiment of the present invention.
FIG. 10 is a diagram illustrating the operation of the blood cell separation unit according to the embodiment of the present invention.
FIG. 11 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 12 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 13 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 14 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 15 is a top view of an analysis disk according to an embodiment of the present invention.
FIG. 16 is a diagram illustrating the operation of the mixing unit according to the embodiment of the present invention.
FIG. 17 is a diagram illustrating the operation of the mixing unit according to the embodiment of the present invention.
FIG. 18 is a partial detailed view of a flow channel portion downstream of a nucleic acid binding site according to an embodiment of the present invention.
FIG. 19 is a view illustrating a flow state of a flow path according to an embodiment of the present invention.
FIG. 20 is a view illustrating a flow state of a flow path according to an embodiment of the present invention.
FIG. 21 is a view showing a flow state of a flow path according to an embodiment of the present invention.
FIG. 22 is a view illustrating a flow state of a flow path according to an embodiment of the present invention.
FIG. 23 is a view illustrating a flow state of a flow channel according to an embodiment of the present invention.
FIG. 24 is a view showing a flow state of a flow path according to an embodiment of the present invention.
FIG. 25 is a partial detailed view of a flow unit according to an embodiment of the present invention.
FIG. 26 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 27 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 28 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 29 is a circuit diagram of a positioning mechanism according to an embodiment of the present invention.
FIG. 30 is a timing chart of a positioning operation according to the embodiment of the present invention.
FIG. 31 is an explanatory diagram showing a procedure of an analysis operation according to the embodiment of the present invention.
FIG. 32 is an explanatory view showing the procedure of the analysis operation according to the embodiment of the present invention continued from FIG. 31;
FIG. 33 is an explanatory diagram showing a procedure of an analysis operation according to the embodiment of the present invention.
FIG. 34 is a configuration diagram of an upper cover of an analysis disk according to an embodiment of the present invention.
FIG. 35 is a configuration diagram of a channel portion of an analysis disk according to an embodiment of the present invention.
FIG. 36 is a configuration diagram of a lower cover of an analysis disk according to an embodiment of the present invention.
FIG. 37 is a diagram showing the correspondence between the upper cover of the analysis disk and the flow path according to the embodiment of the present invention.
FIG. 38 is a partial detailed view of the flow path section downstream of the nucleic acid binding site according to an embodiment of the present invention.
FIG. 39 is a view showing a flow state of a flow path according to an embodiment of the present invention.
FIG. 40 is a view illustrating a flow state of a flow channel according to an embodiment of the present invention.
FIG. 41 is a view illustrating a flow state of a flow channel according to an embodiment of the present invention.
FIG. 42 is a view illustrating a flow state of a flow path according to an embodiment of the present invention.
FIG. 43 is a view illustrating a flow state of a flow path according to an embodiment of the present invention.
FIG. 44 is a view illustrating a flow state of a flow path according to an embodiment of the present invention.
FIG. 45 is a view illustrating a flow state of a flow path according to an embodiment of the present invention.
FIG. 46 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 47 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 48 is a sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 49 is a cross-sectional view of an analysis disk according to an embodiment of the present invention.
FIG. 50 is an explanatory diagram showing a procedure of an analysis operation according to the embodiment of the present invention.
FIG. 51 is an explanatory diagram showing a procedure of an analysis operation according to the embodiment of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Gene analyzer, 2 ... Analysis disk, 11 ... Motor, 12 ... Holding disk, 13 ... Perforator, 14 ... Upper optical device, 15 ... Lower optical device , 16: Position detector, 20: Upper cover, 30: Flow path, 40: Lower cover, 121: Projection, 122: Holding disk optical window, 202: Vent hole, 210: sample inlet, 220: reagent inlet, 221: vent, 301: nucleic acid binding member, 310: sample container, 380: mixing container, 390 ..Eluent recovery container, 460... Positioning hole, 490...

Claims (10)

A nucleic acid adsorbing member for capturing nucleic acid in the sample, and a plurality of reagent holding portions for separately holding a plurality of reagents, wherein a flow path for connecting the nucleic acid adsorbing member and the reagent holding portion is provided inside the structure. In the genetic diagnostic device prepared for
A genetic diagnostic device, wherein the nucleic acid adsorbing member also serves as a nucleic acid amplifier. A rotatable supporting structure, a nucleic acid adsorbing member for capturing nucleic acids in a sample, and a plurality of reagent holding portions for separately holding a plurality of reagents, connecting the nucleic acid adsorbing member and the reagent holding portion In a genetic diagnostic device provided with a flow path for performing inside the structure,
A genetic diagnostic device, wherein the nucleic acid adsorbing member also serves as nucleic acid amplification. A rotatable supporting structure, a nucleic acid adsorbing member for capturing nucleic acids in a sample, and a plurality of reagent holding portions for separately holding a plurality of reagents, connecting the nucleic acid adsorbing member and the reagent holding portion In a genetic diagnostic device provided with a flow path for performing inside the structure,
A gene diagnosis apparatus, comprising: a temperature control mechanism for controlling a temperature of the nucleic acid adsorbing member, wherein an amplification solution is supplied to the nucleic acid adsorbing member. A rotatable supporting structure, a nucleic acid adsorbing member for capturing nucleic acids in a sample, a plurality of reagent holding portions for separately holding a plurality of reagents, and an amplifying portion; In the genetic diagnostic device provided with a flow path for connecting the parts inside the structure,
A gene diagnosis apparatus, further comprising a temperature control mechanism for controlling a temperature of the amplification section, wherein an amplification solution is supplied to the nucleic acid adsorption member. Downstream from the nucleic acid binding member, a first flow path for the flow of the reagent before the amplification liquid is supplied to the nucleic acid adsorption member, and a second flow path for the flow of the amplification liquid after passing through the nucleic acid adsorption member The gene diagnostic apparatus according to claim 3, further comprising a path, wherein a cross-sectional area of a part of the first flow path is reduced. The gene diagnostic apparatus according to claim 5, wherein the nucleic acid binding member and a part of the narrow flow path of the first flow path face a center of rotation. 5. The method according to claim 3, further comprising a detection unit or an amplification unit downstream of the nucleic acid binding site, wherein a part of a flow path connecting the nucleic acid binding site and the detection unit or the amplification unit faces the center of rotation. A genetic diagnostic device according to item 1. The gene diagnosis according to any one of claims 5 to 7, further comprising means for injecting a sealing material into a part of the first flow path or a part of the flow path facing the rotation center direction. apparatus. A reservoir is provided on the flow path connecting the detection unit and the amplification unit, the width of the flow path connecting the reservoir and the detection unit is smaller than the minimum width of the reservoir, and the reservoir has a vent. The genetic diagnostic device according to claim 7, wherein 8. The method according to claim 7, wherein there are a plurality of detection units, wherein the width of the flow path on the inner diameter side of the detection unit is smaller than the minimum width of the detection unit, and the flow paths on the inner diameter side of the detection unit are connected. Genetic diagnostic device.

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