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WO2022219758A1 - Dispositif pour effectuer une analyse par pcr d'un échantillon, réacteur de pcr, système de pcr et procédé de pcr - Google Patents

Dispositif pour effectuer une analyse par pcr d'un échantillon, réacteur de pcr, système de pcr et procédé de pcr Download PDF

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Publication number
WO2022219758A1
WO2022219758A1 PCT/JP2021/015513 JP2021015513W WO2022219758A1 WO 2022219758 A1 WO2022219758 A1 WO 2022219758A1 JP 2021015513 W JP2021015513 W JP 2021015513W WO 2022219758 A1 WO2022219758 A1 WO 2022219758A1
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Prior art keywords
pcr
sample
channel
opening
detection
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English (en)
Japanese (ja)
Inventor
友幸 坂井
祐美子 合志
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Hitachi High Tech Corp
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Hitachi High Tech Corp
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Priority to PCT/JP2021/015513 priority Critical patent/WO2022219758A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to devices, PCR reactors, PCR systems and PCR methods for performing PCR analysis of samples, for example, those capable of temperature regulation and nucleic acid amplification.
  • the PCR (Polymerase Chain Reaction) method is widely used as a method for amplifying nucleic acids.
  • the PCR method is a method of amplifying the DNA of interest by repeating a thermal cycle (temperature cycle), in which one cycle consists of the following three consecutive steps (A) to (C).
  • B Binding of complementary primers to the single-stranded DNA (annealing).
  • C A step of replicating double-stranded DNA by allowing DNA polymerase to synthesize a complementary strand from the primer (elongation).
  • heat denaturation is performed at about 95°C, annealing at about 60-65°C, and extension at about 72°C.
  • a thermal cycler device that automatically controls the reaction temperature and reaction time is widely used in the PCR method.
  • a reaction tube containing a reaction solution is inserted into a well provided in a metal block, and the temperature of the metal block is controlled so as to meet preset conditions.
  • the metal block is designed to accommodate reaction tubes and microplates, and has a volume equal to or greater than that of the microplates to reduce temperature variations. As a result, the heat capacity of the metal block increases, so that it takes time to raise or lower the temperature, and it takes at least 30 minutes, typically 1 to 2 hours, to perform PCR.
  • Patent Document 1 describes a nucleic acid amplification device in which a microchannel chip having a meandering channel is brought into contact with a heat block set to two different temperature zones, and a thermal cycle is performed according to the number of meandering of the channel. ing.
  • a meandering flow path system PCR Such a system is called a meandering flow path system PCR, and it is only necessary to maintain the temperature of each heat block at a predetermined temperature. Therefore, the temperature transition of the reaction liquid is determined by the channel length and the flow rate of the reaction liquid, without depending on the heating and cooling speeds of the heat block.
  • Patent Document 2 a microchannel chip having one channel is brought into contact with a heat block set to two different temperature zones, and the PCR reaction solution in the channel is reciprocated between two different temperature zones.
  • a nucleic acid amplification device is described that performs thermal cycling at .
  • Such a system is called a reciprocating channel system PCR, and it is only necessary to maintain the temperature of each heat block at a preset temperature. Therefore, the temperature transition of the reaction solution is determined by the time the reaction solution is held in the temperature zone, not depending on the heating and cooling speeds of the heat block.
  • Patent Document 3 proposes a technique of an apparatus capable of multiplex nucleic acid measurement, in which a sample introduction inlet, a purification zone, a PCR zone, and a detection zone are provided in a primary flow channel within a cartridge.
  • both the meandering channel system and the reciprocating channel system can achieve high-speed PCR.
  • the contact area of the reaction solution on the channel wall surface is large, so if the initial template gene or the DNA polymerase that causes high costs for the reaction is small, adsorption to the channel wall surface will occur. , there is a possibility that the desired amount of amplification will not be achieved.
  • the reciprocating channel system since the reaction solution reciprocates in the same channel, the contact area of the reaction solution with the wall surface of the channel is smaller than in the meandering channel method, which is advantageous when the initial template gene and DNA polymerase are small. work to Therefore, the reciprocating channel system is considered advantageous for the diagnosis of infectious diseases, which requires low cost and high sensitivity.
  • Patent Document 2 which uses a reciprocating flow path method, employs fluorescence detection as a method for detecting genes amplified by PCR.
  • the number of detection items in multiplex detection depends on the fluorophore species. From the viewpoint of fluorescence wavelength resolution, the number of detection items is limited to about four, and it is difficult to apply detection to a larger number of items.
  • hybridization can be considered as a detection method for increasing the number of detected items.
  • a detection region multiple detection region
  • a DNA probe having a specific sequence is immobilized at a specific position in the region.
  • the probes capture the amplified gene after PCR.
  • the fixed position of the probe is changed depending on the type.
  • the type can be identified by detecting the location where the gene amplified by PCR is captured. Using this method, it is possible to increase the number of item detections.
  • Patent Document 2 when installing the multi-item detection area, a configuration like the device 101 shown in FIG. 1 can be considered.
  • a PCR region 103 capable of gene amplification is arranged between the pressure application section 105 and the pressure application section 106 by reciprocating the sample solution through the channel 102 .
  • a multiple detection region 108 is arranged via the channel 102 for gene detection using the hybridization.
  • a valve 107 is arranged on the channel 102 between the PCR area 103 and the multi-item detection area 108 to turn on/off the flow of liquid and air.
  • the valve 107 is closed, pressure is applied from the pressure application units 105 and 106, and the sample solution introduced from the sample solution introduction unit 104 is sent back and forth in the PCR region 103 to cause gene amplification reaction.
  • the valve 107 is opened, pressure is applied from the pressure applying unit 105, the reaction liquid is sent to the multi-item detection area 108, and the amplified gene is detected.
  • Patent Document 3 introduces a device having a structure in which the primary channel is provided with an inlet, a purification zone, a PCR reaction zone, and a detection zone, and the primary channel is further provided with one or more air vent ports. It is Since a simple opening (port) that can apply pressure is installed on the device, there is no need to install a valve.
  • the PCR reaction zone includes a first temperature control zone and a second temperature control zone, and PCR is performed by reciprocating the sample solution through the two temperature zones.
  • Air vent ports are provided at both ends of the PCR reaction zone, and pressure is applied to the two ports during the reciprocating liquid transfer.
  • an object of the present invention is to suppress an increase in device cost and to accurately transfer a sample solution during PCR in a device, a PCR reaction apparatus, a PCR system, and a PCR method for performing PCR analysis of a sample.
  • An example of a device for performing PCR analysis of a sample comprises: a sample introduction part for introducing a sample; a PCR section for amplifying the samples by transporting the samples a plurality of times to a plurality of temperature zones; a pressure application opening for applying pressure to transport the sample; with The sample introduction section, the PCR section, and the pressure application opening are arranged in this order in one first channel,
  • the first channel has a first branching part that branches into a second channel between the sample introduction part and the PCR part,
  • the second channel comprises a detection unit for independently detecting a plurality of components contained in the sample,
  • the second channel has an openable portion on the opposite side of the first channel with respect to the sensing portion.
  • An example of the PCR reaction device is A PCR reactor configured for use with the device described above, comprising: a temperature adjustment unit that adjusts temperatures in the plurality of temperature zones; a pressure application unit that applies pressure to the pressure application opening; opening means for opening the openable portion; detection means for detecting each component of the sample in the detection unit; Prepare.
  • An example of the PCR system according to the present invention comprises the device described above and the PCR reactor described above.
  • the PCR method according to the present invention is A PCR method using the device described above and the PCR reactor described above, placing the device on the apparatus; introducing a sample through the sample introduction portion of the device; introducing a sample into the first channel of the device by the pressure applying portion of the apparatus; opening the openable portion of the device; transporting the sample to the plurality of temperature zones of the device a plurality of times by the pressure applying unit of the apparatus; conveying a sample to the sensing portion of the device by the pressure applying portion of the apparatus; detecting each component of a sample in the detection portion of the device by the detection means of the apparatus; Prepare.
  • High-speed gene amplification multi-item detection device with configuration modified from conventional technology
  • the top view of the high-speed gene amplification multi-item detection device of Example 1 of the present invention AA sectional view of FIG. 2(a) BB cross-sectional view of FIG. 2(a) Cross-sectional view of the PCR system of Example 1 (corresponding to the AA cross-sectional view in FIG. 2(a)) Cross-sectional view of the PCR system in FIG. 5 (corresponding to the BB cross-sectional view in FIG. 2(a)) Cross-sectional view of a modified example of the PCR system of Example 1 (corresponding to the BB cross-sectional view of FIG.
  • An example of determining a target DNA in a specimen in which multiple types of target DNA may exist is shown. It also includes the case where multiple types of target DNA are present in the sample.
  • FIG. 2(a) is a top view of the device 109, which is a gene amplification multiitem detection device
  • FIG. 2(b) is an enlarged view of the PCR region
  • FIG. 3 is a sectional view of FIG. 2(a) AA.
  • 4 shows a cross-sectional view of FIG. 2(a) BB.
  • the device 109 is a device for performing PCR analysis of samples.
  • the device 109 includes a sample introduction portion 110, a pressure application region 111, a washing solution introduction portion 112, a PCR region 113 (PCR portion), and a multiple detection region 114 (detection portion).
  • Device 109 also includes a groove that connects sample introduction portion 110 , pressure application region 111 , washing solution introduction portion 112 , PCR region 113 , and multi-item detection region 114 .
  • the groove depth is, for example, 0.8 mm.
  • the sample introduction part 110 and the multiple detection area 114 are arranged on the opposite side of the pressure application area 111 with respect to the PCR area 113 .
  • the device 109 is preferably made of a material that is stable against temperature changes. Furthermore, it is preferably made of a material that is optically transparent and has low autofluorescence. Such materials include glass, cycloolefin polymer, acrylic resin, polycarbonate, and the like. Further, when optical measurement is performed from the cover seal side, which will be described later, the material of the device 109 may be an optically opaque material (such as metal).
  • the manufacturing method of the device 109 is not particularly limited, but it is possible using injection molding, 3D printer, cutting, etc., for example.
  • a cover seal 115 is applied to the underside of the device 109 so that the grooves described above form flow channels.
  • the directions of “up” and “down” represent, for example, the vertical upward direction and the vertical downward direction when the device 109 is arranged in use, but the directions can be defined arbitrarily.
  • the cover seal 115 is configured using a film, for example.
  • An adhesive substance may be applied to one side of the cover seal 115, or a material that can be crimped may be used.
  • a material that can be crimped may be used as the material of the cover seal 115.
  • optically transparent resins such as cycloolefin polymer, acrylic resin, and polycarbonate are suitable, but not limited to these.
  • a specific configuration of the device 109 is shown below.
  • the channel 116, the branch part 117 (first branch part), the channel 118, and the PCR area 113 are arranged in this order from the sample introduction part 110 toward the pressure application area 111.
  • first channel including channel 116 and channel 118, sample introduction section 110, PCR region 113, and pressure applying region 111 are arranged in this order.
  • the first flow channel has the above-described branching portion 117 between the sample introduction portion 110 and the PCR region 113, and at this branching portion 117, it branches into another flow channel (flow channel 122, which will be described later).
  • the sample introduction unit 110 is used to introduce a sample into the device 109.
  • the sample introduction part 110 is formed, for example, by providing a through hole in the device 109 and closing one end (lower end in FIG. 3) of the through hole with a cover seal 115 .
  • the PCR region 113 is composed of meandering channels as shown in FIG. 2(b).
  • the device 109 When the device 109 is installed in the apparatus, it is provided in the apparatus main body described later below the two temperature zones of the first temperature zone 119 (denaturation temperature zone) and the second temperature zone 120 (extension/annealing temperature zone). Two heat blocks (heat blocks 201 and 202 in FIG. 5) are arranged. Thus, the device 109 has multiple temperature zones (two in this example).
  • the PCR region 113 is used to amplify the sample by transporting the sample to each of these multiple temperature zones multiple times (details will be described later with reference to FIG. 9 and the like).
  • a pressure application opening 121 which is an opening, is provided on the side of the cover seal 115 of the pressure application region 111.
  • the pressure applying opening 121 is used to apply pressure to transport the sample to the channel.
  • a first channel including channel 116 and channel 118 is connected to multi-item detection area 114 from branch 117 via channel 122 (second channel).
  • One end of the multi-item detection area 114 is connected to the channel 122, and the other end is provided with a multi-item detection area tip 129.
  • a water absorption pad 123 and an opening/closing port 124 are arranged at the multi-item detection area tip portion 129 .
  • channel 122 includes absorbent pad 123 and open/close port 124 on the opposite side of multi-item detection region 114 from the first channel, which includes channel 116 and channel 118 .
  • the water absorbing pad 123 is preferably made of a material that does not decompose by absorbing the solution. Materials for the water absorbent pad 123 include pulp, glass fiber, and cellulose fiber. It is also possible to omit the water absorption pad 123 .
  • the open/close port 124 is formed by providing an opening in the cover seal 115 .
  • the open/close port 124 itself is open, and can be closed by blocking the open/close port 124 from the outside of the device 109 .
  • the open/close port 124 is configured to be openable and closable (that is, openable and closable).
  • a plurality of fixed probes 125 are fixed to the multi-item detection area 114 .
  • Six fixed probes 125 are provided, for example, and each consists of DNA of 30 bases with a different sequence.
  • Each fixed probe is fixed on the bottom surface (upper side in FIG. 4) of the multi-item detection area 114 at intervals of 0.5 mm. Methods for immobilizing DNA probes on resin devices are well known in the art.
  • the multiple detection area 114 is used to independently detect multiple components (eg, target DNA) contained in the sample.
  • the channel 122 includes a branching portion 126 (second branching portion) that branches into a channel 127 (cleaning liquid channel) that is another channel.
  • the channel 122 is connected to the cleaning liquid introduction section 112 via a channel 127 .
  • the channel 127 is a channel for conveying the cleaning liquid.
  • a cleaning liquid port 128 which is an opening, is provided on the side of the cover seal 115 of the cleaning liquid introduction section 112 , and the cleaning liquid can be introduced into the channel of the device 109 from the cleaning liquid port 128 .
  • the width of channel 116, channel 118, channel 122, and channel in PCR region 113 is, for example, 0.8 mm, and the width of channel 127 is a smaller value (eg, 0.2 mm).
  • FIG. 5 shows a cross-sectional view when the device 109 is installed in the PCR reactor. This cross section corresponds to the position AA in FIG. 2(a).
  • a PCR reactor is configured for use with device 109 .
  • the combination of device 109 and PCR reactor is referred to as a PCR system in this example.
  • the apparatus main body 200 that houses the device 109 is preferably made of a material that is highly adiabatic, stable against temperature changes, and resistant to attack by the sample solution used.
  • materials include polyetheretherketone (PEEK) and polycarbonate.
  • Heat blocks 201 and 202 are built into the device main body 200 . These two heat blocks are equipped with heaters and temperature sensors for measuring temperature. The heater and the temperature sensor are connected to a temperature control unit 203, and each heat block is controlled at a constant temperature. Heat blocks 201 and 202 regulate the temperature of first temperature zone 119 and second temperature zone 120, respectively.
  • a pressure channel 204 is formed in the device main body 200 at a position corresponding to the pressure application opening 121 of the device 109 .
  • the pressure channel 204 is connected through a tube 205 to a syringe 206 (pressure applying section).
  • a cylinder within syringe 206 can be driven by actuator 207 . By driving the actuator 207 and pushing/pulling the cylinder, it becomes possible to apply positive pressure or negative pressure to the channel inside the device 109 . Thus, the syringe 206 applies pressure to the pressure applying opening 121 .
  • the syringe 206 and the actuator 207 are used as the pressure applying section, but other means such as a micro fan or diaphragm pump may be used as the pressure applying section. It is preferable that the volume of the channel between the pressure applying opening 121 and the pressure applying part (for example, the syringe 206) is as small as possible. , the tube diameter of the tube 205 is 0.5 mm, and the tube length is 10 mm.
  • the upper part of the device 109 is covered with a device cover 208 made of resin. It is preferable that the device cover 208 is made of a material that has a high thermal insulation property and is resistant to corrosion by the sample solution used. Such materials include PEEK and polycarbonate.
  • the apparatus cover 208 is provided with a cover opening 209 at a portion corresponding to the sample introduction section 110 of the device 109 .
  • the PCR reactor includes a sample inlet cover 210 (sealing member), which can be placed in cover opening 209 .
  • the outer diameter of the sample introduction port cover 210 is smaller than the inner diameter of the cover opening 209 , and the inner diameter of the sample introduction port cover 210 is larger than the opening of the sample introduction part 110 .
  • the sample inlet cover 210 has a cylindrical shape with a bottom, and has a structure in which the sample solution that may adhere to the periphery of the sample introduction part 110 does not adhere to the device cover 208 .
  • the sample introduction port cover 210 is connected to a solenoid 211. By driving the solenoid 211, the sample introduction port cover 210 is driven up and down to seal the sample introduction portion 110 at a predetermined timing.
  • FIG. 6 shows a cross-sectional view of the PCR system of FIG. This cross section corresponds to the position of BB in FIG. 2(a).
  • a detection opening 212 is provided above the device cover 208 at a position corresponding to the multi-item detection area 114 .
  • An irradiation detection unit 213 (detection means) for detecting fluorescence in the multi-item detection region 114 is arranged near the detection aperture 212 . The irradiation detection unit 213 detects each component of the sample in the multi-item detection area 114 .
  • a laser beam oscillated from a laser 214 which is a light source, has its beam width expanded by a beam expander 215, and is reflected by a dichroic mirror 216 that reflects a predetermined wavelength (for example, near the excitation wavelength of a phosphor used for PCR),
  • the light is collected by the objective lens 217 and projected onto the multi-item detection area 114 of the device 109 .
  • Fluorescence excited by laser light in the multi-item detection area 114 and emitted is collected by the objective lens 217, transmitted through the dichroic mirror 216, and cuts below a predetermined wavelength (fluorescence wavelength of the phosphor used for PCR). Background light is removed by the filter 218 and imaged on the CCD camera 220 by the imaging lens 219 .
  • Other light sources such as mercury lamps, xenon lamps, and light emitting diodes may be used as the light source.
  • a heat block 221 containing a heater and a temperature sensor is arranged below the multi-item detection area 114 in the device main body 200. , is set to a predetermined temperature (50° C.).
  • An opening is provided below the opening/closing port 124 in the device main body 200, and an opening/closing cap 222 (sealing member) is arranged in the opening.
  • a cam 224 is arranged below the cap support rod 223 connected to the opening/closing cap 222 .
  • a motor 225 is connected to the cam 224. When the motor 225 rotates, the cam 224 rotates and the cap support rod 223 is driven up and down.
  • the opening/closing port 124 of the device 109 can be opened/closed by the opening/closing cap 222 .
  • the opening/closing cap 222 , the cap support rod 223 , the cam 224 and the motor 225 act as opening means for opening the opening/closing port 124 and sealing means for sealing the opening/closing port 124 .
  • the open/close port 124 is an opening provided in the device 109, and the open/close port 124 is closed or opened by movement of the open/close cap 222.
  • FIG. 7 shows a cross-sectional view of a variant of the openable part. This cross section corresponds to the position of BB in FIG. 2(a).
  • a method of opening the cover seal 115 affixed to the device 109 by a needle 230 is adopted as the opening/closing method of the tip portion 129 of the multi-item detection region.
  • the lower surface of the tip 129 of the multi-item detection area of the device 109 is covered with a cover seal 115 and blocked.
  • a needle 230 is arranged below the tip 129 of the multi-item detection area in the device.
  • a motor 232 with a cam 231 is arranged at the lower end of the needle 230 . Rotation of the motor 232 rotates the cam 231 and drives the needle 230 up and down, allowing it to pierce and open the cover seal 115 under the multi-detection area tip 129 of the device 109 .
  • the openable portion is constructed using a cover seal 115 provided on the device 109 and the opening means for opening this openable portion is a needle 230 which pierces the cover seal 115.
  • the opening means for opening this openable portion is a needle 230 which pierces the cover seal 115.
  • the opening/closing port 124 can be opened and closed, and more flexible control is possible.
  • the device 109 can be manufactured more easily, the initial sealing operation can be omitted, and sealing before opening can be realized more reliably.
  • a cleaning liquid channel 226 is arranged below the cleaning liquid port 128. As shown in FIG.
  • the cleaning liquid flow path 226 is connected to a syringe 228 via a tube 227 .
  • An SSC (Saline Sodium Citrate) buffer for washing is stored in the syringe 228 .
  • a cylinder within the syringe 228 is actuatable by an actuator 229 , and by actuating the actuator 229 and pushing the cylinder, a wash buffer can be introduced into the flow path of the device 109 .
  • Fig. 8 shows a schematic diagram of the primer set used for PCR. Both primers consist of 20-base oligo DNA having a sequence complementary to the target DNA. The 5' end of one primer 300 is modified with a fluorescent substance 301 . A tag sequence 304 is bound to the 5' end of the other primer 302 via a spacer 303 . The tag sequence 304 is composed of 30-base oligo DNA having a sequence complementary to the immobilized probe 125 of the multiple detection region 114 .
  • the spacer 303 has the function of inhibiting the extension reaction by the polymerase.
  • the structure of spacer 303 is not particularly limited as long as it can inhibit the elongation reaction by polymerase, but preferably includes a nucleic acid derivative or a non-nucleic acid derivative. Due to the presence of the spacer 303, the tag sequence portion does not become double-stranded during PCR, and the resulting PCR product can be a single-stranded DNA tag and a fluorescent substance bound together.
  • the primer set is prepared for each target DNA.
  • Each tag sequence of each primer set has a different sequence.
  • Immobilized probes 125 having sequences complementary to each tag sequence are immobilized at different positions of the multiple detection region 114 .
  • a reaction solution is sent to the multi-item detection area 114, hybridization reaction is performed, and the fluorescence measurement position in the multi-item detection area 114 is specified, so that a plurality of components contained in the specimen are detected. (eg, multiple types of target DNA) can be determined.
  • a method including the following steps is performed using device 109 and a PCR reactor.
  • the device 109 is placed in the PCR reactor (for example, in the device main body 200).
  • the temperature controller 203 sets the heat block 201 to the temperature in the denaturation temperature range and the heat block 202 to the temperature in the annealing/extension temperature range.
  • the temperature in the denaturing temperature range is preferably 90 to 100°C, more preferably 95°C.
  • the temperature in the annealing/extension temperature range is preferably 40 to 80°C, more preferably 55 to 65°C.
  • the heat block 221 is set to a temperature (50° C.) suitable for hybridization reaction.
  • the reagent solution contains a mixture of a primer set that specifically reacts with multiple types of target DNA, a thermostable polymerase, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, and dTTP).
  • Fig. 9 shows the operation flow from sample reaction liquid introduction to multi-item detection unit liquid transfer.
  • the motor 225 is driven, the cap support rod 223 is shifted upward, and the opening/closing port 124 is closed by the opening/closing cap 222 (a).
  • the syringe 206 is sucked, negative pressure is applied in the channel of the device 109, and the sample reaction liquid is moved to the denaturation temperature zone (for example, the first temperature zone 119) in the channel of the device 109 (c). That is, the sample is introduced into the first channel of device 109 by means of syringe 206 via pressure application region 111 .
  • the denaturation temperature zone for example, the first temperature zone 119
  • the motor 225 is driven to shift the cap support rod 223 downward and open the open/close port 124 (d).
  • the solenoid 211 is driven to close the sample introduction part 110 with the sample introduction port cover 210 (e). After this step, the sample will not flow back to the sample introduction section 110 when the sample is transported.
  • the sample reaction solution is kept in the elongation/annealing temperature range and waits for a predetermined time B (7 seconds in this example) (h).
  • the syringe 206 is sucked, negative pressure is applied in the channel of the device 109, and the sample reaction liquid is moved to the denaturation temperature zone in the channel of the device 109 (i).
  • the sample reaction solution is kept in the denaturing temperature zone and waits for a predetermined time C (3 seconds in this embodiment) (j).
  • the above operations (g) to (j) are repeated a predetermined number of times (40 times in this embodiment). That is, the syringe 206 conveys the sample to the first temperature zone 119 and the second temperature zone 120 of the device 109 multiple times. In this example, the sample is cycled between first temperature zone 119 and second temperature zone 120 multiple times. A predetermined region of the target DNA is thereby amplified.
  • the syringe 206 is pushed to apply positive pressure to the channel of the device 109 to move the sample reaction liquid to the multi-item detection area 114 (k).
  • the syringe 206 delivers the sample to the multi-item detection area 114 of the device.
  • the cross-sectional area of channel 127 is smaller than the cross-sectional area of channel 122, the sample reaction liquid preferentially flows through channel 122, and the sample reaction liquid that flows into channel 127 is lost. quantity is restrained.
  • step (k) above if the syringe 228 is fixed, the sample reaction liquid is less likely to flow into the channel 127 . Loss can be further reduced by making it smaller than the area.
  • the actuator 229 After maintaining the sample reaction liquid in the multi-item detection area 114 for a certain period of time, the actuator 229 is driven to detect the washing liquid filled in the syringe 228 through the washing liquid introduction part 112, the flow path 127, and the flow path 122. Anything other than the PCR product introduced into the region 114 and hybridized to the immobilized probe 125 is washed away toward the absorbent pad 123 .
  • each component of the sample is detected in the multi-item detection area 114 by the irradiation detection unit 213 .
  • the multi-item detection region 114 and the irradiation detection unit 213 perform hybridization as the gene multiple-item detection means, so the number of detection items can be increased.
  • DNA is used as the target, but in the case of RNA such as viruses, reverse transcription reaction may be performed outside the device and cDNA may be introduced as a sample.
  • a reverse transcription reaction region may be provided in the channel of the device 109, a heat block for reverse transcription reaction may be provided in the apparatus, and the reverse transcription reaction may be performed inside the device 109.
  • fluorescence detection is used as the detection method in this embodiment, other detection methods such as light absorption, reflectance, chemiluminescence, electrochemiluminescence, and light scattering may be used.
  • the device 109 (especially the opening/closing port 124) does not have a valve. Therefore, in multi-item detection using reciprocating flow channel type PCR, stable PCR can be provided by suppressing an increase in cost due to installing a valve in the device and appropriately moving the reaction solution. It should be noted that it is possible to arrange valves provided separately from the device 109 outside the device 109 , for example outside the open/close port 124 .
  • Example 2 provides a simple device configuration that does not require a sample inlet cover. Hereinafter, explanations of parts common to the first embodiment may be omitted.
  • Example 2 Device 109 according to Example 1 (FIG. 2) is the same as Example 1 except that the cross-sectional area (e.g., width and depth) of some channels is different and sample inlet cover 210 is not required.
  • the cross-sectional area e.g., width and depth
  • sample inlet cover 210 is not required.
  • Fig. 10 shows a schematic diagram of the gene amplification multiitem detection device of this embodiment.
  • the channel 130 is a portion that connects the sample introduction portion 110 and the branch portion 117 in the first channel. Width of channel 122 (second channel) provided between branching portion 117 and multi-item detection region 114, and width and depth of channel 130 provided between sample introduction portion 110 and branching portion 117 Other than that, it is the same as Example 1.
  • the channel 118 has a width of 0.8 mm and a depth of 0.8 mm
  • the channel 122 has a width of 2.0 mm and a depth of 0.8 mm
  • the channel 130 has a width of 0.2 mm and a depth of 0.2 mm.
  • FIG. 11 shows the operation flow of liquid transfer from the introduction of the sample reaction liquid to the multi-item detection unit after the device 109 is installed in the PCR reactor. Details will be described below with reference to FIGS. 5 and 7.
  • FIG. 11 shows the operation flow of liquid transfer from the introduction of the sample reaction liquid to the multi-item detection unit after the device 109 is installed in the PCR reactor. Details will be described below with reference to FIGS. 5 and 7.
  • FIG. 11 shows the operation flow of liquid transfer from the introduction of the sample reaction liquid to the multi-item detection unit after the device 109 is installed in the PCR reactor. Details will be described below with reference to FIGS. 5 and 7.
  • FIG. 11 shows the operation flow of liquid transfer from the introduction of the sample reaction liquid to the multi-item detection unit after the device 109 is installed in the PCR reactor. Details will be described below with reference to FIGS. 5 and 7.
  • FIG. 11 shows the operation flow of liquid transfer from the introduction of the sample reaction liquid to the multi-item detection unit after the device 109 is installed in
  • the syringe 206 is sucked, negative pressure is applied in the channel of the device 109, and the sample reaction liquid is moved to the denaturing temperature zone in the channel of the device 109 (b).
  • the motor 232 is driven 1/4 turn to lift the needle 230 to open the cover seal 115, and the motor 232 is driven 3/4 turn to lower the needle 230 to move the multi-item detection area tip 129. Open (c).
  • the sample reaction solution is kept in the elongation/annealing temperature zone and waits for a predetermined time B (7 seconds in this example) (f).
  • the syringe 206 is sucked, negative pressure is applied in the channel of the device 109, and the sample reaction liquid is moved to the denaturation temperature zone in the channel of the device 109 (g).
  • the sample reaction solution is kept in the denaturation temperature zone and waits for a predetermined time C (3 seconds in this example) (h).
  • a predetermined region of the target DNA is amplified by repeating the above operations (e) to (h) a predetermined number of times (40 times in this embodiment).
  • the syringe 206 is pushed to apply positive pressure to the channel of the device 109 to move the sample reaction liquid to the multi-item detection area 114 (i).
  • the distribution amount of the sample reaction liquid to the channels 122 and 130 is approximately proportional to the cross-sectional area (for example, the square of the channel diameter), assuming that the channels are simple tubes.
  • the process after liquid transfer to the multi-item detection area 114 is the same as in the first embodiment.
  • Example 2 as in Example 1, in multi-item detection using reciprocating flow path PCR, the cost increase due to installing a valve in the device can be suppressed, and the reaction solution can be moved appropriately. Stable PCR can be provided.
  • DESCRIPTION OF SYMBOLS 109... Device 110 Sample introduction part 111... Pressure application area
  • Tip of multi-item detection area DESCRIPTION OF SYMBOLS 130... Flow path 200... Apparatus main body 201... Heat block (temperature control part) 202 ... Heat block (temperature control unit) 203... Temperature control part 204... Pressure channel 205... Tube 206... Syringe 207... Actuator 208... Apparatus cover 209... Cover opening 210... Sample inlet cover 211... Solenoid 212... Detection opening 213... Irradiation detection part (detection means ) DESCRIPTION OF SYMBOLS 214... Laser 215... Beam expander 216... Dichroic mirror 217... Objective lens 218... Optical filter 219... Imaging lens 220...
  • CCD camera 221 Heat block 222... Opening/closing cap 223... Cap supporting rod 224... Cam 225... Motor 226... Washing liquid flow path 227 Tube 228 Syringe 229 Actuator 230 Needle 231 Cam 232 Motor 300 Primer 301 Phosphor 302 Primer 303 Spacer 304 Tag arrangement

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Abstract

Le dispositif pour effectuer une analyse par PCR d'un échantillon comprend les éléments suivants : une partie d'introduction d'échantillon pour introduire l'échantillon ; une partie de PCR pour transporter l'échantillon plusieurs fois dans chacune de plusieurs zones de température et amplifier l'échantillon ; et une ouverture d'application de pression pour appliquer une pression pour transporter l'échantillon. La partie d'introduction d'échantillon, la partie de PCR et l'ouverture d'application de pression sont positionnées dans cet ordre dans un premier canal d'écoulement. Le premier canal d'écoulement est doté d'une première section de dérivation se ramifiant en un second canal d'écoulement entre la partie d'introduction d'échantillon et la partie de PCR. Le second canal d'écoulement est pourvu d'une partie de détection pour détecter indépendamment de multiples composants contenus dans l'échantillon. Le second canal d'écoulement est pourvu d'une section pouvant être ouverte sur le côté opposé du premier canal d'écoulement par rapport à la partie de détection.
PCT/JP2021/015513 2021-04-14 2021-04-14 Dispositif pour effectuer une analyse par pcr d'un échantillon, réacteur de pcr, système de pcr et procédé de pcr Ceased WO2022219758A1 (fr)

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PCT/JP2021/015513 WO2022219758A1 (fr) 2021-04-14 2021-04-14 Dispositif pour effectuer une analyse par pcr d'un échantillon, réacteur de pcr, système de pcr et procédé de pcr

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008061129A2 (fr) * 2006-11-14 2008-05-22 University Of Utah Research Foundation Procédés et compositions se rapportant à un gradient pcr thermique à écoulement continu
WO2012094459A2 (fr) * 2011-01-06 2012-07-12 Glezer Eli N Cartouches d'essai et leurs procédés d'utilisation
WO2016006612A1 (fr) * 2014-07-08 2016-01-14 国立研究開発法人産業技術総合研究所 Dispositif ainsi que procédé d'amplification d'acide nucléique, et puce pour amplification d'acide nucléique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008061129A2 (fr) * 2006-11-14 2008-05-22 University Of Utah Research Foundation Procédés et compositions se rapportant à un gradient pcr thermique à écoulement continu
WO2012094459A2 (fr) * 2011-01-06 2012-07-12 Glezer Eli N Cartouches d'essai et leurs procédés d'utilisation
WO2016006612A1 (fr) * 2014-07-08 2016-01-14 国立研究開発法人産業技術総合研究所 Dispositif ainsi que procédé d'amplification d'acide nucléique, et puce pour amplification d'acide nucléique

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