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WO2018143469A1 - Système d'amplification de gène, puce de trajet d'écoulement, mécanisme d'entraînement rotatif, et procédé d'amplification de gène - Google Patents

Système d'amplification de gène, puce de trajet d'écoulement, mécanisme d'entraînement rotatif, et procédé d'amplification de gène Download PDF

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Publication number
WO2018143469A1
WO2018143469A1 PCT/JP2018/003953 JP2018003953W WO2018143469A1 WO 2018143469 A1 WO2018143469 A1 WO 2018143469A1 JP 2018003953 W JP2018003953 W JP 2018003953W WO 2018143469 A1 WO2018143469 A1 WO 2018143469A1
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WO
WIPO (PCT)
Prior art keywords
channel
flow path
meandering
heater
chip
Prior art date
Application number
PCT/JP2018/003953
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English (en)
Japanese (ja)
Inventor
真人 齋藤
高橋　和也
民谷　栄一
Original Assignee
国立大学法人大阪大学
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Application filed by 国立大学法人大阪大学 filed Critical 国立大学法人大阪大学
Priority to JP2018566166A priority Critical patent/JPWO2018143469A1/ja
Publication of WO2018143469A1 publication Critical patent/WO2018143469A1/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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a gene amplification system, a channel chip, a rotation drive mechanism, and a gene amplification method.
  • ddPCR digital droplet PCR
  • PCR polymerase chain reaction
  • Non-Patent Document 1 discloses a method of rotating a microchannel and producing a droplet in a centrifugal field.
  • the microchannel is a channel having a width and depth (height) on the order of micrometers.
  • the microchannel has a width and depth of 1 ⁇ m or more and less than 1 mm.
  • One cycle of PCR includes two steps. Specifically, one cycle of PCR includes a heating step and a cooling step. In the heating step, the PCR solution is heated. In the cooling step, the heated PCR solution is cooled.
  • the present invention has been made in view of the above problems, and an object thereof is to provide a gene amplification system and a gene amplification method capable of performing ddPCR more rapidly. Another object of the present invention is to provide a channel chip and a rotation drive mechanism that can be used in a gene amplification system.
  • the gene amplification system of the present invention includes a flow path chip and a rotation drive mechanism.
  • the channel chip has a meandering channel.
  • the meandering channel meanders to one side and the other side.
  • the rotation drive mechanism has a rotation shaft and rotates the flow channel chip around the rotation shaft.
  • the rotation drive mechanism rotates the flow channel chip to impart buoyancy to the droplets in the meandering channel, thereby moving the droplets along the meandering channel.
  • the rotation driving mechanism includes a heater stage and a driving device.
  • the heater stage has a plurality of heater surfaces set at different temperatures.
  • the drive device rotates the heater stage around the rotation axis.
  • the rotation drive mechanism holds the flow path chip so that the meandering flow path is disposed across the plurality of heater surfaces.
  • the plurality of heater surfaces include a first heater surface and a second heater surface.
  • the first heater surface is set to a first temperature.
  • the second heater surface is set to a second temperature different from the first temperature.
  • the rotational drive mechanism has an end on the one side of the meandering channel facing the first heater surface, and an end on the other side of the meandering channel is the second heater surface.
  • the flow channel chip is held so as to face the surface.
  • the meandering channel includes a first inclined channel and a second inclined channel.
  • the first inclined channel extends to the one side.
  • the second inclined channel extends to the other side.
  • first inclined channel and the second inclined channel are inclined with respect to a radial direction orthogonal to the rotation axis.
  • the meandering channel includes a plurality of the first inclined channels and a plurality of the second inclined channels.
  • first inclined flow path and the second inclined flow path are alternately arranged along the radial direction.
  • the flow channel chip further includes a droplet supply chamber.
  • the droplet supply chamber supplies the droplet to the starting end of the meandering flow path.
  • the droplet supply chamber has a shape whose width becomes narrower toward a start end of the meandering flow path.
  • the droplet supply chamber has a triangular shape.
  • a triangular top of the droplet supply chamber is connected to a starting end of the meandering channel.
  • the flow channel chip further includes a liquid introduction chamber and an introduction flow channel.
  • a liquid is introduced into the liquid introduction chamber.
  • the introduction channel communicates with the liquid introduction chamber and the meandering channel.
  • the introduction flow path includes a meandering flow path at an intermediate portion thereof.
  • the channel chip of the present invention includes a meandering channel.
  • the meandering channel meanders to one side and the other side.
  • buoyancy is applied to the droplets in the meandering channel, and the droplets move along the serpentine channel.
  • the rotation drive mechanism of the present invention rotates the flow channel chip around the rotation axis.
  • the channel chip has a meandering channel.
  • the meandering channel meanders to one side and the other side.
  • the rotational drive mechanism includes a heater stage and a drive device.
  • the heater stage has a plurality of heater surfaces set at different temperatures.
  • the drive device rotates the heater stage around the rotation axis.
  • the rotation drive mechanism holds the flow path chip so that the meandering flow path is disposed across the plurality of heater surfaces.
  • the plurality of heater surfaces include a first heater surface and a second heater surface.
  • the first heater surface is set to a first temperature.
  • the second heater surface is set to a second temperature different from the first temperature.
  • the rotational drive mechanism has an end on the one side of the meandering channel facing the first heater surface, and an end on the other side of the meandering channel is the second heater surface.
  • the flow channel chip is held so as to face the surface.
  • the first heater surface extends along a radial direction orthogonal to the rotation axis.
  • the second heater surface is arranged side by side with the first heater surface and extends along the radial direction.
  • the gene amplification method of the present invention is a method of amplifying a gene using a channel chip.
  • the channel chip has a meandering channel and a liquid introduction chamber.
  • the meandering channel meanders on one side and the other side.
  • the liquid introduction chamber communicates with the meandering flow path.
  • the gene amplification method includes a step of holding the flow channel chip with respect to a heater stage so that the meandering flow channel is disposed across a plurality of heater surfaces, and the plurality of heater surfaces are set to different temperatures.
  • the end portion on the one side of the meandering channel faces the first heater surface, and the other side of the meandering channel is on the other side.
  • the flow path chip is held with respect to the heater stage so that the end faces the second heater surface.
  • the first heater surface is set to a first temperature
  • the second heater surface is set to a second temperature different from the first temperature. .
  • ddPCR can be performed more rapidly.
  • FIG. 14B is a sectional view taken along line XIVB-XIVB shown in FIG. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention.
  • FIG. 6 is a graph showing the relationship between the rotation speed and the movement speed of droplets according to Examples 2 to 4 of the present invention. 6 is a graph showing the relationship between the rotation speed and the time required to complete one cycle of PCR according to Examples 2 to 4 of the present invention.
  • A is a figure which shows the detection result of the fluorescence which concerns on Example 5.
  • FIG. (B) is a figure which shows the detection result of the fluorescence which concerns on a comparative example.
  • FIG. 1 is a plan view of the flow path chip 1.
  • the flow channel chip 1 includes a substrate 11.
  • the substrate 11 includes a liquid introduction chamber 12, an introduction channel 13, a droplet supply chamber 14, a meandering channel 15, and a storage chamber 16.
  • the introduction flow path 13 and the meandering flow path 15 are typically micro flow paths. However, the introduction flow path 13 and the meandering flow path 15 are not limited to micro flow paths.
  • the introduction channel 13 and the meandering channel 15 may have a channel width of 1 mm and a depth (height) of 1 mm, for example.
  • the liquid introduction chamber 12 and the like are formed on the substrate 11 by, for example, a fine processing technique.
  • the liquid introduction chamber 12 and the like can be formed by soft lithography.
  • Soft lithography is a technique for molding a relatively soft material such as silicone rubber.
  • the liquid introduction chamber 12 or the like can be formed by photolithography and a dry process (dry etching) or a wet process (wet etching).
  • the liquid introduction chamber 12 and the like can be formed by injection molding or cutting.
  • the shape and size of the substrate 11 are not particularly limited as long as the liquid introduction chamber 12 and the like can be formed.
  • the substrate 11 has a rectangular shape.
  • the length of the substrate 11 in the longitudinal direction LD is, for example, 70 mm
  • the length of the width direction WD perpendicular to the longitudinal direction LD of the substrate 11 is, for example, 50 mm.
  • the material of the substrate 11 is not particularly limited as long as the liquid introduction chamber 12 and the like can be formed, but is preferably a material that does not inhibit PCR.
  • the material of the substrate 11 is preferably a material that does not hinder the observation (detection) of fluorescence.
  • the substrate 11 may be made of silicone rubber, glass, cycloolefin polymer (COP), or polycarbonate (PC).
  • the liquid introduction chamber 12 is formed at one end of the substrate 11 in the longitudinal direction LD.
  • one side of the substrate 11 in the longitudinal direction LD is the upper side
  • the other side of the substrate 11 in the longitudinal direction LD is the lower side.
  • the liquid introduction chamber 12 has an inlet (opening), and the liquid is introduced into the liquid introduction chamber 12 through the inlet.
  • the inlet is formed in the ceiling of the liquid introduction chamber 12.
  • the liquid is an oil such as fluorine oil and a PCR solution.
  • the PCR solution is introduced after the oil is introduced. More specifically, after the introduction channel 13, the droplet supply chamber 14, and the meandering channel 15 are filled with oil and the oil is supplied to the storage chamber 16, the PCR solution is introduced.
  • the PCR solution typically includes a sample solution, a primer, a DNA polymerase, a DNA molecule to be amplified (target sequence), deoxynucleotide triphosphate (dNTP), and a buffer solution. PCR solutions can also contain fluorescent dyes or probe DNA.
  • the sample liquid contains DNA, and the DNA has DNA molecules to be amplified.
  • the sample liquid is a turbid liquid containing, for example, influenza virus, norovirus, or other infectious disease virus.
  • the sample liquid is a turbid liquid containing drug-resistant bacteria, enterohemorrhagic E. coli, and other infectious bacteria.
  • the sample liquid is a turbid liquid containing microorganisms or animal cells.
  • the sample solution is an extract of DNA or RNA of influenza virus, norovirus, or other infectious disease virus.
  • the sample liquid is an extract of DNA or RNA of drug-resistant bacteria, enterohemorrhagic Escherichia coli, or other infectious bacteria.
  • the sample liquid is an extract of DNA or RNA of microorganisms or animal cells.
  • the PCR solution may be prepared after the reverse transcription process is performed on the sample liquid.
  • a reverse transcription process may be included in the PCR step.
  • the reverse transcription process is a process of synthesizing complementary DNA for RNA by reverse transcriptase.
  • the depth (height) of the liquid introduction chamber 12 is not particularly limited.
  • the liquid introduction chamber 12 may have a depth selected from a range of 10 ⁇ m to 1 mm.
  • the volume of the liquid introduction chamber 12 is preferably larger than the total value of the volume of the introduction channel 13, the volume of the droplet supply chamber 14, and the volume of the meandering channel 15. Under this condition, the introduction flow path 13, the droplet supply chamber 14, and the meandering flow path 15 can be filled with oil, and the oil can be supplied to the storage chamber 16.
  • the introduction channel 13 allows the liquid introduction chamber 12 and the droplet supply chamber 14 to communicate with each other.
  • the start end of the introduction flow path 13 is connected (communication) with the liquid introduction chamber 12, and the end of the introduction flow path 13 is connected (communication) with the droplet supply chamber 14.
  • the introduction channel 13 includes a first introduction channel 13a and a second introduction channel 13b.
  • the first introduction flow path 13 a includes the start end of the introduction flow path 13.
  • the first introduction flow path 13 a extends from the liquid introduction chamber 12 to the other end portion in the longitudinal direction LD of the substrate 11 and then extends in the width direction WD of the substrate 11.
  • the channel width and depth of the first introduction channel 13a are not particularly limited.
  • the first introduction channel 13a may have a channel width and depth selected from a range of 10 ⁇ m to 1 mm.
  • the second introduction channel 13 b includes the end of the introduction channel 13.
  • the second introduction channel 13 b extends from the end of the first introduction channel 13 a in the longitudinal direction LD of the substrate 11 and is connected to the droplet supply chamber 14.
  • the channel width and depth of the second introduction channel 13b may be the same as or different from the channel width and depth of the first introduction channel 13a.
  • the second introduction channel 13b may have a channel width selected from a range of 10 ⁇ m to 1 mm.
  • the second introduction channel 13b has a narrower channel width than the first introduction channel 13a.
  • the second introduction flow path 13b may have a depth selected from a range of 10 ⁇ m to 1 mm.
  • the second introduction channel 13 b has a depth shallower than that of the droplet supply chamber 14.
  • the droplet supply chamber 14 is formed at the other end of the substrate 11 in the longitudinal direction LD.
  • the droplet supply chamber 14 communicates (connects) with the starting end of the meandering channel 15.
  • the droplet supply chamber 14 supplies a PCR solution droplet generated at the terminal end of the introduction channel 13 (near the interface between the introduction channel 13 and the droplet supply chamber 14) to the meandering channel 15.
  • the droplet of the PCR solution is generated when the droplet supply chamber 14 is filled with oil.
  • the shape of the droplet supply chamber 14 is not particularly limited, but preferably includes a shape for guiding the droplet of the PCR solution to the meandering channel 15.
  • the shape of the droplet supply chamber 14 on the side of the meandering channel 15 is a triangle, and the top of the triangle is connected to the starting end of the meandering channel 15. With this shape, a droplet of the PCR solution can be guided to the meandering channel 15.
  • the depth of the droplet supply chamber 14 is not particularly limited.
  • the droplet supply chamber 14 may have a depth selected from a range of 10 ⁇ m to 1 mm.
  • the droplet supply chamber 14 has a deeper depth than the second introduction channel 13b.
  • the meandering flow path 15 extends to one side in the longitudinal direction LD of the substrate 11 while meandering in the width direction WD of the substrate 11.
  • the end of the meandering channel 15 communicates (connects) with the accommodation chamber 16.
  • the meandering flow path 15 is formed at the other end of the substrate 11 in the longitudinal direction LD.
  • the width and depth of the meandering channel 15 are not particularly limited as long as the PCR solution droplets can pass through the meandering channel 15.
  • the meandering channel 15 may have a channel width and depth selected from a range of 10 ⁇ m to 1 mm.
  • the storage chamber 16 is formed between the liquid introduction chamber 12 and the meandering channel 15. Therefore, the liquid introduction chamber 12, the storage chamber 16, and the meandering flow path 15 are arranged in this order along the longitudinal direction LD of the substrate 11.
  • the storage chamber 16 stores oil and droplets of the PCR solution.
  • the storage chamber 16 has an outlet (opening) for discharging gas.
  • the outlet is formed in the ceiling portion of the storage chamber 16.
  • the depth of the storage chamber 16 is not particularly limited.
  • the storage chamber 16 may have a depth selected from a range of 10 ⁇ m to 1 mm.
  • the volume of the storage chamber 16 is typically larger than the liquid introduction chamber 12. Since the storage chamber 16 has a larger volume than the liquid introduction chamber 12, it is possible to suppress leakage of liquid such as oil from the outlet of the storage chamber 16.
  • the flow channel chip 1 can further include at least one spacer 17 disposed in the liquid introduction chamber 12.
  • the flow channel chip 1 may further include at least one spacer 17 disposed in the storage chamber 16.
  • the ceiling of the liquid introduction chamber 12 may bend depending on the area of the liquid introduction chamber 12.
  • the spacer 17 in the liquid introduction chamber 12 it is possible to prevent the ceiling portion of the liquid introduction chamber 12 from being bent.
  • the spacer 17 in the storage chamber 16 it is possible to prevent the ceiling portion of the storage chamber 16 from being bent.
  • FIG. 2 is a front view of the flow path chip 1.
  • the flow channel chip 1 further includes a floor member 18.
  • the floor member 18 is disposed below the substrate 11 and constitutes a floor portion such as the liquid introduction chamber 12.
  • the material of the floor member 18 is not particularly limited, but is preferably a material that does not inhibit PCR.
  • the material of the floor member 18 is preferably a material that does not hinder the observation (detection) of fluorescence.
  • the rigidity of the floor member 18 is preferably higher than the rigidity of the substrate 11.
  • the floor member 18 can be a glass substrate.
  • the rigidity of the floor member 18 is higher than the rigidity of the substrate 11, the floor member 18 supports the substrate 11. Therefore, it is possible to suppress the substrate 11 (flow channel chip 1) from being bent by the floor member 18. In other words, the rigidity of the channel chip 1 can be increased. Therefore, when moving the flow path chip 1 to the fluorescence detection field after the PCR step is completed, it is possible to reduce the risk of liquid (oil etc.) leaking from the flow path chip 1.
  • the rigidity of the floor member 18 may be higher or lower than the rigidity of the substrate 11.
  • the rigidity of the floor member 18 may be the same as the rigidity of the substrate 11.
  • the floor member 18 can be glass, cycloolefin polymer, polycarbonate, or a crimp seal.
  • the flow path chip 1 may include a lid member instead of the floor member 18.
  • the flow path chip 1 includes a lid member, the top and bottom of the substrate 11 are reversed and the lid member is disposed above the substrate 11.
  • the lid member constitutes a ceiling portion of the liquid introduction chamber 12 or the like. Therefore, when the flow channel chip 1 includes the lid member, the inlet of the liquid introduction chamber 12 and the outlet of the storage chamber 16 are formed in the lid member.
  • the material of the lid member is not particularly limited, but is preferably a material that does not inhibit PCR, like the floor member 18.
  • the material of the lid member is preferably a material that does not hinder the observation of fluorescence.
  • FIG. 3 is an enlarged view showing a part of the flow path chip 1. Specifically, FIG. 3 shows an enlarged view of the meandering flow path 15 of the flow path chip 1.
  • the meandering flow path 15 meanders to one side and the other side of the width direction WD of the substrate 11.
  • one side of the width direction WD of the substrate 11 is the right side
  • the other side of the width direction WD of the substrate 11 is the left side.
  • One end of the meandering channel 15 is disposed on the first heater surface 31a
  • the other end of the meandering channel 15 is disposed on the second heater surface 32a.
  • the first heater surface 31a is set to the first temperature.
  • the second heater surface 32a is set to a second temperature different from the first temperature.
  • the first heater surface 31a (first temperature) is set to a temperature for executing the PCR heating step
  • the second heater surface 32a (second temperature) executes the PCR cooling step.
  • Set temperature for. Therefore, the second heater surface 32a is set to a temperature lower than that of the first heater surface 31a.
  • the 1st heater surface 31a (1st temperature) is set to the temperature selected from the range of 90 to 98 degreeC.
  • the temperature of the second heater surface 32a is set according to the amplification target DNA molecule.
  • the 2nd heater surface 32a (2nd temperature) is set to the temperature selected from the range of 50 to 70 degreeC.
  • the droplets of the PCR solution reciprocate between the heating field and the cooling field by moving through the meandering flow path 15.
  • One cycle of PCR is completed when the droplet of PCR solution passes through the heating field and the cooling field once.
  • the meandering flow path 15 shown in FIG. Therefore, the number of PCR cycles is 30.
  • count of folding back the meandering flow path 15 is not limited to 60 times. The number of times the meandering channel 15 is folded back can be changed as necessary.
  • the length L1 of the meandering channel 15 along the longitudinal direction LD of the substrate 11 is changed according to the number of PCR cycles. Specifically, the length L1 of the meandering channel 15 becomes longer as the number of PCR cycles is increased. For example, when the number of PCR cycles is 30, the length L1 of the meandering flow path 15 may be 20 mm.
  • the meandering flow path 15 is formed in the first heater surface 31 a and the second heater surface so that the area overlapping the second heater surface 32 a is larger than the area overlapping the first heater surface 31 a. It is made to oppose 32a (temperature control field).
  • the meandering flow path 15 is disposed such that a portion extending from the center of the width direction WD of the substrate 11 to the other end faces the second heater surface 32 a.
  • FIG. 4 is a diagram showing a configuration of the rotation drive mechanism 3.
  • the rotation drive mechanism 3 includes a heater stage 30, a disk-shaped base 40, and a drive device 50.
  • the heater stage 30 is fixed to the base 40, and the driving device 50 rotates the heater stage 30 and the base 40 about the rotation axis AX (center axis).
  • the heater stage 30 and the base 40 are made of, for example, aluminum or copper, and the driving device 50 is typically a motor.
  • the driving device 50 is preferably capable of controlling the rotation speed (number of rotations).
  • the material of the heater stage 30 and the base 40 is not limited to aluminum or copper, it is preferable that it is a material with high heat conductivity.
  • the heater stage 30 will be further described.
  • the heater stage 30 has the first heater surface 31a and the second heater surface 32a described with reference to FIG. As shown in FIG. 4, the first heater surface 31 a and the second heater surface 32 a extend along a radial direction orthogonal to the rotation axis AX. Moreover, the 1st heater surface 31a and the 2nd heater surface 32a are located in a line at intervals.
  • one end of the meandering channel 15 is disposed on the first heater surface 31a, and the other end of the meandering channel 15 is on the second heater surface 32a. Be placed. Accordingly, the flow channel chip 1 described with reference to FIGS. 1 to 3 is set with respect to the rotational drive mechanism 3 such that the longitudinal direction LD thereof is along the radial direction of the rotational drive mechanism 3. As a result, the liquid introduction chamber 12, the storage chamber 16, and the meandering channel 15 are arranged in this order along the radial direction. Specifically, among the liquid introduction chamber 12, the storage chamber 16, and the meandering channel 15, the liquid introduction chamber 12 is closest to the rotation axis AX (rotation center).
  • the heater stage 30 includes a first heater stage 31 and a second heater stage 32.
  • the first heater stage 31 has a first heater surface 31a
  • the second heater stage 32 has a second heater surface 32a.
  • the first heater stage 31 heats the first heater surface 31a to the first temperature.
  • the second heater stage 32 heats the second heater surface 32a to the second temperature.
  • the first heater stage 31 includes an annular connecting part 311 and a plurality of first heating parts 312.
  • the connecting portion 311 is disposed around the rotation axis AX.
  • the plurality of first heating units 312 extend radially from the coupling unit 311 around the rotation axis AX. Accordingly, each of the first heating units 312 is disposed along the radial direction.
  • the first heater stage 31 shown in FIG. 4 has four first heating units 312.
  • the four first heating units 312 are typically provided with an angular interval of 90 degrees.
  • Each first heating unit 312 includes a protrusion 313 that protrudes upward.
  • the protrusion 313 extends in the radial direction.
  • the ridge portion 313 has a rectangular parallelepiped shape, and the upper surface of the ridge portion 313 constitutes the first heater surface 31a.
  • the length of the protrusion 313 in the radial direction is determined according to the length L1 of the meandering channel 15 described with reference to FIG. For example, when the length L1 of the meandering channel 15 is 20 mm, the length in the radial direction of the protrusion 313 is 20 mm or more.
  • the second heater stage 32 includes a disk-shaped main body 321 and a plurality of second heating units 322.
  • the main body 321 is disposed below the first heater stage 31 and separated from the first heater stage 31.
  • Each of the second heating parts 322 protrudes upward from the main body part 321 and extends in the radial direction.
  • the second heater stage 32 shown in FIG. 4 has four second heating units 322.
  • the four second heating units 322 are typically provided with an angular interval of 90 degrees.
  • Each of the second heating sections 322 has a second heater surface 32a.
  • the 2nd heating part 322 is a rectangular parallelepiped shape, and the upper surface of the 2nd heating part 322 comprises the 2nd heater surface 32a.
  • the length of the second heating unit 322 in the radial direction is determined according to the length L1 of the meandering flow path 15 described with reference to FIG. For example, when the length L1 of the meandering flow path 15 is 20 mm, the radial length of the second heating unit 322 is 20 mm or more.
  • the width of the second heater surface 32a is preferably larger than the width of the first heater surface 31a. Since the second heater surface 32a has a larger width than the first heater surface 31a, the region of the meandering flow path 15 that overlaps the second heater surface 32a is made larger than the region that overlaps the first heater surface 31a. Becomes easier.
  • the separation member 33 is fixed to the center portion of the main body portion 321 of the second heater stage 32, and supports the first heater stage 31 so that the first heater stage 31 is separated from the main body portion 321 of the second heater stage 32.
  • the material of the separation member 33 is not particularly limited, but is preferably a material having high heat resistance.
  • the material of the spacing member 33 may be a resin such as polycarbonate or polyphenylene sulfide (PPS).
  • the separation member 33 includes a large diameter portion 331 and a small diameter portion 332.
  • the small diameter portion 332 is fixed on the large diameter portion 331, and the large diameter portion 331 is fixed to the central portion of the main body portion 321 of the second heater stage 32.
  • a step shape (step) is formed by the large diameter portion 331 and the small diameter portion 332, and the connecting portion 311 of the first heater stage 31 is fixed around the small diameter portion 332 (step).
  • the first heater stage 31 is disposed at a position separated from the main body portion 321 of the second heater stage 32 by the height of the large diameter portion 331.
  • the large diameter portion 331 and the small diameter portion 332 may be formed integrally.
  • the separation member 33 supports the first heater stage 31 so that the first heater surface 31a and the second heater surface 32a are arranged with a space therebetween.
  • the separation member 33 supports the first heater stage 31 such that thermal interference between the adjacent first heater surface 31a and the second heater surface 32a is reduced.
  • the distance between adjacent first heater surface 31a and second heater surface 32a may be 1 mm.
  • FIG. 5 is a schematic diagram showing a part of the gene amplification system 100.
  • the gene amplification system 100 includes the flow path chip 1 described with reference to FIGS. 1 to 3 and the rotation drive mechanism 3 described with reference to FIG.
  • the rotation drive mechanism 3 rotates the channel chip 1 around the rotation center X (around the rotation axis AX).
  • the rotation direction RD is a clockwise direction.
  • the rotation drive mechanism 3 holds the flow channel chip 1 so that the liquid introduction chamber 12 in the liquid introduction chamber 12 and the like is closest to the rotation center X.
  • the gene amplification system 100 rotates the channel chip 1 around the rotation center X in a state where the meandering channel 15 is opposed to the first heater surface 31a and the second heater surface 32a (temperature control field), and ddPCR is performed. Execute.
  • FIG. 6 is a flowchart showing the gene amplification method according to this embodiment.
  • the gene amplification method according to this embodiment includes steps S601 to S607.
  • steps S601 to S607 will be described.
  • the flow path chip 1 is held by the rotation drive mechanism 3 in step S601. After the flow path chip 1 is held by the rotational drive mechanism 3, oil is introduced into the liquid introduction chamber 12 in step S602.
  • step S603 the rotation drive mechanism 3 rotates the flow path chip 1 around the rotation axis AX (rotation center X).
  • the introduction channel 13, the droplet supply chamber 14, and the meandering channel 15 are filled with oil, and the oil is supplied to the storage chamber 16.
  • the temperatures of the first heater surface 31a and the second heater surface 32a are set in step S604. Specifically, the first heater surface 31a is set to a temperature for executing a PCR heating step, and the second heater surface 32a is set to a temperature for executing a PCR cooling step.
  • the PCR solution is introduced into the liquid introduction chamber 12 in step S605.
  • the rotation driving mechanism 3 rotates the flow path chip 1 around the rotation axis AX (rotation center X).
  • AX rotation center X
  • a droplet of the PCR solution is generated at the end portion of the introduction channel 13, and ddPCR is performed in the meandering channel 15.
  • the droplets of the PCR solution that have passed through the meandering flow path 15 are stored in the storage chamber 16.
  • step S607 the channel chip 1 is moved to the fluorescence detection field, and the presence or absence of DNA in each droplet is determined by detecting fluorescence.
  • FIGS. 7 to 11 are schematic diagrams showing a gene amplification method using the gene amplification system 100.
  • FIG. 7 to 11 are schematic diagrams showing a gene amplification method using the gene amplification system 100.
  • the flow path chip 1 is set in the rotation drive mechanism 3 (step S601). Specifically, the flow path is such that one end of the meandering flow path 15 is disposed on the first heater surface 31a and the other end of the meandering flow path 15 is disposed on the second heater surface 32a. The chip 1 is held on the heater stage 30. Thereafter, the oil 71 is introduced into the liquid introduction chamber 12 (step S602). For introducing the oil 71, a pipetter 72 is typically used.
  • the rotation drive mechanism 3 rotates the flow channel chip 1 in the rotation direction RD about the rotation axis AX (rotation center X) (step S603).
  • the oil 71 flows from the liquid introduction chamber 12 to the storage chamber 16.
  • the rotation of the flow path chip 1 is continued until all the oil 71 is discharged from the liquid introduction chamber 12.
  • the first heater surface 31a and the second heater surface 32a are heated, the first heater surface 31a is set to the first temperature, and the second heater surface 32a is set to the second temperature.
  • the first heater surface 31a (first temperature) is set to a temperature selected from the range of 90 ° C. to 98 ° C.
  • the second heater surface 32a (second temperature) is 50 ° C. to 70 ° C. It is set to a temperature selected from the range of °C or less.
  • the PCR solution 91 is introduced into the liquid introduction chamber 12 as shown in FIG. 9 (step S605).
  • a pipetter 92 is typically used for introducing the PCR solution 91.
  • the rotation drive mechanism 3 rotates the flow path chip 1 in the rotation direction RD about the rotation axis AX (rotation center X) (step S606).
  • the PCR solution 91 flows through the introduction channel 13 and becomes a droplet 91 a at the end portion of the introduction channel 13.
  • the droplet 91 a is supplied to the meandering channel 15 via the droplet supply chamber 14 filled with the oil 71, moves in the meandering channel 15 filled with the oil 71, and is accommodated in the accommodation chamber 16.
  • the interface between the oil 71 and the PCR solution 91 is between the oil 71 (oil phase) and the PCR solution 91 (water phase).
  • a pressure difference due to the density difference occurs.
  • a droplet 91a is generated.
  • a pressure based on centrifugal force is applied to the oil 71 and the PCR solution 91. Therefore, the gene amplification system 100 and the gene amplification method according to the present embodiment generate the droplet 91a by the centrifugal field.
  • the droplet 91a moving through the meandering channel 15 is heated by the first heater surface 31a and then cooled by the second heater surface 32a.
  • ddPCR one cycle of PCR
  • the first heater surface 31a heats the droplet 91a
  • the double-stranded DNA contained in the droplet 91a is separated and single-stranded DNA is generated.
  • the second heater surface 32a cools the droplet 91a.
  • the primer binds to the amplification target DNA molecule in the single-stranded DNA.
  • dNTP binds to the primer by DNA polymerase, and a DNA strand extends from the primer to generate double-stranded DNA.
  • the rotation of the channel chip 1 is continued until a desired amount of the PCR solution 91 is discharged from the liquid introduction chamber 12.
  • the flow path chip 1 is removed from the rotation drive mechanism 3 and moved to the fluorescence detection field as shown in FIG. 11 (step S607).
  • the fluorescence detection field the presence or absence of amplified DNA molecules in each droplet 91a is determined by detecting fluorescence. By counting the number of droplets 91a containing DNA, it is possible to measure the absolute quantification of the DNA (or nucleic acid such as RNA) that is statistically likely to be detected.
  • the excitation light emitted from the excitation light source is applied to the accommodation chamber 16.
  • the excitation light excites the fluorescent dye.
  • the excited fluorescent dye emits fluorescence.
  • the emitted fluorescence is detected by a fluorescence detector. Since the droplet 91a that emits fluorescence contains DNA and the droplet 91a that does not emit fluorescence does not contain DNA, the presence or absence of DNA in each droplet 91a can be determined.
  • the excitation light source can be, for example, a laser light source or a light emitting diode (LED).
  • the fluorescence detector includes, for example, a photomultiplier detector, a condenser lens, and a fluorescence filter.
  • Fluorescence detection methods include intercalator method and hybridization method.
  • intercalator method a fluorescent dye (SYBR green I) that specifically inserts into double-stranded DNA and emits fluorescence is used.
  • the PCR solution 91 to which a fluorescent dye is added is used.
  • the TagMan probe method is the most common hybridization method, and a probe DNA in which a fluorescent dye is bound to an oligonucleotide specific to the DNA sequence is used.
  • the PCR solution 91 to which the probe DNA is added is used.
  • the fluorescent dye used in the TagMan probe method can be, for example, FAM (Carboxyfluorescein).
  • the reverse transcription process is performed before the PCR step.
  • the PCR solution may be prepared after the reverse transcription process is performed on the sample liquid.
  • a reverse transcription process may be included in the PCR step.
  • FIG. 12 is an enlarged schematic view showing a part of the meandering flow path 15.
  • the meandering flow path 15 includes a first inclined flow path 15a and a second inclined flow path 15b.
  • the first inclined channel 15a extends to one side of the substrate 11 in the width direction WD and is connected (communicated) to one end of the second inclined channel 15b.
  • the second inclined channel 15b extends to the other side in the width direction WD of the substrate 11 and is connected (communicated) to the other end of the first inclined channel 15a.
  • the first inclined channel 15 a and the second inclined channel 15 b are inclined with respect to the longitudinal direction LD of the substrate 11. In other words, the first inclined channel 15a and the second inclined channel 15b are inclined with respect to the radial direction orthogonal to the rotation axis AX.
  • the first inclined channel 15a and the second inclined channel 15b are alternately arranged along the longitudinal direction LD (radial direction).
  • the buoyancy F toward the rotation axis AX (rotation center X) with respect to the droplet 91a is caused by centrifugal force. Is granted. Since the first inclined flow path 15a and the second inclined flow path 15b are inclined with respect to the radial direction, the droplet 91a has a meandering flow path 15 (first inclined flow path 15a and It moves toward the rotation axis AX (rotation center X) along the second inclined flow path 15b).
  • the magnitude of the buoyancy F depends on the rotational speed (rotational speed) of the drive device 50 described with reference to FIG. Specifically, the greater the number of revolutions, the greater the buoyancy F and the faster the moving speed of the droplet 91a. Therefore, the moving speed of the droplet 91a can be controlled by controlling the rotation speed of the driving device 50. Therefore, by increasing the number of rotations, it is possible to speed up heat exchange with respect to the droplet 91a, that is, speed up PCR (ddPCR). However, depending on the channel lengths of the first inclined channel 15a and the second inclined channel 15b, if the moving speed of the droplet 91a becomes too fast, appropriate heat exchange may not be performed and PCR may not be performed. is there. Therefore, it is preferable to determine the rotation speed of the drive device 50 according to the flow path lengths of the first inclined flow path 15a and the second inclined flow path 15b.
  • FIG. 13 is an enlarged view showing a part of the meandering flow path 15.
  • the first inclined channel 15 a and the second inclined channel 15 b have an inclination of an angle ⁇ with respect to the longitudinal direction LD of the substrate 11.
  • the angle ⁇ is larger than 0 °
  • the droplet 91a moves in the meandering flow path 15 using the buoyancy F described with reference to FIG.
  • the angle ⁇ and the length L2 of the meandering channel 15 in the width direction WD of the substrate 11 are not particularly limited as long as the PCR heating step and the cooling step can be performed.
  • the angle ⁇ can be selected from a range of 10 ° or less.
  • the length L2 of the meandering channel 15 can be selected from a range of 1 mm or more and 20 mm or less.
  • FIG. 14A is an enlarged view showing a part of the substrate 11. Specifically, FIG. 14A shows an enlarged view of the vicinity of the second introduction flow path 13b.
  • FIG. 14B is a cross-sectional view taken along line XIVB-XIVB shown in FIG.
  • the substrate 11 further includes a terrace structure 19.
  • the introduction flow path 13 includes a terrace portion 13c.
  • the terrace portion 13 c is formed between the end of the second introduction flow path 13 b and the droplet supply chamber 14.
  • the terrace structure 19 includes a second introduction flow path 13b (straight line portion) and a terrace portion 13c.
  • the width W2 of the terrace portion 13c along the width direction WD of the substrate 11 is larger than the flow path width W1 of the second introduction flow path 13b.
  • the channel width of the terminal portion of the second introduction channel 13b is expanded by the terrace portion 13c.
  • the depth of the terrace portion 13c is equal to the depth h1 of the second introduction channel 13b, and is shallower than the depth h2 of the droplet supply chamber 14.
  • each part of the terrace structure 19 are not particularly limited as long as the droplet 91a can be generated.
  • the width W2 of the terrace portion 13c can be 100 ⁇ m.
  • the terrace length L3 of the terrace portion 13c along the longitudinal direction LD of the substrate 11 may be 30 ⁇ m.
  • the depth h2 of the droplet supply chamber 14 is 100 ⁇ m
  • the depth h1 of the second introduction flow path 13b and the terrace portion 13c may be 30 ⁇ m.
  • the diameter of the droplet 91a can be controlled by the dimensions of each part of the terrace structure 19. Typically, the diameter of the droplet 91a is not less than 10 ⁇ m and not more than 300 ⁇ m.
  • the channel width W1 of the second introduction channel 13b is 30 ⁇ m
  • the width W2 of the terrace portion 13c is 100 ⁇ m
  • the terrace length L3 is 30 ⁇ m
  • the depth h1 of the second introduction channel 13b and the terrace portion 13c Is 30 ⁇ m, a droplet 91a having a diameter of about 95 ⁇ m is generated.
  • FIGS. 15 to 20 are views showing a part of the introduction flow path 13, the droplet supply chamber 14, and a part of the meandering flow path 15. Specifically, FIGS. 15 to 20 show the state in which the droplet 91a is supplied from the droplet supply chamber 14 to the meandering flow path 15 in time series.
  • the speed at which the droplet 91a moves through the droplet supply chamber 14 is faster than the speed at which the droplet 91a moves through the meandering channel 15. This is because the droplet 91 a moves in the direction of the buoyancy F in the droplet supply chamber 14.
  • the droplet supply chamber 14 has a shape whose width becomes narrower toward the start end of the meandering flow path 15. With this shape, the droplet 91a can be supplied to the meandering channel 15 more smoothly. More preferably, the width of the droplet supply chamber 14 is narrowed along the direction of the buoyancy F. By narrowing the width of the droplet supply chamber 14 along the direction of the buoyancy F, the droplet 91a can be guided more smoothly toward the starting end of the meandering channel 15.
  • the shape for guiding the droplet 91a to the meandering channel 15 is not limited to a triangle.
  • the droplet supply chamber 14 may include an arc shape (a part of the circumferential surface) whose width becomes narrower toward the starting end of the meandering flow path 15.
  • the droplet 91a reciprocates between the heating field (first heater surface 31a) and the cooling field (second heater surface 32a) using the buoyancy F as a driving force. Therefore, ddPCR can be performed more rapidly than the configuration in which the temperature of the temperature control field is transitioned between two temperatures.
  • the moving speed of the droplet 91a can be controlled by controlling the rotation speed. Therefore, ddPCR can be performed more quickly by controlling the number of rotations and increasing the moving speed of the droplet 91a. Furthermore, by controlling the rotation speed, it is possible to control the time during which the droplet 91a moves through the heating field and the time during which the droplet 91a moves through the cooling field. Therefore, ddPCR can be performed reliably.
  • the droplet 91a moves in the meandering flow path 15 using the buoyancy F as a driving force. Therefore, it is not necessary to feed the oil phase (oil 71). As a result, the amount of oil phase (oil 71) used can be reduced. Therefore, the running cost can be reduced. Furthermore, since a solution driving unit such as a micropump is not required, the gene amplification system 100 (ddPCR system) can be downsized.
  • the droplet 91a is surrounded by the oil 71, the adsorption of the droplet 91a to the channel wall surface can be suppressed. Therefore, sample loss can be reduced and efficiency can be increased.
  • the generation of the droplet 91a in the centrifugal field and the PCR in the temperature control field can be achieved by one apparatus (rotation drive mechanism 3). Therefore, ddPCR can be performed simply by dripping the oil 71 and the PCR solution 91 onto the flow path chip 1 and the rotation drive mechanism 3 rotating the flow path chip 1. Can do.
  • the generation of the droplet 91a and the PCR can be performed within one chip. Therefore, ddPCR can be performed more simply and rapidly.
  • the PCR solution 91 is introduced into the liquid introduction chamber 12 after the temperatures of the first heater surface 31 a and the second heater surface 32 a are set, but the PCR solution 91 is introduced into the liquid introduction chamber 12. After that, the temperature of the first heater surface 31a and the second heater surface 32a may be set.
  • the channel chip 1 is rotated in the clockwise direction, but the channel chip 1 may be rotated in the counterclockwise direction.
  • the rotation drive mechanism 3 includes four sets (temperature control fields) of the first heater surface 31a and the second heater surface 32a, but the present invention is not limited to this form.
  • the rotational drive mechanism 3 may include one, two, or three pairs of the first heater surface 31a and the second heater surface 32a, or five pairs of the first heater surface 31a and the second heater surface 32a. You may have more than one.
  • the introduction channel 13 of the channel chip 1 may include a meandering channel 13d as shown in FIG.
  • FIG. 21 is a plan view of a channel chip 1 according to another embodiment of the present invention.
  • the introduction flow channel 13 includes a meandering flow channel 13d.
  • the meandering flow path 13 d is formed between the liquid introduction chamber 12 and the droplet supply chamber 14. More specifically, the meandering flow path 13d is formed in the first introduction flow path 13a.
  • the heater stage 30 has two heater surfaces (the first heater surface 31a and the second heater surface 32a), and the meandering channel 15 is disposed across the two heater surfaces.
  • the heater stage 30 may have three or more heater surfaces. Three or more heater surfaces are set to different temperatures.
  • the meandering channel 15 is disposed across three or more heater surfaces. Since the heater stage 30 has three or more heater surfaces, the temperature gradient of the temperature control field can be controlled more freely.
  • Example 1 First, the flow channel chip 1 used in Example 1 will be described.
  • the flow channel chip 1 shown in FIG. 1 was used.
  • the substrate 11 was made of silicone rubber.
  • the depths of the liquid introduction chamber 12, the droplet supply chamber 14, and the storage chamber 16 were 100 ⁇ m.
  • the channel width and depth of the first introduction channel 13a were both 100 ⁇ m.
  • the terrace structure 19 described with reference to FIGS. 14A and 14B the flow path width W1 of the second introduction flow path 13b is 30 ⁇ m, the width W2 of the terrace portion 13c is 100 ⁇ m, and the terrace length L3 is 30 ⁇ m, the depth h1 of the second introduction flow path 13b and the terrace portion 13c was 30 ⁇ m.
  • the length L1 of the meandering flow path 15 along the longitudinal direction LD of the substrate 11 was 20 mm.
  • the length L2 of the meandering channel 15 along the width direction WD of the substrate 11 was 3.5 mm.
  • the channel width and depth of the meandering channel 15 were both 100 ⁇ m.
  • the angle ⁇ at which the first inclined channel 15a and the second inclined channel 15b are inclined with respect to the radial direction (longitudinal direction LD) was 4 °.
  • Example 1 fluorine oil was used as the oil 71. Further, instead of the PCR solution 91 (aqueous phase), water colored in red was used.
  • the channel chip 1 was rotated around the rotation axis AX at a rotation speed of 440 rpm.
  • FIG. 22 is a diagram showing the results of Example 1.
  • FIG. 22 was obtained by imaging the meandering flow path 15 with a high speed camera. As shown in FIG. 22, it was confirmed that the droplet S was generated by rotating the flow channel chip 1 around the rotation axis AX. Further, it was confirmed that the droplet S moved along the meandering channel 15 toward the rotation axis AX, and the meandering channel 15 was filled with the droplet S. In Example 1, a droplet S having a diameter of about 95 ⁇ m was generated.
  • Example 2 to 4 the moving speed of the droplet S was measured using the channel chip 1 having the same configuration as that of Example 1.
  • fluorine oil was used as the oil 71.
  • PCR solution 91 aqueous phase
  • water colored in red was used instead of the PCR solution 91 (aqueous phase).
  • Example 2 the channel chip 1 was rotated around the rotation axis AX at a rotation speed of 440 rpm as in Example 1.
  • Example 3 the flow channel chip 1 was rotated around the rotation axis AX at a rotation speed of 1000 rpm.
  • Example 4 the flow channel chip 1 was rotated around the rotation axis AX at a rotation speed of 1320 rpm.
  • a droplet S having a diameter of about 95 ⁇ m was also generated.
  • FIG. 23A and FIG. 23B are diagrams illustrating the movement of the droplet S according to the second embodiment.
  • FIG. 23A and FIG. 23B were obtained by imaging the meandering flow path 15 with a high-speed camera.
  • FIG. 23B shows the meandering flow path 15 when 30 seconds have elapsed from the time when FIG. 23A was imaged.
  • the arrow indicates the distance that the droplet S has moved in 30 seconds.
  • FIGS. 24A and 24B are diagrams illustrating movement of the droplet S according to the third embodiment.
  • 24A and 24B are obtained by imaging the meandering flow path 15 with a high-speed camera.
  • FIG. 24B shows the meandering flow path 15 when 30 seconds have elapsed from the time when FIG. 24A was imaged.
  • the arrow indicates the distance that the droplet S has moved in 30 seconds.
  • FIG. 25A and FIG. 25B are diagrams illustrating the movement of the droplet S according to the fourth embodiment.
  • FIG. 25A and FIG. 25B were obtained by imaging the meandering flow path 15 with a high speed camera.
  • FIG. 25B shows the meandering flow path 15 when 30 seconds have elapsed from the time when the image of FIG. In FIG. 25B, the arrow indicates the distance that the droplet S has moved in 30 seconds.
  • the moving distance of the droplet S increases as the rotational speed rpm increases.
  • FIG. 26 is a graph showing the relationship between the rotational speed and the moving speed of the droplet S.
  • the horizontal axis indicates the rotational speed
  • the vertical axis indicates the moving speed of the droplet S.
  • the graph shown in FIG. 26 is created by plotting the moving speed of the droplet S acquired from the imaging results shown in FIGS. 23 (a) and 23 (b) to FIG. 25 (a) and FIG. 25 (b). As shown in FIG. 26, it was confirmed that the moving speed of the droplet S increased as the rotational speed rpm increased.
  • FIG. 27 is a graph showing the relationship between the rotation speed and the time required to complete one cycle of PCR.
  • the horizontal axis indicates the rotation speed.
  • the vertical axis indicates the time required to complete one cycle of PCR.
  • the time required to complete one cycle of PCR indicates the time required for the droplet S to reciprocate once through the meandering channel 15.
  • the time required for the droplet S to make one round trip through the meandering channel 15 is the liquid obtained from the imaging results shown in FIGS. 23 (a) and 23 (b) to 25 (a) and 25 (b). It calculated
  • Example 5 and Comparative Example The channel chip 1 used in Example 5 and the comparative example has the same configuration as that of Example 1 except that it includes six types of thermo seals A to F. Specifically, the thermo seals A to F are embedded in the substrate 11 so as to face the meandering flow path 15. Specifically, the thermo seals A to C are opposed to one end of the meandering flow path 15 in the width direction WD of the substrate 11. In other words, the thermo seals A to C are embedded in the substrate 11 so as to face the first heater surface 31a.
  • the color of the thermo seal A becomes green at a temperature of 100 ° C.
  • the color of the thermo seal B becomes green at a temperature of 95 ° C.
  • the color of the thermo seal C becomes green at a temperature of 90 ° C.
  • thermo seals D to F face the other end of the meandering flow path 15 in the width direction WD of the substrate 11.
  • the thermo seals D to F are embedded in the substrate 11 so as to face the second heater surface 32a.
  • the color of the thermo seal D becomes green at a temperature of 65 ° C.
  • the color of the thermo seal E becomes green at a temperature of 60 ° C.
  • the color of the thermo seal F becomes green at a temperature of 55 ° C.
  • Example 5 ddPCR was performed using PCR-6 as a target, using a PCR solution containing genomic DNA of drug-resistant bacteria (IMP-6 positive). In addition, fluorescence was detected after the ddPCR step.
  • the ddPCR step was performed using a solution to which no DNA was added. The solution used in the comparative example contains the same components as in Example 5 except that no DNA was added. Also in the comparative example, fluorescence was detected after the end of the ddPCR step.
  • Example 5 the flow path chip 1 was rotated around the rotation axis AX at a rotation speed of 800 rpm using fluorine oil.
  • the color of the thermo seal B is green
  • the color of the thermo seal E is green. Therefore, it was confirmed that the temperature (first temperature) of the first heater surface 31a can be controlled to about 95 ° C. Moreover, it has confirmed that the temperature (2nd temperature) of the 2nd heater surface 32a was controllable to about 60 degreeC.
  • FIG. 28 (a) is a diagram showing a fluorescence detection result according to Example 5.
  • FIG. 28B is a diagram showing a fluorescence detection result according to the comparative example.
  • the droplet containing the drug-resistant bacterial genomic DNA (Example 5) is associated with DNA amplification compared to the droplet not containing DNA (Comparative Example). Strong fluorescence was observed (detected). Therefore, it was confirmed that ddPCR can be performed by the gene amplification system described in the embodiment.
  • ddPCR can be performed more rapidly, which is useful for a system for measuring absolute quantification of genes.

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Abstract

La présente invention concerne un système d'amplification de gène (100) comprenant une puce de trajet d'écoulement (1) et un mécanisme d'entraînement rotatif (3). La puce de trajet d'écoulement (1) présente un trajet d'écoulement sinueux (15) qui forme des méandres vers un côté et vers l'autre côté. Le mécanisme d'entraînement rotatif (3) fait tourner la puce de trajet d'écoulement (1) autour d'un axe de rotation. Le mécanisme d'entraînement rotatif (3) amène la puce de trajet d'écoulement (1) à tourner et fait agir une force de flottabilité sur une gouttelette liquide (91a) à l'intérieur du trajet d'écoulement sinueux (15) de sorte que la gouttelette liquide (91a) se déplace le long du trajet d'écoulement sinueux (15). Le mécanisme d'entraînement rotatif (3) comprend un étage de chauffage (30). L'étage de chauffage (30) comporte une pluralité de surfaces chauffantes (31a, 32a). La pluralité des surfaces chauffantes (31a, 32a) sont paramétrées à des températures différentes. Le mécanisme d'entraînement rotatif (3) maintient la puce de trajet d'écoulement (1) de sorte que le trajet d'écoulement sinueux (15) est disposé de manière à chevaucher la pluralité de surfaces chauffantes (31a, 32a).
PCT/JP2018/003953 2017-02-06 2018-02-06 Système d'amplification de gène, puce de trajet d'écoulement, mécanisme d'entraînement rotatif, et procédé d'amplification de gène WO2018143469A1 (fr)

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JP2020174661A (ja) * 2019-04-18 2020-10-29 奎克生技光電股▲フン▼有限公司 熱伝導均一性及び熱履歴整合性を改善するための熱サイクル装置
CN113755316A (zh) * 2021-10-09 2021-12-07 苏州国科均豪生物科技有限公司 可切换的温育模块、pcr扩增检测仪

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