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WO2025032840A1 - Procédé de pcr - Google Patents

Procédé de pcr Download PDF

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Publication number
WO2025032840A1
WO2025032840A1 PCT/JP2023/031944 JP2023031944W WO2025032840A1 WO 2025032840 A1 WO2025032840 A1 WO 2025032840A1 JP 2023031944 W JP2023031944 W JP 2023031944W WO 2025032840 A1 WO2025032840 A1 WO 2025032840A1
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WIPO (PCT)
Prior art keywords
sample
pcr
flow path
temperature region
microfluidic chip
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PCT/JP2023/031944
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English (en)
Japanese (ja)
Inventor
康正 三谷
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Go Foton Inc
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Go Foton Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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

Definitions

  • the present invention relates to a method for polymerase chain reaction (PCR).
  • Gene testing is widely used in various medical fields, for identifying agricultural crops and pathogenic microorganisms, for evaluating food safety, and for testing for pathogenic viruses and various infectious diseases.
  • Methods are known for detecting minute amounts of DNA with high sensitivity, in which a portion of the DNA is amplified and analyzed.
  • the method using PCR is a notable technology that selectively amplifies a certain portion of very small amounts of DNA taken from a living organism.
  • PCR involves subjecting a biological sample containing DNA to a mixture of PCR reagents, such as primers and enzymes, to a prescribed thermal cycle, causing repeated denaturation, annealing and extension reactions to selectively amplify specific portions of the DNA.
  • PCR analysis can be performed in real time by measuring the fluorescent signal as the DNA is amplified and performing quantitative analysis.
  • a given amount of the target sample is typically placed in a PCR tube or a chip such as a microplate (microwell) with multiple holes, and the temperature of the sample is changed using a device called a thermal cycler (see, for example, Patent Document 1).
  • Real-time PCR is possible with conventional PCR methods by extracting and purifying nucleic acids from the blood to remove pigments that inhibit the measurement of fluorescent signals before adding the blood to the PCR reagent, or by diluting the blood, for example, by 10-fold or more and then adding only a trace amount of blood to the PCR reagent, but performing such work is very cumbersome and time-consuming.
  • the present invention was made in consideration of these circumstances, and its purpose is to provide a PCR method that can suitably measure the fluorescent signal associated with PCR amplification without having to carry out cumbersome nucleic acid purification procedures, even when blood is used as a sample.
  • one embodiment of the PCR method of the present invention comprises the steps of preparing a sample by adding blood to a PCR reagent, subjecting the sample to a thermal cycle by repeatedly moving the sample back and forth within a flow channel of a microfluidic chip, and detecting fluorescence from the sample within the flow channel to monitor the progress of PCR of the sample.
  • the present invention provides a PCR method that can suitably measure the fluorescent signal associated with PCR amplification, even when blood is used as a sample, without the need for cumbersome nucleic acid purification procedures.
  • FIG. 1 is a perspective view showing a first surface side of a microfluidic chip applicable to a PCR method according to an embodiment of the present invention.
  • FIG. 2 is a perspective view showing a second surface side of the microfluidic chip.
  • FIG. 2 is a plan view of a first surface of a substrate provided on the microfluidic chip.
  • FIG. 2 is a plan view of a second surface of a substrate provided on the microfluidic chip.
  • FIG. 2 is a conceptual diagram for explaining a cross-sectional structure of a microfluidic chip.
  • FIG. 1 is a schematic diagram for explaining a thermal cycler and a PCR device for performing PCR that can utilize a microfluidic chip. 1 is a flowchart illustrating a PCR method according to the present embodiment.
  • FIG. 2 is a diagram for explaining how a sample changes when the sample is moved within a channel of a microfluidic chip.
  • FIG. 2 is a diagram for explaining how a sample changes when the sample is moved within a channel of a microfluidic chip.
  • FIG. 2 is a diagram for explaining how a sample changes when the sample is moved within a channel of a microfluidic chip.
  • FIG. 2 is a diagram for explaining how a sample changes when the sample is moved within a channel of a microfluidic chip.
  • FIG. 2 is a diagram for explaining how a sample changes when the sample is moved within a channel of a microfluidic chip.
  • FIG. 2 is a diagram for explaining how a sample changes when the sample is moved within a channel of a microfluidic chip.
  • FIG. 2 is a plan view of a first surface of another microfluidic chip applicable to a PCR method according to an embodiment of the present invention.
  • 15(a) and 15(b) are diagrams showing amplification curves obtained when a reciprocal flow type real-time PCR device is used.
  • FIG. 1 shows an amplification curve when a general real-time PCR device is used.
  • 17(a) and 17(b) are diagrams showing amplification curves obtained when a reciprocal flow type real-time PCR device is used.
  • FIG. 1 shows an amplification curve when a general real-time PCR device is used.
  • FIG. 1 is a perspective view showing the first surface side of a microfluidic chip 10 applicable to a PCR method according to an embodiment of the present invention.
  • FIG. 2 is a perspective view showing the second surface side of the microfluidic chip 10.
  • FIG. 3 is a plan view of the first surface 14a of the substrate 14 provided in the microfluidic chip 10.
  • FIG. 4 is a plan view of the second surface 14b of the substrate 14 provided in the microfluidic chip 10.
  • FIG. 5 is a conceptual diagram for explaining the cross-sectional structure of the microfluidic chip 10. Note that FIG. 5 is a diagram for explaining the positional relationship between the flow path, film, and filter and the substrate, and differs from the cross-sectional view of an actual microfluidic chip.
  • the microfluidic chip 10 (also called a “fluid chip,” “(micro)channel chip,” “reaction processing chip,” “reaction processing vessel,” “nucleic acid amplification chip,” “DNA chip,” or “measurement chip”) has a channel 12 through which a sample to be subjected to a thermal cycle or reaction moves.
  • the microfluidic chip 10 has a resin substrate 14 on whose first surface 14a is mainly formed a groove-shaped channel 12 through which the sample moves, a first sealing film 16 and a second sealing film 18 attached to the first surface 14a of the substrate 14, and a third sealing film 20 attached to the second surface 14b of the substrate.
  • the microfluidic chip 10 may have a first filter 28 and a second filter 30 near both ends of the channel 12 of the substrate 14 in order to suppress contamination of the channel 12.
  • the first surface 14a of the substrate 14 of the microfluidic chip 10 is provided with at least a groove-shaped flow channel 12, air communication ports 24, 26 arranged at both ends of the flow channel 12 for connecting the flow channel 12 with the output of a pump that pressurizes or blows air into the flow channel, chamber-shaped chambers 32, 34 provided above the flow channel 12, and filter spaces 36, 38 for placing filters 28, 30 to prevent contamination of the flow channel 12.
  • the groove-shaped flow channel 12 is intended to carry samples to be subjected to PCR or thermal cycling.
  • the second surface 14b of the substrate 14 is provided with groove-shaped air ducts 40, 42 for connecting the air communication ports 24, 26 with the filter spaces 36, 38, and a sample introduction port (sample introduction hole) 44 for introducing a sample into the flow path 12.
  • the groove-shaped air ducts 40, 42 may be intended as a flow path mainly for air to pass through for blowing or pressurizing from a pump or the like connected to the air communication ports 24, 26. Providing the flow path 12 and air ducts 40, 42 in this way on the two opposing main surfaces (first surface 14a and second surface 14b) of the substrate 14 helps to make effective use of the main surfaces of the substrate 14.
  • the substrate 14 of the microfluidic chip 10 is preferably formed from a material that is stable against temperature changes and is not easily damaged by the sample solution used. Furthermore, the substrate 14 is preferably formed from a material that has good moldability, good barrier properties, and low autofluorescence. Such materials include resins such as acrylic, polypropylene, and silicone, and in particular, cyclic polyolefin resins. When the substrate is made of resin, it is possible to produce large quantities of it with high quality by injection molding using a mold.
  • the size of the substrate 14 of the microfluidic chip 10 is not particularly limited, and any size is acceptable as long as it satisfies the chemical and physical actions within the flow paths formed within the substrate. On the other hand, if there is a demand to miniaturize the device in which the microfluidic chip is mounted, the microfluidic chip and the substrate that constitutes it can also be made smaller or thinner.
  • the substrate 14 of the microfluidic chip 10 may be a parallel plate that is easy to mold, portable, and handle.
  • the size of the substrate 14 of the microfluidic chip 10 is, for example, a thickness of 1 mm to 10 mm, preferably 2 mm to 6 mm, and a rectangular shape of W ⁇ L in plan view, where W is 10 mm to 60 mm, preferably 20 mm to 35 mm, and H is 45 mm to 100 mm, preferably 60 mm to 85 mm.
  • the microfluidic chip 10 may also be made of a transparent resin.
  • the microfluidic chip 10 is expected to be used to amplify nucleic acids contained in a sample by real-time PCR.
  • the progress and completion of the reaction can be known by detecting and monitoring the fluorescence emitted from the sample in real time.
  • the microfluidic chip 10 is preferably made of a transparent material.
  • a groove-shaped flow channel 12 is formed on the first surface 14a of the substrate 14.
  • most of the flow channel 12 is formed as a groove exposed on the first surface 14a of the substrate.
  • the groove-shaped air ducts 40, 42 are formed as grooves exposed on the second surface 14b of the substrate 14.
  • the dimensions of the flow channel 12 are, for example, about 0.2 mm to 1.5 mm in width and 0.2 mm to 2 mm in depth, and the dimensions of the flow channel 12 may be 0.7 mm in width and 0.7 mm in depth.
  • the structure of the flow channel 12 may include a side surface that is so-called a "draft taper" and has a certain angle with respect to the first surface 14a and the second surface 14b of the substrate 14.
  • the taper angle may be 1° to 30°, or 3° to 15°.
  • the sealing films 16, 18, 20 for sealing the flow path 12, the sample inlet 44, the air communication port 24, 26, the chambers 32, 34, and the filters 28, 30 may have one of their main surfaces with adhesiveness, or may have a functional layer formed on one of their main surfaces that exhibits adhesiveness or adhesion when pressed, irradiated with energy such as ultraviolet rays, or heated, and have the function of being easily attached to and integrated with the first surface 14a and the second surface 14b of the substrate 14. It is desirable that the sealing films 16, 18, 20 are formed from a material that has low, preferably almost zero, autofluorescence, including the adhesive.
  • a transparent film made of a resin such as cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic is suitable, but is not limited to these.
  • the sealing films 16, 18, 20 may also be formed from plate-shaped glass or resin. In this case, rigidity can be expected, which helps prevent warping and deformation of the microfluidic chip 10.
  • a first filter 28 is provided near one end of the flow path 12.
  • a second filter 30 may be provided near the other end of the flow path 12.
  • the pair of first filter 28 and second filter 30 provided near both ends of the flow path 12 are intended to prevent contamination of the flow path and the sample so as not to interfere with the amplification and detection of the target DNA by PCR or to prevent deterioration of the quality of the target DNA.
  • the dimensions of the first filter 28 and second filter 30 are formed so that they fit snugly into the filter spaces 36, 38 formed on the first surface 14a of the substrate 14.
  • the substrate 14 is formed with a first air communication port 24 that communicates with one end of the flow path 12 via a first air duct 40 and a first filter 28.
  • the substrate 14 is formed with a second air communication port 26 that communicates with the other end of the flow path 12 via a second air duct 42 and a second filter 30.
  • the pair of first air communication port 24 and second air communication port 26 are formed so as to be exposed on the first surface 14a of the substrate 14.
  • a part of the flow channel 12 includes a thermal cycle region between a pair of first filter 28 and second filter 30, in which multiple levels of temperature can be controlled by a PCR device described later.
  • a part of the flow channel 12 of the microfluidic chip 10 is heated by the temperature control system and maintained at the desired temperature.
  • the temperature is controlled so as to be maintained at multiple temperatures required to apply a thermal cycle and cause a reaction such as PCR.
  • the reaction includes a reaction in which the nucleic acid contained in the sample is thermally denatured, an annealing reaction, and an extension reaction.
  • the temperature controlled in the part of the flow channel 12 may be a temperature that causes these reactions.
  • the thermal cycle region includes a first temperature region 46 and a second temperature region 48.
  • the first temperature region 46 is maintained at a relatively high temperature (e.g., about 95°C)
  • the second temperature region 48 is maintained at a lower temperature than the first temperature region 46 (e.g., about 60°C).
  • Nucleic acids contained in the sample may undergo a thermal denaturation reaction in the flow path included in the first temperature region 46, and annealing and extension reactions may occur in the flow path included in the second temperature region 48.
  • the first temperature region 46 and the second temperature region 48 may be referred to as a "high temperature region” and a "medium temperature region", respectively, from the viewpoint of the high and low temperatures.
  • the first temperature region 46 and the second temperature region 48 may also be referred to as a "denaturation region” and an "annealing and extension region” from the viewpoint of the attributes of the reaction.
  • first temperature region 46 is connected to the first chamber 32. Furthermore, the first chamber 32 is connected to the first air communication port 24 via the first filter 28 and the first air duct 40. The other end of the first temperature region 46 is connected to one end of the second temperature region 48 via the connecting flow path 50. The other end of the second temperature region 48 is connected to one end of the third serpentine flow path 52 through the connecting flow path 41. The other end of the third serpentine flow path 52 is connected to the second chamber 34. Furthermore, the second chamber 34 is connected to the second air communication port 26 via the second filter 30 and the second air duct 42.
  • the third serpentine flow path 52 is a flow path that is not directly related to thermal cycling or PCR, and includes a straight flow path and a curved flow path.
  • the sample inlet 44 for introducing a sample into the flow path to be connected to the third serpentine flow path 52, it may be used as a buffer-like flow path for waiting samples to be subjected to PCR.
  • the first temperature region 46 and the second temperature region 48 each include a first serpentine flow channel 54 and a second serpentine flow channel 56 that are a combination of a curved flow channel and a straight flow channel and are continuously folded back. It can also be said that a plurality of curved flow channels are connected by straight flow channels.
  • a serpentine flow channel the limited effective area of a heater or the like that constitutes a temperature control system described later can be effectively used, which contributes to reducing the temperature variation in each temperature region, and has the advantage that the actual size of the microfluidic chip 10 can be reduced, which contributes to the miniaturization of a PCR device.
  • the radius of curvature R2 of the curved flow channel may be 0.3 mm to 10 mm, and preferably 0.5 mm to 6 mm.
  • the connecting flow path 50 may be provided with a region (referred to as the "fluorescence detection region” or simply as the “detection region”) 58 that is irradiated with excitation light in order to detect fluorescence from the sample flowing through the flow path when the microfluidic chip 10 is mounted on a PCR device described below.
  • the flow path included in this fluorescence detection region 58 is referred to as the detection flow path 59.
  • the connection flow path 50 corresponds to a portion where a part of a fluorescence detection device for detecting the fluorescence of a sample is disposed. Therefore, the interval between the first temperature region 46 and the second temperature region 48 may be relatively long, and for example, the interval between the first temperature region 46 and the second temperature region 48 may be longer than the linear flow path included in the first temperature region 46 and/or the second temperature region 48. Since the part of the fluorescence detection device disposed in the connection flow path 50 has a predetermined volume, it is advantageous to set the interval to a certain distance or more in order to dispose some parts of the fluorescence detection device. In addition, the longer the interval between the first temperature region 46 and the second temperature region 48, the smaller the thermal interference between both temperature regions can be. When the thermal interference between the temperature regions is small, the heat provided from the device through a temperature control means such as a heater can be effectively utilized, which contributes to power saving of the device.
  • the distance between the first temperature region 46 and the second temperature region 48 may be shorter than, for example, 1/2 the length L of the long side in the plan view of the microfluidic chip 10.
  • the degree of freedom in designing the microfluidic chip such as mounting other elements and arranging margins and blank spaces, can be improved, making it easier to ensure the size and portability required for the PCR device.
  • the first chamber 32 is disposed between the thermal cycle area and the first air communication port 24. More specifically, the first chamber 32 is disposed between the first temperature area 46 of the thermal cycle area and the first filter 28.
  • the sample in the flow path moves toward the first temperature area 46, for example, by being pressurized or blown with air from the second air communication port 26.
  • the first chamber 32 has the role of preventing the sample from being pushed too hard or from overrunning the first temperature area 46 and reaching the first filter 28.
  • the first chamber 32 has a first connection 32a which is a connection (or opening) with the flow path end of the flow path extending from the first temperature region 46 of the thermal cycle region, and a second connection 32b which is a connection with the flow path end of the flow path leading to the first filter 28.
  • the first chamber 32 has a first structure 32c near the first connection 32a. This first structure 32c prevents the liquid sample that has entered the first chamber 32 from the first connection 32a from moving directly toward the second connection 32b, and contributes to preventing the sample from entering in the direction of the first filter 28.
  • the second chamber 34 is disposed between the thermal cycle region and the second air communication port 26. More specifically, the second chamber 34 is disposed between the third serpentine flow path 52 and the second filter 30.
  • the sample in the flow path is, for example, pressurized or blown from the first air communication port 24, causing the sample to move toward the second temperature region 48.
  • the second chamber 34 serves to prevent the sample from being pushed too hard or from overrunning the second temperature region 48 or the third serpentine flow path 52 and reaching the second filter 30.
  • the second chamber 34 has a third connection 34a which is a connection to the end of the flow path extending from the third serpentine flow path 52, and a fourth connection 34b which is a connection to the end of the flow path leading to the second filter 30.
  • the second chamber 34 has a second structure 34c near the third connection 34a. This second structure 34c prevents the liquid sample that has entered the second chamber 34 from the third connection 34a from moving directly toward the fourth connection 34b, and contributes to preventing the sample from entering in the direction of the second filter 30.
  • the first chamber 32 and the second chamber 34 may have a function other than preventing sample overrun.
  • the first chamber 32 and the second chamber 34 may be used as reaction chambers.
  • a PCR reagent may be held in the first chamber 32 and the second chamber 34 in advance, and a sample may be added to the PCR reagent in the first chamber 32 and the second chamber 34, and then PCR may be performed.
  • a first widening flow passage 60 may be provided between the first serpentine flow passage 54 and the first chamber 32.
  • the first widening flow passage 60 is adjacent to the flow passage belonging to the first temperature region 46, and is provided further back than the first serpentine flow passage 54 when viewed from the connecting flow passage 50.
  • a second widening flow passage 62 may be provided between the second serpentine flow passage 56 and the third serpentine flow passage 52.
  • the second widening flow passage 62 is adjacent to the flow passage belonging to the second temperature region 48, and is formed further back than the flow passage belonging to the second temperature region 48 when viewed from the connecting flow passage 50.
  • the first widening flow channel 60 is provided so that its cross-sectional area is larger than that of the first serpentine flow channel 54.
  • the second widening flow channel 62 is provided so that its cross-sectional area is larger than that of the second serpentine flow channel 56.
  • the first widening flow channel 60 and the second widening flow channel 62 each have the role of exerting a braking effect on the sample flowing through the first serpentine flow channel 54 and the second serpentine flow channel 56. This utilizes Bernoulli's law, which states that the speed of a fluid passing through a flow tube is inversely proportional to the cross-sectional area of the flow tube.
  • a brake is applied to the sample, and a function is added to stop the sample within the first temperature region 46 and the second temperature region 48.
  • a region in which the cross-sectional area changes in a part of the flow channel 12 in this way it is possible to promote stirring and mixing of the sample in that region. This allows the sample to be effectively separated into a solid component and a transparent solution component, as described below.
  • the microfluidic chip 10 may have at least one sample inlet for introducing a sample into the flow channel.
  • the sample inlet is, for example, an inlet for introducing a sample containing a nucleic acid to be amplified into the flow channel.
  • the shape, size, and arrangement of the sample inlet are not important as long as it has a perforated structure that connects from the main surface of the substrate 14 to any of the flow channels.
  • a sample inlet 44 is provided so as to connect to the third serpentine flow channel 52.
  • Figs. 3 and 4 as an example, a sample inlet 44 is provided so as to connect to the third serpentine flow channel 52.
  • the flow channel 12 is provided on surface 14a, and the sample inlet 44 is provided on surface 14b, and they are formed on opposing surfaces of the substrate 14, but they may be provided on the same surface of the substrate 14.
  • a microfluidic chip according to another embodiment may have two sample inlets (a first sample inlet and a second sample inlet).
  • a first sealing film 16 for sealing at least the flow path is attached to the first surface 14a of the substrate 14.
  • the first sealing film 16 may be large enough to cover almost the entire surface of the first surface 14a of the substrate 14.
  • the first sealing film 16 may have holes pre-drilled at positions corresponding to the air communication ports 24, 26 so that the air communication ports 24, 26 are not sealed in order to connect a nozzle leading from the discharge port of a pump of the liquid delivery system.
  • the first sealing film 16 may be in a form in which the air communication ports 24, 26 are sealed.
  • a nozzle leading from the discharge port of a pump used in a liquid delivery system for pressurizing or blowing air into the flow path is attached to the air communication ports 24, 26 when the microfluidic chip 10 is used. If the nozzle end is provided with a hollow needle or the like, the film can be pierced with the tip of the needle to communicate with the output port of the pump and the air communication ports 24, 26.
  • the first surface 14a of the substrate 14 may be provided with a first sealing film 16 for sealing the flow path 12, the filter space 36, 38, and the chambers 32, 34, excluding the air communication ports 24, 26, and may also be provided with an adhesive second sealing film 18, for example, so that only the second air communication port 26 is sealed.
  • the first air communication port 24 may not be provided with a sealing film for sealing.
  • the second sealing film may be peeled off.
  • the first air communication port 24 is open, but the second air communication port 26 and other ports are sealed with a sealing film, so dust, dirt, and other particles are less likely to enter through the first air communication port 24, making it possible to suppress contamination.
  • the second surface 14b of the substrate 14 is provided with groove-shaped air ducts 40, 42 that connect the air communication ports 24, 26 with the filter spaces 36, 38, and a sample inlet 44 for introducing a sample into the flow path.
  • a third sealing film 20 for sealing the groove-shaped air ducts 40, 42 may be attached to the second surface 14b of the substrate 14.
  • a fourth sealing film for sealing the sample inlet 44 may be attached to the second surface 14b of the substrate 14. If the air ducts 40, 42 and the sample inlet 44 are close to each other, the third sealing film 20 may simultaneously seal the air ducts 40, 42 and the sample inlet 44. In this case, the fourth sealing film is not necessary.
  • Figures 1 and 2 show an example in which the substrate 14 and each of these sealing films 16, 18, and 20 are attached and integrated.
  • Figure 2 shows an example of the third sealing film 20 that simultaneously seals the air ducts 40, 42 and the sample inlet 44.
  • a measured amount of sample is placed into the flow path 12 through the sample introduction port 44 using a pipette, dropper, syringe, etc.
  • the pipette, dropper, syringe, etc. may be further manipulated to push the sample until it reaches the first temperature region 46 or the second temperature region 48 of the thermal cycle region.
  • the position reached by the sample can be used as the initial position of the sample during PCR or thermal cycling.
  • the introduced sample pushes air of approximately the same volume as the sample in the flow path, and this is released from the open first air communication port 24. At this time, the first air communication port 24 becomes an exhaust port when the sample is introduced.
  • the third sealing film 20 is attached back to its original position. It is preferable that the third sealing film 20 has properties that enable it to withstand multiple cycles of attachment and removal. This is advantageous from the standpoint of maintaining the cleanliness of the microfluidic chip 10 during distribution and of convenience in the sample introduction process.
  • the second sealing film 18 and the third sealing film 20 may be provided with tabs 18a, 20a with non-adhesive front and back at the ends of the film so that they can be easily peeled off (and possibly reattached) when in use, from the initial state when they are attached to the main surface of the substrate 14. This helps to improve the convenience of the operator in opening the sample introduction port 44 and the air communication ports 24, 26 of the microfluidic chip 10.
  • the third sealing film 20 may have a corner portion 20b between the sample introduction port 44 and the air duct 42.
  • This corner portion 20b serves as a guide for the limit to which the operator should peel off the third sealing film 20 when opening only the sample introduction port 44.
  • a roughly circular cut may be made in the corner portion to emphasize the guide, or the film may be peeled off partway to make it less likely to tear.
  • FIG. 6 is a schematic diagram illustrating a PCR device (also called a “thermal cycle device,” “thermal cycler,” or “reaction processing device”) 100 for performing thermal cycling and PCR that can utilize the microfluidic chip 10.
  • a PCR device also called a "thermal cycle device,” “thermal cycler,” or “reaction processing device” 100 for performing thermal cycling and PCR that can utilize the microfluidic chip 10.
  • the PCR device 100 includes a mounting section 101 on which the microfluidic chip 10 is mounted, a first heater 102 for heating the first temperature region 46 of the flow path 12, which is controlled to a relatively high temperature, a second heater 104 for heating the second temperature region 48 of the flow path, and a temperature control system including a temperature sensor (not shown), such as a thermocouple, for measuring the actual temperature of each reaction region.
  • a temperature sensor such as a thermocouple
  • Each heater may be, for example, a resistive heating element or a Peltier element.
  • the first temperature region 46 in the flow path 12 of the microfluidic chip 10 is maintained at approximately 95°C, and the second temperature region 48 is maintained at approximately 60°C by the control device 110, which is composed of these heaters, an appropriate heater driver, and a microcomputer, a microprocessor, an electronic circuit, etc., and the temperature of each reaction region in the thermal cycle region required for performing PCR is set.
  • the PCR device 100 includes a battery built into the device and a power supply unit (neither of which is shown) for receiving power from an external source in order to operate each of these parts appropriately. Explanations and descriptions of the drivers for driving each part are omitted.
  • the PCR device 100 further includes a fluorescence detection device 106.
  • a specific fluorescent probe is added to the sample S.
  • the intensity of the fluorescent signal emitted from the sample S increases, and the intensity value of the fluorescent signal can be used as an indicator for determining the progress and completion of PCR.
  • the optical fiber fluorescence detector FLE-510 manufactured by Nippon Sheet Glass Co., Ltd. can be used, which has a very compact optical system, can perform rapid measurements, and can detect fluorescence regardless of whether it is in a bright or dark place.
  • This optical fiber fluorescence detector can be tuned to have its excitation light/fluorescence wavelength characteristics suitable for the fluorescence characteristics emitted by sample S, making it possible to provide an optimal optical/detection system for samples with various characteristics.
  • the small diameter of the light beam provided by the optical fiber fluorescence detector it is suitable for detecting fluorescence from samples present in small or narrow areas such as flow channels, and has excellent response speed.
  • the optical fiber type fluorescence detection device 106 includes an optical head 108, a fluorescence detection device main body (including a driver) 112, and an optical fiber 114 that connects the optical head 108 and the fluorescence detection device main body 112.
  • the fluorescence detection device main body 112 includes an excitation light source (LED, laser, or other light source adjusted to emit a specific wavelength), an optical fiber type multiplexer/demultiplexer, and a photoelectric conversion element (photodetector such as PD, APD, or photomultiplier) (none of which are shown), and is composed of a driver and a microcomputer for controlling these.
  • the optical head 108 is composed of an optical system such as a lens, and is responsible for the functions of directional irradiation of the excitation light to the sample and collecting the fluorescence emitted from the sample.
  • the collected fluorescence is separated from the excitation light by the optical fiber type multiplexer/demultiplexer in the fluorescence detector driver through the optical fiber 114, and is converted into an electrical signal by the photoelectric conversion element.
  • an optical fiber type fluorescence detector one described in JP 2010-271060 A can be used.
  • the optical fiber type fluorescence detection device 106 can be further modified to enable coaxial detection of multiple wavelengths using a single or multiple optical heads.
  • the invention described in International Publication No. 2014/003714 can be utilized for a fluorescence detector for multiple wavelengths and its signal processing.
  • an optical head 108 is positioned so that it can detect the fluorescence from the sample S in the detection flow path 59.
  • the sample S is repeatedly moved back and forth within the thermal cycle area in the flow path, causing the reaction to proceed and amplifying the specified DNA contained in the sample S.
  • the output value from the fluorescence detection device 106 is used to control the movement of the sample S.
  • the output value from the fluorescence detection device 106 may be sent to the control device 110 and used as a parameter for controlling the liquid delivery system 120 described below.
  • the fluorescence detection device 106 is not limited to an optical fiber type fluorescence detection device, as long as it has the function of detecting fluorescence from the sample.
  • the fluorescence detection device 106 can be provided with multiple sets of excitation light/fluorescence.
  • the first fluorescence detection device may be set to irradiate the sample with blue excitation light and detect green fluorescence emitted from the sample
  • the second fluorescence detection device may be set to irradiate the sample with green excitation light and detect red fluorescence.
  • fluorescence detection devices may be arranged that are preset with combinations of excitation light/fluorescence wavelength characteristics according to combinations of nucleic acids and fluorescent probes to be detected.
  • the optical head 108 of the fluorescence detection device 106 is positioned to detect fluorescence from the sample in a connecting flow path 50 in the thermal cycle region.
  • the connecting flow path 50 is a flow path that connects the first temperature region 46 and the second temperature region 48.
  • the region in which fluorescence from the sample is expected to be detected is referred to as the fluorescence detection region 58.
  • the flow path belonging to the fluorescence detection region 58 is referred to as the detection flow path 59.
  • the detection flow path 59 is provided on the first surface 14a of the substrate 14 on which the flow paths are formed.
  • the PCR device 100 further includes a liquid delivery system 120 for moving and stopping the sample S within the flow path of the microfluidic chip 10.
  • the liquid delivery system 120 can move the sample S in one direction within the flow path by sending (blowing) air through either the first air communication port 24 or the second air communication port 26.
  • the liquid delivery system 120 can stop the sample S at a predetermined position by stopping the blowing of air into the flow path or equalizing the pressure on both sides of the sample S within the flow path.
  • the liquid delivery system 120 in this embodiment includes a liquid delivery pump 128, a first three-way valve 122, a second three-way valve 124, and a pressurized chamber 126. Furthermore, the liquid delivery system 120 may include a tube and a nozzle that connect the discharge port of the liquid delivery pump 128 to the air communication port.
  • the pressurized chamber 126 forms a space with a certain volume inside.
  • the pressurized chamber 126 is configured to be able to change the pressure in its internal space.
  • the pressure inside the pressurized chamber 126 is maintained at a pressure (e.g., 1.05 to 1.3 atm) that is at least higher than the atmospheric pressure in the surrounding environment of the PCR device 100 while PCR is being performed.
  • the PCR device 100 may include, but is not limited to, a pressure pump, an air pump, a blower, a micropump, a syringe pump, etc. (not shown) whose output port is connected to the pressurized chamber 126.
  • the atmospheric pressure in the surrounding environment of the PCR device 100 refers to the pressure (or atmospheric pressure) in the location where the PCR device 100 is installed, the location where PCR is performed by the PCR device 100, or, if the PCR device 100 is installed in a location partitioned from the surroundings, in that partitioned location.
  • the pressure in the pressurized chamber 126 should be set to a level that can prevent significant sample evaporation and the generation of bubbles that could affect the PCR reaction process, even if the sample is repeatedly exposed to high temperatures (approximately 95°C).
  • the pressurized chamber 126 is provided with an atmospheric pressure release valve (leak valve) 127.
  • the atmospheric pressure release valve 127 is controlled so that the pressure in the liquid delivery system 120 and the flow path 12 of the microfluidic chip 10 becomes equal to atmospheric pressure when the microfluidic chip 10 is attached or detached. This makes it possible to prevent the sample S from suddenly moving or jumping out.
  • the liquid delivery pump 128 is placed in the pressurized chamber 126.
  • the liquid delivery pump 128 may be a blower or microblower that equalizes the pressure on the primary side (air intake side) and secondary side (air exhaust side) when stopped. Note that the liquid delivery pump 128 has the role of sending air into the flow path, and the liquid sample moves in response to the blowing air and increased pressure, but is not a pump that actually delivers the liquid itself.
  • the first port A of the first three-way valve 122 is connected to the output port (discharge port) 128a of the liquid delivery pump 128.
  • the second port B of the first three-way valve 122 is connected to the internal space of the pressurized chamber 126.
  • the third port C of the first three-way valve 122 is connected to one end of a first tube 130, and the other end of the first tube 130 is connected to the first air communication port 24 of the microfluidic chip 10 as the first output port 131 of the liquid delivery system 120.
  • the first three-way valve 122 is switchable between a state in which the first air communication port 24 of the microfluidic chip 10 is connected to the output port 128a of the liquid delivery pump 128 and a state in which the first air communication port 24 of the microfluidic chip 10 is open to the internal space of the pressurized chamber 126.
  • the first three-way valve 122 is controlled to a state in which port A and port C are connected.
  • the first three-way valve 122 is controlled to a state in which port B and port C are connected.
  • the first port A of the second three-way valve 124 is connected to the output port 128a of the liquid delivery pump 128.
  • the second port B of the second three-way valve 124 is connected to the internal space of the pressurized chamber 126.
  • the third port C of the second three-way valve 124 is connected to the second air communication port 26 of the microfluidic chip 10.
  • the third port C of the second three-way valve 124 is connected to one end of the second tube 132, and the other end of the second tube 132 is connected to the second air communication port 26 of the microfluidic chip 10 as the second output port 133 of the liquid delivery system 120.
  • the second three-way valve 124 is switchable between a state in which the second air communication port 26 of the microfluidic chip 10 is connected to the output port 128a of the liquid delivery pump 128 and a state in which the second air communication port 26 of the microfluidic chip 10 is opened to the internal space of the pressurized chamber 126.
  • the second three-way valve 124 is controlled to a state in which port A and port C are connected.
  • the second three-way valve 124 is controlled to a state in which port B and port C are connected.
  • the liquid delivery pump 128, the first three-way valve 122, and the second three-way valve 124 can be operated by the control device 110 through appropriate drivers.
  • the optical head 108 arranged as described above can transmit output values based on the obtained fluorescent signal to the control device 110, and have the control device 110 recognize the position of the sample S in the flow path and its passage, thereby controlling the liquid delivery system 120.
  • the liquid delivery pump 128 is controlled to an operating state (ON).
  • the first three-way valve 122 is controlled to a state where the first port A and the third port C are connected, and the second three-way valve 124 is controlled to a state where the second port B and the third port C are connected.
  • the first air communication port 24 of the microfluidic chip 10 is connected to the output port 128a of the liquid delivery pump 128, and the second air communication port 26 of the microfluidic chip 10 is opened to the internal space of the pressurized chamber 126.
  • the liquid delivery pump 128 since the liquid delivery pump 128 is disposed in the pressurized chamber 126, when air is discharged from the liquid delivery pump 128, the pressure of the first air communication port 24 of the microfluidic chip 10 becomes higher than the second air communication port 26, and the sample S moves from the first temperature region 46 to the second temperature region 48.
  • the first three-way valve 122 is controlled so that the second port B is in communication with the third port C
  • the second three-way valve 124 is controlled so that the second port B is in communication with the third port C.
  • the first air communication port 24 and the second air communication port 26 are both open to the internal space of the pressurized chamber 126, so that the sample S stops in the second temperature region 48.
  • the first three-way valve 122 and the second three-way valve 124 can be operated in the reverse order to that described above.
  • the sample S can be continuously moved back and forth within the flow path between the first temperature region 46 and the second temperature region 48, thereby providing the sample S with an appropriate thermal cycle.
  • a liquid delivery pump 128 is disposed in the internal space of a pressurized chamber 126 set at a pressure (for example, 1.3 atm) higher than the atmospheric pressure of the surrounding environment, and the second port B of the first three-way valve 122 and the second three-way valve 124 is configured to be open to the internal space of the pressurized chamber 126. Therefore, during the reaction process, the entire flow path 12 of the microfluidic chip 10 is maintained at a pressure higher than the atmospheric pressure of the surrounding environment. This is particularly advantageous when the PCR device 100 is used, for example, at high altitudes such as Mexico City or inside an aircraft during flight.
  • the first temperature of the first temperature region 46 is 95°C or close to it.
  • the pressure at high altitudes and inside an aircraft is lower than 1 atm, there is a possibility that a part of the sample S will boil even at 95°C.
  • a liquid delivery system 120 equipped with the pressurized chamber 126 of the PCR device 100 it is possible to suppress undesirable boiling of the sample in such a low atmospheric pressure environment.
  • the PCR device 100 can be used without problems even in environments other than low atmospheric pressure.
  • the liquid delivery system may adopt a form other than that shown in FIG. 6.
  • a syringe pump, diaphragm pump, micro-blower, or fan may be used. Since two pumps are used, it may be necessary to adjust for individual differences between the pumps in order to stop them accurately at the desired temperature range.
  • an air delivery pump consisting of a fan or micro-blower that equalizes the pressure on the primary side (air intake side) and secondary side (air discharge side) when the two pumps are stopped may be used.
  • air delivery pump consisting of a fan or micro-blower that equalizes the pressure on the primary side (air intake side) and secondary side (air discharge side) when the two pumps are stopped may be used.
  • the PCR device 100 to which the microfluidic chip 10 having the flow channel 12 can be applied is also called a reciprocal flow type real-time PCR device.
  • FIG. 7 is a flowchart for explaining the PCR method according to this embodiment.
  • the PCR method according to this embodiment is a real-time PCR method using blood as a sample.
  • blood is added to a PCR reagent to prepare a sample (S10).
  • blood means "whole blood”.
  • “adding blood to a PCR reagent” means adding whole blood directly to the PCR reagent as is, and adding nucleic acid purified from blood to a PCR reagent or adding crude blood that has been simply purified to a PCR reagent is not referred to as the act of adding blood to a PCR reagent.
  • adding such blood to a PCR reagent is sometimes expressed as "adding blood to a PCR reagent”.
  • the PCR reagents include primers and a probe.
  • the probe may be a TaqMan probe (TaqMan is a registered trademark of Roche Diagnostics Deutschen mit Beschlenktel Kunststoff).
  • a sample is prepared by adding a predetermined amount of blood to a predetermined amount of PCR reagent.
  • the blood content in the sample may be 0.01% to 20%, preferably 0.1% to 10%, and more preferably 1% to 5%.
  • a 20 ⁇ L sample with a blood content of 5% can be prepared by adding 1 ⁇ L of whole blood to 19 ⁇ L of PCR reagent.
  • the microfluidic chip 10 is prepared, and the prepared sample is introduced into the flow path 12 in the microfluidic chip 10 (S12).
  • the sample is introduced into the flow path 12 through the sample introduction port 44 of the microfluidic chip 10 using a pipette, dropper, syringe, etc.
  • the sample is prepared and then introduced into the microfluidic chip 10, but the sample may also be prepared within the microfluidic chip 10.
  • PCR reagents may be held in advance within the first chamber 32 and the second chamber 34, and blood may be added to the PCR reagents within the first chamber 32 and the second chamber 34.
  • the sample is repeatedly moved back and forth between the first temperature region 46 and the second temperature region 48 of the microfluidic chip 10 to subject the sample to thermal cycling (S14).
  • the target nucleic acid is amplified.
  • a fluorescent signal emitted from the sample in the flow path 12 is detected (S16) to monitor the progress of PCR of the sample.
  • Real-time PCR analysis is then performed based on the detected fluorescent signal (S18).
  • an amplification curve is created with the number of cycles (C) on the horizontal axis and the fluorescent signal intensity on the vertical axis.
  • FIGS. 8 to 13 are diagrams for explaining how the sample S changes when it is moved within the flow channel 12 of the microfluidic chip 10.
  • FIGS. 8 to 13 show a schematic diagram of the flow channel 12 formed on the first surface 14a of the microfluidic chip 10.
  • the sample S is made by directly adding blood to a PCR reagent.
  • Figure 8 shows the state immediately after the sample S is introduced into the flow channel 12 from the sample introduction port 44.
  • the sample S immediately after introduction is located in the third serpentine flow channel 52 as shown in Figure 8.
  • the sample S immediately after introduction is red and transparent.
  • FIG. 9 shows the state in which the sample S has been moved from the third serpentine flow path 52 through the second temperature region 48 and the connecting flow path 50 to the first temperature region 46.
  • the sample can be moved within the flow path 12 by pressurizing or blowing air through the air communication ports 24, 26.
  • the sample In the first temperature region 46, the sample is heated to approximately 95° C. for 15 seconds. This causes the sample S to change from a transparent red to an opaque light brown color.
  • FIG. 10 shows the state in which the sample S has been moved from the first temperature region 46 through the connecting flow path 50 to the second temperature region 48.
  • the second temperature region 48 it is heated to approximately 60°C for 15 seconds.
  • the sample S gradually begins to separate into a solid component (precipitate) and a clear solution component.
  • the solid component of the sample S is light brown and opaque, and the clear solution component is light brown and transparent.
  • FIG. 11 shows the state in which the sample S has been moved from the second temperature region 48 through the connecting flow path 50 to the first temperature region 46.
  • the first temperature region 46 it is heated to approximately 95°C for 15 seconds. This promotes aggregation of the solid components of the sample S, and further promotes separation of the colored solid components from the clear solution components.
  • Figure 12 shows the state of the sample S after it has been moved from the first temperature region 46 through the connecting flow path 50 to the second temperature region 48. In the second temperature region 48, it is heated to approximately 60°C for 15 seconds. This causes further aggregation of the solid components of the sample S, and as shown in Figure 12, the separation of the colored solid components and the clear solution components becomes clear.
  • the solid components of the sample S change from a light brown, opaque color to a dark brown, opaque color.
  • the clear solution component is light brown, transparent.
  • FIG. 13 shows the state in which the sample S has been moved from the second temperature region 48 through the connecting flow path 50 to the first temperature region 46.
  • the first temperature region 46 it is heated to approximately 95°C for 15 seconds. This causes further aggregation of the solid components of the sample S, and further separation of the colored solid components (dark brown) from the clear solution components (light brown, clear).
  • the inventors have found that, as described above, the blood specimen S is separated into an opaque colored solid component and a transparent solution component by several reciprocating movements between the first temperature region 46 and the second temperature region 48 at the beginning of the PCR.
  • separation of blood components does not occur after the blood is denatured by heating, making it difficult to accurately measure the intensity of the fluorescent signal due to PCR amplification.
  • the sample S is separated into a colored solid component and a transparent solution component, making it possible to accurately measure the intensity of the fluorescent signal emitted from the transparent solution component, and therefore real-time PCR analysis can be suitably performed.
  • the phenomenon in which the sample separates into a solid component and a clear solution component in the flow channel 12 is presumably due to the sample being stirred and mixed as it moves through the flow channel 12.
  • the first temperature region 46 and the second temperature region 48 include a first serpentine flow channel 54 and a second serpentine flow channel 56 that combine a straight flow channel and a curved flow channel.
  • the microfluidic chip may be configured so that the cross-sectional area of one portion of the flow channel 12 is different from the cross-sectional area of the other portions. In this case, the unevenness of the portion where the cross-sectional area changes will further agitate and mix the sample, promoting the separation of the fixed components of the sample from the clear solution components. An example of this is shown below.
  • FIG. 14 is a plan view of the first surface 14a of another microfluidic chip 70 applicable to a PCR method according to an embodiment of the present invention.
  • the cross-sectional area of the detection channel 59 in the connection channel 50 is larger than the cross-sectional areas of the first serpentine channel 54 and the second serpentine channel 56.
  • the unevenness of the portions where the cross-sectional area of the channels changes causes the sample to be more agitated and mixed, facilitating the separation of the fixed component of the sample from the clear solution component.
  • the PCR reagent may contain a surfactant.
  • a surfactant When a surfactant is added to the PCR reagent, the sample can be mixed appropriately while flowing through the flow path 12.
  • the surfactant may be any of cationic surfactants, anionic surfactants, zwitterionic surfactants, and nonionic surfactants, and both high molecular weight and low molecular weight surfactants may be used.
  • surfactants include pure soap, alpha sulfo fatty acid ester salts, linear alkylbenzene sulfonates, alkyl sulfate ester salts, alkyl ether sulfate ester salts, alpha olefin sulfonates, alkyl sulfonates, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, fatty acid alkanolamides, alkyl glucosides, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, alkyl amino fatty acid salts, alkyl betaines, alkyl amine oxides, alkyl trimethyl ammonium salts, and dialkyl dimethyl ammonium salts, specifically, N,N-Bis (3-D-gluconamidopropyl) cholamide [BIGCHAP], N,N-B
  • the PCR reagent may contain an additive.
  • an additive When an additive is added to the PCR reagent, the sample can be mixed appropriately while flowing through the flow path 12. This is because the proteins in the blood are denatured and do not interfere with the PCR.
  • it is effective to add an appropriate amount of additives such as ammonium sulfate, bovine serum albumin, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), betaine, formamide, gelatin, glycerol, PEG6000, SDS, spermidine, Triton, Triton X-100, urea, etc.
  • alkaline solution examples include, but are not limited to, solutions of alkali metal hydroxides, such as sodium hydroxide solution and potassium hydroxide solution.
  • the alkaline concentration (OH-1 concentration) can be 0.1 mM or more, or 1 mM or more, preferably 10 mM or more, and more preferably 30 mM or more.
  • the pH can be 7 or higher, preferably 8 or higher, and more preferably 9 or higher.
  • the alkaline concentration and pH can be any concentration and pH (e.g., 10 M or lower) as long as they do not inhibit the subsequent detection or amplification step.
  • a reciprocal flow real-time PCR device and a general real-time PCR device using a PCR tube were used to prepare samples by directly adding blood to a PCR reagent, and real-time PCR measurements were performed.
  • PCR reagent was prepared by adding primers (0.9 ⁇ M each) described in SEQ ID NO: 1 and 2 for amplifying horse mackerel genes, a probe (0.4 ⁇ M) described in SEQ ID NO: 3, and 10 5 copies of template DNA described in SEQ ID NO: 4 so that the 2xPCR reagent of GoTaq (registered trademark) Enviro qPCR Systems (manufactured by Promega) was diluted 2-fold. 1 ⁇ L of whole blood or ddH 2 O was added thereto to prepare 20 ⁇ L of sample, which was used as a sample per reaction for each device. The sequences are shown below.
  • the real-time PCR device and PCR conditions are shown below.
  • the reciprocal flow type measurement chip and real-time PCR device correspond to the microfluidic chip 10 and PCR device 100 described above.
  • (Reciprocal flow real-time PCR device and PCR conditions) Measuring equipment: PicoGene PCR1100 (GoPhoton) Measurement chip: MCP2000 (GoPhoton) Measurement conditions: Set value on PCR1100 Hot Start 95°C, 15sec. Denaturation 95°C, 3.5sec. Annealing & Extension 60°C,15sec.
  • Cycle 50 (General real-time PCR device and PCR conditions) Measurement equipment: CFX96 Touch Deep Well (manufactured by Bio-Rad) Measurement tube: 8-tube series Measurement conditions: Actual measured value of CFX96 Touch Deep Well (same as PCR1100) Hot Start 95°C, 15sec. Denaturation 95°C, 3.5sec. Annealing & Extension 60°C,15sec. Cycle 50
  • a sample in which blood has been added to PCR reagent is sent alternately through the flow path 12 to a first temperature region 46 of 95°C and a second temperature region 48 of 60°C, stirring and mixing the sample while the blood coagulates, separating the solid components from the clear solution components, making it possible to measure the fluorescent signal of the clear solution components.
  • Figures 15(a) and 15(b) show amplification curves obtained when using a reciprocal flow real-time PCR device.
  • Figure 15(a) is a real-time amplification curve for a sample with added blood
  • Figure 15(b) is a real-time amplification curve for a sample without added blood.
  • similar amplification curves can be obtained for samples with and without added blood, making it very easy to determine the PCR amplification.
  • a typical real-time PCR device measurements were performed using a PCR tube.
  • the PCR reaction proceeds while the sample remains stationary in the PCR tube.
  • the sample In samples to which no blood was added, the sample was transparent both before and during PCR, and the fluorescent signal associated with PCR amplification could be adequately measured.
  • the sample In samples to which blood was added, the sample was red and transparent before PCR, but during PCR, the blood components browned due to heat denaturation at the beginning of PCR, and the fluorescent transmittance of the sample decreased. As a result, it became impossible to adequately measure the fluorescent signal associated with PCR amplification.
  • Figure 16 shows the amplification curves when a typical real-time PCR device is used. As shown in Figure 16, the amplification curve for a sample to which no blood has been added has a clear rise in the curve, making it possible to clearly determine PCR amplification. On the other hand, the amplification curve for a sample to which blood has been added has an unclear rise in the curve, making it very difficult to determine PCR amplification.
  • Second Example In the second embodiment, a reciprocal flow real-time PCR device and a general real-time PCR device using PCR tubes were used to prepare samples by directly adding blood to an RT-PCR reagent, and real-time RT-PCR measurements were performed.
  • Samples were prepared under the following conditions. A predetermined amount of reverse transcriptase included with GoTaq (registered trademark) Enviro RT-qPCR Systems (manufactured by Promega) was added, and 19 ⁇ L of RT-PCR reagent was prepared by adding primers (0.9 ⁇ M each) described in SEQ ID NO: 5 and 6 for amplifying the SFTS gene, a probe (0.3 ⁇ M) described in SEQ ID NO: 7, and 10 5 copies of template RNA described in SEQ ID NO: 8 so that the 2xPCR reagent was diluted 2-fold. 1 ⁇ L of whole blood or ddH 2 O was added thereto to prepare 20 ⁇ L of sample, which was used as a sample per reaction for each device.
  • GoTaq registered trademark
  • Enviro RT-qPCR Systems manufactured by Promega
  • the real-time PCR device and PCR conditions are shown below.
  • the reciprocal flow type measurement chip and real-time PCR device correspond to the microfluidic chip 10 and PCR device 100 described above.
  • (Reciprocal flow real-time PCR device and PCR conditions) Measuring equipment: PicoGene PCR1100 (GoPhoton) Measurement chip: MCP2000 (GoPhoton) Measurement conditions: Set value on PCR1100 RT 50°C,5min. Hot Start 95°C, 15sec. Denaturation 95°C, 3.5sec. Annealing & Extension 60°C,20sec.
  • Cycle 45 (General real-time PCR device and PCR conditions) Measurement equipment: CFX96 Touch Deep Well (manufactured by Bio-Rad) Measurement tube: 8-tube series Measurement conditions: Actual measured value of CFX96 Touch Deep Well (same as PCR1100) RT 50°C,5min. Hot Start 95°C, 15sec. Denaturation 95°C, 3.5sec. Annealing & Extension 60°C,20sec. Cycle 45
  • the sample in which blood is added to the RT-PCR reagent, undergoes a reverse transcription reaction at 50°C for 5 minutes in the flow path 12, and is then alternately sent to a first temperature region 46 at 95°C and a second temperature region 48 at 60°C.
  • a reverse transcription reaction at 50°C for 5 minutes in the flow path 12
  • a first temperature region 46 at 95°C and a second temperature region 48 at 60°C.
  • the sample is stirred and mixed, blood coagulation progresses, and the solid components are separated from the clear solution components, making it possible to measure the fluorescent signal of the clear solution components.
  • Figures 17(a) and 17(b) show amplification curves when using a reciprocal flow real-time PCR device.
  • Figure 17(a) is a real-time amplification curve for a sample with added blood
  • Figure 17(b) is a real-time amplification curve for a sample without added blood.
  • similar amplification curves can be obtained for samples with and without added blood, making it very easy to determine the PCR amplification.
  • the RT-PCR reaction proceeds while the sample remains stationary within the PCR probe.
  • the sample In samples to which no blood was added, the sample was transparent both before and during PCR, and the fluorescent signal associated with PCR amplification could be adequately measured.
  • the sample In samples to which blood was added, the sample was red and transparent before PCR, but during PCR, the blood components browned due to heat denaturation at the beginning of PCR, reducing the fluorescent transmittance of the sample. As a result, it became impossible to adequately measure the fluorescent signal associated with RT-PCR amplification.
  • Figure 18 shows the amplification curves when a typical real-time PCR device is used. As shown in Figure 18, the amplification curve for a sample to which no blood has been added has a clear rise in the curve, making it possible to clearly determine PCR amplification. On the other hand, the amplification curve for a sample to which blood has been added has an unclear rise in the curve, making it very difficult to determine PCR amplification.
  • the PCR method according to this embodiment can suitably measure the fluorescent signal associated with PCR amplification for samples in which blood has been directly added to the PCR reagent, and can appropriately perform real-time PCR analysis. Since the PCR method according to this embodiment uses samples in which blood has been directly added to the PCR reagent, there is no need for cumbersome nucleic acid purification procedures, etc. This makes it possible to perform rapid real-time PCR without any hassle.
  • the present invention can be used for PCR.
  • SEQ ID NO: 1 primer SEQ ID NO: 2: primer SEQ ID NO: 3: probe SEQ ID NO: 4: template DNA SEQ ID NO: 5: primer SEQ ID NO: 6: primer SEQ ID NO: 7: probe SEQ ID NO: 8: template RNA

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Abstract

Procédé de PCR comportant les étapes suivantes : (S10) ajout de sang à un réactif de PCR pour préparer un échantillon ; (14) mouvement alternatif répété de l'échantillon dans une puce microfluidique pour appliquer un cycle thermique à l'échantillon ; et (S16) détection de la fluorescence de l'échantillon dans la voie d'écoulement pour suivre la progression de la PCR sur l'échantillon.
PCT/JP2023/031944 2023-08-04 2023-08-31 Procédé de pcr Pending WO2025032840A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010503413A (ja) * 2006-09-14 2010-02-04 ディエヌエイ・ポリメラーゼ・テクノロジー・インコーポレイテッド Ｐｃｒ阻害物質の存在下におけるｄｎａ増幅のためのｔａｑポリメラーゼ変異体酵素の使用
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
JP2017108736A (ja) * 2015-12-11 2017-06-22 東洋紡株式会社 核酸増幅用組成物及び核酸増幅法
JP2018507694A (ja) * 2015-03-05 2018-03-22 ビージー リサーチ エルティーディーＢｇ Ｒｅｓｅａｒｃｈ Ｌｔｄ 血液を含むサンプルからの直接の核酸標的の多重検出
CN111073795A (zh) * 2020-01-10 2020-04-28 苏州新海生物科技股份有限公司 一种用于全血荧光定量pcr的反应容器及方法
JP2021532770A (ja) * 2018-07-31 2021-12-02 ビージー リサーチ エルティーディーBg Research Ltd 微生物およびウイルス粒子を検出するための方法および組成物

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010503413A (ja) * 2006-09-14 2010-02-04 ディエヌエイ・ポリメラーゼ・テクノロジー・インコーポレイテッド Ｐｃｒ阻害物質の存在下におけるｄｎａ増幅のためのｔａｑポリメラーゼ変異体酵素の使用
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
JP2018507694A (ja) * 2015-03-05 2018-03-22 ビージー リサーチ エルティーディーＢｇ Ｒｅｓｅａｒｃｈ Ｌｔｄ 血液を含むサンプルからの直接の核酸標的の多重検出
JP2017108736A (ja) * 2015-12-11 2017-06-22 東洋紡株式会社 核酸増幅用組成物及び核酸増幅法
JP2021532770A (ja) * 2018-07-31 2021-12-02 ビージー リサーチ エルティーディーBg Research Ltd 微生物およびウイルス粒子を検出するための方法および組成物
CN111073795A (zh) * 2020-01-10 2020-04-28 苏州新海生物科技股份有限公司 一种用于全血荧光定量pcr的反应容器及方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MANAGE, D. P. ET AL.: "On-chip PCR amplification of genomic and viral templates in unprocessed whole blood", MICROFLUIDICS AND NANOFLUIDICS, vol. 10, 2011, pages 697 - 702, XP019883093, DOI: 10.1007/s10404-010-0702-4 *

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