PCR reaction container

Abstract

To provide a PCR reaction container free from problems associated with residual sample droplets.SOLUTION: A PCR reaction container includes a substrate, a flow channel formed in the substrate, a pair of filters provided at both ends of the flow channel, a pair of air communication ports communicating with the flow channel through the filters, a thermal cycle region formed between the pair of filters in the flow channel, and a sample injection port through which a sample can be injected into the flow channel from above, wherein the sample injection port in the surface of the substrate has an area of 0.7 to 1.8 mm2.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a PCR reaction vessel used in a polymerase chain reaction (PCR), a PCR device using the PCR reaction vessel, and a PCR method. [Background technology]

[0002] General-purpose PCR and real-time PCR thermal cyclers take a long time to change temperature due to their huge heat capacity, and PCR reactions take 1 to 2 hours, but the present inventors have already developed a method to speed up the thermal cycle by repeatedly sending liquid to multiple temperature zones using a microfluidic chip (Patent Document 1). Furthermore, the present inventors have proposed a mechanism that does not require metering and prevents liquid leakage by using a structure that combines branched flow paths along the plane that constitutes the PCR reaction vessel as the sample introduction section (Patent Document 2).

[0003] In the method proposed in Patent Document 2, some residual sample droplets remain in the branched flow path when the sample is introduced, and during the subsequent thermal cycle of back-and-forth liquid transport, there is a possibility that these residual droplets may accidentally enter the main flow path and interfere with the transport.

[0004] Patent document 3 discloses a method in which the temperature of the dispensing area is raised above room temperature using a heater, the sample is then moved to a thermal cycle area, and then the temperature of the dispensing area is lowered to cool and contract the air, thereby pulling back the remaining droplets in the dispensing area from the main flow path. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6226284 [Patent Document 2] WO2017 / 094674 [Patent Document 3] Patent Publication 2018-19606 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a PCR reaction vessel in which no problems occur in the transfer of liquid through the main flow path during thermal cycling, even if there are residual droplets of sample. [Means for solving the problem]

[0007] The present invention provides the following PCR reaction vessel. [1] A substrate; A flow path formed in the substrate; A pair of filters provided at both ends of the flow path; A pair of air communication ports communicating with the flow path through the filter; a thermal cycle region formed between the pair of filters in the flow path; A sample injection port is provided for injecting a sample into the flow channel from above, The surface area of ​​the substrate at the sample injection port is 0.7 to 1.8 mm 2 characterized in that PCR reaction vessel. [2] The reaction vessel according to claim 1, wherein the sample injection port is circular, elliptical or polygonal. [3] The reaction vessel according to [1] or [2], wherein the width of the flow path is 300 to 1000 μm. [4] The reaction vessel according to any one of [1] to [3], characterized in that the tip of a sample injection member having a circular or polygonal tubular shape used separately for sample injection reaches the inside of the flow channel. [5] The reaction vessel according to any one of [1] to [4], further comprising a sample injection port having a volume (a space from the surface of the substrate to the flow channel) of 7.5 μL or less. [6] The reaction vessel according to any one of [1] to [5], wherein after sample injection, the upper opening of the sample injection port is sealed by a seal or the sample injection member, etc. [7] The reaction vessel according to any one of [1] to [6], which has a thickness of 3 to 5 mm. Effect of the Invention

[0008] In the present invention, a sample injection port is provided on the flow channel without using a branch flow channel as in the prior art. When a sample is injected through a branch flow channel, the sample remains in the branch flow channel, and the remaining sample enters the main flow channel during thermal cycling, which is a problem. However, when the sample injection port is provided on the flow channel, the sample remaining in the space above the flow channel at the sample injection port is retained in the space during thermal cycling, so there is no need to use a heater as described in Reference 3. [Brief description of the drawings]

[0009] [Figure 1] 1(a) and (b) are diagrams illustrating a PCR reaction vessel according to a first embodiment of the present invention. [Diagram 2] 2 is a cross-sectional view of the PCR reaction vessel shown in FIG. 1(a) taken along line AA. [Diagram 3] FIG. 2 is a cross-sectional view showing a sample injection port. [Figure 4] The results of rapid PCR using E. coli uidA are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The following describes a PCR reaction vessel and a PCR device according to an embodiment of the present invention. The same or equivalent components, members, and processes shown in each drawing are given the same reference numerals, and duplicated descriptions are omitted as appropriate. In addition, the embodiments do not limit the invention but are merely examples, and all the features and combinations thereof described in the embodiments are not necessarily essential to the invention. The PCR reaction vessel of the present invention can be used as a chip for amplifying nucleic acids.

[0011] Figures 1(a) and 1(b) are diagrams for explaining a PCR reaction vessel 10 according to a first embodiment of the present invention. Figure 1(a) is a plan view of the PCR reaction vessel 10, and Figure 1(b) is a front view of the PCR reaction vessel 10. Figure 2 is a cross-sectional view of the PCR reaction vessel shown in Figure 1(a) taken along line AA. Figure 3 is a diagram showing a state in which a disposable tip of a pipette is inserted into the sample injection port.

[0012] The PCR reaction vessel 10 consists of a resin substrate 14 having a groove-shaped flow path 12 formed on its bottom surface 14a, a flow path sealing film 16 for sealing the flow path 12 attached to the bottom surface 14a of the substrate 14, and three sealing films (a first sealing film 18, a second sealing film 20 and a third sealing film 22) attached to the top surface 14b of the substrate 14.

[0013] The substrate 14 is preferably made of a material that has good thermal conductivity, is stable against temperature changes, and is not easily affected by the sample solution used. Furthermore, the substrate 14 is preferably made of a material that has good moldability, good transparency and barrier properties, and low autofluorescence. Such materials include inorganic materials such as glass and silicon, as well as resins such as acrylic, polyester, and silicone, and among these, cycloolefin is preferable. An example of the dimensions of the substrate 14 is a long side of 70 mm, a short side of 42 mm, and a thickness of 3 mm. An example of the dimensions of the flow channel 12 formed on the lower surface 14a of the substrate 14 is a width of 0.5 mm and a depth of 0.5 mm.

[0014] A groove-shaped flow path 12 is formed on the lower surface 14a of the substrate 14, and this flow path 12 is sealed by a flow path sealing film 16 (see FIG. 2). A first air communication port 24 is formed at the position of one end 12a of the flow path 12 in the substrate 14. A second air communication port 26 is formed at the position of the other end 12b of the flow path 12 in the substrate 14. The pair of first air communication port 24 and second air communication port 26 are formed so as to be exposed on the upper surface 14b of the substrate 14. Such a substrate can be manufactured by injection molding or cutting processing using an NC processing machine or the like. The width of the flow path is preferably 300 to 1000 μm. The depth of the flow path is preferably 300 to 1000 μm.

[0015] A first filter 28 is provided between the first air communication port 24 in the substrate 14 and one end 12a of the flow path 12 (see FIG. 2). A second filter 30 is provided between the second air communication port 26 in the substrate 14 and the other end 12b of the flow path 12. The pair of first filter 28 and second filter 30 provided at both ends of the flow path 12 have good low impurity characteristics, and also allow only air to pass through, preventing contamination so that the quality of DNA amplified by PCR is not deteriorated. As the filter material, polyethylene, PTFE, etc. are suitable, and may be porous or hydrophobic. The dimensions of the first filter 28 and the second filter 30 are formed to fit snugly into the filter installation space formed in the substrate 14.

[0016] The substrate 14 is provided with a sample injection port 133 between the first filter 28 and the thermal cycle region 12e or between the second filter 30 and the thermal cycle region 12e. The sample injection port 133 is formed so as to be exposed on the upper surface 14b of the substrate 14.

[0017] The portion of the flow channel 12 between the first filter 28 and the second filter 30 forms a thermal cycle region 12e, which is scheduled to have a high temperature region and a medium temperature region, in order to thermally cycle the sample. The thermal cycle region 12e of the flow channel 12 includes a serpentine flow channel. This is to efficiently provide the heat provided from the PCR device to the sample in the PCR process, and to ensure that the volume of the sample that can be used for PCR is a certain amount or more (for example, 25 μL or more). The PCR reaction vessel 10 is intended to be placed in a PCR device, to thermally cycle the sample, and to measure optical properties such as fluorescence emitted from the sample. Therefore, the arrangement of each element, including the flow channel and branching points, may be arbitrarily selected, taking into consideration the arrangement of a temperature control unit and a fluorescence detection probe, which will be described later.

[0018] In the PCR reaction vessel 10 according to the first embodiment, most of the flow channel 12 is formed in a groove shape exposed on the lower surface 14a of the substrate 14. This is to facilitate molding by injection molding using a mold or the like. In order to utilize this groove as a flow channel, a flow channel sealing film 16 is attached to the lower surface 14a of the substrate 14. The flow channel sealing film 16 may have adhesiveness on one main surface, or a functional layer that exerts adhesiveness or adhesion by pressing may be formed on one main surface, and has a function of easily adhering to and integrating with the lower surface 14a of the substrate 14. It is desirable that the flow channel sealing film 16 is formed from a material having low autofluorescence, including the adhesive. In this respect, a transparent film made of a resin such as a cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic is suitable, but is not limited to these. The flow channel sealing film 16 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 PCR reaction vessel 10.

[0019] In the PCR reaction container 10 according to the first embodiment, the first air communication port 24, the second air communication port 26, the first filter 28, the second filter 30, and the sample injection port 133 are exposed on the upper surface 14b of the substrate 14. Therefore, the first sealing film 18 is attached to the upper surface 14b of the substrate 14 to seal the first air communication port 24 and the first filter 28. In addition, the second sealing film 20 is attached to the upper surface 14b of the substrate 14 to seal the second air communication port 26 and the second filter 30. In addition, the third sealing film 22 is attached to the upper surface 14b of the substrate 14 to seal the sample injection port 133.

[0020] The first sealing film 18 has a size capable of simultaneously sealing the first air communication port 24 and the first filter 28, and the second sealing film 20 has a size capable of simultaneously sealing the second air communication port 26 and the second filter 30. A pressurizing pump (described later) is connected to the first air communication port 24 and the second air communication port 26 by perforating the first air communication port 24 and the second air communication port 26 with a hollow needle (a syringe needle with a sharp tip) provided at the tip of the pump. For this reason, the first sealing film 18 and the second sealing film 20 are preferably films made of a material and thickness that can be easily perforated by a needle. In the first embodiment, a sealing film of a size capable of simultaneously sealing the corresponding air communication port and the filter has been described, but a mode in which these are sealed separately is also possible. Also, a sealing film capable of sealing the first air communication port 24, the first filter 28, the second air communication port 26, and the second filter 30 together (as one piece) may be used.

[0021] The third sealing film 22 is of a size capable of sealing the sample injection port 133. The injection of the sample into the flow channel 12 through the sample injection port 133 is performed by once peeling the third sealing film 22 from the substrate 14, and after a predetermined amount of sample is injected, the third sealing film 22 is returned and attached to the upper surface 14b of the substrate 14 again. For this reason, the third sealing film 22 is preferably a film having an adhesiveness that can withstand several cycles of attachment / peel-off. The third sealing film 22 may be of a type in which a new film is attached after the sample is injected, in which case the importance of the characteristics relating to attachment / peel-off may be alleviated.

[0022] In addition, when injecting a sample, either the first sealing film 18 or the second sealing film 20 must be peeled off once, similar to the third sealing film 22. This is because the sample will not enter the flow path unless an air outlet is created. For this reason, the first sealing film 18 and the second sealing film 20 are desirably films having adhesiveness that can withstand several cycles of attachment / detachment. Also, a new film may be attached after the sample is injected. It is also possible to provide an air outlet in addition to the air communication ports 24 and 26, and to inject a sample into the flow channel by attaching / removing the fourth sealing film.

[0023] The first sealing film 18, the second sealing film 20, and the third sealing film 22 may have an adhesive layer formed on one main surface, or a functional layer that exerts adhesiveness or adhesion by pressing, similar to the flow path sealing film 16. The first sealing film 18, the second sealing film 20, and the third sealing film 22 are preferably formed from a material having low autofluorescence, including the adhesive. In this respect, a transparent film made of a resin such as cycloolefin, polyester, polypropylene, polyethylene, or acrylic is suitable, but is not limited to these. As described above, it is desirable that the adhesiveness and other properties do not deteriorate to a degree that affects use even after multiple attachment / detachment, but in the case of an embodiment in which a new film is attached after peeling and injection of a sample, the importance of the properties related to attachment / detachment may be mitigated.

[0024] Next, a method of using the PCR reaction vessel 10 configured as above will be described. First, a sample to be amplified by thermal cycling is prepared. The sample may be a mixture containing two or more types of DNA, to which a plurality of types of primers, a heat-resistant enzyme, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) are added as PCR reagents. Next, the first sealing film 18 and the third sealing film 22 are peeled off from the substrate 14, and the first air communication port 24 and the sample injection port 133 are opened. If the first sealing film 18 is of a size that can simultaneously seal the first air communication port 24 and the first filter 28, the first sealing film 18 may be completely peeled off from the substrate 14 to expose the first air communication port 24 and the first filter 28 to the atmosphere, but by opening only the first air communication port 24 without completely peeling off the first sealing film 18 from the substrate 14, the first filter 28 is not exposed to the atmosphere, which is effective in preventing contamination. Similarly, when a sealing film capable of sealing the first air communication port 24 and the first filter 28 separately is used, the first filter 28 is not exposed to the atmosphere, which is effective in preventing contamination.

[0025] Next, the sample is injected into the sample injection port 133 from a thin, conical disposable tip (sample injection member) attached to the tip of the micropipette. A certain amount of sample can be injected into the flow path 12 from the disposable tip by using the micropipette. The micropipette can discharge a certain amount of sample by pressing the push button down to the first stop. All sample remaining in the disposable tip can be discharged by pressing the push button once stopped at the first stop further down to the second stop. Since the disposable tip is thin and long, it is inserted directly downward from the top of the sample injection port 133 toward the flow path 12, but is fixed by abutting against the top of the sample injection port at any position on the pipette attachment side of the tip, and the sample is injected from there. If the diameter of the top of the sample injection port is too large, the tip of the disposable pipette will reach the flow path, and if a liquid sample is injected in this state, the sample will overflow without entering the flow path, which is not preferable. Also, if the diameter of the top of the sample injection port is too small, the tip of the disposable tip is only slightly inserted into the sample injection port, and in this state the sample will overflow from the injection port. Therefore, there is an optimum range for the size of the sample injection port. The preferred size of the sample injection port is about 1 to 1.5 mm in diameter when the injection port is cylindrical.

[0026] When a sample is injected from a disposable pipette attached to the tip of a micropipette through a sample inlet of appropriate diameter, by pressing the push button firmly down to the second stop, all of the sample in the disposable tip can be expelled and pushed into the flow channel.

[0027] On the other hand, if the push button of the micropipette is only pressed down to the first stop, there is a risk that liquid sample will remain in the space of sample injection port 133 on flow channel 12. It is conceivable that the liquid sample in the space of sample injection port 133 will flow into flow channel 12 due to gravity during the thermal cycle, but in reality, the amount of liquid sample in the space of sample injection port 133 is the same before and after the thermal cycle, and the liquid sample in this space does not adversely affect PCR.

[0028] Therefore, the reaction vessel of the present invention makes it possible to carry out PCR regardless of the injection method used by the user. In order to carry out PCR without adverse effects, the area of ​​the sample injection port 133 (the area of ​​the opening on the surface of the substrate) should be within the range of 0.7 to 1.8 mm 2 and more preferably 0.9 to 1.7 mm. 2 and particularly preferably 1.3 to 1.6 mm 2 The upper limit of the area of ​​the sample injection port 133 is 1.8 mm 2 Less than 1.7mm is preferable 2 Less than 1.6mm is more preferable. 2 Less than 1.5mm is preferable. 2 Less than 1.4mm is more preferable. 2 The lower limit of the area of ​​the sample injection port 133 is preferably 0.7 mm or less. 2 More than 0.9mm is preferable. 2 More preferably, 1.0 mm or more 2 More preferably, 1.3 mm or more 2 The above is even more preferable.

[0029] Moreover, the volume of the sample injection port (the space from the surface of the substrate to the channel) is preferably 7.5 μL or less, and more preferably 3 to 7.5 μL.

[0030] The shape of the sample injection port is not particularly limited, but is preferably a tubular shape such as a circle, an ellipse, or a polygon, and is particularly preferably a circle.

[0031] Next, the first sealing film 18 and the third sealing film 22 are attached back to the substrate 14, and the first air communication port 24 and the sample injection port 133 are sealed. As described above, new first sealing film 18 and third sealing film 22 may be attached. This completes the injection of the sample 70 into the PCR reaction vessel 10. After the sample is injected, a predetermined number of PCR thermal cycles are performed according to a conventional method, and the amplified DNA can be detected by fluorescence or the like. EXAMPLES

[0032] The present invention will be described below based on examples, but the present invention is not limited to these examples. Example 1 1. The PCR device used was a reciprocating PCR reaction vessel (4 mm thick) with a single flow path to transport PCR reagents alternately between two temperature zones, enabling high-speed thermal cycling. 2. A through hole with a diameter of 0.9 to 1.6 mm was drilled from the top surface of the resin substrate of the PCR reaction vessel so as to be perpendicular to the central axis of the channel formed in the substrate, and a reagent injection port was created. After removing excess burrs and dirt, all sealing films, including the channel sealing film, were bonded, and the PCR verification was performed. 3. PCR reagents were prepared as follows:

[0033] [Table 1]

[0034] 4. Using a micropipette equipped with a disposable pipette tip (Molecular BioProducts ART 100E (100 μL)), 20 μl of the prepared PCR reagent was aspirated, and with the tip of the pipette tip inserted into the reagent inlet, the entire aspirated amount of PCR reagent was injected into the flow path of the PCR reaction vessel. 5. When PCR reagent is discharged from a micropipette, the discharged solution is usually cut off at the tip of the disposable pipette tip. Therefore, depending on the diameter of the reagent inlet, it is possible that the tip of the pipette tip does not reach the inside of the flow channel completely and stops inside the reagent inlet. In this case, the rear end of the PCR reagent that has been injected into the flow channel and formed a plug-like shape will remain inside the reagent inlet, and some of the PCR reagent will remain in the reagent inlet when the liquid is sent in the subsequent PCR. 6. On the other hand, when injecting this PCR reagent, by pushing the micropipette in excessively, after discharging all of the aspirated PCR reagent, it is possible to continue pushing the air into the flow path, and inject the PCR reagent completely into the flow path, including the rear end of the plug. In this way, the condition in which the entire amount of PCR reagent is pushed completely into the flow path by the micropipette is expressed as "with reagent pushing," while the case in which the PCR reagent is not pushed into the flow path by general micropipette operation will hereafter be expressed as "without reagent pushing." 7. The PCR reaction vessels, which were filled with PCR reagents and sealed with a sealing film, were attached to a device equipped with a temperature range of 98°C and 61°C, a pump for reciprocating liquid, and a fluorescence detector for quantifying the amplified DNA in the flow path, and real-time PCR was performed. The PCR conditions were as follows.

[0035]

number

[0036] Results and Discussion: 1. Table 2 summarizes the positions that the end of the pipette tip reaches when it is inserted into the sample injection port.

[0037] [Table 2]

[0038] 2. Table 3 summarizes the liquid height at the rear end of the PCR reagent plug in the reagent inlet before PCR in the pattern where the reagent was not pushed in.

[0039] [Table 3]

[0040] 3. For the 0.9 mm diameter sample, the pipette tip could not be inserted into the sample injection port, making it impossible to inject PCR reagents. On the other hand, for the 1.6 mm diameter sample, the diameter of the sample injection port was wider than the tip of the pipette tip, so PCR reagents overflowed from the top of the sample injection port, making it impossible to inject. 4. As described above, if the pipette tip does not fit into the reagent injection port, or if the hole diameter is larger than the tip of the pipette tip, injection is not possible and the PCR reagent overflows. 5. When the diameter of the reagent injection port was 1.5 mm, the tip of the pipette tip reached the inside of the flow channel, but when the reagent was not pushed in, the rear end of the PCR reagent plug entered the reagent injection port. This is thought to be because the pipette tip was pulled back when it was pulled out after the reagent was injected. 6. Next, the amplification curves obtained by performing real-time PCR are shown in FIG. 7. The difference of about 2 cycles in Ct value was within the range of uncertainty due to the measurement device, and no significant difference was confirmed. 8. Based on the above results, Table 4 summarizes the evaluation of whether reagent injection and PCR are possible for each size of drill used to form the reagent injection port.

[0041] [Table 4]

[0042] 9. When PCR reagents were pushed into a 1.5 mm diameter reagent injection port using a pipette, the pressure of the micropipette caused leakage from the entrance of the sample injection port, and sample injection could not be performed normally. 10. However, under all conditions where the PCR reagents were injected normally, there was no effect on PCR liquid flow, and real-time PCR was possible as shown in Figure 4. 11. The amount of liquid remaining in the sample inlet after PCR is summarized in Table 5. There was almost no change in the amount of reagent remaining in the reagent inlet before and after PCR.

[0043] [Table 5]

[0044] 12. As described above, when injecting the solution directly through the sample injection port without providing a branch flow path as in this embodiment, rather than injecting the sample through a branch flow path, it was predicted that if some solution remained in the reagent injection port located at the top of the flow path, it would leak out due to gravity during the liquid transfer, blocking the flow path and preventing normal back-and-forth liquid transfer. However, it was confirmed that normal PCR liquid transfer was possible without any leakage from the sample injection port. [Explanation of symbols]

[0045] 10 PCR reaction vessels 12 Flow Path 14 Substrate 16 Flow path sealing film 18 First sealing film 20 Second sealing film 22 Third sealing film 24 First air communication port 26 Second air vent 28 First Filter 30 Second Filter 133 Sample injection port [Industrial Applicability]

[0046] The PCR device achieved by the present invention realizes rapid testing and is useful as a first response device for pandemics such as highly pathogenic influenza. In addition, it can be applied to genetic testing technology for tailored medical treatment based on genetic information, and in clinical settings, it can quickly determine the effectiveness of treatment by quantitative PCR, so it has a high market advantage, especially in the medical field.

Claims

[Claim 1] A substrate; A flow path formed in the substrate; A pair of filters provided at both ends of the flow path; A pair of air communication ports communicating with the flow path through the filter; a thermal cycle region formed between the pair of filters in the flow path; A sample injection port is provided for injecting a sample into the flow channel from above, The surface area of ​​the substrate at the sample injection port is 0.7 to 1.8 mm 2 characterized in that PCR reaction vessel.

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