WO2022168150A1

WO2022168150A1 - Channel device and nucleic acid amplification reactor

Info

Publication number: WO2022168150A1
Application number: PCT/JP2021/003702
Authority: WO
Inventors: 祐美子合志; 友幸坂井
Original assignee: 株式会社日立ハイテク
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2022-08-11

Abstract

This channel device comprises a sample introduction port for introducing a sample, a water-absorbing portion provided around the sample introduction port, a non-water-absorbing portion between the sample introduction port and the water-absorbing portion, a nucleic acid amplification reaction portion, and a channel for leading the sample introduced from the sample introduction port to the nucleic acid amplification reaction portion. This nucleic acid amplification reactor comprises a channel device, a temperature control mechanism for forming a temperature zone used in a nucleic acid amplification reaction, and a liquid feed control mechanism for moving a sample within the channel device.

Description

Flow path device and nucleic acid amplification reactor

The present invention relates to a channel device and a nucleic acid amplification reactor.

Methods for amplifying nucleic acids include isothermal amplification methods such as the PCR (Polymerase Chain Reaction) method and the LAMP (Loop-Mediated Isothermal Amplification) method. The PCR method consists of DNA denaturation (dissociation of double-stranded DNA into single-stranded DNA), annealing (complementary primer binding to single-stranded DNA), and elongation (double-stranded DNA replication by complementary strand synthesis). is repeated at different temperatures to amplify the target DNA. Typically, heat denaturation is performed at about 95°C, annealing at about 60-65°C, and extension at about 72°C. In isothermal amplification methods, the reactions corresponding to these cycles are carried out at a constant temperature of the order of 35-65°C.

In recent years, rapid genetic testing systems using nucleic acid amplification/detection equipment and disposable dedicated devices have been proposed. Nucleic acid amplification/detection mechanisms and dedicated devices have various forms, but a typical test flow includes the step of collecting a specimen, the step of introducing a solution (sample) from which the nucleic acid of a pathogen is extracted into a dedicated device, and the step of nucleic acid amplification. It consists of a step of amplifying the target gene in a reactor and a step of detecting the amplified product. A microchannel (microchannel chip) formed on a substrate has been proposed as a dedicated device for nucleic acid amplification reaction. By moving the sample inside the microfluidic chip installed on heat blocks set to different temperature zones, each heat block can be maintained at a preset temperature to achieve the temperature changes necessary for PCR. can.

JP 2018-20283 A JP 2019-198335 A JP 2004-358635 A

With conventional technology, there was the problem that it was difficult to precisely control the amount of sample liquid to be introduced into the device for nucleic acid amplification reaction.

The success or failure of the nucleic acid amplification reaction is greatly affected by the concentration of the reagent composition. It is also beneficial to reduce sample temperature variations. For this reason, it is extremely important to precisely control and maintain a constant amount of the sample liquid introduced into the device for nucleic acid amplification reaction.

This can usually be achieved in environments where skilled operators are allowed to use precision weighing instruments such as micropipettes. However, in order to make a testing system that can be introduced into many medical sites including clinics, it is necessary that non-experts can achieve the above with simple operations without using special weighing instruments.

Patent Document 1 describes a method of aspirating a container containing a sample with a special pipette provided with a structure for releasing liquid that has been aspirated in excess of a target volume and introducing it into a device. On the other hand, biological specimens such as nasal secretions and blood contain numerous nucleic acid amplification reaction inhibitors such as mucin and blood components. step is often required.

Filter filtration of the sample is a simple way to remove reaction inhibitors. Since this process requires a pressure of about several kPa, it is impossible to obtain sufficient pressure by manual suction operation, and a dedicated filtering device is required. In addition, when passing through the filter, liquid residue tends to remain on the filter, and the entire amount of the introduced liquid cannot be recovered at a later stage, so even if it is weighed before passing through the filter, the desired amount cannot be introduced into the device. Therefore, in this method, after the sample is filtered, it is necessary to transfer the sample to a separate device for weighing and introduce it into the chip, which complicates the operator's operation multiple times.

In Patent Document 2, as a method of dispensing a predetermined amount of sample in a microchannel chip, two sample introduction ports are opened on the chip, and the channel volume of the dispensing channel provided between them is measured. A structure is described that defines a sample volume that In this method, the sample can be introduced by connecting a pipette or dropper to the sample inlet and pressurizing it. Need to check carefully. In addition, because of the structure in which the surplus sample is discharged to one of the sample inlets, contamination of the surplus sample may contaminate the apparatus.

In addition, Patent Document 3 describes a microvalve used for controlling a trace amount of fluid sample.

As described above, current genetic tests based on nucleic acid amplification reactions are difficult to precisely control the amount of sample liquid introduced into nucleic acid amplification reaction devices, which limits the human and time resources of clinics. It is not an acceptable form under the environment where it is used.

The present invention has been made to solve such problems, and provides a channel device and a nucleic acid amplification reaction apparatus that can more precisely control the amount of sample liquid to be introduced into the device for nucleic acid amplification reaction. With the goal.

An example of the flow path device according to the present invention is
a sample introduction port for introducing a sample;
a water absorbing portion provided around the sample inlet;
a non-water-absorbing region between the sample inlet and the water-absorbing portion;
a nucleic acid amplification reaction section;
a channel for guiding the sample introduced from the sample introduction port to the nucleic acid amplification reaction section;
Prepare.

An example of the nucleic acid amplification reaction device according to the present invention is
A nucleic acid amplification reaction apparatus comprising the flow channel device described above,
a temperature control mechanism that forms a temperature zone used for nucleic acid amplification reaction;
a liquid transfer control mechanism for moving the sample in the channel device;
Prepare.

According to the flow path device and the nucleic acid amplification reaction apparatus according to the present invention, it is possible to more precisely control the amount of sample liquid to be introduced into the device for nucleic acid amplification reaction.

For this reason, for example, it is possible to provide a nucleic acid amplification reaction apparatus that enables accurate sample weighing with simple operations and that can be stably operated.

Problems, configurations and effects other than the above will be clarified by the following description of the embodiments.

Device configuration diagram of Example 1 Device sectional view of Example 1 Configuration diagram of device 101 for nucleic acid amplification reaction Sectional view of device 101 for nucleic acid amplification reaction Enlarged view around the sample inlet 106 Cross-sectional view around the sample inlet 106 Work flow of Example 1 Work flow of Example 1 in endpoint nucleic acid detection system External view of nucleic acid amplification reaction device 201 according to Example 2 Device configuration diagram of a nucleic acid amplification reaction device 201 according to a modification of Example 2 Sectional view of device 101 for nucleic acid amplification reaction according to Example 3 A configuration diagram of a device 101 for nucleic acid amplification reaction according to Example 4. Configuration diagram of device 101 for nucleic acid amplification reaction according to Example 5 Sectional view for explaining the sample weighing mechanism used in Example 6 Sectional view for explaining an undesirable reference example for weighing a sample in Example 6 Cross-sectional view around the sample introduction port 106 according to the modification of the sixth embodiment Cross-sectional view around the sample introduction port 106 of the embodiment shown in Example 7 Graph showing the relationship between the water absorption pad distance and the amount of dripping liquid in Examples 1 to 7 Graph showing the relationship between water absorption pad distance and residual liquid amount in Examples 1 to 7 Graph showing the relationship between the amount of dripping liquid and the amount of residual liquid in Examples 1 to 7

Embodiments of the present invention will be described below with reference to the drawings.

The nucleic acid amplification reaction device 201 related to Example 1 of the present invention will be described below. FIG. 1 shows the device configuration of the nucleic acid amplification reaction device 201 . 2 shows a cross-sectional view of the nucleic acid amplification reaction device 201. As shown in FIG. FIG. 2 is a cross-sectional view taken along line AA of FIG.

The nucleic acid amplification reaction apparatus 201 includes a nucleic acid amplification reaction device 101 and a device holding section 202 . The nucleic acid amplification reaction device 101 is placed in the device holding section 202 . The nucleic acid amplification reaction apparatus 201 includes a device holding section 202 made of PEEK (polyetheretherketone), a cover 203 made of PC (polycarbonate), and a liquid delivery device 213 . Device holder 202 includes pump connection 204 and

external heat sources

206 and 207 . The cover 203 has a role of preventing the nucleic acid amplification reaction device 101 placed on the device holding portion 202 from floating and fixing it at a predetermined position.

A material other than PEEK can be used as the material of the device holding portion 202, but a material with a heat resistance temperature of 100°C or higher and a low thermal conductivity is preferable. It is preferable that the material of the cover 203 has a heat resistance temperature of about 70° C. and a low thermal conductivity.

The

external heat sources

206 and 207 each contain a thermocouple and a heater. The nucleic acid amplification reaction device 201 has a temperature controller 208 . A temperature controller 208 controls the

external heat sources

206 and 207 to keep the surface at a predetermined temperature. Thus, temperature controller 208 and

external heat sources

206 and 207 function as a temperature control mechanism that forms a temperature zone used for nucleic acid amplification reaction.

In this example, in order to speed up PCR, a method called 2-step PCR was adopted in which denaturation, annealing, and elongation were repeated in two temperature ranges. Using nickel-plated copper

external heat sources

206, 207, the target temperature of the first temperature zone is the annealing and extension temperature zone (eg, 68 ° C.), and the target temperature of the second temperature zone is the denaturation temperature zone (eg, 98 ° C.). ). The first temperature range may be the denaturation temperature range and the second temperature range may be the annealing and extension temperature range.

The liquid delivery device 213 incorporates a syringe pump 205 and functions as a liquid delivery control mechanism for moving the sample in the nucleic acid amplification reaction device 101 . The operation of the syringe pump 205 moves the sample within the channel. A diaphragm pump, a microblower, or the like may be used for the liquid delivery device 213 . The liquid delivery device 213 and the pump connection section 204 are connected by a tube 210 .

The fluorescence detection unit 209 is composed of an objective lens (not shown), a dichroic mirror 215, and a photodetector 216, and is connected to the excitation light irradiation unit 214 and the signal detection circuit 211.

Next, the nucleic acid amplification reaction device 101 related to Example 1 will be described. FIG. 3 shows a plan view of the nucleic acid amplification reaction device 101, and FIG. 4 shows a cross-sectional view taken along line AA of FIG. The nucleic acid amplification reaction device 101 includes a sample introduction port 106 , a sample introduction channel 107 , a nucleic acid amplification reaction section 108 , an air channel 109 and a vent port 110 . The sample introduction channel 107 is a channel that guides the sample introduced from the sample introduction port 106 to the nucleic acid amplification reaction section 108 . Thus, the nucleic acid amplification reaction device 101 functions as a channel device.

The sample inlet is a configuration for introducing samples. The sample inlet 106 and vent 110 are located at the end points of the channel and are processed as through holes on the substrate 102 . A sealing material 105 is adhered to the lower surface of the substrate 102 . The sample inlet 106 and the vent 110 are opened on the upper surface of the substrate 102 (that is, the surface opposite to the surface to which the sealing material 105 is adhered).

The shape of the sample inlet 106 may not be cylindrical, and may be conical or countersunk, for example. It is preferable that the shape of the sample inlet 106 when viewed from above is a shape without corners (circular, elliptical, etc.). The shape is not limited to these, and any shape may be employed as long as the sample 115 (see FIG. 6) at the sample introduction port 106 can be drawn into the sample introduction channel 107 .

Since the sample inlet 106 has no corners (for example, a circular shape) as viewed from above, it is possible to prevent unexpected leakage of the sample from the corners, and the force acting on the dropped sample is isotropic. and stable weighing can be achieved.

The air flow path 109 that connects the air vent 110 and the nucleic acid amplification reaction section 108 has a filter 111 that does not allow passage of nucleic acids in the middle.

As the nucleic acid amplification reaction device 101, a substrate 102 made of polycarbonate (PC) of 80 mm × 30 mm × 3 mm in thickness is provided with a sample introduction channel 107 of 0.3 mm in width and 0.3 mm in depth, and a sample introduction channel 107 of 0.8 mm in width and 0.8 mm in depth. The flow path of the nucleic acid amplification reaction section 108 and the air flow path 109 of 0.6 mm, the sample introduction port 106 of 3 mm in diameter, and the air vent 110 of 1 mm in diameter were manufactured by cutting. Thereafter, a sealing material 105 made of polyolefin (PO) having a thickness of 0.1 mm was crimped to the entire cut surface of the substrate 102 to form a flow path.

The material of the substrate 102 does not have to be PC, and other resins such as acrylic resin (PMMA) and cycloolefin polymer (COP) may be used. The material of the sealing material 105 may not be PO, and may be another resin such as polypropylene.

In Example 1, the sample does not enter the sample introduction port 106 or the sample introduction channel 107 of the nucleic acid amplification reaction device 101 prior to a certain position, and remains at a certain position from the time the sample is dropped until the weighing is completed. It is preferable to keep it. Therefore, it is beneficial to provide a valve mechanism in the sample introduction channel 107 .

This may be controlled by, for example, installing a microvalve as in Patent Document 3 in the channel. Alternatively, as in this embodiment, the thickness of the sample introduction channel 107 is about 1/10 or less of the depth of the sample introduction port 106, and the sample does not enter the sample introduction channel 107 unless a constant pressure is applied. This can also be achieved by constructing a valve mechanism that does not allow When configuring such a valve mechanism, the minimum pressure (water intrusion pressure) at which the sample 115 at the sample introduction port 106 flows into the sample introduction channel 107 at the interface between the sample introduction port 106 and the sample introduction channel 107 ) is greater than the atmospheric pressure.

The channel of the nucleic acid amplification reaction section 108 is a single channel without branches, and has a first temperature region 113 and a second temperature region 114 . The first temperature region 113 is in contact with the external heat source 206, and the second temperature region 114 is in contact with the external heat source 207, respectively. Vent 110 is connected to liquid delivery device 213 via pump connection 204 .

FIG. 5 shows an enlarged view of the vicinity of the sample introduction port 106, and FIG. 6 shows a cross-sectional view of the sample introduction port 106 when the sample is dropped. A water-absorbing pad 103 as a water-absorbing portion was provided around the sample inlet 106 with a non-water-absorbing region 104 having a width of 2 mm. The non-absorbent area 104 is the area between the sample inlet 106 and the absorbent pad 103 .

The water absorbing pad 103 is ring-shaped with an inner diameter of 7 mm and an outer diameter of 20 mm. Filter paper, porous resin, or the like can be used as the water absorbing pad 103 . It is preferable that the water absorbing pad 103 has sufficient water absorbing ability to absorb the entire amount of the sample that can be dripped. The outer shape may be rectangular instead of circular. The water absorption pad 103 may be installed on the surface of the device for nucleic acid amplification reaction 101 with a double-sided tape or the like, or may be simply left standing.

In this example, one or more types of DNA to be detected, a primer that specifically reacts with the target DNA, a fluorescent dye, a thermostable enzyme, and four types of deoxyribonucleoside triphosphates (dATP, dCTP , dGTP, dTTP) was used as a sample. A PCR reaction system was constructed using 23 μL of sample. Fluorescent dyes for nucleic acid detection are, for example, TaqMan probes and Cycleave probes that specifically bind to nucleic acids to be detected, and fluorescent labeled probes called molecular beacons. Also, an intercalator such as SYBR Green may be used. A real-time PCR reagent kit or the like can be used as these reagent systems.

The sample introduction in this example was performed with the nucleic acid amplification reaction device 101 installed in the device holding portion 202 . FIG. 7 shows an operation flow from sample introduction to target gene detection. Each operation step will be described below with reference to FIG.

In this embodiment, it is assumed that a sample exceeding the target liquid volume is dropped into the sample introduction port 106 . Also, the amount of the sample to be dropped must exceed the volume of the sample introduction port 106 and must reach the water absorption pad 103 at least once. Specifically, in the case of this embodiment, about 60 to 300 μL of the sample may be dropped with respect to the target liquid volume of 23 μL.

When about 60 to 300 μL of sample is dropped into the sample inlet 106, the volume of the dropped sample inlet 106 is exceeded and touches the non-water-absorbing region 104 on the surface of the substrate 102, reaching the water-absorbing pad 103 (FIG. 7a). The sample overflowing into the non-absorbing region 104 is sucked into the water-absorbing pad 103 by the suction force based on the capillary action of the water-absorbing pad 103 (Fig. 7b).

Even if more sample is dropped after this, as long as the water absorption capacity of the water absorption pad 103 is not exceeded, the surplus sample will be sucked into the water absorption pad 103 again, and a certain amount of sample will remain in the sample inlet 106. As a result, the amount of sample remaining in the sample introduction port 106 is constant regardless of the amount of dropped sample if the ambient environment is constant.

Thus, according to the nucleic acid amplification reaction device 101 according to Example 1, it is possible to more precisely control the amount of sample liquid to be introduced.

After the dripping operation is finished and the suction of the sample by the water absorption pad 103 is stopped, the sample remaining in the sample introduction port 106 is drawn into the sample introduction channel 107 by the suction operation of the syringe pump 205 (Fig. 7c), and the nucleic acid amplification reaction is started. Pump up to the second temperature zone 114 of the part 108 (Fig. 7d).

Next, the flow of sample reciprocation for nucleic acid amplification reaction will be explained. After the sample is moved to the second temperature region 114 by the initial introduction operation (Fig. 7d), the sample is waited for a certain period of time (15 seconds in this embodiment) to raise the temperature of the sample (Fig. 7e).

After that, the discharge operation of the syringe pump 205 applies pressure in the flow channel to move the sample to the first temperature region 113 (Fig. 7f). Annealing and elongation reactions are performed by holding the sample in the first temperature region 113 for a certain period of time (7 seconds in this example) (FIG. 7g).

After that, the suction operation of the syringe pump 205 moves the sample to the second temperature region 114 (Fig. 7h). When the sample passes through the detection unit 112 (FIG. 1) provided between the first temperature region 113 and the second temperature region 114, fluorescence derived from nucleic acid amplification is detected by the fluorescence detection unit 209 (FIG. 2). The fluorescence intensity detected by the fluorescence detection unit 209 is converted into an electric signal by the signal detection circuit 211 and analyzed to measure the amount of amplified nucleic acid.

After that, the denaturation reaction is performed by holding the sample in the second temperature region 114 for a certain period of time (3 seconds in this example) (Fig. 7i). By repeating the operation from FIG. 7f to FIG. 7i a predetermined number of times (40 times in this embodiment), a specific region of DNA to be detected is amplified. This allows real-time PCR to be performed in about 8 minutes.

In Example 1 above, the nucleic acid detection method is a real-time PCR method in which nucleic acid amplification is sequentially detected in each cycle using a reagent containing a fluorescent dye, but the method is not limited to this. It is also possible to construct an endpoint detection system that detects the final amount of nucleic acid after the nucleic acid amplification reaction is completed.

When an endpoint detection system is used, the configuration of the nucleic acid amplification reaction device 201 does not include the fluorescence detection unit 209, the excitation light irradiation unit 214, the dichroic mirror 215, the photodetector 216, and the signal detection circuit 211. Different from Example 1.

Fig. 8 shows the operation flow from sample introduction to target gene detection when using the endpoint detection system. The operation flow from the dropping of the sample (FIG. 8a) to the temperature rise of the sample (FIG. 8e) is the same as that of the first embodiment (FIG. 7).

After that, the discharge operation of the syringe pump 205 applies pressure in the flow channel to move the sample to the first temperature region 113 (Fig. 8f). Annealing and elongation reactions are performed by holding the sample in the first temperature region 113 for a certain period of time (7 seconds in this example) (FIG. 8g). The suction action of the syringe pump 205 moves the sample to the second temperature zone 114 (Fig. 8h). A denaturation reaction is performed by holding the sample in the second temperature region 114 for a certain period of time (3 seconds in this example) (Fig. 8i).

By repeating the operations from (f) to (i) a predetermined number of times (40 times in this embodiment), the specific region of the DNA to be detected is amplified. Thereafter, the sample is moved to the sample introduction port 106 by the discharge operation of the syringe pump 205 (Fig. 8j). The sample returned to the sample inlet is recovered, and the amplified nucleic acid is detected by electrophoresis, hybridization, or the like (Fig. 8k). Thereby, the nucleic acid amplification reaction can be performed in about 8 minutes, and the post-reaction sample can be measured by any method.

As described above, according to Example 1, the amount of sample liquid to be introduced into the device for nucleic acid amplification reaction can be controlled more precisely.

Example 2 of the present invention will be described below. It should be noted that descriptions of parts common to the first embodiment may be omitted.

FIG. 9 is an external view of a nucleic acid amplification reaction device 201 equipped with a guide mechanism for easily determining the sample dropping position. The device pullout part 217 has a holding part that holds the nucleic acid amplification reaction device 101 at a fixed position and a dropping guide 218, and can be pulled out from the nucleic acid amplification reaction apparatus 201 by a fixed distance.

The dropping guide 218 functions as a guide part for guiding the dropping of the sample into the sample introduction port 106 . Drop guide 218 is provided, for example, above sample inlet 106 . The drip guide 218 is a circular opening in this embodiment and is arranged to be movable to a position coaxial with the sample inlet 106 .

When the pull-out button 219 is pressed once, the device pull-out portion 217 is automatically pulled out, and the drop guide 218 stops at a position where the dropper for sample dropping can be installed. The dropping guide 218 is configured so that the dropper for sample dropping can be fixed to the sample introduction port 106 when dropping the sample. Next, the nucleic acid amplification reaction device 101 is installed in the device drawer 217, and the dropper for sample dropping is placed on the dropping guide 218 to drop the sample.

When the withdrawal button 219 is pressed again after the dropping operation is completed, the device withdrawal section 217 is pulled back into the nucleic acid amplification reaction apparatus 201 and the nucleic acid amplification reaction device 101 is installed on the device holding section 202 . As a result, the sample can be dropped from the sample introduction port 106 without deviating.

The sample dropping operation can also be performed outside the nucleic acid amplification reaction device 201. FIG. 10 shows the nucleic acid when a sample is introduced into the nucleic acid amplification reaction device 101 outside the nucleic acid amplification reaction device 201 and then the nucleic acid amplification reaction device 101 is installed in the nucleic acid amplification reaction device 201 in Example 2. 1 is a configuration diagram of an amplification reaction device 201. FIG.

It differs from the configuration shown in FIG. 1 in that a valve 212 is provided. A valve 212 is provided between the liquid transfer device 213 and the nucleic acid amplification reaction device 101 . Valve 212 can be open to the atmosphere.

If the water absorption pad 103 and the sample introduction port 106 are greatly inclined when introducing the sample, the weighing characteristics may change due to the influence of gravity. preferably.

The installation of the nucleic acid amplification reaction device 101 is performed with the valve 212 open to the atmosphere in order to prevent the air in the channel from being discharged to the sample introduction port 106 and air bubbles from entering the sample. If the cover 203 is closed to fix the device 101 for nucleic acid amplification reaction, and the valve 212 is closed before the sample is sucked into the sample introduction channel 107, liquid transfer can be started.

Example 3 of the present invention will be described below. Note that the description of the parts common to the first or second embodiment may be omitted.

As Example 3 of the present invention, a nucleic acid amplification reaction device 101 capable of mixing a sample-derived nucleic acid extract and a PCR reagent in a channel device will be described. FIG. 11 shows a configuration diagram of the vicinity of the sample introduction port 106 of the device 101 for nucleic acid amplification reaction according to Example 3. As shown in FIG. Parts that do not appear in FIG. 11 can be the same as those in the first embodiment, for example.

The nucleic acid amplification reaction device 101 stores reagents 116 (for example, reaction reagents) required for PCR in the channel. The reagent 116 includes a primer that specifically reacts with the target DNA, a fluorescent dye, a thermostable enzyme, four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP), and the like.

A reagent storage section 117 having a width of 0.8 mm, a length of 1.5 mm, and a depth of 1.5 mm was prepared at the end of the sample introduction channel 107 of the device for nucleic acid amplification reaction 101, and a reagent 116 was placed in the reagent storage section 117. solidified. The reagent storage section 117 functions as a reagent installation section for installing a reagent for nucleic acid amplification reaction.

The reagent 116 is solid or gel, and may be molded into a bead shape as shown in FIG. It is preferable to use a composition that is readily soluble and easily mixed with a sample that is a solution component.

The sample dropped into the sample introduction port 106 in this embodiment was a nucleic acid extract of a specimen (nasal discharge, etc.). A certain amount of the sample was weighed out from the dropped sample based on the same principle as in Example 1, and then the weighed sample was introduced into the sample introduction channel 107 . When the sample passes through the reagent storage section 117 in the middle of the step of moving the sample to the nucleic acid amplification reaction section, the reagent 116 dissolves and the reagent components diffuse within the sample. Thus, the reagent storage section 117 also functions as a reagent mixing section that mixes the sample and the reagent.

Then, nucleic acid amplification and detection are performed according to the flow of Figures 7d-i or the flow of Figures 8d-k. As a result, a nucleic acid amplification reaction can be achieved without the need for a pretreatment of mixing PCR reagents with the sample to be dropped, and at the same time, the use of reagents can be minimized.

A step of reciprocating the sample several times by suction and pressure operation of the syringe pump 205 in the vicinity of the reagent storage unit 117 or in the first temperature region 113 or the second temperature region 114 before starting the PCR cycle to mix the reagent components. may be added.

In Example 3, as described above, the single reagent storage unit 117 functions as a reagent installation unit for installing reagents for nucleic acid amplification reactions, and also as a reagent mixing unit for mixing samples and reagents. Function. Alternatively, they may be configured separately. For example, the reagent installing section and the reagent mixing section may be provided as different spaces.

The fourth embodiment will be explained below. Note that descriptions of parts common to the first to third embodiments may be omitted.

As Example 4 of the present invention, a nucleic acid amplification reaction device 101 and a nucleic acid amplification reaction apparatus 201 including a reverse transcription (RT) reaction of a nucleic acid to be detected will be described. FIG. 12(a) shows a nucleic acid amplification reaction device 101 having a nucleic acid reverse transcription reaction region. FIG. 12(b) shows a cross-sectional view (taken along line AA in FIG. 12(a)) when the nucleic acid amplification reaction device 101 is installed in the nucleic acid amplification reaction apparatus 201. As shown in FIG.

Compared to the nucleic acid amplification reaction device 101 of Examples 1 to 3, the third temperature region 118 is provided between the sample introduction channel 107 and the first temperature region 113, and the nucleic acid amplification reaction device 201 also has the third temperature It differs in that an external heat source 220 corresponding to the region 118 is provided. The third temperature region 118 is kept at the RT temperature (45° C. in this embodiment) by the external heat source 220 .

In this example, the sample consisted of one type of DNA to be detected, a primer that specifically reacts with the target DNA, a thermostable enzyme, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP ) and further including reverse transcriptase. An RT-PCR reaction system was constructed using this sample.

First, a sample is introduced into the nucleic acid amplification reaction device 101 in the same flow as in FIGS. 7a to 7c. The introduced sample is moved to the third temperature region 118 and stopped for a certain period of time (60 seconds in this embodiment). As a result, even if the nucleic acid to be detected is RNA, it is reverse transcribed into DNA, so nucleic acid amplification and detection can be carried out according to the flow of FIGS.

Moreover, as shown in FIG. 1, a detection unit 112 is provided, a reagent system containing a fluorescent dye is used, and a nucleic acid amplification reaction apparatus 201 is provided with a fluorescence detection unit 209 as shown in FIG. 2 to provide a real-time fluorescence detection system. For example, after the RT reaction, the amplified nucleic acid can be detected according to the flow of FIGS. 7d to 7i.

The third temperature region 118 may be arranged between the first temperature region 113 and the second temperature region 114 or may be arranged between the second temperature region 114 and the filter 111 .

Example 5 of the present invention will be described below. Note that descriptions of parts common to the first to fourth embodiments may be omitted.

As Example 5 of the present invention, a nucleic acid amplification reaction device 101 and a nucleic acid amplification reaction apparatus 201 based on the LAMP isothermal amplification method will be described. FIG. 13(a) shows a nucleic acid amplification reaction device 101 based on the isothermal amplification method, and FIG. 13(b) is a sectional view when the nucleic acid amplification reaction device 101 is installed in the nucleic acid amplification reaction apparatus 201 (FIG. 13(a)). AA line sectional view).

　Compared to the nucleic acid amplification reaction device 101 of Examples 1 to 4, it differs in that only one pair of the temperature control region and the external heat source is provided.

In this example, the sample consisted of one type of DNA to be detected, four types of primers that specifically react with the target DNA, a thermostable enzyme, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) are mixed.

The first temperature region 113 is kept at the isothermal amplification reaction temperature (65° C. in this embodiment) by the external heat source 206 . First, a sample is introduced into the nucleic acid amplification reaction device 101 in the same flow as in FIGS. 7a to 7c. The introduced sample is moved to the first temperature region 113 and stopped for a certain period of time (for example, 10 minutes). As a result, the primers are annealed to the target DNA, the double strands dissociate, and an amplification reaction progresses in which new double strands are extended.

After that, sample collection and nucleic acid detection can be performed according to the flow of Figures 8j and k. Further, if the nucleic acid amplification reaction apparatus 201 is configured to have a real-time fluorescence detection system such as the fluorescence detection unit 209, it is possible to detect the amplified nucleic acid during the nucleic acid amplification reaction.

Even when the nucleic acid amplification reaction device 101 and the nucleic acid amplification reaction apparatus 201 shown in Examples 1 to 4 are used, the first temperature region 113 and the second temperature region 114 (and the configuration of Example 4) In this case, if the third temperature region 118) is all isothermal (for example, 65° C.), the nucleic acid amplification reaction can be carried out by the isothermal amplification method as in the present embodiment.

Example 6 of the present invention shows the specific operation of the sample weighing mechanism in Examples 1-5.

14(a) to (d) are schematic cross-sectional views of the vicinity of the cylindrical sample inlet 106, showing the operation from the contact of the sample 115 with the water-absorbing pad 103 to the end of the water-absorbing operation and reaching a steady state. represents

When the sample 115 is dripped from above the sample introduction port 106, the sample 115 accumulates in the sample introduction port 106 and spreads to the non-water absorbing region 104 (Fig. 14(a)).

When the sample 115 comes into contact with the absorbent pad 103, a capillary force acts on the contact area, the balance of the intermolecular forces within the sample 115 is disrupted, and the sample 115 begins to be absorbed by the absorbent pad 103 (Fig. 14(b)).

In Example 6, the upper end of the water-absorbing pad 103 is at a height higher than the upper end of the sample introduction port 106, so only the sample overflowing above the sample introduction port 106 can be efficiently absorbed. However, it is also possible to arrange the upper end of the absorbent pad 103 at a lower height than the upper end of the sample inlet 106 .

The liquid surface of the sample 115 gradually decreases, and the moment the liquid surface contacts the end of the sample inlet 106, the sample 115 in the sample inlet 106 and the sample 115 on the non-water-absorbing region 104 rupture (Fig. 14(c)).

After that, the sample 115 that was present on the non-absorbing region 104 when the sample 115 was torn is quickly absorbed by the water absorbing pad 103, and a certain amount of the sample 115 remains in the sample inlet 106 (Fig. 14 ( d)).

If the sample 115 is continued to drop further, the phenomena shown in FIGS. 14(a) to 14(d) will occur repeatedly as long as the water absorbing pad 103 has sufficient sample retention force. Thus, when the sample 115 in the vicinity of the sample inlet 106 reaches a certain amount or more, the sample is collected by the water absorption pad 103, and the amount of liquid remaining in the sample inlet 106 is always about the volume of the sample inlet 106. It should be noted that changing the target weighing liquid volume can be easily achieved by simply changing the volume of the sample inlet 106 .

The amount of sample 115 remaining on sample inlet 106 and non-absorbing region 104 until contacting water-absorbing pad 103 depends on the distance to water-absorbing pad 103, the wettability of the material of non-absorbing region 104, the surface tension of sample 115, etc. The smaller the distance from the sample inlet 106 to the water absorption pad 103, the more the amount of the sample 115 that needs to be dropped before the start of water absorption and the amount of the sample liquid remaining in the sample inlet 106 after the water absorption is complete. The amount of sample 115 required to reach volume is less.

By changing the distance from the sample introduction port 106 to the water absorption pad 103 in this way, the amount of the dripping liquid until the start of water absorption can be adjusted.

FIG. 15 is an example of a cross-sectional schematic diagram of the vicinity of the sample introduction port 106 at the end of sample dropping when the weighing of the present invention fails. In this way, if the dropping operation is terminated without the dropped sample 115 coming into contact with the water absorbent pad 103, more sample 115 than the weighing target will remain.

In order to prevent this, it is desirable that the amount of liquid dropped from the first time onwards is greater than or equal to the amount that the sample touches the absorbent pad 103 (Fig. 14(a)). In addition, it is desirable that the amount of liquid dropped from the second time onwards is larger than the difference between the amount of liquid at the moment when the absorbent pad 103 is touched (FIG. 14(a)) and the target amount of liquid (FIG. 14(d)).

The amount of liquid remaining in the sample introduction port 106 is approximately determined by the volume of the sample introduction port 106, but is also affected by the wettability of the sample introduction port 106. Regarding the modification shown in FIG. 16, the relationship between the shape of the sample inlet and the volume of the sample remaining in the sample inlet 106 after water absorption by the water absorbent pad is completed (FIG. 14(d)) will be described.

Let φ be the angle formed by the non-water-absorbing region 104 (more precisely, its surface) and the side wall surface of the sample inlet 106 (angle φ in FIG. 16). If one of the surfaces is a curved surface (for example, a cylindrical surface or a conical surface), the angle formed by the lines appearing in the vertical cross section can be φ, as shown in FIG.

The liquid surface of the sample remaining in the sample introduction port 106 makes a certain contact angle with the side wall surface of the sample introduction port 106 . When this contact angle is smaller than (180°-φ), the sample liquid surface is convex downward, and when it is larger than (180°-φ), the sample liquid surface is convex upward.

For example, when using a general polycarbonate (PC) cylindrical sample introduction port 106 as shown in Example 1, φ is 90°. On the other hand, when the sample to be dropped is pure water, the contact angle of the side wall surface of the sample inlet 106 is about 95°, so that the sample liquid surface becomes nearly horizontal and slightly convex when the water absorption is completed.

Taking such a liquid surface shape into consideration, when φ is large, when the sample inlet 106 is made of a highly hydrophobic material, or when the surface tension of the sample is large, the volume of the sample inlet 106 is reduced by the amount of nucleic acid amplification. By designing the sample volume to be equal to or less than the volume of the sample introduced into the reaction section, the volume of the sample liquid to be weighed can be controlled with high accuracy. Conversely, when φ is small, when the sample inlet 106 is made of a highly hydrophilic material (for example, PC whose surface is plasma-treated), or when the surface tension of the sample is small, the volume of the sample inlet 106 is By designing the volume of the sample to be introduced into the nucleic acid amplification reaction part or more, the volume of the sample liquid to be weighed can be controlled with high accuracy.

That is, as shown in FIG. 16, when the contact angle between the liquid surface of the sample and the side wall surface of the sample introduction port 106 is (180°−φ) or less, the volume of the sample introduction port 106 is used for nucleic acid amplification. It is preferable that the sample volume be equal to or larger than the volume of the sample introduced into the reaction section 108 . Similarly, when the contact angle between the liquid surface of the sample and the side wall surface of the sample introduction port 106 is (180°−φ) or more, the volume of the sample introduction port 106 is introduced into the nucleic acid amplification reaction section 108. A sample volume or less is preferable.

When the non-water-absorbing region 104 is made of a material with high wettability, or when the surface tension of the sample 115 is small, the contact angle of the liquid on the non-water-absorbing region 104 becomes small, so the amount of dripping liquid until the start of water absorption is small. Become. However, under the condition that the distance from the sample inlet 106 to the absorbent pad 103 is more than a certain distance, the amount of liquid remaining in the sample inlet 106 is constant regardless of these conditions. Therefore, highly robust weighing is possible with respect to physical properties such as the surface state of the non-water absorbing region 104 and the surface tension of the sample 115 .

As a reference example, a similar water absorption phenomenon occurs when a sample drop portion is determined on the substrate plane without providing a recessed structure like the sample inlet 106, and a water absorption pad is placed at a certain distance from the sample drop portion. . However, since the amount of liquid remaining in the sample dripping portion is smaller than the amount of dripped liquid compared to the case of having a concave structure, the difference between the amount of dripped liquid until it touches the water absorbing pad 103 and the target liquid amount becomes large. Cheap. In addition, since the amount of liquid remaining on the substrate is determined only by the wettability of the substrate or the distance of the absorbent pad, the degree of freedom in design is low. On the other hand, with the structure having the sample introduction port 106 as in Examples 1 to 6, as described above, the material of the substrate 102, the shape of the sample introduction port 106, etc. can be selected more freely, and the target liquid volume to be weighed can be increased. Can be set freely.

Example 7 of the present invention will be described below. Note that descriptions of parts common to the first to sixth embodiments may be omitted.

In Example 7, as shown in FIG. 17( a ), the substrate 102 has a groove 119 for installing the water absorbing pad 103 , and the bottom surface of the water absorbing pad 103 is positioned lower than the upper end of the sample inlet 106 . The width (radial dimension) of the groove 119 may be equal to or greater than the width (radial dimension) of the water absorbent pad 103 .

In addition, the non-water-absorbing region 104 exists not only around the upper end of the sample introduction port 106 but also partially below the upper end of the sample introduction port 106 (the bottom of the groove 119).

　The sample weighing mechanism shown in FIG. Therefore, the lower surface of the water absorbing pad 103 may be positioned lower or higher than the upper end of the sample inlet 106 as shown in FIG. 17(a).

With the configuration of FIG. 17( a ), when the thickness of the nucleic acid amplification reaction device 101 (the axial dimension of the water absorption pad 103 ) needs to be reduced due to the convenience of designing the nucleic acid amplification reaction device 201 , Even if the area where the water absorbing pad 103 can be installed is limited, it is possible to install the water absorbing pad 103 having a water retention capacity capable of sufficiently absorbing the dropped sample. In addition, the groove 119 serves as a positioning guide when installing the water absorbing pad 103, and the nucleic acid amplification reaction device 101 can be easily manufactured.

In FIG. 17A, the groove 119 is formed in the substrate 102 having the same thickness as the sample introduction port 106, but as a modification, the sample introduction port 106 and the non-water absorbing region 104 protrude in the thickness direction. A shape in which the peripheral substrate 102 is thin may be used. As a result, the thickness of the substrate 102 other than the sample inlet 106, non-water-absorbing region 104, and water-absorbing pad 103 can be reduced, and the size and/or weight of the nucleic acid amplification reaction device 101 can be reduced.

FIG. 17(b) shows an embodiment in which a portion of the side wall of the sample inlet 106 is processed into a conical surface, and the non-water-absorbing region 104 is sloped. The non-water-absorbing region 104 may be provided with a certain width or more between the upper end of the sample introduction port 106 and the water-absorbing pad 103 . Also, the non-water absorbing region 104 does not have to be a horizontal surface. According to the embodiment as shown in FIG. 17(b), the non-absorbing area 104 can be arranged at a lower position, so the amount of dripping liquid is smaller than that of the embodiment in which the non-absorbing area 104 having the same width is provided horizontally. , the weighing of the present invention can be carried out.

Example 8 of the present invention is an experimental example related to the sample weighing mechanism of Examples 1-7.

<Experiment>
A through-hole (corresponding to the sample introduction port) with a diameter of 3 mm corresponding to the sample introduction port 106 was made in the center of a hydrophobic polycarbonate (PC) resin substrate of 50 mm x 50 mm x 3 mm thickness. A polyolefin (PO) seal having a thickness of 0.1 mm was crimped to one side of the substrate, and the weight was measured. After that, the substrate was placed on a horizontal table with the surface to which the seal was crimped down, and a 40 mm × 40 mm water absorption pad (Atto blotting filter paper, CB-20A) with a hole was placed so that it was concentric with the through hole of the substrate. was placed on the substrate for a period of time.

Using a micropipette, 5 μL of pure water was dropped toward the center of the through-hole of the substrate. Dropping was stopped when pure water absorption by the water-absorbing pad started, and the total amount of dropped liquid until the start of water absorption was recorded. After confirming that pure water absorption by the water-absorbing pad had stopped and that the movement of pure water in the vicinity of the through-hole of the substrate had stopped, the water-absorbing pad was gently removed, and the weight of the substrate with the seal attached was measured. The amount of liquid remaining in the through-hole was calculated from the difference in weight before and after pure water was dropped.

<Results>
FIG. 18 shows the distance from the through-hole (strictly speaking, its upper edge) to the water-absorbing pad (for example, the difference between the inner diameter of the through-hole and the inner diameter of the hole in the water-absorbing pad), and the amount of pure water until the water-absorbing pad starts to absorb water. and the total amount of dripping liquid.

As shown in FIG. 18, as the distance from the through hole (strictly speaking, its upper edge) to the absorbent pad increases, the total amount of dripping liquid until the start of water absorption increases, and when the distance to the absorbent pad is 1 mm, the average amount of dripping liquid is 40 μL. , 60 μL on average at 2 mm, and 100 μL on average at 2.5 mm.

Fig. 19 shows the relationship between the distance from the through-hole (strictly speaking, its upper edge) to the absorbent pad and the amount of liquid remaining in the through-hole. When the distance to the water-absorbing pad was 1 mm or more, the amount of liquid remaining in the through-hole was approximately 23 μL±1 μL regardless of the distance to the water-absorbing pad. From these results, it can be seen that the method of the present invention can achieve sufficient weighing accuracy to carry out stable nucleic acid amplification reactions.

Example 9 of the present invention is an experimental example related to the sample weighing mechanism of Examples 1-7.

<Experiment>
A through hole with a diameter of 3 mm corresponding to the sample inlet 106 was made in the center of a substrate of hydrophobic polycarbonate (PC) resin of 50 mm×50 mm×thickness of 3 mm. A polyolefin (PO) seal having a thickness of 0.1 mm was crimped to one side of the substrate, and the weight was measured. After that, the substrate was placed on a horizontal table with the crimped side of the seal facing down, and a 15 mm × 15 mm water-absorbing pad (Atto blotting filter paper, CB-20A) with a hole of 8 mm diameter was placed concentrically with the through-hole of the substrate. It was left still on the substrate so as to be

Using a micropipette, pure water was dropped toward the center of the through-hole of the substrate. After confirming that the movement of the pure water in the vicinity of the through-hole of the substrate stopped (for example, fluctuation of the liquid surface), the water absorption pad was gently removed, and the weight of the substrate with the seal attached was measured. The amount of liquid remaining in the through-hole was calculated from the difference in weight before and after pure water was dropped.

<Results>
FIG. 20 shows the relationship between the amount of sample liquid dropped and the amount of liquid remaining in the through-hole. If the amount of liquid does not reach the absorbent pad, the amount of liquid remaining in the through-holes is the same as the amount of liquid dropped. When the amount of liquid that exceeded the volume of the through-hole and contacted the water-absorbing pad was put in, the amount of liquid remaining in the through-hole was approximately 23 μL±0.4 μL regardless of the distance to the water-absorbing pad.

Although not shown in FIG. 20, when the amount of the dripping liquid was 300 μL or more, the sample that could not be fully absorbed remained in the lower part of the water absorbing pad. From this result, according to Examples 1 to 7, as long as the water absorbing power of the water absorbing pad is within a sufficient range, it is possible to achieve sufficient weighing accuracy to carry out a stable nucleic acid amplification reaction regardless of the amount of the dropped liquid. Recognize. If it is desired to accurately weigh a larger amount of dripping liquid, it is possible to use a water absorbing pad with higher water absorption or increase the area and thickness of the water absorbing pad.

Example 10 of the present invention is an experimental example related to the sample weighing mechanism of Examples 1-7.

<Experiment>
Two hydrophobic polycarbonate (PC) resin substrates of 50 mm×50 mm×thickness 3 mm were prepared, and a through-hole (corresponding to the sample introduction port) of 3 mm in diameter was formed in the center of each substrate. For each substrate, 2 μL of pure water was dropped on the surface of the substrate away from the through hole, and the contact angle with the substrate was measured with a contact angle meter (Surfgauge, ST-1).

Next, the entire surface of one of the substrates was irradiated with ultraviolet light (excimer lamp, wavelength 172 nm) for 6 minutes, and after irradiation, 2 μL of pure water was dropped again on the substrate surface, and the contact angle was measured. After measuring the contact angle, the dripped pure water was immediately removed.

A polyolefin (PO) seal with a thickness of 0.1 mm was crimped to one side of each substrate, and the weight was measured.

After that, the substrate was placed on a horizontal table with the surface to which the seal was crimped downward, and a 15 mm × 15 mm water absorption pad (Atto blotting filter paper, CB-20A) with a hole of 6 mm in diameter was placed in a concentric circle with the through hole of the substrate. It was left still on the substrate so that

Using a micropipette, pure water was dropped toward the center of the through-hole of each substrate. After confirming that the pure water stopped moving near the through-hole of the substrate, the water absorption pad was gently removed, and after observing the surface of the pure water remaining in the through-hole, the weight of the substrate with the seal attached was measured. . The amount of liquid remaining in the through-hole was calculated from the difference in weight before and after pure water was dropped.

<Results>
Before irradiation with ultraviolet light, the contact angle was about 95° with any substrate. On the other hand, the contact angle of the substrate irradiated with ultraviolet light was about 60°. In the substrate that was not irradiated with ultraviolet light, the liquid surface remaining in the through-hole was almost horizontal and slightly convex, and the liquid volume was about 23 μL±0.4 μL. On the other hand, in the substrate irradiated with ultraviolet light, the surface of the liquid remaining in the through hole was convex downward, and the amount of liquid was about 19.5±0.2 μL. From these results, when the through-hole volume is the same size, when the contact angle between the substrate and the sample is large, the amount of liquid remaining is larger than the through-hole volume, and when the contact angle is small, the amount of liquid remaining is smaller than the through-hole volume. Recognize. Under certain conditions, highly accurate weighing is possible regardless of the wettability of the substrate.

In the nucleic acid amplification reaction apparatus according to Examples 1 to 7, the nucleic acid amplification reaction can be selected from the following.
- polymerase chain reaction (PCR)
- reverse transcription PCR (RT-PCR)
- Multiplex PCR
- Multiplex RT-PCR
- real-time PCR
- real-time RT-PCR
- DNA isothermal amplification

101 Device for nucleic acid amplification reaction 102 Substrate 103 Water absorbing pad (water absorbing portion)
104 Non-water absorbing area 105 Sealing material 106 Sample introduction port 107 Sample introduction channel 108 Nucleic acid amplification reaction section 109 Air channel 110 Vent 111 Filter 112 Detection section 113 First temperature zone 114 Second temperature zone 115 Sample 116 Reagent 117 Reagent storage Section (reagent installation section, reagent mixing section)
118 Third temperature region 119 Groove 201 Nucleic acid amplification reaction device 202 Device holding part 203 Cover 204 Pump connecting part 205 Syringe pump 206 External heat source (temperature control mechanism)
207 external heat source (temperature control mechanism)
208 temperature control unit (temperature control mechanism)
209 fluorescence detection unit 210 tube 211 signal detection circuit 212 valve 213 liquid sending device (liquid sending control mechanism)
214 excitation light irradiation section 215 dichroic mirror 216 photodetector 217 device drawer section 218 dropping guide (guide section)
219 drawer button 220 external heat source

Claims

a sample introduction port for introducing a sample;
a water absorbing portion provided around the sample inlet;
a non-water-absorbing region between the sample inlet and the water-absorbing portion;
a nucleic acid amplification reaction section;
a channel for guiding the sample introduced from the sample introduction port to the nucleic acid amplification reaction section;
A flow path device with
a reagent installation unit for installing a reagent for nucleic acid amplification reaction;
a reagent mixing unit for mixing the sample and the reagent;
The flow path device of claim 1, further comprising:
The flow channel device according to claim 1, wherein the distance from the sample introduction port to the water absorption part is 1 mm or more.
The flow channel device according to claim 1, wherein the upper end of the water absorbing portion is at a height equal to or higher than the upper end of the sample inlet.
The flow path device according to claim 1, wherein the sample inlet has a circular shape when viewed from above.
The contact angle between the liquid surface of the sample and the side wall surface of the sample inlet is (180°−φ) or less, where φ is the angle formed by the non-water-absorbing region and the side wall surface of the sample inlet. 2. The flow path device according to claim 1, wherein in some cases, the volume of said sample introduction port is equal to or greater than the volume of the sample introduced into said nucleic acid amplification reaction section.
The contact angle between the liquid surface of the sample and the side wall surface of the sample inlet is (180°−φ) or more, where φ is the angle formed by the non-water-absorbing region and the side wall surface of the sample inlet. 2. The flow path device according to claim 1, wherein in some cases, the volume of said sample introduction port is equal to or less than the volume of the sample to be introduced into said nucleic acid amplification reaction section.
A nucleic acid amplification reaction apparatus comprising the flow path device according to claim 1,
a temperature control mechanism that forms a temperature zone used for nucleic acid amplification reaction;
a liquid transfer control mechanism for moving the sample in the channel device;
A nucleic acid amplification reaction device comprising:
The nucleic acid amplification reaction device according to claim 8, comprising a guide part for guiding the dropping of the sample into the sample introduction port.
A nucleic acid amplification reaction apparatus comprising the flow path device according to claim 1,
Nucleic acid amplification reaction
A nucleic acid amplification reactor selected from polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), multiplex PCR, multiplex RT-PCR, real-time PCR, real-time RT-PCR, isothermal DNA amplification.
The nucleic acid amplification reaction apparatus according to claim 8, comprising a valve capable of opening to the atmosphere between the liquid transfer control mechanism and the channel device.