CN211339446U

CN211339446U - PCR reaction system

Info

Publication number: CN211339446U
Application number: CN201921682904.1U
Authority: CN
Inventors: 胡军荣; 徐强; 崔相民
Original assignee: Shenyi Biotech Hangzhou Co Ltd
Current assignee: Shenyi Biotech Hangzhou Co Ltd
Priority date: 2019-07-01
Filing date: 2019-10-09
Publication date: 2020-08-25
Anticipated expiration: 2029-10-09
Also published as: CN112175786A

Abstract

The utility model provides a PCR reaction system, this system includes: a sample-containing unit provided with a first liquid outlet/inlet; a diluent containing unit provided with a diluent outlet; the PCR reaction unit is provided with a cracked sample mixed liquid inlet and a cracked PCR reaction liquid outlet, and the PCR reaction liquid outlet is connected with the diluent outlet through a fourth pipeline; and the piston unit comprises an injection chamber and a piston, the injection chamber is provided with a second liquid outlet/inlet, the second liquid outlet/inlet is connected with the first liquid outlet/inlet through a first pipeline, the second liquid outlet/inlet is connected with the diluent outlet through a second pipeline, and the second liquid outlet/inlet is connected with the cracked sample mixed liquid inlet through a third pipeline. The system realizes full-automatic operation.

Description

PCR reaction system

Technical Field

The utility model relates to a molecular biology field, specifically, the utility model relates to a PCR reaction system.

Background

The traditional PCR reaction process is generally carried out separately, namely, required nucleic acid is extracted through a nucleic acid extraction kit, then the extracted nucleic acid and a reagent are mixed and added into a PCR reaction tube, and finally the PCR reaction tube is placed into a PCR instrument for PCR amplification reaction to obtain a final result. The traditional PCR reaction process has complicated operation steps and low working efficiency.

Therefore, the simple and efficient PCR reaction process needs further research and development.

SUMMERY OF THE UTILITY MODEL

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

the traditional PCR reaction process has complicated operation steps and low working efficiency. For this reason, the inventors found that each step of the conventional PCR reaction process generally requires a professional to perform the operation, and the operations generally need to be performed by different instruments such as a nucleic acid purification instrument, a fully automatic workstation, etc., and the operation of the whole process needs to be performed in a standard PCR laboratory environment. Based on the problems, the extraction of nucleic acid, the mixing of nucleic acid and reagents and the final PCR reaction are all integrated into one system through a microfluidic pipeline, the system realizes real full-automatic operation, solves the problem that the traditional PCR experiment process needs to be operated by professionals in a professional experiment environment, reduces errors caused by manual operation, greatly improves the working efficiency of PCR reaction, and greatly saves the cost of human resources.

To this end, in a first aspect of the present invention, the present invention provides a PCR reaction system. According to the utility model discloses an embodiment, the system includes: a sample containing unit in which a lysis raw material freeze-dried powder is disposed, and which has a first liquid outlet/inlet; a diluent containing unit in which a diluent is disposed and which has a diluent outlet; the PCR reaction unit is internally provided with reverse transcriptase and PCR raw material freeze-dried powder and is provided with a PCR reaction liquid outlet and a cracked sample mixed liquid inlet; and a piston unit including an injection chamber and a piston, the injection chamber having a second liquid outlet/inlet; the second liquid outlet/inlet is connected with the first liquid outlet/inlet through a first pipeline, the second liquid outlet/inlet is connected with the diluent outlet through a second pipeline, the second liquid outlet/inlet is connected with the cracked sample mixed liquid inlet through a third pipeline, and the PCR reaction liquid outlet is connected with the diluent outlet through a fourth pipeline.

According to the PCR reaction system provided by the embodiment of the utility model, the sample containing unit, the diluent containing unit, the PCR reaction unit and the piston unit are connected together through the microfluidic pipeline; meanwhile, each unit is an independently arranged unit so as to store different reactants before use, and the long-term storage of the reactants under the condition of nonuse is facilitated. For example, the independent arrangement of the sample accommodating unit facilitates the independent addition of the sample, simplifies the sample addition operation, and also facilitates the long-term preservation of the sample. First, the piston is pulled outward to a position so that a part of the diluent in the diluent accommodating unit flows to the injection chamber; then the piston is moved back and forth, so that the diluent in the injection chamber enters the sample containing unit and is uniformly mixed with the lysis freeze-dried powder and the sample in the sample containing unit; heating the sample accommodating unit to a set temperature, and fully cracking the sample in the sample accommodating unit at the set temperature; after the cracking is finished, the piston is pulled outwards to a certain position again, so that the cracked sample mixed liquid in the sample containing unit flows to the injection chamber; then the piston is moved in a reciprocating manner, so that the sample mixed liquid cracked in the injection chamber returns to the diluent containing unit and is uniformly mixed with the rest diluent in the diluent containing unit, and therefore the cracked sample mixed liquid is diluted, and the impurity concentration in the sample mixed liquid is reduced; then, the piston is pulled outwards to a certain position again, so that the diluted sample mixed liquid in the diluent containing unit flows to the injection chamber; then the piston is moved in a reciprocating way, so that the diluted sample mixed solution in the injection chamber enters the PCR reaction unit and is uniformly mixed with the reverse transcriptase in the PCR reaction unit and the freeze-dried powder of the PCR raw material; and finally, carrying out PCR temperature heating control on the PCR reaction unit so as to finally finish the PCR amplification reaction. According to the utility model discloses PCR reaction system has realized real full automatization operation, has solved traditional PCR experimentation and need carry out the difficult problem of operating by the professional under professional experimental environment, has reduced the error that the manual operation brought, has greatly improved PCR reaction's work efficiency, has practiced thrift the human resource cost greatly.

Optionally, the system further comprises: a sample control valve provided on the first pipeline for controlling a communication state of the first liquid outlet/inlet and the second liquid outlet/inlet; a dilution control valve provided on the second pipeline for controlling a communication state of the diluent outlet and the second liquid outlet/inlet; the first PCR control valve is arranged on the third pipeline and is used for controlling the communication state of the inlet of the sample mixed liquid after the cracking and the outlet/inlet of the second liquid; and the fourth pipeline is provided with the second PCR control valve which is used for controlling the communication state of the diluent outlet and the PCR reaction liquid outlet. From this, utilize the basis the utility model discloses the PCR reaction that the PCR reaction system goes on is gone on under inclosed environment, has reduced the pollution to system's environment, has improved the credibility of experiment, and operates more portably.

Optionally, the system further comprises: the buffer unit is provided with a PCR reaction liquid inlet and an air vent, the fourth pipeline is provided with the buffer unit, the second PCR control valve is connected with the PCR reaction liquid inlet, and the diluent outlet is connected with the air vent. Therefore, the PCR reaction carried out by the PCR reaction system can solve the overflow problem of the PCR reagent when expanding at high temperature.

Optionally, the system further comprises: a sample-accommodating unit sealing member provided on the first liquid outlet/inlet surface for performing a first sealing process on the sample-accommodating unit; and a diluent containing unit sealing member provided on a surface of the diluent outlet and configured to perform a second sealing process on the diluent containing unit. The inventor finds that the sample containing unit sealing element and the diluent containing unit sealing element can isolate reactants in each independent unit, so that the reactants can be conveniently stored for a long time under the condition of nonuse, the storage time of each reactant in each unit is greatly prolonged, the pollution of each reactant to a PCR reaction system is avoided, and the service life of the PCR reaction system is prolonged.

Optionally, the system further comprises: a sample receiving unit seal piercing device for subjecting the sample receiving unit seal to a first piercing process; and a diluent containing unit seal piercing device for subjecting the diluent containing unit seal to a second piercing process. Therefore, according to the utility model discloses be in the connected state between sample among the PCR reaction system and the diluent holds unit and the micro-fluidic pipeline, be convenient for going on of follow-up process.

Optionally, at least one of the diluent containing cell seal and the sample containing cell seal is a sealing membrane.

Optionally, the sealing film is formed from at least one of tinfoil paper, plastic packaging film, or kraft paper.

Optionally, the sealing film has a thickness of 0.01 to 0.2mm, such as 0.03mm, 0.05mm, 0.07mm, 0.09mm, 0.1mm, 0.13mm, 0.15mm, 0.17mm, or 0.19 mm. The inventors have found that if the thickness of the sealing film is too small, permeation is likely to occur, and if the thickness of the sealing film is too large, piercing is difficult. In some embodiments, the sealing film has a thickness of 0.05 to 0.1 mm.

Drawings

FIG. 1 is a schematic diagram of a PCR system according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of a PCR system according to another embodiment of the present invention; and

FIG. 3 is a schematic diagram of a PCR system according to another embodiment of the present invention.

Reference numerals:

100: sample containing unit

110: first liquid outlet/inlet

200: diluent containing unit

210: diluent outlet

300: PCR reaction unit

310: sample mixed liquid inlet after cracking

320: PCR reaction liquid outlet

400: piston unit

410: injection chamber

411: second liquid inlet/outlet

420: piston

500: buffer unit

510: PCR reaction solution inlet

520: vent port

610: sample containment unit seal

620: diluent containment unit seal

710: sample containment unit seal piercing device

720: diluent containing unit seal piercing device

810: sample control valve

820: dilution control valve

830: first PCR control valve

840: second PCR control valve

910: first pipeline

920: second pipeline

930: third pipeline

940: fourth pipeline

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are exemplary and intended to be used for explaining the present invention, and should not be construed as limiting the present invention.

PCR reaction system

In a first aspect of the present invention, the present invention provides a PCR reaction system. According to an embodiment of the present invention, referring to fig. 1, the system includes: a sample-containing unit 100, in which a lysis material lyophilized powder is disposed in the sample-containing unit 100, and the sample-containing unit 100 is provided with a first liquid outlet/inlet 110; a diluent accommodating unit 200, in which a diluent is disposed within the diluent accommodating unit 200, and the diluent accommodating unit 200 is provided with a diluent outlet 210; the PCR reaction unit 300 is internally provided with reverse transcriptase and PCR raw material freeze-dried powder, the PCR reaction unit 300 is provided with a cracked sample mixed liquid inlet 310 and a PCR reaction liquid outlet 320, and the PCR reaction liquid outlet 320 is connected with the diluent outlet 210 through a fourth pipeline 940; and a piston unit 400, wherein the piston unit 400 includes an injection chamber 410 and a piston 420, the injection chamber 410 is provided with a second liquid inlet/outlet 411, the second liquid inlet/outlet 411 is connected to the first liquid inlet/outlet 110 through a first pipeline 910, the second liquid inlet/outlet 411 is connected to the diluent outlet 210 through a second pipeline 920, and the second liquid inlet/outlet 411 is connected to the lysed sample mixed liquid inlet 310 through a third pipeline 930.

According to the embodiment of the present invention, the PCR reaction system connects the sample accommodating unit 100, the diluent accommodating unit 200, the PCR reaction unit 300, and the piston unit 400 to each other through the microfluidic circuit; meanwhile, each unit is an independently arranged unit so as to store different reactants before use, and the long-term storage of the reactants under the condition of nonuse is facilitated. For example, the independent arrangement of the sample-accommodating unit 100 facilitates the separate addition of the sample, simplifies the sample addition operation, and also facilitates the long-term preservation of the sample. Referring to fig. 1, first, the piston 420 is pulled outward to a position such that the diluent in the diluent containing unit 200 flows toward the injection chamber 410; then, the piston 420 is moved back and forth, so that part of the diluent in the injection chamber 410 enters the sample containing unit 100 and is uniformly mixed with the lysis freeze-dried powder and the sample in the sample containing unit 100; heating the sample accommodating unit 100 to a set temperature, so that the sample in the sample accommodating unit 100 is fully cracked at the set temperature; after the lysis is completed, the piston 420 is pulled outward to a certain position again, so that the lysed sample mixture in the sample-containing unit 100 flows to the injection chamber 410; then, the piston 420 is moved back and forth, so that the lysed sample mixed liquid in the injection chamber 410 returns to the diluent containing unit 200, and is uniformly mixed with the diluent remaining in the diluent containing unit 200, thereby diluting the lysed sample mixed liquid and reducing the concentration of impurities therein; thereafter, the piston 420 is pulled outward to a certain position again, so that the diluted sample mixture in the diluent accommodating unit 200 flows to the injection chamber 410; then, the piston 420 is moved back and forth, so that the diluted sample mixed solution in the injection chamber 410 enters the PCR reaction unit 300 and is uniformly mixed with the reverse transcriptase in the PCR reaction unit 300 and the freeze-dried powder of the PCR raw material; and finally, performing PCR temperature heating control on the PCR reaction unit 300 so as to finally complete the PCR amplification reaction. According to the utility model discloses in the PCR reaction system, PCR reaction liquid export with the diluent export links to each other through the fourth pipeline, has formed the pressure system intercommunication of PCR reaction liquid export and diluent export to make too much reaction liquid can flow out the fourth pipeline through the reaction liquid export smoothly in the PCR reaction unit. Further, in the PCR reaction system according to the embodiment of the present invention, a valve or other switch may be flexibly designed at a suitable position of the microfluidic circuit, so as to control the communication state of the piston unit 400 with the sample accommodating unit 100, the diluent accommodating unit 200, or the PCR reaction unit 300. In addition, the movement of the piston and the control of valves or other switches can be flexibly designed into other mechanical devices for automation. From this, according to the utility model discloses PCR reaction system holds the unit with the sample with the piston unit respectively, diluent holds unit and PCR reaction unit and couples together, realized extracting from sample nucleic acid to mix with reagent, the full-automatic process of PCR reaction arrives again at last, the difficult problem that traditional PCR experimentation needs to be operated by the professional under professional experimental environment has been solved, it can accomplish to need not the professional, and the error that manual operation brought has been reduced, the work efficiency of PCR reaction has greatly been improved, the human resource cost has been practiced thrift greatly.

A system according to an embodiment of the invention is described in further detail below with reference to the accompanying drawings:

according to an embodiment of the present invention, referring to fig. 2, the system further comprises: a sample control valve 810, the sample control valve 810 being provided on the first pipe 910 for controlling a communication state of the second liquid inlet/outlet 411 and the first liquid inlet/outlet 110; a dilution control valve 820, wherein the dilution control valve 820 is disposed on the second pipeline 920 and is used for controlling the communication state between the second liquid inlet/outlet 411 and the diluent outlet 210; a first PCR control valve 830, wherein the first PCR control valve 830 is disposed on the third pipeline 930, and is used for controlling the communication state between the second liquid inlet/outlet 411 and the lysed sample mixture inlet 310; and a second PCR control valve 840, wherein the second PCR control valve 840 is disposed on the fourth pipeline 940, and is used for controlling the communication state between the PCR reaction solution outlet 320 and the diluent outlet 210.

According to an embodiment of the present invention, referring to fig. 2, first, the first PCR control valve 830, the second PCR control valve 840 and the sample control valve 810 are closed, and the dilution control valve 820 is opened; then the piston 420 is pulled outward to a position such that the diluent in the diluent containing unit 200 flows toward the injection chamber 410; then the dilution control valve 820 is closed and the sample control valve 810 is opened; then, the piston 420 is moved back and forth, so that part of the diluent in the injection chamber 410 enters the sample containing unit 100 and is uniformly mixed with the lysis freeze-dried powder and the sample in the sample containing unit 100; heating the sample accommodating unit 100 to a set temperature, so that the sample in the sample accommodating unit 100 is fully cracked at the set temperature; after the lysis is completed, the piston 420 is pulled outward to a certain position again, so that the lysed sample mixture in the sample-containing unit 100 flows to the injection chamber 410; thereafter, the sample control valve 810 is closed and the dilution control valve 820 is opened; then, the piston 420 is moved back and forth, so that the lysed sample mixed liquid in the injection chamber 410 returns to the diluent containing unit 200, and is uniformly mixed with the diluent remaining in the diluent containing unit 200, thereby diluting the lysed sample mixed liquid and reducing the concentration of impurities therein; thereafter, the piston 420 is pulled outward to a certain position again, so that the diluted sample mixture in the diluent accommodating unit 200 flows to the injection chamber 410; then, the dilution control valve 820 is closed, and the first PCR control valve 830 and the second PCR control valve 840 are opened; then, the piston 420 is moved back and forth, so that the diluted sample mixed solution in the injection chamber 410 enters the PCR reaction unit 300 and is uniformly mixed with the reverse transcriptase in the PCR reaction unit 300 and the freeze-dried powder of the PCR raw material; and finally, performing PCR temperature heating control on the PCR reaction unit 300, wherein the PCR temperature heating control comprises a preliminary constant temperature stage for activating enzyme and a temperature cycle control stage, and before performing temperature cycle control, closing the first PCR control valve 830 and the second PCR control valve 840 so as to finally complete the PCR amplification reaction. According to the utility model discloses each unit and each valve perfect adaptation in the PCR reaction system, the cooperative play effect for the pollution of experimental result to the environment and the pollution of environment to the experimentation all reduce, are favorable to realizing the full automatization, need not the professional and carry out manual operation. From this, utilize the basis the utility model discloses the PCR reaction that the PCR reaction system goes on is gone on under airtight environment, has reduced the pollution to system's environment, has improved the credibility of experiment, and operates more simple accurate.

According to an embodiment of the present invention, referring to fig. 3, the system further includes: buffer unit 500, buffer unit 500 is provided with PCR reaction liquid inlet 510 and vent 520, buffer unit 500 sets up on the fourth pipeline 940, PCR reaction liquid inlet 510 with second PCR control valve 840 links to each other, vent 520 with diluent export 210 links to each other.

According to an embodiment of the present invention, referring to fig. 3, first, the first PCR control valve 830, the second PCR control valve 840 and the sample control valve 810 are closed, and the dilution control valve 820 is opened; then the piston 420 is pulled outward to a position such that a part of the diluent in the diluent containing unit 200 flows to the injection chamber 410; then the dilution control valve 820 is closed and the sample control valve 810 is opened; then, the piston 420 is moved back and forth, so that the diluent in the injection chamber 410 enters the sample containing unit 100 and is uniformly mixed with the lysis freeze-dried powder in the sample containing unit 100 and the sample; heating the sample accommodating unit 100 to a set temperature, so that the sample in the sample accommodating unit 100 is fully cracked at the set temperature; after the lysis is completed, the piston 420 is pulled outward to a certain position again, so that the lysed sample mixture in the sample-containing unit 100 flows to the injection chamber 410; thereafter, the sample control valve 810 is closed and the dilution control valve 820 is opened; then, the piston 420 is moved back and forth, so that the lysed sample mixed liquid in the injection chamber 410 returns to the diluent containing unit 200, and is uniformly mixed with the diluent remaining in the diluent containing unit 200, thereby diluting the lysed sample mixed liquid and reducing the concentration of impurities therein; thereafter, the piston 420 is pulled outward to a certain position again, so that the diluted sample mixture in the diluent accommodating unit 200 flows to the injection chamber 410; thereafter, the dilution control valve 820 is closed, and the first PCR control valve 830 and the second PCR control valve 840 are opened; then, the piston 420 is moved back and forth, so that the diluted sample mixed solution in the injection chamber 410 enters the PCR reaction unit 300 and is uniformly mixed with the reverse transcriptase in the PCR reaction unit 300 and the freeze-dried powder of the PCR raw material; and finally, performing PCR temperature heating control on the PCR reaction unit 300, activating a preliminary constant temperature section of enzyme in PCR amplification, wherein the mixed solution of the PCR reaction unit 300 expands due to high temperature, overflowing liquid in the expansion process can flow into the buffer unit 500, and after the constant temperature section is finished, closing the first PCR control valve 830 and the second PCR control valve 840, and starting to perform temperature cycle control on the PCR reaction unit 300 so as to finally complete the PCR amplification reaction. From this, utilize the basis the utility model discloses PCR reaction that PCR reaction system goes on can solve the problem that overflows of PCR reagent when the high temperature inflation, and PCR reaction goes on under airtight environment, has reduced the pollution to system's environment, has improved the credibility of experiment, and the operation is simple accurate more.

According to an embodiment of the present invention, referring to fig. 3, the system further includes: a sample-accommodating unit sealing member 610, the sample-accommodating unit sealing member 610 being provided on a surface of the first liquid outlet/inlet port 110, for performing a first sealing process on the sample-accommodating unit 100; and a diluent receiving unit sealing member 620, the diluent receiving unit sealing member 620 being provided on a surface of the diluent outlet 210 for performing a second sealing process on the diluent receiving unit 200.

According to the embodiment of the present invention, referring to fig. 3, in the initial state, the sample containing unit 100 contains the lysis material existing in the form of freeze-dried powder, the PCR reaction unit 300 contains the reverse transcriptase and the PCR material existing in the form of freeze-dried powder, and the diluent containing unit 200 contains the appropriate diluent. The sample accommodating unit 100 and the diluent accommodating unit 200 are sealed by a sample accommodating unit sealing member 610 and a diluent accommodating unit sealing member 620 at the positions communicating with the microfluidic circuit, so that the lysis material of the sample accommodating unit 100, the diluent of the diluent accommodating unit 200, and the reverse transcriptase and PCR material of the PCR reaction unit 300 are isolated from each other, and the piston 420 is located at the topmost end of the injection chamber 410 (i.e., the injection chamber is in a state of being filled with the piston). It should be noted that, on the premise that the PCR reaction system has the sample containing unit sealing member and the diluent containing unit sealing member, the PCR reaction system can also have the function of isolating each unit without arranging a PCR reaction sealing device, and even if a small amount of reactants in the PCR reaction unit enter the pipeline, the whole reaction is not greatly affected. The inventor finds that the sample containing unit sealing element and the diluent containing unit sealing element can isolate reactants in each independent unit, so that the reactants can be conveniently stored for a long time under the condition of nonuse, the storage time of each reactant in each unit is greatly prolonged, the pollution of each reactant to a PCR reaction system is avoided, and the service life of the PCR reaction system is prolonged.

According to an embodiment of the present invention, referring to fig. 3, the system further includes: a sample-containing cell seal piercing device 710, said sample-containing cell seal piercing device 710 for subjecting said sample-containing cell seal 610 to a first piercing process; and a diluent containing unit seal piercing means 720, said diluent containing unit seal piercing means 720 being for subjecting said diluent containing unit seal 620 to a second piercing process. According to the embodiment of the present invention, after the sample is added to the sample holding unit 100, the system starts to work, and first, the piercing device 710/720 pierces the sample holding unit sealing member 610 and the diluent holding unit sealing member 620, so as to make the units and the microfluidic pipeline in a communicating state, thereby facilitating the subsequent processes.

According to an embodiment of the present invention, at least one of the sample receiving cell seal 610 and/or the diluent receiving cell seal 620 is a sealing membrane.

According to an embodiment of the invention, the sealing membrane is prepared from at least one of a tin foil paper, a plastic film or a kraft paper.

According to an embodiment of the present invention, the thickness of the sealing membrane is 0.01-0.2 mm, such as 0.03mm, 0.05mm, 0.07mm, 0.09mm, 0.1mm, 0.13mm, 0.15mm, 0.17mm or 0.19 mm. The inventors have found that if the thickness of the sealing film is too small, permeation is likely to occur, and if the thickness of the sealing film is too large, piercing is difficult. In some embodiments, the sealing film has a thickness of 0.05 to 0.1 mm.

The invention is further described below by means of specific examples.

Example 1

The structure of the system is as follows:

referring to fig. 3, the structure of the system includes: a sample-receiving unit 100 designed as a sample chamber, a diluent-receiving unit 200 designed as a dilution chamber, an injection chamber 410, a piston 420, a PCR reaction unit 300 designed as a PCR chamber, a buffer unit 500 designed as a buffer chamber, a sample-receiving unit seal 610 designed as a sample sealing membrane, a diluent-receiving unit seal 620 designed as a diluent sealing membrane, a microfluidic circuit, a sample control valve 810, a diluent control valve 820, a first PCR control valve 830, a second PCR control valve 840, which are connected by the microfluidic circuit to form an associated circuit.

The working principle of the system is as follows:

referring to FIG. 3, in the initial state, the sample chamber contains lysis material in the form of lyophilized powder, the PCR chamber contains reverse transcriptase and PCR material in the form of lyophilized powder, and the dilution chamber contains an appropriate diluent. The sample chamber and the diluting chamber are sealed by a sample sealing film and a diluting sealing film at the position communicated with the microfluidic pipeline, so that the cracking raw material of the sample chamber is mutually isolated from the diluting liquid of the diluting chamber and the reverse transcriptase and the PCR raw material of the PCR chamber. The piston is at the very top of the injection chamber (the injection chamber is in a piston filled state).

The system starts when the sample chamber has added a sample. First, the sample sealing film and the dilution sealing film are pierced by the piercing means 710/720, so that the cells and the micro flow hole line are in communication with each other. And secondly, closing the sample control valve, the first PCR control valve and the second PCR control valve, and moving the piston to pull outwards to a certain position, so that the diluent in the dilution chamber flows to the injection chamber through the dilution control valve. And thirdly, closing the dilution control valve, opening the sample control valve, reciprocating the piston, and allowing part of the diluent in the injection chamber to enter the sample chamber through the sample control valve, wherein in the process of reciprocating the piston, the cracking freeze-dried powder in the sample chamber, the diluent and the added sample are fully and uniformly mixed. Fourthly, heating the sample chamber to the set temperature is started, so that the sample in the sample chamber is fully cracked at the set temperature. Fifthly, after the lysis is finished, the movable piston is pulled outwards to a certain position, so that the lysed sample mixed liquid in the sample chamber flows to the injection chamber through the sample control valve. Sixthly, closing the sample control valve, opening the dilution control valve, reciprocating the piston, returning the sample mixed liquid cracked in the injection chamber to the dilution chamber, and fully and uniformly mixing the cracked sample mixed liquid with the rest diluent in the dilution chamber in the process of reciprocating the piston, so that the cracked sample mixed liquid is diluted, and the impurity concentration in the sample mixed liquid is reduced; seventhly, the piston is pulled outwards to a certain position again, so that the sample mixed liquid after being diluted in the diluting chamber flows to the injection chamber; and eighthly, closing the dilution control valve, opening the first PCR control valve and the second PCR control valve, reciprocating the piston, enabling the sample mixed liquid in the injection chamber to enter the PCR chamber through the PCR control valve, and fully and uniformly mixing the diluted sample mixed liquid with the reverse transcriptase and the PCR raw material freeze-dried powder in the PCR chamber in the process of reciprocating the piston. Ninth, begin to carry out PCR temperature heating control to the PCR room, the earlier stage thermostatted segment of activation enzyme in PCR amplifications, the mixed liquor in the PCR room can produce the inflation because of high temperature, and liquid overflow in the inflation process can flow to the buffer chamber through second PCR control valve in, after the thermostatted segment ended, closed first PCR control valve and second PCR control valve, begin to carry out temperature cycle control to the PCR room, finally accomplish the PCR amplification experiment.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," and "fixed" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally formed; may be mechanically coupled, may be electrically coupled or may be in communication with each other; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meaning of the above terms in the present invention can be understood according to specific situations by those skilled in the art.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art without departing from the scope of the present invention.

Claims

1. A PCR reaction system, comprising:

a sample containing unit in which a lysis raw material freeze-dried powder is disposed, and which has a first liquid outlet/inlet;

a diluent containing unit in which a diluent is disposed and which has a diluent outlet;

the PCR reaction unit is internally provided with reverse transcriptase and PCR raw material freeze-dried powder and is provided with a PCR reaction liquid outlet and a cracked sample mixed liquid inlet; and

a piston unit including an injection chamber and a piston, the injection chamber having a second liquid outlet/inlet;

the second liquid outlet/inlet is connected with the first liquid outlet/inlet through a first pipeline,

the second liquid outlet/inlet is connected with the diluent outlet through a second pipeline,

the second liquid outlet/inlet is connected with the cracked sample mixed liquid inlet through a third pipeline,

and the PCR reaction liquid outlet is connected with the diluent outlet through a fourth pipeline.

2. The system of claim 1, further comprising:

a sample control valve provided on the first pipeline for controlling a communication state of the first liquid outlet/inlet and the second liquid outlet/inlet;

a dilution control valve provided on the second pipeline for controlling a communication state of the diluent outlet and the second liquid outlet/inlet;

the first PCR control valve is arranged on the third pipeline and is used for controlling the communication state of the inlet of the sample mixed liquid after the cracking and the outlet/inlet of the second liquid; and

and the fourth pipeline is provided with the second PCR control valve which is used for controlling the communication state of the diluent outlet and the PCR reaction liquid outlet.

3. The system of claim 2, further comprising:

the buffer unit is provided with a PCR reaction liquid inlet and an air vent, the fourth pipeline is provided with the buffer unit, the second PCR control valve is connected with the PCR reaction liquid inlet, and the diluent outlet is connected with the air vent.

4. The system of claim 1, further comprising:

a sample-accommodating unit sealing member provided on the first liquid outlet/inlet surface for performing a first sealing process on the sample-accommodating unit; and

and a diluent containing unit sealing member provided on a surface of the diluent outlet and configured to perform a second sealing process on the diluent containing unit.

5. The system of claim 4, further comprising:

a sample receiving unit seal piercing device for subjecting the sample receiving unit seal to a first piercing process; and

a diluent containment unit seal piercing device for subjecting the diluent containment unit seal to a second piercing process.

6. The system of claim 4, wherein at least one of the diluent containing cell seal and the sample containing cell seal is a sealing membrane.

7. The system of claim 6, wherein the sealing membrane is formed from at least one of tinfoil paper, plastic film, or kraft paper.

8. The system of claim 6, wherein the sealing membrane has a thickness of 0.01 to 0.2 mm.

9. The system of claim 6, wherein the sealing membrane has a thickness of 0.05 to 0.1 mm.