CN112175786A - PCR reaction system - Google Patents

Abstract

The present invention provides a PCR reaction system, comprising: 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 invention relates to the field of molecular biology, in particular 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.

Disclosure of Invention

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 invention, the invention proposes a PCR reaction system. According to an embodiment of the invention, the system comprises: 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 invention, 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. The PCR reaction system provided by the embodiment of the invention realizes real full-automatic operation, solves the problem that the traditional PCR experiment process needs to be operated by professional people 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.

According to an embodiment of the present invention, the system may further include at least one of the following additional features:

according to an embodiment of the invention, 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. Therefore, the PCR reaction carried out by using the PCR reaction system provided by the embodiment of the invention is carried out in a closed environment, the pollution to the system environment is reduced, the experimental reliability is improved, and the operation is simpler and more convenient.

According to an embodiment of the invention, 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. Thus, the PCR reaction using the PCR reaction system according to the embodiment of the present invention can solve the problem of overflow of PCR reagents when the PCR reagents swell at high temperature.

According to an embodiment of the invention, 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.

According to an embodiment of the invention, 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, the sample containing unit and the diluent containing unit in the PCR reaction system according to the embodiment of the invention are in a communicated state with the microfluidic pipeline, so that the subsequent process is facilitated.

According to an embodiment of the invention, at least one of the diluent containing cell seal and the sample containing cell seal is a sealing membrane.

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

According to an embodiment of the invention, 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 illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

PCR reaction system

In a first aspect of the invention, a PCR reaction system is presented. According to an embodiment of the invention, with reference to fig. 1, the system comprises: 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.

The PCR reaction system according to the embodiment of the present invention connects thesample accommodating unit 100, the diluentaccommodating unit 200, thePCR reaction unit 300, and thepiston unit 400 to each other through a 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-accommodatingunit 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 PCR reaction system provided by the embodiment of the invention, the PCR reaction liquid outlet is connected with the diluent outlet through the fourth pipeline, so that the pressure system communication between the PCR reaction liquid outlet and the diluent outlet is formed, and excessive reaction liquid in the PCR reaction unit can smoothly flow out to the fourth pipeline through the reaction liquid outlet. 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 a communication state of thepiston unit 400 with the sample-accommodatingunit 100, the diluent-accommodatingunit 200, or thePCR 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. Therefore, according to the PCR reaction system provided by the embodiment of the invention, the piston unit is respectively connected with the sample containing unit, the diluent containing unit and the PCR reaction unit, so that a full-automatic process from sample nucleic acid extraction to reagent mixing and finally to PCR reaction is realized, the problem that a professional person needs to operate in a professional experiment environment in the traditional PCR experiment process is solved, the PCR reaction can be completed without the professional person, errors caused by manual operation are reduced, the working efficiency of PCR reaction is greatly improved, and the cost of human resources is greatly saved.

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: asample control valve 810, thesample control valve 810 being provided on thefirst pipe 910 for controlling a communication state of the second liquid inlet/outlet 411 and the first liquid inlet/outlet 110; adilution control valve 820, wherein thedilution control valve 820 is disposed on thesecond pipeline 920 and is used for controlling the communication state between the second liquid inlet/outlet 411 and thediluent outlet 210; a firstPCR control valve 830, wherein the firstPCR control valve 830 is disposed on thethird pipeline 930, and is used for controlling the communication state between the second liquid inlet/outlet 411 and the lysedsample mixture inlet 310; and a secondPCR control valve 840, wherein the secondPCR control valve 840 is disposed on thefourth pipeline 940, and is used for controlling the communication state between the PCRreaction solution outlet 320 and thediluent 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 PCR reaction system provided by the embodiment of the invention, each unit and each valve are perfectly matched and play a role in a synergistic manner, so that the pollution of the experimental product to the environment and the pollution of the environment to the experimental process are reduced, the full automation is favorably realized, and the manual operation of professionals is not needed. Therefore, the PCR reaction carried out by using the PCR reaction system according to the embodiment of the invention is carried out in a closed environment, the pollution to the system environment is reduced, the experimental reliability is improved, and the operation is simpler and more convenient.

According to an embodiment of the present invention, referring to fig. 3, the system further comprises:buffer unit 500,buffer unit 500 is provided with PCR reactionliquid inlet 510 and vent 520,buffer unit 500 sets up on thefourth pipeline 940, PCR reactionliquid inlet 510 with secondPCR control valve 840 links to each other, vent 520 withdiluent 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. Therefore, the PCR reaction carried out by using the PCR reaction system provided by the embodiment of the invention can solve the problem of overflow of the PCR reagent during high-temperature expansion, and the PCR reaction is carried out in a closed environment, so that the pollution to the system environment is reduced, the experimental reliability is improved, and the operation is simpler and more convenient.

According to an embodiment of the present invention, referring to fig. 3, the system further comprises: a sample-accommodatingunit sealing member 610, the sample-accommodatingunit 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-accommodatingunit 100; and a diluent receivingunit sealing member 620, the diluent receivingunit sealing member 620 being provided on a surface of thediluent outlet 210 for performing a second sealing process on thediluent receiving unit 200.

According to an embodiment of the present invention, referring to fig. 3, in an initial state, the sample-accommodatingunit 100 contains lysis material in a freeze-dried powder form, thePCR reaction unit 300 contains reverse transcriptase and PCR material in a freeze-dried powder form, and the diluent-accommodatingunit 200 contains a suitable diluent. Thesample accommodating unit 100 and the diluentaccommodating unit 200 are sealed by a sample accommodatingunit sealing member 610 and a diluent accommodatingunit sealing member 620 at the positions communicating with the microfluidic circuit, so that the lysis material of thesample accommodating unit 100, the diluent of the diluentaccommodating unit 200, and the reverse transcriptase and PCR material of thePCR reaction unit 300 are isolated from each other, and thepiston 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 comprises: a sample-containing cellseal piercing device 710, said sample-containing cellseal piercing device 710 for subjecting said sample-containingcell 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 containingunit seal 620 to a second piercing process. According to the embodiment of the present invention, when the sample is added to the sample-containingcell 100, the system starts to operate, and first, the sample-containingcell seal 610 and the diluent-containingcell seal 620 are pierced by the piercingdevice 710/720, so that each cell is in communication with the microfluidic circuit, thereby facilitating the subsequent processes.

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

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

According to an embodiment of the invention, 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.

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-receivingunit 100 designed as a sample chamber, a diluent-receivingunit 200 designed as a dilution chamber, aninjection chamber 410, apiston 420, aPCR reaction unit 300 designed as a PCR chamber, abuffer unit 500 designed as a buffer chamber, a sample-receivingunit seal 610 designed as a sample sealing membrane, a diluent-receivingunit seal 620 designed as a diluent sealing membrane, a microfluidic circuit, asample control valve 810, adiluent control valve 820, a firstPCR control valve 830, a secondPCR 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," "secured," and the like are to be construed broadly and can, 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 meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

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 above, 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 within the scope of the present invention.

Claims (9)

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.

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