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WO2021000750A1 - 利用pcr综合反应系统进行pcr反应的新方法 - Google Patents

利用pcr综合反应系统进行pcr反应的新方法 Download PDF

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Publication number
WO2021000750A1
WO2021000750A1 PCT/CN2020/097142 CN2020097142W WO2021000750A1 WO 2021000750 A1 WO2021000750 A1 WO 2021000750A1 CN 2020097142 W CN2020097142 W CN 2020097142W WO 2021000750 A1 WO2021000750 A1 WO 2021000750A1
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WIPO (PCT)
Prior art keywords
diluent
sample
pcr
unit
pcr reaction
Prior art date
Application number
PCT/CN2020/097142
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English (en)
French (fr)
Inventor
胡军荣
韩巧玲
徐强
崔相民
Original Assignee
申翌生物科技(杭州)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201921682904.1U external-priority patent/CN211339446U/zh
Priority claimed from CN201910954116.1A external-priority patent/CN112175787B/zh
Priority claimed from CN201910953826.2A external-priority patent/CN112176038A/zh
Priority claimed from CN201910954105.3A external-priority patent/CN112301097B/zh
Application filed by 申翌生物科技(杭州)有限公司 filed Critical 申翌生物科技(杭州)有限公司
Priority to EP20834221.2A priority Critical patent/EP3995563A4/en
Publication of WO2021000750A1 publication Critical patent/WO2021000750A1/zh
Priority to US17/551,153 priority patent/US20220106626A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
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    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
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    • B01L2200/0621—Control of the sequence of chambers filled or emptied
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    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0672—Integrated piercing tool
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2300/08—Geometry, shape and general structure
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    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
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    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/125—Specific component of sample, medium or buffer
    • C—CHEMISTRY; METALLURGY
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/629—Detection means characterised by use of a special device being a microfluidic device

Definitions

  • the present invention relates to the field of molecular biology, in particular, the present invention relates to a method for performing PCR reaction using a PCR reaction system.
  • the traditional PCR reaction process is generally carried out separately, that is, first extract the required nucleic acid through a nucleic acid extraction kit, and then mix the extracted nucleic acid and reagents into the PCR reaction tube, and finally put the PCR reaction tube into the PCR reaction tube.
  • the PCR amplification reaction is carried out in the PCR instrument to get the final result.
  • the traditional PCR reaction process has complicated operation steps and low work efficiency.
  • the traditional PCR reaction process has complicated operation steps and low work efficiency. Investigating the reason, the inventor found that each step in the traditional PCR reaction process generally requires professional operations, and these operations generally need to be performed separately through different instruments such as nucleic acid purifiers and fully automated workstations. In addition, the operation of the whole process also needs to be carried out in a standard PCR laboratory environment. Based on the above problems, the inventor integrated the extraction of nucleic acid, the mixing of nucleic acid and reagents, and the final PCR reaction into one system through a microfluidic pipeline. The method of using this system to perform PCR reactions realized a truly fully automated operation. It solves the problem that the traditional PCR experiment process needs to be operated by professionals in a professional experimental environment, reduces the error caused by human operation, greatly improves the working efficiency of the PCR reaction, and greatly saves the cost of human resources.
  • the present invention proposes a method for performing PCR reactions using a PCR comprehensive reaction system.
  • the PCR reaction system includes: a sample containing unit in which the lysed lyophilized powder and a sample are arranged, and the sample containing unit has a first liquid inlet/outlet; and a diluent containing Unit, the diluent accommodating unit is provided with a diluent, and the diluent accommodating unit has a diluent outlet; a PCR reaction unit, the PCR reaction unit is provided with reverse transcriptase and PCR raw material freeze-dried powder, and
  • the PCR reaction unit has an outlet for the PCR reaction solution and an inlet for the lysed sample mixture; and a piston unit, the piston unit includes an injection chamber and a piston, and the injection chamber has a second liquid outlet/inlet, the second liquid The outlet/inlet is connected to the first liquid outlet/inlet through a first pipeline
  • the method includes: subjecting the piston to a first movement process so as to Part of the diluent enters the injection chamber, and the diluent is set in the diluent containing unit; the piston is subjected to a second movement process, so that the diluent and the lysed lyophilized powder entering the injection chamber are combined
  • the sample is subjected to a first mixing process, the lysed lyophilized powder and the sample are set in the sample holding unit, and the first mixing process is performed in the sample holding unit;
  • the first mixed process product is subjected to Lysis treatment, the lysis treatment is performed in the sample holding unit;
  • the piston is subjected to a third movement treatment so that the lysis treatment product enters the injection chamber;
  • the piston is subjected to a fourth movement treatment to The lysis treatment product entering the injection chamber and the remaining part of the
  • the PCR reaction system connects the sample holding unit, the diluent holding unit, the PCR reaction unit, and the piston unit to each other through a microfluidic pipeline; at the same time, each unit is an independent unit to make Each unit stores different reactants before use, which is conducive to long-term storage of reactants when not in use.
  • the independent setting of the sample holding unit facilitates the separate addition of samples, simplifies the operation of adding samples, and also facilitates long-term preservation of samples.
  • the method according to the embodiment of the present invention realizes a truly fully automated operation, solves the problem that the traditional PCR experiment process needs to be operated by professionals in a professional experimental environment, reduces errors caused by human operation, and greatly improves the PCR reaction
  • the work efficiency greatly saves the cost of human resources.
  • the above method may further include at least one of the following additional technical features:
  • the PCR temperature cycle amplification treatment includes: subjecting the third mixed treatment product to a constant temperature treatment; and subjecting the constant temperature treatment product to a temperature cycle treatment.
  • the PCR reaction system further includes: a sample control valve.
  • the sample control valve is provided on the first pipeline for controlling the first liquid inlet/outlet and the second liquid The connection state of the inlet/outlet; the dilution control valve, which is provided on the second pipeline, is used to control the communication state of the dilution liquid inlet and the second liquid outlet/inlet; the first PCR control A valve, where the first PCR control valve is provided on the third pipeline for controlling the communication state between the lysed sample mixture inlet and the second liquid outlet/inlet; and a second PCR control valve, The second PCR control valve is provided on the fourth pipeline to control the communication state between the diluent outlet and the PCR reaction solution outlet; the method further includes: before the first moving process, Close the sample control valve, the first PCR control valve, and the second PCR control valve, open the dilution control valve; after the first movement process and before the second movement process, close the Dilution control valve, open the sample control valve; after the third
  • the PCR reaction system further includes: a buffer unit having a PCR reaction solution inlet and a vent, the buffer unit is provided on the fourth pipeline, and the second PCR control
  • the valve is connected to the PCR reaction solution inlet, and the diluent outlet is connected to the vent. Therefore, the method according to the embodiment of the present invention can solve the overflow problem of PCR reagents when expanding at high temperature, and the method is carried out in a closed environment, which reduces the pollution of the system environment and improves the reliability of the experiment. It's easier.
  • the PCR reaction system further includes: a sample containing unit seal, and the sample containing unit seal is provided on the first liquid inlet/outlet surface for performing the first liquid A sealing treatment; a diluent containing unit seal, the diluent containing unit seal is provided on the surface of the diluent outlet, for performing a second sealing treatment on the diluent containing unit; the method further includes: The piercing treatment, the pre-piercing treatment includes subjecting the sample holding unit seal to a first piercing treatment, and subjecting the diluent holding unit seal to a second piercing treatment.
  • the sample containment unit seal and the diluent containment unit seal can not only isolate the reactants in each independent unit, but also facilitate long-term storage when not in use, greatly improving the storage time of each reactant in each unit. Moreover, the contamination of the PCR reaction system caused by the reactants is avoided, and the service life of the PCR reaction system is improved. Furthermore, when the sample containing unit is added with a sample, it is only necessary to pierce the seal of the sample containing unit and the seal of the diluent containing unit to make the sample containing unit, the diluent containing unit and the microfluidic pipeline in a communication state , The system can start working. Therefore, the operation of the method according to the embodiment of the present invention is more convenient.
  • the first piercing process is performed by the sample holding unit seal piercing device, and the second piercing process is performed by the diluent holding unit seal piercing device.
  • At least one of the diluent containing unit seal and the sample containing unit seal is a sealing film.
  • the sealing film is formed of at least one of tin foil, plastic film, and kraft paper.
  • the thickness of the sealing film is 0.01-0.2mm, such as 0.03mm, 0.05mm, 0.07mm, 0.09mm, 0.1mm, 0.13mm, 0.15mm, 0.17mm or 0.19mm.
  • the thickness of the sealing film is 0.05 to 0.1 mm.
  • the present invention provides a PCR reaction system.
  • the system includes: a sample containing unit in which the lysed raw material freeze-dried powder is arranged, and the sample containing unit has a first liquid inlet/outlet; a diluent containing unit, The diluent containing unit is provided with a diluent, and the diluent containing unit has a diluent outlet; a PCR reaction unit, the PCR reaction unit is provided with reverse transcriptase and PCR raw material freeze-dried powder, and the PCR
  • the reaction unit has an outlet for the PCR reaction solution and an inlet for the lysed sample mixture; and a piston unit, the piston unit includes an injection chamber and a piston, the injection chamber has a second liquid outlet/inlet; the second liquid outlet/inlet Connected to the first liquid outlet/inlet through a first pipeline, the second liquid outlet/inlet is connected to the diluent outlet through
  • the PCR reaction system connects the sample holding unit, the diluent holding unit, the PCR reaction unit, and the piston unit to each other through a microfluidic pipeline; at the same time, each unit is an independent unit to make Each unit stores different reactants before use, which is conducive to long-term storage of reactants when not in use.
  • the independent setting of the sample holding unit facilitates the separate addition of samples, simplifies the operation of adding samples, and also facilitates long-term preservation of samples.
  • the PCR reaction system according to the embodiment of the present invention realizes a truly fully automated operation, solves the problem that the traditional PCR experiment process needs to be operated by professionals in a professional experimental environment, reduces errors caused by human operation, and greatly improves The working efficiency of the PCR reaction greatly saves the cost of human resources.
  • the aforementioned system may further include at least one of the following additional technical features:
  • the system further includes: a sample control valve, where the sample control valve is provided on the first pipeline for controlling the first liquid inlet/outlet and the second liquid outlet/outlet/ The communication state of the inlet; a dilution control valve, where the dilution control valve is provided on the second pipeline for controlling the communication state of the diluent outlet and the second liquid inlet/outlet; the first PCR control valve, The first PCR control valve is provided on the third pipeline for controlling the communication state between the inlet of the lysed sample mixture and the second liquid outlet/inlet; and a second PCR control valve, located in the The second PCR control valve is provided on the fourth pipeline to control the communication state between the diluent outlet and the PCR reaction solution outlet. Therefore, the PCR reaction performed by the PCR reaction system according to the embodiment of the present invention is performed in a closed environment, which reduces the pollution to the system environment, improves the reliability of the experiment, and the operation is more convenient.
  • the system further includes: a buffer unit having a PCR reaction solution inlet and a vent, the buffer unit is provided on the fourth pipeline, and the second PCR control valve is connected to The PCR reaction solution inlet is connected, and the diluent outlet is connected to the vent. Therefore, the PCR reaction performed by the PCR reaction system according to the embodiment of the present invention can solve the problem of overflow of the PCR reagent when the PCR reagent expands at high temperature.
  • the system further includes: a sample containing unit sealing member, the sample containing unit sealing member is provided on the first liquid inlet/outlet surface for first sealing the sample containing unit Processing; and a diluent accommodating unit seal, the diluent accommodating unit seal is provided on the diluent outlet surface, for performing a second sealing treatment on the diluent accommodating unit.
  • a sample containment unit seal and the diluent containment unit seal can not only isolate the reactants in each independent unit, but also facilitate long-term storage when not in use, greatly improving the storage time of each reactant in each unit. Moreover, the contamination of the PCR reaction system caused by the reactants is avoided, and the service life of the PCR reaction system is improved.
  • the system further includes: a sample containing unit seal piercing device, the sample containing unit seal piercing device is used to perform a first piercing process on the sample containing unit seal; and Diluent accommodating unit seal piercing device, the diluent accommodating unit seal piercing device is used to perform a second piercing process on the diluent accommodating unit seal. Therefore, the sample containing unit and the diluent containing unit in the PCR reaction system according to the embodiment of the present invention are in a communication state with the microfluidic pipeline, which facilitates the subsequent process.
  • At least one of the diluent containing unit seal and the sample containing unit seal is a sealing film.
  • the sealing film is formed of at least one of tin foil, plastic film or kraft paper.
  • the thickness of the sealing film is 0.01-0.2mm, such as 0.03mm, 0.05mm, 0.07mm, 0.09mm, 0.1mm, 0.13mm, 0.15mm, 0.17mm or 0.19mm.
  • the thickness of the sealing film is 0.05 to 0.1 mm.
  • the lyophilized powder of the lysis raw material includes a metal ion chelator, sodium lauryl sulfate, saponin, proteinase K, polyethylene glycol 3350, Tris-HCl, water; the reverse transcriptase And PCR raw material freeze-dried powder includes mannitol, sucrose, chloride, bovine serum albumin, dNTP, polyoxyethylene lauryl ether, HEPES, DNA polymerase, reverse transcriptase and RNase inhibitor, water.
  • the metal ion chelating agent is EDTA and EGTA.
  • the chloride salt is potassium chloride and magnesium chloride.
  • the diluent includes: polyol, chloride salt, Tris-HCl, surfactant and water.
  • the polyol is glycerol.
  • the preparation and dispensing of fluorescent quantitative PCR reagents are required to be carried out in the standard reagent preparation room to prevent sample contamination, and the preparation and dispensing operations are complicated and require specially trained personnel.
  • the inventor has researched and developed a composition in which the first component can be effectively used for sample lysis and nucleic acid extraction, and the second component can be effectively used for PCR amplification reaction.
  • the first component has significantly reduced toxicity and pollution, significantly improved safety and stability, is advantageous for transportation and storage, and has reduced requirements for samples;
  • the second component The stability is significantly improved, which is beneficial to transportation and storage, and reduces the requirements on the experimental environment; the combined use of the first component and the second component can make the lysis of the sample, the extraction of nucleic acid and the preparation of fluorescent quantitative PCR reagents unnecessary Professionals can complete it, reducing the technical requirements for operators, and the PCR amplification effect is excellent.
  • the present invention provides a composition for PCR reaction.
  • the composition includes: a first component, the first component includes a metal ion chelator, sodium dodecyl sulfate (SDS), saponin, proteinase K ), polyethylene glycol 3350 (PEG 3350), Tris-HCl, water; and/or a second component including mannitol, sucrose, chloride, bovine serum albumin (BSA), dNTP, polyoxyethylene lauryl ether (Brij 35), HEPES, DNA polymerase, reverse transcriptase and RNase inhibitors, water.
  • SDS sodium dodecyl sulfate
  • PEG 3350 polyethylene glycol 3350
  • Tris-HCl water
  • a second component including mannitol, sucrose, chloride, bovine serum albumin (BSA), dNTP, polyoxyethylene lauryl ether (Brij 35), HEPES, DNA polymerase, reverse transcriptase and RNase inhibitors, water
  • the metal ion chelating agent is EDTA and EGTA.
  • the chloride salt is potassium chloride and magnesium chloride.
  • the second component is stable and effective, which is beneficial to transportation and storage, and reduces the requirements for transportation and storage temperature; the first component and the second
  • the combined use of the components can make the lysis of the sample, the extraction of nucleic acid and the preparation of the fluorescent quantitative PCR reagents to be completed without professionals, which reduces the technical requirements for the operators, and the PCR amplification effect is excellent.
  • the above composition may further include at least one of the following additional technical features:
  • the concentration of the EDTA is 0.1-10 mmol/L, such as 0.5, 0.7, 1.0, 2.0, 3.0 or 4.0 mmol/L
  • the EGTA The concentration of proteinase K is 0.1-15 mmol/L, such as 0.5, 0.7, 3, 5, 7, 10, or 13 mmol/L, and the concentration of proteinase K is 5-150 U/mL, such as 7, 10, 15, 30, 45, 60, 75, 90 or 120 U/mL, the concentration of the sodium lauryl sulfate is 0.1-3.0%, such as 0.3, 0.5, 1.0, 1.5, 2.0 or 2.5%, and the concentration of the saponin is 0.1 ⁇ 3.0%, such as 0.3, 0.5, 1.0, 1.5, 2.0 or 2.5%, the concentration of the polyethylene glycol 3350 is 0.1-5.0%, such as 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or 4.
  • the concentration of the sodium lauryl sulfate, saponins and polyethylene glycol 3350 is the mass volume concentration, which refers to the sodium lauryl sulfate, saponins or polyethylene glycol per 100 mL of solution
  • the mass of 3350, the unit is g.
  • the concentration of the sodium lauryl sulfate is 0.1-3.0%, which means that the mass of the sodium lauryl sulfate in 100 mL of the first component is 0.1-3.0 g.
  • the concentration of the EDTA is 0.5-5 mmol/L
  • the concentration of the EGTA is 0.5-10 mmol/L
  • the concentration of proteinase K is 10 ⁇ 100U/mL
  • the concentration of the sodium lauryl sulfate is 0.5-2.5%
  • the concentration of the saponin is 0.5-2.5%
  • the concentration of the polyethylene glycol 3350 is 0.5-4.5%.
  • the Tris-HCl is provided in a water-soluble form. It should be noted that Tris-HCl is a type of buffer material commonly used in this field, which can be configured by itself or purchased directly.
  • the pH of the solution formed by dissolving the Tris-HCl in water is 7.5-8.2, such as 7.6, 7.7, 7.8, 7.9, 8.0 or 8.1. It should be noted that the pH does not refer to the pH of the first component, but the pH of a solution formed by dissolving Tris-HCl in water. According to an embodiment of the present invention, when the pH of the solution formed by dissolving Tris-HCl in water is 7.5-8.2, Tris-HCl has a better buffering effect on the first component, which is more conducive to sample lysis and nucleic acid extraction , The stability is better, and the subsequent PCR amplification effect is better. In some embodiments, the pH of the solution formed by dissolving Tris-HCl in water is 7.6.
  • the concentration of Tris-HCl is 1-25 mmol/L, such as 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24mmol/L.
  • the concentration of Tris-HCl is 1-25 mmol/L, Tris-HCl has a better buffering effect on the first component, which is more conducive to sample lysis and nucleic acid extraction, and has better stability. Good, furthermore, the subsequent PCR amplification effect is better.
  • the concentration of Tris-HCl is 5-20 mmol/L.
  • the concentration of potassium chloride is 10-150 mmol/L, such as 15, 20, 30, 40, 60, 80, 100 or 120 mmol/L L
  • the concentration of the magnesium chloride is 0.5-10.0 mmol/L, such as 0.7, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, or 10.0 mmol/L
  • the concentration of the dNTP is 150-250 ⁇ mol/L, such as 180, 200, or 230 ⁇ mol/L
  • the concentration of the DNA polymerase is 10-250 U/mL, such as 13, 15, 18, 20, 30, 50, 80, 100, 120, 150, 180, 200, or 230 U/mL mL
  • the concentration of the reverse transcriptase is 5-100 U/mL, such as 7, 10, 20, 30, 40, 50, 70 or 90 U/mL
  • the concentration of the RNase inhibitor is 100-1000 U/mL mL, such as 150, 200, 300, 500, 700 or
  • the concentration of the sucrose and polyoxyethylene lauryl ether is mass volume concentration, which refers to the mass of the sucrose or polyoxyethylene lauryl ether per 100 mL of the solution, and the unit is g.
  • concentration of sucrose is 0.1-10%, which means that the mass of sucrose per 100 mL of the second component is 0.1-10 g.
  • concentration of mannitol is 0.1-10%, which means that the volume of mannitol per 100 mL of the second component is 0.1-10 mL.
  • the concentration of the potassium chloride is 20-100 mmol/L
  • the concentration of the magnesium chloride is 1.0-5.0 mmol/L
  • the concentration of the dNTP The concentration of the DNA polymerase is 200 ⁇ mol/L
  • the concentration of the DNA polymerase is 20 to 200 U/mL
  • the concentration of the reverse transcriptase is 10 to 50 U/mL
  • the concentration of the RNase inhibitor is 200 to 1000 U/mL.
  • the concentration of mannitol is 0.5-8%
  • the concentration of sucrose is 0.5-8%
  • the concentration of bovine serum albumin is 0.1-1 mg/mL
  • the concentration of polyoxyethylene lauryl ether is 0.05 %.
  • the HEPES is provided in a water-soluble form. It should be noted that HEPES is a type of buffer material commonly used in this field, which can be configured by itself or purchased directly.
  • the pH of the solution formed by dissolving HEPES in water is 8.0-8.5, such as 8.1, 8.2, 8.25, 8.3 or 8.4. It should be noted that the pH does not refer to the pH of the second component, but the pH of the solution formed by dissolving HEPES in water. According to an embodiment of the present invention, when the pH of the solution formed by dissolving HEPES in water is 8.0-8.5, HEPES has a better buffering effect on the second component, which is more conducive to the PCR amplification reaction, and the PCR amplification effect is better , And higher stability. In some embodiments, the pH of the solution of HEPES dissolved in water is 8.25.
  • the concentration of the HEPES is 5 to 55 mmol/L, such as 10, 15, 20, 25, 30, 35, 40, 45 or 50 mmol/L L.
  • the concentration of the HEPES is 10-50 mmol/L.
  • the saponins include at least one selected from the group consisting of tea saponin, ginsenosides, saponins, and soybean saponin.
  • the DNA polymerase includes at least one selected from the group consisting of Taq enzyme and Tth DNA polymerase.
  • the reverse transcriptase includes at least one selected from M-MLV reverse transcriptase and AMV reverse transcriptase.
  • the RNase inhibitor includes at least one selected from the group consisting of diethyl pyrophosphate, guanidine isothiocyanate, vanadyl ribonucleoside complex, RNasin, urea, and diatomaceous earth.
  • the invention provides a composition.
  • the composition includes: a first component, based on the total volume of the first component, the first component includes EDTA at a concentration of 0.5-5 mmol/L, and a concentration of 0.5- 10mmol/L EGTA, proteinase K with a concentration of 10-100 U/mL, sodium lauryl sulfate with a concentration of 0.5-2.5%, saponin with a concentration of 0.5-2.5%, polyethylene with a concentration of 0.5-4.5% Alcohol 3350, Tris-HCl with a concentration of 5-20 mmol/L, and water; and/or a second component, based on the total volume of the second component, the second component including a concentration of 20-100 mmol/L L potassium chloride, magnesium chloride with a concentration of 1.0-5.0 mmol/L, dNTP with a concentration of 200 ⁇ mol/L, DNA polymerase with a concentration of 20-200 U/
  • the present invention provides a freeze-dried powder.
  • the lyophilized powder is prepared from the composition described in any one of the above, and the lyophilized powder is suitable for use in the PCR reaction method and PCR reaction using a PCR reaction system in the above embodiment System. Specifically, it can be set in the sample holding unit.
  • the inventor found that when the first component and/or the second component are in the form of freeze-dried powder, the stability is greatly improved, and the storage and transportation can be carried out under normal temperature conditions, which greatly reduces the need for storage and transportation. It can be reconstituted after mixing the lyophilized powder with a suitable buffer, while maintaining the original function.
  • sample lysis diluent and PCR diluent are very different in formula and composition.
  • the sample lysis diluent is dedicated to sample lysis and sample nucleic acid extraction, while the PCR diluent is dedicated to PCR amplification. To increase the reaction, the two diluents cannot be used in common.
  • the inventors studied and developed a diluent composition through a large number of experimental investigations, which can simultaneously play a buffer role in the sample lysis and PCR amplification reaction, thereby providing a good solution for sample nucleic acid extraction and fluorescent PCR reaction.
  • the integration provides technical support.
  • the present invention proposes a diluent.
  • the diluent includes: polyol, chloride salt, Tris-HCl, surfactant and water.
  • the polyol is glycerol; the diluent is suitable for use in the PCR reaction method and PCR reaction system using the PCR reaction system in the foregoing embodiments. Specifically, it can be arranged in the diluent containing unit.
  • the diluent according to the embodiment of the present invention has a certain buffering effect on the sample lysis solution and the PCR reaction system. After the buffer dilution of the buffer solution, the lysis solution after the sample lysis does not need to be purified and can be directly added to the PCR reaction system to complete
  • the PCR reaction provides technical support for the integration of sample nucleic acid extraction and PCR detection.
  • the aforementioned diluent may further include at least one of the following additional technical features:
  • the surfactant includes selected from Tween 20 (Tween 20), Tween 80, polyethylene glycol octyl phenyl ether, sodium dodecyl sulfate (SDS), dodecyl At least one of sodium benzene sulfonate, sodium dioctyl succinate sulfonate, and sodium glycocholate.
  • the chloride salt includes at least one selected from potassium chloride, sodium chloride, and magnesium chloride.
  • the chloride salt is magnesium chloride and sodium chloride.
  • the concentration of the magnesium chloride is 0.5-15 mmol/L, such as 1.0, 1.5, 2.0, 3.0, 5.0, 6.0, 8.0, 10.0 or 12.0mmol/L
  • the concentration of the sodium chloride is 1 ⁇ 150mmol/L, such as 2, 4, 5, 10, 20, 40, 50, 60, 80, 100 or 120mmol /L
  • the concentration of the surfactant is 0.1 to 7%, such as 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 or 7.0%
  • the concentration of the glycerol is 2 -20%, such as 3, 5, 7, 8, 9, 12, 15 or 18%.
  • the concentration of the surfactant is the mass volume concentration, which refers to the mass of the surfactant per 100 mL of the solution, and the unit It is g.
  • the concentration of the sodium lauryl sulfate is 0.1 to 7%, which means that the mass of the sodium lauryl sulfate in 100 mL of the diluent is 0.1 to 7 g.
  • the surfactant is a liquid such as polyethylene glycol octyl phenyl ether or Tween 20
  • the concentration of the surfactant is the volume concentration, which refers to the volume of the surfactant per 100 mL of the solution.
  • the unit is mL.
  • the concentration of the polyethylene glycol octyl phenyl ether or Tween 20 is 0.1 to 7%, which means that the polyethylene glycol octyl phenyl ether or Tween 20 in 100 mL of diluent
  • the volume is 0.1-7mL.
  • the concentration of glycerol 2-20% is the volume concentration, which means that the volume of glycerol per 100 mL of the diluted solution is 2-20 mL. The inventor found that when the concentration of each component in the diluent is within this range, the diluent can more effectively play a buffering role in both sample lysis and PCR amplification reactions.
  • the chloride salt is magnesium chloride and sodium chloride. Based on the total volume of the diluent, the concentration of the magnesium chloride is 1.5-10 mmol/L, and the concentration of the sodium chloride is 5- 100mmol/L, the concentration of the surfactant is 0.1 to 5%, and the concentration of the glycerol is 5 to 10%.
  • the Tris-HCl is provided in a water-soluble form, which means that the diluent of the present invention is added in the form of a Tris-HCl aqueous solution when the diluent is configured.
  • the Tris-HCl aqueous solution is a type of buffer material commonly used in the field, and it can be configured by itself. For example, it can be slowly adjusted to a predetermined pH with Tris base and hydrochloric acid, or it can be purchased directly.
  • the pH of the solution formed by dissolving Tris-HCl in water is 7.5-8.0, such as 7.6, 7.7, 7.8, 7.9 or 8.0. It should be noted that the pH does not refer to the pH of the diluent, but the pH of a solution formed by dissolving Tris-HCl in water. According to an embodiment of the present invention, when the pH of the solution formed by dissolving the Tris-HCl in water is 7.5-8.0, the buffering effect of Tris-HCl on the diluent is better. In some embodiments, the pH of the solution formed by dissolving Tris-HCl in water is 7.6.
  • the concentration of the Tris-HCl is 1-100 mmol/L, such as 3, 5, 7, 10, 20, 30, 40, 50, 70, 90 or 100mmol/L. It should be noted that the concentration of Tris-HCl can be understood according to the conventional knowledge of those skilled in the art. According to an embodiment of the present invention, when the concentration of Tris-HCl is 1-100 mmol/L, Tris-HCl has a better buffering effect on the diluent. In some embodiments, the concentration of Tris-HCl is 5-50 mmol/L.
  • the invention provides a diluent.
  • the diluent includes: magnesium chloride with a concentration of 1.5-10 mmol/L, sodium chloride with a concentration of 5-100 mmol/L, and a concentration of 0.1-5% Surfactant, glycerol with a concentration of 5-10%, Tris-HCl with a concentration of 5-50mmol/L, and water.
  • the surfactant is Tween 20, sodium lauryl sulfate or polyethylene Glycol octyl phenyl ether;
  • the diluent is suitable for use in the PCR reaction method and PCR reaction system using the PCR reaction system in the above embodiment. Specifically, it can be arranged in the diluent containing unit.
  • the diluent according to the embodiment of the present invention can more effectively play a buffering role in both sample lysis and PCR amplification reaction, and therefore, can also be used as a buffer for PCR reaction.
  • Fig. 1 is a schematic flow chart of a method for performing a PCR reaction using a PCR reaction system according to an embodiment of the present invention
  • FIG. 2 is a schematic flow chart of a method for performing PCR reaction using a PCR reaction system according to another embodiment of the present invention
  • Fig. 3 is a schematic flow chart of a method for performing a PCR reaction using a PCR reaction system according to another embodiment of the present invention
  • FIG. 4 is a schematic flow chart of a method for performing PCR reaction using a PCR reaction system according to another embodiment of the present invention.
  • Figure 5 is a schematic structural diagram of a PCR system according to an embodiment of the present invention.
  • Fig. 6 is a schematic structural diagram of a PCR system according to another embodiment of the present invention.
  • Fig. 7 is a schematic structural diagram of a PCR system according to another embodiment of the present invention.
  • FIG. 9 is a schematic diagram of test results according to Embodiment 3 of the present invention.
  • Figure 10 is a schematic diagram of the test results of Comparative Example 1 according to the present invention.
  • FIG. 11 is a schematic diagram of the test result of Comparative Example 2 according to the present invention.
  • Figure 13 is a schematic diagram of the test results of Comparative Example 4 according to the present invention.
  • Example 15 is a schematic diagram of the property test results according to Example 4 of the present invention.
  • FIG. 17 is a schematic diagram of the property test result according to Embodiment 6 of the present invention.
  • Example 7 is a schematic diagram of the test results of the properties of Example 7 according to the present invention.
  • FIG. 20 is a schematic diagram of the property test results of Comparative Example 7 according to the present invention.
  • the PCR reaction system includes: a sample containing unit 100, the sample containing unit 100 is provided with a lysate and a sample, and the sample containing unit 100 is provided with a first liquid Outlet/inlet 110; diluent containing unit 200, the diluent containing unit 200 is provided with a diluent, and the diluent containing unit 200 is provided with a diluent outlet 210; PCR reaction unit 300, the PCR reaction unit 300 is provided with reverse transcriptase and PCR raw material freeze-dried powder, and the PCR reaction unit 300 is provided with a lysed sample mixture inlet 310 and a PCR reaction solution outlet 320, the PCR reaction solution outlet 320 and the dilution
  • the liquid outlet 210 is connected by a fourth pipeline 940; and a piston unit 400,
  • the method includes: subjecting the piston 420 to a first movement processing S100, so that a part of the diluent enters the injection chamber 410, and the diluent is set in the diluent containing unit 200;
  • the piston 420 performs a second movement process S200, so that the diluent entering the injection chamber 410, the lysed lyophilized powder and the sample are subjected to a first mixing process, and the lysed lyophilized powder and the sample are set in the sample containing unit
  • the first mixing process is performed in the sample holding unit 100;
  • the first mixed processing product is subjected to a lysis process S300, and the lysis process is performed in the sample holding unit 100;
  • the piston 420 is subjected to a third movement process S400 to allow the pyrolysis treatment product to enter the injection chamber 410;
  • the piston 420 is subjected to a fourth movement treatment S500 to dilute the cleavage treatment product entering the injection chamber 410 with
  • the PCR reaction system connects the sample holding unit 100, the diluent holding unit 200, the PCR reaction unit 300, and the piston unit 400 to each other through a microfluidic pipeline; at the same time, each unit is independently set Units, so that each unit can store different reactants before use, which is conducive to long-term storage of reactants when not in use.
  • each unit is independently set Units, so that each unit can store different reactants before use, which is conducive to long-term storage of reactants when not in use.
  • the independent setting of the sample holding unit 100 facilitates the separate addition of samples, simplifies the operation of adding samples, and also facilitates long-term preservation of samples.
  • the PCR reaction solution outlet and the diluent outlet are connected through a fourth pipeline, forming a pressure system connecting the PCR reaction solution outlet and the diluent outlet, so that the PCR reaction unit The excess reaction liquid inside can smoothly flow out to the fourth pipeline through the reaction liquid outlet.
  • a valve or other switch can be flexibly designed at a suitable position of the microfluidic pipeline to control the piston unit 400 and the sample containing unit 100, the diluent containing unit 200 or the PCR reaction The connection status of the unit 300.
  • the movement of the piston and the control of valves or other switches can also be flexibly designed with other mechanical devices to achieve automation.
  • the lysed sample mixture is returned to the diluent accommodating unit and mixed evenly with the remaining diluent in the diluent accommodating unit.
  • the lysed sample mixture can be diluted to The concentration of impurities is significantly reduced, so as to prevent the impurities generated after lysis from adversely affecting the subsequent PCR amplification reaction, which is beneficial to the automation of the PCR reaction. It should be noted that those skilled in the art can design the specific dosage ratio of the two diluents according to actual needs.
  • the PCR reaction method according to the embodiment of the present invention realizes the fully automatic process from sample nucleic acid extraction to mixing with reagents, and finally to PCR reaction, which solves the need for the traditional PCR experiment process to be operated by professionals in a professional experimental environment
  • the problem can be completed without professionals, and the error caused by human operation is reduced, the working efficiency of the PCR reaction is greatly improved, and the cost of human resources is greatly saved.
  • the PCR temperature cycle amplification treatment S800 includes: subjecting the third mixed treatment product to a constant temperature treatment S810; and subjecting the constant temperature treatment product to a temperature cycle treatment S820.
  • the PCR reaction system further includes: a sample control valve 810, the sample control valve 810 is disposed on the first pipeline 910, and is used to control the second liquid The connection state of the inlet/outlet 411 and the first liquid outlet/inlet 110; a dilution control valve 820, which is arranged on the second pipeline 920 and is used to control the outlet/inlet of the second liquid The connection state of the inlet 411 and the diluent outlet 210; the first PCR control valve 830, the first PCR control valve 830 is arranged on the third pipeline 930, and is used to control the second liquid outlet/inlet 411 and the lysed sample mixture inlet 310; and the second PCR control valve 840, the second PCR control valve 840 is provided on the fourth pipeline 940, for controlling the PCR reaction The communication state of the liquid outlet 320 and the diluent outlet 210;
  • the method further includes: S910: before the first movement processing, closing the sample control valve, the first PCR control valve, and the second PCR control valve, and diluting the The control valve is opened; S920: after the first movement and before the second movement, the dilution control valve is closed, and the sample control valve is opened; S930: After the third movement process and before the fourth movement process, the sample control valve is closed and the dilution control valve is opened; S940: after the fifth movement process and before the sixth movement process , The dilution control valve is closed, and the first PCR control valve and the second PCR control valve are opened; S950: after the constant temperature treatment and before the temperature cycle treatment, the first The PCR control valve and the second PCR control valve are closed.
  • the first PCR control valve 830, the second PCR control valve 840, and the sample control valve 810 close the first PCR control valve 830, the second PCR control valve 840, and the sample control valve 810, and open the dilution control valve 820; then pull the piston 420 outward to Certain position, so that part of the diluent in the diluent holding 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 reciprocated to make the diluent in the injection chamber 410 enter the sample holding
  • the unit 100 is uniformly mixed with the lysed lyophilized powder and the sample in the sample holding unit 100; then the sample holding unit 100 is heated to a set temperature, so that the sample in the sample holding unit 100 is fully heated at the set temperature.
  • Lysis after the lysis is completed, pull the piston 420 outward again to a certain position, so that the lysed sample mixture in the sample holding unit 100 flows to the injection chamber 410; then, close the sample control valve 810 and open the dilution control valve 820; The piston 420 is moved back and forth to return the lysed sample mixture in the injection chamber 410 to the diluent holding unit 200, and at the same time, it is evenly mixed with the remaining diluent in the diluent holding unit 200 to dilute the lysed sample mixture.
  • the impurity concentration therein is reduced; afterwards, the piston 420 is pulled outward again to a certain position, so that the diluted sample mixture in the diluent holding unit 200 flows to the injection chamber 410; afterwards, the dilution control valve 820 is closed, and the first PCR is opened Control valve 830 and the second PCR control valve 840; then reciprocate the piston 420, so that the diluted sample mixture in the injection chamber 410 enters the PCR reaction unit 300, and at the same time with the reverse transcriptase in the PCR reaction unit 300 and PCR raw materials
  • the freeze-dried powder is mixed uniformly; finally, the PCR temperature heating control is performed on the PCR reaction unit 300, including the prophase constant temperature stage of activating the enzyme and the temperature cycle control stage.
  • the first PCR control valve 830 and the second PCR control are closed Valve 840 to finally complete the PCR amplification reaction.
  • each unit and each valve are perfectly coordinated to work together, so that the environmental pollution of the experimental product and the environmental pollution to the experimental process are reduced, which is beneficial to realize full automation, and does not require manual labor by professionals. operating. Therefore, the PCR reaction method according to the embodiment of the present invention is performed in a closed environment, which reduces the pollution to the system environment, improves the reliability of the experiment, and the operation is more convenient and easy to implement.
  • the PCR reaction system further includes: a buffer unit 500, the buffer unit 500 is provided with a PCR reaction solution inlet 510 and a vent 520, and the buffer unit 500 is provided On the fourth pipeline 940, the PCR reaction solution inlet 510 is connected to the second PCR control valve 840, and the vent 520 is connected to the diluent outlet 210.
  • the first PCR control valve 830, the second PCR control valve 840, and the sample control valve 810 close the first PCR control valve 830, the second PCR control valve 840, and the sample control valve 810, and open the dilution control valve 820; then pull the piston 420 outward to Certain position, so that part of the diluent in the diluent holding 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 reciprocated to make the diluent in the injection chamber 410 enter the sample holding
  • the unit 100 is uniformly mixed with the lysed lyophilized powder and the sample in the sample holding unit 100; then the sample holding unit 100 is heated to a set temperature, so that the sample in the sample holding unit 100 is fully heated at the set temperature.
  • Lysis after the lysis is completed, pull the piston 420 outward again to a certain position, so that the lysed sample mixture in the sample holding unit 100 flows to the injection chamber 410; then, close the sample control valve 810 and open the dilution control valve 820; The piston 420 is moved back and forth to return the lysed sample mixture in the injection chamber 410 to the diluent holding unit 200, and at the same time, it is evenly mixed with the remaining diluent in the diluent holding unit 200 to dilute the lysed sample mixture.
  • the impurity concentration therein is reduced; afterwards, the piston 420 is pulled outward again to a certain position, so that the diluted sample mixture in the diluent holding unit 200 flows to the injection chamber 410; afterwards, the dilution control valve 820 is closed, and the first PCR is opened Control valve 830 and the second PCR control valve 840; then reciprocate the piston 420, so that the diluted sample mixture in the injection chamber 410 enters the PCR reaction unit 300, and at the same time with the reverse transcriptase in the PCR reaction unit 300 and PCR raw materials
  • the freeze-dried powder is mixed uniformly; finally, the PCR temperature heating control is performed on the PCR reaction unit 300, and the prophase constant temperature section of the enzyme is activated during PCR amplification.
  • the PCR reaction method according to the embodiment of the present invention can solve the overflow problem of PCR reagents during high temperature expansion, and the PCR reaction is carried out in a closed environment, which reduces the pollution of the system environment and improves the reliability of the experiment. It's easier and easier.
  • the PCR reaction system further includes: a sample containing unit sealing member 610, the sample containing unit sealing member 610 is disposed on the surface of the first liquid inlet/outlet 110 for The sample containing unit 100 is subjected to a first sealing process; and a diluent containing unit seal 620 is provided on the surface of the diluent outlet 210 and is used to hold the diluent containing unit 200 for the second sealing treatment;
  • the method further includes: a pre-piercing process S1000: the pre-piercing process includes pre-performing the sample containing unit seal to a first piercing process, and subjecting the diluent holding unit seal to a second Piercing treatment.
  • the sample containing unit 100 contains the lysing material in the form of freeze-dried powder
  • the PCR reaction unit 300 contains the reverse transcription in the form of freeze-dried powder.
  • Enzymes and PCR materials, and the diluent holding unit 200 contains an appropriate diluent.
  • the sample accommodating unit 100 and the diluent accommodating unit 200 are sealed with a sample accommodating unit seal 610 and a diluent accommodating unit seal 620 where the sample accommodating unit 100 and the diluent accommodating unit 200 communicate with the microfluidic pipeline, so that the lysis material and the diluent of the sample accommodating unit 100
  • the diluent of the unit 200 and the reverse transcriptase and PCR materials of the PCR reaction unit 300 are isolated from each other, and the piston 420 is at the top of the injection chamber 410 (that is, the injection chamber is in a state of being filled with the piston).
  • the PCR reaction system has a sample containing unit seal and a diluent containing unit seal
  • there is no need to set up a PCR reaction sealing device and it can already play the role of isolating each unit, and even if PCR A small amount of reactants in the reaction unit enters the pipeline, which has little effect on the overall reaction.
  • the sample containment unit seal and the diluent containment unit seal can not only isolate the reactants in each independent unit, facilitate long-term storage when not in use, greatly increase the storage time of each reactant in each unit, and avoid The contamination of the PCR reaction system caused by the reactants increases the service life of the PCR reaction system.
  • the sample containing unit when the sample containing unit is added with a sample, it is only necessary to pierce the seal of the sample containing unit and the seal of the diluent containing unit to make the sample containing unit, the diluent containing unit and the microfluidic pipeline in a communication state , The system can start working. Therefore, the operation of the method according to the embodiment of the present invention is more convenient.
  • the first piercing process is performed by the sample holding unit seal piercing device 710, and the second piercing process is performed by the diluent holding unit seal.
  • Piercing device 720 is performed.
  • At least one of the sample containment unit seal and the diluent containment unit seal is a sealing film.
  • the sealing film is formed of at least one of tin foil, plastic film, and kraft paper.
  • the thickness of the sealing film is 0.01-0.2mm, such as 0.03mm, 0.05mm, 0.07mm, 0.09mm, 0.1mm, 0.13mm, 0.15mm, 0.17mm or 0.19mm.
  • the thickness of the sealing film is 0.05 to 0.1 mm.
  • the structure of the system includes: a sample containing unit 100 designed as a sample chamber, a diluent containing unit 200 designed as a dilution chamber, an injection chamber 410, a piston 420, a PCR reaction unit 300 designed as a PCR chamber,
  • the first PCR control valve 830 and the second PCR control valve 840 are connected by microfluidic pipelines to form an associated circuit.
  • the sample chamber contains the lysis material in the form of freeze-dried powder
  • the PCR chamber contains the reverse transcriptase and the PCR material in the form of freeze-dried powder
  • the dilution chamber contains an appropriate diluent.
  • the sample chamber and the dilution chamber are sealed with a sample sealing film and a dilution sealing film where they communicate with the microfluidic pipeline, so that the lysis material in the sample chamber and the diluent in the dilution chamber and the reverse transcriptase and PCR materials in the PCR chamber are mutually connected. Isolated.
  • the piston is at the top of the injection chamber (the injection chamber is in a state where the piston is filled).
  • the system starts to work.
  • the lyophilized powder in the sample chamber is fully lysed Mix well with diluent and added sample.
  • Fourth start to heat the sample chamber to a set temperature, so that the sample in the sample chamber is fully lysed at the set temperature.
  • Fifth after the lysis is completed, the moving piston is pulled outward to a certain position, so that the lysed sample mixture in the sample chamber flows to the injection chamber through the sample control valve.
  • Sixth close the sample control valve, open the dilution control valve, and move the piston back and forth to return the lysed sample mixture in the injection chamber to the dilution chamber. In the process of reciprocating the piston, fully connect the lysed sample mixture to the dilution chamber.
  • the prophase constant temperature of the enzyme is activated.
  • the mixed liquid in the PCR chamber will expand due to the high temperature.
  • the liquid overflow during the expansion process can be controlled by the second PCR
  • the valve flows into the buffer chamber. After the end of the constant temperature section, the first PCR control valve and the second PCR control valve are closed, the temperature cycle control of the PCR chamber is started, and the PCR amplification experiment is finally completed.
  • composition and lyophilized powder of the PCR reaction as described above are described in detail below. It should be noted that, unless otherwise specified, the meaning of each component and each concentration in the present invention can be understood according to conventional interpretations in the art, such as EDTA and EGTA. In addition, Amplification Plot represents the amplification curve; Cycle represents the number of cycles. It should also be noted that, except that the sample lysis and nucleic acid extraction composition or lyophilized powder of the present invention are the inventor’s research and development results, unless otherwise specified, other relevant reagents used in the following steps can be purchased or consulted Obtained from existing technology. Those skilled in the art can purchase relevant reagents according to actual needs, or consult the prior art to obtain relevant reagents.
  • EDTA is 0.5mM-5mM
  • EGTA is 0.5mM-10mM
  • sodium lauryl sulfate is 0.5%-2.5 %
  • saponin is 0.5%-2.5%
  • proteinase K is 10-100U/mL
  • polyethylene glycol 3350 is 0.5-4.5%
  • Tris-HCl is 5mM-20mM
  • water is configured to a predetermined volume
  • the Tris-HCl solution drawn The pH is 7.5 ⁇ 8.2.
  • mannitol is 0.5%-8%
  • sucrose is 0.5%-8%
  • potassium chloride is 20-100mM
  • Magnesium chloride is 1.0mM-5mM
  • bovine serum albumin is 0.1-1mg/mL
  • dNTP is 200 ⁇ M
  • Brij 35 is 0.05%
  • HEPES is 10-50mM
  • DNA polymerase is 20-200U/mL
  • M-MLV reverse transcription The enzyme is 10-50 U/mL
  • the RNase inhibitor is 200-1000 U/mL
  • the water is configured to a predetermined volume
  • the pH of the drawn HEPES solution is 8.0-8.5.
  • composition of the lyophilized powder for sample lysis and nucleic acid extraction does not contain toxic substances such as phenol, chloroform, guanidine hydrochloride or guanidine isothiocyanate, and has low toxicity.
  • Fluorescence quantitative PCR reagent freeze-dried powder can be stored at room temperature with good stability.
  • FluA-Prob CY3-CAAGACCAATCCTGTCACCTCTGA-BHQ2 (SEQ ID NO: 3)
  • the mixed solution EDTA is 2mM
  • EGTA is 2mM
  • sodium lauryl sulfate is 1%
  • saponin is 1.0%
  • proteinase K is 20U /mL
  • polyethylene glycol 3350 is 1%
  • Tris-HCl is 10mM
  • the pH of the absorbed Tris-HCl solution is 7.6.
  • Pipette 50 ⁇ L of the above mixed solution add it to an eight-tube tube, centrifuge to the bottom of the tube, and freeze overnight at -80°C. Take it out and put it in a lyophilizer for lyophilization overnight, cover it after lyophilization, and store at room temperature. Ensure that the temperature of the lyophilizer is below -45°C, the vacuum pressure is ⁇ 450 Torr, and the sample in the test tube is placed on dry ice for at least 30 minutes.
  • mannitol is 4%
  • sucrose is 1.5%
  • potassium chloride is 80mM
  • magnesium chloride is 3.5mM
  • bovine serum albumin is 0.5mg/mL
  • dNTP is 200 ⁇ M
  • Brij 35 is 0.05%
  • HEPES is 20mM
  • DNA polymerase is 40U/mL
  • M-MLV reverse transcriptase is 20U/mL
  • RNase inhibitor is 500U/mL
  • the pH of the drawn HEPES solution is 8.25.
  • EDTA is 4mM
  • EGTA is 4mM
  • sodium lauryl sulfate is 2%
  • saponin is 2%
  • proteinase K is 80U /mL
  • Polyethylene Glycol 3350 is 4%
  • Tris-HCl is 18mM
  • water is configured to a predetermined volume
  • the pH of the aspirated Tris-HCl solution is 7.6.
  • Pipette 50 ⁇ L of the above mixed solution add it to an eight-tube tube, centrifuge to the bottom of the tube, and freeze overnight at -80°C. Take it out and put it in a lyophilizer for lyophilization overnight, cover it after lyophilization, and store at room temperature. Ensure that the temperature of the lyophilizer is below -45°C, the vacuum pressure is ⁇ 450 Torr, and the sample in the test tube is placed on dry ice for at least 30 minutes.
  • mannitol is 6%
  • sucrose is 3%
  • potassium chloride is 40mM
  • magnesium chloride is 5mM
  • bovine serum albumin is 1mg /mL
  • dNTP is 200 ⁇ M
  • Brij 35 is 0.05%
  • HEPES is 40mM
  • DNA polymerase is 100U/mL
  • M-MLV reverse transcriptase is 40U/mL
  • RNase inhibitor is 800U/mL
  • water is used to prepare Volume
  • the pH of the drawn HEPES solution is 8.25.
  • the raw material ratio is different from that in Example 1 only: the proteinase K in the first component is 2 U/mL, and the other component ratios remain unchanged.
  • the test method is the same as in Example 1.
  • Example 1 The raw material ratio is only different from that in Example 1: The concentration of EDTA and EGTA in the first component are both 0 mM, and the ratio of other components remains unchanged.
  • the test method is the same as in Example 1.
  • the raw material ratio is different from that in Example 1 only: the concentration of saponin in the first component is 10%, and the ratio of other components remains unchanged.
  • the test method is the same as in Example 1.
  • the raw material ratio is different from that of Example 1 only: the concentration of magnesium chloride in the second component is 0.2 mM, and the other component ratios remain unchanged.
  • the test method is the same as in Example 1.
  • Example 1 The raw material ratio is different from that in Example 1 only: the concentration of HEPES in the second component is 2 mM, and the ratio of other components remains unchanged.
  • the test method is the same as in Example 1.
  • Amplification Plot represents the amplification curve
  • Cycle represents the number of cycles.
  • a predetermined volume of diluent in which: magnesium chloride is 0.5-15mmol/L, sodium chloride is 1-150mmol/L, surfactant is 0.1-7%, The content of glycerol is 2-20%, and it is prepared to a predetermined volume with water.
  • the concentration of the absorbed Tris-HCl solution is 1-100 mM and the pH is 7.5-8.0.
  • Surfactants are Tween 20, Tween 80, polyethylene glycol octyl phenyl ether, sodium lauryl sulfate, sodium dodecylbenzene sulfonate, sodium dioctyl succinate sulfonate or glycocholic acid sodium.
  • Tris-HCl 5% glycerol, 2mM magnesium chloride, 5mM sodium chloride, 5mM Tris-HCl, 0.5% Tween20, the pH of the added Tris-HCl is 7.6.
  • Line 1 indicates that the solution (the original lysis solution) after the sample is lysed is directly added to the amplification result of the subsequent PCR reaction system;
  • Line 2 represents the result of amplification by adding the same PCR reaction system after the above-mentioned lysis stock solution is diluted by the dilution buffer (diluent);
  • Line 3 represents the result of amplification by adding the same PCR reaction system after the above-mentioned lysis stock solution is diluted by H2O by equal multiples;
  • Line 4 represents negative control, that is, the amplification result of the same PCR reaction system is added with the dilution buffer (diluent) as a template.
  • Line 1 shows that the solution after the sample is lysed is directly added to the subsequent PCR reaction system and the amplification result shows that the amplification curve cannot be amplified.
  • Line 2 shows that the lysis stock solution is diluted by the dilution buffer and added to the same PCR reaction system, showing that a positive amplification curve can be obtained.
  • Line 3 shows that the above-mentioned lysis stock solution is diluted by H2O by equal multiples, indicating that the sample is amplified, but the Ct value is significantly higher than that of the dilution buffer (the dilution of the present invention) diluted sample, indicating that the buffering effect of the dilution buffer is better than that of H2O.
  • Line 4 is the dilution buffer as a template added to the same PCR reaction system as a negative control, and the negative control has no amplification curve, indicating that the dilution buffer can effectively buffer the lysis buffer and the PCR reaction system.
  • Tris-HCl 8% glycerol, 6mM magnesium chloride, 50mM sodium chloride, 40mM Tris-HCl, 3% Tween20, the pH of the added Tris-HCl is 8.0.
  • Line 1 indicates that the solution (the original lysis solution) after the sample is lysed is directly added to the amplification result of the subsequent PCR reaction system;
  • Line 2 represents the result of amplification by adding the same PCR reaction system after the above-mentioned lysis stock solution is diluted by the dilution buffer (diluent);
  • Line 3 represents the result of amplification by adding the same PCR reaction system after the above-mentioned lysis stock solution is diluted by H2O by equal multiples;
  • Line 4 represents the negative control, and the dilution buffer is the template and the amplification result of the same PCR reaction system is added.
  • Line 1 shows that the solution after the sample is lysed is directly added to the subsequent PCR reaction system and the amplification result shows that the amplification curve cannot be amplified.
  • Line 2 shows that the lysis stock solution is diluted by the dilution buffer and added to the same PCR reaction system, showing that a positive amplification curve can be obtained.
  • Line 3 shows that the above-mentioned lysis stock solution is diluted by H2O by equal multiples, indicating that the sample is amplified, but the Ct value is significantly higher than that of the dilution buffer (the dilution of the present invention) diluted sample, indicating that the buffering effect of the dilution buffer is better than that of H2O.
  • Line 4 is the dilution buffer as a template added to the same PCR reaction system as a negative control, and the negative control has no amplification curve, indicating that the dilution buffer can effectively buffer the lysis buffer and the PCR reaction system.
  • Tris-HCl 1% sodium lauryl sulfate
  • the pH of the added Tris-HCl is 8.0.
  • Line 1 indicates that the solution (the original lysis solution) after the sample is lysed is directly added to the amplification result of the subsequent PCR reaction system;
  • Line 2 represents the result of amplification by adding the same PCR reaction system after the above-mentioned lysis stock solution is diluted by the dilution buffer (diluent);
  • Line 3 represents the result of amplification by adding the same PCR reaction system after the above-mentioned lysis stock solution is diluted by H2O by equal multiples;
  • Line 4 represents the negative control, and the dilution buffer is the template and the amplification result of the same PCR reaction system is added.
  • Line 1 shows that the solution after the sample is lysed is directly added to the subsequent PCR reaction system and the amplification result shows that the amplification curve cannot be amplified.
  • Line 2 shows that the lysis stock solution is diluted by the dilution buffer and added to the same PCR reaction system, showing that a positive amplification curve can be obtained.
  • Line 3 shows that the above-mentioned lysis stock solution is diluted by H2O by equal multiples, indicating that the sample is amplified, but the Ct value is significantly higher than that of the dilution buffer (the dilution of the present invention) diluted sample, indicating that the buffering effect of the dilution buffer is better than that of H2O.
  • Line 4 is the dilution buffer as a template added to the same PCR reaction system as a negative control, and the negative control has no amplification curve, indicating that the dilution buffer can effectively buffer the lysis buffer and the PCR reaction system.
  • Tris-HCl 5% glycerol, 2mM magnesium chloride, 5mM sodium chloride, 5mM Tris-HCl, 0.1% polyethylene glycol octylphenyl ether, the pH of the added Tris-HCl is 8.0.
  • Line 1 indicates that the solution (the original lysis solution) after the sample is lysed is directly added to the amplification result of the subsequent PCR reaction system;
  • Line 2 represents the result of amplification by adding the same PCR reaction system after the above-mentioned lysis stock solution is diluted by the dilution buffer (diluent);
  • Line 3 represents the result of amplification by adding the same PCR reaction system after the above-mentioned lysis stock solution is diluted by H2O by equal multiples;
  • Line 4 represents the negative control, and the dilution buffer is the template and the amplification result of the same PCR reaction system is added.
  • Line 1 shows that the solution after the sample is lysed is directly added to the subsequent PCR reaction system and the amplification result shows that the amplification curve cannot be amplified.
  • Line 2 shows that the lysis stock solution is diluted by the dilution buffer and added to the same PCR reaction system, showing that a positive amplification curve can be obtained.
  • Line 3 shows that the above-mentioned lysis stock solution is diluted by H2O by equal multiples, indicating that the sample is amplified, but the Ct value is significantly higher than that of the dilution buffer (the dilution of the present invention) diluted sample, indicating that the buffering effect of the dilution buffer is better than that of H2O.
  • Line 4 is the dilution buffer as a template added to the same PCR reaction system as a negative control, and the negative control has no amplification curve, indicating that the dilution buffer can effectively buffer the lysis buffer and the PCR reaction system.
  • Example 1 Only the raw material ratio is different from Example 1: The concentration of Tween20 is 8%, and the rest is the same as Example 1.
  • Example 1 The only difference between the raw material ratio and Example 1 is that the concentration of glycerol is 30%, and the rest is the same as Example 1.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, "a plurality of” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the terms “installed”, “connected”, “connected”, “fixed” and other terms should be understood in a broad sense, for example, it can be a fixed connection or a detachable connection. , Or integrated; it can be mechanically connected, or it can be electrically connected or can communicate with each other; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal communication of two components or the interaction relationship between two components, Unless otherwise clearly defined.
  • the specific meaning of the above-mentioned terms in the present invention can be understood according to specific circumstances.

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Abstract

利用PCR反应系统进行PCR反应的方法和PCR反应系统，以及组合物、冻干粉和稀释液。其中，PCR反应方法包括：将所述活塞进行第一移动处理；将所述活塞进行第二移动处理，以便使进入注射室的所述稀释液与裂解冻干粉和样本进行第一混合处理；将第一混合处理产物进行裂解处理；将所述活塞进行第三移动处理；将所述活塞进行第四移动处理，以便使进入注射室的所述裂解处理产物与剩余部分稀释液进行第二混合处理；将所述活塞进行第五移动处理，以便使第二混合处理产物进入所述注射室；将所述活塞进行第六移动处理，以便使进入注射室的所述第二混合处理产物与反转录酶和PCR原料冻干粉进行第三混合处理；以及将第三混合处理产物进行PCR温度循环扩增处理。

Description

利用PCR综合反应系统进行PCR反应的新方法 技术领域
本发明涉及分子生物学领域，具体地，本发明涉及利用PCR反应系统进行PCR反应的方法。
背景技术
传统的PCR反应过程一般都是分开进行的，即先通过核酸提取试剂盒提取所需要的核酸，然后再将提取出的核酸和试剂混合添加到PCR反应管中，最后再将PCR反应管放入PCR仪器中进行PCR扩增反应，才能得出最终结果。传统的PCR反应过程操作步骤繁琐，工作效率低。
因此，简便高效的PCR反应过程还需要进一步研究开发。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识做出的：
传统的PCR反应过程操作步骤繁琐，工作效率低。究其原因，发明人发现，传统的PCR反应过程中的每一步一般都需要专业人士进行操作，并且这些操作一般都需要通过不同的仪器如核酸纯化仪、全自动工作站等分别进行。另外，整个过程的操作也需要在标准的PCR实验室环境下才能进行。基于上述问题，发明人通过微流控管路将核酸的提取、核酸和试剂的混合以及最终PCR的反应全部整合到了一个系统中，利用该系统进行PCR反应的方法实现了真正的全自动化操作，解决了传统PCR实验过程需要在专业实验环境下由专业人士进行操作的难题，减少了人为操作带来的误差，极大地提高了PCR反应的工作效率，大大节约了人力资源成本。
为此，在本发明的一个方面，本发明提出了一种利用PCR综合反应系统进行PCR反应的方法。根据本发明的实施例，所述PCR反应系统包括：样本容纳单元，所述样本容纳单元内设置有裂解冻干粉和样本，并且所述样本容纳单元具有第一液体出/入口；稀释液容纳单元，所述稀释液容纳单元内设置有稀释液，并且所述稀释液容纳单元具有稀释液出口；PCR反应单元，所述PCR反应单元内设置有反转录酶和PCR原料冻干粉，并且所述PCR反应单元具有PCR反应液出口和裂解后的样本混合液入口；以及活塞单元，所述活塞单元包括注射室和活塞，所述注射室具有第二液体出/入口，所述第二液体出/入口通过第一管路与所述第一液体出/入口相连，所述第二液体出/入口通过第二管路与所述稀释液出口相连，所述第二液体出/入口通过第三管路与所述裂解后的样本混合液入口相连，所述PCR反应液出口通过第四管路与所述稀释液出口相连；所述方法包括：将所述活塞进行第一移动处理，以便使部分稀释液进入所述注射室，所述稀释液设置在所述稀释液容纳单元内；将所述活塞进行第二移动处理，以便使进入注射室的所述稀释液与裂解冻干粉和样本进行第一混合处理，所述裂解冻干粉和所述样本设置在所述样本容纳单元内，所述第一混合处理是在所述样本容纳单元中进行的；将第一混合处理产物进行裂解处理，所述裂解处理是在所述样本容纳单元中进行的；将所述活塞进行第三移动处理，以便使裂解处理产物进入所述注射室；将所述活塞进行第四移动处理，以便使进入注射室的所述裂解处理产物与剩余部分稀释液进行第二混合处理，所述第二混合处理是在所述稀释液容纳单元中进行的；将所述活塞进行第五移动处理，以便使第二混合处理产物进入所述注射室；将所述活塞进行第六移动处理，以便使进入注射室的所述第二混合处理产物与反转录酶和PCR原料冻干粉进行第三混合处理，所述反转录酶和PCR原料冻干粉设置在所述PCR反应单元内，所述第三混合处理是在所述PCR反应单元中进行的；以及将第三混合处理产物进行PCR温度循环扩增处理，所述PCR温度循环扩增处理是在所述PCR反应单元中进行的。
根据本发明实施例的PCR反应系统通过微流控管路将样本容纳单元、稀释液容纳单元、PCR反应单元以及活塞单元相互连接在一起；同时，各单元又分别为独立设置的单元，以便使各单元在使用前存放不同的反应物，有利于反应物在不使用的情况进行长久保存。例如，样本容纳单元的独立设置有利于样本的单独添加，使样本的添加操作简单化，同时也有利于样本的长久保存。首先，将活塞向外拉动到一定位置，从而使稀释液容纳单元中的部分稀释液流向注射室；之后往复移动活塞，使注射室中的稀释液进入样本容纳单元，同时与样本容纳单元中的裂解冻干粉以及样本混合均匀；之后对样本容纳单元进行加热升温到设定温度，使样本容纳单元中的样本在设定的温度下进行充分裂解；裂解完成后，再次将活塞向外拉动到一定位置，从而使样本容纳单元中已裂解的样本混合液流向注射室；之后往复移动活塞，使注射室中已裂解的样本混合液返回稀释液容纳单元，同时与稀释液容纳单元中剩余的稀释液混合均匀，从而将已裂解的样本混合液进行稀释，将其中的杂质浓度降低；之后，再次将活塞向外拉动到一定位置，从而使稀释液容纳单元中稀释后的样本混合液流向注射室；随后往复移动活塞，使注射室中稀释后的样本混合液进入PCR反应单元，同时与PCR反应单元中的反转录酶以及PCR原料的冻干粉混合均匀；最后对PCR反应单元进行PCR温度加热控制，以便最终完成PCR扩增反应。根据本发明实施例的方法实现了真正的全自动化操作，解决了传统PCR实验过程需要在专业实验环境下由专业人士进行操作的难题，减少了人为操作带来的误差，极大地提高了PCR反应的工作效率，大大节约了人力资源成本。
根据本发明的实施例，上述方法还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述PCR温度循环扩增处理包括：将所述第三混合处理产物进行恒温处理；以及将恒温处理产物进行温度循环处理。
根据本发明的实施例，所述PCR反应系统进一步包括：样本控制阀，在所述第一管路上设置所述样本控制阀，用于控制所述第一液体出/入口与所述第二液体出/入口的连通状态；稀释控制阀，在所述第二管路上设置所述稀释控制阀，用于控制所述稀释液入口与所述第二液体出/入口的连通状态；第一PCR控制阀，在所述第三管路上设置所述第一PCR控制阀，用于控制所述裂解后的样本混合液入口与所述第二液体出/入口的连通状态；以及第二PCR控制阀，在所述第四管路上设置所述第二PCR控制阀，用于控制所述稀释液出口与所述PCR反应液出口的连通状态；所述方法进一步包括：在所述第一移动处理前，关闭所述样本控制阀、所述第一PCR控制阀以及所述第二PCR控制阀，开启所述稀释控制阀；在所述第一移动处理后以及所述第二移动处理前，关闭所述稀释控制阀，开启所述样本控制阀；在所述第三移动处理后以及所述第四移动处理前，关闭所述样本控制阀，开启所述稀释控制阀；在所述第五移动处理后以及所述第六移动处理前，关闭所述稀释控制阀，开启所述第一PCR控制阀以及所述第二PCR控制阀；在所述恒温处理后以及温度循环处理前，关闭所述第一PCR控制阀以及所述第二PCR控制阀。由此，根据本发明实施例的方法在密闭的环境下进行，减少了对系统环境的污染，提高了实验的可信度，且操作更加简便。
根据本发明的实施例，所述PCR反应系统进一步包括：缓冲单元，所述缓冲单元具有PCR反应液入口和通气口，在所述第四管路上设置所述缓冲单元，所述第二PCR控制阀与所述PCR反应液入口相连，所述稀释液出口与所述通气口相连。由此，根据本发明实施例的方法可以解决PCR试剂在高温膨胀时的溢出问题，且所述方法在密闭的环境下进行，减少了对系统环境的污染，提高了实验的可信度，操作更加简便。
根据本发明的实施例，所述PCR反应系统进一步包括：样本容纳单元密封件，在所述第一液体出/入口表面设置所述样本容纳单元密封件，用于将所述样本容纳单元进行第一密封处理；稀释液容纳单元密封件，在所述稀释液出口表面设置所述稀释液容纳单元密封件，用于将所述稀释液容 纳单元进行第二密封处理；所述方法进一步包括：预先刺穿处理，所述预先刺穿处理包括预先将所述样本容纳单元密封件进行第一刺穿处理，将所述稀释液容纳单元密封件进行第二刺穿处理。发明人发现，样本容纳单元密封件和稀释液容纳单元密封件不仅可以隔绝各独立单元内的反应物，便于不使用的情况下长时间保存，大大提高了各单元中各反应物的存储时间，而且避免了各反应物对PCR反应系统造成的污染，提高了PCR反应系统的使用寿命。进而，当样本容纳单元添加了样本后，只需要将样本容纳单元密封件和稀释液容纳单元密封件刺穿，以便使样本容纳单元以及稀释液容纳单元与微流控管路之间处于连通状态，系统便可以开始工作。由此，根据本发明实施例的方法操作更加简便。
根据本发明的实施例，所述第一刺穿处理是通过样本容纳单元密封件刺穿装置进行的，所述第二刺穿处理是通过稀释液容纳单元密封件刺穿装置进行的。
根据本发明的实施例，所述稀释液容纳单元密封件和所述样本容纳单元密封件的至少之一为密封膜。
根据本发明的实施例，所述密封膜是由锡箔纸、塑封膜、牛皮纸的至少之一形成。
根据本发明的实施例，所述密封膜的厚度为0.01～0.2mm，如为0.03mm、0.05mm、0.07mm、0.09mm、0.1mm、0.13mm、0.15mm、0.17mm或0.19mm。发明人发现，若密封膜的厚度过小，则可能渗透，若密封膜的厚度过大，又难以刺穿。在一些实施例中，所述密封膜的厚度为0.05～0.1mm。
在本发明的另一方面，本发明提出了一种PCR反应系统。根据本发明的实施例，所述系统包括：样本容纳单元，所述样本容纳单元内设置有裂解原料冻干粉，并且所述样本容纳单元具有第一液体出/入口；稀释液容纳单元，所述稀释液容纳单元内设置有稀释液，并且所述稀释液容纳单元具有稀释液出口；PCR反应单元，所述PCR反应单元内设置有反转录酶和PCR原料冻干粉，并且所述PCR反应单元具有PCR反应液出口和裂解后的样本混合液入口；以及活塞单元，所述活塞单元包括注射室和活塞，所述注射室具有第二液体出/入口；所述第二液体出/入口通过第一管路与所述第一液体出/入口相连，所述第二液体出/入口通过第二管路与所述稀释液出口相连，所述第二液体出/入口通过第三管路与所述裂解后的样本混合液入口相连，所述PCR反应液出口通过第四管路与所述稀释液出口相连。
根据本发明实施例的PCR反应系统通过微流控管路将样本容纳单元、稀释液容纳单元、PCR反应单元以及活塞单元相互连接在一起；同时，各单元又分别为独立设置的单元，以便使各单元在使用前存放不同的反应物，有利于反应物在不使用的情况进行长久保存。例如，样本容纳单元的独立设置有利于样本的单独添加，使样本的添加操作简单化，同时也有利于样本的长久保存。首先，将活塞向外拉动到一定位置，从而使稀释液容纳单元中的部分稀释液流向注射室；之后往复移动活塞，使注射室中的稀释液进入样本容纳单元，同时与样本容纳单元中的裂解冻干粉以及样本混合均匀；之后对样本容纳单元进行加热升温到设定温度，使样本容纳单元中的样本在设定的温度下进行充分裂解；裂解完成后，再次将活塞向外拉动到一定位置，从而使样本容纳单元中已裂解的样本混合液流向注射室；之后往复移动活塞，使注射室中已裂解的样本混合液返回稀释液容纳单元，同时与稀释液容纳单元中剩余的稀释液混合均匀，从而将已裂解的样本混合液进行稀释，将其中的杂质浓度降低；之后，再次将活塞向外拉动到一定位置，从而使稀释液容纳单元中稀释后的样本混合液流向注射室；随后往复移动活塞，使注射室中稀释后的样本混合液进入PCR反应单元，同时与PCR反应单元中的反转录酶以及PCR原料的冻干粉混合均匀；最后对PCR反应单元进行PCR温度加热控制，以便最终完成PCR扩增反应。根据本发明实施例的PCR反应系统实现了真正的全自动化操 作，解决了传统PCR实验过程需要在专业实验环境下由专业人士进行操作的难题，减少了人为操作带来的误差，极大地提高了PCR反应的工作效率，大大节约了人力资源成本。
根据本发明的实施例，上述系统还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述系统进一步包括：样本控制阀，在所述第一管路上设置所述样本控制阀，用于控制所述第一液体出/入口与所述第二液体出/入口的连通状态；稀释控制阀，在所述第二管路上设置所述稀释控制阀，用于控制所述稀释液出口与所述第二液体出/入口的连通状态；第一PCR控制阀，在所述第三管路上设置所述第一PCR控制阀，用于控制所述裂解后的样本混合液入口与所述第二液体出/入口的连通状态；以及第二PCR控制阀，在所述第四管路上设置所述第二PCR控制阀，用于控制所述稀释液出口与所述PCR反应液出口的连通状态。由此，利用根据本发明实施例的PCR反应系统进行的PCR反应在密闭的环境下进行，减少了对系统环境的污染，提高了实验的可信度，且操作更加简便。
根据本发明的实施例，所述系统进一步包括：缓冲单元，所述缓冲单元具有PCR反应液入口和通气口，在所述第四管路上设置所述缓冲单元，所述第二PCR控制阀与所述PCR反应液入口相连，所述稀释液出口与所述通气口相连。由此，利用根据本发明实施例的PCR反应系统进行的PCR反应可以解决PCR试剂在高温膨胀时的溢出问题。
根据本发明的实施例，所述系统进一步包括：样本容纳单元密封件，在所述第一液体出/入口表面设置所述样本容纳单元密封件，用于将所述样本容纳单元进行第一密封处理；以及稀释液容纳单元密封件，在所述稀释液出口表面设置所述稀释液容纳单元密封件，用于将所述稀释液容纳单元进行第二密封处理。发明人发现，样本容纳单元密封件和稀释液容纳单元密封件不仅可以隔绝各独立单元内的反应物，便于不使用的情况下长时间保存，大大提高了各单元中各反应物的存储时间，而且避免了各反应物对PCR反应系统造成的污染，提高了PCR反应系统的使用寿命。
根据本发明的实施例，所述系统进一步包括：样本容纳单元密封件刺穿装置，所述样本容纳单元密封件刺穿装置用于将所述样本容纳单元密封件进行第一刺穿处理；以及稀释液容纳单元密封件刺穿装置，所述稀释液容纳单元密封件刺穿装置用于将所述稀释液容纳单元密封件进行第二刺穿处理。由此，根据本发明实施例的PCR反应系统中的样本容纳单元以及稀释液容纳单元与微流控管路之间处于连通状态，便于后续过程的进行。
根据本发明的实施例，所述稀释液容纳单元密封件和所述样本容纳单元密封件的至少之一为密封膜。
根据本发明的实施例，所述密封膜是由锡箔纸、塑封膜或牛皮纸的至少之一形成。
根据本发明的实施例，所述密封膜的厚度为0.01～0.2mm，如为0.03mm、0.05mm、0.07mm、0.09mm、0.1mm、0.13mm、0.15mm、0.17mm或0.19mm。发明人发现，若密封膜的厚度过小，则可能渗透，若密封膜的厚度过大，又难以刺穿。在一些实施例中，所述密封膜的厚度为0.05～0.1mm。
根据本发明的实施例，所述裂解原料冻干粉包括金属离子螯合剂，十二烷基硫酸钠，皂苷，蛋白酶K，聚乙二醇3350，Tris-HCl，水；所述反转录酶和PCR原料冻干粉包括甘露醇，蔗糖，氯化盐，牛血清白蛋白，dNTP，聚氧乙烯十二烷醚，HEPES，DNA聚合酶，反转录酶和RNA酶抑制剂，水。在一些实施例中，所述金属离子螯合剂为EDTA和EGTA。在一些实施例中，所述氯化盐为氯化钾和氯化镁。
根据本发明的实施例，所述稀释液包括：多元醇，氯化盐，Tris-HCl，表面活性剂以及水。在一些实施例中，所述多元醇为丙三醇。
另外，发明人发现，一方面，在样本提取期间使用的试剂苯酚、氯仿、盐酸胍、乙醇、异硫氰 酸胍等不仅对人体有害，严重的甚至致癌；而且有些试剂是易燃品，对实验室环境和安全风险控制的要求较高；另外，这些试剂也容易造成大气和水源的污染。另一方面，荧光定量PCR试剂的配制和分装要求在标准的试剂准备间进行，以防造成样本污染，且配制和分装的操作复杂，需要特定培训后的人员才可以进行。基于上述问题，发明人研究开发了一种组合物，该组合物中的第一组分可以有效用于样本的裂解和核酸的提取，第二组分可以有效用于PCR扩增反应，该第一组分与现有试剂相比，毒性和污染性显著降低，安全性、稳定性显著提高，对运输和储存有利，且对样本的要求降低；该第二组分与现有技术相比，稳定性显著提高，对运输和储存有利，且降低了对实验环境的要求；第一组分与第二组分的联合使用，可以使样本的裂解、核酸的提取以及荧光定量PCR试剂的配制无需专业人士即可完成，降低了对操作人员的技术要求，同时PCR扩增效果优异。
为此，在本发明的另一方面，本发明提出了一种用于PCR反应的组合物。根据本发明的实施例，所述组合物包括：第一组分，所述第一组分包括金属离子螯合剂，十二烷基硫酸钠(SDS)，皂苷(saponin)，蛋白酶K(proteinase K)，聚乙二醇3350(PEG 3350)，Tris-HCl，水；和/或第二组分，所述第二组分包括甘露醇，蔗糖，氯化盐，牛血清白蛋白(BSA)，dNTP，聚氧乙烯十二烷醚(Brij 35)，HEPES，DNA聚合酶，反转录酶和RNA酶抑制剂，水。在一些实施例中，所述金属离子螯合剂为EDTA和EGTA。在一些实施例中，所述氯化盐为氯化钾和氯化镁。根据本发明实施例的组合物中的第一组分配合其他仪器的加热功能可以有效用于样本的裂解和核酸的提取，第二组分可以有效用于PCR扩增反应，发明人发现，所述第一组分与现有试剂相比，毒性和污染性显著降低，安全性显著提高，且利用第一组分裂解样本后得到的混合液不需要再单独进行纯化，即可直接用于后续的PCR反应，对样本的要求降低；所述第二组分与现有技术相比，稳定有效，对运输和储存有利，且降低了对运输和储存温度的要求；第一组分与第二组分的联合使用，可以使样本的裂解、核酸的提取以及荧光定量PCR试剂的配制无需专业人士即可完成，降低了对操作人员的技术要求，同时PCR扩增效果优异。
根据本发明的实施例，上述组合物还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，基于所述第一组分的总体积，所述EDTA的浓度为0.1～10mmol/L，如为0.5、0.7、1.0、2.0、3.0或4.0mmol/L，所述EGTA的浓度为0.1～15mmol/L，如为0.5、0.7、3、5、7、10或13mmol/L，所述蛋白酶K的浓度为5～150U/mL，如为7、10、15、30、45、60、75、90或120U/mL，所述十二烷基硫酸钠的浓度为0.1～3.0％，如为0.3、0.5、1.0、1.5、2.0或2.5％，所述皂苷的浓度为0.1～3.0％，如为0.3、0.5、1.0、1.5、2.0或2.5％，所述聚乙二醇3350的浓度为0.1～5.0％，如为0.3、0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0或4.5％。需要说明的是，所述十二烷基硫酸钠、皂苷以及聚乙二醇3350的浓度为质量体积浓度，指的是每100mL溶液中所述十二烷基硫酸钠、皂苷或聚乙二醇3350的质量，单位为g。例如，所述十二烷基硫酸钠的浓度为0.1～3.0％，指的是每100mL的第一组分中十二烷基硫酸钠的质量为0.1～3.0g。发明人发现，所述第一组分中各成分的浓度在该范围内时，第一组分可以进一步有效用于样本的裂解和核酸的提取，同时毒性和污染性更低，安全性更高，对样本的要求更低，进而，后续的PCR扩增效果更优。
根据本发明的实施例，基于所述第一组分的总体积，所述EDTA的浓度为0.5～5mmol/L，所述EGTA的浓度为0.5～10mmol/L，所述蛋白酶K的浓度为10～100U/mL，所述十二烷基硫酸钠的浓度为0.5～2.5％，所述皂苷的浓度为0.5～2.5％，所述聚乙二醇3350的浓度为0.5～4.5％。发明人发现，所述第一组分中各成分的浓度在该范围内时，第一组分可以进一步有效用于样本的裂解和核酸的提取，同时毒性和污染性更低，安全性更高，对样本的要求更低，进而，后续的PCR扩增效果更优。
根据本发明的实施例，所述Tris-HCl是以溶于水的形式提供的。需要说明的是，Tris-HCl是本领域常用的一类缓冲物质，可以自己配置，也可以直接购买。
根据本发明的实施例，所述Tris-HCl溶于水形成的溶液的pH为7.5～8.2，如7.6、7.7、7.8、7.9、8.0或8.1。需要说明的是，所述pH并不是指第一组分的pH，而是Tris-HCl溶于水形成的溶液的pH。根据本发明的实施例，所述Tris-HCl溶于水形成的溶液的pH为7.5～8.2时，Tris-HCl对第一组分的缓冲效果更好，更有利于样本的裂解和核酸的提取，稳定性更好，进而，后续的PCR扩增效果更优。在一些实施方案中，所述Tris-HCl溶于水形成的溶液的pH为7.6。
根据本发明的实施例，基于所述第一组分的总体积，所述Tris-HCl的浓度为1～25mmol/L，如为2、4、5、6、8、10、12、14、16、18、20、22或24mmol/L。根据本发明的实施例，所述Tris-HCl的浓度为1～25mmol/L时，Tris-HCl对第一组分的缓冲效果更好，更有利于样本的裂解和核酸的提取，稳定性更好，进而，后续的PCR扩增效果更优。在一些实施方案中，所述Tris-HCl的浓度为5～20mmol/L。
根据本发明的实施例，基于所述第二组分的总体积，所述氯化钾的浓度为10～150mmol/L，如为15、20、30、40、60、80、100或120mmol/L，所述氯化镁的浓度为0.5～10.0mmol/L，如为0.7、1.0、2.0、3.0、4.0、5.0、7.0或10.0mmol/L，所述dNTP的浓度为150～250μmol/L，如为180、200或230μmol/L，所述DNA聚合酶的浓度为10～250U/mL，如为13、15、18、20、30、50、80、100、120、150、180、200或230U/mL，所述反转录酶的浓度为5～100U/mL，如为7、10、20、30、40、50、70或90U/mL，所述RNA酶抑制剂的浓度为100～1000U/mL，如为150、200、300、500、700或900U/mL，所述甘露醇的浓度为0.1～10％，如为0.2、0.4、0.5、1.0、3.0、5.0、7.0或9.0％，所述蔗糖的浓度为0.1～10％，如为0.2、0.4、0.5、1.0、3.0、5.0、7.0或9.0％，所述牛血清白蛋白的浓度为0.1～5mg/mL，如为0.2、0.4、0.6、0.8、1.0、2.0、3.0或4.0mg/mL，所述聚氧乙烯十二烷醚的浓度为0.01～0.10％，如为0.02、0.03、0.04、0.05、0.06、0.07、0.08或0.09％。需要说明的是，所述蔗糖以及聚氧乙烯十二烷醚的浓度为质量体积浓度，指的是每100mL溶液中所述蔗糖或聚氧乙烯十二烷醚的质量，单位为g。例如，所述蔗糖的浓度为0.1～10％，指的是每100mL的第二组分中蔗糖的质量为0.1～10g。所述甘露醇的浓度0.1～10％，指每100mL的第二组分中甘露醇的体积为0.1～10mL。发明人发现，所述第二组分中各成分的浓度在该范围内时，所述第二组分可以进一步有效用于PCR扩增反应，PCR扩增效果更好，同时稳定性更高。
根据本发明的实施例，基于所述第二组分的总体积，所述氯化钾的浓度为20～100mmol/L，所述氯化镁的浓度为1.0～5.0mmol/L，所述dNTP的浓度为200μmol/L，所述DNA聚合酶的浓度为20～200U/mL，所述反转录酶的浓度为10～50U/mL，所述RNA酶抑制剂的浓度为200～1000U/mL，所述甘露醇的浓度为0.5～8％，所述蔗糖的浓度为0.5～8％，所述牛血清白蛋白的浓度为0.1～1mg/mL，所述聚氧乙烯十二烷醚的浓度为0.05％。发明人发现，所述第二组分中各成分的浓度在该范围内时，所述第二组分可以进一步有效用于PCR扩增反应，PCR扩增效果更好，同时稳定性更高。
根据本发明的实施例，所述HEPES是以溶于水的形式提供的。需要说明的是，HEPES是本领域常用的一类缓冲物质，可以自己配置，也可以直接购买。
根据本发明的实施例，所述HEPES溶于水形成的溶液的pH为8.0～8.5，如为8.1、8.2、8.25、8.3或8.4。需要说明的是，所述pH并不是指第二组分的pH，而是HEPES溶于水形成的溶液的pH。根据本发明的实施例，所述HEPES溶于水形成的溶液的pH为8.0～8.5时，HEPES对第二组分的缓冲效果更好，更有利于PCR扩增反应，PCR扩增效果更好，同时稳定性更高。在一些实施方案中，所述HEPES溶于水形成的溶液的pH为8.25。
根据本发明的实施例，基于所述第二组分的总体积，所述HEPES的浓度为5～55mmol/L，如为10、15、20、25、30、35、40、45或50mmol/L。根据本发明的实施例，所述HEPES的浓度为5～55mmol/L时，HEPES更有利于PCR扩增反应，PCR扩增效果更好，同时稳定性更高。在一些实施方案中，所述HEPES的浓度为10～50mmol/L。
根据本发明的实施例，所述皂苷包括选自茶皂素、人参皂苷、重楼皂苷、大豆皂苷的至少之一。
根据本发明的实施例，所述DNA聚合酶包括选自Taq酶、Tth DNA聚合酶的至少之一。
根据本发明的实施例，所述反转录酶包括选自M-MLV反转录酶、AMV反转录酶的至少之一。
根据本发明的实施例，所述RNA酶抑制剂包括选自焦磷酸二乙酯、异硫氰酸胍、氧钒核糖核苷复合物、RNasin、尿素、硅藻土的至少之一。
在本发明另一方面，本发明提出了一种组合物。根据本发明的实施例，所述组合物包括：第一组分，基于所述第一组分的总体积，所述第一组分包括浓度为0.5～5mmol/L的EDTA，浓度为0.5～10mmol/L的EGTA，浓度为10～100U/mL的蛋白酶K，浓度为0.5～2.5％的十二烷基硫酸钠，浓度为0.5～2.5％的皂苷，浓度为0.5～4.5％的聚乙二醇3350，浓度为5～20mmol/L的Tris-HCl，以及水；和/或第二组分，基于所述第二组分的总体积，所述第二组分包括浓度为20～100mmol/L的氯化钾，浓度为1.0～5.0mmol/L的氯化镁，浓度为200μmol/L的dNTP，浓度为20～200U/mL的DNA聚合酶，浓度为10～50U/mL的反转录酶，浓度为200～1000U/mL的RNA酶抑制剂，浓度为0.5～8％的甘露醇，浓度为0.5～8％的蔗糖，浓度为0.1～1mg/mL的牛血清白蛋白，浓度为0.05％的聚氧乙烯十二烷醚，浓度为10～50mmol/L的HEPES，以及水。发明人发现，第一组分与第二组分的联合使用，可以使样本的裂解、核酸的提取以及荧光定量PCR试剂的配制无需专业人士即可完成，降低了对操作人员的技术要求，同时PCR扩增效果优异。
在本发明的另一方面，本发明提出了一种冻干粉。根据本发明的实施例，所述冻干粉是由上述任一项所述的组合物制备的，该冻干粉适于应用在上述实施例的利用PCR反应系统进行PCR反应的方法和PCR反应系统中。具体的，可以设置在样本容纳单元内。发明人发现，所述第一组分和/或所述第二组分以冻干粉的形式存在时，稳定性大大提升，可以在常温条件下进行储存和运输，大大降低了对储存和运输的环境要求，且将冻干粉与合适的缓冲液混合后便可以复溶，同时保持了原有的功能。
另外，发明人发现，现有的样本裂解稀释液与PCR稀释液在配方和组分上有很大的不同，样本裂解稀释液专用于样本裂解和样本核酸提取，而PCR稀释液专用于PCR扩增反应，两种稀释液不能通用。基于上述问题，发明人通过大量的实验探究，研究开发了一种稀释液组合物，该组合物能够同时在样本裂解和PCR扩增反应中发挥缓冲作用，从而为样本的核酸提取和荧光PCR反应的一体化提供了技术支持。
为此，在本发明的另一方面，本发明提出了一种稀释液。根据本发明的实施例，所述稀释液包括：多元醇，氯化盐，Tris-HCl，表面活性剂以及水。在一些实施例中，所述多元醇为丙三醇；该稀释液适于应用在上述实施例的利用PCR反应系统进行PCR反应的方法和PCR反应系统中。具体的，可以设置在稀释液容纳单元内。根据本发明实施例的稀释液对样本裂解液和PCR反应体系均有一定的缓冲作用，经过所述缓冲溶液的缓冲稀释，样本裂解后的裂解液不需要纯化即可直接加入PCR反应体系中完成PCR反应，为样本的核酸提取与PCR检测一体化提供了技术支持。
根据本发明的实施例，上述稀释液还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述表面活性剂包括选自吐温20(Tween20)、吐温80、聚乙二醇辛基苯基醚、十二烷基硫酸钠(SDS)、十二烷基苯磺酸钠、二辛基琥珀酸磺酸钠、甘胆酸钠的至少 之一。
根据本发明的实施例，所述氯化盐包括选自氯化钾、氯化钠、氯化镁的至少之一。
根据本发明的实施例，所述氯化盐为氯化镁和氯化钠，基于所述稀释液的总体积，所述氯化镁的浓度为0.5～15mmol/L，如为1.0、1.5、2.0、3.0、5.0、6.0、8.0、10.0或12.0mmol/L，所述氯化钠的浓度为1～150mmol/L，如为2、4、5、10、20、40、50、60、80、100或120mmol/L，所述表面活性剂的浓度为0.1～7％，如为0.1、0.2、0.4、0.5、1.0、2.0、3.0、4.0、5.0、6.0或7.0％，所述丙三醇的浓度为2～20％，如为3、5、7、8、9、12、15或18％。需要说明的是，所述表面活性剂为十二烷基硫酸钠等固体类时，所述表面活性剂的浓度为质量体积浓度，指的是每100mL溶液中所述表面活性剂的质量，单位为g，例如，所述十二烷基硫酸钠的浓度为0.1～7％，指的是每100mL稀释液中十二烷基硫酸钠的质量为0.1～7g。所述表面活性剂为聚乙二醇辛基苯基醚或吐温20等液体类时，所述表面活性剂的浓度为体积浓度，指的是每100mL溶液中所述表面活性剂的体积，单位为mL，例如，所述聚乙二醇辛基苯基醚或吐温20的浓度为0.1～7％，指的是每100mL稀释液中聚乙二醇辛基苯基醚或吐温20的体积为0.1～7mL。另外，所述丙三醇的浓度2～20％为体积浓度，指的是每100mL稀释液中丙三醇的体积为2～20mL。发明人发现，所述稀释液中各成分的浓度在该范围时，所述稀释液能够更加有效地同时在样本裂解和PCR扩增反应中发挥缓冲作用。
根据本发明的实施例，所述氯化盐为氯化镁和氯化钠，基于所述稀释液的总体积，所述氯化镁的浓度为1.5～10mmol/L，所述氯化钠的浓度为5～100mmol/L，所述表面活性剂的浓度为0.1～5％，所述丙三醇的浓度为5～10％。发明人发现，所述稀释液中各成分的重量份在该范围时，所述稀释液能够更加有效地同时在样本裂解和PCR扩增反应中发挥缓冲作用。因而，也可以作为PCR反应的缓冲液。
根据本发明的实施例，所述Tris-HCl是以溶于水的形式提供的，指的是配置本发明的稀释液时是以Tris-HCl水溶液的形式进行添加的。需要说明的是，Tris-HCl水溶液是本领域常用的一类缓冲物质，可以自己配置，例如可以用Tris base和盐酸慢慢调节到预定pH，也可以直接购买。
根据本发明的实施例，所述Tris-HCl溶于水形成的溶液(Tris-HCl水溶液)的pH为7.5～8.0，如7.6、7.7、7.8、7.9或8.0。需要说明的是，所述pH并不是指所述稀释液的pH，而是Tris-HCl溶于水形成的溶液的pH。根据本发明的实施例，所述Tris-HCl溶于水形成的溶液的pH为7.5～8.0时，Tris-HCl对所述稀释液的缓冲效果更好。在一些实施方案中，所述Tris-HCl溶于水形成的溶液的pH为7.6。
根据本发明的实施例，基于所述稀释液的总体积，所述Tris-HCl的浓度为1～100mmol/L，如为3、5、7、10、20、30、40、50、70、90或100mmol/L。需要说明的是，Tris-HCl的浓度按照本领域技术人员的常规认知进行理解即可。根据本发明的实施例，所述Tris-HCl的浓度为1～100mmol/L时，Tris-HCl对所述稀释液的缓冲效果更好。在一些实施方案中，所述Tris-HCl的浓度为5～50mmol/L。
在本发明的另一方面，本发明提出了一种稀释液。根据本发明的实施例，基于所述稀释液的总体积，所述稀释液包括：浓度为1.5～10mmol/L的氯化镁，浓度为5～100mmol/L的氯化钠，浓度为0.1～5％的表面活性剂，浓度为5～10％的丙三醇，浓度为5～50mmol/L的Tris-HCl，以及水，所述表面活性剂为吐温20、十二烷基硫酸钠或聚乙二醇辛基苯基醚；该稀释液适于应用在上述实施例的利用PCR反应系统进行PCR反应的方法和PCR反应系统中。具体的，可以设置在稀释液容纳单元内。根据本发明实施例的稀释液能够更加有效地同时在样本裂解和PCR扩增反应中发挥缓冲作用，因而，也可以作为PCR反应的缓冲液。
附图说明
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：
图1是根据本发明实施例的利用PCR反应系统进行PCR反应的方法流程示意图；
图2是根据本发明另一实施例的利用PCR反应系统进行PCR反应的方法流程示意图；
图3是根据本发明另一实施例的利用PCR反应系统进行PCR反应的方法流程示意图；
图4是根据本发明另一实施例的利用PCR反应系统进行PCR反应的方法流程示意图；
图5是根据本发明实施例的PCR系统结构示意图；
图6是根据本发明另一实施例的PCR系统结构示意图；
图7是根据本发明另一实施例的PCR系统结构示意图。
图8是根据本发明实施例2的测试结果示意图；
图9是根据本发明实施例3的测试结果示意图；
图10是根据本发明对比例1的测试结果示意图；
图11是根据本发明对比例2的测试结果示意图；
图12是根据本发明对比例3的测试结果示意图；
图13是根据本发明对比例4的测试结果示意图；
图14是根据本发明对比例5的测试结果示意图；
图15是根据本发明实施例4的性质测试结果示意图；
图16是根据本发明实施例5的性质测试结果示意图；
图17是根据本发明实施例6的性质测试结果示意图；
图18是根据本发明实施例7的性质测试结果示意图；
图19是根据本发明对比例6的性质测试结果示意图；
图20是根据本发明对比例7的性质测试结果示意图。
附图标记：
100：样本容纳单元；
110：第一液体出/入口；
200：稀释液容纳单元；
210：稀释液出口；
300：PCR反应单元；
310：裂解后的样本混合液入口；
320：PCR反应液出口；
400：活塞单元；
410：注射室；
411：第二液体出/入口；
420：活塞；
500：缓冲单元；
510：PCR反应液入口；
520：通气口；
610：样本容纳单元密封件；
620：稀释液容纳单元密封件；
710：样本容纳单元密封件刺穿装置；
720：稀释液容纳单元密封件刺穿装置；
810：样本控制阀；
820：稀释控制阀；
830：第一PCR控制阀；
840：第二PCR控制阀；
910：第一管路；
920：第二管路；
930：第三管路；
940：第四管路。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
本发明提出了一种利用PCR反应系统进行PCR反应的方法以及相应的PCR反应系统。根据本发明的实施例，参考图5，该PCR反应系统包括：样本容纳单元100，所述样本容纳单元100内设置有裂解冻干粉和样本，并且所述样本容纳单元100设置有第一液体出/入口110；稀释液容纳单元200，所述稀释液容纳单元200内设置有稀释液，并且所述稀释液容纳单元200内设置有稀释液出口210；PCR反应单元300，所述PCR反应单元300内设置有反转录酶和PCR原料冻干粉，并且所述PCR反应单元300设置有裂解后的样本混合液入口310和PCR反应液出口320，所述PCR反应液出口320与所述稀释液出口210通过第四管路940相连；以及活塞单元400，所述活塞单元包括注射室410和活塞420，所述注射室410设置有第二液体出/入口411，所述第二液体出/入口411与所述第一液体出/入口110通过第一管路910相连，所述第二液体出/入口411与所述稀释液出口210通过第二管路920相连，所述第二液体出/入口411与所述裂解后的样本混合液入口310通过第三管路930相连；
参考图1，该方法包括：将所述活塞420进行第一移动处理S100，以便使部分稀释液进入所述注射室410，所述稀释液设置在所述稀释液容纳单元200内；将所述活塞420进行第二移动处理S200，以便使进入注射室410的所述稀释液与裂解冻干粉以及样本进行第一混合处理，所述裂解冻干粉以及所述样本设置在所述样本容纳单元100内，所述第一混合处理是在所述样本容纳单元100中进行的；将第一混合处理产物进行裂解处理S300，所述裂解处理是在所述样本容纳单元100中进行的；将所述活塞420进行第三移动处理S400，以便使裂解处理产物进入所述注射室410；将所述活塞420进行第四移动处理S500，以便使进入注射室410的所述裂解处理产物与剩余部分稀释液进行第二混合处理，所述第二混合处理是在所述稀释液容纳单元200中进行的；将所述活塞420进行第五移动处理S600，以便使第二混合处理产物进入所述注射室410；将所述活塞420进行第六移动处理S700，以便使进入注射室410的所述第二混合处理产物与反转录酶及PCR原料冻干粉进行第三混合处理，所述反转录酶及PCR原料冻干粉设置在所述PCR反应单元300内，所述第三混 合处理是在所述PCR反应单元300中进行的；以及将第三混合处理产物进行PCR温度循环扩增处理S800，所述PCR温度循环扩增处理是在所述PCR反应单元300中进行的。
根据本发明实施例的PCR反应系统通过微流控管路将样本容纳单元100、稀释液容纳单元200、PCR反应单元300以及活塞单元400相互连接在一起；同时，各单元又分别为独立设置的单元，以便使各单元在使用前存放不同的反应物，有利于反应物在不使用的情况进行长久保存。例如，样本容纳单元100的独立设置有利于样本的单独添加，使样本的添加操作简单化，同时也有利于样本的长久保存。参考图1和图5，首先，将活塞420向外拉动到一定位置，从而使稀释液容纳单元200中的部分稀释液流向注射室410；之后往复移动活塞420，使注射室410中的稀释液进入样本容纳单元100，同时与样本容纳单元100中的裂解冻干粉以及样本混合均匀；之后对样本容纳单元100进行加热升温到设定温度，使样本容纳单元100中的样本在设定的温度下进行充分裂解；裂解完成后，再次将活塞420向外拉动到一定位置，从而使样本容纳单元100中已裂解的样本混合液流向注射室410；之后往复移动活塞420，使注射室410中已裂解的样本混合液返回稀释液容纳单元200，同时与稀释液容纳单元200中剩余的稀释液混合均匀，从而将已裂解的样本混合液进行稀释，将其中的杂质浓度降低；之后，再次将活塞420向外拉动到一定位置，从而使稀释液容纳单元200中稀释后的样本混合液流向注射室410；随后往复移动活塞420，使注射室410中稀释后的样本混合液进入PCR反应单元300，同时与PCR反应单元300中的反转录酶以及PCR原料的冻干粉混合均匀；最后对PCR反应单元300进行PCR温度加热控制，以便最终完成PCR扩增反应。根据本发明实施例的PCR反应系统中，所述PCR反应液出口与所述稀释液出口通过第四管路相连，形成了PCR反应液出口与稀释液出口的压力系统连通，从而使得PCR反应单元内过多的反应液可以顺利通过反应液出口流出到第四管路。进一步地，根据本发明实施例的PCR反应系统中，可以在微流控管路的合适位置灵活设计阀门或者其他开关，以便控制活塞单元400与样本容纳单元100、稀释液容纳单元200或PCR反应单元300的连通状态。另外，活塞的移动以及各阀门或其他开关的控制也可以灵活设计其他机械装置来实现自动化。根据本发明实施例的PCR反应方法中，将已裂解的样本混合液返回稀释液容纳单元，与稀释液容纳单元中剩余的稀释液混合均匀，可以将已裂解的样本混合液进行稀释，将其中的杂质浓度显著降低，从而避免裂解后产生的杂质对后续PCR扩增反应造成不利影响，有利于PCR反应的自动化。需要说明的是，本领域技术人员可以根据实际需求设计两部分稀释液的具体用量比例。由此，根据本发明实施例的PCR反应方法实现了从样本核酸提取到与试剂混合，最后再到PCR反应的全自动过程，解决了传统PCR实验过程需要在专业实验环境下由专业人士进行操作的难题，无需专业人士即可完成，且减少了人为操作带来的误差，极大地提高了PCR反应的工作效率，大大节约了人力资源成本。
下面参考附图，对根据本发明实施例的方法进行进一步详细描述：
根据本发明的另一实施例，参考图2，该PCR温度循环扩增处理S800包括：将所述第三混合处理产物进行恒温处理S810；以及将恒温处理产物进行温度循环处理S820。
根据本发明的另一实施例，参考图6，该PCR反应系统进一步包括：样本控制阀810，所述样本控制阀810设置在所述第一管路910上，用于控制所述第二液体出/入口411与所述第一液体出/入口110的连通状态；稀释控制阀820，所述稀释控制阀820设置在所述第二管路920上，用于控制所述第二液体出/入口411与所述稀释液出口210的连通状态；第一PCR控制阀830，所述第一PCR控制阀830设置在所述第三管路930上，用于控制所述第二液体出/入口411与所述裂解后的样本混合液入口310的连通状态；以及第二PCR控制阀840，所述第二PCR控制阀840设置在所述第四管路940上，用于控制所述PCR反应液出口320与所述稀释液出口210的连通状态；
参考图3，该方法进一步包括：S910：在所述第一移动处理前，将所述样本控制阀、所述第一PCR控制阀以及所述第二PCR控制阀进行关闭处理，将所述稀释控制阀进行开启处理；S920：在所述第一移动处理后以及所述第二移动处理前，将所述稀释控制阀进行关闭处理，将所述样本控制阀进行开启处理；S930：在所述第三移动处理后以及所述第四移动处理前，将所述样本控制阀进行关闭处理，将所述稀释控制阀进行开启处理；S940：在所述第五移动处理后以及第六移动处理前，将所述稀释控制阀进行关闭处理，将所述第一PCR控制阀以及所述第二PCR控制阀进行开启处理；S950：在所述恒温处理后以及温度循环处理前，将所述第一PCR控制阀以及所述第二PCR控制阀进行关闭处理。
根据本发明的实施例，参考图3和图6，首先，关闭第一PCR控制阀830、第二PCR控制阀840以及样本控制阀810，打开稀释控制阀820；然后将活塞420向外拉动到一定位置，从而使稀释液容纳单元200中的部分稀释液流向注射室410；之后关闭稀释控制阀820，打开样本控制阀810；之后往复移动活塞420，使注射室410中的稀释液进入样本容纳单元100，同时与样本容纳单元100中的裂解冻干粉以及样本混合均匀；之后对样本容纳单元100进行加热升温到设定温度，使样本容纳单元100中的样本在设定的温度下进行充分裂解；裂解完成后，再次将活塞420向外拉动到一定位置，从而使样本容纳单元100中已裂解的样本混合液流向注射室410；之后，关闭样本控制阀810，打开稀释控制阀820；之后往复移动活塞420，使注射室410中已裂解的样本混合液返回稀释液容纳单元200，同时与稀释液容纳单元200中剩余的稀释液混合均匀，从而将已裂解的样本混合液进行稀释，将其中的杂质浓度降低；之后，再次将活塞420向外拉动到一定位置，从而使稀释液容纳单元200中稀释后的样本混合液流向注射室410；之后，关闭稀释控制阀820，打开第一PCR控制阀830和第二PCR控制阀840；随后往复移动活塞420，使注射室410中稀释后的样本混合液进入PCR反应单元300，同时与PCR反应单元300中的反转录酶以及PCR原料的冻干粉混合均匀；最后对PCR反应单元300进行PCR温度加热控制，包括激活酶的前期恒温阶段以及温度循环控制阶段，在进行温度循环控制之前，关闭第一PCR控制阀830和第二PCR控制阀840，以便最终完成PCR扩增反应。根据本发明实施例的PCR反应系统中各单元与各阀门完美配合，协同发挥作用，使得实验产物对环境的污染以及环境对实验过程的污染均减少，有利于实现全自动化，无需专业人员进行人工操作。由此，根据本发明实施例的PCR反应方法在密闭环境下进行，减少了对系统环境的污染，提高了实验的可信度，且操作更加简便易行。
根据本发明的另一实施例，参考图3和图7，该PCR反应系统进一步包括：缓冲单元500，所述缓冲单元500设置有PCR反应液入口510和通气口520，所述缓冲单元500设置在所述第四管路940上，所述PCR反应液入口510与所述第二PCR控制阀840相连，所述通气口520与所述稀释液出口210相连。
根据本发明的实施例，参考图3和图7，首先，关闭第一PCR控制阀830、第二PCR控制阀840以及样本控制阀810，打开稀释控制阀820；然后将活塞420向外拉动到一定位置，从而使稀释液容纳单元200中的部分稀释液流向注射室410；之后关闭稀释控制阀820，打开样本控制阀810；之后往复移动活塞420，使注射室410中的稀释液进入样本容纳单元100，同时与样本容纳单元100中的裂解冻干粉以及样本混合均匀；之后对样本容纳单元100进行加热升温到设定温度，使样本容纳单元100中的样本在设定的温度下进行充分裂解；裂解完成后，再次将活塞420向外拉动到一定位置，从而使样本容纳单元100中已裂解的样本混合液流向注射室410；之后，关闭样本控制阀810，打开稀释控制阀820；之后往复移动活塞420，使注射室410中已裂解的样本混合液返回稀释液容纳单元200，同时与稀释液容纳单元200中剩余的稀释液混合均匀，从而将已裂解的样本混合液进 行稀释，将其中的杂质浓度降低；之后，再次将活塞420向外拉动到一定位置，从而使稀释液容纳单元200中稀释后的样本混合液流向注射室410；之后，关闭稀释控制阀820，打开第一PCR控制阀830和第二PCR控制阀840；随后往复移动活塞420，使注射室410中稀释后的样本混合液进入PCR反应单元300，同时与PCR反应单元300中的反转录酶以及PCR原料的冻干粉混合均匀；最后对PCR反应单元300进行PCR温度加热控制，在PCR扩增中激活酶的前期恒温段，PCR反应单元300的混合液会因为高温而产生膨胀，膨胀过程中的液体溢出可以流入缓冲单元500中，恒温段结束后，关闭第一PCR控制阀830和第二PCR控制阀840，开始对PCR反应单元300进行温度循环控制，以便最终完成PCR扩增反应。由此，根据本发明实施例的PCR反应方法可以解决PCR试剂在高温膨胀时的溢出问题，且PCR反应在密闭环境下进行，减少了对系统环境的污染，提高了实验的可信度，操作更加简便易行。
根据本发明的另一实施例，参考图7，该PCR反应系统进一步包括：样本容纳单元密封件610，所述样本容纳单元密封件610设置在所述第一液体出/入口110表面，用于将所述样本容纳单元100进行第一密封处理；以及稀释液容纳单元密封件620，所述稀释液容纳单元密封件620设置在所述稀释液出口210表面，用于将所述稀释液容纳单元200进行第二密封处理；
参考图4，该方法进一步包括：预先刺穿处理S1000：所述预先刺穿处理包括预先将所述样本容纳单元密封件进行第一刺穿处理，将所述稀释液容纳单元密封件进行第二刺穿处理。
根据本发明的实施例，参考图4和图7，初始状态下，样本容纳单元100中含有以冻干粉状存在的裂解原料，PCR反应单元300中含有以冻干粉状存在的反转录酶和PCR原料，稀释液容纳单元200中含有适当的稀释液。样本容纳单元100以及稀释液容纳单元200在与微流控管路连通之处用样本容纳单元密封件610和稀释液容纳单元密封件620密封住，使样本容纳单元100的裂解原料与稀释液容纳单元200的稀释液以及PCR反应单元300的反转录酶及PCR原料相互隔绝，同时活塞420处于注射室410的最顶端(即注射室处于被活塞填满的状态)。需要说明的是，PCR反应系统在具有样本容纳单元密封件和稀释液容纳单元密封件的前提下，不需要再设置PCR反应密封装置，也已经可以起到各单元隔绝设置的作用，并且即使PCR反应单元中的反应物有少量进入管路中，对整体反应也影响不大。样本容纳单元密封件和稀释液容纳单元密封件不仅可以隔绝各独立单元内的反应物，便于不使用的情况下长时间保存，大大提高了各单元中各反应物的存储时间，而且避免了各反应物对PCR反应系统造成的污染，提高了PCR反应系统的使用寿命。进而，当样本容纳单元添加了样本后，只需要将样本容纳单元密封件和稀释液容纳单元密封件刺穿，以便使样本容纳单元以及稀释液容纳单元与微流控管路之间处于连通状态，系统便可以开始工作。由此，根据本发明实施例的方法操作更加简便。
根据本发明的实施例，参考图4和图7，所述第一刺穿处理是通过样本容纳单元密封件刺穿装置710进行的，所述第二刺穿处理是通过稀释液容纳单元密封件刺穿装置720进行的。
根据本发明的实施例，所述样本容纳单元密封件和所述稀释液容纳单元密封件的至少之一为密封膜。
根据本发明的实施例，所述密封膜是由锡箔纸、塑封膜、牛皮纸的至少之一形成。
根据本发明的实施例，所述密封膜的厚度为0.01～0.2mm，如为0.03mm、0.05mm、0.07mm、0.09mm、0.1mm、0.13mm、0.15mm、0.17mm或0.19mm。发明人发现，若密封膜的厚度过小，则可能渗透，若密封膜的厚度过大，又难以刺穿。在一些实施例中，所述密封膜的厚度为0.05～0.1mm。
下面通过具体的实施例对本发明提出的利用PCR反应系统进行PCR反应的方法和PCR反应系 统做出进一步描述。
实施例1
系统的结构：
参考图7，该系统的结构包括：设计为样本室的样本容纳单元100，设计为稀释室的稀释液容纳单元200，注射室410，活塞420，设计为PCR室的PCR反应单元300，设计为缓冲室的缓冲单元500，设计为样本密封膜的样本容纳单元密封件610，设计为稀释密封膜的稀释液容纳单元密封件620，微流控管路，样本控制阀810，稀释控制阀820，第一PCR控制阀830，第二PCR控制阀840，各单元通过微流控管路连接形成一个相关联的回路。
系统的工作原理：
参考图7，初始状态下，样本室含有以冻干粉状存在的裂解原料，PCR室含有以冻干粉状存在的反转录酶和PCR原料，稀释室含有适当稀释液。样本室及稀释室在与微流控管路连通之处用样本密封膜和稀释密封膜密封住，使样本室的裂解原料与稀释室的稀释液以及PCR室的反转录酶及PCR原料相互隔绝。活塞处于注射室的最顶端(注射室处于活塞填满状态)。
当样本室添加了样本后，该系统开始工作。首先，通过刺穿装置710/720将样本密封膜和稀释密封膜刺穿，从而使各单元和微流孔管路之间处于连通状态。第二，关闭样本控制阀、第一PCR控制阀、第二PCR控制阀，移动活塞向外拉动到一定位置，从而使稀释室的稀释液通过稀释控制阀流向注射室。第三，关闭稀释控制阀，打开样本控制阀，往复移动活塞，将注射室的部分稀释液通过样本控制阀进入到样本室，在往复移动活塞的过程中，充分将样本室的裂解冻干粉与稀释液及加入的样本混合均匀。第四，开始对样本室进行加热升温到设定温度，使得样本室中的样本在设定的温度下进行充分裂解。第五，裂解完成后，移动活塞向外拉动到一定位置，从而使样本室的已裂解的样本混合液通过样本控制阀流向注射室。第六，关闭样本控制阀，打开稀释控制阀，往复移动活塞，将注射室中已裂解的样本混合液返回稀释室，在往复移动活塞的过程中，充分将已裂解的样本混合液与稀释室中剩余的稀释液混合均匀，从而将已裂解的样本混合液进行稀释，将其中的杂质浓度降低；第七，再次将活塞向外拉动到一定位置，从而使稀释室中稀释后的样本混合液流向注射室；第八，关闭稀释控制阀，打开第一PCR控制阀与第二PCR控制阀，往复移动活塞，将注射室的样本混合液通过PCR控制阀进入到PCR室，在往复移动活塞的过程中，充分将已稀释的样本混合液与PCR室的反转录酶及PCR原料冻干粉混合均匀。第九，开始对PCR室进行PCR温度加热控制，在PCR扩增中激活酶的前期恒温段，PCR室中的混合液会因为高温而产生膨胀，膨胀过程中的液体溢出可经过第二PCR控制阀流到缓冲室中，恒温段结束后，关闭第一PCR控制阀与第二PCR控制阀，开始对PCR室进行温度循环控制，最终完成PCR扩增实验。
下面对如前所述的PCR反应的组合物和冻干粉进行详细描述。需要说明的是，如无特别说明，本发明中各成分以及各浓度的含义按照本领域的常规解释进行理解即可，如EDTA和EGTA。另外，Amplification Plot表示扩增曲线；Cycle表示循环数。还需要说明的是，除本发明的样本裂解以及核酸提取组合物或其冻干粉为发明人的研发成果外，如无特别说明，下述步骤中使用到的其他相关试剂均可以购买或者查阅现有技术获得。本领域技术人员可依据实际需要购买相关试剂，或查阅现有技术获得相关试剂。
一、样本裂解以及核酸提取冻干粉的制备
1、称量或吸取一定量的目标成分，以便配置预定体积的混合溶液，该混合溶液中：EDTA为0.5mM-5mM，EGTA为0.5mM-10mM，十二烷基硫酸钠为0.5％-2.5％，皂苷为0.5％-2.5％，蛋白酶 K为10-100U/mL，聚乙二醇3350为0.5-4.5％，Tris-HCl为5mM-20mM，用水配置到预定体积，吸取的Tris-HCl溶液的pH为7.5～8.2。
2、吸取50μL上述混合溶液，按照常规冻干方法(如干冰上冻干，放入冻干机，-30℃干燥升华)冻干，制备获得一定量的样本裂解以及核酸提取冻干粉。
二、荧光定量PCR试剂冻干粉的制备
1、称量或吸取一定量的目标成分，以便配置预定体积的混合溶液，该混合溶液中：甘露醇为0.5％-8％，蔗糖为0.5％-8％，氯化钾为20-100mM，氯化镁为1.0mM-5mM，牛血清白蛋白为0.1-1mg/mL，dNTP为200μM，Brij 35为0.05％，HEPES为10-50mM，DNA聚合酶为20-200U/mL，M-MLV反转录酶为10-50U/mL，RNA酶抑制剂为200-1000U/mL，用水配置到预定体积，吸取的HEPES溶液的pH为8.0～8.5。
2、将上述混合溶液过滤，并按照常规冻干方法冻干，制备获得一定量的荧光定量PCR试剂冻干粉。
三、性质测试
1)毒性
样本裂解以及核酸提取冻干粉的配置成分不包含苯酚、氯仿、盐酸胍或异硫氰酸胍等有毒物质，毒性低。
2)稳定性
荧光定量PCR试剂冻干粉室温保存即可，稳定性好。
3)样本裂解和核酸提取以及荧光定量PCR的验证测试
样本裂解：
将Flu A甲型流感病毒以一定比例，加入裂解液中。并于95℃温浴一定时间，进行样本裂解。
荧光定量PCR验证测试：
将上述裂解样本以一定比例加入复溶后的荧光定量PCR反应体系中，并加入对应的Flu A引物与探针。上机进行荧光定量PCR反应。反应程序为：50℃，5min；95℃2min；95℃15s，60℃1min，40Cycles。其中，引物和探针的序列如下所示：
FluA-Forward：CAGAGACTTGAAGATGTTTTTGC(SEQ ID NO：1)
FluA-Reverse：CTACGCTGCAGTCCTCGCTC(SEQ ID NO：2)
FluA-Prob：CY3-CAAGACCAATCCTGTCACCTCTGA-BHQ2(SEQ ID NO：3)
实施例2
1、样本裂解以及核酸提取冻干粉的制备
称量或吸取一定量的目标成分，以便配置预定体积的混合溶液，该混合溶液中：EDTA为2mM，EGTA为2mM，十二烷基硫酸钠为1％，皂苷为1.0％，蛋白酶K为20U/mL，聚乙二醇3350为1％，Tris-HCl为10mM，用水配置到预定体积，吸取的Tris-HCl溶液的pH为7.6。
吸取50μL上述混合溶液，加入八连管中，离心到管底，放入-80℃冰冻过夜。取出放入冻干机进行过夜冻干，冻干后盖盖，放在室温保存。确保冻干机温度低于-45℃，真空压力<450Torr，试管中样本在干冰上放置至少30分钟。
2、荧光定量PCR试剂冻干粉的制备
称量或吸取一定量的目标成分，以便配置预定体积的混合溶液，该混合溶液中：甘露醇为4％，蔗糖为1.5％，氯化钾为80mM，氯化镁为3.5mM，牛血清白蛋白为0.5mg/mL，dNTP为200μM，Brij 35为0.05％，HEPES为20mM，DNA聚合酶为40U/mL，M-MLV反转录酶为20U/mL，RNA酶抑制剂为500U/mL，用水配置到预定体积，吸取的HEPES溶液的pH为8.25。
吸取18μL上述混合溶液，加入八连管中，离心到管底，放入-80℃冰冻过夜。取出放入冻干机进行过夜冻干，冻干后盖盖，放在室温保存。确保冻干机温度低于-45℃，真空压力<450Torr，试管中样本在干冰上放置至少30分钟。
3、样本裂解和核酸提取以及荧光定量PCR的验证测试
按照上述方法进行样本裂解和核酸提取以及荧光定量PCR的试剂配制和冻干。室温存储3个月后，加水复溶后加入Flu A病毒进行样本裂解，进而将裂解后样本加入上述比例的复溶后加入Flu A引物和探针的荧光定量PCR反应体系中，上机行荧光定量PCR验证测试，测试结果如图8所示。
结论：由图8可以看出，冻干粉试剂，室温保存3个月后，复溶，仍能正常扩增。说明第一组分、第二组分试剂配方在冻干后重新复溶仍保持样本裂解和PCR检测活性。
实施例3
1、样本裂解以及核酸提取冻干粉的制备
称量或吸取一定量的目标成分，以便配置预定体积的混合溶液，该混合溶液中：EDTA为4mM，EGTA为4mM，十二烷基硫酸钠为2％，皂苷为2％，蛋白酶K为80U/mL，聚乙二醇3350为4％，Tris-HCl为18mM，用水配置到预定体积，吸取的Tris-HCl溶液的pH为7.6。
吸取50μL上述混合溶液，加入八连管中，离心到管底，放入-80℃冰冻过夜。取出放入冻干机进行过夜冻干，冻干后盖盖，放在室温保存。确保冻干机温度低于-45℃，真空压力<450Torr，试管中样本在干冰上放置至少30分钟。
2、荧光定量PCR试剂冻干粉的制备
称量或吸取一定量的目标成分，以便配置预定体积的混合溶液，该混合溶液中：甘露醇为6％，蔗糖为3％，氯化钾为40mM，氯化镁为5mM，牛血清白蛋白为1mg/mL，dNTP为200μM，Brij 35为0.05％，HEPES为40mM，DNA聚合酶为100U/mL，M-MLV反转录酶为40U/mL，RNA酶抑制剂为800U/mL，用水配置到预定体积，吸取的HEPES溶液的pH为8.25。
吸取18μL上述混合溶液，加入八连管中，离心到管底，放入-80℃冰冻过夜。取出放入冻干机进行过夜冻干，冻干后盖盖，放在室温保存。确保冻干机温度低于-45℃，真空压力<450Torr，试管中样本在干冰上放置至少30分钟。
3、样本裂解和核酸提取以及荧光定量PCR的验证测试
按照上述方法进行样本裂解和核酸提取以及荧光定量PCR的试剂配制和冻干。室温存储3个月后，加水复溶后加入Flu A病毒进行样本裂解，进而将裂解后样本加入上述比例的复溶后加入Flu A引物和探针的荧光定量PCR反应体系中，上机行荧光定量PCR验证测试，测试结果如图9所示。
结论：由图9可以看出，冻干粉试剂，室温保存3个月后，复溶，仍能正常扩增。说明第一组分、第二组分试剂配方在冻干后重新复溶仍保持样本裂解和PCR检测活性。
对比例1
原料配比与实施例1仅区别于：第一组分中蛋白酶K为2U/mL，其他组分配比不变。测试方法与实施例1相同。
扩增结果如图10所示。
结论：由图10可以看出，当蛋白酶K的浓度过低时，会影响第一组分裂解样本的效果，进而 影响PCR扩增结果。说明蛋白酶K在第一组分中的配比浓度很重要。
对比例2
原料配比与实施例1仅区别于：第一组分中EDTA、EGTA浓度均为0mM，其他组分配比不变。测试方法与实施例1相同。
扩增结果如图11所示。
结论：由图11可以看出，当去掉EDTA、EGTA成分，会影响第一组分裂解样本的效果，进而影响PCR扩增结果。说明EDTA、EGTA在第一组分中的作用很重要。
对比例3
原料配比与实施例1仅区别于：第一组分中皂苷的浓度为10％，其他组分配比不变。测试方法与实施例1相同。
扩增结果如图12所示。
结论：由图12可以看出，当皂苷的浓度过高后，会影响第一组分裂解样本的效果，进而影响PCR扩增结果。说明皂苷在第一组分中的配比浓度很重要。
对比例4
原料配比与实施例1仅区别于：第二组分中氯化镁的浓度为0.2mM，其他组分配比不变。测试方法与实施例1相同。
扩增结果如图13所示。
结论：由图13可以看出，当氯化镁的浓度过低，会影响第二组PCR反应结果。说明氯化镁在第二组分中的配比浓度很重要。
对比例5
原料配比与实施例1仅区别于：第二组分中HEPES的浓度为2mM，其他组分配比不变。测试方法与实施例1相同。
扩增结果如图14所示。
结论：由图14可以看出，当HEPES的浓度过低，会影响第二组PCR反应结果。说明HEPES在第二组分中的配比浓度很重要。
下面对如前所述的PCR反应的组合物和稀释液进行详细描述。需要说明的是，如无特别说明，本发明中各成分的含义按照本领域的常规解释进行理解即可，如Tris-HCl和吐温20。另外，Amplification Plot表示扩增曲线；Cycle表示循环数。
一、稀释液的制备
称量或吸取一定量的目标成分，以便配置预定体积的稀释液，该稀释液中：氯化镁为0.5～15mmol/L，氯化钠为1～150mmol/L，表面活性剂为0.1～7％，丙三醇为2～20％，用水配置到预定体积，吸取的Tris-HCl溶液的浓度为1～100mM，pH为7.5～8.0。表面活性剂为吐温20、吐温80、聚乙二醇辛基苯基醚、十二烷基硫酸钠、十二烷基苯磺酸钠、二辛基琥珀酸磺酸钠或甘胆酸钠。
二、性质测试
将处理完样本的裂解液10μL加入到90μL的dilution buffer(上述稀释液)中稀释，将稀释液 5μL加入到PCR反应体系中，进行扩增反应，读取Ct值。
实施例4
1、稀释液的配方组成
5％的丙三醇、2mM的氯化镁、5mM的氯化钠、5mM的Tris-HCl、0.5％的Tween20，加入的Tris-HCl的pH为7.6。
PCR反应体系配方组成
Taq酶1U/反应、dNTP 4mM/反应、18s rRNA Assay(Thermo Fisher)1uL/反应、模板5uL/反应。
2、性质测试结果
按照上述方法进行性质测试，读取Ct值。
测试结果如图15所示。其中：
线1表示样本进行裂解处理后的溶液(裂解原液)直接加入后续的PCR反应体系扩增结果；
线2表示上述裂解原液经过该dilution buffer(稀释液)稀释后，加入相同的PCR反应体系扩增结果；
线3表示上述裂解原液经H2O稀释相等倍数后，加入相同的PCR反应体系扩增结果；
线4表示阴性质控，即以dilution buffer(稀释液)为模板加入相同PCR反应体系扩增结果。
结论：
线1为样本进行裂解处理后的溶液直接加入后续的PCR反应体系扩增结果，显示无法扩增出扩增曲线。线2为上述裂解原液经过该dilution buffer稀释后加入相同的PCR反应体系中，显示能得到阳性的扩增曲线。线3为上述裂解原液经H2O稀释相等倍数，显示样本有扩增，但Ct值显著高于dilution buffer(本发明稀释液)稀释样本，说明dilution buffer的缓冲效果优于H2O。线4为dilution buffer为模板加入相同PCR反应体系中，作为阴性质控，阴性质控无扩增曲线，说明该dilution buffer能有效缓冲裂解液与PCR反应体系。
实施例5
1、稀释液的配方组成
8％的丙三醇、6mM的氯化镁、50mM的氯化钠、40mM的Tris-HCl、3％的Tween20，加入的Tris-HCl的pH为8.0。
PCR反应体系配方组成
Taq酶1U/反应、dNTP 4mM/反应、18s rRNA Assay(Thermo Fisher)1uL/反应、模板5uL/反应。
2、性质测试结果
按照上述方法进行性质测试，读取Ct值。
测试结果如图16所示。其中：
线1表示样本进行裂解处理后的溶液(裂解原液)直接加入后续的PCR反应体系扩增结果；
线2表示上述裂解原液经过该dilution buffer(稀释液)稀释后，加入相同的PCR反应体系扩增结果；
线3表示上述裂解原液经H2O稀释相等倍数后，加入相同的PCR反应体系扩增结果；
线4表示阴性质控，dilution buffer(稀释液)为模板加入相同PCR反应体系扩增结果。
结论：
线1为样本进行裂解处理后的溶液直接加入后续的PCR反应体系扩增结果，显示无法扩增出扩增曲线。线2为上述裂解原液经过该dilution buffer稀释后加入相同的PCR反应体系中，显示能得到阳性的扩增曲线。线3为上述裂解原液经H2O稀释相等倍数，显示样本有扩增，但Ct值显著高于dilution buffer(本发明稀释液)稀释样本，说明dilution buffer的缓冲效果优于H2O。线4为dilution buffer为模板加入相同PCR反应体系中，作为阴性质控，阴性质控无扩增曲线，说明该dilution buffer能有效缓冲裂解液与PCR反应体系。
实施例6
1、稀释液的配方组成
8％的丙三醇、6mM的氯化镁、50mM的氯化钠、40mM的Tris-HCl、1％的十二烷基硫酸钠，加入的Tris-HCl的pH为8.0。
PCR反应体系配方组成
Taq酶1U/反应、dNTP 4mM/反应、18s rRNA Assay(Thermo Fisher)1uL/反应、模板5uL/反应。
2、性质测试结果
按照上述方法进行性质测试，读取Ct值。
测试结果如图17所示。其中：
线1表示样本进行裂解处理后的溶液(裂解原液)直接加入后续的PCR反应体系扩增结果；
线2表示上述裂解原液经过该dilution buffer(稀释液)稀释后，加入相同的PCR反应体系扩增结果；
线3表示上述裂解原液经H2O稀释相等倍数后，加入相同的PCR反应体系扩增结果；
线4表示阴性质控，dilution buffer(稀释液)为模板加入相同PCR反应体系扩增结果。
结论：
线1为样本进行裂解处理后的溶液直接加入后续的PCR反应体系扩增结果，显示无法扩增出扩增曲线。线2为上述裂解原液经过该dilution buffer稀释后加入相同的PCR反应体系中，显示能得到阳性的扩增曲线。线3为上述裂解原液经H2O稀释相等倍数，显示样本有扩增，但Ct值显著高于dilution buffer(本发明稀释液)稀释样本，说明dilution buffer的缓冲效果优于H2O。线4为dilution buffer为模板加入相同PCR反应体系中，作为阴性质控，阴性质控无扩增曲线，说明该dilution buffer能有效缓冲裂解液与PCR反应体系。
实施例7
1、稀释液的配方组成
5％的丙三醇、2mM的氯化镁、5mM的氯化钠、5mM的Tris-HCl、0.1％的聚乙二醇辛基苯基醚，加入的Tris-HCl的pH为8.0。
PCR反应体系配方组成
Taq酶1U/反应、dNTP 4mM/反应、18s rRNA Assay(Thermo Fisher)1uL/反应、模板5uL/反应。
2、性质测试结果
按照上述方法进行性质测试，读取Ct值。
测试结果如图18所示。其中：
线1表示样本进行裂解处理后的溶液(裂解原液)直接加入后续的PCR反应体系扩增结果；
线2表示上述裂解原液经过该dilution buffer(稀释液)稀释后，加入相同的PCR反应体系扩增结果；
线3表示上述裂解原液经H2O稀释相等倍数后，加入相同的PCR反应体系扩增结果；
线4表示阴性质控，dilution buffer(稀释液)为模板加入相同PCR反应体系扩增结果。
结论：
线1为样本进行裂解处理后的溶液直接加入后续的PCR反应体系扩增结果，显示无法扩增出扩增曲线。线2为上述裂解原液经过该dilution buffer稀释后加入相同的PCR反应体系中，显示能得到阳性的扩增曲线。线3为上述裂解原液经H2O稀释相等倍数，显示样本有扩增，但Ct值显著高于dilution buffer(本发明稀释液)稀释样本，说明dilution buffer的缓冲效果优于H2O。线4为dilution buffer为模板加入相同PCR反应体系中，作为阴性质控，阴性质控无扩增曲线，说明该dilution buffer能有效缓冲裂解液与PCR反应体系。
对比例6
仅原料配比与实施例1区别于：Tween20的浓度为8％，其余与实施例1相同。
测试结果如图19所示。
分析与结论：
更改配方后，发现该dilution Buffer稀释后样本无法扩增出阳性结果(线2)，而水稀释的样本仍能扩增出阳性(线1)，说明样本有效，只是因为该配方比例时的稀释液无法扩增出结果。表明Tween20的比例对本申请稀释液组合物的技术效果具有显著影响。
对比例7
仅原料配比与实施例1区别于：丙三醇的浓度为30％，其余与实施例1相同。
测试结果如图20所示。
分析与结论：
更改配方后，发现该dilution Buffer稀释后样本无法扩增出阳性结果(线2)，而水稀释的样本仍能扩增出阳性(线1)，说明样本有效，只是因为该配方比例时的稀释液无法扩增出结果。表明丙三醇的比例对本申请稀释液组合物的技术效果具有显著影响。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接或彼此可通讯；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系，除非另有明确的限定。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一 些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (21)

  1. 一种利用PCR反应系统进行PCR反应的方法，其特征在于，
    所述PCR反应系统包括：
    样本容纳单元，所述样本容纳单元内设置有裂解冻干粉和样本，并且所述样本容纳单元具有第一液体出/入口，
    稀释液容纳单元，所述稀释液容纳单元内设置有稀释液，并且所述稀释液容纳单元具有稀释液出口，
    PCR反应单元，所述PCR反应单元内设置有反转录酶和PCR原料冻干粉，并且所述PCR反应单元具有PCR反应液出口和裂解后的样本混合液入口，
    活塞单元，所述活塞单元包括注射室和活塞，所述注射室具有第二液体出/入口，
    所述第二液体出/入口通过第一管路与所述第一液体出/入口相连，
    所述第二液体出/入口通过第二管路与所述稀释液出口相连，
    所述第二液体出/入口通过第三管路与所述裂解后的样本混合液入口相连，
    所述PCR反应液出口通过第四管路与所述稀释液出口相连；
    所述方法包括：
    将所述活塞进行第一移动处理，以便使部分稀释液进入所述注射室，所述稀释液设置在所述稀释液容纳单元内，
    将所述活塞进行第二移动处理，以便使进入注射室的所述稀释液与裂解冻干粉和样本进行第一混合处理，所述裂解冻干粉和所述样本设置在所述样本容纳单元内，所述第一混合处理是在所述样本容纳单元中进行的，
    将第一混合处理产物进行裂解处理，所述裂解处理是在所述样本容纳单元中进行的，
    将所述活塞进行第三移动处理，以便使裂解处理产物进入所述注射室，
    将所述活塞进行第四移动处理，以便使进入注射室的所述裂解处理产物与剩余部分稀释液进行第二混合处理，所述第二混合处理是在所述稀释液容纳单元中进行的，
    将所述活塞进行第五移动处理，以便使第二混合处理产物进入所述注射室，
    将所述活塞进行第六移动处理，以便使进入注射室的所述第二混合处理产物与反转录酶和PCR原料冻干粉进行第三混合处理，所述反转录酶和PCR原料冻干粉设置在所述PCR反应单元内，所述第三混合处理是在所述PCR反应单元中进行的，以及
    将第三混合处理产物进行PCR温度循环扩增处理，所述PCR温度循环扩增处理是在所述PCR反应单元中进行的。
  2. 根据权利要求1所述的方法，其特征在于，所述PCR温度循环扩增处理包括：
    将所述第三混合处理产物进行恒温处理；以及
    将恒温处理产物进行温度循环处理。
  3. 根据权利要求2所述的方法，其特征在于，
    所述PCR反应系统进一步包括：
    样本控制阀，在所述第一管路上设置所述样本控制阀，用于控制所述第一液体出/入口与所述第二液体出/入口的连通状态，
    稀释控制阀，在所述第二管路上设置所述稀释控制阀，用于控制所述稀释液入口与所述第二液体出/入口的连通状态，
    第一PCR控制阀，在所述第三管路上设置所述第一PCR控制阀，用于控制所述裂解后的样本混合液入口与所述第二液体出/入口的连通状态，以及
    第二PCR控制阀，在所述第四管路上设置所述第二PCR控制阀，用于控制所述稀释液出口与所述PCR反应液出口的连通状态；
    所述方法进一步包括：
    在所述第一移动处理前，关闭所述样本控制阀、所述第一PCR控制阀以及所述第二PCR控制阀，开启所述稀释控制阀，
    在所述第一移动处理后以及所述第二移动处理前，关闭所述稀释控制阀，开启所述样本控制阀，
    在所述第三移动处理后以及所述第四移动处理前，关闭所述样本控制阀，开启所述稀释控制阀，
    在所述第五移动处理后以及所述第六移动处理前，关闭所述稀释控制阀，开启所述第一PCR控制阀以及所述第二PCR控制阀，
    在所述恒温处理后以及温度循环处理前，关闭所述第一PCR控制阀以及所述第二PCR控制阀。
  4. 根据权利要求3所述的方法，其特征在于，所述PCR反应系统进一步包括：
    缓冲单元，所述缓冲单元具有PCR反应液入口和通气口，在所述第四管路上设置所述缓冲单元，所述第二PCR控制阀与所述PCR反应液入口相连，所述稀释液出口与所述通气口相连。
  5. 根据权利要求1所述的方法，其特征在于，
    所述PCR反应系统进一步包括：
    样本容纳单元密封件，在所述第一液体出/入口表面设置所述样本容纳单元密封件，用于将所述样本容纳单元进行第一密封处理，
    稀释液容纳单元密封件，在所述稀释液出口表面设置所述稀释液容纳单元密封件，用于将所述稀释液容纳单元进行第二密封处理；
    所述方法进一步包括：
    预先刺穿处理，所述预先刺穿处理包括预先将所述样本容纳单元密封件进行第一刺穿处理，将所述稀释液容纳单元密封件进行第二刺穿处理。
  6. 根据权利要求5所述的方法，其特征在于，所述第一刺穿处理是通过样本容纳单元密封件刺穿装置进行的，所述第二刺穿处理是通过稀释液容纳单元密封件刺穿装置进行的。
  7. 根据权利要求5所述的方法，其特征在于，所述稀释液容纳单元密封件和所述样本容纳单元密封件的至少之一为密封膜。
  8. 根据权利要求7所述的方法，其特征在于，所述密封膜是由锡箔纸、塑封膜、牛皮纸的至少之一形成。
  9. 根据权利要求7所述的方法，其特征在于，所述密封膜的厚度为0.01～0.2mm。
  10. 根据权利要求7所述的方法，其特征在于，所述密封膜的厚度为0.05～0.1mm。
  11. 一种PCR反应系统，其特征在于，包括：
    样本容纳单元，所述样本容纳单元内设置有裂解原料冻干粉，并且所述样本容纳单元具有第一液体出/入口；
    稀释液容纳单元，所述稀释液容纳单元内设置有稀释液，并且所述稀释液容纳单元具有稀释液出口；
    PCR反应单元，所述PCR反应单元内设置有反转录酶和PCR原料冻干粉，并且所述PCR反应单元具有PCR反应液出口和裂解后的样本混合液入口；以及
    活塞单元，所述活塞单元包括注射室和活塞，所述注射室具有第二液体出/入口；
    所述第二液体出/入口通过第一管路与所述第一液体出/入口相连，
    所述第二液体出/入口通过第二管路与所述稀释液出口相连，
    所述第二液体出/入口通过第三管路与所述裂解后的样本混合液入口相连，
    所述PCR反应液出口通过第四管路与所述稀释液出口相连。
  12. 根据权利要求11所述的系统，其特征在于，进一步包括：
    样本控制阀，在所述第一管路上设置所述样本控制阀，用于控制所述第一液体出/入口与所述第二液体出/入口的连通状态；
    稀释控制阀，在所述第二管路上设置所述稀释控制阀，用于控制所述稀释液出口与所述第二液体出/入口的连通状态；
    第一PCR控制阀，在所述第三管路上设置所述第一PCR控制阀，用于控制所述裂解后的样本混合液入口与所述第二液体出/入口的连通状态；以及
    第二PCR控制阀，在所述第四管路上设置所述第二PCR控制阀，用于控制所述稀释液出口与所述PCR反应液出口的连通状态。
  13. 根据权利要求12所述的系统，其特征在于，进一步包括：
    缓冲单元，所述缓冲单元具有PCR反应液入口和通气口，在所述第四管路上设置所述缓冲单元，所述第二PCR控制阀与所述PCR反应液入口相连，所述稀释液出口与所述通气口相连。
  14. 根据权利要求11所述的系统，其特征在于，进一步包括：
    样本容纳单元密封件，在所述第一液体出/入口表面设置所述样本容纳单元密封件，用于将所述样本容纳单元进行第一密封处理；以及
    稀释液容纳单元密封件，在所述稀释液出口表面设置所述稀释液容纳单元密封件，用于将所述稀释液容纳单元进行第二密封处理。
  15. 根据权利要求14所述的系统，其特征在于，进一步包括：
    样本容纳单元密封件刺穿装置，所述样本容纳单元密封件刺穿装置用于将所述样本容纳单元密封件进行第一刺穿处理；以及
    稀释液容纳单元密封件刺穿装置，所述稀释液容纳单元密封件刺穿装置用于将所述稀释液容纳单元密封件进行第二刺穿处理。
  16. 根据权利要求14所述的系统，其特征在于，所述稀释液容纳单元密封件和所述样本容纳单元密封件的至少之一为密封膜。
  17. 根据权利要求16所述的系统，其特征在于，所述密封膜是由锡箔纸、塑封膜或牛皮纸的至少之一形成。
  18. 根据权利要求16所述的系统，其特征在于，所述密封膜的厚度为0.01～0.2mm。
  19. 根据权利要求16所述的系统，其特征在于，所述密封膜的厚度为0.05～0.1mm。
  20. 根据权利要求11～19任一项所述的系统，其特征在于，
    所述裂解原料冻干粉包括金属离子螯合剂，十二烷基硫酸钠，皂苷，蛋白酶K，聚乙二醇3350，Tris-HCl，水；
    所述反转录酶和PCR原料冻干粉包括甘露醇，蔗糖，氯化盐，牛血清白蛋白，dNTP，聚氧乙烯十二烷醚，HEPES，DNA聚合酶，反转录酶和RNA酶抑制剂，水；
    优选地，所述金属离子螯合剂为EDTA和EGTA；
    优选地，所述氯化盐为氯化钾和氯化镁。
  21. 根据权利要求11～19任一项所述的系统，其特征在于，
    所述稀释液包括：多元醇，氯化盐，Tris-HCl，表面活性剂以及水；
    任选地，所述多元醇为丙三醇。
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