WO2021237396A1 - 一种一体化自助式核酸检测装置及其使用方法 - Google Patents

一种一体化自助式核酸检测装置及其使用方法 Download PDF

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WO2021237396A1
WO2021237396A1 PCT/CN2020/092008 CN2020092008W WO2021237396A1 WO 2021237396 A1 WO2021237396 A1 WO 2021237396A1 CN 2020092008 W CN2020092008 W CN 2020092008W WO 2021237396 A1 WO2021237396 A1 WO 2021237396A1
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chamber
reaction
nucleic acid
reagent
piston
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PCT/CN2020/092008
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English (en)
French (fr)
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刘鹏
白净卫
李寅青
耿智
刘册
袁国华
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杭州梓晶生物有限公司
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Priority to PCT/CN2020/092008 priority Critical patent/WO2021237396A1/zh
Publication of WO2021237396A1 publication Critical patent/WO2021237396A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the invention relates to a nucleic acid detection device and a method of use thereof, in particular to an integrated self-service nucleic acid detection device and a method of use thereof that can simply and conveniently realize nucleic acid amplification and detection at home or any other place.
  • Nucleic acid detection is the most direct, reliable and sensitive method in pathogen detection, and has important applications in the fields of disease diagnosis, epidemic prevention and control, and health monitoring. Nucleic acid detection detects the genetic material (DNA or RNA) of the pathogen, and can determine the type and concentration of the pathogen within 1 hour, avoiding the interference of factors such as the disease infection window.
  • DNA or RNA genetic material
  • the nucleic acid detection process generally includes the following steps: sample pretreatment, nucleic acid extraction, nucleic acid amplification, and amplification result detection.
  • the traditional detection method requires professionals to complete in a laboratory with professional conditions. It not only requires the use of pipettes and centrifuge tubes to complete a large number of tedious liquid transfer, mixing, centrifugation and other operations, but also requires the help of a temperature cycler, Special equipment such as capillary electrophoresis instrument has high requirements for operators and operating environment. In the face of sudden biological safety incidents such as epidemic prevention and control, false positives and false negatives caused by limited laboratory resources and professionals, or operating errors are likely to occur frequently, and traditional laboratory nucleic acid detection methods are difficult to quickly provide Accurate results for large batches of samples.
  • the one-step nucleic acid amplification reaction usually has a low detection rate and insufficient sensitivity; especially when the input sample has not been purified by nucleic acid In the case of the sample, the one-step nucleic acid amplification reaction will be inhibited by the contaminant proteins and small molecules in the sample, and the amplification efficiency will be affected.
  • amplification nucleic acid detection technology such as one-step PCR (polymerase chain reaction), RPA (recombinase polymerase amplification), or LAMP (loop-mediated isothermal amplification), etc., and it is necessary to test the sample first Only carry out nucleic acid purification can carry out the amplification reaction. Therefore, the high threshold of traditional nucleic acid detection technology limits the huge application prospects of this technology.
  • one of the objectives of the present invention is to provide an integrated self-service nucleic acid detection device, which integrates reaction reagents and control systems in a microfluidic chip, and realizes the full integration of the pathogen nucleic acid detection process, so that it can be It is simple and convenient to realize nucleic acid amplification detection at home or any other place; another object of the present invention is to provide a method for using the integrated self-service nucleic acid detection device.
  • an integrated self-service nucleic acid detection device including a microfluidic chip
  • the microfluidic chip includes: a cartridge with a plurality of hole-shaped cavities formed thereon, Each of the hole-shaped cavities respectively forms a piston chamber provided with a piston push rod, a sample receiving chamber for accommodating a sample to be tested, a reagent chamber for accommodating liquid reagents, and a reagent chamber for recovering amplification products
  • An outlet is formed at the bottom of the piston chamber and the sample receiving chamber, an inlet and an outlet are formed at the bottom of the reagent chamber, and an inlet is formed at the bottom of the recovery chamber
  • a reaction chip, at least one reaction pool is formed on the upper surface of the reaction pool, and reaction reagents are pre-stored in the reaction pool
  • an elastic membrane is arranged between the cartridge and the reaction chip for bonding the reaction chip to the The bottom of the cartridge, and the elastic membrane is provided with through holes with the same number of inlets and outlets
  • a hydrophobic membrane is provided at the through hole of the elastic membrane between the piston chamber and the reaction cell, and the hydrophobic membrane can allow air to pass through, but Block the passage of liquids.
  • the piston chamber includes a first piston chamber and a second piston chamber, and the first piston chamber and the second piston chamber are respectively provided with a first piston chamber.
  • a mixing chamber for accommodating liquid reagents is formed on the cartridge, and an outlet and an inlet are also formed at the bottom of the mixing chamber,
  • the outlet and inlet of the mixing chamber and the adjacent through holes on the elastic membrane respectively form a first one-way valve structure and a second one-way valve structure, and the inlet of the mixing chamber passes through the second one-way valve structure.
  • the valve structure is communicated with the second reaction tank, and the outlet of the mixing chamber is communicated with the inlet of the recovery chamber through the connecting flow channel on the reaction chip after passing through the first one-way valve structure.
  • the elastic membrane is a double-sided adhesive base material and has undergone a partial de-tackification treatment; the hydrophobic membrane is a PTFE membrane.
  • the reaction reagents are solid or liquid RPA reagents, LAMP reagents, PCR reagents, NASBA reagents, cas12-mediated specific sequence signal amplification reaction reagents or cas13-mediated The specific sequence signal amplification reaction reagent.
  • the microfluidic chip adopts a fully enclosed form, that is, a groove is processed on one side of the cartridge, and then a single-sided glue is used to connect the frame of the cartridge They are attached and sealed along the frame through a heat-sealing process; at the same time, the top outlets of each chamber of the cartridge are sealed by a sealing cover or a rubber plug, wherein the first piston chamber and the second piston chamber
  • the sealing cover or rubber plug of the chamber is provided with through holes for the first piston push rod and the second piston push rod to pass through; in addition, the first piston chamber, the second piston chamber, and the sample receiving
  • the side walls of the chamber, the reagent chamber and the recovery chamber are all provided with air guide holes.
  • the microfluidic chip is placed in a housing that matches its shape, and the microfluidic chip is enclosed in the housing by a cover plate, and at the same time
  • a heating unit mainly composed of PI film, resistance wire and Peltier is provided at the bottom of the microfluidic chip; two push rod limiting grooves are opened on the front side of the housing to respectively limit the first piston The pull-up distance of the push rod and the second piston push rod; a reaction result observation area is provided on the side of the housing for displaying the results of the test strip; a buzzer and a status indicator are also provided on the front of the housing , For prompting the user to operate; a circuit control unit connected with the heating unit, buzzer and status indicator is provided in the housing for controlling heating, alarming and status indication.
  • a method for using the above-mentioned integrated self-service nucleic acid detection device includes the following steps:
  • Step 1 Add or form a solution containing the nucleic acid to be tested in the sample receiving chamber;
  • Step 2 Pull up the first piston push rod, the solution in the sample receiving chamber enters the first reaction tank through the first one-way valve underneath it under negative pressure, and is mixed with the reagents pre-existing in the first reaction tank ;
  • Step 3 Heat the reaction chip to the set temperature to start the first step of the amplification reaction. After a certain time of reaction, push down the first plunger and the amplified solution after the reaction passes through the second one-way valve under positive pressure Enter the reagent chamber and mix with the liquid reagent inside;
  • Step 4 Pull up the second piston push rod, the amplification solution in the reagent chamber enters the second reaction tank through the first one-way valve under the negative pressure, and is mixed with the reaction reagents pre-existing in the second reaction tank ;
  • Step 5 Heat the reaction chip to the set temperature again to start the second step of the amplification reaction. After a certain time of reaction, push down the second plunger and the amplified solution after the reaction passes through the second one-way under the action of positive pressure.
  • the valve enters the mixing chamber and mixes with the liquid reagent inside;
  • Step 6 Continue to push down the second piston push rod.
  • the pressure in the closed space above the mixing chamber reaches a critical value, the mixed amplification solution breaks through the first one-way valve and flows out, and then enters the recovery through the second one-way valve Chamber
  • Step 7 The amplification solution entering the recovery chamber undergoes a color reaction with the test strip placed in the recovery chamber, and the nucleic acid detection result is obtained according to the color result of the test strip.
  • adding or forming a solution containing the nucleic acid to be tested into the sample receiving chamber is divided into two situations: 1) The nucleic acid to be tested is in the form of single-stranded or double-stranded free in the lysis solution Add the sample receiving chamber, and enter the subsequent reaction directly through operation at this time; 2) Part or all of the nucleic acid to be tested is present in the cells, tissues, virus particles, and bacteria added to the liquid. At this time, it needs to be heated or lysed after the lysis solution Add to the sample receiving chamber.
  • the present invention has the following advantages due to the above technical scheme: 1.
  • the present invention integrates the reaction reagents and control system in the microfluidic chip, which does not depend on the control and operation of any external instrument, and only requires 4 simple steps.
  • the entire process of nucleic acid detection can be completed by pushing and pulling the rod. Not only does it require professional training and special laboratory conditions, but also does not require pipettes, temperature cycling equipment and other equipment, which greatly simplifies the operating steps of nucleic acid detection and reduces the need for operators and operations.
  • the requirements of the venue have the advantages of simplicity, speed, safety and reliability, and can be widely used in homes and community clinics to realize real-time screening and testing during epidemic prevention and control, and personal daily health monitoring. 2.
  • the present invention uses a hydrophobic membrane to realize the fluid limit during the push-pull process of the piston push rod.
  • the hydrophobic membrane and the reaction cell on the microfluidic chip realize the accurate quantification of the micro-upgrading liquid, which solves the problem of quantitative quantification on the microfluidic chip. problem. 3.
  • liquid and gas can be introduced into the closed mixing chamber successively, and different reagents can be mixed when liquid is introduced, and gas compression and liquid pump can be realized when gas is introduced. Therefore, the two steps of reagent mixing and transfer can be realized by pressing down the push rod in one step, which simplifies the operation of fluid control of the microfluidic chip. 4.
  • the nucleic acid detection device provided by the present invention realizes the communication and sealing of the reaction chamber through the air guide hole and the sealing membrane, and can realize the first step amplification reaction and the second step amplification reaction, and the second step amplification reaction and the lateral layer.
  • the seamless connection of analysis test paper detection can effectively avoid false positive test results caused by aerosol pollution. 5.
  • the present invention can further amplify the target nucleic acid signal on the basis of the first step of the amplification reaction, so that high-sensitivity detection can be achieved even if a sample that has not undergone the nucleic acid purification step is used ,
  • the sensitivity can reach 300 copies/mL, which is equivalent to QPCR (Real-time Quantitative PCR Detection System, real-time fluorescent quantitative nucleic acid amplification and detection system).
  • Figure 1 is a schematic structural diagram of an integrated self-service nucleic acid detection device in an embodiment of the present invention
  • Figure 2 (a) and Figure 2 (b) are schematic diagrams of the structure of an integrated self-service nucleic acid detection device in another embodiment of the present invention.
  • Figure 3(a)-(e) is a working flow chart of the device of the present invention in application;
  • Fig. 4 is an experimental result diagram of an application example of the present invention.
  • the number in the figure is the number of pseudoviruses detected by the device, namely copies/rxn; NC is a negative control, that is, a sample that has not participated in the pseudovirus.
  • the integrated self-service nucleic acid detection device provided by the present invention includes a microfluidic chip 100.
  • the microfluidic chip 100 includes: a cassette 1 on which 5 hole-shaped cavities (only Take 5 as an example, but it is not limited to 5), in which two cavities respectively form the first piston chamber 11 and the second piston chamber 12, and the first piston push rod 13 and the second piston push rod 13 and the second piston push rod are respectively provided inside.
  • the rod 14 and the other three cavities respectively form a sample receiving chamber 15 for accommodating a sample to be tested, a reagent chamber 16 for accommodating liquid reagents, and a recovery chamber 17 for recovering amplification products, and An outlet is formed at the bottom of a piston chamber 11, a second piston chamber 12 and a sample receiving chamber 15, an inlet and an outlet are formed at the bottom of the reagent chamber 16, and an inlet is formed at the bottom of the recovery chamber 17
  • the reaction chip 2, the upper surface of which is formed with a first reaction tank 21 and a second reaction tank 22, the first reaction tank 21 and the second reaction tank 22 are pre-stored with reaction reagents; the elastic membrane 3 is set in the cartridge 1 and The reaction chips 2 are used to bond the reaction chips 2 to the bottom of the cassette 1, and the elastic membrane 3 is provided with through holes with the same number of inlets and outlets as the bottom of the cassette 1.
  • the outlets of the first piston chamber 11 and the second piston chamber 12 respectively coincide with the two through holes on the elastic membrane 3, so that the first piston chamber 11 and the second piston chamber 12 react with the first piston chamber 11 and the second piston chamber 12 respectively.
  • the cell 21 is in communication with the second reaction cell 22; the outlet of the sample receiving chamber 15, the inlet and outlet of the reagent chamber 16, and the inlet of the recovery chamber 17 do not coincide with the through hole on the elastic membrane 3, so that the sample is received
  • the outlets of the chamber 15 and the reagent chamber 16 and the adjacent through holes on the elastic membrane 3 constitute a first one-way valve structure that can only go out and cannot enter.
  • the inlets of the reagent chamber 16 and the recovery chamber 17 are connected to the elastic membrane 3
  • the adjacent through holes constitute a second one-way valve structure that can only enter but not exit.
  • the outlet of the sample receiving chamber 15 and the outlet of the reagent chamber 16 are respectively communicated with the first reaction tank 21 through the first one-way valve structure, and the inlet of the reagent chamber 16 and the inlet of the recovery chamber 17 respectively pass through the first one-way valve structure.
  • the two one-way valve structure is connected with the second reaction tank 22 to ensure that the fluid or air completes the directional transfer under a specific operation.
  • the principle of the structure of the one-way valve is as follows: because the inlet at the bottom of the cartridge 1 (for example, the inlet of the reagent chamber 16) does not overlap with the through hole on the elastic membrane 3, there is no key. Therefore, the positive pressure can push the elastic membrane 3 there so that the fluid in the first reaction cell 21 flows into the reagent chamber 16, while the reverse pressure will press the elastic membrane 3 against the bottom of the cartridge 1 to achieve closure.
  • the first one-way valve structure can ensure that the fluid is one-way empty from the cartridge
  • the cavity enters the reaction chip 2, and its structure is equivalent to the second one-way valve structure upside down, and the principle is the same.
  • the through hole of the elastic membrane 3 between the first piston chamber 11 and the first reaction cell 21 and the elastic membrane between the second piston chamber 12 and the second reaction cell 22 3 through holes are provided with a hydrophobic membrane 4, the hydrophobic membrane 4 can allow air to pass, but block the passage of liquid.
  • the hydrophobic membrane 4 is preferably a PTFE membrane.
  • a closed mixing chamber 18 is further formed on the cartridge 1, and an outlet and an inlet are also formed at the bottom of the mixing chamber 18, and the outlet and the inlet of the mixing chamber 18 are respectively formed.
  • the first one-way valve structure and the second one-way valve structure are formed with the adjacent through holes on the elastic membrane 3, and the inlet of the mixing chamber 18 is connected to the second reaction tank 22 through the second one-way valve structure for mixing.
  • the outlet of the chamber 18 is communicated with the inlet of the recovery chamber 17 through the connecting flow channel on the reaction chip 2 after passing through the first one-way valve structure.
  • the elastic film 3 can be a double-sided adhesive substrate, and after partial de-tackification treatment, it can not only realize the one-way valve function, but also realize the bonding of the cartridge 1 and the reaction chip 2.
  • the reaction reagents are solid or liquid RPA reagents, LAMP reagents, PCR reagents, NASBA (nucleic acid sequence-dependent amplification) reagents, cas12-mediated specific sequence signal amplification reaction reagents or cas13-mediated reagents. Guided specific sequence signal amplification reaction reagent.
  • the microfluidic chip 100 can adopt a fully enclosed form, that is, a groove is processed on one side of the card box 1, and then a single-sided glue 5 is used to adhere to the frame 19 of the card box 1, and pass
  • the heat sealing process seals along the frame 19; at the same time, the top outlets of each chamber of the cartridge 1 are sealed by a sealing cover or a rubber plug 23, and the sealing covers or rubbers of the first piston chamber 11 and the second piston chamber 12
  • the plug 23 is provided with through holes for the first piston push rod 13 and the second piston push rod 14 to pass through; in addition, the first piston chamber 11, the second piston chamber 12, the sample receiving chamber 15, and the reagent
  • the side walls of the chamber 16 and the recovery chamber 17 are provided with air guide holes 20, so that the gas will not leak from the upper outlet of the cartridge 1, but it can be separated from the air guide holes 20 on the side wall of the chamber and the inside of the card box 1.
  • the sealed area performs gas exchange and realizes pressure communication, thereby realizing the complete sealing
  • the microfluidic chip 100 can also be placed in a housing 24 matching its shape, and the microfluidic chip 100 can be enclosed in the housing 24 by a cover 25 Inside, at the same time, a heating unit (not shown in the figure) mainly composed of elements such as PI film, resistance wire, and Peltier is provided at the bottom of the microfluidic chip 100.
  • Two push rod limiting grooves 26 are opened on the front of the housing 24 to limit the pulling distance of the first piston push rod 13 and the second piston push rod 14 respectively; a reaction result observation area 27 is provided on the side of the housing 24 , Used to display the results of the test strip; on the front of the housing 24 is also provided with a buzzer 28 and a status indicator 29 to prompt the user to operate; the housing 24 is provided with a heating unit, a buzzer 28 and a status indicator The circuit control unit 30 connected to the lamp 29 is used to control heating, alarm and status indication.
  • the microfluidic chip 100 adopts a shape similar to a tape cartridge, but without departing from the essence of the invention, the microfluidic chip 100 can also be modified for specific practical applications.
  • the structure can have the following several variants: 1The shape of the cartridge 1 can be adjusted according to the processing method and functional requirements, specifically it can be a combination of rectangular parallelepiped, cylinder, truncated cone, etc.; 2The chambers on the cartridge 1 are arranged The sequence can be adjusted according to functional requirements; 3For the order of the chambers on the cartridge 1, the arrangement of the reaction cells and fluid channels on the reaction chip 2 can also be adjusted accordingly; 4 the piston chamber on the cartridge 1 The number can be adjusted according to requirements, and the number of reaction cells on the reaction chip 2 can be adjusted according to requirements.
  • the present invention also proposes a method for using the integrated self-service nucleic acid detection device, which includes the following steps:
  • Step 1 The initial state of the device is shown in Figure 43(a). Add or form a solution containing the nucleic acid to be tested into the sample receiving chamber 15.
  • Step 2 Pull up the first piston push rod 13, the solution in the sample receiving chamber 15 enters the first reaction tank 21 through the first one-way valve A below it under the action of negative pressure, and is combined with the pre-existing first reaction tank
  • the reaction reagents in 21 are mixed (such as RPA or LAMP dry powder); at the same time, due to the barrier of the hydrophobic membrane 4, the mixed solution will not enter the first piston chamber 11 (as shown in 3(b)).
  • Step 3 Heat the reaction chip 2 to 37-45°C to start the first step of the amplification reaction. After reacting for a certain period of time (such as 10 minutes), push down the first plunger 13 and the amplified solution after the reaction is under positive pressure It enters the reagent chamber 16 through the second one-way valve B, and mixes with the liquid reagent inside (as shown in 3(c)).
  • a certain period of time such as 10 minutes
  • Step 4 Pull up the second piston push rod 14, the amplification solution in the reagent chamber 16 enters the second reaction tank 22 through the first one-way valve C below it under negative pressure, and is combined with the pre-existing second reaction tank
  • the reaction reagents such as RPA or LAMP dry powder
  • the amplification solution will not enter the second piston chamber 12 (as shown in 3(d)).
  • Step 5 Heat the reaction chip 2 to 37-45°C again to start the second step of the amplification reaction. After a certain period of time (such as 10 minutes), push down the second plunger 14 and the amplified solution after the reaction is at positive pressure Under the action, it enters the mixing chamber 18 through the second one-way valve D, and mixes with the internal liquid reagent (as shown in 3(e)).
  • Step 6 Continue to push down the second piston push rod 14.
  • the pressure in the closed space above the mixing chamber 18 reaches a critical value, the mixed amplification solution breaks through the first one-way valve E and flows out, and then passes through the second one-way The valve F enters the recovery chamber 17.
  • Step 7 The amplification solution entering the recovery chamber 17 undergoes a color reaction with the test strip placed in the recovery chamber 17, and the nucleic acid detection result is obtained according to the color result of the test strip.
  • adding or forming a solution containing the nucleic acid to be tested into the sample receiving chamber 15 can be divided into two situations: 1) The nucleic acid to be tested (RNA or DNA) is free from the monomer in the lysis solution. The chain or double-strand form is added to the sample receiving chamber 15, at this time, the subsequent reaction can be directly entered through the operation; 2) Part or all of the nucleic acid to be tested is present in the cells, tissues, virus particles, and bacteria added to the liquid. After lysing by heating or lysing solution, it is added to the sample receiving chamber 15.
  • the purpose of the second step of the amplification reaction is to further improve the specificity of nucleic acid detection sensitivity.
  • the selectable reactions include RPA reaction, LAMP reaction, cas12-mediated specific sequence signal amplification reaction or cas13 Mediated specific sequence signal amplification reaction.
  • the nested RPA reaction with probes and the specific sequence signal amplification reaction mediated by Lbcas12a are used, and the nested RPA reaction with probes is: inside the amplified sequence in the first step of the amplification reaction (Does not contain the first step primer sequence, or only contains the 1-10nt first step amplification primer) Design the second step amplification primer, and design the specific probe in the sequence amplified by the second step amplification primer (Does not contain the second step primer sequence, or only contains the 1-10 nt second step amplification primer).
  • the specific sequence signal amplification reaction mediated by Cas12 is composed of Cas12 protein, Cas12 binding small RNA and Cas12 reaction buffer.
  • the Cas12 protein can be, but is not limited to, LbCas12a, AsCas12a, FnCas12a, and Cas12 mutants containing mutations.
  • Cas12 binding small RNA contains two sequences, one of which binds to the corresponding Cas12 protein (for example, the sequence corresponding to the LbCas12a protein is AAUUUCUACUAAGU(U/G)UAGA), and the other is similar to the sequence in the RPA amplicon interval ( Contain at least 15 identical base sequences), which is a targeting sequence.
  • the 5'upstream of the targeting sequence is the PAM sequence corresponding to Cas12, for example, the PAM corresponding to LbCas12a is TTTV.
  • crRNA can be obtained by RNA synthesis or in vitro transcription, such as in vitro transcription reaction based on T7 RNA polymerase.
  • the composition of Cas12 reaction buffer should contain more than 2mM Mg 2+ ions, and not contain more than 200mM monovalent ions, such as Na + , K + , such as 50 mM potassium acetate, 20 mM Tris acetic acid, 10 mM magnesium acetate, 100 micrograms per milliliter of BSA, The pH is 7.9 at 25 degrees.
  • the pre-reaction reagent of the nested RPA reaction with probe contains a primer with one kind of chemical modification (such as biotin, FAM, FITC, DIG, etc.) and a primer with a different kind of Chemically modified (such as FAM, FITC, DIG, biotin, etc.) probes;
  • the positive reaction product contains a nucleic acid with a chemical modification (such as biotin, FAM, FITC, DIG, etc.) on one end and another on the other end
  • the double-stranded DNA of chemically modified molecules can be detected with lateral chromatography test paper by the colloidal gold sandwich method.
  • the signal band of colloidal gold is composed of a substance that can be combined with one of the chemical modifications.
  • the band is composed of streptavidin; or when the substance is FAM or FITC, the band is composed of FAM. Or FITC antibody composition.
  • the cas12-mediated specific sequence signal amplification reaction reagent contains single-stranded DNA.
  • One end of the single-stranded DNA has a chemical modification (such as biotin, FAM, FITC, DIG, etc.), and the other end has a different chemical modification (such as FAM, FITC, DIG, biotin, etc.).
  • these DNA probes are cleaved by the enzyme, and the product contains a chemical modification (such as biotin, FAM, FITC, DIG, etc.) or another chemical modification (such as FAM, FITC, etc.) at the end.
  • the signal band of colloidal gold is composed of a substance that can be combined with one of the chemical modifications.
  • the band is composed of streptavidin; or when the substance is FAM or FITC, the band is composed of FAM. Or FITC antibody composition.
  • the signal band of colloidal gold is composed of proteins that bind to antibodies on gold particles, such as protein A or protein G.
  • SARS-CoV-2 The new coronavirus (SARS-CoV-2) pseudovirus was purchased from Fu Baiao Biotechnology Co., Ltd. (article number: FNV-2019-ncov-abEN);
  • the 1st RPA reaction kit was purchased from TwistDX Company, UK, and the product model is BASIC RT KIT;
  • the 2nd RPA reaction kit was purchased from TwistDX Company, UK, and the product model is NFO KIT;
  • Virus preservation solution brand Youkang, Item No.: MT0301, main ingredients: based on Hanks solution added with gentamicin, fungal antibiotics, BSA, cryoprotectant, biological buffer, amino acid and other ingredients;
  • the main components of the virus release agent Tris-His 400mM, sodium chloride 150mM, Tween-200.8%, Triton X-1001.2%, ethylphenyl polyethylene glycol 1.5%, potassium chloride 6mg/mL, hydrogen Sodium oxide 15mg/mL;
  • RPA-F ATGTACTCATTCGTTTCGGAAGAGACAGG;
  • RPA-R AGACCAGAAGATCAGGAACTCTAGAAGAA
  • 2nd RPA-F TTCTTTTTCTTGCTTTCGTGGTATTCTTGC;
  • the 1st RPA and 2nd RPA reaction system solutions are added to the first reaction tank 21 and the second reaction tank 22 of the reaction chip 2 respectively, and the freeze-drying process is performed.
  • reaction chip 2 Assemble the reaction chip 2 containing the freeze-dried reagents into a nucleic acid detection device for testing according to FIG. 1, and add 250ul and 200 ⁇ L DEPC water into the reagent chamber 16 and the mixing chamber 18, respectively.
  • the integrated self-service nucleic acid detection device provided by the present invention and the two-step RPA reagent, 4 copies of virus particles in a 50ul reaction system can be stably detected.
  • the volume of the solution actually entering the 1st RPA reaction system is 25ul (the other 25ul volume is the virus release solution)
  • the overall detection sensitivity of the integrated self-service nucleic acid detection device is 0.16 copies/ul.

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Abstract

本发明提供一种一体化自助式核酸检测装置及其使用方法，该装置包括：卡盒，其上形成有活塞腔室、样本接收腔室、试剂腔室和回收腔室；反应芯片，其上表面形成有两个反应池；弹性膜，将反应芯片键合在卡盒底部；样本接收腔室和试剂腔室通过第一单向阀结构与一反应池相连通，试剂腔室和回收腔室通过第二单向阀结构与另一反应池相连通。利用该装置只需4步简单的推杆推拉动作即可完成核酸检测的全部流程，不仅无需专业培训和特殊实验室条件，也无需移液枪、温度循环仪器等设备，大大简化了核酸检测的操作步骤，降低了对操作人员和操作场所的要求，可在家庭、社区诊所进行普及应用，实现疫情防控期间的实时筛查检测、以及个人日常健康状况监测。

Description

一种一体化自助式核酸检测装置及其使用方法 技术领域
本发明涉及一种核酸检测装置及其使用方法，具体是关于一种可以在家庭或其他任何场所简单、方便地实现核酸扩增检测的一体化自助式核酸检测装置及其使用方法。
背景技术
核酸检测是病原体检测中最直接、最可靠、最灵敏的方法，在疾病诊断、疫情防控、健康监测等领域具有重要的应用。核酸检测通过对病原体的遗传物质(DNA或RNA)进行检测，可以在1小时内确定病原体的种类、浓度等信息，避免疾病感染窗口期等因素的干扰。
核酸检测过程一般包括以下步骤：样本前处理、核酸提取、核酸扩增、扩增结果检测。传统的检测方法需要有专业人员在具有专业条件的实验室中完成，不仅需要使用移液枪、离心管完成大量繁琐的液体转移、混匀、离心等操作，而且还需要借助于温度循环仪、毛细管电泳仪等专用仪器设备，对于操作人员和操作环境有很高的要求。在面对疫情防控等突发生物安全事件时，由于实验室资源、专业人员有限，或操作失误造成的假阳性、假阴性等问题易频发，传统的实验室核酸检测方法难以快速给出大批量样本的准确结果。
目前，对于检测低拷贝数的核酸样本(比如1000copy/mL以下的SARS-CoV-2样本)，一步核酸扩增反应通常检出率较低，灵敏度不够；特别是当输入样本为没有经过核酸提纯的样本时，一步核酸扩增反应会受到样本中的杂蛋白、小分子等抑制，扩增效率受到影响。因此，通常只有一步扩增的核酸检测技术，例如一步PCR(聚合酶链式反应)、RPA(重组酶聚合酶扩增)或LAMP(环介导等温扩增)等，需要先对代测样本进行核酸提纯才能进行扩增反应。因此，传统的核酸检测技术的高门槛，限制了这一技术的巨大应用前景。
发明内容
针对上述问题，本发明的其中一个目的是提供一种一体化自助式核酸检测装置，其将反应试剂及控制系统集成在微流控芯片内，实现了病原体核酸检测过程的全集成，从而可以在家庭或其他任何场所简单、方便地实现核酸扩增检测；本发明的另一个目的是提供一种该一体化自助式核酸检测装置的使用方法。
为实现上述目的，本发明采取以下技术方案：一种一体化自助式核酸检测装置，包括微流控芯片，所述微流控芯片包括：卡盒，其上形成有多个孔状空腔，各所述孔状空腔分别形成内部设置有活塞推杆的活塞腔室、用于容置待测样本的样本接收腔室、用于容置液体试剂的试剂腔室以及用于回收扩增产物的回收腔室，且所述活塞腔室和样本接收腔室的底部均形成有一个出口，所述试剂腔室的底部形成有一个进口和一个出口，所述回收腔室的底部形成有一个进口；反应芯片，其上表面形成有至少一个反应池，所述反应池内预存有反应试剂；弹性膜，设置在所述卡盒和反应芯片之间，用于将所述反应芯片键合在所述卡盒底部，且所述弹性膜上开设有与所述卡盒底部进口和出口数量相同的通孔；其中，所述活塞腔室的出口与所述弹性膜上对应的通孔重合，以使所述活塞腔室与所述反应池相连通；所述样本接收腔室的出口、试剂腔室的进口和出口以及所述回收腔室的进口均与所述弹性膜上的通孔不重合，以使所述样本接收腔室和试剂腔室的出口与所述弹性膜上的相邻通孔构成只能出不能进的第一单向阀结构，所述试剂腔室和回收腔室的进口与所述弹性膜上的相邻通孔构成只能进不能出的第二单向阀结构；由此，所述样本接收腔室的出口和试剂腔室的出口分别通过所述第一单向阀结构与所述反应池相连通，所述试剂腔室的进口和回收腔室的进口分别通过所述第二单向阀结构与所述反应池相连通，从而保证流体或空气在特定操作下完成定向转移。
所述的一体化自助式核酸检测装置，优选地，在位于所述活塞腔室与所述反应池之间的所述弹性膜通孔处设置有疏水膜，所述疏水膜可供空气通过，但阻挡液体通过。
所述的一体化自助式核酸检测装置，优选地，所述活塞腔室包括第一活塞腔室和第二活塞腔室，所述第一活塞腔室和第二活塞腔室内部分别设置有第一活塞推杆和第二活塞推杆；所述反应芯片的上表面形成有第一反应池和第二反应池，所述第一活塞腔室与所述第一反应池相连通，所述第二活塞腔室与所述第二活塞腔室相连通，所述样本接收腔室和试剂腔室分别通过第一单向阀结构与所述第一反应池相连通，所述试剂腔室和回收腔室分别通过第二单向阀结构与所述第二反应池相连通。
所述的一体化自助式核酸检测装置，优选地，所述卡盒上还形成有用于容置液体试剂的混匀腔室，所述混匀腔室的底部亦形成有一个出口和一个进口，所述混匀腔室的出口和进口分别与所述弹性膜上的相邻通孔形成第一单向阀结构和第二单向阀结构，所述混匀腔室的进口通过第二单向阀结构与所述第二反应池相连通，所述混匀腔室的出口通过第一单向阀结构后经所述反应芯片上的连接流道与所述回收腔室的进口相连通。
所述的一体化自助式核酸检测装置，优选地，所述弹性膜选用双面胶基材，经 过局部去粘性处理；所述疏水膜为PTFE膜。
所述的一体化自助式核酸检测装置，优选地，所述反应试剂为固状或液状的RPA试剂、LAMP试剂、PCR试剂、NASBA试剂、cas12介导的特异序列信号放大反应试剂或cas13介导的特异序列信号放大反应试剂。
所述的一体化自助式核酸检测装置，优选地，所述微流控芯片采用全封闭形式，即在所述卡盒的一侧加工凹槽，然后用单面胶与所述卡盒的边框贴合，并通过热封工艺沿所述边框进行密封；同时，所述卡盒的各腔室顶端出口处均通过密封盖或胶塞密封，其中所述第一活塞腔室和第二活塞腔室的密封盖或胶塞上均设有过孔，以供所述第一活塞推杆和第二活塞推杆穿过；此外，所述第一活塞腔室、第二活塞腔室、样本接收腔室、试剂腔室和回收腔室的侧壁上均设有导气孔。
所述的一体化自助式核酸检测装置，优选地，所述微流控芯片置于与其形状相适配的外壳内，并通过盖板将所述微流控芯片封闭在所述外壳内，同时在所述微流控芯片底部设置主要由PI膜、电阻丝和帕尔贴组成的加热单元；在所述外壳的正面开设有两个推杆限位槽，用于分别限制所述第一活塞推杆和第二活塞推杆的拉升距离；在所述外壳的侧面设置有反应结果观察区，用于显示试纸条结果；在所述外壳的正面还设置有蜂鸣器和状态指示灯，用于提示用户操作；在所述外壳内设置有与所述加热单元、蜂鸣器和状态指示灯连接的电路控制单元，用于控制加热、报警和状态指示。
一种上述一体化自助式核酸检测装置的使用方法，包括以下步骤：
步骤1：在样本接收腔室内加入或形成含待测核酸的溶液；
步骤2：拉起第一活塞推杆，样本接收腔室内的溶液在负压作用下通过其下方的第一单向阀进入第一反应池，并与预存在第一反应池中的反应试剂混合；
步骤3：将反应芯片加热至设定温度以开始第一步扩增反应，反应一定时间后，推下第一活塞推杆，反应后的扩增溶液在正压作用下经由第二单向阀进入试剂腔室内，与内部的液体试剂混匀；
步骤4：拉起第二活塞推杆，试剂腔室内的扩增溶液在负压作用通过其下方的第一单向阀进入第二反应池，并与预存在第二反应池中的反应试剂混合；
步骤5：再次将反应芯片加热至设定温度以开始第二步扩增反应，反应一定时间后，推下第二活塞推杆，反应后的扩增溶液在正压作用下经由第二单向阀进入混匀腔室内，与内部的液体试剂混匀；
步骤6：继续推下第二活塞推杆，当混匀腔室上方密闭空间内压强达到临界值后，混匀的扩增溶液突破第一单向阀流出，进而通过第二单向阀进入回收腔室内；
步骤7：进入回收腔室的扩增溶液与放置在回收腔室内的试纸条发生显色反应， 根据试纸条的颜色结果得到核酸检测结果。
所述的使用方法，优选地，在所述样本接收腔室内加入或形成含待测核酸的溶液分为两种情况：1)待测核酸以游离在裂解溶液中的单链或者双链的形式加入样本接收腔室，此时直接通过操作进入后续的反应；2)待测核酸部分或者全部的存在于液体加入的细胞、组织、病毒颗粒、细菌，此时需要通过加热裂解或裂解液裂解后加入样本接收腔室中。
本发明由于采取以上技术方案，其具有以下优点：1、本发明将反应试剂及控制系统集成在微流控芯片内，可以不依赖于外部任何仪器的控制与操作，只需4步简单的推杆推拉动作即可完成核酸检测的全部流程，不仅无需专业培训和特殊实验室条件，也无需移液枪、温度循环仪器等设备，大大简化了核酸检测的操作步骤，降低了对操作人员和操作场所的要求，具有简单、快速、安全可靠的优势，可在家庭、社区诊所进行普及应用，实现疫情防控期间的实时筛查检测、以及个人日常健康状况监测。2、本发明使用疏水膜实现活塞推杆推拉过程中的流体限位，通过疏水膜与微流控芯片上的反应池实现微升级别液体的准确定量，解决了微流控芯片上定量困难的问题。3、本发明可以在第二活塞推杆下压时，向密闭的混匀腔室内先后通入液体和气体，通入液体时实现不同试剂的混匀，通入气体时实现气体压缩及液体泵出，可以通过一步的推杆下压实现试剂混匀和转移两个步骤，简化了微流控芯片流体控制的操作。4、本发明提供的核酸检测装置通过导气孔和密封膜实现反应腔室的连通和封闭，可以实现第一步扩增反应与第二步扩增反应以及第二步扩增反应与侧向层析试纸检测的无缝衔接，从而可以有效避免由于气溶胶污染而造成的假阳性检测结果。5、本发明通过第二步扩增反应，可以在第一步扩增反应的基础上进一步对目标核酸信号进行放大，这样即使使用没有经过核酸提纯步骤的样本，也可以实现高灵敏度的检出，灵敏度可以达到300copies/mL，与QPCR(Real-time Quantitative PCR Detecting System，实时荧光定量核酸扩增检测系统)相当。
附图说明
图1是本发明一实施例中一体化自助式核酸检测装置的结构示意图；
图2(a)和图2(b)是本发明另一实施例中一体化自助式核酸检测装置的结构示意图；
图3(a)-(e)是本发明装置在应用时的工作流程图；
图4是本发明一应用实例的实验结果图，图中数字为装置所检测的假病毒个数，即copies/rxn；NC为阴性对照，即未参入假病毒的样本。
具体实施方式
以下将结合附图对本发明的较佳实施例进行详细说明，以便更清楚理解本发明的目的、特点和优点。应理解的是，附图所示的实施例并不是对本发明范围的限制，而只是为了说明本发明技术方案的实质精神。
如图1所示，本发明提供的一体化自助式核酸检测装置包括微流控芯片100，该微流控芯片100包括：卡盒1，卡盒1上形成有5个孔状空腔(仅以5个为例，但并不限于5个)，其中两个空腔分别形成第一活塞腔室11和第二活塞腔室12，内部分别设置有第一活塞推杆13和第二活塞推杆14，另外三个空腔分别形成用于容置待测样本的样本接收腔室15、用于容置液体试剂的试剂腔室16以及用于回收扩增产物的回收腔室17，且第一活塞腔室11、第二活塞腔室12和样本接收腔室15的底部均形成有一个出口，试剂腔室16的底部形成有一个进口和一个出口，回收腔室17的底部形成有一个进口；反应芯片2，其上表面形成有第一反应池21和第二反应池22，第一反应池21和第二反应池22内均预存有反应试剂；弹性膜3，设置在卡盒1和反应芯片2之间，用于将反应芯片2键合在卡盒1底部，且弹性膜3上开设有与卡盒1底部进口和出口数量相同的通孔。
其中，第一活塞腔室11和第二活塞腔室12的出口分别与弹性膜3上的两个通孔重合，以使第一活塞腔室11和第二活塞腔室12分别与第一反应池21和第二反应池22相连通；样本接收腔室15的出口、试剂腔室16的进口和出口以及回收腔室17的进口均与弹性膜3上的通孔不重合，以使样本接收腔室15和试剂腔室16的出口与弹性膜3上的相邻通孔构成只能出不能进的第一单向阀结构，试剂腔室16和回收腔室17的进口与弹性膜3上的相邻通孔构成只能进不能出的第二单向阀结构。由此，样本接收腔室15的出口和试剂腔室16的出口分别通过第一单向阀结构与第一反应池21相连通，试剂腔室16的进口和回收腔室17的进口分别通过第二单向阀结构与第二反应池22相连通，从而保证流体或空气在特定操作下完成定向转移。
在上述实施例中，需要说明的是，构成单向阀结构的原理如下：由于卡盒1底部的进口(例如试剂腔室16的进口)与弹性膜3上的通孔不重合的部分没有键合，因此正向压强可以顶开该处的弹性膜3使第一反应池21内的流体流入试剂腔室16内，而反向压强则会将弹性膜3压在卡盒1底部以实现封闭作用，从而形成只能进不能出的第二单向阀结构，保证流体单向地由反应芯片2进入卡盒腔室内；反之，第一单向阀结构可保证流体单向地由卡盒空腔进入反应芯片2，其结构相当于第二单向阀结构上下颠倒，原理相同。
在上述实施例中，优选地，在位于第一活塞腔室11与第一反应池21之间的弹 性膜3通孔处以及第二活塞腔室12与第二反应池22之间的弹性膜3通孔处均设置有疏水膜4，该疏水膜4可供空气通过，但阻挡液体通过。在本实施例中，疏水膜4优选为PTFE膜。
在上述实施例中，优选地，卡盒1上还形成有密闭的混匀腔室18，混匀腔室18的底部亦形成有一个出口和一个进口，混匀腔室18的出口和进口分别与弹性膜3上的相邻通孔形成第一单向阀结构和第二单向阀结构，混匀腔室18的进口通过第二单向阀结构与第二反应池22相连通，混匀腔室18的出口通过第一单向阀结构后经反应芯片2上的连接流道与回收腔室17的进口相连通。
在上述实施例中，优选地，弹性膜3可选用双面胶基材，经过局部去粘性处理，既可实现单向阀功能，又能实现卡盒1和反应芯片2的键合。
在上述实施例中，优选地，反应试剂为固状或液状的RPA试剂、LAMP试剂、PCR试剂、NASBA(核酸序列依赖性扩增)试剂、cas12介导的特异序列信号放大反应试剂或cas13介导的特异序列信号放大反应试剂。
在上述实施例中，优选地，微流控芯片100可以采用全封闭形式，即在卡盒1的一侧加工凹槽，然后用单面胶5与卡盒1的边框19贴合，并通过热封工艺沿边框19进行密封；同时，卡盒1的各腔室顶端出口处均通过密封盖或胶塞23密封，其中第一活塞腔室11和第二活塞腔室12的密封盖或胶塞23上均设有过孔，以供第一活塞推杆13和第二活塞推杆14穿过；此外，第一活塞腔室11、第二活塞腔室12、样本接收腔室15、试剂腔室16和回收腔室17的侧壁上均设有导气孔20，由此气体不会从卡盒1上部出口泄漏，但可以由腔室侧壁上的导气孔20与卡盒1内的密封区域进行气体交换，实现压强连通，从而实现微流控芯片100的全封闭。
在上述实施例中，优选地，如图2所示，还可以将微流控芯片100置于与其形状相适配的外壳24内，并通过盖板25将微流控芯片100封闭在外壳24内，同时在微流控芯片100底部设置主要由PI膜、电阻丝和帕尔贴等元件组成的加热单元(图中未示出)。在外壳24的正面开设有两个推杆限位槽26，用于分别限制第一活塞推杆13和第二活塞推杆14的拉升距离；在外壳24的侧面设置有反应结果观察区27，用于显示试纸条结果；在外壳24的正面还设置有蜂鸣器28和状态指示灯29，用于提示用户操作；在外壳24内设置有与加热单元、蜂鸣器28和状态指示灯29连接的电路控制单元30，用于控制加热、报警和状态指示等。
需要指出的是，在上述实施例中，微流控芯片100采用类似磁带卡盒形状，但在不脱离本发明创造本质的情况下，微流控芯片100也可以针对特定实际应用进行变体，例如在结构上可以有以下几种变体：①卡盒1的形状可因加工方式、功能需求进行调整，具体可为长方体、圆柱体、圆台等形状组合；②卡盒1上各腔室排列 顺序可因功能需求进行相应的调整；③针对卡盒1上各腔室排列顺序，反应芯片2上的反应池和流体通道排布也可进行相应的调整；④卡盒1上的活塞腔室数目可以根据需求进行调整，而反应芯片2上的反应池数目可以根据需求进行调整。
基于上述实施例提供的一体化自助式核酸检测装置，本发明还提出了该一体化自助式核酸检测装置的使用方法，包括以下步骤：
步骤1：装置初始状态如图43(a)所示，在样本接收腔室15内加入或形成含待测核酸的溶液。
步骤2：拉起第一活塞推杆13，样本接收腔室15内的溶液在负压作用下通过其下方的第一单向阀A进入第一反应池21，并与预存在第一反应池21内的反应试剂混合(如RPA或LAMP干粉)；同时由于疏水膜4的阻隔，混合溶液不会进入第一活塞腔室11(如3(b)所示)。
步骤3：将反应芯片2加热至37-45℃，开始第一步扩增反应，反应一定时间(如10min)后，推下第一活塞推杆13，反应后的扩增溶液在正压作用下经由第二单向阀B进入试剂腔室16内，与内部的液体试剂混匀(如3(c)所示)。
步骤4：拉起第二活塞推杆14，试剂腔室16内的扩增溶液在负压作用通过其下方的第一单向阀C进入第二反应池22，并与预存在第二反应池22中的反应试剂(如RPA或LAMP干粉)混合；同时由于疏水膜4的阻隔，扩增溶液不会进入第二活塞腔室12(如3(d)所示)。
步骤5：再次将反应芯片2加热至37-45℃，开始第二步扩增反应，反应一定时间(如10min)后，推下第二活塞推杆14，反应后的扩增溶液在正压作用下经由第二单向阀D进入混匀腔室18内，与内部的液体试剂混匀(如3(e)所示)。
步骤6：继续推下第二活塞推杆14，当混匀腔室18上方密闭空间内压强达到临界值后，混匀的扩增溶液突破第一单向阀E流出，进而通过第二单向阀F进入回收腔室17内。
步骤7：进入回收腔室17的扩增溶液与放置在回收腔室17内的试纸条发生显色反应，根据试纸条的颜色结果得到核酸检测结果。
在上述实施例中，优选地，在样本接收腔室15内加入或形成含待测核酸的溶液可以分为两种情况：1)待测核酸(RNA或者DNA)以游离在裂解溶液中的单链或者双链的形式加入样本接收腔室15，此时可以直接通过操作进入后续的反应；2)待测核酸部分或者全部的存在于液体加入的细胞、组织、病毒颗粒、细菌，此时需要通过加热裂解或裂解液裂解后加入样本接收腔室15中。
在上述实施例中，优选地，第二步扩增反应的目的是进一步提高核酸的检测灵 敏度的特异性，可以选择的反应有RPA反应、LAMP反应、cas12介导的特异序列信号放大反应或cas13介导的特异序列信号放大反应。在本实施例中，采用带有探针的巢式RPA反应和Lbcas12a介导的特异序列信号放大反应，其中含有探针的巢式RPA反应为：在第一步扩增反应所扩增序列内部(不含有第一步引物序列，或者仅含有1-10nt的第一步扩增引物)设计第二步扩增引物，并在第二步扩增引物所扩增的序列中设计特异性探针(不含有第二步引物序列，或者仅含有1-10nt的第二步扩增引物)。其中，Cas12介导的特异序列信号放大反应组成部分由Cas12蛋白、Cas12结合小RNA和Cas12反应缓冲液构成。Cas12蛋白可以为但不限于LbCas12a、AsCas12a、FnCas12a和含有突变的Cas12突变体等。Cas12结合小RNA包含两段序列，其中一段与对应的Cas12蛋白结合(如对应LbCas12a蛋白的序列为AAUUUCUACUAAGU(U/G)UAGA)，另一段与在RPA扩增子区间中的序列具有相似性(含有至少15个相同碱基序列)，该序列为靶向序列。靶向序列的5’端上游为Cas12对应的PAM序列，如LbCas12a对应的PAM为TTTV。当靶向序列与RPA扩增引物有重叠时，重叠部分不多于9个碱基。crRNA可以通过RNA合成或体外转录获得，如基于T7RNA聚合酶的体外转录反应。Cas12反应缓冲液构成应含有高于2mM的Mg 2+离子，不含有超过200mM的单价离子，如Na +,K +，比如50mM乙酸钾，20mM Tris乙酸，10mM乙酸镁，100微克每毫升BSA，25度时pH 7.9。
在上述实施例中，优选地，带有探针的巢式RPA反应的反应前试剂中含有带有一种化学修饰(比如biotin,FAM，FITC，DIG等)的引物和带有另一种不同的化学修饰(比如FAM,FITC,DIG，biotin等)探针；阳性反应产物中含有一段核酸，其一端带有一种化学修饰(比如biotin，FAM，FITC，DIG等)，另一端带有另一种化学修饰(比如FAM，FITC，DIG，biotin等)分子的双链DNA，可以通过胶体金夹心法实现侧向层析试纸的检测。胶体金的信号条带由可以结合其中一种化学修饰的物质构成，比如，当这种物质为biotin时，条带由streptavidin构成；或当这种物质为FAM或FITC时，条带由结合FAM或FITC的抗体构成。
在上述实施例中，优选地，cas12介导的特异序列信号放大反应试剂中含有单链DNA。该单链DNA的一个末端带有一种化学修饰(比如biotin，FAM，FITC，DIG等)，另一末端带有另一种不同的化学修饰(比如FAM，FITC，DIG，biotin等)。在含有阳性检测物的反应过程中，这些DNA探针被酶切割，产物中含有末端带有一种化学修饰(比如biotin，FAM，FITC，DIG等)或另一种化学修饰(比如FAM，FITC，DIG，biotin等)的单链DNA，通过胶体金竞争法实现侧向层析试纸的检测。胶体金的信号条带由可以结合其中一种化学修饰的物质构成，比如，当这种物质为biotin时，条带由streptavidin构成；或当这种物质为FAM或FITC时，条带由结 合FAM或FITC的抗体构成。胶体金的信号条带由结合金颗粒上抗体的蛋白构成，比如protein A或protein G。
下面通过一个具体的应用实例来验证本发明的技术效果。
一、试剂准备：
①新冠病毒(SARS-CoV-2)假病毒购买自复百澳生物科技有限公司(货号：FNV-2019-ncov-abEN)；
②1st RPA反应试剂盒购自英国TwistDX公司，产品型号为
Figure PCTCN2020092008-appb-000001
BASIC RT KIT；
③2nd RPA反应试剂盒购自英国TwistDX公司，产品型号为
Figure PCTCN2020092008-appb-000002
NFO KIT；
④胶体金试纸购自北京库尔科技有限公司；
⑤病毒保存液品牌：友康，货号：MT0301，主要成分：基于Hanks液基础上添加庆大霉素，真菌抗生素，BSA，冷冻保护剂，生物缓冲剂和氨基酸等成分；
⑥病毒释放剂主要成分：Tris-His 400mM，氯化钠150mM，吐温-200.8％，曲拉通X-1001.2％，乙基苯基聚乙二醇1.5％，氯化钾6mg/mL，氢氧化钠15mg/mL；
⑦实验用的引物探针购自生工生物工程(上海)股份有限公司，具体名称序列如下：
1st RPA-F:ATGTACTCATTCGTTTCGGAAGAGACAGG；
1st RPA-R:AGACCAGAAGATCAGGAACTCTAGAAGAA；
2nd RPA-F:TTCTTTTTCTTGCTTTCGTGGTATTCTTGC；
2nd RPA-R:biotin-AAGAAGGTTTTACAAGACTCACGTTAACAAT；
Probe：
FAM-TTACACTAGCCATCCTTACTGCGCTTCGAT[thf]GTGTGCGTACTGCTG[C3spacer]。
二、实验步骤：
1)试剂冻干
按照以下表格分别配制1st RPA和2nd RPA反应体系。
①1st RPA反应体系配置(
Figure PCTCN2020092008-appb-000003
Basic RT kit)
Figure PCTCN2020092008-appb-000004
Figure PCTCN2020092008-appb-000005
②2nd RPA反应体系配置(
Figure PCTCN2020092008-appb-000006
nfo kit)
Figure PCTCN2020092008-appb-000007
然后，将1st RPA和2nd RPA反应体系溶液分别加入到反应芯片2的第一反应池21和第二反应池22中，并进行冻干处理。
2)将含有冻干试剂的反应芯片2按照图1组装为测试用的核酸检测装置，并在试剂腔室16和混匀腔室18中分别加入250ul和200μL DEPC水。
3)将假病毒溶液用友康保存液稀释至合适浓度，分别取40copies，20copies，8copies，4copies，0copies病毒颗粒加入到EP管中，用友康保存液补充体积至50ul；然后向EP管中加入50ul病毒释放液，用移液器吹打混匀，立刻将全部100ul溶液加入到样本接收腔室15中。
4)向上拉起第一活塞推杆13，使50ul样品(分别含有20，10，4，2，0copy假病毒颗粒)进入到第一反应池21，溶解冻干的反应试剂；然后将核酸检测装置置于加热板上，37℃加热10min。
5)向下推下第一活塞推杆13使第一反应池21中的反应产物进入试剂腔室16，与其内预存的DEPC水混合，直至少量气泡产生。
6)向上拉起第二活塞推杆14，使试剂腔室16中的液体进入第二反应池22，溶解冻干的反应试剂；然后再次将核酸检测装置置于加热板上，37℃加热10min。
7)向下推下第二活塞推杆14使第二反应池22中的液体进入混匀腔室18，与其内预存的DEPC水混合，直至少量气泡产生。
8)继续按下第二活塞推杆14，气体进入混匀腔室18中直至其内部气压达到临界值，使混匀腔室18内混匀后的液体进入回收腔室17，并与放置在回收腔室17内的试纸条接触后发生显色反应。
9)2min后待层析结束，眼睛读取显色结果。
三、实验结果如图4所示。
四、实验结论：
通过本发明提供的一体化自助式核酸检测装置配合两步法RPA试剂，可以稳定检测出50ul反应体系中4copies病毒颗粒。考虑到实际进入1st RPA反应体系溶液的体积为25ul(另外25ul体积为病毒释放液)，因此该一体化自助式核酸检测装置的整体检测灵敏度为0.16copies/ul。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (10)

  1. 一种一体化自助式核酸检测装置，其特征在于，包括微流控芯片(100)，所述微流控芯片(100)包括：
    卡盒(1)，其上形成有多个孔状空腔，各所述孔状空腔分别形成内部设置有活塞推杆的活塞腔室、用于容置待测样本的样本接收腔室(15)、用于容置液体试剂的试剂腔室(16)以及用于回收扩增产物的回收腔室(17)，且所述活塞腔室和样本接收腔室(15)的底部均形成有一个出口，所述试剂腔室(16)的底部形成有一个进口和一个出口，所述回收腔室(17)的底部形成有一个进口；
    反应芯片(2)，其上表面形成有至少一个反应池，所述反应池内预存有反应试剂；
    弹性膜(3)，设置在所述卡盒(1)和反应芯片(2)之间，用于将所述反应芯片(2)键合在所述卡盒(1)底部，且所述弹性膜(3)上开设有与所述卡盒(1)底部进口和出口数量相同的通孔；
    其中，所述活塞腔室的出口与所述弹性膜(3)上对应的通孔重合，以使所述活塞腔室与所述反应池相连通；所述样本接收腔室(15)的出口、试剂腔室(16)的进口和出口以及所述回收腔室(17)的进口均与所述弹性膜(3)上的通孔不重合，以使所述样本接收腔室(15)和试剂腔室(16)的出口与所述弹性膜(3)上的相邻通孔构成只能出不能进的第一单向阀结构，所述试剂腔室(16)和回收腔室(17)的进口与所述弹性膜(3)上的相邻通孔构成只能进不能出的第二单向阀结构；
    由此，所述样本接收腔室(15)的出口和试剂腔室(16)的出口分别通过所述第一单向阀结构与所述反应池相连通，所述试剂腔室(16)的进口和回收腔室(17)的进口分别通过所述第二单向阀结构与所述反应池相连通，从而保证流体或空气在特定操作下完成定向转移。
  2. 根据权利要求1所述的一体化自助式核酸检测装置，其特征在于，在位于所述活塞腔室与所述反应池之间的所述弹性膜(3)通孔处设置有疏水膜(4)，所述疏水膜(4)可供空气通过，但阻挡液体通过。
  3. 根据权利要求2所述的一体化自助式核酸检测装置，其特征在于，所述活塞腔室包括第一活塞腔室(11)和第二活塞腔室(12)，所述第一活塞腔室(11)和第二活塞腔室(12)内部分别设置有第一活塞推杆(13)和第二活塞推杆(14)；所述反应芯片(2)的上表面形成有第一反应池(21)和第二反应池(22)，所述第一活塞腔室(11)与所述第一反应池(21)相连通，所述第二活塞腔室(12)与所 述第二活塞腔室(12)相连通，所述样本接收腔室(15)和试剂腔室(16)分别通过第一单向阀结构与所述第一反应池(21)相连通，所述试剂腔室(16)和回收腔室(17)分别通过第二单向阀结构与所述第二反应池(22)相连通。
  4. 根据权利要求3所述的一体化自助式核酸检测装置，其特征在于，所述卡盒(1)上还形成有用于容置液体试剂的混匀腔室(18)，所述混匀腔室(18)的底部亦形成有一个出口和一个进口，所述混匀腔室(18)的出口和进口分别与所述弹性膜(3)上的相邻通孔形成第一单向阀结构和第二单向阀结构，所述混匀腔室(18)的进口通过第二单向阀结构与所述第二反应池(22)相连通，所述混匀腔室(18)的出口通过第一单向阀结构后经所述反应芯片(2)上的连接流道与所述回收腔室(17)的进口相连通。
  5. 根据权利要求4所述的一体化自助式核酸检测装置，其特征在于，所述弹性膜(3)选用双面胶基材，经过局部去粘性处理；所述疏水膜(4)为PTFE膜。
  6. 根据权利要求3到5任一项所述的一体化自助式核酸检测装置，其特征在于，所述反应试剂为固状或液状的RPA试剂、LAMP试剂、PCR试剂、NASBA试剂、cas12介导的特异序列信号放大反应试剂或cas13介导的特异序列信号放大反应试剂。
  7. 根据权利要求3到5任一项所述的一体化自助式核酸检测装置，其特征在于，所述微流控芯片(100)采用全封闭形式，即在所述卡盒(1)的一侧加工凹槽，然后用单面胶(5)与所述卡盒(1)的边框(19)贴合，并通过热封工艺沿所述边框(19)进行密封；
    同时，所述卡盒(1)的各腔室顶端出口处均通过密封盖或胶塞(23)密封，其中所述第一活塞腔室(11)和第二活塞腔室(12)的密封盖或胶塞(23)上均设有过孔，以供所述第一活塞推杆(13)和第二活塞推杆(14)穿过；
    此外，所述第一活塞腔室(11)、第二活塞腔室(12)、样本接收腔室(15)、试剂腔室(16)和回收腔室(17)的侧壁上均设有导气孔(20)。
  8. 根据权利要求3到5任一项所述的一体化自助式核酸检测装置，其特征在于，所述微流控芯片(100)置于与其形状相适配的外壳(24)内，并通过盖板(25)将所述微流控芯片(100)封闭在所述外壳(24)内，同时在所述微流控芯片(100)底部设置主要由PI膜、电阻丝和帕尔贴组成的加热单元；
    在所述外壳(24)的正面开设有两个推杆限位槽(26)，用于分别限制所述第一活塞推杆(13)和第二活塞推杆(14)的拉升距离；
    在所述外壳(24)的侧面设置有反应结果观察区(27)，用于显示试纸条结果；
    在所述外壳(24)的正面还设置有蜂鸣器(28)和状态指示灯(29)，用于提示用户操作；
    在所述外壳(24)内设置有与所述加热单元、蜂鸣器(28)和状态指示灯(29)连接的电路控制单元(30)，用于控制加热、报警和状态指示。
  9. 一种基于权利要求3到8任一项所述一体化自助式核酸检测装置的使用方法，其特征在于，包括以下步骤：
    步骤1：在样本接收腔室(15)内加入或形成含待测核酸的溶液；
    步骤2：拉起第一活塞推杆(13)，样本接收腔室(15)内的溶液在负压作用下通过其下方的第一单向阀(A)进入第一反应池(21)，并与预存在第一反应池(21)中的反应试剂混合；
    步骤3：将反应芯片(2)加热至设定温度以开始第一步扩增反应，反应一定时间后，推下第一活塞推杆(13)，反应后的扩增溶液在正压作用下经由第二单向阀(B)进入试剂腔室(16)内，与内部的液体试剂混匀；
    步骤4：拉起第二活塞推杆(14)，试剂腔室(16)内的扩增溶液在负压作用通过其下方的第一单向阀(C)进入第二反应池(22)，并与预存在第二反应池(22)中的反应试剂混合；
    步骤5：再次将反应芯片(2)加热至设定温度以开始第二步扩增反应，反应一定时间后，推下第二活塞推杆(14)，反应后的扩增溶液在正压作用下经由第二单向阀(D)进入混匀腔室(18)内，与内部的液体试剂混匀；
    步骤6：继续推下第二活塞推杆(14)，当混匀腔室(18)上方密闭空间内压强达到临界值后，混匀的扩增溶液突破第一单向阀(E)流出，进而通过第二单向阀(F)进入回收腔室(17)内；
    步骤7：进入回收腔室(17)的扩增溶液与放置在回收腔室(17)内的试纸条发生显色反应，根据试纸条的颜色结果得到核酸检测结果。
  10. 根据权利要求9所述的使用方法，其特征在于，在所述样本接收腔室(15)内加入或形成含待测核酸的溶液分为两种情况：
    1)待测核酸以游离在裂解溶液中的单链或者双链的形式加入样本接收腔室(15)，此时直接通过操作进入后续的反应；
    2)待测核酸部分或者全部的存在于液体加入的细胞、组织、病毒颗粒、细菌，此时需要通过加热裂解或裂解液裂解后加入样本接收腔室(15)中。
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CN107964504B (zh) * 2017-10-28 2023-10-27 深圳职业技术学院 一种封闭式样本一体化检测装置及检测方法
CN109929735A (zh) * 2019-04-25 2019-06-25 湖南工业大学 一种柱塞式核酸检测一体化卡盒及其检测方法
CN114480096A (zh) * 2021-12-29 2022-05-13 清华大学 按压式微流控芯片、微流控装置及细菌检测方法
CN114317234B (zh) * 2022-01-14 2023-06-27 西安交通大学 基于模块结构的多重病原快速检测体式微流控系统
CN114317234A (zh) * 2022-01-14 2022-04-12 西安交通大学 基于模块结构的多重病原快速检测体式微流控系统
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WO2023206093A1 (zh) * 2022-04-26 2023-11-02 广州国家实验室 样本提取装置
CN114574324A (zh) * 2022-04-26 2022-06-03 广州国家实验室 样本提取装置
CN115521862A (zh) * 2022-09-14 2022-12-27 大连理工大学 一种结合通用试纸条的核酸检测芯片通用结构及使用方法
CN115340946B (zh) * 2022-10-19 2023-02-03 天津智善生物科技有限公司 用于核酸检测的扩增装置及基于扩增装置的检测方法
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WO2024103297A1 (zh) * 2022-11-16 2024-05-23 广州国家实验室 样品提取卡盒和样品提取方法及核酸检测设备
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