WO2024098324A1 - 一种核酸提纯及检测反应液配制装置和方法 - Google Patents

一种核酸提纯及检测反应液配制装置和方法 Download PDF

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WO2024098324A1
WO2024098324A1 PCT/CN2022/131112 CN2022131112W WO2024098324A1 WO 2024098324 A1 WO2024098324 A1 WO 2024098324A1 CN 2022131112 W CN2022131112 W CN 2022131112W WO 2024098324 A1 WO2024098324 A1 WO 2024098324A1
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Prior art keywords
nucleic acid
pool
magnetic rod
magnetic
sleeve
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French (fr)
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喻学锋
周文华
舒伟良
惠允
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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Priority to PCT/CN2022/131112 priority Critical patent/WO2024098324A1/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
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Definitions

  • the present application relates to the field of biomedical technology, and in particular to a device and method for preparing nucleic acid purification and detection reaction solution.
  • POCT point-of-care diagnostic tests
  • PCR Polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • RCA rolling circle amplification
  • RPA recombinant polymerase amplification
  • centrifugal column method or magnetic bead method is usually used for nucleic acid extraction, which generally requires steps such as lysis, binding, and elution, making the entire "sample in, result out" fully automated instrument very difficult to achieve.
  • steps such as lysis, binding, and elution, making the entire "sample in, result out" fully automated instrument very difficult to achieve.
  • manual transfer is mostly used in the prior art, which is not only cumbersome, time-consuming and labor-intensive, but also difficult to fully and efficiently transfer samples. Manual operation can easily lead to unstable results and difficulty in detection.
  • the prior art basically uses complex liquid circuit valves to transfer liquids with different functions. Large-scale adoption will increase the cost of consumables, and the control of technology will greatly increase the difficulty and instability of operation.
  • PCR technology has the characteristics of exponential amplification of templates, the existing open consumables make the entire operation process prone to PCR aerosol contamination.
  • a Chinese patent application with publication number CN101452003A discloses a liquid pool micropump fully integrated in a microfluidic chip.
  • the liquid pool micropump consists of a closed filtration liquid pool, a pump liquid pool and a microchannel integrated on the microfluidic chip. It can only realize part of the functions of nucleic acid detection, and does not take into account the actual needs of clinical users, so it is difficult to put into practical use.
  • One purpose of the present application is to provide a nucleic acid purification and detection reaction liquid preparation device, which can realize the full process operation of sample lysis, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction liquid in a fully enclosed environment, and the overall structure of the device is simple, the operation is simple, and it can be compatible with various forms of samples.
  • nucleic acid purification and detection reaction solution preparation device comprising:
  • the upper cover layer comprises an upper cover body and a sample sealing cover and a silicone sleeve which are spaced apart from the upper cover body;
  • a reagent storage layer is rotatably disposed under the upper cover layer, and comprises a reagent storage body and a plurality of liquid pools spaced apart on the reagent storage layer, the liquid pools being used to store a treatment liquid for treating the sample nucleic acid;
  • the sample sealing cover is used to deliver samples to the lysis liquid pool of the reagent storage layer and maintain the sealing of the reagent storage layer;
  • the silicone sleeve is an elastic telescopic tubular structure, which is used to stretch up and down to cooperate with the inserted magnetic rod and magnetic rod sleeve to achieve the attraction and transfer of magnetic beads adsorbed with nucleic acid in the corresponding liquid pool, thereby realizing fully enclosed nucleic acid purification processing and preparation of nucleic acid detection reaction solution.
  • the reagent storage layer and the upper cover layer are rotatably connected using a transmission gear structure; the rotation angle range of each reagent storage layer is 15 to 90 degrees.
  • the upper cover layer also includes a convex portion having a central groove extending from the upper cover body, and the silicone sleeve is installed in the central groove of the convex portion;
  • the reagent storage body has a cylindrical structure and a columnar structure extending from the cylindrical structure, the diameter of the columnar structure is smaller than the cylindrical structure, and the columnar structure corresponds one-to-one to the liquid pool.
  • the multiple liquid pools include a magnetic bead preservation liquid pool for storing magnetic beads that adsorb nucleic acids, a lysis liquid pool for storing sample lysis liquid, a cleaning liquid pool for storing nucleic acid cleaning liquid, and an eluent and detection reagent preservation liquid pool for storing nucleic acid eluent and paraffin-wrapped detection reagent freeze-dried balls.
  • the magnetic bead preservation liquid pool, the lysis liquid pool, the first cleaning liquid pool, the second cleaning liquid pool, the third cleaning liquid pool and the eluent and detection reagent preservation liquid pool are sequentially arranged at even intervals along the circumference of the reagent storage layer.
  • the silicone sleeve is an elastic telescopic tubular structure, and a sealing silicone pad is arranged between the upper cover layer and the reagent storage layer.
  • the upper cover body and the reagent storage layer are both prepared by injection molding.
  • the sample is sputum, tissue, blood or feces
  • the detection reagent in the detection reagent freeze-dried ball is PCR, RPA or LAMP reagent
  • the overall length of the nucleic acid purification and detection reaction liquid preparation device is 35 mm, the width is 35 mm, and the height is 43 mm.
  • the present application also provides a method for nucleic acid purification and detection reaction solution preparation, comprising the following steps performed by the nucleic acid purification and detection reaction solution preparation device:
  • each rotation angle of the reagent storage layer is set to 15 to 90 degrees.
  • step S2 specifically includes the following steps:
  • the reagent storage layer further includes a lysis solution pool for storing sample lysis solution, a cleaning solution pool for storing nucleic acid cleaning solution, and a nucleic acid eluent and an eluent and a detection reagent preservation solution pool for storing paraffin-wrapped detection reagent freeze-dried balls, and the cleaning solution pool includes a first cleaning solution pool, a second cleaning solution pool, and a third cleaning solution pool arranged in sequence; step S3 includes the steps of:
  • step S1 further includes the steps of: heating and ultrasonically treating the lysis solution pool during sample lysis, and the sample lysis time is 1 to 5 minutes.
  • the duration of the nucleic acid adsorption process is 1 to 10 minutes.
  • the cleaning time of the magnetic beads in the first cleaning solution pool, the second cleaning solution pool and the third cleaning solution pool is 1 to 2 minutes respectively, and the magnetic beads are dried for 1 to 5 minutes after cleaning in the first cleaning solution pool, the second cleaning solution pool and the third cleaning solution pool.
  • step S36 the nucleic acid elution process takes 1 to 5 minutes.
  • the nucleic acid purification and detection reaction liquid preparation device of the present application can realize the full process operation of sample lysis, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction liquid in a fully closed environment, which is conducive to saving the pre-processing time of nucleic acid detection and improving the efficiency of nucleic acid detection.
  • the present application realizes a closed environment of multiple liquid pools by combining the upper cover layer and the reagent storage layer, thereby ensuring that sample lysis, nucleic acid binding, cleaning, elution and preparation of the detection reaction solution can be achieved in a completely closed state, ensuring the airtightness of the nucleic acid extraction and expansion process, reducing the interference of external aerosols on the test results, and preventing the sample from infecting the test personnel.
  • the reagent storage layer of the present application includes multiple liquid pools, and the type of processing liquid in the liquid pool can be set according to the requirements of nucleic acid purification and reaction liquid preparation of different samples, and can be compatible with the requirements of nucleic acid purification and detection reaction liquid preparation of various forms of samples.
  • the present application through the design of a rotating main body, can reduce the setting of the flow path switch control valve, and realize the transfer of magnetic beads between different liquid storage chambers by rotating the reagent storage main structure, thereby providing a sample nucleic acid purification and detection reaction liquid preparation device with simple structure and simple operation.
  • the nucleic acid purification and detection reaction liquid preparation device of the present application places the eluent and the detection reagent freeze-dried balls in a liquid pool, which solves the need to transfer the nucleic acid eluent during the detection reaction liquid preparation process. This saves the liquid path of the device and avoids the operation of liquid control.
  • the present application uses freeze-dried balls to preserve the detection reagents, and wraps a layer of paraffin on the outer surface of the detection reagent freeze-dried balls, thereby ensuring that the detection reagent freeze-dried balls can be placed in a liquid pool with the nucleic acid eluent, solving the problem of coexistence of the detection reagent and the nucleic acid eluent.
  • the nucleic acid purification and detection reaction liquid preparation device of the present application is prepared by injection molding, which can realize batch production and has the advantages of small batch difference, high output and low cost.
  • the nucleic acid purification and detection reaction solution preparation device of the present application can be used in combination with existing external supporting instruments such as PCR, LAMP, RCA and RPA, which is conducive to realizing a fully automated detection process of sample input and result output.
  • FIG1 is a schematic diagram of the three-dimensional structure of the nucleic acid purification and detection reaction solution preparation device according to a preferred embodiment of the present application;
  • FIG2 is an explosion diagram of the nucleic acid purification and detection reaction solution preparation device shown in FIG1 ;
  • FIG3 is a schematic top view of the nucleic acid purification and detection reaction solution preparation device shown in FIG1 ;
  • FIG4 is a schematic cross-sectional view of the nucleic acid purification and detection reaction solution preparation device shown in FIG3 along the A-A axis;
  • FIG. 5 is a schematic top view of a reagent storage layer of the nucleic acid purification and detection reaction solution preparation device shown in FIG. 1 .
  • nucleic acid purification and detection reaction liquid preparation device 100 nucleic acid purification and detection reaction liquid preparation device 100; upper cover layer 10; upper cover body 11; sample sealing cover 12; silicone sleeve 13; convex portion 14; central groove 14; reagent storage layer 20; reagent storage body 21; cylindrical structure 211; columnar structure 212; liquid pool 22; magnetic bead preservation liquid pool 221; lysis liquid pool 222; cleaning liquid pool 223; first cleaning liquid pool 2231; second cleaning liquid pool 2232; third cleaning liquid pool 2233; eluent and detection reagent preservation liquid pool 224.
  • one should be understood as “at least one” or “one or more”, that is, in one embodiment, the number of an element may be one, while in another embodiment, the number of the element may be multiple, and the term “one” should not be understood as a limitation on the quantity.
  • the terms “installed”, “connected”, and “connected” should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or mutual communication; it can be a direct connection, or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements.
  • installed should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or mutual communication; it can be a direct connection, or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements.
  • the nucleic acid purification and detection reaction solution preparation device 100 is used for extraction and purification of sample nucleic acid, and for preparing detection reaction solution.
  • the device can be used in combination with external supporting instruments to achieve full-process operation of sample processing and detection.
  • the external supporting instruments can be instruments such as PCR, LAMP, RCA and RPA, and this application does not limit this.
  • the detection reaction solution prepared by the device is PCR, LAMP, RCA and RPA detection reaction solution, and this application does not limit this.
  • the nucleic acid purification and detection reaction liquid preparation device 100 of the present application can realize the full process operation of sample lysis, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction liquid in a fully closed environment, which is conducive to saving pre-processing time of detection and improving detection efficiency.
  • the nucleic acid purification and detection reaction liquid preparation device 100 is composed of a two-layer structure, namely an upper cover layer 10 and a reagent storage layer 20 rotatably arranged under the upper cover layer 10. Through the cooperation of the upper cover layer 10 and the reagent storage layer 20, sample processing and detection reaction liquid preparation can be achieved in a fully enclosed environment.
  • the present application can reduce the setting of the flow path switch control valve through the design of the rotating reagent storage layer.
  • the magnetic beads can be transferred between different liquid storage chambers, thereby achieving different treatments of nucleic acids, and providing a sample nucleic acid purification and detection reaction liquid preparation device 100 with a simple structure and simple operation.
  • the reagent storage layer 20 includes a reagent storage body 21 and a plurality of liquid pools 22 spaced apart in the reagent storage layer 20.
  • the plurality of liquid pools 22 include a magnetic bead preservation liquid pool 221 for storing magnetic beads that adsorb nucleic acids, a lysis liquid pool 222 for storing sample lysis liquid, a cleaning liquid pool 223 for storing nucleic acid cleaning liquid, and an eluent and detection reagent preservation liquid pool 224 for storing nucleic acid eluent and detection reagent freeze-dried balls wrapped in paraffin.
  • the liquid pool 22 may also be a liquid pool containing other types of processing liquids, and may be adjusted according to actual sample processing and detection requirements, and the present application does not impose any limitation on this.
  • the nucleic acid purification and detection reaction liquid preparation device 100 of the present application places the eluent and the detection reagent freeze-dried balls in a liquid pool, which solves the need to transfer the nucleic acid eluent during the preparation of the detection reaction liquid. This saves the liquid circuit of the device and avoids the operation of liquid control.
  • the present application uses freeze-dried balls to preserve the detection reagents, and wraps a layer of paraffin on the outer surface of the detection reagent freeze-dried balls, so as to prevent the detection reagent freeze-dried balls from dissolving in the nucleic acid eluent, thereby ensuring that the detection reagent freeze-dried balls can be placed in a liquid pool with the nucleic acid eluent, solving the problem of coexistence of the detection reagent and the nucleic acid eluent.
  • paraffin wax also has the function of solving the problem of sample evaporation during the PCR heating process and preventing PCR aerosol contamination.
  • the paraffin wax wrapped around the outside of the freeze-dried ball can be dissolved and float on the solution, thereby sealing the solution below and preventing sample evaporation and aerosol contamination after heating.
  • freeze-dried balls can be changed as needed, including the reagent system and probe system used in RPA, LAMP and other PCR methods, and this application does not impose any restrictions on this.
  • the upper cover layer 10 includes an upper cover body 11 and a sample sealing cover 12 and a silicone sleeve 13 spaced apart from the upper cover body 11, wherein the sample sealing cover 12 is used to deliver samples to the lysis liquid pool 222 of the reagent storage layer 20 and maintain the sealing of the reagent storage layer 20; the silicone sleeve 13 is used to insert a magnetic rod and a magnetic rod sleeve, and is used to extend and retract up and down to cooperate with the magnetic rod and the magnetic rod sleeve to realize the attraction and transfer of magnetic beads in the corresponding liquid pool.
  • a transmission tooth structure can be used to form a rotatable connection between the reagent storage layer 20 and the upper cover layer 10; according to the number of liquid pools 22 of the reagent storage layer 20, the reagent storage layer 20 is also provided with different rotation angles, and each rotation angle range of the reagent storage layer 20 is 15 to 90°.
  • the silicone sleeve 13 is a retractable tubular structure with elasticity, so it can be extended and retracted up and down.
  • the silicone sleeve 13 can continue to move downward and penetrate into the liquid pool of the reagent storage layer 20.
  • the magnetic rod and the magnetic rod sleeve move upward to the initial position of the silicone sleeve 13, rotate the reagent storage layer 20, and turn the next liquid pool to be reacted to the bottom of the silicone sleeve 13.
  • the magnetic rod and the magnetic rod sleeve will drive the silicone sleeve 13 to move downward to the corresponding treatment liquid for reaction.
  • the magnetic rod and the magnetic rod sleeve will be operated separately, the magnetic rod moves upward, the magnetic rod sleeve remains stationary, and the magnetic beads adsorbed outside the silicone sleeve 13 will be dispersed into the liquid due to the disappearance of the magnetic field, so as to react more fully.
  • the nucleic acid purification and detection reaction liquid preparation device 100 opens the sample sealing cover 12 to deliver the sample into the lysis liquid pool 222 to achieve sample lysis, and rotates the reagent storage layer 20 to align the silicone sleeve 13 with the corresponding liquid pool, and further combines the up and down extension and contraction of the silicone sleeve 13 with the magnetic rod extraction method to achieve the adsorption of nucleic acids by magnetic beads, as well as the processing and transfer of the magnetic beads adsorbed with nucleic acids in the corresponding liquid pool, thereby fully enclosedly performing the nucleic acid purification process of the sample and the preparation of the detection reaction liquid.
  • the upper cover layer 10 also includes a protrusion 14 extending from the upper cover body 11 and having a central groove 14; that is, the protrusion 14 is a hollow cylindrical structure, which is used to define the installation space of the silicone sleeve 13, that is, the silicone sleeve 13 is installed in the central groove 14 of the protrusion 14.
  • the cleaning liquid pool 223 may also be other quantities, such as four, five or more than five.
  • the present application does not impose any limitation on this and it can be set according to actual detection requirements.
  • the number of liquid pools provided in the reagent storage layer 20 can be set according to actual needs, and the type of treatment liquid in the liquid pool can be set according to the nucleic acid purification and reaction liquid preparation requirements of different samples, so that the nucleic acid purification and detection reaction liquid preparation device 100 of the present application can be compatible with the nucleic acid purification and detection reaction liquid preparation requirements of various forms of samples.
  • the sample is any one of sputum, tissue, blood or feces.
  • a sealing silicone pad is also provided between the upper cover layer 10 and the reagent storage layer 20 to further ensure the sealing of the nucleic acid purification and detection reaction solution preparation device 100 .
  • the upper cover layer 10 and the reagent storage layer 20 also have corresponding central perforations, and the liquid storage body has a two-part structure, that is, the upper half is an integrated cylindrical structure, and the lower half is a columnar structure with a smaller diameter formed by extending downward from the cylindrical structure 211.
  • the number of the columnar structures 212 corresponds to the number of the liquid pools 22, and the positions correspond one to one.
  • the columnar structure 212 can be cylindrical or conical, and the present application does not impose any restrictions on this.
  • the present application realizes a closed environment of multiple liquid pools through the combination of the upper cover layer 10 and the reagent storage layer 20, and through the design of the rotatable reagent storage layer 20 combined with the magnetic sleeve extraction method, it can ensure that sample lysis, nucleic acid binding, cleaning, elution and preparation of the detection reaction solution are achieved in a completely closed state, thereby ensuring the airtightness of the nucleic acid extraction and expansion process, reducing the interference of external aerosols on the test results, and preventing sample leakage from causing infectious hazards to testers.
  • the upper cover body 11 and the reagent storage layer 20 are both prepared by injection molding.
  • the overall length of the nucleic acid purification and detection reaction solution preparation device 100 is 35 mm, the width is 35 mm, and the height is 43 mm. According to actual needs, the device can also be set to other sizes, and the present application does not limit this.
  • nucleic acid purification and detection reaction solution preparation device 100 of the present application The following will take the combination of the nucleic acid purification and detection reaction solution preparation device 100 of the present application and a PCR instrument to realize nucleic acid detection of samples as an example to specifically illustrate the method of using the nucleic acid purification and detection reaction solution preparation device 100 of the present application.
  • the collected samples include sputum, tissue, blood, feces, etc.
  • the pre-processing of these samples is relatively complicated and requires compatibility with multiple processing methods, such as chemical lysis solution guanidine isothiocyanate, biological enzyme lysis solution proteinase K, as well as heating and mechanical shearing, and different methods are configured for different objects.
  • the sample sealing cover 12 is covered. Under the action of the lysis liquid in the lysis liquid pool 222, the sample is lysed. During the lysis process, the outside of the liquid pool can be heated to 70°C, and ultrasound is applied during the reaction to further promote the lysis of the sample. This process takes 1 to 5 minutes.
  • the magnetic rod and the magnetic rod sleeve move downward, extend into the silicone sleeve 13 and drive the silicone sleeve 13 to slowly enter the magnetic bead preservation solution pool 221, and attract the magnetic beads in the magnetic bead preservation solution pool 221 to the top of the silicone sleeve 13.
  • the magnetic rod and the magnetic rod sleeve move upward to the initial state position of the silicone sleeve 13, and the reagent storage layer 20 is rotated 60° clockwise through the transmission of the external supporting device (i.e., the PCR instrument), so that the lysis solution pool 222 is located directly below the silicone sleeve 13.
  • the magnetic rod and the magnetic rod sleeve move downward with the silicone sleeve 13 and enter the lysis solution in the lysis solution pool 222.
  • the magnetic rod moves upward to leave the magnetic rod sleeve, that is, the magnetic rod is pulled out of the magnetic rod sleeve. Due to the disappearance of the magnetic field, the magnetic beads outside the silicone sleeve 13 will be fully dispersed into the lysis solution under the vibration of the magnetic rod sleeve, and the adsorption of nucleic acids is achieved during the mixing process. This process is waited for 1 to 10 minutes.
  • the magnetic rod and the magnetic rod cover slowly move into the lysate with the silicone cover 13, and absorb the magnetic beads in the lysate to the top of the silicone cover 13. Then they move upward together to the initial state of the silicone cover 13, and the reagent storage layer 20 is rotated 60° clockwise through the transmission of the external matching device, so that the first cleaning liquid pool 2231 is located directly below the silicone cover 13.
  • the magnetic rod and the magnetic rod sleeve move downward with the silicone sleeve 13 and enter the first cleaning solution, and the magnetic rod moves upward and leaves the magnetic rod sleeve.
  • the magnetic beads outside the silicone sleeve 13 will be fully dispersed into the first cleaning solution under the vibration of the magnetic rod sleeve. Wait for 1 to 2 minutes and continue the above operation to adsorb the magnetic beads to the top of the silicone sleeve 13, and then rotate the reagent storage layer 20 to perform the cleaning operation of the magnetic beads in the second cleaning solution pool 2232 and the third cleaning solution pool 2233. After completing the cleaning of the nucleic acid, the magnetic beads are adsorbed on the top of the silicone sleeve 13, and after moving to the initial state position of the silicone sleeve 13, they are dried for 1 to 5 minutes.
  • the reagent storage layer 20 is rotated 60° clockwise through the transmission of an external supporting device, so that the eluent and detection reagent preservation liquid pool 224 are located directly below the silicone sleeve 13, and the magnetic rod sleeve moves downward with the silicone sleeve 13 into the eluent, and the magnetic rod sleeve is vibrated to fully disperse the magnetic beads into the eluent, and the nucleic acid adsorbed by the magnetic beads is eluted with the eluent to obtain a nucleic acid purification solution; after waiting for 1 to 5 minutes, the magnetic rod moves downward, and after completing the magnetic absorption, the magnetic rod and the magnetic rod sleeve move upward with the silicone sleeve 13 to leave the nucleic acid purification solution, and the nucleic acid extraction is completed.
  • the heating module of the external supporting instrument is attached to the outside of the elution solution and the detection reagent preservation solution pool 224, and the temperature is raised to 70°C to dissolve the paraffin wrapping the PCR detection reagent freeze-dried ball.
  • the dissolved paraffin will float to the top layer, and the PCR detection reagent freeze-dried ball will dissolve in the nucleic acid purification solution.
  • the PCR freeze-dried ball contains primers for amplifying the target fragment, detection probes and other required components, thereby completing the preparation of the PCR reaction solution.
  • the corresponding heating program is set, and the PCR reaction solution is detected through the corresponding detection module of the PCR instrument.
  • the corresponding fluorescence detection is performed according to the fluorescence of the detection probe of the reaction. This process is consistent with the conventional qPCR.
  • the present application actually also provides a fully enclosed nucleic acid extraction and amplification qPCR detection device, which is equipped with the required nucleic acid extraction and detection reagents. After the sample is added, fully enclosed automated processing is achieved, and a fully automated detection process of sample input and result output is realized. The detection efficiency is high, and the whole process is closed, which is conducive to ensuring the accuracy and safety of the detection.
  • the nucleic acid purification and detection reaction liquid preparation device 100 of the present application is suitable for the detection of various clinical samples, such as urine, blood, nasopharyngeal swabs, sputum, tissues, feces and other samples, and the reagent formula can be adjusted and the processing steps can be increased or decreased according to the specific samples.
  • the nucleic acid purification and detection reaction liquid preparation device 100 of the present application uses a silicone sleeve to achieve the closed operation of conventional magnetic rod method for extracting nucleic acid. It is a fully enclosed nucleic acid detection reagent card box with excellent airtightness. It is not connected to the outside air during use, which can greatly avoid the influence of the external environment on the test results and protect the health of the test personnel.
  • the nucleic acid purification and detection reaction liquid preparation device 100 of the present application can realize the full process operation of sample lysis, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction liquid in a fully enclosed environment through the design of a rotatable main body and the combination of silicone sleeve and magnetic sleeve extraction method. Moreover, the device has a simple overall structure and simple operation, and is compatible with various forms of samples.
  • the reagent storage layer 20 of the present application includes multiple liquid pools 22, and the processing liquid type of the liquid pool can be set according to the nucleic acid purification and reaction liquid preparation requirements of different samples, and can be compatible with the nucleic acid purification and detection reaction liquid preparation requirements of various forms of samples.
  • the present application can reduce the setting of the flow path switch control valve through the design of a rotating body, and realize the transfer of magnetic beads in different liquid storage chambers by rotating the reagent storage body 21 structure, thereby providing a sample nucleic acid purification and detection reaction liquid preparation device 100 with simple structure and simple operation.
  • the nucleic acid purification and detection reaction liquid preparation device 100 of the present application places the eluent and the detection reagent freeze-dried balls in a liquid pool, which solves the need to transfer the nucleic acid eluent during the detection reaction liquid preparation process. This saves the liquid path of the device and avoids the operation of liquid control.
  • the present application uses freeze-dried balls to store detection reagents, and wraps a layer of paraffin on the outer surface of the detection reagent freeze-dried balls, thereby ensuring that the detection reagent freeze-dried balls can be placed in a liquid pool with the nucleic acid eluent, solving the problem of coexistence of the detection reagent and the nucleic acid eluent.
  • the nucleic acid purification and detection reaction solution preparation device 100 of the present application is prepared by injection molding, which can realize batch production and has the advantages of small batch difference, high output and low cost.

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Abstract

一种核酸提纯及检测反应液配制装置和方法，该装置包括上盖层（10）和可转动地设置在上盖层（10）之下的试剂储存层（20），上盖层（10）包括上盖主体（11）和间隔设置于上盖主体（11）的样本密封盖（12）和硅胶套（13）；试剂储存层（20）包括试剂储存主体（21）和间隔设置于试剂储存层（20）的多个用于储存对样本核酸进行处理的处理液的液池（22）；该装置通过可旋转式主体的设计以及硅胶套与磁套提取法的结合，能够在全封闭式环境下实现样本裂解、核酸吸附、核酸清洗、核酸洗脱以及检测反应液的全流程操作，而且该装置整体结构简单，操作简单，可以兼容多种形式的样本。

Description

一种核酸提纯及检测反应液配制装置和方法 技术领域
本申请涉及生物医学技术领域，特别是涉及一种核酸提纯及检测反应液配制装置和方法。
背景技术
早期，快速和准确的疾病检测对于最大限度地提高危机管理效率、治疗效果和经济稳定至关重要。然而，目前的检测做法主要局限于集中中央实验室，通常病人的样本会被带到医院或诊所进行检测，检测结果会在几天内反馈，在发展中国家或地区，由于缺乏熟练的人员和医疗基础设施，疾病检测往往会更久。因此，对便携、易于使用和即时诊断检测(POCT)的需求正在迅速增加。
分子诊断学正在革新感染性疾病的临床检测，其中一个显著的进步是减少了诊断感染性疾病所需的时间。聚合酶链反应(PCR)是最常用的分子诊断技术，但热循环需要一个耗能的仪器，为了简化及缩短热循环的时间，有发展出各种等温扩增反应，如环介导等温扩增(LAMP)、滚环扩增(RCA)和重组聚合酶扩增(RPA)，在这些技术的基础上，开发了许多商业化试剂盒，可以实现高灵敏和快速的检查病原体。然而，这些试剂盒在样本制备和核酸提取方面的操作步骤比较多，限制了其在临床诊断中的广泛应用。因此，开发一种将分子诊断的所有步骤都集成到一个小型化设备中的片上实验室系统已成为一种趋势。在POCT诊断中，患者可以自己收集样本，无需医务人员的帮助，采集的样本可以在采样点立即进行分析和疾病筛查，而且只需要非常小的样本量 就可以实现对生物标志物的检测。
在现有技术中通常使用离心柱法或磁珠法进行核酸提取，一般需要进行裂解、结合、洗脱等步骤，使得整个“样本进结果出”的全自动化仪器非常难以实现。就各步骤中有效成分的转移而言，现有技术中多采用手动转移的方式，不仅操作繁琐、费时费力，且样本很难充分、高效第转移，人工操作极易导致结果不稳定，检测实现难度大。而且，现有技术都基本是采用复杂的液路阀门转移不同功能的液体，对于大规模采用会增加耗材的使用成本，对于技术的把控也会大大增加操作难度和不稳定性。此外，分子检测的主流技术为荧光定量PCR技术，由于PCR技术具有指数级扩增模板的特点，现有的开放式耗材使整个操作过程容易造成PCR气溶胶污染。例如公开号为CN101452003A的中国专利申请公开了一种完全集成于微流控芯片的液池微泵，该液池微泵由集成于微流控芯片上的密闭过滤液池、泵液池以及微通道组成，其只能实现核酸检测的部分功能，且没有考虑临床用户的实际需求，很难实用化。
发明内容
本申请的一目的是，提供一种核酸提纯及检测反应液配制装置，该装置能够在全封闭式环境下实现样本裂解、核酸吸附、核酸清洗、核酸洗脱以及检测反应液的全流程操作，且该装置整体结构简单，操作简单，可以兼容多种形式的样本。
本申请在一方面提供了一种核酸提纯及检测反应液配制装置，包括：
所述上盖层包括上盖主体和间隔设置于所述上盖主体的样本密封盖和硅胶套；和
试剂储存层，所述试剂储存层可转动地设置在所述上盖层之下，包括试剂储存主体和间隔设置于所述试剂储存层的多个液池，所述液池用于储存对样本核酸进行处理的处理液；
其中所述样本密封盖用于向所述试剂储存层的所述裂解液池投送样本并保持所述试剂储存层的密封性；所述硅胶套为具有弹性的伸缩管状结构，用于上下伸缩来与插入的磁棒和磁棒套配合实现吸附有核酸的磁珠在对应液池中的吸引和转移，从而实现全封闭式的核酸提纯处理和核酸检测反应液的配制。
在本申请的一实施例中，所述试剂储存层与所述上盖层之间采用传动卡齿的结构形成可转动连接；所述试剂储存层的每次转动角度范围为15～90°。
在本申请的一实施例中，所述上盖层还包括延伸自所述上盖主体的具有中心槽的凸部，所述硅胶套安装于所述凸部的中心槽内；所述试剂储存主体具有圆柱状结构和延伸自所述圆柱状结构的柱状结构，所述柱状结构的直径小于所述圆柱状结构，所述柱状结构与所述液池一一对应。
在本申请的一实施例中，多个所述液池包括用于储存吸附核酸的磁珠的磁珠保存液池、用于储存样本裂解液的裂解液池、用于储存核酸清洗液的清洗液池和用于储存核酸洗脱液与由石蜡包裹的检测试剂冻干球的洗脱液及检测试剂保存液池。
在本申请的一实施例中，所述清洗液池为三个，包括依次设置的第一清洗液池、第二清洗液池以及第三清洗液池。
在本申请的一实施例中，其中所述磁珠保存液池、所述裂解液池、所述第一清洗液池、所述第二清洗液池、所述第三清洗液池和所述洗脱液及检测试剂保存液池依次沿所述试剂储存层的周向均匀间隔设置。
在本申请的一实施例中，所述硅胶套为具有弹性的伸缩管状结构，所述上 盖层和所述试剂储存层之间设置有密封硅胶垫。
在本申请的一实施例中，所述上盖主体和所述试剂储存层均通过注塑成型的方式制备得到。
在本申请的一实施例中，所述样本为痰液、组织、血液或粪便；所述检测试剂冻干球中的检测试剂为PCR、RPA或LAMP试剂；所述核酸提纯及检测反应液配制装置的整体长度为35mm，宽度为35mm，高度为43mm。
本申请在另一方面还提供了一种核酸提纯及检测反应液配制方法，包括采用所述核酸提纯及检测反应液配制装置执行的以下步骤：
S1、打开样本密封盖在裂解液池中投送样本，实现样本裂解；
S2、在上盖层的硅胶套中插入磁棒和磁棒套，通过磁棒将磁珠保存液中的磁珠吸引在硅胶套上；
S3、依次按照预设角度转动所述试剂储存层，使得所述硅胶套依次与所述试剂储存层的对应液池对齐，并在所述硅胶套每次与对应的液池对齐时，通过从磁棒套中抽离磁棒的方式，实现磁珠在对应的液池中的释放，然后再次通过插入磁棒并带动所述硅胶套伸入对应的液池中的方式，实现对磁珠的吸引，然后以此通过重复对磁珠的释放和吸引操作，依次实现磁珠对应液池中的转移，从而依次实现磁珠对样本核酸的吸附、核酸清洗、核酸洗脱以及核酸检测反应液制备的过程。
在本申请的一实施例中，所述试剂储存层的每次转动角度设置为15～90°。
在本申请的一实施例中，所述步骤S2具体包括步骤：
S21、将磁棒及磁棒套插入上盖层的硅胶套中，通过磁棒和磁棒套带动所述硅胶套向下伸缩进入磁珠保存液池中，将磁珠吸引到所述硅胶套的顶部，完 成吸引后，将磁棒及磁棒套往上运动至所述硅胶套的初始位置状态。
在本申请的一实施例中，所述试剂储存层还包括用于储存样本裂解液的裂解液池、用于储存核酸清洗液的清洗液池和用于储存核酸洗脱液与由石蜡包裹的检测试剂冻干球的洗脱液及检测试剂保存液池，所述清洗液池包括依次设置的第一清洗液池、第二清洗液池以及第三清洗液池；步骤S3包括步骤：
S31、将所述试剂储存层转动60°，使得所述硅胶套位于所述裂解液池正上方，将磁棒和磁棒套带动所述硅胶套向下伸缩进入所述裂解液池中，将磁棒抽离磁棒套，使得磁场消失而使得所述硅胶套吸附的磁珠释放进所述裂解液池中，通过磁珠吸附所述裂解液池中的核酸；
S32、再次将磁棒插入磁棒套并带动所述硅胶套伸入所述裂解液池，将所述裂解液池中的吸附有核酸的磁珠吸引到所述硅胶套的顶部，完成吸引后，将磁棒及磁棒套往上运动至所述硅胶套的初始位置状态；
S33、将所述试剂储存层转动60°，使得插有磁棒及磁棒套的所述硅胶套位于第一清洗液池正上方，将磁棒和磁棒套带动所述硅胶套向下伸缩进入所述第一清洗液池中，将磁棒抽离磁棒套，使得磁场消失而使得所述硅胶套吸附的磁珠进入所述清洗液池中，实现核酸清洗；
S34、再次将磁棒插入磁棒套并带动所述硅胶套伸入所述第一清洗液池，将清洗后的磁珠吸引到所述硅胶套的顶部，完成吸引后，将磁棒及磁棒套往上运动至所述硅胶套的初始位置状态；
S35、按照与步骤S33和S34同样的操作，完成磁珠在所述第二清洗液池和所述第三清洗液池中的清洗过程；
S36、将所述试剂储存层转动60°，使得所述硅胶套位于洗脱液及检测试剂保存液池正上方，再次通过磁棒和磁棒套带动所述硅胶套向下伸缩进入所述 洗脱液及检测试剂保存液池中，将磁棒抽离磁棒套，使得磁场消失而使得所述硅胶套吸附的磁珠进入所述洗脱液及检测试剂保存液池中，实现核酸洗脱；
S37、再次将磁棒插入磁棒套并带动所述硅胶套伸入所述洗脱液及检测试剂保存液池，将洗脱出核酸的磁珠吸引到所述硅胶套的顶部，完成吸引后，将磁棒及磁棒套往上运动至所述硅胶套的初始位置状态；
S38、加热所述洗脱液及检测试剂保存液池，使得所述洗脱液及检测试剂保存液池中包裹检测试剂冻干球的石蜡溶解，溶解后的石蜡漂浮到所述洗脱液及检测试剂保存液池最上层，检测试剂冻干球溶解到核酸洗脱液中对核酸进行处理，以此完成检测反应液的配制。
在本申请的一实施例中，步骤S1还包括步骤：在样本裂解时，还对所述裂解液池进行加热和超声处理，样本裂解时间为1～5min。
在本申请的一实施例中，在步骤S31中，核酸吸附过程的时间为1～10min。
在本申请的一实施例中，磁珠在所述第一清洗液池、所述第二清洗液池以及所述第三清洗液池中的清洗时间分别为1～2min，磁珠在在所述第一清洗液池、所述第二清洗液池以及所述第三清洗液池中清洗后干燥1～5min。
在本申请的一实施例中，在步骤S36中，核酸洗脱过程的时间为1～5min。
本申请具有以下有益效果：
(1)本申请的所述核酸提纯及检测反应液配制装置能够在全封闭式环境下实现样本裂解、核酸吸附、核酸清洗、核酸洗脱以及检测反应液的全流程操作，有利于节省核酸检测的前处理时间，提高核酸检测效率。
(2)本申请通过上盖层和试剂储存层的结合实现了多个液池的封闭环境，以此能够确保在完全封闭的状态下实现样本裂解、核酸结合、清洗、洗脱以及检测反应液的配制，保证了核酸提取及扩张过程的密闭性，减少外界气溶胶对 检测结果的干扰，以及防止样本对检测人员的传染危害。
(3)本申请的试剂储存层包括多个液池，可以根据不同样本的核酸提纯和反应液配制需求设置液池的处理液类型，可以兼容多种形式的样本的核酸提纯及检测反应液配制需求。
(4)本申请通过旋转式主体的设计，能够缩减流路开关控制阀门的设置，通过转动试剂储存主体结构，实现磁珠在不同储液腔的转移，提供了一种结构简单、操作简单的样本的核酸提纯及检测反应液配制装置。
(5)本申请的所述核酸提纯及检测反应液配制装置将洗脱液和检测试剂冻干球放置在一个液池内，解决了检测反应液配制过程中需要将核酸洗脱液转移的操作，这样可以省去该装置的液路，避免了液体控制的操作。
(6)本申请采用冻干球的方式保存检测试剂，并在检测试剂冻干球的外表面包裹一层石蜡，从而确保检测试剂冻干球可以与核酸洗脱液放置在一个液池内，解决了检测试剂和核酸洗脱液共存的问题。
(7)本申请的所述核酸提纯及检测反应液配制装置采用注塑成型的方式实现制备，可以实现批量化生产，具有批间差小，产量高，成本低的优势。
(8)本申请的所述核酸提纯及检测反应液配制装置可以与现有的PCR、LAMP、RCA以及RPA等外部配套仪器结合使用，有利于实现样本进结果出的全自动化检测过程。
通过对随后的描述和附图的理解，本申请进一步的目的和优势将得以充分体现。
附图说明
图1为本申请的一优选实施例的所述核酸提纯及检测反应液配制装置的立体结构示意图；
图2为图1所示的所述核酸提纯及检测反应液配制装置的爆炸示意图；
图3为图1所示的所述核酸提纯及检测反应液配制装置的俯视示意图；
图4为图3所示的所述核酸提纯及检测反应液配制装置沿A-A轴的剖视示意图；
图5为图1所示的所述核酸提纯及检测反应液配制装置的试剂储存层的俯视示意图。
附图标号说明：核酸提纯及检测反应液配制装置100；上盖层10；上盖主体11；样本密封盖12；硅胶套13；凸部14；中心槽14；试剂储存层20；试剂储存主体21；圆柱状结构211；柱状结构212；液池22；磁珠保存液池221；裂解液池222；清洗液池223；第一清洗液池2231；第二清洗液池2232；第三清洗液池2233；洗脱液及检测试剂保存液池224。
具体实施方式
以下描述用于揭露本申请以使本领域技术人员能够实现本申请。以下描述中的优选实施例只作为举例，本领域技术人员可以想到其他显而易见的变型。在以下描述中界定的本申请的基本原理可以应用于其他实施方案、形变方案、改进方案、等同方案以及没有背离本申请的精神和范围的其他技术方案。
本领域技术人员应理解的是，在本申请的揭露中，术语“竖向”、“横向”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”等指示的方位或位置关系是基于附图所示的方位或位置关系，其仅是为了便于描述本申请和简化描述，而不是指示 或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此上述术语不能理解为对本申请的限制。
可以理解的是，术语“一”应理解为“至少一”或“一个或多个”，即在一个实施例中，一个元件的数量可以为一个，而在另外的实施例中，该元件的数量可以为多个，术语“一”不能理解为对数量的限制。
在本申请的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接或可以相互通讯；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。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申请中的具体含义。
如图1至图5所示，根据本申请的一优选实施例的一种核酸提纯及检测反应液配制装置100的具体结构被阐明。所述核酸提纯及检测反应液配制装置100用于对样本核酸的提取和纯化，并用于配制检测反应液，该装置可以与外部配套仪器组合使用，以实现样本处理和检测的全流程操作。外部配套仪器可以为PCR、LAMP、RCA以及RPA等仪器，本申请对此不作限制。相对应地，该装置用于配制的检测反应液为PCR、LAMP、RCA以及RPA检测反应液，本申请对此也不作限制。
本申请的所述核酸提纯及检测反应液配制装置100能够在全封闭式环境下实现样本裂解、核酸吸附、核酸清洗、核酸洗脱以及检测反应液的全流程操作，有利于节省检测的前处理时间，提高检测效率。
具体地，所述核酸提纯及检测反应液配制装置100共由两层结构组成，分别是上盖层10和可转动地设置在所述上盖层10之下的试剂储存层20，通过所 述上盖层10和所述试剂储存层20的配合，可以实现在全封闭式环境下对样本的处理和检测反应液配制。
可以理解的是，本申请通过旋转式试剂储存层的设计，能够缩减流路开关控制阀门的设置，通过转动所述试剂储存层20，实现磁珠在不同储液腔的转移，从而实现对核酸的不同处理，提供了一种结构简单、操作简单的样本的核酸提纯及检测反应液配制装置100。
更具体地，所述试剂储存层20包括试剂储存主体21和间隔设置于所述试剂储存层20的多个液池22，在这一优选实施例中，多个所述液池22包括用于储存吸附核酸的磁珠的磁珠保存液池221、用于储存样本裂解液的裂解液池222、用于储存核酸清洗液的清洗液池223和用于储存核酸洗脱液与由石蜡包裹的检测试剂冻干球的洗脱液及检测试剂保存液池224。
应该理解的是，在本申请的一些实施例中，所述液池22还可以为包含其他类型处理液的液池，可以根据实际的样本处理和检测需求进行调整，本申请对此不作限制。
可以理解的是，本申请的所述核酸提纯及检测反应液配制装置100将洗脱液和检测试剂冻干球放置在一个液池内，解决了检测反应液配制过程中需要将核酸洗脱液转移的操作，这样可以省去该装置的液路，避免了液体控制的操作。
而且，本申请采用冻干球的方式保存检测试剂，并在检测试剂冻干球的外表面包裹一层石蜡，从而避免检测试剂冻干球溶解在核酸洗脱液中，以此确保检测试剂冻干球可以与核酸洗脱液放置在一个液池内，解决了检测试剂和核酸洗脱液共存的问题。
另外，采用石蜡还有一个作用是解决PCR加热过程中样本蒸发的问题以及防止PCR气溶胶污染，通过在检测反应液制备过程中对所述洗脱液及检测 试剂保存液池224进行加热的方式，能够使得冻干球外面包裹的石蜡溶解并漂浮在溶液上面，起到对下面的溶液密封的作用，防止加热后样本蒸发以及气溶胶污染。
值得一提的是，冻干球内容物可以根据需要进行变更，包括RPA、LAMP及其他PCR方法所用到的试剂体系和探针体系，本申请对此不作限制。
具体地，所述上盖层10包括上盖主体11和间隔设置于所述上盖主体11的样本密封盖12和硅胶套13，所述样本密封盖12用于向所述试剂储存层20的所述裂解液池222投送样本并保持所述试剂储存层20的密封性；所述硅胶套13用于插入磁棒和磁棒套，并用于上下伸缩来与磁棒和磁棒套配合实现磁珠在对应的液池中的吸引和转移。
值得一提的是，所述试剂储存层20与所述上盖层10之间可以采用传动卡齿的结构形成可转动连接；根据所述试剂储存层20的液池22的数量的不同，所述试剂储存层20也设置有不同的转动角度，所述试剂储存层20的每次转动角度范围为15～90°。
特别地，所述硅胶套13为具有弹性的伸缩管状结构，因此能够上下伸缩，当外部配套仪器的磁棒和磁棒套插入所述硅胶套13后，可以带着所述硅胶套13继续往下运动，深入到所述试剂储存层20的液池内，当需要更换不同处理液时，磁棒和磁棒套向上运动到所述硅胶套13的初始位置，转动所述试剂储存层20，把下一个需反应的液池转到硅胶套13正下方。等待完成转动后，磁棒和磁棒套会带动所述硅胶套13往下运动到对应的处理液中进行反应。需要将磁珠释放到处理液的时候，磁棒和磁棒套会分开操作，磁棒往上运动，磁棒套保持不动，而硅胶套13外吸附的磁珠由于磁场的消失会分散到液体中，进行更充分的反应。
也就是说，所述核酸提纯及检测反应液配制装置100通过打开所述样本密封盖12在所述裂解液池222中投送样本，实现样本裂解，并通过转动所述试剂储存层20来使得所述硅胶套13与对应的液池对齐，进一步结合所述硅胶套13的上下伸缩与磁棒提取法来实现磁珠对核酸的吸附，以及将吸附有核酸的磁珠在对应的液池中的处理和转移，从而全封闭式地进行样本的核酸的提纯处理及检测反应液配制。
值得一提的是，所述上盖层10还包括延伸自所述上盖主体11并具有中心槽14的凸部14；即所述凸部14为中空的圆柱状结构，用于界定形成所述硅胶套13的安装空间，也就是说，所述硅胶套13安装在所述凸部14的中心槽14内。
还值得一提的是，在本申请的一优选实施例中，所述清洗液池223可以为三个，包括依次设置的第一清洗液池2231、第二清洗液池2232以及第三清洗液池2233；其中所述磁珠保存液池221、所述裂解液池222、所述第一清洗液池2231、所述第二清洗液池2232、所述第三清洗液池2233和所述洗脱液及检测试剂保存液池224依次沿所述试剂储存层20的周向均匀间隔设置，即在这一优选实施例中，所述试剂储存层20设置有六个液池，六个液池均匀间隔排布，以此所述试剂储存层20通过每次转动60°的方式，即可以使得所述硅胶套13与对应的液池相对齐。
在本申请的一些实施例中，所述清洗液池223也可以为其他数量，如四个、五个或五个以上，本申请对此不作限制，可根据实际的检测需求进行设置。
也就是说，所述试剂储存层20设置的液池数量可以根据实际需求进行设置，而且可以根据不同样本的核酸提纯和反应液配制需求设置液池的处理液类型，以此本申请的所述核酸提纯及检测反应液配制装置100可以兼容多种形式 的样本的核酸提纯及检测反应液配制需求。可选地，所述样本为痰液、组织、血液或粪便中的任一种。
值得一提的是，所述上盖层10和所述试剂储存层20之间还设置有密封硅胶垫，以进一步确保所述核酸提纯及检测反应液配制装置100的密封性。
还值得一提的是，所述上盖层10和所述试剂储存层20还具有相对应的中心穿孔，且所述储液主体具有两部分的结构，即上半部为一体式的圆柱状结构，下半部为由所述圆柱状结构211向下延伸形成的直径更小的柱状结构，所述柱状结构212的数量与所述液池22的数量相对应，且位置一一对应，所述柱状结构212可以为圆柱状，也可以为圆锥状，本申请对此不作限制。
可以理解的是，本申请通过所述上盖层10和所述试剂储存层20的结合实现了多个液池的封闭环境，并通过可转动式的所述试剂储存层20的设计与结合磁套提取法，能够确保在完全封闭的状态下实现样本裂解、核酸结合、清洗、洗脱以及检测反应液的配制，保证了核酸提取及扩张过程的密闭性，减少外界气溶胶对检测结果的干扰，同时防止了样本泄漏而对检测人员造成传染危害。
值得一提的是，在本申请的这一优选实施例中，所述上盖主体11和所述试剂储存层20均通过注塑成型的方式制备得到。所述核酸提纯及检测反应液配制装置100的整体长度为35mm，宽度为35mm，高度为43mm，根据实际需要，该装置也可以设置为其他尺寸，本申请对此不作限制。
下面将以本申请的所述核酸提纯及检测反应液配制装置100与PCR仪器结合实现样本的核酸检测为例，具体说明本申请的所述核酸提纯及检测反应液配制装置100的使用方法。
样本前处理和PCR扩增检测：
一、样本前处理
A)裂解样本：
采集的样本包括痰液、组织、血液、粪便等，这些样本的前处理是比较复杂的，需要兼容多种处理方式，如化学裂解液异硫氰酸胍、生物酶裂解液蛋白酶K、以及加热和机械剪切，对应不同的对象配置不同的方式。
样本通过所述样本密封盖12加入到所述裂解液池222后，盖上所述样本密封盖12，在所述裂解液池222内裂解液的作用下，实现样本的裂解，在裂解的过程中可以对液池外部进行加热70℃,并在反应的过程中施加超声，进一步促进样本的裂解，此过程等待1～5min。
B)核酸结合：
待样本裂解完成后，磁棒及磁棒套往下运动，伸入所述硅胶套13中并带动所述硅胶套13缓慢进入到所述磁珠保存液池221中，将所述磁珠保存液池221中的磁珠吸引到所述硅胶套13的顶部。完成磁珠的吸引后，磁棒及磁棒套往上运动至所述硅胶套13的初始状态位置，通过外部配套装置(即PCR仪器)的传动将所述试剂储存层20顺时针旋转60°，使所述裂解液池222处于所述硅胶套13的正下方位置。磁棒和磁棒套带着所述硅胶套13往下运动，进入到所述裂解液池222的裂解液中，磁棒往上运动离开磁棒套，即将磁棒从磁棒套中抽离，由于磁场消失，硅胶套13外部的磁珠会在磁棒套的振动下充分分散到裂解液中，在混合的过程中实现核酸的吸附，此过程等待1～10min。
核酸清洗：
待磁珠完成吸附核酸后，磁棒和磁棒套带着所述硅胶套13一起缓慢运动到裂解液中，将裂解液中的磁珠吸附到所述硅胶套13的顶部位置。再一起往上运动到硅胶套13的初始状态位置，通过外部配套装置的传动将所述试剂储存层20顺时针旋转60°，使所述第一清洗液池2231处于所述硅胶套13的正 下方位置。磁棒和磁棒套带着所述硅胶套13往下运动，进入到所述第一清洗液中，磁棒往上运动离开磁棒套，由于磁场消失，所述硅胶套13外部的磁珠会在磁棒套的振动下充分分散到所述第一清洗液中，等待1～2min，继续以上操作，将磁珠吸附到硅胶套13顶部，再转动所述试剂储存层20，进行磁珠在所述第二清洗液池2232和所述第三清洗液池2233中的清洗操作，完成核酸的清洗后，将磁珠吸附在所述硅胶套13的顶部，并运动到所述硅胶套13的初始状态位置后，干燥1～5min。
C)核酸洗脱：
待磁珠干燥完成后，通过外部配套装置的传动将所述试剂储存层20顺时针旋转60°，使所述洗脱液及检测试剂保存液池224处于硅胶套13的正下方位置，磁棒套带着所述硅胶套13往下运动到洗脱液中，振动磁棒套使磁珠充分分散到洗脱液中，通过洗脱液将磁珠吸附的核酸洗脱出来，得到核酸纯化液；等待1～5min后，磁棒往下，完成吸磁后，磁棒和磁棒套带着所述硅胶套13往上运动离开核酸纯化液，完成核酸的提取。
二、PCR扩增检测模块
A)配制PCR反应液
待完成样本的核酸提取后，将外部配套仪器的加热模块和所述洗脱液及检测试剂保存液池224的外部贴合，升温到70℃使包裹PCR检测试剂冻干球的石蜡溶解，溶解后的石蜡会漂浮到最上层，PCR检测试剂冻干球会溶解到核酸纯化液中，PCR冻干球包含有扩增目的片段的引物和检测的探针及其它所需的成分，以此完成PCR反应液的配制。
B)PCR反应及检测
按照常规PCR设置相应的加热程序，通过PCR仪器的相应检测模块对PCR 反应液进行检测，在PCR反应的过程中，根据反应的检测探针荧光进行相应的荧光检测，此过程同常规qPCR一致。
可以理解的是，通过所述核酸提纯及检测反应液配制装置100与PCR仪器的结合，本申请实际还提供了一种全封闭式核酸提取及扩增的qPCR检测装置，配备了所需的核酸提取和检测试剂，样本添加后，实现全封闭自动化处理，实现样本进结果出的全自动化检测过程，检测效率高，而且全过程封闭，有利于确保检测的准确性和安全性。
本申请的所述核酸提纯及检测反应液配制装置100适合多种临床样本，如尿液、血液、鼻咽拭子、痰液、组织、粪便等样本的检测，可以根据具体的样本调整试剂配方及增减处理步骤。
本申请的所述核酸提纯及检测反应液配制装置100采用硅胶套的方式实现了常规磁棒法提取核酸的密闭性操作，是一种全封闭式的核酸检测试剂卡盒，密闭性非常好，使用过程中不与外部空气连通，能极大地避免外部环境对检测结果的影响，也能保护检测人员的健康。
本申请的所述核酸提纯及检测反应液配制装置100通过可旋转式主体的设计以及硅胶套与磁套提取法的结合，能够在全封闭式环境下实现样本裂解、核酸吸附、核酸清洗、核酸洗脱以及检测反应液的全流程操作，而且该装置整体结构简单，操作简单，可以兼容多种形式的样本。
本申请的试剂储存层20包括多个液池22，可以根据不同样本的核酸提纯和反应液配制需求设置液池的处理液类型，可以兼容多种形式的样本的核酸提纯及检测反应液配制需求。
本申请通过旋转式主体的设计，能够缩减流路开关控制阀门的设置，通过转动试剂储存主体21结构，实现磁珠在不同储液腔的转移，提供了一种结构 简单、操作简单的样本的核酸提纯及检测反应液配制装置100。
本申请的所述核酸提纯及检测反应液配制装置100将洗脱液和检测试剂冻干球放置在一个液池内，解决了检测反应液配制过程中需要将核酸洗脱液转移的操作，这样可以省去该装置的液路，避免了液体控制的操作。
本申请采用冻干球的方式保存检测试剂，并在检测试剂冻干球的外表面包裹一层石蜡，从而确保检测试剂冻干球可以与核酸洗脱液放置在一个液池内，解决了检测试剂和核酸洗脱液共存的问题。
本申请的所述核酸提纯及检测反应液配制装置100采用注塑成型的方式实现制备，可以实现批量化生产，具有批间差小，产量高，成本低的优势。
以上实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上实施例仅表达了本申请的优选的实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (16)

  1. 一种核酸提纯及检测反应液配制装置，其特征在于，包括：
    所述上盖层包括上盖主体和间隔设置于所述上盖主体的样本密封盖和硅胶套；和
    试剂储存层，所述试剂储存层可转动地设置在所述上盖层之下，包括试剂储存主体和间隔设置于所述试剂储存层的多个液池，所述液池用于储存对样本核酸进行处理的处理液；
    其中所述样本密封盖用于向所述试剂储存层的所述裂解液池投送样本并保持所述试剂储存层的密封性；所述硅胶套为具有弹性的伸缩管状结构，用于上下伸缩来与插入的磁棒和磁棒套配合实现吸附有核酸的磁珠在对应液池中的吸引和转移，从而实现全封闭式的核酸提纯处理和核酸检测反应液的配制。
  2. 根据权利要求1所述的核酸提纯及检测反应液配制装置，其特征在于，所述试剂储存层与所述上盖层之间采用传动卡齿的结构形成可转动连接；所述试剂储存层的每次转动角度范围为15～90°。
  3. 根据权利要求2所述的核酸提纯及检测反应液配制装置，其特征在于，所述上盖层还包括延伸自所述上盖主体的具有中心槽的凸部，所述硅胶套安装于所述凸部的中心槽内；所述试剂储存主体具有圆柱状结构和延伸自所述圆柱状结构的柱状结构，所述柱状结构的直径小于所述圆柱状结构，所述柱状结构与所述液池一一对应。
  4. 根据权利要求3所述的核酸提纯及检测反应液配制装置，其特征在于，多个所述液池包括用于储存吸附核酸的磁珠的磁珠保存液池、用于储存样本裂解液的裂解液池、用于储存核酸清洗液的清洗液池和用于储存核酸洗脱液与由石蜡包裹的检测试剂冻干球的洗脱液及检测试剂保存液池；所述清洗液池为三 个，包括依次设置的第一清洗液池、第二清洗液池以及第三清洗液池。
  5. 根据权利要求4所述的核酸提纯及检测反应液配制装置，其特征在于，其中所述磁珠保存液池、所述裂解液池、所述第一清洗液池、所述第二清洗液池、所述第三清洗液池和所述洗脱液及检测试剂保存液池依次沿所述试剂储存层的周向均匀间隔设置。
  6. 根据权利要求1至5中任一项所述的核酸提纯及检测反应液配制装置，其特征在于，所述上盖层和所述试剂储存层之间设置有密封硅胶垫。
  7. 根据权利要求1至5中任一项所述的核酸提纯及检测反应液配制装置，其特征在于，所述上盖主体和所述试剂储存层均通过注塑成型的方式制备得到，所述核酸提纯及检测反应液配制装置的整体长度为35mm，宽度为35mm，高度为43mm。
  8. 根据权利要求1至5中任一项所述的核酸提纯及检测反应液配制装置，其特征在于，所述样本为痰液、组织、血液或粪便；所述检测试剂冻干球中的检测试剂为PCR、RPA或LAMP试剂。
  9. 一种核酸提纯及检测反应液配制方法，其特征在于，包括采用根据权利要求1至8中任一项所述的核酸提纯及检测反应液配制装置执行的以下步骤：
    S1、打开样本密封盖在裂解液池中投送样本，实现样本裂解；
    S2、在上盖层的硅胶套中插入磁棒和磁棒套，通过磁棒将磁珠保存液中的磁珠吸引在硅胶套上；
    S3、依次按照预设角度转动所述试剂储存层，使得所述硅胶套依次与所述试剂储存层的对应液池对齐，并在所述硅胶套每次与对应的液池对齐时，通过从磁棒套中抽离磁棒的方式，实现磁珠在对应的液池中的释放，然后再次通过插入磁棒并带动所述硅胶套伸入对应的液池中的方式，实现对磁珠的吸引，然后以此通过重复对磁珠的释放和吸引操作，依次实现磁珠对应液池中的转移， 从而依次实现磁珠对样本核酸的吸附、核酸清洗、核酸洗脱以及核酸检测反应液制备的过程。
  10. 根据权利要求9所述的核酸提纯及检测反应液配制方法，其特征在于，所述试剂储存层的每次转动角度设置为15～90°。
  11. 根据权利要求10所述的核酸提纯及检测反应液配制方法，其特征在于，所述步骤S2具体包括步骤：
    S21、将磁棒及磁棒套插入上盖层的硅胶套中，通过磁棒和磁棒套带动所述硅胶套向下伸缩进入磁珠保存液池中，将磁珠吸引到所述硅胶套的顶部，完成吸引后，将磁棒及磁棒套往上运动至所述硅胶套的初始位置状态。
  12. 根据权利要求11所述的核酸提纯及检测反应液配制方法，其特征在于，所述试剂储存层还包括用于储存样本裂解液的裂解液池、用于储存核酸清洗液的清洗液池和用于储存核酸洗脱液与由石蜡包裹的检测试剂冻干球的洗脱液及检测试剂保存液池，所述清洗液池包括依次设置的第一清洗液池、第二清洗液池以及第三清洗液池；步骤S3包括步骤：
    S31、将所述试剂储存层转动60°，使得所述硅胶套位于所述裂解液池正上方，将磁棒和磁棒套带动所述硅胶套向下伸缩进入所述裂解液池中，将磁棒抽离磁棒套，使得磁场消失而使得所述硅胶套吸附的磁珠释放进所述裂解液池中，通过磁珠吸附所述裂解液池中的核酸；
    S32、再次将磁棒插入磁棒套并带动所述硅胶套伸入所述裂解液池，将所述裂解液池中的吸附有核酸的磁珠吸引到所述硅胶套的顶部，完成吸引后，将磁棒及磁棒套往上运动至所述硅胶套的初始位置状态；
    S33、将所述试剂储存层转动60°，使得插有磁棒及磁棒套的所述硅胶套位于第一清洗液池正上方，将磁棒和磁棒套带动所述硅胶套向下伸缩进入所述第一清洗液池中，将磁棒抽离磁棒套，使得磁场消失而使得所述硅胶套吸附的磁 珠进入所述清洗液池中，实现核酸清洗；
    S34、再次将磁棒插入磁棒套并带动所述硅胶套伸入所述第一清洗液池，将清洗后的磁珠吸引到所述硅胶套的顶部，完成吸引后，将磁棒及磁棒套往上运动至所述硅胶套的初始位置状态；
    S35、按照与步骤S33和S34同样的操作，完成磁珠在所述第二清洗液池和所述第三清洗液池中的清洗过程；
    S36、将所述试剂储存层转动60°，使得所述硅胶套位于洗脱液及检测试剂保存液池正上方，再次通过磁棒和磁棒套带动所述硅胶套向下伸缩进入所述洗脱液及检测试剂保存液池中，将磁棒抽离磁棒套，使得磁场消失而使得所述硅胶套吸附的磁珠进入所述洗脱液及检测试剂保存液池中，实现核酸洗脱；
    S37、再次将磁棒插入磁棒套并带动所述硅胶套伸入所述洗脱液及检测试剂保存液池，将洗脱出核酸的磁珠吸引到所述硅胶套的顶部，完成吸引后，将磁棒及磁棒套往上运动至所述硅胶套的初始位置状态；
    S38、加热所述洗脱液及检测试剂保存液池，使得所述洗脱液及检测试剂保存液池中包裹检测试剂冻干球的石蜡溶解，溶解后的石蜡漂浮到所述洗脱液及检测试剂保存液池最上层，检测试剂冻干球溶解到核酸洗脱液中对核酸进行处理，以此完成检测反应液的配制。
  13. 根据权利要求12所述的核酸提纯及检测反应液配制方法，其特征在于，步骤S1还包括步骤：在样本裂解时，还对所述裂解液池进行加热和超声处理，样本裂解时间为1～5min。
  14. 根据权利要求12所述的核酸提纯及检测反应液配制方法，其特征在于，在步骤S31中，核酸吸附过程的时间为1～10min。
  15. 根据权利要求12所述的核酸提纯及检测反应液配制方法，其特征在于，磁珠在所述第一清洗液池、所述第二清洗液池以及所述第三清洗液池中的 清洗时间分别为1～2min，磁珠在在所述第一清洗液池、所述第二清洗液池以及所述第三清洗液池中清洗后干燥1～5min。
  16. 根据权利要求12所述的核酸提纯及检测反应液配制方法，其特征在于，在步骤S36中，核酸洗脱过程的时间为1～5min。
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