WO2022088472A1 - Poste de travail intégré pour la détection des acides nucléiques - Google Patents

Poste de travail intégré pour la détection des acides nucléiques Download PDF

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Publication number
WO2022088472A1
WO2022088472A1 PCT/CN2020/140654 CN2020140654W WO2022088472A1 WO 2022088472 A1 WO2022088472 A1 WO 2022088472A1 CN 2020140654 W CN2020140654 W CN 2020140654W WO 2022088472 A1 WO2022088472 A1 WO 2022088472A1
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WIPO (PCT)
Prior art keywords
nucleic acid
assembly
tube
detection
sample
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PCT/CN2020/140654
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English (en)
Chinese (zh)
Inventor
陈华云
杨迎宾
邹天桥
薛儒冰
肖湘文
刘淑园
文素珍
曾烨
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广州和实生物技术有限公司
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Publication of WO2022088472A1 publication Critical patent/WO2022088472A1/fr

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    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples

Definitions

  • the present application relates to the technical field of molecular diagnosis, for example, to an integrated nucleic acid detection workstation.
  • Biological sample pretreatment, nucleic acid extraction and purification, and nucleic acid amplification detection are important experimental methods in molecular biology research.
  • Traditional experimental methods are performed manually or by machines in stages, including but not limited to sample pretreatment, Nucleic acid extraction and purification, preparation and distribution of nucleic acid amplification reagents, sample addition of nucleic acid solutions, sealing of amplification reagent tubes, completion of detection in a fluorescence PCR instrument or other chemical reaction instruments that can be used for nucleic acid amplification, and then results in other ways
  • Analysis and interpretation are time-consuming, prone to biological sample contamination or amplification product contamination, and experimental results are easily affected by individual differences in the operation of the experimenter, resulting in unreliable experimental results and serious false positives and false negatives. Influence the practical application of molecular detection technology.
  • the instruments and equipment for nucleic acid extraction, amplification and detection commercially used in diagnosis are all discrete, such as sample preparation instruments for nucleic acid extraction, nucleic acid extraction instruments and quantitative PCR instruments for amplification and detection. Or other nucleic acid amplification detectors, etc.
  • the specific process is to manually add the sample to the automatic nucleic acid extraction instrument, use the automatic nucleic acid extraction instrument to extract and purify the nucleic acid of the biological sample, manually transfer it to the nucleic acid amplification detection instrument, and use the nucleic acid amplification detection instrument to amplify and detect the extract. , all the transfer operations in the middle need to be done manually by the experimenter.
  • the present application provides an integrated nucleic acid detection workstation, which can integrate sample pretreatment, nucleic acid extraction and purification, amplification and detection into one, and realize automated and efficient nucleic acid detection.
  • An embodiment provides an integrated nucleic acid detection workstation, including a housing and a workstation body disposed inside the housing, where the workstation body includes a reagent rack assembly, consumables, a robotic arm assembly, a magnetic frame assembly, and a fluorescence detection assembly.
  • the consumable includes a plurality of test tubes, and several of the test tubes are used as carriers for processing the samples.
  • the reagent rack assembly includes a consumables support for placing the consumables and a temperature control unit, the temperature control unit is arranged below the consumables support, and the temperature control unit is arranged to provide the sample with the sample during sample processing. required temperature.
  • the robotic arm assembly is disposed above the reagent rack assembly, and the robotic arm assembly includes a movably arranged pipetting head, and the pipetting head is configured to suck liquid and mix liquid by blowing and beating during sample processing.
  • the magnetic frame assembly is configured to extract and separate nucleic acid from the sample.
  • the fluorescence detection component includes a movably arranged detection unit, and the detection unit is configured to perform fluorescence detection and analysis on the amplified nucleic acid.
  • FIG. 1 is a schematic perspective view of an integrated nucleic acid detection workstation according to an embodiment of the application
  • FIG. 2 is a schematic side view of a consumable according to an embodiment of the application.
  • FIG. 3 is one of the three-dimensional schematic diagrams of the workstation body according to an embodiment of the application.
  • FIG. 4 is the second three-dimensional schematic diagram of the workstation body according to an embodiment of the application.
  • Fig. 5 is an exploded view of the workstation body in Fig. 1;
  • FIG. 6 is a schematic perspective view of an amplification tube capping assembly according to an embodiment of the present application.
  • FIG. 7 is a schematic perspective view of a pipetting head according to an embodiment of the application.
  • Fig. 8 is the three-dimensional schematic diagram of the pipetting head body in Fig. 7;
  • FIG. 9 is a schematic cross-sectional view of a guide sleeve according to an embodiment of the application.
  • FIG. 10 is a three-dimensional schematic diagram of a magnetic stand assembly according to an embodiment of the application.
  • FIG. 11 is one of the three-dimensional schematic diagrams of the reagent rack assembly according to an embodiment of the application.
  • Figure 12 is an exploded view of the reagent rack assembly of Figure 11;
  • FIG. 13 is the second schematic perspective view of the reagent rack assembly according to an embodiment of the application.
  • FIG. 14 is a schematic perspective view of a fluorescence detection assembly according to an embodiment of the present application.
  • Robot arm assembly 41, pipette head; 411, protective shell; 412, pipette head body; 4121, tip rod; 4122, fixing bracket; 4123, guide sleeve; 41231, through hole; 4124, second motor ; 4125, screw; 4126, nut; 42, syringe pump; 43, the first driver; 44, the second driver;
  • Magnetic frame assembly 51, Support plate; 52, Lifting unit; 53, Magnet holder; 54, Magnet; 6, Fluorescence detection assembly; 61, Detection unit; 62, Slider; 63, Guide rail; 64, Drive unit 7. Workstation base; 71. Bottom plate; 72. Main frame; 8. Battery; 9. Amplification tube cover assembly; 91. The first motor; Two fixed seat; 95, pressing block.
  • a first feature "on” or “under” a second feature may include the first and second features in direct contact, or may include that the first and second features are not Direct contact but through additional features between them.
  • a first feature being "above” a second feature includes that the first feature is directly above and obliquely above the second feature, or simply means that the first feature is at a higher level than the second feature.
  • the first feature being “below” the second feature includes that the first feature is directly below and diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.
  • first, second, etc. are only used for the purpose of description, and should not be construed as indicating or implying relative importance or implying that Indicates the number of technical characteristics.
  • a feature defined as “first” or “second” may expressly or implicitly include one or more of that feature.
  • the integrated nucleic acid detection workstation provided by this application utilizes physical and chemical means in related technologies to realize "sample in and result out” through a variety of automated tools, and realizes all molecular diagnosis processes in one instrument and equipment, which is the current leader in the field of nucleic acid detection focus.
  • the integrated nucleic acid detection workstation includes a housing 1 and a workstation body disposed inside the housing 1 .
  • the workstation body includes a reagent rack assembly 2 , consumables 3 , a robotic arm assembly 4 , and a magnetic stand Component 5 and fluorescence detection component 6.
  • the consumables 3 can be placed on the reagent rack assembly 2, and the consumables 3 include a plurality of test tubes, and the plurality of test tubes are used as carriers for processing samples.
  • the reagent rack assembly 2 includes a temperature control unit, which provides the required temperature for sample processing.
  • the robotic arm assembly 4 includes a movably arranged pipetting head 41, and the pipetting head 41 is configured to suck liquid and mix the liquid during sample processing; the magnetic frame assembly 5 is configured to Extraction and separation are performed; the fluorescence detection component 6 is configured to perform fluorescence detection and analysis on the amplified nucleic acid.
  • the integrated nucleic acid detection workstation of the present application is provided with a reagent rack assembly 2, consumables 3, a robotic arm assembly 4, a magnetic frame assembly 5 and a fluorescence detection assembly 6 in the casing 1.
  • the consumables 3 include a plurality of test tubes, and the plurality of test tubes are used as a pair of samples.
  • the carrier for processing includes sample pretreatment, nucleic acid extraction and purification of the sample.
  • Consumables 3 can be selected in the form of excluding nucleic acid amplification tubes.
  • the nucleic acid amplification tubes configured in the detection kit are directly used for nucleic acid amplification.
  • the temperature control unit in the reagent rack assembly 2 provides the required temperature for the nucleic acid extraction, purification and nucleic acid amplification process of the sample, and the pipette head 41 movably set in the robotic arm assembly 4 can be used for multiple After the nucleic acid amplification is completed, the fluorescence detection component 6 can perform fluorescence detection and analysis. Therefore, the operator only needs to remove the consumables 3 containing the samples. After the nucleic acid amplification tube and the nucleic acid amplification tube are placed in the reagent rack assembly 2, the entire nucleic acid detection process will be carried out automatically inside the shell 1 without manual operation, which not only greatly improves the efficiency of nucleic acid detection, but also reduces the risk of contamination and improves nucleic acid detection. accuracy.
  • the workstation body further includes a workstation base 7 and a battery 8 , and the reagent rack assembly 2 , the robotic arm assembly 4 , the magnetic frame assembly 5 and the fluorescence detection assembly 6 are all mounted on the workstation base 7 .
  • the workstation base 7 includes a base plate 71 and a main frame 72 disposed on the base plate 71
  • the reagent rack assembly 2 is disposed on the main frame 72 and is fixedly connected to the base plate 71
  • the robotic arm assembly 4 is disposed on the main frame 72 .
  • the magnetic frame assembly 5 and the fluorescence detection assembly 6 are both arranged on the bottom plate 71 .
  • the battery 8 is fixed on the bottom plate 71, so that the integrated nucleic acid detection workstation of this embodiment can operate without plugging in.
  • the plurality of test tubes include a sample tube 31 , a reagent tube 32 and a reaction tube 33 .
  • the reagent tube 32 contains reagents such as proteinase K and magnetic beads, and the sample passes through the pipette head 41 from the sample tube. 31 is transferred to the reagent tube 32 for lysis, and then the mixed solution containing proteinase K, magnetic beads and samples is transferred to the reaction tube 33 containing the magnetic bead binding solution, so that the magnetic beads are combined with free nucleic acid, nucleic acid. After purification and elution, the magnetic beads are separated from the magnetic beads by the magnetic rack assembly 5 and transferred to the nucleic acid amplification tube for amplification.
  • the temperature control unit in the reagent rack assembly 2 heats or cools the reaction tube 33 and the nucleic acid amplification tube.
  • the fluorescence detection component 6 performs fluorescence detection and analysis on the amplified nucleic acid in the nucleic acid amplification tube.
  • the entire processing process of the sample is automatically completed by means of the pipetting head 41 in various test tubes of the consumables 3 placed on the reagent rack assembly 2 . The whole process is efficient and convenient, and is suitable for clinical testing.
  • the integrated nucleic acid detection workstation further includes an amplification tube capping assembly 9, and the amplification tube capping assembly 9 includes a first motor 91, a rotating shaft 92, a first fixing seat 93, The second fixing seat 94 and the plurality of pressing blocks 95, the two ends of the rotating shaft 92 are respectively connected with the first fixing seat 93 and the second fixing seat 94, and the first fixing seat 93 and the second fixing seat 94 are arranged on the reagent rack assembly 2
  • a plurality of pressing blocks 95 are arranged on the rotating shaft 92, and the first motor 91 is arranged on one end of the rotating shaft 92 and can drive the rotating shaft 92 to rotate, so that the pressing blocks 95 selectively seal the nucleic acid amplification tube.
  • nucleic acid aerosols are tiny water droplets containing nucleic acid fragments of different lengths, and these tiny droplets settle. Into other samples can lead to aerosol contamination, resulting in inaccurate test results.
  • the amplification tube capping assembly 9 seals the nucleic acid amplification tube when the pipetting operation of the nucleic acid amplification tube is not required, thereby greatly reducing the possibility of aerosol contamination and improving the accuracy of the detection result.
  • the consumable 3 further includes a base plate 34 and a plurality of suction heads 35 .
  • the base plate 34 is provided with a plurality of suction head placement grooves 36 , a waste liquid groove 37 and a washing liquid pipe 38 .
  • the suction head 35 is placed in the suction head placement groove 36, and the waste liquid groove 37 is arranged close to the reaction tube 33 and is spaced from the reaction tube 33.
  • the consumable 3 in this embodiment adopts an integrated structure, that is, the sample tube 31, the reagent tube 32, and the reaction tube. 33.
  • the base plate 34, the suction head placement groove 36, the waste liquid groove 37 and the washing liquid tube 38 are integrally formed, and many types of test tubes will not shake or fall, which is convenient for daily use.
  • the pipette head 41 includes a plurality of pipette rods 4121 that can move in a vertical direction.
  • the pipette rods 4121 are configured to be inserted into the pipette tips 35 in the pipette tip placement grooves 36 , and the washing liquid tube 38 is installed inside.
  • There is a washing solution or an eluent and the washing solution tube 38 is set for the purification of nucleic acid and the separation of magnetic beads and nucleic acid. Setting the waste liquid tank 37 close to the reaction tube 33 can reduce the moving distance when the pipette head 41 discharges the waste liquid in the reaction tube 33.
  • the groove 37 is spaced from other test tubes to provide enough space for the magnetic stand assembly 5 to move.
  • the suction head placement groove 36 allows the suction heads 35 to be placed neatly, which is convenient for the suction rod 4121 to insert the suction head 35.
  • a sealing film can also be plastic-sealed on the base plate 34 , and the film is a plastic film, an aluminum film or a paper film, which is used for sealing and transportation protection of the reagent loaded in the consumables 3 .
  • the pipette head 41 includes a protective shell 411 and a pipette head body 412 , and the pipette head body 412 includes a fixing bracket 4122 and a guide sleeve in addition to the tip rod 4121 .
  • the second motor 4124, the lead screw 4125 and the nut 4126, the guide sleeve 4123 and the second motor 4124 are arranged on the fixing bracket 4122, the lead screw 4125 is connected with the second motor 4124 and can rotate under the driving of the second motor 4124,
  • the screw rod 4125 is set vertically, the nut 4126 is sleeved on the screw rod 4125 and can move along the screw rod 4125, the first end of the suction rod 4121 is fixedly connected with the nut 4126, and the guide sleeve 4123 is provided with a plurality of suction head rods
  • the second end of the 4121 is inserted into a through hole 41231 that moves up and down.
  • the through hole 41231 is a stepped hole, and the through hole 41231 can limit the suction head 35 .
  • the transmission mode of the ball screw has the characteristics of high precision and stability, so the precise positioning of the tip rod 4121 can be achieved.
  • the through hole 41231 opened in the guide sleeve 4123 is a stepped hole.
  • the robotic arm assembly 4 further includes a syringe pump 42 , a first driving member 43 and a second driving member 44 , and one end of the plurality of suction rods 4121 away from the guide sleeve 4123 is connected to
  • the syringe pump 42 is connected by a hose (not shown in the figure)
  • the first driving member 43 is connected with the pipetting head 41
  • the first driving member 43 makes the pipetting head 41 move in the first direction in the horizontal plane
  • the first driving The second driving member 44 is arranged on the second driving member 44
  • the second driving member 44 moves the first driving member 43 in a second direction in the horizontal plane, and the first direction is perpendicular to the second direction.
  • the robotic arm assembly 4 is also provided with a number of sensors (not shown in the figure), so as to accurately monitor the distance and direction of the displacement, and ensure that the tip rod 4121 is always in place. be in the correct position.
  • the magnetic stand assembly 5 includes a support plate 51 , a lifting unit 52 , a magnet fixing seat 53 and a plurality of magnets 54 , the lifting unit 52 is arranged on the supporting plate 51 , and the magnet fixing seat 53 is connected to the support plate 51 .
  • the lifting unit 52 is connected, and a plurality of magnets 54 are arranged on the magnet holder 53 at intervals.
  • the magnet holder 53 is set in a relatively long and narrow shape, which can be smoothly inserted into the side of the reaction tube 33, so that the magnets 54 arranged on the magnet holder 53 can adsorb the magnetic beads in the reaction tube 33.
  • the reaction tube 33 usually has more The number of magnets 54 is arranged at intervals, and each magnet 54 corresponds to one reaction tube 33, which can ensure a better magnetic bead adsorption effect.
  • the elevating unit 52 can also adopt a ball screw driving mode similar to that in the pipetting head 41 , so as to realize the precise movement of the position of the magnet 54 .
  • the reagent rack assembly 2 further includes a plurality of consumables supports 21 , a positioning plate 22 , a plurality of fans 23 and a fixing plate 24 , and the consumables 3 are placed on the consumables support 21 .
  • the bracket 21 is placed on the positioning plate 22, the plurality of fans 23, the positioning plate 22 and the temperature control unit are all connected to the fixing plate 24, the fixing plate 24 is fixed on the bottom plate 71 by a plurality of fixing rods 26, and the temperature control unit includes a first temperature control unit.
  • the control 251 and the second temperature control 252, the first temperature control 251 is set to heat the reaction tube 33, the second temperature control 252 is set to heat or cool the nucleic acid amplification tube, and the second temperature control 252 includes a plurality of semiconductors
  • the cooling fin 2521, the plurality of heat sinks 2522, and the plurality of fans 23 are arranged to assist in cooling the second temperature control 252 and the nucleic acid amplification tube.
  • the consumables holder 21 can be removed from the positioning plate 22 as a whole, and the consumables holder 21 is provided with an opening 211 which is convenient for human hands to grasp, which facilitates the insertion and removal of the consumables holder 21 and the consumables 3 placed on the consumables holder 21 .
  • the nucleic acid amplification reaction needs to be heated first and then cooled down, and there are certain requirements for the cooling rate. Therefore, the nucleic acid amplification tube is heated or cooled by using a semiconductor cooling chip 2521 that can be heated and cooled, and several heat sinks 2522 and 2522 are used to heat or cool the nucleic acid amplification tube. Several fans 23 perform rapid cooling.
  • the fluorescence detection assembly 6 includes two detection units 61 , a slider 62 , a guide rail 63 and a drive unit 64 .
  • the detection unit 61 is disposed on the slider 62 , and the drive unit 64 can drive the slider.
  • the block 62 reciprocates linearly along the guide rail 63, so that the detection unit 61 is configured to emit excitation light and collect reflected light, so as to perform fluorescence detection on the nucleic acid amplification tube.
  • the number of detection units 61 is at least one.
  • the fluorescence detection assembly 6 of this embodiment includes two detection units 61, so two different nucleic acid amplification tubes can be detected at the same time.
  • the driving unit 64 can also adopt a transmission mode similar to the ball screw in the pipetting head 41 to realize the stability of the moving process of the detection unit 61 .
  • a ventilation filter device (not shown in the figure) and an ultraviolet lamp disinfection device (not shown in the figure) are also provided inside the casing 1 .
  • the exhaust filter device can play the role of heat dissipation and ventilation, and at the same time can prevent external pollutants from entering the interior of the housing 1, and the ultraviolet lamp disinfection device can reduce or prevent the contamination of at least one of biological samples and nucleic acids during the detection process.
  • the manual operation part includes:
  • Automated operations include:
  • nucleic acid extraction is performed, including the following steps:
  • the pressing block 95 is opened, the nucleic acid amplification tube is opened, the quantitative nucleic acid is taken from the reaction tube 33 into the nucleic acid amplification tube, and pipetting is repeated, and then the nucleic acid amplification detection step is performed.
  • Nucleic acid amplification detection includes the following steps:
  • the amplification tube capping assembly 9 is activated to seal the nucleic acid amplification tube, and the second temperature control 252 starts to heat to provide the temperature required for the nucleic acid amplification reaction to ensure that the nucleic acid reaction proceeds;
  • the detection unit 61 in the fluorescence detection assembly 6 sequentially detects a plurality of nucleic acid amplification tubes, and obtains detection results through real-time fluorescent signals.
  • the UV lamp is automatically turned on for UV disinfection.
  • This detection step imitates the manual operation process of human beings, which integrates extraction, amplification and detection. The operator only needs to perform simple preparation actions to complete the entire detection process and obtain the results. Convenient features, suitable for clinical testing.

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Abstract

Poste de travail intégré pour la détection d'acides nucléiques, comprenant un boîtier et un corps de poste de travail logé à l'intérieur du boîtier. Le corps de poste de travail comprend un ensemble porte-réactif, des produits consommables, un ensemble bras robotique, un ensemble cadre magnétique et un ensemble de détection par fluorescence. Les produits de consommation comprennent une pluralité de tubes de test utilisés en tant que supports pour traiter des échantillons. L'ensemble porte-réactif comprend une unité de régulation de température configurée pour fournir la température requise pour les échantillons pendant le traitement d'échantillon. L'ensemble bras robotique comprend une pipette mobile configurée pour aspirer et mélanger un liquide pendant le traitement d'échantillon. L'ensemble cadre magnétique est conçu pour extraire et séparer l'acide nucléique des échantillons. L'ensemble de détection par fluorescence est configuré pour effectuer une analyse de détection par fluorescence sur l'acide nucléique amplifié.
PCT/CN2020/140654 2020-10-27 2020-12-29 Poste de travail intégré pour la détection des acides nucléiques WO2022088472A1 (fr)

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CN202011166955.6A CN112175809A (zh) 2020-10-27 2020-10-27 一体化核酸检测工作站
CN202011166955.6 2020-10-27

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