CN115197833A - Automatic warehouse building instrument and method for step sampling - Google Patents
Automatic warehouse building instrument and method for step sampling Download PDFInfo
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- CN115197833A CN115197833A CN202211009479.6A CN202211009479A CN115197833A CN 115197833 A CN115197833 A CN 115197833A CN 202211009479 A CN202211009479 A CN 202211009479A CN 115197833 A CN115197833 A CN 115197833A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
- C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
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Abstract
The invention discloses an automatic warehouse building instrument and a warehouse building method for step sampling, which comprise a refrigeration module, a thermal cycle module, a normal temperature reagent storage module, a gun head module, a heating oscillation module, an optical detection module, a magnetic suction module and a mechanical moving module; the gun head module comprises a plurality of gun head frames which can be stacked up and down, a plurality of gun head holes for storing gun heads are arranged on the gun head frames, the gun head frames are provided with positioning connection mechanisms, and the upper gun head frame and the lower gun head frame are connected through the positioning connection mechanisms; the mechanical moving module is arranged on the moving guide rail component, the moving guide rail component drives the mechanical moving module to move along the X-axis direction and the Y-axis direction, and the mechanical moving module is provided with a pipettor capable of moving up and down and a manipulator component capable of moving up and down. The invention can replace manual work to realize automatic warehouse building operation, saves time and labor and has high operation efficiency.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an automatic warehouse building instrument and a warehouse building method for step sampling.
Background
The automatic storehouse building instrument is generally used for reagent refrigeration in a second-generation storehouse building, automatic liquid transfer, sample thermal circulation, heating oscillation, magnetic absorption purification and other library preparation works, and has the advantages of high flux, semi-automatic or full-automatic performance, man-machine interaction in intermediate process alternation and the like. The application of nanopore sequencing technology is more and more extensive, in order to obtain complete nucleic acid information, a library establishment mode aiming at long-fragment nucleic acid sequencing is mainly based on manual experiment library establishment at present, and a scientific research worker needs to repeatedly operate for a long time in the experiment process to obtain a high-quality sequencing library.
The existing main defects are as follows: the existing automatic library building instrument is a short-fragment processing automatic working machine developed based on a second-generation NGS library building scheme, and cannot be suitable for library building of nanopore single-molecule long-fragment sequencing, so that the library building work of nanopore single-molecule long-fragment sequencing is mainly finished manually, the automatic operation cannot be realized, the workload of personnel is large, and the efficiency is low.
Disclosure of Invention
The invention aims to provide an automatic library building instrument and a library building method for step sampling, which can simulate a manual library building mode, realize the automatic construction of a nanopore single-molecule long-fragment sequencing library, reduce the workload of personnel and improve the operation efficiency.
The purpose of the invention is realized by the following technical scheme: an automatic warehouse building instrument for stepped sampling comprises a refrigeration module, a thermal cycle module, a normal temperature reagent storage module, a gun head module, a heating oscillation module, an optical detection module, a magnetic suction module and a mechanical movement module;
the gun head module comprises a plurality of gun head frames which can be stacked up and down, a plurality of gun head holes for storing gun heads are arranged on the gun head frames, the gun head frames are provided with positioning connection mechanisms, and the upper gun head frame and the lower gun head frame are connected through the positioning connection mechanisms;
the mechanical movement module is arranged on the movement guide rail component, the movement guide rail component drives the mechanical movement module to move along the X-axis direction and the Y-axis direction, and a pipettor capable of moving up and down and a manipulator component capable of moving up and down are arranged on the mechanical movement module.
Preferably, the refrigeration module, the thermal cycle module and the normal temperature reagent storage module are arranged on one side of the gun head module, and the heating oscillation module, the magnetic suction module and the optical detection module are arranged on the other side of the gun head module.
Preferably, the magnetic suction module comprises a supporting seat, a PCR plate bracket and a magnetic column bottom plate support, wherein the PCR plate bracket is arranged on the supporting seat, a lifting driving device for driving the magnetic column bottom plate support to move up and down is arranged on the PCR plate bracket, a magnetic column fixing plate is arranged on the magnetic column bottom plate support, and a plurality of magnetic columns are arranged on the magnetic column fixing plate; positioning grooves are arranged on two sides of the upper end of the PCR plate bracket; when the PCR plate is placed on the PCR plate bracket, the two sides of the PCR plate are clamped into the positioning grooves on the PCR plate bracket, and the magnetic column fixing plate is positioned below the PCR plate.
Preferably, the positioning connection mechanism comprises a positioning sleeve arranged on the upper side of the gun head frame and a positioning inserted rod arranged below the gun head frame and corresponding to the positioning sleeve, and when the gun head frames are vertically stacked, the positioning inserted rod on the gun head frame above is inserted into the positioning sleeve on the gun head frame below.
Preferably, the mechanical movement module comprises a mounting base plate, the mounting base plate is connected with the movable guide rail part, a lifting frame is connected onto the mounting base plate in a sliding manner, and a lifting driving mechanism for driving the lifting frame to move is arranged on the mounting base plate; the pipettor and the manipulator component are arranged on the lifting frame, and the lifting frame is also provided with a pipettor lifting driving device for driving the pipettor to move up and down and a manipulator component lifting driving device for driving the manipulator component to move up and down.
Preferably, the lifting driving mechanism comprises a lifting driving motor and a first guide rail, the lifting driving motor is arranged on the mounting base plate, the first guide rail is vertically arranged on the mounting base plate, a first sliding block is connected to the first guide rail, and the lifting frame is arranged on the first sliding block; the mounting base plate is rotatably connected with a screw rod, the screw rod is parallel to the first guide rail and is in transmission connection with a lifting driving motor, a threaded hole corresponding to the screw rod is formed in the lifting frame, and the screw rod is in threaded connection with the threaded hole.
Preferably, the lifting frame is provided with a fixed plate, the fixed plate is provided with a second guide rail, the second guide rail is arranged along the vertical direction, the second guide rail is connected with a second sliding block, and the manipulator part is arranged on the second sliding block; but the manipulator part lift drive is equipped with vertical mobile's first output shaft on, and the manipulator part is connected to the one end of first output shaft.
Preferably, the pipettor lifting driving device is provided with a second output shaft capable of vertically moving, and one end of the second output shaft is connected with the pipettor.
The library building method adopts the automatic library building instrument for stepped sampling to realize the automatic library building of a nanopore monomolecular long fragment sequencing platform, covers long fragment amplicons and builds a library by gene composition, and comprises the following steps:
placing samples with unknown concentration in 1-3 rows of areas of a PCR plate, and moving the unknown samples to an optical detection module to obtain the initial concentration of the unknown samples;
separating the terminal repair enzyme premix liquid into 4-6 rows of areas of a PCR plate of a thermal cycle module, transferring a set volume of unknown samples from 1-3 rows of areas based on the initial concentration, adding the unknown samples into 4-6 rows of areas to obtain a uniform mixing liquid, and incubating the uniform mixing liquid by regulating and controlling the temperature of the thermal cycle module; transferring a magnetic bead with a set volume from a normal-temperature reagent storage module, adding the magnetic bead into the incubated mixing liquid, transferring a PCR plate to a heating oscillation module, transferring the PCR plate to a magnetic absorption module after oscillation incubation is finished, and adding a cleaning reagent after supernatant is removed when the magnetic absorption module keeps a magnetic absorption state; keeping the magnetic attraction state, sucking the supernatant, adding a cleaning reagent repeatedly, and removing the supernatant again; keeping the magnetic attraction state, naturally drying for 30s, transferring the PCR plate to a heating oscillation module, adding a nucleic acid eluent on a normal-temperature reagent storage module for incubation, transferring the PCR plate again to the magnetic attraction module to separate magnetic beads, and transferring supernatant to the 7-9 rows of areas of the PCR plate;
absorbing nucleic acid molecular labels from the refrigeration module, adding the nucleic acid molecular labels into 7-9 rows of areas of the PCR plate, and adding T/A ligase premix liquid absorbed from the refrigeration module to obtain uniform mixing liquid; transferring the PCR plate to a thermal cycle module, then incubating at controlled temperature, transferring a set volume of magnetic beads from a normal-temperature reagent storage module, adding the magnetic beads into the incubated mixing liquid, transferring the PCR plate to a heating oscillation module, transferring the PCR plate to a magnetic absorption module after oscillation incubation is finished, removing supernatant when the magnetic absorption module keeps a magnetic absorption state, then adding a cleaning reagent, keeping the magnetic absorption state, absorbing the supernatant, and removing the supernatant again after the cleaning reagent is added repeatedly; keeping the magnetic attraction state, naturally drying for 30s, transferring the PCR plate to a heating and oscillating module, adding the nucleic acid eluent on the normal-temperature reagent storage module, incubating for 3 minutes, and transferring the PCR plate to the magnetic attraction module again to separate magnetic beads; sucking the separated samples to an optical detection area to detect the secondary concentration of the samples, calculating the volume of the mixed samples required by each sample based on the secondary concentration of the samples, and sucking each sample of the volume of the mixed samples to 10 rows of A hole areas of a PCR plate to realize high-flux mixed samples;
adding a sequencing joint with a specific volume, a fast ligase and a buffer solution into the hole A region of 10 rows of the PCR plate, uniformly mixing, transferring to a thermal cycle module for temperature-controlled incubation, transferring a set volume of magnetic beads from a normal-temperature reagent storage module, adding the incubated uniform mixing liquid, transferring the PCR plate to a heating oscillation module, transferring the PCR plate to a magnetic absorption module after oscillation incubation is finished, and removing a supernatant when the magnetic absorption module keeps a magnetic absorption state; transferring the PCR plate to a heating and oscillating module, adding a joint cleaning reagent sucked from a refrigerating module, uniformly mixing, transferring the PCR plate to a magnetic absorption module to separate a liquid phase from magnetic beads, and removing a supernatant; repeatedly transferring the PCR plate to the heating and oscillating module for one time, adding a joint cleaning reagent sucked from the refrigerating module, uniformly mixing, transferring the PCR plate to the magnetic absorption module to separate a liquid phase from magnetic beads, and removing a supernatant; and (3) keeping a magnetic attraction state, naturally air-drying for 30s, transferring the PCR plate to a heating oscillation module, adding a joint nucleic acid desorption solution absorbed from a refrigeration module, oscillating, uniformly mixing, incubating for 10 minutes, transferring the PCR plate to the magnetic attraction module to separate a liquid phase from magnetic beads, and absorbing supernatant to 10 rows of E hole areas of the PCR plate to obtain a qualified high-quality multi-sample library based on the nanopore single molecule sequencing platform.
The invention has the beneficial effects that: the invention can replace manual work to realize automatic warehouse building operation, saves time and labor and has high operation efficiency.
Drawings
FIG. 1 is a schematic structural diagram of the present invention.
Fig. 2 is a top view of the present invention.
Fig. 3 is a schematic structural diagram of the lance tip module.
Fig. 4 is a front view of the mechanical movement module.
Fig. 5 is a side view of the mechanical movement module.
Fig. 6 is a schematic view of the mechanical movement module mounting eight pipette tips.
Fig. 7 is a schematic view of the mechanical movement module mounting a single pipette tip.
FIG. 8 is a schematic view of the mechanical movement module grasping the PCR plate.
FIG. 9 is a schematic diagram of the mechanical movement module grasping the nucleic acid quantifying turntable.
Fig. 10 is a schematic diagram of the mechanical movement module grasping the headstock.
Fig. 11 is a schematic structural view of a heating oscillation module.
FIG. 12 is a schematic view of a magnetic module.
Fig. 13 is a front view of the magnetic attraction module.
FIG. 14 is a schematic diagram of one of the structures of the PCR plate.
FIG. 15 is another schematic diagram of the structure of the PCR plate.
FIG. 16 is a schematic view of the PCR plate placed on the magnetic attraction module and the magnetic force column near the PCR plate.
FIG. 17 is a schematic view of the PCR plate placed on the magnetic module and the magnetic columns away from the PCR plate.
FIG. 18 is a schematic structural diagram of an optical inspection module.
FIG. 19 is an electrophoretogram of different fragments.
FIG. 20 is a statistical graph of the distribution of lengths of the pooled sequenced fragments.
FIG. 21 is a statistical data of 1 hour of banking.
In the figure: 1. y-axis moving guide rails, 2X-axis moving guide rails, 3 mechanical moving modules, 4 refrigeration modules, 5 normal temperature reagent storage modules, 6 thermal cycle modules, 8 gun heads, 11 heating oscillation modules, 12 magnetic attraction modules, 13 optical detection modules, 14 waste consumable storage frames, 15 racks, 16 gun head frames, 17 positioning insertion rods, 18 positioning sleeves, 19 positioning sleeves, bases, 20 gun head holes, 21 mounting base plates, 22 first guide rails, 23 first sliding blocks, 24 lifting driving motors, 25 screw rods, 26 lifting frames, 27 pipettors, 28, a pipettor lifting drive device, 29, a gun head, 30, a manipulator component, 31, a fixing plate, 32, a second guide rail, 33, a second slide block, 34, a manipulator component lifting drive device, 35, a PCR plate, 36, a nucleic acid quantitative turntable, 40, a PCR plate heat conduction frame, 41, a bottom plate, 42, a support column, 43, a PCR plate bracket, 44, a linear motor, 45, a positioning groove, 46, a guide rod, 47, a sliding sleeve, 48, a magnetic column bottom plate bracket, 49, a magnetic column fixing plate, 50, a magnetic column, 52, a grabbing part, 53, a nucleic acid quantitative reagent storage frame, 54 and a quantitative turntable temporary suspension frame.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
It will be understood by those skilled in the art that in the present disclosure, the terms "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in an orientation or positional relationship indicated in the drawings for ease of description and simplicity of description, and do not indicate or imply that the referenced devices or components must be constructed and operated in a particular orientation and thus are not to be considered limiting.
It is understood that the terms "a" and "an" should be interpreted as meaning that a number of one element or element is one in one embodiment, while a number of other elements is one in another embodiment, and the terms "a" and "an" should not be interpreted as limiting the number.
As shown in fig. 1 to 18, an automatic warehouse building instrument for step sampling comprises a rack 15, a refrigeration module 4, a thermal cycle module 6, a normal temperature reagent storage module 5, a gun head module 8, a heating oscillation module 11, an optical detection module 13, a magnetic suction module 12, a mechanical movement module 3, and a waste consumable storage frame 14. Refrigeration module 4, thermal cycle module 6, normal atmospheric temperature reagent storage module 5 are the inline and set up in one side of rifle head module 8, and the heating is shaken module 11, is inhaled module 12 and optical detection module 13 and abandonment consumptive material and is deposited frame 14 and be located the opposite side of rifle head module 8.
The gun head module 8 comprises a plurality of gun head frames 16 which can be stacked up and down, and a plurality of gun head holes 20 for storing gun heads 29 are formed in the gun head frames 16. The gun head frame is provided with a positioning connecting mechanism, and the upper gun head frame and the lower gun head frame are connected through the positioning connecting mechanism. In this embodiment, each of the gun head frames 16 is provided with 96 gun head holes 20, and the gun head holes 20 are arranged on the gun head frame 16 in an 8-by-12 manner. The gun heads 29 are inserted into the gun head holes 20 on the gun head frames 16, and each gun head frame 16 can accommodate 96 gun heads 29.
The gun head frame 8 is provided with a positioning connection mechanism, and when the gun head frame 8 is vertically stacked, the upper gun head frame 6 and the lower gun head frame 6 are connected through the positioning connection mechanism. Specifically, the headstock 8 is provided with a positioning inserted bar 17 and a positioning sleeve 18 corresponding to the positioning inserted bar 17, and the positioning inserted bar 17 and the positioning sleeve 18 are respectively arranged on the upper side and the lower side of the headstock 8. In the application, each gun head frame 16 is provided with four positioning insertion rods 17 and four positioning sleeves 18, wherein the positioning sleeves 18 are arranged on the upper side of the gun head frame 16 and are positioned around the gun head frame 16; the positioning plunger 17 is located on the underside of the headstock 16 and around the headstock 16. When the gun head frame 16 is vertically stacked, the positioning inserted rod 17 on the upper gun head frame 16 is inserted into the positioning sleeve 18 on the lower gun head frame 16, so that the gun head frame is stably stacked, and the gun head frame is prevented from overturning. A plurality of gun head frames 16 are vertically stacked to form a vertically stacked group, and the gun head storage module can be composed of one or more vertically stacked groups.
Further, the gun head storage module further comprises a base 19, a placing groove is formed in the base 19 and matched with the gun head frame 16 in shape, the gun head frame 16 located at the lowest position is placed on the base 19, and the gun head frame 16 is embedded into the placing groove in the base 19, so that stable placement of the whole vertical stacking group is guaranteed.
The mechanical movement module 3 is provided on a movement guide member, the movement guide member drives the mechanical movement module 3 to move in the X-axis direction and the Y-axis direction, and the mechanical movement module 3 is provided with a pipettor 27 capable of moving up and down and a manipulator member 30 capable of moving up and down.
The movable guide rail component comprises an X-axis movable guide rail 2 and a Y-axis movable guide rail 1,Y, wherein the X-axis movable guide rail 2 is provided with an axis movable guide rail 1, and the mechanical movable module 3 is arranged on the Y-axis movable guide rail 1. The movement of the mechanical movement module in the X-axis direction and the Y-axis direction is realized through the X-axis movement guide rail and the Y-axis movement guide rail 2.
Specifically, the mechanical movement module 3 includes a mounting substrate 21, the mounting substrate 21 is connected to the movable guide rail part, a lifting frame 26 is slidably connected to the mounting substrate 21, and a lifting driving mechanism for driving the lifting frame to move is provided on the mounting substrate 21. The pipettor 27 and the manipulator unit 30 are provided on the crane 26, and the crane 26 is further provided with a pipettor elevation driving device 28 for driving the pipettor 27 to move up and down and a manipulator unit elevation driving device 34 for driving the manipulator unit 30 to move up and down.
Wherein, lift actuating mechanism is including setting up lift driving motor 24, the first guide rail 22 on mounting substrate 21, and first guide rail 22 is vertical to be set up on mounting substrate 21, is connected with first slider 23 on the first guide rail 22, first slider 23 and first guide rail 22 sliding connection, and crane 26 installs on first slider 23, and crane 26 can be along first guide rail 22 vertical migration. The mounting base plate 21 is rotatably connected with a screw rod 25, the screw rod 25 is parallel to the first guide rail 22, the screw rod 25 is in transmission connection with a lifting driving motor 24, a threaded hole corresponding to the screw rod 25 is formed in the lifting frame 26, and the screw rod 25 is in threaded connection with the threaded hole.
In this embodiment, the lifting driving motor 24 is connected to the screw rod 25 through a belt. Wherein, the output shaft of the lifting driving motor 24 and one end of the screw rod 25 are respectively provided with a belt pulley, a belt is connected between the two belt pulleys, and the transmission between the lifting driving motor and the screw rod is realized through the belt. The lifting driving motor and the screw rod can also adopt gear connection or other transmission modes. The lead screw is driven to rotate by the lifting driving motor, and the lifting frame is driven to move up and down when the lead screw rotates.
The lifting frame 26 is provided with a fixing block 31, the fixing block 31 is provided with a second guide rail 32, the second guide rail 32 is arranged along the vertical direction, the second guide rail 32 is connected with a second sliding block 33, and the manipulator part 30 is arranged on the second sliding block 33. The robot member elevation driving device 34 is provided with a first output shaft that is vertically movable, and one end of the first output shaft is connected to the robot member 30. In this embodiment, the robot member elevation driving device 34 is an electric push rod. The robot member 30 is driven by the robot member elevation drive device 34 to move up and down. The robot part 30 is provided with two jaws which can be opened or closed. As shown in FIGS. 8 to 10, the robot part can grasp the PCR plate 35, the nucleic acid quantitative turntable 36 and the gun head rack 16, and the above-mentioned devices can be transported by the mechanical moving module. Wherein, the nucleic acid quantitative rotating disk 36 is provided with a grabbing part 52 which is convenient for the manipulator part 30 to grab the nucleic acid quantitative rotating disk 36; when the robot member 30 grips the nucleic acid quantitative turn table 36, the gripping jaws grip both sides of the gripping section 52. When the PCR plate 35 is gripped, the grip jaws of the robot part 30 grip both ends of the PCR plate 35. When the gun head holder 16 is grasped, the two jaws of the robot part 30 grip both ends of the gun head holder 16.
Further, the inner side of the clamping jaw is provided with a clamping groove 35, when the device is used for grabbing, the grabbing part 52 or the PCR plate 35 or two ends of the gun head frame 16 are clamped into the clamping groove, so that the device is stably grabbed.
The pipettor lifting drive device 28 is provided with a second output shaft capable of moving vertically, and one end of the second output shaft is connected with the pipettor 27. In this embodiment, the pipette lift driving device 28 is an electric push rod. Preferably, the pipette 27 is an eight-channel pipette, and a maximum of eight tips 29 can be connected to the pipette 27, so that eight liquid samples can be simultaneously pipetted.
Be equipped with the deep hole board on the normal atmospheric temperature reagent storage module 5, be equipped with a plurality of deep hole that is used for depositing normal atmospheric temperature reagent on the deep hole board, normal atmospheric temperature reagent is stored in each deep hole on the deep hole board. The pipettor can suck the required normal temperature reagent from the deep hole plate. The normal temperature reagent comprises the following components: magnetic beads, nucleic acid eluents, and the like.
The refrigeration module 4 is prior art and is used for storing some low temperature reagents and for dispensing the reagents under low temperature conditions.
The heating oscillation module 11 is used for heating and oscillating and mixing the detection sample. The heating oscillation module 11 is provided with a PCR plate heat conduction frame 40. The PCR plate heat-conducting frame 40 is provided with holes matched with the PCR plate. When using, place the PCR board on PCR board heat conduction frame 40, the module 11 is vibrate and the heating to the PCR board to vibrate the process of hatching. Wherein, the module 11 adopts prior art is vibrate in the heating, and the concrete structure and the principle of module 11 are vibrate in the heating can refer to the utility model patent that the publication is CN209865901U, and the name of this utility model patent is that the vibration that has heating and refrigeration function shakes even device.
The magnetic module 12 includes a support base, a PCR plate bracket 45 disposed on the support base, and a magnetic pillar base plate bracket 48. Wherein, the supporting seat comprises a bottom plate 41 and a supporting column 42 arranged on the bottom plate 41, and a PCR plate bracket 45 is fixed at the upper end of the supporting column 42. The PCR plate bracket 45 is U-shaped, and the PCR plate bracket 45 is provided with a lifting driving device 44 for driving the magnetic column bottom plate bracket 48 to move up and down. Wherein, the lifting driving device 44 is arranged at the lower end of the PCR plate bracket 45, the lifting driving device 44 is vertically arranged, the lifting driving device 44 is provided with an output shaft which can move up and down, and the magnetic column bottom plate bracket 48 is fixedly arranged at the upper end of the output shaft of the lifting driving device 44. In this embodiment, the lifting driving device 44 is a linear push rod motor. The magnetic column bottom plate support 48 is provided with a magnetic column fixing plate 49, and the magnetic column fixing plate 49 is provided with a plurality of magnetic columns 50. The magnetic columns 50 are arranged in a rectangular array on the magnetic column securing plate 49. Positioning grooves are arranged on two sides of the upper end of the PCR plate bracket 45. When the PCR plate 35 is placed on the PCR plate holder 45, both sides of the PCR plate 35 are caught in the positioning grooves on the PCR plate holder 45, and the magnetic column fixing plate is positioned below the PCR plate. As shown in fig. 14 and 15, the conventional PCR plate generally has two structures, including a half skirt PCR plate and a full skirt PCR plate, the positioning groove of the magnetic module in the present invention can be adapted to both the half skirt PCR plate and the full skirt PCR plate, when in use, both sides of the half skirt PCR plate and the full skirt PCR plate can be adapted to the positioning groove of the magnetic module, and both the half skirt PCR plate and the full skirt PCR plate can fall into the positioning groove.
After the PCR plate containing the detection sample and the magnetic beads is placed on the PCR plate bracket, the distance between the magnetic column and the PCR plate can be changed by the lifting driving device 44; carry out high-order magnetism when the purification and inhale, ensure to detect liquid phase separation in the sample for reduce the loss of sample when siphoning away the waste liquid, can reduce the low level magnetic pole again when the elution, guarantee to pass through all magnetic beads with minimum eluant, reduce the experiment system, can reduce the reagent quantity of later stage experiment simultaneously, make the experiment cost reduce by a wide margin. Wherein, PCR plate bracket can be placed with the compatibility of half shirt rim PCR board and full skirtboard PCR on the current market, and the influence that the difference in height brought can be eliminated to the magnetic column of deuterogamy to guarantee the uniformity of experimental result.
As shown in fig. 18, the optical detection module 13 of the present invention adopts the prior art, and the optical detection module is used for performing optical detection on a detection sample. Wherein, the optical detection module 13 is provided with a nucleic acid quantitative reagent storage rack 53. When detecting, the detection sample is firstly placed on the nucleic acid quantitative rotating disc 36, then the nucleic acid quantitative rotating disc 36 with the detection sample is placed at the corresponding position on the optical detection module 13 through the mechanical movement module 3, and the optical detection is performed on the detection sample. After the detection, the turntable 36 can be moved away by the mechanical moving module 3, and then the next batch is replaced for detection.
Furthermore, a quantitative turntable temporary support 54 is disposed beside the optical detection module 13, wherein two placing positions for placing the nucleic acid quantitative turntable 36 are disposed on the quantitative turntable temporary support 54. In practical use, the nucleic acid quantitative rotary disk 36 with the detection sample can be temporarily placed on the quantitative rotary disk temporary holding frame 54 for queue detection, and after the detection sample on the nucleic acid quantitative rotary disk 36 on the optical detection module 13 is completely finished, the nucleic acid quantitative rotary disk on the quantitative rotary disk temporary holding frame 54 is transferred to the optical detection module 13 for detection through the mechanical movement module.
The PCR plate 35 used in the present invention is provided with 96 well sites, which are 96 well plates, arranged in a manner of 8 by 12. The PCR plate has 1-12 rows of wells from left to right, and there are 8 rows of wells from top to bottom, i.e., a row of wells C. The 10-row A hole area is a hole position where the 10 th-row holes and the A-row holes are crossed and corresponding; the 10 columns of E hole areas are hole areas corresponding to the 10 th column of holes and the E rows of holes in a crossed mode.
In the present invention, the mechanical movement module 3 can replace manual work to transfer the detection sample and the reagent, and can transport the PCR plate 35, the nucleic acid quantitative turntable 36, and the gun head rack 16. Because the pipette head 29 needs to be changed continuously in the process of transferring the detection sample and the reagent, the pipette head module 8 is arranged in the invention, and a certain number of pipette heads 29 are stored in the pipette head module 8 and are used for changing the pipette. The gun head module 8 is in a three-dimensional storage mode, and compared with a traditional gun head rack single-layer tiled storage mode, on the premise of storing the same number of gun heads, the storage device occupies a smaller tiled area, is more compact in arrangement, and reduces the occupied area of an automatic warehouse building instrument. And due to the reduction of the tiling area of the gun head frame, the stroke span of the pipette in the horizontal direction is reduced when the pipette head is taken, so that the arrangement length of the guide rail can be shortened, and the cost of equipment is reduced.
According to the invention, the gun head frames are vertically stacked and can be compatible with the pollution-free gun head with the filter element, and when the gun head is taken and used, the height fall between the upper gun head frame and the lower gun head frame is large, so that when the gun head is taken and used, a liquid transfer device is required to have a large displacement stroke in the vertical direction. According to the invention, the lifting frame moves up and down under the driving of the lifting driving mechanism, and the lifting frame drives the pipettor and the mechanical clamping jaw to realize primary lifting when moving up and down; the pipettor lifting driving device can drive the pipettor to move up and down, so that the pipettor can be lifted up and down on the basis of one-level lifting, the lifting stroke of the pipettor is effectively improved, and the taking of the gun heads at different height positions is met. Similarly, the manipulator part lifting driving device can drive the manipulator part to move up and down, so that the manipulator part can be lifted up and down at two levels on the basis of lifting at one level, the moving stroke of the manipulator part in the vertical direction is effectively improved, and the carrying requirements on the gun head frames with different heights are met.
The method for taking the gun heads comprises the following steps:
1) Establishing a gun head storage module: the gun head is placed in a gun head hole in the gun head frame, then the gun head frames are sequentially stacked from bottom to top to form a vertical stacking group, then the vertical stacking groups are sequentially arranged from front to back along the horizontal direction, and the gun head frames on the top of the vertical stacking groups are located at the same height.
2) Taking a gun head: taking a gun head from a gun head frame on the uppermost layer; when the gun head is taken, the mechanical moving module is driven to reach the position above the taking position through the moving guide rail component, then the pipettor descends, a gun head butt joint port on the pipettor is in butt joint with a gun head on the gun head frame, so that the gun head is connected to the pipettor, then the pipettor ascends, and therefore the taking of the gun head is completed; when all the gun heads on the gun head frame are used up, the gun head frame is grabbed and moved out through a mechanical hand part on the mechanical moving module, and then the gun heads are used on the gun head frame at the top of the rest of the vertically stacked groups in the same way; and when all the gun head frames on the uppermost layer are used up, returning the mechanical movement module to the rearmost vertical stacking group, taking the gun heads from the gun head frames on the second layer in the same manner, and so on until all the gun heads are taken up.
The automatic library building instrument for step sampling provided by the invention can be applied to the library automatic construction of a nano single-molecule long fragment sequencing platform, and performs enzyme incubation and purification on nucleic acids with different fragment lengths so as to reduce the interruption of nucleic acid fragments in the mechanical treatment process; and the automatic warehouse building instrument for the step sampling is suitable for any samples from 1 to 24.
The invention provides a library building method, which comprises the following specific steps:
1. quantification of samples of unknown concentration
Taking an unknown sample and placing the unknown sample in 1-3 rows of areas of a PCR plate, and sucking and moving the unknown sample to an optical detection module by a mechanical moving module according to a built-in program to obtain the initial concentration of the unknown sample, wherein the bottom of the optical detection module is provided with a light-emitting acquisition device for acquiring the initial concentration of the unknown sample.
Specifically, a sample (referring to any double-stranded DNA) is added into the 1-3-column position area of the PCR plate, and the sample on the PCR plate is sucked by a liquid shifter on the mechanical movement module and transferred to the optical detection module for detection, so that the concentration of the sample is obtained. During detection, samples are placed on the nucleic acid quantitative rotating disc 36, when one sample is detected, the nucleic acid quantitative rotating disc 36 can rotate to switch one position, and the bottom of the nucleic acid quantitative rotating disc is provided with a light-emitting collecting device for collecting the concentration of the initial sample.
2. End repair with A tail and purification
Separating the terminal repair enzyme premix liquid into 4-6 rows of areas of a PCR plate of a thermal cycle module, transferring a set volume of unknown samples from 1-3 rows of areas based on the initial concentration, adding the unknown samples into 4-6 rows of areas to obtain a uniform mixing liquid, and incubating the uniform mixing liquid by regulating and controlling the temperature of the thermal cycle module;
transferring a magnetic bead with a set volume from a normal-temperature reagent storage module, adding the magnetic bead into the incubated mixing liquid, transferring a PCR plate to a heating oscillation module, transferring the PCR plate to a magnetic absorption module after oscillation incubation is finished, and adding a cleaning reagent after supernatant is removed when the magnetic absorption module keeps a magnetic absorption state;
sucking the supernatant when the magnetic module keeps the magnetic state, adding the cleaning reagent again, and then removing the supernatant again; keeping the magnetic attraction state and naturally drying for 30 s;
transferring the PCR plate to a heating oscillation module, adding the nucleic acid eluent on a normal-temperature reagent storage module for incubation, transferring the PCR plate again to a magnetic adsorption module to separate magnetic beads, and transferring the supernatant to a 7-9 row position area of the PCR plate.
Specifically, the method comprises the following steps:
s1: preparing a terminal repair enzyme premix solution for building a warehouse in a refrigeration module, placing a PCR plate on a thermal cycle module, separating the prepared terminal repair enzyme premix solution onto the PCR plate, calculating the required sample volume according to the collected sample concentration, sequentially transferring the samples to the hole sites of the separated terminal repair enzyme premix solution, and uniformly mixing. Wherein the terminal-repair enzyme premix was dispensed onto the 4-6 column position area on the PCR plate.
S2: controlling the thermal circulation module to realize incubation at 20 ℃ for 5-10min, and then incubating at 65 ℃ for 5min.
S3: absorb the magnetic bead and the mixing from normal atmospheric temperature reagent storage module through the pipettor on the mechanical movement module, then according to the sample number, correspond absorb on the equal volumn magnetic bead transfers the PCR board on the thermal cycle module and with the sample mixing, then shift the PCR board to the heating through the manipulator part on the mechanical movement module and vibrate the module, vibrate and incubate.
S4: after the oscillation incubation is finished, transferring the PCR plate on the oscillation module to a magnetic suction module through a manipulator component on the mechanical moving module; the magnetic beads in the liquid phase are enriched to a specific position as much as possible by raising and lowering the magnetic column to a specific height and then slowly lowering. The specific height is the height from the magnetic end face to the bottom of the PCR plate according to the application scheme, and the height from the magnetic end face to the bottom of the PCR plate in the optimal separation state of magnetic beads and liquid phase set during the transfer of consumables and liquid in different specifications and in the addition volume of liquid.
S5: keeping the magnetic attraction state, sucking the supernatant through a liquid transfer device on the mechanical moving module, adding a cleaning reagent, sucking the supernatant again, and repeating the process once.
S6: transferring the PCR plate to a heating oscillation module through a mechanical hand part on the mechanical movement module, absorbing nucleic acid eluent on the normal-temperature reagent storage module, adding the nucleic acid eluent into a sample, incubating magnetic beads for 3min, and recovering the nucleic acid sample treated by enzyme.
S7: the PCR plate is transferred to the magnetic suction module through a manipulator component on the mechanical movement module, liquid phase and magnetic bead separation is carried out at a specific height through lifting the magnetic column, and supernatant in a sample is transferred to 7-9 rows of position areas on the PCR plate through a liquid transfer device on the mechanical movement module.
Barcode ligation and purification
Absorbing nucleic acid molecular labels from the refrigeration module, adding the nucleic acid molecular labels into 7-9 rows of areas of the PCR plate, and adding T/A enzyme premix of the refrigeration module to obtain uniformly mixed liquid; transferring the PCR plate to a thermal cycle module, then performing temperature control incubation, transferring a set volume of magnetic beads from a normal temperature reagent storage module, adding the magnetic beads into the incubated mixing liquid, transferring the PCR plate to a heating oscillation module, transferring the PCR plate to a magnetic absorption module after oscillation incubation is finished, and adding a cleaning reagent after supernatant is removed when the magnetic absorption module keeps a magnetic absorption state;
sucking the supernatant when the magnetic module keeps the magnetic state, adding the cleaning reagent again, and then removing the supernatant again; keeping the magnetic attraction state and naturally drying for 30 s; transferring the PCR plate to a heating oscillation module, adding the nucleic acid eluent on the normal-temperature reagent storage module, incubating, and transferring the PCR plate to a magnetic module component separation magnetic bead again; transferring the separated samples to an optical detection area to detect the secondary concentration of the samples, calculating the volume of the mixed samples required by each sample based on the secondary concentration of the samples, and sucking each sample of the volume of the mixed samples to the 10 rows of A hole areas of the PCR plate to realize high-flux mixed samples; the method specifically comprises the following steps:
s1: and absorbing corresponding nucleic acid molecule labels from the refrigeration module, adding the nucleic acid molecule labels into 7-9 rows of position areas on the PCR plate, absorbing T/A enzyme premix liquid for label connection on the refrigeration module, adding the T/A enzyme premix liquid and uniformly mixing the T/A enzyme premix liquid and the PCR plate.
S2: the robotic assembly on the mechanical movement module transferred the PCR plate to the thermal cycling module for incubation at a temperature of 20 ℃.
S3: magnetic beads on the normal temperature reagent storage module are sucked and mixed through a liquid transfer device on the mechanical movement module, the equal-volume magnetic beads are added and mixed correspondingly according to the number of samples, then a PCR plate is transferred to the heating oscillation module through a manipulator component on the mechanical movement module, and oscillation incubation is carried out.
S4: after the oscillation incubation is finished, the PCR plate on the heating oscillation module is transferred to the magnetic suction module through the mechanical clamping jaw. By raising and lowering the magnetic column to a specific height and then slowly lowering, the magnetic beads in the liquid phase are enriched to a specific position as much as possible.
S5: keeping the magnetic attraction state, sucking the supernatant through a liquid transfer device on the mechanical moving module, adding a cleaning reagent, sucking the supernatant again, and repeating the process once.
S6: and transferring the PCR plate to a heating oscillation module by a manipulator component on the mechanical movement module, absorbing nucleic acid eluent, adding incubated magnetic beads, and recovering the nucleic acid sample treated by the enzyme.
S7: and transferring the PCR plate to a magnetic suction module through a manipulator component on the mechanical movement module to separate a liquid phase from magnetic beads.
S8: and sucking the nucleic acid sample of the PCR plate by a liquid shifter on the mechanical moving module, transferring the nucleic acid sample to an optical detection area for detection, and collecting the concentration of the nucleic acid sample.
S9: converting the concentration of the secondarily collected nucleic acid sample into a sample mixing volume required by each sample, keeping a magnetic attraction state, and sucking each sample of the sample mixing volume to 10 rows of A hole areas of the PCR plate to realize high-throughput sample mixing;
4. add nanopore sequencing adaptor and purify
Adding a sequencing joint with a specific volume, a quick ligase and a buffer solution into the hole A region of 10 rows of the PCR plate, uniformly mixing, transferring to a thermal cycle module for temperature-controlled incubation, transferring a set volume of magnetic beads from a normal-temperature reagent storage module, adding the incubated uniform mixing liquid, transferring the PCR plate to a heating oscillation module, transferring the PCR plate to a magnetic absorption module after oscillation incubation is finished, and removing a supernatant when the magnetic absorption module keeps a magnetic absorption state; transferring the PCR plate to a heating and oscillating module, adding a joint cleaning reagent sucked from a refrigerating module, uniformly mixing, transferring the PCR plate to a magnetic absorption module to separate a liquid phase from magnetic beads, and removing a supernatant; repeatedly transferring the PCR plate to the heating and oscillating module for one time, adding a joint cleaning reagent sucked from the refrigerating module, uniformly mixing, transferring the PCR plate to the magnetic absorption module to separate a liquid phase from magnetic beads, and removing a supernatant; and keeping the magnetic attraction state, naturally air-drying for 30s, transferring the PCR plate to a heating oscillation module, adding a joint nucleic acid washing stripping solution sucked from a refrigeration module, oscillating, uniformly mixing and incubating for 10 minutes, transferring the PCR plate to the magnetic attraction module to separate a liquid phase from magnetic beads, and sucking supernatant to 10 rows of E hole sites of the PCR plate to obtain a qualified high-quality multi-sample library based on the nanopore monomolecular sequencing platform. The method specifically comprises the following steps:
s1, adding a specific volume of a joint, a rapid ligase and a buffer solution into a sample, and uniformly mixing.
And S2, transferring the PCR plate to a thermal cycle module through a manipulator part on the mechanical moving module, and incubating at the online temperature controlled by 20 ℃.
S3, sucking magnetic beads with the volume of 0.5 time of that of the sample by a liquid shifter on the mechanical moving module, uniformly mixing the magnetic beads with the sample, transferring the PCR plate to a heating and vibrating module by a manipulator part on the mechanical moving module, and vibrating and incubating.
S4: after the oscillation incubation is finished, transferring the PCR plate on the heating oscillation module to a magnetic suction module through a mechanical clamping jaw; by raising and lowering the magnetic column to a specific height and then slowly lowering, the magnetic beads in the liquid phase are enriched to a specific position as much as possible.
S5: keeping the magnetic attraction state, sucking the supernatant through a liquid transfer device on the mechanical movement module, adding a connector cleaning reagent sucked from the refrigeration module, sucking the supernatant again, and repeating the process once.
And S6, transferring the PCR plate to a heating and vibrating module by a manipulator part on the mechanical moving module, adding a joint nucleic acid eluting solution sucked from the refrigerating module into the incubation magnetic beads, and recovering the nucleic acid sample treated by the enzyme.
And S7, transferring the PCR plate to a magnetic suction module through a mechanical clamping jaw to separate a liquid phase from magnetic beads, and sucking the supernatant in the sample to a new hole position on the PCR plate to obtain the high-quality library.
The library construction method provided by the scheme can cover long-fragment amplicon and gene composition library construction, realizes 1-24 arbitrary sample library construction, and provides some specific embodiments of the scheme as follows:
example 1:400bp amplification product
And (3) performing electrophoresis on one amplified product to confirm the length of the DNA fragment, adding the other amplified product into a PCR 96 pore plate, putting reagents and consumables according to the library building method, taking out the PCR 96 pore plate after operation, loading the library into a nanopore sequencing chip for sequencing, and performing fragment length analysis according to 50M fastq data of each experimental sample sequencing.
Example 2:760bp amplification product
And (3) performing electrophoresis on one part of the amplified product to confirm the length of the DNA fragment, adding the other part of the amplified product into a PCR 96 pore plate, putting reagents and consumables according to the library building method, taking out the PCR 96 pore plate after the operation is finished, loading the library into a nanopore sequencing chip for sequencing, and analyzing according to 50M fastq data of each experimental sample sequencing.
Example 3:1200bp amplification product
And (3) performing electrophoresis on one part of the amplified product to confirm the length of the DNA fragment, adding the other part of the amplified product into a PCR 96 pore plate, putting reagents and consumables according to the library building method, taking out the PCR 96 pore plate after operation, loading the library into a nanopore sequencing chip for sequencing, and analyzing according to 50M fastq data of each experimental sample sequencing.
Example 4:16s full-length 1500bp amplification product
And (3) performing electrophoresis on one part of the amplified product to confirm the length of the DNA fragment, adding the other part of the amplified product into a PCR 96 pore plate, putting reagents and consumables according to the library building method, taking out the PCR 96 pore plate after operation, loading the library into a nanopore sequencing chip for sequencing, and analyzing according to 50M fastq data of each experimental sample sequencing.
Example 5: its-23s 3200bp amplification product
And (3) performing electrophoresis on one part of the amplified product to confirm the length of the DNA fragment, adding the other part of the amplified product into a PCR 96 pore plate, putting reagents and consumables according to the library building method, taking out the PCR 96 pore plate after operation, loading the library into a nanopore sequencing chip for sequencing, and analyzing according to 50M fastq data of each experimental sample sequencing.
Example 6: automatic library construction for extracting nucleic acid from bacterial genome
And (3) performing electrophoresis on one part of nucleic acid extracted by the column membrane method, confirming the length of the DNA fragment, adding the other part of nucleic acid into a PCR 96 pore plate, putting a reagent and consumables according to the library building method, taking out the PCR 96 pore plate after operation, loading the library into a nanopore sequencing chip for sequencing, and analyzing according to 50M fastq data of each experimental sample sequencing.
The electrophorograms of the different fragments of examples 1 to 6 are shown in FIG. 19, the statistical graph of the length distribution of the pooled sequenced fragments is shown in FIG. 20, and the statistical data of 1 hour of pooling is shown in FIG. 21.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.
Claims (10)
1. An automatic warehouse building instrument for stepped sampling is characterized by comprising a refrigeration module, a thermal cycle module, a normal temperature reagent storage module, a gun head module, a heating oscillation module, an optical detection module, a magnetic suction module and a mechanical movement module;
the gun head module comprises a plurality of gun head frames which can be stacked up and down, a plurality of gun head holes for storing gun heads are arranged on the gun head frames, the gun head frames are provided with positioning connecting mechanisms, and the upper gun head frame and the lower gun head frame are connected through the positioning connecting mechanisms;
the mechanical moving module is arranged on the moving guide rail component, the moving guide rail component drives the mechanical moving module to move along the X-axis direction and the Y-axis direction, and the mechanical moving module is provided with a pipettor capable of moving up and down and a manipulator component capable of moving up and down.
2. The automatic warehouse building instrument for the stepped sampling according to claim 1, wherein the refrigeration module, the thermal cycle module and the normal temperature reagent storage module are arranged on one side of the gun head module, and the heating oscillation module, the magnetic absorption module and the optical detection module are arranged on the other side of the gun head module.
3. The automatic warehouse building instrument for stepped sampling according to claim 1, wherein the magnetic suction module comprises a support base, a PCR plate bracket and a magnetic column base plate bracket, the PCR plate bracket is arranged on the support base, the PCR plate bracket is provided with a lifting driving device for driving the magnetic column base plate bracket to move up and down, the magnetic column base plate bracket is provided with a magnetic column fixing plate, and the magnetic column fixing plate is provided with a plurality of magnetic columns; positioning grooves are arranged on two sides of the upper end of the PCR plate bracket; when the PCR plate is placed on the PCR plate bracket, the two sides of the PCR plate are clamped into the positioning grooves on the PCR plate bracket, and the magnetic column fixing plate is positioned below the PCR plate.
4. The automatic warehouse building instrument for the stepped sampling according to claim 1, wherein the positioning connection mechanism comprises a positioning sleeve arranged on the upper side of the gun head frame and a positioning insertion rod arranged below the gun head frame and corresponding to the positioning sleeve, and when the gun head frames are vertically stacked, the positioning insertion rod on the upper gun head frame is inserted into the positioning sleeve on the lower gun head frame.
5. The automatic warehouse building instrument for the step sampling according to claim 1, wherein the mechanical moving module comprises a mounting base plate, the mounting base plate is connected with the moving guide rail part, a lifting frame is connected on the mounting base plate in a sliding manner, and a lifting driving mechanism for driving the lifting frame to move is arranged on the mounting base plate; the pipettor and the manipulator component are arranged on the lifting frame, and the lifting frame is also provided with a pipettor lifting driving device for driving the pipettor to move up and down and a manipulator component lifting driving device for driving the manipulator component to move up and down.
6. The automatic warehouse building instrument for the stepped sampling according to claim 5, wherein the lifting driving mechanism comprises a lifting driving motor and a first guide rail which are arranged on the mounting base plate, the first guide rail is vertically arranged on the mounting base plate, a first sliding block is connected to the first guide rail, and the lifting frame is arranged on the first sliding block; the mounting base plate is rotatably connected with a screw rod, the screw rod is parallel to the first guide rail, the screw rod is in transmission connection with a lifting driving motor, a threaded hole corresponding to the screw rod is formed in the lifting frame, and the screw rod is in threaded connection with the threaded hole.
7. The automatic warehouse building instrument for the stepped sampling according to claim 5, wherein the lifting frame is provided with a fixed plate, the fixed plate is provided with a second guide rail, the second guide rail is arranged along the vertical direction, a second sliding block is connected to the second guide rail, and the manipulator part is installed on the second sliding block; but the manipulator part lift drive is equipped with vertical mobile's first output shaft on, and the manipulator part is connected to the one end of first output shaft.
8. The automatic warehouse building instrument for the step sampling according to claim 1, wherein the pipettor lifting drive device is provided with a second output shaft capable of moving vertically, and one end of the second output shaft is connected with the pipettor.
9. A library construction method, which is characterized in that the library construction of the nanopore monomolecular long fragment sequencing platform is automatically realized by using the step-sampling automatic library construction instrument of any one of claims 1 to 8, and a long fragment amplicon and a genome construction library are covered.
10. The library building method of claim 9, comprising:
placing samples with unknown concentration in 1-3 rows of areas of a PCR plate, and moving the unknown samples to an optical detection module to obtain the initial concentration of the unknown samples;
separating the terminal repair enzyme premix liquid into 4-6 rows of areas of a PCR plate of a thermal cycle module, transferring a set volume of unknown samples from 1-3 rows of areas based on the initial concentration, adding the unknown samples into 4-6 rows of areas to obtain a uniform mixing liquid, and incubating the uniform mixing liquid by regulating and controlling the temperature of the thermal cycle module; transferring a magnetic bead with a set volume from a normal-temperature reagent storage module, adding the magnetic bead into the incubated mixing liquid, transferring a PCR plate to a heating oscillation module, transferring the PCR plate to a magnetic absorption module after oscillation incubation is finished, and adding a cleaning reagent after supernatant is removed when the magnetic absorption module keeps a magnetic absorption state; keeping the magnetic attraction state, sucking the supernatant, adding a cleaning reagent repeatedly, and removing the supernatant again; keeping the magnetic attraction state, naturally drying for 30s, transferring the PCR plate to a heating oscillation module, adding a nucleic acid eluent on a normal-temperature reagent storage module for incubation, transferring the PCR plate again to the magnetic attraction module to separate magnetic beads, and transferring supernatant to the 7-9 rows of areas of the PCR plate;
absorbing nucleic acid molecular labels from the refrigeration module, adding the nucleic acid molecular labels into 7-9 rows of areas of the PCR plate, and adding T/A ligase premixed liquid absorbed from the refrigeration module to obtain uniformly mixed liquid; transferring the PCR plate to a thermal cycle module, then incubating at a controlled temperature, transferring a set volume of magnetic beads from a normal-temperature reagent storage module, adding the magnetic beads into the incubated mixed liquid, transferring the PCR plate to a heating oscillation module, after oscillating incubation, transferring the PCR plate to a magnetic absorption module, removing supernatant when the magnetic absorption module keeps a magnetic absorption state, then adding a cleaning reagent, keeping the magnetic absorption state, absorbing the supernatant, adding the cleaning reagent again, and removing the supernatant again after adding the cleaning reagent again; keeping the magnetic attraction state, naturally drying for 30s, transferring the PCR plate to a heating and oscillating module, adding the nucleic acid eluent on the normal-temperature reagent storage module, incubating for 3 minutes, and transferring the PCR plate to the magnetic attraction module again to separate magnetic beads; sucking the separated samples to an optical detection area to detect the secondary concentration of the samples, calculating the volume of the mixed samples required by each sample based on the secondary concentration of the samples, and sucking each sample of the volume of the mixed samples to 10 rows of A hole areas of a PCR plate to realize high-flux mixed samples;
adding a specific volume sequencing joint, fast ligase and buffer solution into a 10-row A hole area of a PCR plate, uniformly mixing, transferring to a thermal cycle module for temperature-controlled incubation, transferring a set volume of magnetic beads from a normal-temperature reagent storage module, adding the incubated mixed liquid, transferring the PCR plate to a heating oscillation module, transferring the PCR plate to a magnetic absorption module after oscillation incubation is completed, and removing supernatant when the magnetic absorption module keeps a magnetic absorption state; transferring the PCR plate to a heating oscillation module, adding a joint cleaning reagent sucked from a refrigeration module, uniformly mixing, transferring the PCR plate to a magnetic absorption module to separate a liquid phase from magnetic beads, and removing a supernatant; repeatedly transferring the PCR plate to the heating and oscillating module for one time, adding a joint cleaning reagent sucked from the refrigerating module, uniformly mixing, transferring the PCR plate to the magnetic absorption module to separate a liquid phase from magnetic beads, and removing a supernatant; and keeping the magnetic attraction state, naturally air-drying for 30s, transferring the PCR plate to a heating oscillation module, adding a joint nucleic acid washing and removing solution sucked from a refrigeration module, oscillating, uniformly mixing and incubating for 10 minutes, transferring the PCR plate to the magnetic attraction module to separate a liquid phase from magnetic beads, and sucking supernatant to 10 rows of E hole areas of the PCR plate to obtain a qualified high-quality multi-sample library based on the nanopore monomolecular sequencing platform.
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| CN117467532A (en) * | 2023-10-07 | 2024-01-30 | 浙江基预科技有限公司 | Kit based on nanopore sequencing and control method thereof |
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| CN107557356A (en) * | 2017-09-05 | 2018-01-09 | 苏州普川生物科技有限公司 | A kind of small multi-channel library construction instrument and the method for building library |
| WO2022088477A1 (en) * | 2020-10-27 | 2022-05-05 | 广州和实生物技术有限公司 | Nucleic acid detector and nucleic acid detection method |
| CN112175943A (en) * | 2020-10-29 | 2021-01-05 | 绍兴迅敏康生物科技有限公司 | Continuous sample introduction sequencing library building instrument and operation method thereof |
| CN216473243U (en) * | 2021-12-02 | 2022-05-10 | 杭州奥盛仪器有限公司 | Automatic sample preparation system with fluorescence detection module |
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| CN117467532A (en) * | 2023-10-07 | 2024-01-30 | 浙江基预科技有限公司 | Kit based on nanopore sequencing and control method thereof |
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