CN112175943A - Continuous sample introduction sequencing library building instrument and operation method thereof - Google Patents
Continuous sample introduction sequencing library building instrument and operation method thereof Download PDFInfo
- Publication number
- CN112175943A CN112175943A CN202011176704.6A CN202011176704A CN112175943A CN 112175943 A CN112175943 A CN 112175943A CN 202011176704 A CN202011176704 A CN 202011176704A CN 112175943 A CN112175943 A CN 112175943A
- Authority
- CN
- China
- Prior art keywords
- module
- pcr
- magnetic rod
- reaction tube
- magnetic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- 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
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
The invention provides a sequencing library building instrument for continuous sample introduction and an operation method thereof, wherein the sequencing library building instrument at least comprises: the working space, the bottom side of working space sets up the module of getting ready in succession, the TIP head module, module and two at least PCR modules are deposited to the reagent, move liquid module and bar magnet cover module activity and arrange in the working space, and move liquid module reciprocal at the module of getting ready in succession, the TIP head module, the module is deposited to the reagent and two at least PCR module place space removal, bar magnet cover module is at least at PCR module place space reciprocating motion, wherein the module of getting ready in succession includes the reaction tube of getting ready of multirow interval arrangement at least, wherein bar magnet cover module includes bar magnet subassembly and bar magnet cover subassembly, the absorption and the release of magnetic bead are realized to the cooperation motion of bar magnet subassembly and bar magnet cover subassembly, it can realize the continuous sampling of many samples, and reduce the use of consumptive material.
Description
Technical Field
The invention relates to the field of gene sequencing library construction, in particular to a sequencing library construction instrument for continuous sample introduction and an operation method thereof.
Background
The rapid acquisition of gene sequence information is very important for the research of life science, and the most common means at present is nucleic acid high-throughput gene sequencing, and the technology can perform sequence determination on hundreds of thousands to hundreds of millions of DNA molecules at one time. In order to enable the DNA extracted from a living body to meet the requirement of sequencing, the operations of end repair, joint addition, purification and the like are required to be carried out on the broken DNA, the experimental process is called library construction, the aim is to connect the joints required at the two ends of a target DNA fragment, the library structure can be sequenced only when meeting the on-machine requirement of a high-throughput sequencer, and the library construction process is divided into several steps: 1. performing end repair on the broken DNA; 2. ligating an adaptor to the fragment of interest; 3. purifying the obtained product by magnetic beads; 4. carrying out PCR on the purified library to obtain a large amount of libraries; 5. and purifying the library after PCR to obtain the library capable of being subjected to computer sequencing.
Although the current automatic sequencing library builder can also complete the library construction of nucleic acid samples, there are several fatal problems: 1. the method can only realize sample loading and library building of 16 channels at most, consumables and configuration parameters in a library building instrument need to be replaced again if sample loading detection of more samples needs to be carried out, and 2-3 hours are consumed for reconfiguring a sequencing library building instrument each time, so that the library building time is long, and the requirement of mixed library building of multiple samples is difficult to meet; 2. the consumable cost is high, a large number of TIP heads need to be consumed to complete sample loading and library building at one time, and the sample and magnetic bead transfer process is complex and easily causes cross contamination. 3. At present, a library building instrument in the market comprises a PCR instrument, so that the cost is high, and the later maintenance is difficult. 4. Most libraries are built only up to the PCR library purification process, which is not included in the pre-machine library dilutions and mixes.
Disclosure of Invention
The invention aims to provide a sequencing library building instrument for continuous sample introduction and an operation method thereof, the sequencing library building instrument can realize continuous sample loading of multiple samples (32 channels), the library building efficiency is improved, meanwhile, the use of consumables is greatly reduced by adopting a magnetic rod sleeve to transfer magnetic beads, and in addition, the independent operation and matching of the magnetic rod sleeve and a liquid transfer module can also avoid cross contamination between reagents and samples.
In order to achieve the above object, in a first aspect, the present technical solution provides a sequencing and library building apparatus with continuous sample injection, which at least includes: working space, the bottom side of working space sets up the module of appearance of going up in succession, the TIP head module, module and two at least PCR modules are deposited to the reagent, move liquid module and bar magnet cover module activity and arrange in working space, and move liquid module reciprocal at the module of appearance of going up in succession, the TIP head module, the module is deposited to the reagent and two at least PCR module place space removal, bar magnet cover module is at least at PCR module place space reciprocating motion, wherein the module of going up in succession includes the reaction tube that takes out a sample of multirow interval arrangement at least, wherein bar magnet cover module includes bar magnet subassembly and bar magnet cover subassembly, the absorption and the release of magnetic bead are realized in the cooperation motion of bar magnet subassembly and bar magnet cover subassembly.
In a second aspect, an operating method of the sequencing library building instrument is provided, which includes the following steps: adding a sample into a sample feeding reaction tube of the continuous sample feeding module; after the liquid transfer module is driven to transfer a sample from the sample loading reaction tube to a PCR reaction tube of the PCR module, the liquid transfer module is driven to move to the TIP head module to replace the TIP head, the liquid transfer module is driven to move to the reagent storage module to absorb reaction liquid and magnetic beads to be added into the corresponding PCR reaction tube, and the reaction tube is set in the PCR reaction tube according to a program to build a library; driving the magnetic rod sleeve module to extend into the corresponding PRC reaction tube to adsorb magnetic beads; and driving the magnetic rod sleeve module to transfer the magnetic beads to the second reaction tube of the PCR module for cleaning, taking out and airing, and driving the magnetic rod sleeve module to transfer the magnetic beads to the third reaction tube of the PCR module for elution and release of the magnetic beads.
Compared with the prior art, the technical scheme has the following characteristics and beneficial effects:
1. the continuous sample loading and library building of the 4 x 8 32 channels of samples can be realized, the sample loading does not influence the proceeding of the preorder and the subsequent library building process every time, and the traditional multi-sample library building time is shortened manyfold while the library building process of the samples is not influenced.
2. The magnetic rod sleeve matched with the PCR is configured to realize the integration of magnetic bead extraction and transfer, the magnetic rod sleeve and the magnetic rod are matched to improve the adsorption rate and the release rate of the magnetic beads, in addition, the magnetic rod sleeve is driven by a driving part to realize automatic magnetic bead transfer, and the artificial pollution possibly generated in the manual transfer process is avoided.
3. The liquid transfer module replaces the TIP head after the sample and the reagent are transferred, so that cross contamination to the sample and the reagent in the liquid transfer process is avoided; and the PCR auxiliary reagent can be configured in the EP pipe area and can also be independently transferred to a PCR module for PCR treatment of samples, and the continuous sample introduction structure is reasonable and compact in arrangement and cannot interfere with each other.
4. Each PCR module is provided with an independent parallel reaction tube, a movable magnetic rod sleeve is matched with the same PCR module to wash and elute magnetic beads, the elution liquid with nucleic acid is sequenced, and each PCR module independently finishes all work, thereby finishing the PCR treatment of samples with 32 channels.
Drawings
FIG. 1 is a schematic diagram of the overall structure of a continuous sample injection sequencing library builder with a housing cover according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of the overall structure of a continuous sample injection sequencing library building instrument for extracting the housing cover according to an embodiment of the invention.
FIG. 3 is a front view of a serial sample injection sequencing library builder with housing shells removed according to an embodiment of the present invention.
FIG. 4 is a side view of a sequential sample injection sequencing library builder with housing cover removed according to an embodiment of the present invention.
Fig. 5 is a schematic structural diagram of a magnetic rod sleeve module according to an embodiment of the invention.
Fig. 6 is a partial structure enlarged view based on fig. 5.
Fig. 7 is a schematic structural diagram of a pipetting module according to an embodiment of the invention.
FIG. 8 is a schematic diagram of a partial structure of a continuous-injection sequencing and library building apparatus according to an embodiment of the present invention.
In the figure: 10-shell, 100-working space, 11-outer shell cover, 111-sample introduction opening, 12-base, 13-movable base, 131-sliding space, 14-magnetic rod sleeve module slide rail, 20-air filtration module, 21-air filter, 22-base air outlet, 23-side wall air outlet, 30-continuous sample loading module, 31-sample loading reaction tube, 32-sample loading reaction tube storage base, 40-nucleic acid extraction module, 50-TIP head module, 51-TIP head, 52-TIO head storage base, 60-reagent storage module, 61-reagent bottle placement area, 62-reaction tube placement area, 70-magnetic rod sleeve module, 71-magnetic rod sleeve driving component, 711-transverse moving plate, 712-magnetic rod vertical driving component, 713-magnetic rod sleeve driving part, 72-magnetic rod assembly, 721-magnetic rod seat, 722-magnetic rod, 73-magnetic rod sleeve assembly, 731-magnetic rod sleeve base, 732-magnetic rod sleeve, 80-pipetting module, 81-pipetting piece, 82-pipetting piece driving part, 821-pipetting piece vertical driving part, 822-pipetting piece transverse driving part, 90-PCR module, 91-PCR reaction tube, 92-second reaction tube and 93-third reaction tube.
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.
According to the first aspect of the invention, a sequencing library building instrument for continuous sample introduction is provided, which can be used for nucleic acid high-throughput gene sequencing, and can realize continuous sample introduction different from the conventional sequencing library building instrument, the conventional sequencing library building instrument can only realize sample introduction detection of 16 channels at most, while the sequencing library building instrument provided by the scheme can realize sample introduction detection of 32 channels, and the samples of the scheme do not influence preorders and subsequent sample library building during sample introduction; in addition, the sequencing library building instrument provided by the scheme adopts the movable control magnetic rod sleeve module and the liquid transfer module to carry out automatic library building treatment, the equipment arrangement is compact and reasonable, and the efficiency of nucleic acid gene sequencing library building can be greatly improved.
Specifically, the sequencing library building instrument comprises at least a working space 100, a continuous sample loading module 30, a TIP head module 50, a reagent storage module 60 and at least two PCR modules 90 are arranged at the bottom side of the working space 100, a liquid transfer module 80 and a magnetic rod sleeve module 70 are movably arranged in the working space 100, and the pipetting module 80 reciprocates in the spaces where the consecutive loading module 30, the TIP module 50, the reagent storage module 60 and the at least two PCR modules 90 are located, the bar magnet housing module 70 reciprocates in the spaces where the PCR modules 90 are located, wherein the continuous sample loading module 30 at least comprises a plurality of rows of sample loading reaction tubes 31 which are arranged at intervals, each PCR module 90 at least comprises a PCR reaction tube 91, a second reaction tube 92 and a third reaction tube 93 which are arranged at intervals, wherein the magnetic rod sleeve module 70 comprises a magnetic rod assembly 72 and a magnetic rod sleeve assembly 73, and the magnetic rod assembly 72 and the magnetic rod sleeve assembly 73 are matched to move to realize the adsorption and release of magnetic beads.
The sequencing library building instrument comprises a shell 10, a working space 100 is formed in the shell 10, and a sample is placed in the working space 100 to be built. The sample in which the present scheme can be sequenced and the library is built comprises biological liquid (such as case liquid or clinical sample), and the liquid sample or the fluid sample can be derived from solid or semisolid samples, including excrement and biological tissues. The solid or semi-solid sample may be converted to a liquid sample by any suitable method, such as mixing, triturating, macerating, incubating, dissolving, or enzymatically digesting a solid sample in a suitable solution (e.g., water, phosphate solution, or other buffered solution). "biological samples" include samples derived from animals, plants and food, including, for example, urine, saliva, blood and components thereof, vaginal secretions, sperm, feces, sweat, secretions, tissues, organs, tumors, cultures of tissues and organs, cell cultures and media derived from humans or animals.
Specifically, the bottom side of the casing 10 is provided with a base 12, a working space 100 is formed above the base 12, and the consecutive loading module 30, the TIP module 50, the reagent storage module 60, and at least two PCR modules 90 are placed on the base 12. In one embodiment of the present embodiment, as shown in fig. 2, in order to make the structure of the sequencing library builder more compact and reasonable, the continuous loading module 30 and the reagent storage module 60 are disposed at the corner positions of the working space 100, the TIP module 50 is disposed side by side between the continuous loading module 30 and the reagent storage module 60, the PCR module 90 is disposed inside the TIP module 50, and the continuous loading module 30, the TIP module 50, the reagent storage module 60, and at least two PCR modules 90 are independently disposed. The advantages of such a position arrangement are: after the pipetting module 80 samples the sample from the continuous sample loading module 30 to the PCR reaction tube 91, the TIP head can be quickly replaced at the TIP head module 50; after taking out the reaction reagent from the reagent storage module 60 into the PCR reaction tube 91, the TIP head can be quickly replaced at the position of the TIP head module 50; in addition, the mode that the PCR module 90 is arranged at the inner side of the working space 100 is convenient for the magnetic rod sleeve module 70 to move, the mutual interference between the magnetic rod sleeve module 70 and the liquid-transferring module 80 in the moving process is reduced, the structural arrangement of the sequencing library building instrument can be compact, and the size of the sequencing library building instrument is reduced.
In one embodiment of the present disclosure, the moving tracks of the pipetting module 80 and the magnetic rod sleeve module 70 are separately disposed in different space for movement, so that the pipetting module 80 and the magnetic rod sleeve module 70 can move independently, and the pipetting module 80 is disposed in the upper space of the working space 100 for movement because the moving space of the pipetting module 80 is larger than that of the magnetic rod sleeve module 70.
Specifically, as shown in fig. 3, a movable seat 13 is provided at a position of the working space 100 of the housing 10 close to the top side, that is, the movable seat 13 is spaced from the top side of the working space 100 by a certain distance, and the pipetting module 80 is movably placed on the movable seat 13 to move with a set moving track, wherein the pipetting module 80 can freely move transversely and longitudinally on the movable seat 13; the base 12 is provided with a magnetic bar sleeve module slide rail 14, the magnetic bar sleeve module slide rail 14 is disposed on the inner side of the working space 100, and the magnetic bar sleeve module 70 is disposed on the magnetic bar sleeve module slide rail 14 and moves along the track direction of the magnetic bar sleeve module slide rail 14.
In one embodiment of the present disclosure, the PCR modules 90 are arranged side by side, and the bar sleeve module slide 14 is disposed inside the PCR modules 90 and horizontally arranged, so that the bar sleeve module 70 can perform a horizontal stroke motion on the bar sleeve module slide 14.
Specifically, the magnetic rod sleeve module 70 includes a magnetic rod sleeve driving assembly 71, a magnetic rod assembly 72, and a magnetic rod sleeve assembly 73, and the magnetic rod sleeve driving assembly 71 drives the magnetic rod assembly 72 and the magnetic rod sleeve assembly 73 to move along the magnetic rod sleeve module slide rail 14, and drives the magnetic rod assembly 72 and the magnetic rod sleeve assembly 73 to move vertically by independent movement strokes. The magnetic rod sleeve driving assembly 71 further comprises a transverse moving plate 711, a magnetic rod vertical driving member 712 and a magnetic rod sleeve driving member 713, the transverse moving plate 711 is vertically arranged on the magnetic rod sleeve module slide rail 14 and is connected with a motor to be driven by stroke change, the magnetic rod assembly 72 and the magnetic rod sleeve assembly 73 are arranged on the transverse moving plate 711, the magnetic rod vertical driving member 712 is connected with and controls the magnetic rod assembly 72 to move vertically, and the magnetic rod sleeve driving member 713 is connected with and controls the magnetic rod sleeve assembly 73 to move vertically.
It should be noted that the magnetic rod assembly 72 is disposed above the magnetic rod sleeve assembly 73 and is located on the same axis with the magnetic rod sleeve assembly 73, and the magnetic rod vertical driving member 712 drives the magnetic rod assembly 72 to move up and down relative to the magnetic rod sleeve assembly 73. The magnetic rod vertical driving part 712 is sleeved on the magnetic rod sleeve vertical driving part 713, and the magnetic rod sleeve vertical driving part 713 can drive the magnetic rod sleeve assembly 73 and the magnetic rod assembly 72 to move up and down.
As shown in fig. 6, the magnetic rod assembly 72 includes a magnetic rod seat 721 and a magnetic rod 722 vertically connected to the magnetic rod seat 721, wherein the magnetic rod assembly 72 includes a plurality of rows of magnetic rods 722 arranged at intervals; correspondingly, the bar magnet sleeve assembly 73 includes a bar magnet sleeve base 731 and a bar magnet sleeve 732 vertically connected to the bar magnet sleeve base 731, wherein the bar magnet sleeve assembly 73 includes a plurality of rows of bar magnet sleeves 732 arranged at intervals, and the bar magnet 722 and the bar magnet sleeve 732 are arranged in opposite positions so that the bar magnet 722 can be inserted into the bar magnet sleeve 732 in the corresponding position.
The release process of the magnetic beads is as follows: when the magnetic rod sleeve 732 is inserted into a corresponding reaction tube, the magnetic rod 722 is removed from the magnetic rod sleeve 732, the magnetic rod sleeve 732 vibrates along the axial direction, and the vibration of the magnetic rod sleeve 732 is communicated with the liquid tension of the reaction liquid so that the magnetic beads are released from the surface of the magnetic rod sleeve 732 into the reaction tube.
The adsorption process of the magnetic beads is as follows: after the magnetic rod 722 is inserted into the magnetic rod sleeve 732, the magnetic rod sleeve 732 with the magnetic rod 722 is driven to slowly move up and down, the upper limit position of the movement of the magnetic rod sleeve 732 is that the bottom of the magnetic rod sleeve 732 reaches the liquid level, and the lower limit position of the movement of the magnetic rod sleeve 732 is that the magnetic rod sleeve 732 does not touch the bottom wall and the side wall of the reaction tube, so that the magnetic beads suspended in the reaction tube are sufficiently adsorbed.
The transfer process of the magnetic beads is as follows: the magnetic rod sleeve 732 with the magnetic beads is driven by the magnetic rod sleeve driving assembly 71 to move horizontally into another reaction tube after being taken out of the reaction tube, and the magnetic rod sleeve 732 should move slowly to avoid the magnetic beads from falling off during the transferring process.
As shown in FIGS. 3 and 4, the cross-sectional area of the movable base 13 is not smaller than the area of the continuous loading module 30, the TIP head module 50, the reagent storage module 60, and the at least two PCR modules 90, and a slide space 131 is provided in the movable base 13, and the pipetting module 80 is placed in the slide space 131 to move forward, backward, leftward, and rightward.
Specifically, the pipetting module 80 comprises a vertically arranged pipetting piece 81 and a pipetting piece driving assembly 82 connected to control the pipetting piece 81, wherein the pipetting piece driving assembly 82 is arranged in the sliding space 131 for sliding. Wherein the pipetting member driving assembly 82 comprises a pipetting member vertical driving member 821 for driving the pipetting member 81 to move up and down, and a pipetting member transverse driving member 822 arranged in the sliding space 131, wherein the pipetting member transverse driving member 822 is connected with the fixing member through a conveyor belt to realize the forward and backward stroke movement in the sliding space 131.
As shown in fig. 8, the continuous loading module 30 includes a loading reaction tube storage base 32 and loading reaction tubes 31 arranged side by side on the loading reaction tube storage base 31. It is particularly worth mentioning that the continuous sample loading module 30 of the present embodiment may be provided with 4 × 8 32 channels of sample loading reaction tubes 31, so that the sequencing library building instrument can realize 32 channels of sample loading detection because the present embodiment is provided with at least two PCR modules 90, and a sample for each sample loading can be placed in an independent PCR module 90 for reaction. Correspondingly, at least four PCR modules 90 are arranged side by side, and the samples in each row of loading reaction tubes 31 correspond to the individual PCR modules 90.
The TIP module 50 includes a TIP storage base 52 and TIP heads 52 disposed side by side on the TIP storage base 52.
The reagent storage module 60 includes a reagent bottle placement area 61 and a reaction tube placement area 62, which are independently arranged, wherein reagents required for an experiment, including (injector, ligand, DNA ligand, Amplification Mix, etc.), are placed in the reagent bottle placement area 61, and a plurality of reaction tubes are placed in the reaction tube placement area 62, so that the pipetting module 80 can move the required reagents into the reaction tubes for pretreatment (such as mixing, standing, resuspending, etc.).
The PCR module 90 comprises a plurality of rows of PCR reaction tubes 91, a second reaction tube 92 and a third reaction tube 93 which are arranged at intervals, wherein a temperature control component is arranged below the PCR module 90, a sample can be placed in the PCR reaction tube 91 for operations such as breaking, connecting, amplifying and purifying, and the like, and the PCR reaction in the PCR reaction tube 91 is the prior art and is not described herein; 85% ethanol for cleaning magnetic beads is placed in the second reaction tube 92, and in some schemes, at least two rows of second reaction tubes 92 are required to be arranged so as to clean the magnetic beads twice; an eluent is placed in the third reaction tube 93 for eluting the magnetic beads and releasing the magnetic beads, so that the entire set of pooling operations can be completed in each individual PCR module 90.
For example, the sample loading reaction tube storage base 31 of the present embodiment is provided with four horizontal rows of sample loading reaction tubes 31, and each horizontal row is provided with eight sample loading reaction tubes 31; correspondingly, a plurality of rows of TIP heads are arranged on the TIP head storage base 52, and eight TIP heads are arranged in each vertical row; correspondingly, eight PCR reaction tubes 91 are arranged on each vertical row of the PCR module 90, and the arrangement can ensure that each transfer position of the pipetting module 70 has a corresponding hole position.
In some embodiments, the sequencing library builder further comprises a nucleic acid extraction module, wherein the nucleic acid extraction module can be used for extracting nucleic acid from the sample, and the sample after nucleic acid extraction is transferred to the PCR module 90 for reaction. Since magnetic bead purification is also required in the nucleic acid extraction module, the nucleic acid extraction module of this embodiment is disposed at the side of the PCR module 90, and preferably, the nucleic acid extraction module and the PCR module 90 are disposed in the same row. The magnetic sleeve module slide 14 is disposed inside the nucleic acid extraction module and the PCR module 90. The process of nucleic acid extraction is prior art and will not be described herein in great detail.
In order to collect waste liquid and waste tips, the sequencing library building instrument comprises a waste tip box and a waste tip box which are arranged on a base 12, and the waste tip box are arranged at the side position of a working space 100.
In addition, in some embodiments, the casing 10 of the sequencing library building instrument includes a casing 11 disposed outside the working space 100, wherein the casing 11 is provided with a sample inlet opening 111, and the sample inlet opening 111 is disposed corresponding to a position of the continuous sample injection module 30, so that a user can realize continuous sample injection through the sample inlet opening 111.
In addition, in order to guarantee that the sequencing of sample advances the appearance process and arranges in under the pollution-free environment and go on, the sequencing of this scheme builds storehouse appearance and further includes the filtration module 20 of arranging in workspace 100 top side, wherein filtration module 20 includes air cleaner 21, in order to filter external air, it is corresponding, the four sides side on base 12 surface is equipped with base air outlet 22, the avris of casing 11 is equipped with lateral wall air outlet 23, the setting of this mode can make the air after air cleaner 21 filters oppress downwards with the mode of laminar flow, prevent that external air from getting into workspace 100, in order to reduce the production of pollution.
It is particularly worth mentioning that, because the sequencing library building instrument of this scheme can carry out the continuous sampling, promptly, user's accessible introduction opening 111 adds new sample to in the continuous sampling module 30, and the air filter module 20 of this scheme cooperates the setting of lateral wall air outlet 23 and base air outlet 22, plays the effect with the air escape in the workspace 100, can avoid the pollution that the application of sample in-process probably brought.
According to a second aspect of the present invention, there is provided a method for operating more than one continuous sample injection sequencing library building instrument, that is, a method for performing sequencing library building by using the above sequencing library building instrument, the method for operating the continuous sample injection sequencing library building instrument includes the following steps:
adding a sample into a sample feeding reaction tube 31 of the continuous sample feeding module 30;
after the pipetting module 80 is driven to remove a sample from the sample loading reaction tube 31 and add the sample to the PCR reaction tube 91 of the PCR module 90, the pipetting module 80 is driven to move to the TIP head module 50 to replace the TIP head 51 of the pipetting piece 81, the pipetting module 80 is driven to move to the reagent storage module 60 to suck reaction liquid and add the reaction liquid to the corresponding PCR reaction tube 91, the PCR reaction tube 91 is set according to the program for building a library, and after the library building is finished, the pipetting module 80 is driven to the reagent storage module 60 to suck magnetic beads into the PCR reaction tube 31;
the magnetic rod sleeve module 70 is driven to extend into the corresponding PRC reaction tube 91 to adsorb magnetic beads, and at the moment, the magnetic rod assembly 72 is sleeved in the corresponding magnetic rod sleeve assembly 73;
and driving the magnetic rod sleeve module 70 to transfer the magnetic beads to the second reaction tube 92 for cleaning, taking out and drying, and driving the magnetic rod sleeve module 70 to transfer the magnetic beads to the third reaction tube 93 for elution and release of the magnetic beads.
It should be noted that the sample injection of the next batch of samples can be performed in the library establishment process of the previous-sequence samples, the above steps are repeated, and each batch of samples is placed in the independent PCR module 90 for library establishment. In the operation method of the continuous sample introduction sequencing library building instrument, an operator can add 8 sample loading samples to the sample loading reaction tube 31 every time, and the consumable materials in the sequencing library building instrument are replaced after the samples are added for 4 times, so that the library building time of the samples can be greatly saved.
In the process of driving the pipetting module 80 to move to the reagent storage module 60 to suck the reaction solution and the magnetic beads to be added into the corresponding PCR reaction tubes 91, the pipetting module 80 can firstly pipette the reagents in the reagent bottle placing region 61 into the reaction tubes in the reaction tube placing region 62 for mixing, standing and the like, and then transfer the reagents into the PCR reaction tubes 91. It should be noted that the pipetting module 80 moves to the TIP head module 50 to replace the corresponding TIP head after transferring the reagent each time, so as to avoid cross contamination during pipetting.
In addition, pipetting module 80 moves to waste tips box and the position of waste tips box during pipetting and discards waste fluids and waste tips.
In the process of driving the magnetic rod sleeve module 70 to extend into the corresponding PRC reaction tube 91 to adsorb the magnetic beads: after the magnetic rod assembly 72 is first inserted into the corresponding magnetic rod sleeve assembly 73, the magnetic rod sleeve 732 with the magnetic rod 722 is driven to be inserted into the corresponding PCR reaction tube 91 for adsorbing magnetic beads.
In the process of driving the magnetic rod sleeve module 70 to transfer the magnetic beads to the second reaction tube 92 for cleaning, taking out and airing: firstly, the pipetting module 80 is driven to pipette 85% ethanol from the reagent storage module 60 and add the ethanol into the second reaction tube 92, and the magnetic rod sleeve assembly 73 is driven to extend into the 85% ethanol in the second reaction tube 92 for cleaning. In some embodiments, two rows of second reaction tubes 92 are provided to repeat the washing of the magnetic beads twice.
And driving the magnetic rod sleeve module 70 to transfer the magnetic beads to the third reaction tube 93 for elution and release of the magnetic beads: firstly, the pipetting module 80 is driven to pipette eluent from the reagent storage module 60 and add the eluent into the third reaction tube 93, the magnetic rod sleeve component 73 is driven to extend into the eluent in the third reaction tube 93 for elution, a magnetic block is arranged below the third reaction tube 93, and the magnetic beads are released in a manner described in the release process of the magnetic beads.
In some embodiments, if the sample requires nucleic acid extraction, the method of operating the continuous feed sequencing library builder comprises the steps of:
adding a sample into a sample feeding reaction tube 31 of the continuous sample feeding module 30;
the liquid-transferring module 80 is driven to transfer a sample from the sample-loading reaction tube 31 and add the sample into the nucleic acid extraction module for nucleic acid extraction, the middle cracking liquid hole is used for nucleic acid extraction,
and the nucleic acid extraction module is preloaded with lysis solution and washing solution, the magnetic rod sleeve module 70 is firstly driven to be transferred into the lysis solution of the nucleic acid extraction module, and then the magnetic rod sleeve module 70 is driven to extend into the corresponding lysis solution to adsorb magnetic beads. In the process, after the magnetic rod assembly 72 is firstly driven to be inserted into the corresponding magnetic rod sleeve assembly 73, the magnetic rod sleeve 732 with the magnetic rod 722 is driven to be inserted into the corresponding lysis solution to adsorb magnetic beads, then the magnetic rod sleeve module 70 is driven to be transferred into the hole where the first washing solution is located, the magnetic rod sleeve module 70 is driven to be transferred into the hole where the second washing solution is located after standing for 30s, after the magnetic rod sleeve module 70 is standing for 30s, the magnetic rod sleeve module 70 is lifted and suspended over the hole where the second washing solution is located for 3min, the magnetic rod sleeve module 70 is transferred to the PCR module 91 (the PCR tube of the module contains eluent), after the magnetic beads are taken out, the magnetic beads are released to the eluent by oscillating and mixing for standing for 5min, after the magnetic rod 722 is inserted into the magnetic rod sleeve 732, the magnetic rod sleeve module 70 is driven to be transferred to the second washing solution hole after the magnetic beads are adsorbed, after the magnetic beads are diluted. And then the pipetting module 80 is driven to transfer the sample to the PCR reaction tube 91, and then the PCR reaction tube 91 of the PCR module 90 is driven.
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. A sequencing and library building instrument for continuous sample introduction is characterized by at least comprising: a working space (100), wherein the bottom side of the working space (100) is provided with a continuous sample loading module (30), a TIP head module (50), a reagent storage module (60) and at least two PCR modules (90), a liquid transfer module (80) and a magnetic rod sleeve module (70) are movably arranged in the working space (100), the liquid transfer module (80) reciprocates in the space where the continuous sample loading module (30), the TIP head module (50), the reagent storage module (60) and the at least two PCR modules (90) are located, the magnetic bar sleeve module (70) reciprocates in the space where the PCR modules (90) are located, wherein the continuous sample loading module (30) at least comprises a plurality of rows of sample loading reaction tubes (31) which are arranged at intervals, the magnetic rod sleeve module (70) comprises a magnetic rod assembly (72) and a magnetic rod sleeve assembly (72), and magnetic beads are adsorbed and released through the matching motion of the magnetic rod assembly (72) and the magnetic rod sleeve assembly (73).
2. The continuous-feed sequencing library builder of claim 1, wherein the continuous-feed modules (30) comprise at least 4-by-8 sets of feed reaction tubes (31), at least four PCR modules (90) are arranged side by side, and the samples in each set of feed reaction tubes (31) correspond to the individual PCR modules (90).
3. The continuous-sampling sequencing library building instrument according to claim 1, wherein the moving tracks of the pipetting module (80) and the magnetic rod sleeve module (70) are respectively arranged in different space motions.
4. The continuous sample introduction sequencing library building instrument according to claim 1, wherein a movable seat (13) is arranged at a position of the working space (100) close to the top side, and the pipetting module (80) is placed on the movable seat (13) to move randomly in the transverse direction and the longitudinal direction; the magnetic bar sleeve module sliding rail (14) is arranged at the bottom side of the working space (100), and the magnetic bar sleeve module (70) is arranged on the magnetic bar sleeve module sliding rail (14) and moves along the rail direction of the magnetic bar sleeve module sliding rail (14).
5. The continuous-sampling sequencing library building instrument according to claim 3, wherein the magnetic rod sleeve module (70) comprises a magnetic rod sleeve driving assembly (71), a magnetic rod assembly (72) and a magnetic rod sleeve assembly (73), the magnetic rod sleeve driving assembly (71) drives the magnetic rod assembly (72) and the magnetic rod sleeve assembly (73) to move along the magnetic rod sleeve module slide rail (14) and drives the magnetic rod assembly (72) and the magnetic rod sleeve assembly (73) to move vertically by independent movement strokes.
6. The continuous sample introduction sequencing library building instrument according to claim 1, wherein each PCR module (90) at least comprises a PCR reaction tube (91), a second reaction tube (92) and a third reaction tube (93) which are arranged at intervals, wherein a cleaning solution for cleaning magnetic beads is placed in the second reaction tube (92), and an eluent for eluting magnetic beads is placed in the third reaction tube (93).
7. The continuous sample injection sequencing library builder as claimed in claim 1, comprising a nucleic acid extraction module disposed at the side of the PCR module (90), wherein the nucleic acid extraction module and the PCR module (90) are disposed in the same row. The magnetic rod sleeve module slide rail (14) is arranged on the inner sides of the nucleic acid extraction module and the PCR module (90).
8. A method of operating a continuous sample sequencing library builder as claimed in any one of claims 1 to 7, comprising the steps of: adding a sample into a sample feeding reaction tube (31) of a continuous sample feeding module (30); after a sample is moved from the sample loading reaction tube (31) to a PCR reaction tube (91) of the PCR module (90) by driving the liquid moving module (30), the liquid moving module (80) is driven to move to the TIP head module (50) to replace the TIP head (51), the liquid moving module (80) is driven to move to the reagent storage module (60) to absorb reaction liquid and add the reaction liquid into the corresponding PCR reaction tube (91), the reaction liquid is set in the PCR reaction tube (91) according to a program to build a library, and after the library is built, the liquid moving module (80) is driven to the reagent storage module (60) to absorb magnetic beads into the PCR reaction tube (91); the magnetic rod sleeve module (70) is driven to extend into the corresponding PRC reaction tube (91) to adsorb magnetic beads; and driving the magnetic rod sleeve module (70) to transfer the magnetic beads to a second reaction tube (92) of the PCR module (90) for cleaning, taking out and airing, and driving the magnetic rod sleeve module (70) to transfer the magnetic beads to a third reaction tube (93) of the PCR module (90) for elution and release of the magnetic beads.
9. The method of claim 8, wherein the next sample is injected during the process of preparing the library of the previous sample, and the above steps are repeated, and each sample is placed in an independent PCR module (90) for preparing the library.
10. The method of claim 8, comprising the steps of, before transferring the sample to the PCR reaction tube (91): adding a sample into a sample feeding reaction tube (31) of a continuous sample feeding module (30); the pipetting module (80) is driven to remove samples from the sample loading reaction tube (31) and add the samples to the lysis solution holes in the nucleic acid extraction module for nucleic acid extraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011176704.6A CN112175943B (en) | 2020-10-29 | 2020-10-29 | Continuous sample introduction sequencing library building instrument and operation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011176704.6A CN112175943B (en) | 2020-10-29 | 2020-10-29 | Continuous sample introduction sequencing library building instrument and operation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112175943A true CN112175943A (en) | 2021-01-05 |
| CN112175943B CN112175943B (en) | 2022-07-15 |
Family
ID=73916217
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011176704.6A Active CN112175943B (en) | 2020-10-29 | 2020-10-29 | Continuous sample introduction sequencing library building instrument and operation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112175943B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115197833A (en) * | 2022-08-22 | 2022-10-18 | 杭州柏炬科技有限公司 | Automatic warehouse building instrument and method for step sampling |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103562727A (en) * | 2011-05-30 | 2014-02-05 | 株式会社日立高新技术 | Sample treatment device, sample treatment method, and reaction container for use therein |
| CN106029885A (en) * | 2014-02-10 | 2016-10-12 | 基因细胞生物系统有限公司 | Composite liquid cell (clc) mediated nucleic acid library preparation device, and methods for using the same |
| US20170022558A1 (en) * | 2007-10-30 | 2017-01-26 | Complete Genomics, Inc. | Integrated system for nucleic acid sequence and analysis |
| CN108034565A (en) * | 2017-12-29 | 2018-05-15 | 广州市金圻睿生物科技有限责任公司 | Nucleic acid sequencing pre-treatment instrument |
| CN108410859A (en) * | 2018-04-28 | 2018-08-17 | 绍兴迅敏康生物科技有限公司 | Method for extracting nucleic acid |
| CN108441423A (en) * | 2018-04-28 | 2018-08-24 | 绍兴迅敏康生物科技有限公司 | Instrument for extracting nucleic acid |
| CN108603155A (en) * | 2016-09-23 | 2018-09-28 | 深圳华大智造科技有限公司 | A kind of system being used to prepare gene sequencing sample and its application |
| CN108642045A (en) * | 2018-05-08 | 2018-10-12 | 绍兴迅敏康生物科技有限公司 | A kind of bar magnet set |
| CN109957492A (en) * | 2017-12-26 | 2019-07-02 | 安诺优达基因科技(北京)有限公司 | A kind of automatic fluid processing workstation for two generations sequencing DNA library building |
| CN110846225A (en) * | 2019-10-10 | 2020-02-28 | 长春市博坤生物科技有限公司 | Automatic change nucleic acid extraction appearance |
| CN111440703A (en) * | 2020-05-19 | 2020-07-24 | 广州高盛生物科技股份有限公司 | Double-extraction-mode nucleic acid extraction workstation and nucleic acid extraction method |
-
2020
- 2020-10-29 CN CN202011176704.6A patent/CN112175943B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170022558A1 (en) * | 2007-10-30 | 2017-01-26 | Complete Genomics, Inc. | Integrated system for nucleic acid sequence and analysis |
| CN103562727A (en) * | 2011-05-30 | 2014-02-05 | 株式会社日立高新技术 | Sample treatment device, sample treatment method, and reaction container for use therein |
| CN106029885A (en) * | 2014-02-10 | 2016-10-12 | 基因细胞生物系统有限公司 | Composite liquid cell (clc) mediated nucleic acid library preparation device, and methods for using the same |
| CN108603155A (en) * | 2016-09-23 | 2018-09-28 | 深圳华大智造科技有限公司 | A kind of system being used to prepare gene sequencing sample and its application |
| CN109957492A (en) * | 2017-12-26 | 2019-07-02 | 安诺优达基因科技(北京)有限公司 | A kind of automatic fluid processing workstation for two generations sequencing DNA library building |
| CN108034565A (en) * | 2017-12-29 | 2018-05-15 | 广州市金圻睿生物科技有限责任公司 | Nucleic acid sequencing pre-treatment instrument |
| CN108410859A (en) * | 2018-04-28 | 2018-08-17 | 绍兴迅敏康生物科技有限公司 | Method for extracting nucleic acid |
| CN108441423A (en) * | 2018-04-28 | 2018-08-24 | 绍兴迅敏康生物科技有限公司 | Instrument for extracting nucleic acid |
| CN108642045A (en) * | 2018-05-08 | 2018-10-12 | 绍兴迅敏康生物科技有限公司 | A kind of bar magnet set |
| CN110846225A (en) * | 2019-10-10 | 2020-02-28 | 长春市博坤生物科技有限公司 | Automatic change nucleic acid extraction appearance |
| CN111440703A (en) * | 2020-05-19 | 2020-07-24 | 广州高盛生物科技股份有限公司 | Double-extraction-mode nucleic acid extraction workstation and nucleic acid extraction method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115197833A (en) * | 2022-08-22 | 2022-10-18 | 杭州柏炬科技有限公司 | Automatic warehouse building instrument and method for step sampling |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112175943B (en) | 2022-07-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110564607B (en) | Full-automatic nucleic acid extraction amplification micro-fluidic chip dynamic quantitative detection integrated device | |
| US9187725B2 (en) | Automatic biological sample purification apparatus equipped with magnetic filed applying part, method of extracting target substance from biological sample, and protein expression and purification method | |
| JP5650056B2 (en) | Sample processing apparatus and sample processing method | |
| CN202830011U (en) | Automated nucleic acid extraction platform | |
| CN102492603B (en) | Automated instrument for nucleic acid extraction and mass spectrum sample application | |
| CN206566549U (en) | Full-automatic solid phase extraction cleaning system | |
| JP2004229657A (en) | Kit for separating and purifying nucleic acid or various biological materials, and system for automated operation of the same | |
| EP1882948A2 (en) | Sample processing device | |
| CN205590667U (en) | Biological sample preparation system | |
| CN107345199A (en) | Biological sample preparation system | |
| CN112175943B (en) | Continuous sample introduction sequencing library building instrument and operation method thereof | |
| CN208964916U (en) | A kind of magnetic beads for purifying instrument of enrichment method | |
| CN108034565A (en) | Nucleic acid sequencing pre-treatment instrument | |
| KR101282841B1 (en) | Apparatus and method for isolating nucleicacids or biological materials | |
| CN109675649A (en) | A kind of liquid transcription device | |
| WO2016031705A1 (en) | Magnetic particle reaction control apparatus utilizing variable-pitch dispensing apparatus and reaction control method therefor | |
| CN213680637U (en) | Continuous sample introduction sequencing library building instrument | |
| CN210458215U (en) | Instrument for extracting and purifying nucleic acid by magnetic bead method | |
| CN108315243B (en) | Automatic sample adding system | |
| WO2023284844A1 (en) | Automatic extraction device and use method therefor | |
| CN211170706U (en) | Nucleic acid extraction instrument for batch samples | |
| CN114410427A (en) | Large-volume full-automatic nucleic acid extraction method and device | |
| CN115178315A (en) | Full-automatic high-flux workstation integration device | |
| CN115197833A (en) | Automatic warehouse building instrument and method for step sampling | |
| CN112011533A (en) | Integrated system for extracting and detecting nucleic acid and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |