CN112844151A - Nucleic acid extraction equipment and mixing device based on eccentric oscillation - Google Patents
Nucleic acid extraction equipment and mixing device based on eccentric oscillation Download PDFInfo
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- CN112844151A CN112844151A CN202011355163.3A CN202011355163A CN112844151A CN 112844151 A CN112844151 A CN 112844151A CN 202011355163 A CN202011355163 A CN 202011355163A CN 112844151 A CN112844151 A CN 112844151A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
- B01F31/20—Mixing the contents of independent containers, e.g. test tubes
- B01F31/201—Holders therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
- B01F31/20—Mixing the contents of independent containers, e.g. test tubes
- B01F31/265—Mixing the contents of independent containers, e.g. test tubes the vibrations being caused by an unbalanced rotating member
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F2101/00—Mixing characterised by the nature of the mixed materials or by the application field
- B01F2101/23—Mixing of laboratory samples e.g. in preparation of analysing or testing properties of materials
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to nucleic acid extraction equipment and a mixing device based on eccentric oscillation, and belongs to the technical field of nucleic acid extraction. The blending device comprises a mounting bracket and at least a pipe mounting support used for mounting a blending pipe in an unloading way; the mixing vibration device is arranged on the mounting bracket, is positioned on the lower side of the pipe mounting support and comprises a vibration transmission sleeve seat sleeved on the lower end part of the mixing pipe and a vibration generator for applying vibration to the mixing pipe through the vibration transmission sleeve seat; the vibration generator comprises a rotary driving shaft and an eccentric transmission shaft which are vertically arranged, and the eccentric transmission shaft comprises a first external connection shaft part and a second external connection shaft part which are fixedly connected into an integral structure; the central axes of the installation circumferential surfaces of the first outer coupling shaft part and the second outer coupling shaft part have eccentric distance at the fixed connection part of the first outer coupling shaft part and the second outer coupling shaft part, and the central axes of the first outer coupling shaft part and the second outer coupling shaft part form an acute angle. The mixing device can effectively improve the mixing effect and efficiency of nucleic acid, and can be widely applied to the field of epidemic prevention and control such as detection of new coronavirus.
Description
Technical Field
The invention relates to the technical field of nucleic acid extraction, in particular to nucleic acid extraction equipment and a mixing device which can be used for constructing the nucleic acid extraction equipment and is based on eccentric oscillation.
Background
At present, two detection methods for the novel coronavirus mainly exist, and specifically include a rapid detection method and a PCR detection method; wherein, PCR (polymerase chain reaction) detection method can be used for detecting the genetic material fragment of pathogen or microorganism, and is used for detecting whether a person is infected at the beginning of epidemic situation, and the RT-PCR detection of the nucleic acid positive of the novel coronavirus and the virus gene sequencing homology are listed as the determination standard of the novel coronavirus in the diagnosis scheme (trial seventh edition) issued by Weijian Commission of China; for the rapid detection method based on antibody detection, it can perform rapid detection, and is easy to operate and widely used, but it usually requires 1-2 weeks after infection to detect the result, and it is necessary to make a diagnosis of a part of patients who are negative in nucleic acid detection using clinical symptoms and image representation.
In the PCR detection method, nucleic acid needs to be separated and purified, namely, the nucleic acid needs to be extracted; at present, the extraction mode of nucleic acid mainly comprises a strong salt method, an organic solvent extraction method, a density gradient centrifugation method, an adsorption material combination method and the like; the adsorption material combination method mainly comprises a siliceous material method, an anion exchange resin method and a magnetic bead method, wherein the magnetic bead method is widely applied in the adsorption material combination methods, the working mechanism of the magnetic bead combination method is that magnetic bead particles are utilized to wrap specific groups so as to specifically adsorb a target object, and then the magnetic bead particles are separated from a biological sample so as to achieve the purpose of extracting specific nucleic acid.
If the magnetic bead method is used for extracting nucleic acid from a sample, the time consumption is long if the nucleic acid is completely manually operated, the manual labor intensity is high, and the nucleic acid is required to be operated by professional personnel, so that the requirement on rapid detection of the novel coronavirus is not met; nucleic acid extraction equipment is usually adopted for automatic extraction, so that the time consumption of nucleic acid extraction is greatly shortened; for example, an automatic nucleic acid extraction instrument disclosed in patent document No. CN103897987A is used, which includes a base, a motor, a magnetic rod clamping mechanism, a stirring sleeve clamping mechanism, a deep hole plate, a control circuit, and an upper computer; in the working process, adding lysis solution and a sample into a lysis groove of a deep hole plate, adding magnetic beads into a magnetic bead groove, adding washing solution into a washing groove, and adding eluent into an elution groove; then inserting the stirring sleeve into the stirring sleeve clamp; the method comprises the steps that through driving a deep hole plate to move transversely, solutions carried by different grooves in the deep hole plate are moved to the side located right below a stirring sleeve, the solutions are stirred based on the stirring sleeve to be uniformly mixed and the cracking speed is improved, and after nucleic acid is combined with the magnetic beads, magnetic rods sleeved in the stirring sleeve are used for attracting the magnetic beads, so that the nucleic acid is extracted from nucleic acid cracking liquid; the nucleic acid extraction equipment improves the cracking speed based on the stirring of the magnetic rod, but also has the problem of poor mixing effect and mixing efficiency.
In addition, the aforementioned devices also generally have the problem of low automation level, and require more manual operations during the detection process.
Disclosure of Invention
The invention mainly aims to provide a blending device based on eccentric oscillation, so that a reagent contained in a blending pipe can vibrate in an omnidirectional space in the blending process, and the blending effect and the blending efficiency of target objects such as nucleic acid, magnetic beads and the like are improved;
the second purpose of the invention is to provide a nucleic acid extraction device constructed by the blending device;
it is a third object of the present invention to provide a nucleic acid extraction apparatus with an improved structure to improve the degree of automation of nucleic acid extraction.
In order to achieve the main purpose, the blending device provided by the invention comprises a mounting bracket and at least a pipe mounting support which is used for detachably mounting a blending pipe; the mixing vibration device is arranged on the mounting bracket, is positioned on the lower side of the pipe mounting support and comprises a vibration transmission sleeve seat sleeved on the lower end part of the mixing pipe and a vibration generator for applying vibration to the mixing pipe through the vibration transmission sleeve seat; the vibration generator comprises a rotary driving shaft and an eccentric transmission shaft which are vertically arranged, and the eccentric transmission shaft comprises a first external connection shaft part and a second external connection shaft part which are fixedly connected into an integral structure; the central axes of the installation circumferential surfaces of the first outer coupling shaft part and the second outer coupling shaft part have eccentric distance at the fixed connection part of the first outer coupling shaft part and the second outer coupling shaft part; the first external coupling shaft part is in transmission connection with the rotary driving shaft, and the second external coupling shaft part is rotatably sleeved and connected with the vibration transmission sleeve seat through a bearing; and the central axes of the two outer coupling shaft parts form an acute angle beta.
In above-mentioned technical scheme, based on eccentric drive shaft and the contained angle setting that has for the acute angle, the central axis of two outer antithetical couplet axial regions promptly has the interval in the axial region junction, two central axis nonparallels set up, thereby can make and have eccentric interval between the axis of target reagent pipe and the axis of rotation drive axle, and the two nonparallel arranges and has contained angle beta to be the acute angle, thereby can apply omnidirectional vibration to the mixing pipe, in order to improve the schizolysis/mixing speed of drawing the in-process effectively, thereby improve mixing effect and mixing efficiency effectively, in order to improve nucleic acid extraction speed.
The specific scheme is that the value range of the acute angle beta is more than 0 and less than or equal to gamma,
wherein R is the inner diameter of the mixing pipe, rho is the average density of substances in the mixing pipe, R is the driving rotating speed of the rotating driving shaft, t is the sufficient mixing time, m is the preset eccentric distance, and c is a proportionality constant. Tests prove that even though the included angle is larger than 0, the blending effect and efficiency can be effectively improved, but when the value range of the technical scheme is adopted, the blending effect and efficiency can be obviously improved compared with the value arrangement of other parts.
More specifically, the value range of the acute angle beta is more than 0 and less than or equal to 3.6 degrees. When the included angle is within the range, the preferable blending efficiency and blending effect can be ensured, the inclination angle of the blending pipe in the blending oscillation process can be effectively reduced, and the effective capacity ratio in the blending process is ensured.
The preferred proposal is that the value range of the acute angle beta is more than or equal to 1.5 degrees and less than or equal to 3.6 degrees.
A preferred embodiment has a proportionality constant C of 1.3.
The preferred solution is that the tube mounting support is used for mounting the mixing tube in a suspended manner. The technical scheme is convenient for placing and taking out the reagent tube in the nucleic acid extraction process, thereby further improving the extraction speed of the nucleic acid.
The preferred arrangement is a coplanar arrangement of the central axes of the two outer coupling parts.
The first external coupling shaft part is provided with a shaft hole for sleeving the outside of the rotary driving shaft, and the circumferential surface of the shaft hole forms the installation circumferential surface of the first external coupling shaft part; the second external coupling shaft part is sleeved on an inner ring of the bearing, and the lower end part of the transfer sleeve seat is provided with a sleeve hole sleeved on an outer ring of the bearing; the sleeving ring surface of the inner ring forms the installation circumferential surface of the second outer coupling shaft part.
Preferably, the rotary drive shaft is a rotor shaft of a rotary drive motor.
The preferable scheme is that the second outer coupling shaft part and a sleeve hole which is arranged on the vibration transmission sleeve seat and used for sleeving the target reagent tube are arranged on the same central axis.
In order to achieve the second object, the nucleic acid extraction device provided by the invention performs detection based on a magnetic bead method, and specifically comprises a bracket, and a mixing device and a magnetic attraction device which are arranged on the bracket; wherein, the blending device is the blending device described in any one of the above technical schemes; the magnetic attraction device comprises a magnetic attraction module and a displacement module, wherein the displacement module is used for driving the magnetic attraction module to be close to or far away from the outer wall surface of the mixing pipe. In the technical scheme, based on the matching of the blending device and the magnetic suction device, the separation operation scheme can be effectively simplified, and the magnetic rod sleeve does not need to be replaced.
In order to achieve the third object, the invention provides a specific scheme that the nucleic acid extraction equipment comprises a liquid transfer system arranged on a bracket, wherein the liquid transfer system is used for adding a reagent into a mixing tube and sucking the reagent from the mixing tube; the liquid transfer system comprises a suction pipe joint for detachably mounting the suction head and a lifting driving mechanism for driving the suction pipe joint to lift relative to the bracket. The automation degree in the operation process can be effectively improved.
The more specific scheme is that the liquid transfer system comprises an unloading mechanism, the unloading mechanism is used for pushing the suction heads sleeved on the suction pipe connectors down, the unloading mechanism comprises a material pushing plate and a linear displacement output device, the linear displacement output device is used for driving the material pushing plate to reciprocate vertically, and the material pushing plate is provided with a sleeving hole sleeved outside the suction pipe connectors. Thus, the disposable tip can be automatically unloaded after the nucleic acid extraction is completed, and the automation degree of the nucleic acid extraction can be further improved.
The preferable scheme is that the pipe mounting support can be rotatably mounted on the mounting bracket around a first vertical axis; a plurality of sleeving openings which are arranged around the first vertical axis are distributed on the outer peripheral part of the pipe mounting support and are used for hanging the mixing pipe, other reagent pipes and the suction head in a manner of being pulled out from the upper side; the blending device comprises a rotary driver for driving the tube mounting support to rotate around a first vertical axis, and the tube mounting support can rotate until the reagent tubes and the suction heads carried by the tube mounting support are positioned under the suction pipe joint one by one; the shifting module is used for driving the magnetic suction module to be close to or far away from the outer wall surface of the mixing pipe which is positioned right below the suction pipe joint in a rotating mode.
In the above technical solution, a reagent tube group including a reagent tube and a pipette tip, which is composed of a sample tube, a lysis solution tube, a waste solution tube, and the like required for nucleic acid extraction, is mounted on a tube mounting support and is circumferentially arranged around a first vertical axis, and in the extraction process, the tube mounting support is driven by a rotary driver to rotate until a target reagent tube is positioned below the pipette tip, and the pipette tip sleeved on the pipette tip is used for sucking or injecting a target solution from or into the target reagent tube, thereby completing the pipetting work required for nucleic acid extraction, and the relative position adjustment of the pipetting process is established based on the rotation setting of the reagent tube group and the lifting setting of the pipette tip, compared with the prior art, the whole size can be effectively reduced, and the nucleic acid extraction of a small amount of samples is facilitated; the cracking/mixing speed in the extraction process can be improved based on the vibration applied by the mixing oscillation device, and the nucleic acid extraction speed is effectively improved; in the waste liquid absorbing process, the magnetic suction module is convenient to separate from the test tube by approaching or separating from the outer side of the test tube wall, and the displacement module can move with only a single degree of freedom at least so as to be convenient to position and control.
Drawings
FIG. 1 is a side view of a nucleic acid extraction apparatus in an embodiment of the present invention;
FIG. 2 is a perspective view of a nucleic acid extraction apparatus according to an embodiment of the present invention;
FIG. 3 is a top view of a nucleic acid extraction apparatus according to an embodiment of the present invention;
FIG. 4 is a perspective view of the nucleic acid extraction apparatus of the embodiment of the present invention, with the reagent tube holder and the mixing oscillator omitted;
FIG. 5 is an enlarged view of a portion A of FIG. 4;
FIG. 6 is a perspective view of a magnetic attraction device in an embodiment of the present invention;
FIG. 7 is a rear view of the magnetic attraction device, the mixing oscillation device, the reagent tube rack, and the reagent tube and reagent tube sheathing trays mounted on the reagent tube rack in the embodiment of the present invention;
FIG. 8 is a perspective view of an eccentric drive shaft in an embodiment of the present invention;
FIG. 9 is a side view of the magnetic attraction device, the mixing oscillation device, the reagent tube rack, and the reagent tube and reagent tube sleeve tray mounted on the reagent tube rack according to the embodiment of the present invention;
fig. 10 is a perspective view of a rotation angle detection mechanism of the kneading oscillation device, the reagent tube holder, and the disc-shaped cassette in the embodiment of the present invention;
FIG. 11 is a structural diagram of a mixing oscillator and a mixing tube sleeved in a vibration transmission sleeve seat thereof according to an embodiment of the present invention;
FIG. 12 is a bottom side view of a reagent tube housing tray with a plurality of reagent tubes and a single pipette tip mounted thereon according to an embodiment of the present invention;
fig. 13 is an axial sectional view of an eccentric drive shaft in an embodiment of the invention;
FIG. 14 is an enlarged view of a portion B of FIG. 11;
FIG. 15 is an axial cross-sectional view of a pipetting system in an embodiment of the invention;
FIG. 16 is a perspective view of a nucleic acid isolation apparatus according to an embodiment of the present invention, and a reagent cartridge, a tip, and a reagent cartridge tray mounted thereon.
Detailed Description
The invention is further illustrated by the following examples and figures.
Examples
The nucleic acid extraction equipment of the invention extracts nucleic acid from a sample based on a magnetic bead method, and the specific structure is shown in fig. 1-5 and fig. 16, the nucleic acid extraction equipment 1 comprises a bracket 2, a shell, a control unit, a reagent tube rack 3 arranged on the bracket 2, a liquid transfer system 4, a uniform mixing oscillation device 5 and a magnetic suction device 6. Most of the surface areas of the bracket 2, the reagent tube rack 3, the liquid transferring system 4, the blending oscillation device 5 and the magnetic suction device 6 are covered in the shell, and the shell is provided with an opening and closing door for protecting an operation area; the control unit comprises a processor, a memory electrically connected with the processor and a touch screen, wherein the touch screen is arranged on the shell and used for receiving operation instructions and displaying relevant parameters, such as a nucleic acid extraction process and the like. Wherein, the reagent tube frame 3 and the mixing oscillation device 5 together form the nucleic acid mixing device in the embodiment.
As shown in fig. 1 to 4, the bracket 2 includes a horizontal mounting base plate 20 and a vertical mounting plate 21 arranged vertically, and the two are fixedly connected at a joint by fasteners such as bolts or by welding; in this embodiment, the bracket 2 is formed by welding a plurality of steel plate members, and the cross section of the vertical mounting plate 21 parallel to the horizontal plane is of a zigzag structure. A control circuit board 10 containing a processor and a memory is mounted on the rear plate surface of the vertical mounting plate 21.
As shown in fig. 1 to 6, the pipetting system 4 includes an elevating platform 40 vertically reciprocally and elevatably mounted on the front plate surface of the vertical mounting plate 21 by a rail slider mechanism 46, a pipette tip 41 fixedly mounted on the elevating platform 40 and detachably mounting the pipette tip 01, a linear displacement output device 42 for driving the elevating platform 40 to move up and down relative to the rack 2, a discharge mechanism 43 for discharging and pushing out the pipette tip 01 detachably mounted on the pipette tip 41, a pipetting pump 44 for providing a suction force and a pumping force to the pipette tip 41, and an air pipe 45 for communicating the pipette tip 41 with the pipetting pump 44. Wherein, the suction head is also called TIP head, which is in interference fit with the suction connector 41 and is detachably connected in a sleeving way.
The lifting platform 40, the guide rail slider mechanism 41 and the linear displacement output device 42 together form a lifting driving mechanism in this embodiment, for driving the suction pipe joint 41 to move up and down relative to the support 2. The linear displacement output device 42 may be constructed by a cylinder, an oil cylinder, a linear motor, or other linear displacement output devices, or by a rotation driving motor cooperating with a rack or a four-bar nut mechanism, in this embodiment, in order to be constructed by a lead screw 421 and a lead screw nut 422 driven by a rotation driving motor 420, the lead screw nut 422 is fixedly connected with the lifting table 40; the rail slider mechanism 41 may be constructed using a linear rail, or may be constructed using a guide rod in cooperation with a guide hole provided on the elevating table 40.
The liquid transfer pump 44 can be constructed by a bidirectional pump capable of sucking air and pumping air, and is constructed by a peristaltic pump and a flexible pump pipe which rotate in two directions; in the present embodiment, in order to be constructed using the gas cylinder and the linear output device 442, the gas cylinder includes a cylinder body 441 having a movable piston built therein and a piston rod 440 for driving the piston to move in the cylinder body 441; the linear output device 442 is mounted on the front plate surface of the vertical mounting plate 21, and is specifically constructed by adopting a rotary driving motor and a feed screw nut mechanism, the lower end of the piston rod 440 is fixed on the transverse mounting plate 20, the cylinder 441 is fixedly connected with a feed screw nut of the feed screw nut mechanism, and the feed screw nut is vertically movably mounted on the vertical mounting plate 21 through a linear guide rail slider mechanism.
As shown in fig. 2 and 4, the unloading mechanism 43 is used for pushing down the suction head 01 sleeved on the suction pipe connector 41, and the unloading mechanism 43 specifically includes a material pushing plate 430 and a linear displacement output device 431 for driving the material pushing plate 430 to reciprocate vertically; the stator of the linear displacement output device 431 is fixed on the lifting table 40, and the mover 4310 is fixedly connected with the material pushing plate 430; the material pushing plate 430 is provided with a sleeving hole 4300 sleeved outside the suction pipe connector 41; the specific structure of the linear displacement output device 431 can be constructed by a linear motor or the like, and can also be constructed by an electromagnet and an armature, wherein the armature is driven by a return spring to move upwards to pull the material pushing plate 430 to move upwards to exit from the unloading position, and moves downwards to enter into the material pushing position under the action of the electromagnet to force the suction head 01 to exit from the suction pipe connector 41.
As shown in fig. 7, 9, 10, 12 and 16, the reagent tube rack 3 includes a rotary holder 7 and a rotary driver 30; in this embodiment, the rotary driver 30 is a rotary driving motor, which may be specifically constructed by a servo motor or a stepping motor; the rotary support 7 comprises a disc-shaped clamping seat 70 and a circular transmission seat 71 which are arranged in parallel, the circular transmission seat 71 is arranged below the disc-shaped clamping seat 70 at a preset interval and fixedly connected with the disc-shaped clamping seat 70 through a plurality of supporting transmission columns 72 so as to reduce the interference between a reagent tube and a suction head hung on the rotary support; the lower end part of the circular transmission seat 71 is fixedly provided with a rotating shaft 73 which is rotatably arranged on the door-shaped mounting seat 22 through a bearing; as shown in fig. 1, fig. 2 and fig. 4, the n-shaped mounting seat 22 is fixed on the transverse mounting plate 20, the rotary driver 30 is disposed in the n-shaped mounting seat 22, and a rotor shaft thereof is fixedly connected with the rotating shaft 73, or is in transmission connection with the rotating shaft through a coupling, or is in transmission connection with the rotating shaft through a speed reducer. The disc-shaped clamping seat 70 constitutes a tube mounting support in this embodiment, and the circular transmission seat 71 constitutes a mounting bracket in this embodiment.
On the outer peripheral edge of the disk-shaped cassette 70, a plurality of reagent tube set mouths 701-708 and tip set mouths 700 are arranged circumferentially around the first vertical axis 100, that is, the central axes of the set mouths are distributed on a cylindrical surface with the first vertical axis 100 as the central axis, so that reagent tubes and tips suspended thereon can be rotated one by one to a position right below the tip 01 by rotating the disk-shaped cassette 70; wherein, the caliber of the reagent tube sleeve opening 701 positioned right above the blending oscillation device 5 is larger than the calibers of other reagent tube sleeve openings; the caliber of the sucker sleeve opening 700 is smaller than the calibers of all reagent tube sleeve openings, so that the sucker sleeve opening can be used as a reference for sleeving the reagent tube and the sucker; in the present embodiment, the reagent tube housing port has a notch-like structure disposed on the outer peripheral edge of the disk-shaped cassette 70, and may be formed by using a through hole disposed on the disk-shaped cassette 70.
As shown in FIG. 7, FIG. 9 and FIG. 12, the consumable set 02 of the nucleic acid isolation apparatus 1 of the present invention includes a reagent tube housing tray 03 having a plurality of housing holes, and a reagent tube set and a tip 01 mounted in the housing holes and suspended on the reagent tube housing tray 03; in this embodiment, the reagent tube set includes a mixing tube 041, an elution tube 042, a waste tube 043 and 045, a cleaning solution tube 046, a product tube 047, a lysis tube 048, and a sample tube 049. In the process of mounting the consumable component 02 on the reagent tube rack 3, the mixing tube 041 is sleeved in the reagent tube sleeve opening 701 with a larger caliber in an aligned manner, and the suction head 01 is sleeved in the suction head sleeve opening 700 with a smaller caliber, so that the reagent tube sleeve tray 03 is supported on the disc-shaped clamping seat 70 in a sleeved manner, and the fastening connection mechanism 709 which passes through the central hole of the reagent tube sleeve tray 03 is matched with the bayonet on the disc-shaped clamping seat 70 to releasably and tightly press and fixedly connect the reagent tube sleeve tray 03 supported on the disc-shaped clamping seat 70; as for the fastening connection mechanism 709, detachable connection may be performed based on a screw connection, a snap structure.
Therefore, in the working process, the rotating support 7 is at least used for mounting a plurality of reagent tubes which are circumferentially arranged around the first vertical axis 100 and driven by the rotating driver 30 to rotate to the position that the reagent tubes carried by the rotating support are positioned under the pipette connector 41 one by one, in the process, in order to improve the positioning accuracy, the photoelectric sensor 15 is fixedly arranged on the door-shaped mounting seat 22, and the shielding piece 16 for shielding the light path of the photoelectric sensor 15 is fixedly arranged on the circular transmission seat 71, so that the rotating support 7 is used for positioning the rotating position in the weekly movement, the positioning function can be achieved based on the control of the rotation of the motor, and the more accurate positioning can be performed based on the encoder arranged on the rotating shaft; and the rotary support 7 is specifically used for hanging and installing the reagent tube, thereby facilitating the one-time installation and one-time extraction of the consumable assembly 02. Wherein the reagent tube set together with the tip 01 form a tube set in the present embodiment, which tube set is arranged circumferentially around the first vertical axis 100, i.e. their central axes are located on a cylindrical surface with the first vertical axis 100 as central axis; so that during operation the rotary support 7 is driven by the rotary drive 30 to rotate until the tube sets carried by it are located one by one directly below the suction attachment 41.
As shown in fig. 1, fig. 2 and fig. 10, the mixing oscillator 5 is disposed below the rotating support 7 and can rotate synchronously with the rotating support 7, specifically, the mixing oscillator 5 is fixed on the circular transmission seat 71 and located right below the reagent tube set port 701 for applying vibration to the mixing tube 041, thereby promoting the mixing and cracking processes and increasing the nucleic acid extraction speed.
As shown in fig. 8, 10, and 11, the kneading oscillation device 5 includes a vibration transmission holder 50 for fitting to the lower end portion of the target reagent tube, i.e., the kneading tube 041, and a vibration generator 51 for applying vibration to the kneading tube 041 via the vibration transmission holder 50. In this embodiment, the vibration transmission sleeve seat 50 is a cylindrical structure, and is sleeved on the lower end of the blending pipe 041 to form a supporting pipe for supporting, so as to transmit vibration at multiple angles.
The vibration generator 51 includes a rotation driving motor 52 and an eccentric transmission shaft 53 arranged in a vertical direction; among them, the rotor shaft 5200 of the rotation driving motor 52 constitutes a rotation driving shaft in this embodiment. The eccentric transmission shaft 53 includes a first externally coupled shaft portion 54 and a second externally coupled shaft portion 55 which are fixedly connected as an integral structure; the central axes 542 of the mounting circumferential surfaces of the first and second outer coupling shaft portions 54 and 55 are separated from the central axis 550 by a predetermined eccentric distance m where they are fixedly connected, and the central axes 542 and the central axis 550 are sandwiched by an acute angle β, i.e., are not arranged in parallel, in this embodiment, the central axes 542 and the central axis 550 are arranged in a coplanar manner; the first outer coupling shaft part 54 is provided with a shaft hole 540 for sleeving the rotor shaft of the rotary driving motor 52, and the connection relationship between the first outer coupling shaft part and the rotary driving motor is fixedly connected through a pin penetrating through the pin hole 541; the second outer coupling portion 55 is fitted over the inner race of the bearing 56, and the lower end portion of the transmission sleeve holder 50 is provided with a sleeve hole 500 fitted over the outer race of the bearing 56, so that the second outer coupling portion 55 is rotatably fitted over the vibration transmission sleeve holder 50 via the bearing 56.
After the assembly is completed, the second outer coupling portion 55 is arranged coaxially with a trepan hole for sheathing the target reagent tube on the vibration transmission hub 50.
In the embodiment, the included angle β between the two central axes is 0 < β ≦ γ,
wherein R is the inner diameter of the target reagent tube and the unit is mm; rho is the average density of the substances in the target reagent tube, and the unit is g/L; r is the driving speed of the rotary driving shaft, and the unit is revolution/min; t is the time of mixing, unit s; m is a preset eccentric distance and is in mm; c is a proportionality constant related to the mass of the oscillation device and the mixing tube.
The applicant has shown, through simulation and experiments, that under otherwise identical conditions, as shown in table 1 below, the time for thorough mixing is significantly shortened when the included angle β is greater than zero, and it should be noted that when the angle γ is greater than 3.6 °, the time is not significantly shortened, mainly because there is a minimum detection limit for the methodology used to test the time for thorough mixing, as detailed in table 2 below.
Table 1 experimental parameter settings
Wherein M1 is the mass of the mixing tube 01; v is the liquid volume in the mixing tube.
TABLE 2 blending time as a function of the angle
The inventor discovers through a large number of experiments and creative assumptions that under the condition of sufficient mixing time being fixed, the preset angle beta is positively correlated with the inner diameter R of the mixing pipe and the average density rho of substances in the mixing pipe, and is negatively correlated with the rotating speed R of the motor and the eccentricity m of the rotating shaft of the motor and the mixing pipe in the vertical direction. As shown in table 3 below, when the time t for thorough mixing was 10s, R, ρ, R, m, and γ were all actually measured, and the following relational expressions were obtained by analysis
Table 3: gamma 1 and gamma calculation results
As can be seen from the above table, γ is 1.3 × γ 1.
Meanwhile, the inventor of the present invention further verifies that as shown in table 4, when the allowable sufficient blending time is gradually increased, the value γ 1 calculated according to the actually measured R, ρ, R, m still satisfies
And has a relationship of 1.3 x γ 1 with the measured value γ.
TABLE 4
Based on experimental results, the included angle beta between the two central axes is more than 0 and less than or equal to 3.6 degrees, the cost and the blending efficiency are high under the condition, and the preferable value is that beta is 1.5 degrees.
As shown in fig. 5, 7, 9 and 15, the magnetic attraction device 6 is fixed on the door-shaped mounting base 22, and includes a magnetic attraction module 63 and a displacement module for driving the magnetic attraction module 63 to approach or separate from the target reagent tube rotated to be located right below the straw connector, in this embodiment, the magnetic attraction module 63 is constructed by using permanent magnets; the displacement module comprises an L-shaped first mounting bracket 60 fixedly connected with the door-shaped mounting seat 22, a swing arm 61 which can be arranged on the first mounting bracket 60 in a swinging manner around a first swinging axis 6000, a swinging driver 62 for driving the swing arm 61 to swing, and trigger sensors 64 and 65 which are arranged on the first mounting bracket 60 and are used for carrying out in-place detection on the positions, close to the target reagent tube, and the positions, far away from the target reagent tube, of the magnetic absorption module 63, wherein the trigger sensors 64 and 65 are constructed by adopting travel switches; in the present embodiment, the first swing axis 6000 is orthogonal to the first vertical axis 100, i.e., the first swing axis 6000 is arranged in the horizontal direction.
In this embodiment, the magnetic attracting device 6 is disposed beside the side directly below the tip interface 41, so that during the operation, the shifting module can drive the magnetic attracting module to approach or separate from the side wall of the target reagent tube rotated to the position directly below the tip interface 41, so as to attract the magnetic beads loaded in the target reagent tube to the tube wall, thereby facilitating the sucking of the liquid from the tip 01 without taking away the magnetic beads and the nucleic acids adsorbed on the magnetic beads.
Based on the nucleic acid extraction apparatus 1 configured as described above, the control method in the extraction process thereof includes the steps of storing a computer program in a memory of the control unit, the computer program being executable by a processor of the control unit to implement the steps of:
and a suction head mounting step S1, wherein the rotary driver 30 is controlled to drive the rotary support 7 to rotate around the first vertical axis 100 until the suction head 01 suspended on the rotary support is positioned right below the suction pipe joint 41, the lifting mechanism is controlled to drive the suction pipe joint 41 to descend and be detachably sleeved and fixedly connected with the upper port of the suction head 01, and then the lifting mechanism is controlled to drive the suction pipe joint 41 to lift the suction head 01 to the upper side of the rotary support 7, namely to the position avoiding the rotation process of the test tube rack 3.
In the sample sucking step S2, the rotary driver 30 is controlled to rotate the rotary support 7 around the first vertical axis 100 until the sample tube 049 suspended thereon is positioned right below the pipette tip 41, the lifting mechanism is controlled to drive the pipette tip 41 carrying the pipette tip 01 to descend so that the pipette tip is inserted into the sample tube 049, the liquid transfer pump 44 is controlled to suck the sample to be extracted through the pipette tip 01, and the lifting mechanism is controlled to drive the pipette tip 41 carrying the pipette tip 01 to ascend to the side above the rotary support 7.
And a sample injection step S3, controlling the rotary driver to drive the rotary support 7 to rotate around the first vertical axis 100 until the mixing pipe 041 suspended on the rotary support is positioned right below the suction connector 41, controlling the lifting mechanism to drive the suction connector 41 to carry the suction head 01 to descend so as to insert the suction head into the mixing pipe, controlling the liquid transfer pump 44 to inject a sample through the suction head, and controlling the lifting mechanism to drive the suction connector 41 to carry the suction head to ascend to the side positioned above the rotary support 7.
And a lysate sucking step S4, controlling the rotary driver to drive the rotary support 7 to rotate around the first vertical axis 100 until a lysate pipe 048 suspended on the rotary support is positioned right below the straw connector 41, controlling the lifting mechanism to drive the straw connector 41 to carry the pipette tip 01 to descend so that the pipette tip 01 is inserted into the lysate pipe 048, controlling the liquid transfer pump 44 to suck the lysate through the pipette tip 01, and then controlling the lifting mechanism to drive the straw connector 41 to carry the pipette tip 01 to ascend to the side above the rotary support 7.
And a lysate injection step S5, controlling the rotary driver to drive the rotary support 7 to rotate around the first vertical axis 100 until the mixing pipe 041 suspended on the rotary support is positioned right below the suction pipe connector 41, controlling the lifting mechanism to drive the suction pipe connector 41 to carry the suction head 01 to descend so that the suction head 01 is inserted into the mixing pipe 041, controlling the liquid transfer pump 44 to inject the lysate through the suction head 01, and then controlling the lifting mechanism to drive the suction pipe connector 41 to carry the suction head 01 to ascend to the upper side of the rotary support 7.
And an oscillation blending step S6, controlling a vibration generator of the blending oscillation device 5 to apply vibration for a preset time to a blending pipe 041 with the lower end part sleeved in the seat of the vibration transmission sleeve 50.
A waste liquid suction step S7, controlling the magnetic attraction device 6 to drive the magnetic attraction module 63 to approach the tube sidewall of the mixing tube 041, and magnetically attracting the magnetic beads to the tube sidewall; controlling the lifting mechanism to drive the suction pipe joint 41 to carry the suction head 01 to descend so as to enable the suction head 01 to be inserted into the uniform mixing pipe 041, and controlling the liquid transfer pump 44 to suck waste liquid through the suction head 01; then the lifting mechanism is controlled to drive the suction pipe joint 41 to lift the suction head 01 to the upper side of the rotary support 7, and the magnetic suction module is controlled to be far away from the mixing pipe.
And a waste liquid discharging step S8, controlling the rotary driver to rotate the rotary support 7 around the first vertical axis 100 until the waste liquid pipe suspended thereon is located right below the suction pipe joint 41, controlling the lifting mechanism to drive the suction pipe joint 41 to carry the suction head 01 to descend so that the suction head 01 is inserted into the waste liquid pipe, controlling the liquid transferring pump 44 to inject waste liquid through the suction head 01, and then controlling the lifting mechanism to drive the suction pipe joint 41 to carry the suction head 01 to ascend to the upper side of the rotary support 7.
A cleaning step S9, controlling the rotary driver to drive the rotary support 7 to rotate around the first vertical axis 100 until a cleaning liquid pipe 046 hung on the rotary support is positioned right below the suction pipe joint 41, then controlling the lifting mechanism to drive the suction pipe joint 41 to carry the suction head 01 to descend so that the suction head 01 is inserted into the cleaning liquid pipe 046, controlling the liquid transfer pump 44 to suck cleaning liquid through the suction head 01, and then controlling the lifting mechanism to drive the suction pipe joint 41 to carry the suction head 01 to ascend to the upper side of the rotary support 7; and when the waste liquid pipe rotates under the waste liquid pipe, the waste liquid pipe is discharged into the waste liquid pipe, and the cleaning of the suction head is completed.
And an eluent sucking step S10, wherein the rotary driver is controlled to drive the rotary support 7 to rotate around the first vertical axis 100 until the eluent tube 042 suspended on the rotary support is positioned right below the suction connector 41, the lifting mechanism is controlled to drive the suction connector 41 to carry the suction head 01 to descend so that the suction head 01 is inserted into the eluent tube 042, the liquid transferring pump 44 is controlled to suck eluent through the suction head 01, and then the lifting mechanism is controlled to drive the suction connector 41 to carry the suction head 01 to ascend to the upper side of the rotary support 7.
And an eluent injecting step S11, controlling the rotary driver to drive the rotary support 7 to rotate around the first vertical axis 100 until the mixing pipe 041 suspended on the rotary support is positioned right below the suction connector 41, controlling the lifting mechanism to drive the suction connector 41 to carry the suction head 01 to descend so that the suction head 01 is inserted into the mixing pipe 041, controlling the liquid transfer pump 44 to inject eluent through the suction head 01, and then controlling the lifting mechanism to drive the suction connector 41 to carry the suction head 01 to ascend to the upper side of the rotary support 7.
The step S12 is repeated, and at least the washing step S9, the waste liquid suction step S7, the waste liquid discharge step S8, the eluent suction step S10 and the eluent injection step S11 are repeated in this order, and the waste liquid suction step S7, the waste liquid discharge step S8 and the washing step S9 are repeated again.
And a finished product sucking step S13, controlling the rotary driver to drive the rotary support 7 to rotate around the first vertical axis 100 until the mixing pipe 041 suspended on the rotary support is positioned right below the suction pipe connector 41, controlling the lifting mechanism to drive the suction pipe connector 41 to carry the suction head 01 to descend so as to insert the suction head 01 into the washing and mixing pipe, controlling the liquid transferring pump 44 to suck magnetic beads through the suction head 01, and then controlling the lifting mechanism to drive the suction pipe connector 41 to carry the suction head 01 to ascend to the upper side of the rotary support 7.
And a finished product injecting step S14, controlling the rotary driver to drive the rotary support 7 to rotate around the first vertical axis 100 until the finished product tube 047 suspended on the rotary support is positioned right below the suction connector 41, controlling the lifting mechanism to drive the suction connector 41 to carry the suction head 01 to descend so as to insert the suction head 01 into the finished product tube, controlling the liquid transferring pump 44 to inject magnetic beads through the suction head 01, and then controlling the lifting mechanism to drive the suction connector 41 to carry the suction head 01 to ascend to the upper side of the rotary support 7.
And a suction head 01 dismounting step S15, controlling the rotary driver to drive the rotary support 7 to rotate around the first vertical axis 100 until the suction head 01 suspended on the rotary support is positioned right below the suction pipe joint 41, controlling the lifting mechanism to drive the suction pipe joint 41 to descend and be detachably sleeved and fixedly connected with the upper port of the suction head 01, and then controlling the lifting mechanism to drive the suction pipe joint 41 to lift the suction head 01 to be positioned on the upper side of the rotary support 7.
As can be seen from the above steps, the pipetting system 4 is used to transfer target solutions between the individual reagent vessels during operation.
In the above-described embodiment, the "angle β between the central axis of the first outer coupling shaft portion 54 and the central axis of the second outer coupling shaft portion 55" is configured as one of the largest projected angles of the two central axes in projection in a vertical plane, which is the extending arrangement direction of the rotary drive shafts; when they are coplanar, they are included in the coplanar plane.
Claims (10)
1. A blending device based on eccentric oscillation comprises an installation support and at least one pipe installation support used for installing a blending pipe in an uninstallable way; the method is characterized in that:
the mounting bracket is provided with a blending oscillation device, the blending oscillation device is positioned on the lower side of the pipe mounting support and comprises a vibration transmission sleeve seat which is sleeved on the lower end part of the blending pipe and a vibration generator which is used for applying vibration to the blending pipe through the vibration transmission sleeve seat;
the vibration generator comprises a rotating driving shaft and an eccentric transmission shaft which are vertically arranged, and the eccentric transmission shaft comprises a first external connection shaft part and a second external connection shaft part which are fixedly connected into an integral structure; the central axes of the installation circumferential surfaces of the first outer coupling shaft part and the second outer coupling shaft part have eccentric distance at the fixed connection part of the first outer coupling shaft part and the second outer coupling shaft part; the first external coupling shaft part is in transmission connection with the rotary driving shaft, and the second external coupling shaft part is rotatably sleeved and connected with the vibration transmission sleeve seat through a bearing; and the central axes of the two outer coupling shaft parts form an acute angle beta.
2. The blending device of claim 1, wherein:
the value range of the acute angle beta is more than 0 and less than or equal to gamma,
wherein R is the inner diameter of the mixing pipe, rho is the average density of substances in the mixing pipe, R is the driving rotating speed of the rotating driving shaft, t is the sufficient mixing time, m is the preset eccentric distance, and c is a proportionality constant.
3. The blending device of claim 2, wherein:
the value range of the acute angle beta is more than 0 and less than or equal to 3.6 degrees.
4. The blending device of claim 2 or 3, wherein:
the value range of the acute angle beta is more than or equal to 1.5 degrees and less than or equal to 3.6 degrees;
the proportionality constant C is 1.3.
5. The blending apparatus according to any one of claims 1 to 4, wherein:
the pipe mounting support is used for mounting the blending pipe in a hanging manner.
6. The blending apparatus of any one of claims 1 to 5, wherein:
the central axes of the two outer coupling parts are arranged in a coplanar manner;
the first outer coupling shaft part is provided with a shaft hole for sleeving the outer part of the rotary driving shaft, and the circumferential surface of the shaft hole forms the installation circumferential surface of the first outer coupling shaft part; the second external coupling shaft part is sleeved on an inner ring of the bearing, and a sleeve hole sleeved on an outer ring of the bearing is formed in the lower end part of the transfer sleeve seat; the sleeving surface of the inner ring forms the installation circumferential surface of the second outer coupling shaft part;
the rotary driving shaft is a rotor shaft of a rotary driving motor;
the second external shaft part and a trepan boring used for sleeving the target reagent tube on the vibration transmission sleeve seat are arranged on the same central axis.
7. A nucleic acid extraction device based on a magnetic bead method comprises a bracket, and a mixing device and a magnetic attraction device which are arranged on the bracket; the method is characterized in that:
the blending device is as claimed in any one of claims 1 to 6;
the magnetic attraction device comprises a magnetic attraction module and a displacement module, wherein the displacement module is used for driving the magnetic attraction module to be close to or far away from the outer wall surface of the mixing pipe.
8. The nucleic acid extraction apparatus according to claim 7, characterized in that:
the nucleic acid extraction equipment comprises a liquid transfer system arranged on the bracket, and the liquid transfer system is used for adding reagents into the mixing tube and sucking the reagents from the mixing tube;
the liquid transfer system comprises a suction pipe joint for detachably mounting the suction head and a lifting driving mechanism for driving the suction pipe joint to lift relative to the bracket.
9. The nucleic acid extraction apparatus according to claim 8, characterized in that:
the liquid transfer system comprises an unloading mechanism, the unloading mechanism is used for pushing the suction heads sleeved on the suction pipe joints down, the unloading mechanism comprises a material pushing plate and a linear displacement output device used for driving the material pushing plate to vertically reciprocate, and the material pushing plate is provided with a sleeving hole sleeved outside the suction pipe joints.
10. The nucleic acid extraction apparatus according to claim 8 or 9, characterized in that:
the tube mounting support is rotatably mounted on the mounting bracket about a first vertical axis; a plurality of sleeving openings which are arranged around the first vertical axis are distributed on the outer peripheral edge of the pipe mounting support and are used for hanging the mixing pipe, other reagent pipes and the suction head in a manner of being pulled out from the upper side;
the blending device comprises a rotary driver for driving the tube mounting support to rotate around the first vertical axis, and the tube mounting support can rotate to a position under the pipette joint one by one, wherein the reagent tubes and the pipette heads are carried by the tube mounting support;
the shifting module is used for driving the magnetic suction module to be close to or far away from the outer wall surface of the mixing pipe which is positioned right below the suction pipe joint in a rotating mode.
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| CN112852611A (en) * | 2020-11-26 | 2021-05-28 | 珠海启奥生物技术有限公司 | Nucleic acid extraction equipment and control method thereof |
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| CN112844151B (en) | 2022-12-20 |
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Address after: 201112 Room 202, building 11, No. 789, Puxing Road, Minhang District, Shanghai Applicant after: Shanghai Jinnuo Aomei Biotechnology Co.,Ltd. Address before: 104c, block a, Zhuhai International Health port testing office building, 628 Airport West Road, Sanzao Town, Jinwan District, Zhuhai City, Guangdong Province, 519090 Applicant before: Zhuhai Qiao Biotechnology Co.,Ltd. |
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Inventor after: Chen Qiyue Inventor after: Deng Jing Inventor after: Liu Jun Inventor after: Jin Xiaojun Inventor after: Sun Zhiwei Inventor after: Zhang Binbin Inventor before: Chen Qiyue Inventor before: Deng Jing Inventor before: Liu Jun Inventor before: Jin Xiaojun Inventor before: Sun Zhiwei Inventor before: Zhang Binbin |
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